Download 博士論文 Analysis of gene function involved in plant organ

博士論文 （要約）
Analysis of gene function involved in plant organ development using
Arabidopsis thaliana mutants.
シロイヌナズナ変異体を用いた植物器官形成に関わる遺伝子の解析
平成 25 年 1 月 博士(理学)申請
鎌田 直子
Contents
Acknowledgement
2
List of Abbreviation
3
Abstract
6
General Introduction
9
Chapter I. Stunted growth of acaulis1 (acl1) mutants is dependent on constitutive
activation of defense response pathways
Introduction
12
Results
15
Discussion
31
Materials and Methods
42
Tables
49
Figures
Chapter II. Mutations in epidermis-specific HD-ZIP IV genes affect floral organ identity
in Arabidopsis thaliana.
Introduction
62
Results
65
Discussion
72
Materials and Methods
77
Tables
82
Figures
General Discussion
84
References
86
1
Acknowledgement
I thank Dr. Jane E. Parker for kindly providing eds1-2 seeds in Col background, Dr. Ken
Shirasu for rar1-21 and sid2-2 seeds, and Dr. Goro Horiguchi for 2080 seeds. I am also
grateful to Dr. Tetsuya Kurata for being helpful in performing the next-generation
sequencing, Dr. Minako Ueda for providing the pBarMap vector, Prof. Hiroo Fukuda
for supporting experiments using confocal microscopy, Dr. Hiroyasu Motose for useful
advice and Ms. Hitomi Okada for supporting the experiments. I am also grateful to Dr.
Sarah-mi Ishida for critical reading of the manuscript, and Dr. Susan D. Fischer for
kindly giving comments on English writing. I want to express sincere gratitude to Prof.
Taku Takahashi for critical reading of the manuscripts and providing materials and lab
space to perform most of the experiments described in the chapter II.
2
List of abbreviations
acl
acaulis
ACT8
ACTIN8
AG
AGAMOUS
AP3
APETALA3
ATML1
ARABIDOPSIS THALIANA MERISTEM LAYER1
BDG
BODYGUARD
CAPS
cleaved-amplified polymorphic sequence
CC
coiled-coil
Col
Columbia
EDS1
ENHANCED DISEASE SUSCEPTIBILITY1
ETI
Effector-triggered immunity
FDH
FIDDELHEAD
GA
gibberellin
GL2
GLABRA2
HD-ZIP IV
class IV homeodomain-leucine zipper
HDG
HOMEODIMAIN GLABROUS
HSP90
HEAT SHOCK PROTEIN90
KLU
KLUH
LFY
LEAFY
LTP
LIPID TRANSFER PROTEIN
3
Ler
Landsberg erecta
MS
Murashige and Skoog
NBS-LRR
nucleotide binding site-leucine-rich repeat
NPR1
NONEXPRESSOR OF PR GENES1
OCL
OUTER CELL LAYER
PAD4
PHYTOALEXIN-DEFICIENT4
PCR
polymerase chain reaction
PDF1.2
PLANT DEFENSIN1.2
PDF2
PROTODERMAL FACTOR2
PI
PISTILATA
PR
pathogenesis-related
R
Resistance
RAR1
REQUIRED FOR MLA12 RESISTANCE1
RPP
recognition of Peronospora parasitica
RPT
regulatory particle triple-ATPase
RT
reverse transcriptase
SA
salicylic acid
SAD
START-adjacent
SAR
systemic acquired resistance
SEP3
SEPALLATA3
SGT1
SUPPRESSOR OF THE G2 ALLELE OF SKP1
SID
SA INDUCTION DEFICIENT
4
SNC1
SUPPRESSOR OF NPR1, CONSTITUTIVE1
SRFR1
SUPPRESSOR OF rps4-RLD
SSLP
simple sequence length polymorphism
START
steroidogenic acute regulatory protein-related lipid transfer
TIR
Toll/interleukin-1 receptor
TPR
tetratricopeptide repeat
UFO
UNUSUAL FLORAL ORGANS
ZLZ
zipper-loop-zipper
5
Abstract
Chapter I
acaulis1 (acl1) mutants are isolated in order to explore novel genetic factors
that regulate elongation of inflorescence stem. In the first part, I report on the
identification of an inversion mutation in the original acl1-1 plants. Compared to the
original acl1-1 plants, the “genuine” acl1-1 plants, which is without the inversion, grew
larger and their inflorescence stems grew longer at 22°C and also at 24˚C. In the acl1-1
plants with the inversion, two genes that locate at each end of the inversion were
disrupted and full-length transcripts were not detected, and expressions of some genes
within and adjacent to the inversion were also altered. These results suggest the
possibility that the expression of multiple genes is involved in the enhancement of the
acl1-1 phenotype by the inversion .
In the second part, I further investigated the acl1 mutants using the acl1-1
mutant line without the inversion. I found Col accession-specific Resistance (R) gene,
SUPPRESSOR OF NPR1, CONSTITUTIVE1 (SNC1) as an essential gene for the acl1
growth phenotype. Moreover, I identified the acl1 mutations in SUPPRESSOR OF
rps4-RLD (SRFR1), which is known as a negative regulator of defense responses and
SNC1 activity. These results suggest that the loss of negative regulation of SNC1 causes
stunted growth in the acl1 mutants. Consistent with the srfr1 mutants, which have
already been described, acl1 mutants showed constitutive activation of defense-related
genes at 22˚C. I further observed the acl1 phenotype at intermediate temperatures
6
between 22˚C and 28˚C, at which the srfr1 phenotype was reported to be suppressed. It
was revealed that both stunted growth and increased expression of defense-related genes
were gradually repressed as temperature increases and almost completely suppressed at
temperatures above 26˚C. Double mutant analysis revealed that the acl1 plant growth
depends not only on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) but also on
PHYTOALEXIN-DEFICIENT4 (PAD4) and REQUIRED FOR MLA12 RESISTANCE1
(RAR1). However, salicylic acid accumulation and NONEXPRESSOR OF PR GENES1
(NPR1)-dependent pathways were not essential for the acl1 growth phenotype. I also
discovered that higher ammonium concentration in the growth media alleviates the acl1
phenotype. Nitrogen might be involved in the growth regulation of the plants with
induced defense responses.
Chapter II
Development of the epidermis involves members of the class IV
homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis
HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR2
(PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) play an
indispensable role in the differentiation of shoot epidermal cells. However, the functions
of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis
remain not fully elucidated. Construction of double mutant combinations of these
HD-ZIP IV mutant alleles showed that the double mutants of pdf2-1 with homeodomain
glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2, produced abnormal flowers with
7
sepaloid petals and carpelloid stamens in association with reduced expression of the
petal and stamen identity gene APETALA3 (AP3). Expression of another petal and
stamen identity gene PISTILATA (PI) was less affected in these mutants. I confirmed
that the AP3 expression in pdf2-1 hdg2-3 was normally induced at initial stages of
flower development but attenuated both in the epidermis and internal cell layers of
developing flowers. Since the expression of PDF2 and these HD-ZIP IV genes during
floral organ formation is exclusively limited to the epidermal cell layer, these double
mutations may have non-cell-autonomous effects on the AP3 expression in the internal
cell layers. My results suggest that cooperative functions of PDF2 and other members
of the HD-ZIP IV family in the epidermis are crucial for normal development of floral
organs in Arabidopsis.
Cited and revised from Summary of Kamata et al., 2013. “Mutations in
epidermis-specific HD-ZIP IV genes affect floral organ identity in Arabidopsis
thaliana” Plant Journal, 75, 430-440.
8
General Introduction
Arabidopsis thaliana is a model plant that widely used for plant genome
analysis (Somerville and Koornneef, 2002). Since a large collection of mutants and
transgenic plants, whose growth and development have been disrupted, is available, a
forward genetic approach has been practical for discovering genetic factors that regulate
morphogenesis in A. thaliana. In the year 2000, the whole genome of the A. thaliana
has been sequenced, enabling to presume the function of the genes that are not yet
experimentally verified. Thus, the reverse genetic approach is also being a powerful tool
to search and investigate whether and how the gene in concern is related to the plant
growth and development.
Most organogenesis in plants, as well as in A. thaliana, is occurred
postembryonically, unlike animals, in which the most of the organs are formed during
embryogenesis (Carles and Fletcher, 2003). Both shoot and root apical meristems of
plants maintain their activity and give rise to new organs after the germination, allowing
plants to continue growing and developing their bodies through their lifetime. Although
the postembryonic development of plant body is primarily genetically regulated, it is
also highly flexible to the environmental factors, including biotic factors such as heat,
cold, light, drought and nutrient condition, and biotic factors like attacks from pests and
pathogens, to adapt to the environmental changes. The effects from the environmental
changes on the plant growth and development cannot be ignored, as it has been
suggested that environmental stresses can reduce average yields by as much as >50%
for most major crop plants (Wang et al., 2003).
9
acaulis1 (acl1) mutants, which were previously isolated in a genetic screen
for defective plant morphology, were further discovered to be an unique type of mutants
that their growth phenotype is fully suppressed at higher growth temperatures (Tsukaya
et al., 1993). Even though the growth of Arabidopsis plants is affected by higher
temperature, the morphological modification in the acl1 plants is so drastic that it
cannot be explained by general effects from higher growth temperature (Thingnaes et
al., 2003). Thus I thought that acl1 would be more than a tool for investigating a genetic
factor that regulate normal plant growth, and give us some more insight into a
relationship between a growth regulatory pathway and the environmental factors, such
as temperature. And in the first chapter of this thesis, I will report on the analysis on the
acl1 mutants. Since the ACL1 gene had not been isolated, the biggest issue on the acl1
analysis was to identify the ACL1 gene, and to make it clear which genetic pathways are
involved in the acl1 plant growth. I identified some genetic factors that affect the acl1
phenotype, and eventually identified the acl1 mutations in a negative regulator of
defense responses against pathogens.
As shown in the research on the acl1 mutants, and also in some other
acl1-like mutants (Gou and Hua, 2012), responses against pathogen attacks can
dramatically modify the plant morphology. When plants are exposed to such pathogens,
epidermis, the outermost cell layer that covers the plant body, plays the critical roles for
the defense and resistance. Shoot epidermis is also important for organ separation and
defense responses against drought or other environmental stresses as well as in the
integrity of organs. Moreover, it has been reported that epidermis-specific genes are
10
involved in the regulation of organ development in plants (Savaldi-Goldstein et al.,
2007; Eriksson et al., 2010). Therefore, it is likely that epidermis is an important cell
layer for regulating both responses against environmental factors and plant growth. In
the second chapter, I adopted reverse genetic approach to investigate the effects of
T-DNA insertion mutations in the class IV homeodomain-leucine zipper (HD-ZIP IV)
gene family, most of which are confirmed to be specifically expressed in epidermis
(Nakamura et al., 2006), on plant growth and development.
11
Chapter I. Stunted growth of acaulis1 (acl1) mutants is dependent on
constitutive activation of defense response pathways.
Introduction
For exploring genetic factors that are essential for proper morphogenesis of
Arabidopsis thaliana, a forward genetic approach has been a useful tool. Tsukaya et al.
(1993) previously screened for mutants defective in the elongation of the inflorescence
stem and able to identify five complementary groups of mutants, termed acaulis (acl),
for their “stalkless” morphology. Short inflorescence stems and reduced number of
flowers in the acl mutants were due to early proliferative arrest of apical inflorescence
meristems. The number of rosette leaves in the acl mutants is approximately the same as
in wild-type plants, indicating that the timing of transition from vegetative to
reproductive phase is not affected. However, except for the acl5 mutant, the acl mutants
exhibited more or fewer defects in leaf morphology (Tsukaya et al., 1993; Hanzawa et
al., 1997; Akamatsu et al., 1999). Since the acl phenotype cannot be rescued by the
exogenous addition of several growth regulators and phytohormones, the stunted
growth of the acl1 mutants is considered to be different from the dwarfism of known
phytohormone-related mutants (Tsukaya et al., 1993; Hanzawa et al., 1997; Akamatsu
et al., 1999). The only ACL gene identified was ACL5, which encodes thermospermine
synthase (Hanzawa et al., 2000; Kakehi et al., 2008). However, it is still uncertain
which genetic pathway is involved in the regulation of plant growth and development of
12
other acl mutants.
acl1-1 is the most severely stunted mutant among the acl mutants. Cell
elongation and maturation are likely to be inhibited soon after the cells differentiate in
the acl1-1 plants, as implied by the drastic reduction of cell length in the acl1-1 stems
and the loss of intercellular spaces in the acl1-1 leaves (Tsukaya et al., 1993). In order
to determine the role of the ACL1 gene within the developmental network of
inflorescences, double mutants were generated between the acl1-1 mutant and some
developmental mutations that affect the morphology of inflorescences and/or flowers.
However, the function of the ACL1 gene has been shown to be genetically independent
of the shoot- and inflorescence-development genes, such as APETALA1 (AP1),
CLAVATA (CLV1), LEAFY (LFY) and TERMINAL FLOWER1 (TFL1) (Tsukaya et al.,
1993).
It has also been reported by Tsukaya et al. (1993) that stunted growth of the
acl1 mutants is restored at higher temperature, such as 28˚C. Ambient temperature is
known to influence aspects of the appearances of plants, such as leaf size and stem
length (Thingnaes et al., 2003; Atkin et al., 2006). However, the dramatic alteration in
morphology of the acl1 phenotype at high temperature cannot be explained by general
developmental variations controlled by ambient temperature. Temperature-sensitive
stunted phenotypes similar to the acl1 mutants are often observed in suppressor of
NPR1, constitutive1-1 (snc1-1), suppressor of npr1-5-based salicylic acid insensitivity4
(ssi4), bonzai1 (bon1) and constitutive expresser of PR genes 30 (cpr30), all of which
have constitutive activation of defense responses against pathogens (Hua et al., 2001;
13
Shirano et al., 2002; Yang and Hua, 2004; Gou et al., 2009). Effector-triggered
immunity (ETI) is a major defense response in plants that is induced by direct or
indirect recognition of pathogen avirulence effectors by plant resistance (R) proteins
(Jones and Dangl 2006; Alcazár and Parker 2011). In Arabidopsis, a majority of R
proteins possess a nuclotide binding site and leucine-rich repeat (NBS-LRR) motif
either with a Toll/interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain at
the N terminus (Dangl and Jones, 2001). For the activation of downstream defense
response pathways, TIR-NBS-LRR type R proteins require ENHANCED DISEASE
SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN-DEFICIENT4 (PAD4) genes, both of
which encode a protein with homology to lipases/acyl hydrolases (Falk et al., 1999;
Jirage et al., 1999; Feys et al., 2001). On the other hand, CC-NBS-LRR type R proteins
require NON RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), which encodes a
putative membrane-bound protein (Century et al., 1995; 1997; Aarts et al., 1998). ETI is
usually accompanied by a hypersensitive response involving rapid and local
programmed cell death, which restricts further spread of the pathogen. Furthermore,
plants produce salicylic acid (SA) and accumulate defense molecules such as
pathogenesis-related (PR) proteins through systemic acquired resistance (SAR). SAR
provides the plants with long-lasting protection against a broad spectrum of pathogens
(Ryals et al., 1996; van Loon, 1997).
The cloning of the ACL1 gene will give us the answer how the acl1 plant
growth is regulated. During molecular mapping of the acl1 mutations, I identified an
inversion mutation in the original acl1-1 mutant line, and a Col accession-specific
14
TIR-NBS-LRR R gene, SUPPRESSOR OF NPR1, CONSTITUTIVE1 (SNC1) as an
indispensable gene for the acl1 phenotype to be exhibited in the presence of the acl1
mutations. I eventually identified the acl1 mutations in SUPPRESSOR OF rps4-RLD
(SRFR1), a negative regulator of defense responses, suggesting that the acl1 phenotype
is caused by a constitutive activation of defense response pathways. I further analyzed
the acl1 mutants and will report on several phenotypes that yet have not been described
in previous studies in the acl1/srfr1 mutants.
Results
Part 1
An inversion identified in acl1-1 mutant functions as an enhancer of
the acl1-1 phenotype
Identification of an inversion mutation in the original acl1-1 mutant and isolation
of novel acl1-1 line without the inversion
Previous research showed that the acl1-1 mutation locates close to
AGAMOUS (AG) on chromosome 4 (Tsukaya et al., 1993). My early results from
molecular mapping suggested a linkage of the acl1-1 phenotype between a
cleaved-amplified polymorphic sequence (CAPS) marker, SC5, and the polymorphism
MASC04642 on chromosome 4 (Figure I-1a). The acl1-1 mutant was induced by X-ray
irradiation and exhibits a more severe phenotype than the EMS-mutagenized acl1-3
15
mutant. Thus, I suspected that the chromosomes of the acl1-1 mutant might be seriously
disrupted. In order to explore chromosome disruptions, approximately 260 kb of the
genomic region from At4g22290, the gene nearest to MASC04642, to At4g21690 was
examined by amplifying fragments covering the genomic regions by PCR. As the result,
two PCR products including At4g21960, which encodes a peroxidase (Apel and Hirt,
2004; Welinder et al., 2002), and At4g22250, which encodes a zinc finger protein
(Kosarev et al., 2002), were absent in the acl1-1 mutant (Figures I-1a,b). The genomic
regions between these two genes, which are located at a distance of approximately 120
kb, were found to exist in the acl1-1 mutant (data not shown). Every PCR product was
present in the acl1-3 mutant, which indicated that the absence of the two PCR products
was specific to the acl1-1 mutant. One possibility was that the genomic region between
At4g21960 and At4g22250 was inverted. To investigate this possibility, PCR
experiments were performed using combinations of primers at At4g21960 and
At4g22250. Novel DNA fragments were amplified in the acl1-1 mutant and their
sequences suggested that At4g21960 and At4g22250 were cleaved, inverted and fused
each other (Figure I-1c,d). I also cloned At4g21960 and At4g22250 from the acl1-3
plants and found that there was no mutation. Since the T-DNA insertion lines in
At4g21960 and At4g22250 had no obvious growth defects (data not shown), the loss of
either of At4g21960 or At4g22250 is unlikely to be responsible for the acl1-1
phenotype.
I crossed the original acl1-1 plants to Col-0 to evaluate the segregation of the
acl1-1 phenotype and the inversion. The F2 population was segregated into 496 (73%)
16
wild-type plants and 180 (27%) acl1-1 plants at a 3:1 ratio (χ2 = 0.716). I further
examined 371 wild-type plants and 146 acl1-1 plants for the inversion (Table I-1).
Regarding the inversion, the segregation at +/+: inv/+: inv/inv at the ratio of 1:2:1 was
less reliable (χ2 0.025 (2) = 7.38 < χ2 = 9.07 < χ2 0.010 (2) = 9.21). The acl1-1
phenotype did not necessarily cosegregate with the inversion, and the inversion is
independent of the cause of the acl1-1 phenotype. The recombination rate between the
acl1-1 phenotype and the inversion was estimated to be 15.4% according to the
segregation of the F2 plants. To simplify descriptions, I refer to the newly isolated
acl1-1 plants without the inversion as acl1-1 +/+, the original acl1-1 plants with the
inversion as acl1-1 inv/inv, wild-type plants as Col +/+, and the wild-type phenotype
(ACL1/ACL1) plants with the inversion as Col inv/inv.
Expressions of genes related to the inversion
Inversions can disrupt a gene at one of its breakpoints, and furthermore, it is
expected that inversions alter the expression of a gene near a breakpoint because of a
change in its chromosomal environment. Expression of some genes, which locate within
and adjacent to the inversion, differed among the four plant strains: Col +/+, Col inv/inv,
acl1-1 +/+, and acl1-1 inv/inv (Figure I-2a). I observed a decrease in expression of
At4g22270 (IMMUTANS) (Wu et al., 1999), an increase in At4g22214 (defensin like
protein) (Silverstein et al., 2005), and a slight increase in At4g22235 (defensin like
protein) in two inv/inv plants, Col inv/inv and acl1-1 inv/inv. In the acl1-1 inv/inv plants,
we found increased expression of At4g22050 (aspartyl protease family protein) and
17
At4g22070 (WRKY DNA-BINDING PROTEIN 31) (Eulgem et al., 2000) and decreased
expression of At4g21990 (APS REDUCTASE 3) (Houston et al., 2005), At4g22010
(SKU5 SIMILAR 4) (Sedbrook et al., 2002) and At4g22212 (defensin like protein).
Among the genes investigated, At4g22080 (pectate lyase family protein), At4g22090
(pectate lyase family protein), At4g22210 (Cys-rich protein), At4g22217 (defensin like
protein) and At4g22230 (defensin like protein) decreased in both the acl1-1 plants,
regardless of the inversion. The expression of At4g22030 (F-box family protein) and
At4g22100 (glycosyl hydrolase family 1 protein) was decreased in the Col inv/inv plants
and the acl1-1 +/+ plants, and even further decreased in the acl1-1 inv/inv plants
compared to the Col +/+ plants. I wondered if the increase or the decrease in the
expression level of these genes is due to mutations in their genomic sequences.
However, the genomic sequences of these genes cloned from acl1-1 plants were
identical to the wild-type.
Although full-length transcripts of At4g21960 and At4g22250 were not
detected in the inv/inv plants (Figure I-1e), partial transcripts from the fused fragments
of At4g21960 and At4g22250 were detected in the inv/inv plants (Figure I-2b). In the
case of At4g22250, the partial transcripts were rather increased in the plants with the
inversion. Taken together, these results from expression analysis suggest that the
inversion has an influence on the expression of a wide range of genes.
In the acl1-3 mutants, the expression patterns of the genes related to the
inversion, including At4g21960 and At4g22250, were similar to those of the Col +/+
plants (data not shown).
18
The acl1-1 phenotype was enhanced by the inversion
While there was no apparent difference between Col inv/inv plants and Col
+/+ plants (Figure I-3a, Table I-2) and between acl1-3 inv/inv and acl1-3 +/+ plants
(Figure I-5), the comparison of the acl1-1 +/+ plants and the acl1-1 inv/inv plants made
me realize the difference between these two genotypes. Both the acl1-1 +/+ plants and
the acl1-1 inv/inv plants exhibited the acl1-1 phenotype with short inflorescence stems
and small curly leaves. However, the rosettes of the acl1-1 inv/inv plants appeared
slightly smaller than those of the acl1-1 +/+ plants (Figure I-3a). The height of the
acl1-1 +/+ plants was significantly different to that of the acl1-1 inv/inv plants,
indicating that the growth defects of the acl1-1 mutants are enhanced by the inversion
(Table I-2).
The reduction of the cell length was significant in acl1-1 background in all
types of tissues examined; cells in the epidermis, the outermost layer of cortex, and the
pith (Table I-3, Figure I-4a). The reduction in the size of the epidermal cells was most
severe, and the cells in pith also were significantly affected by the acl1-1 mutation. The
acl1-1 inv/inv plants exhibited more severe reduction in the cell length than the acl1-1
+/+ plants. Unlike cells in epidermis and pith, the length of cortex cells was less
affected. In addition to the severe reduction in length, the differentiation of cells
appeared to be inhibited in the inflorescence stems of the acl1-1 +/+ plants and the
acl1-1 inv/inv plants.
Both the acl1-1 +/+ plants and the acl1-1 inv/inv plants were able to restore
19
their plant morphology when grown at 28˚C as previously reported (Tsukaya et al.,
1993). Plants tend to become more slender at higher temperatures, as the result of the
general effects of higher temperature. These general effects of higher temperature were
observed in the Col inv/inv plants as well as in the Col +/+ plants throughout the
experiments. Correspondingly, the cells became longer at higher temperature (Figures
I-4b,c). The restoration of the acl1-1 phenotype to wild-type was not complete at the
intermediated temperature 24˚C (Figure I-3b). However, the difference between the
acl1-1 +/+ plants and the acl1-1 inv/inv plants became more obvious at 24˚C. The
inflorescence stems of acl1-1 +/+ plants elongated to approximately 5 cm in length
(average ± standard deviation, 5.2 ± 3.4 cm, n = 12). On the other hand, the
inflorescence stem of acl1-1 inv/inv plants was as short as that grown at 22˚C. At 24˚C
the length of cells, including those in the cortex, became significantly different between
the acl1-1 +/+ plants and the acl1-1 inv/inv plants (Table I-3). While neither the acl1-1
+/+ plants nor the acl1-1 inv/inv plants fully restored the acl1-1 phenotype at 24˚C, the
inflorescence stems of both the acl1-1 +/+ plants and the acl1-1 inv/inv plants elongated
to a similar length to those of Col plants at 26˚C and showed complete restoration to
wild type (Table I-4, Figures I-4b,c). There was no difference between the acl1-1 +/+
plants and the acl1-1 inv/inv plants at temperatures exceeding 26˚C (t = 0.014 for plant
height at 26˚C).
20
Part 2
Stunted growth of acaulis1 (acl1) mutants is dependent on
constitutive activation of defense response pathways induced by
SUPPRESSOR OF NPR1, CONSTITUTIVE1 (SNC1)
本パートについては最終項を除いて、5年以内に雑誌等で刊行予定のため非公開。
acl1 phenotype requires a Col accession-specific R gene, SNC1
Crude mapping analysis using F2 generation from a cross between acl1-3 and
Landsberg erecta (Ler) showed that the acl1 phenotype linked to the middle to lower
arm of chromosome 4 (Table I-5), consistent with the previous studies (Tsukaya et al.,
1993; Kamata and Komeda, 2008). Four mapping markers, SC5, AG, CIW7 and CAT2,
which span an interval of 7.5 Mb on chromosome 4 has shown equally low
recombination frequencies. After I noticed the presence of the inversion mutation in the
original acl1-1 line (Chapter 1) (Kamata and Komeda, 2008), I used only acl1-1 +/+ for
further analysis of the acl1-1 mutants. I crossed the acl1-1 plant, which is without the
inversion, to Ler and the derived F2 plants showed a similar result (data not shown).
During the mapping process, I found that the ratio of the acl1 phenotype to wild type in
F2 progeny was significantly lower than the expected ratio for a single recessive locus.
In the case of acl1-1, only 14.4% of the F2 progeny showed the acl1-1 phenotype, while
the acl1-1 phenotype was segregated in a Mendelian fashion when crossed with Col
(Table I-6). This implies the existence of a natural modifier of the acl1 phenotype in
Ler.
21
No recombination was found at G4539, which is located between SC5 and
AG, in the F3 acl1-1 plants derived from the F2 lines heterozygous for acl1-1 (Figure
I-6). For finer mapping, F3 and F4 plants were selected, and additional mapping
markers were generated between SC5 and AG (Table I-7). The region tightly linked to
the acl1-1 phenotype was further narrowed to an approximately 159-kb region between
the polymorphisms PERL0772901 and PERL0776597 (Figure I-6b). The genetic region
between PERL0772901 and PERL0776597 in the acl1 mutants was amplified and
sequenced. However, there was no mutation in any genes within this region compared
to Col wild type.
I further investigated the relationship between the acl1 phenotype and the
region identified by the mapping analysis. The 159-kb region in question includes RPP5
(for recognition of Peronospora parasitica 5) gene cluster that consists of multiple
copies of TIR-NB-LRR R genes homologous to the RPP5 gene of Ler (Figure I-6b,c)
(Parker et al., 1997; Noël et al., 1999). The genomic sequence of the RPP5 gene cluster
in Col differs from that in Ler, and synteny is lost in this region between these two
accessions (Stokes et al., 2002). I found that the expression level of RPP4 (At4g16860),
SNC1 (At4g16890), At4g16950 and At4g16960 was increased in the acl1-1 mutant at
22˚C (Figure I-6d). SNC1 and At4g16960 were also increased in the acl1-3 mutant,
though not at as high a level as in acl1-1 in the case of At4g16960. The expression of
At4g16920 was slightly increased in both acl1-1 and acl1-3 mutants. At4g16990, which
is located next to the RPP5 gene cluster, seemed to be expressed at similar levels
between the acl1 mutants and wild-type plants.
22
A gain-of-function mutation of SNC1 such as snc1-1 is known to cause severe
stunted phenotype similar to acl1-1, and many of the Arabidopsis mutants exhibiting
stunted growth with auto-activation of defense responses are SNC1-dependent (Li et al.,
2001; Gou and Hua, 2012). I therefore generated double mutants between acl1 and
snc1-11, the loss-of-function mutation in SNC1, to examine whether the expression of
SNC1 is responsible for the acl1 phenotype. The overall appearance of acl1-1 snc1-11
and acl1-3 snc1-11 double mutants was identical to that in wild-type plants. Moreover, a
heterozygous snc1-11 mutation was enough to cause the restoration of plant growth of
acl1 mutants, even in the more severe acl1-1 allele (Figure I-6e,f). RPP4 is another
RPP5 homologue whose gain-of-function mutant can also cause a severe dwarfism in
plants (Huang et al., 2010). However, I identified 8 plants having homozygous rpp4
mutation among 137 acl1-1 phenotyped plants in F2 generation derived from the cross
between acl1-1 and rpp4. These acl1-1 rpp4 plants were fully identical to the acl1
single mutants. Above results demonstrated that SNC1 is essential for and mainly
responsible for the stunted growth of the acl1 mutants.
ACL1 encodes a tetratricopeptide repeat domain containing protein
Taking into account that the acl1 phenotype is observable only in the presence
of homozygous Col-specific SNC1 locus, the ACL1 gene is calculated to be located 24.2
cM apart from the SNC1 locus (Table I-6). Next-generation sequencing identified a
point mutation in At4g37460 that is located 8.1 Mb, which is equivalent to
approximately 25 cM, apart from SNC1 in each acl1-1 and acl1-3 genome (Figure I-7a).
23
The acl1-1 mutant had a G to A substitution in the conserved AG dinucleotide of the 3’
acceptor splice site at the end of the 10th intron (Reddy, 2007). I obtained the full-length
At4g37460 cDNA from the acl1-1 mutant and found a deletion of single G at the first
base of the 11th exon. Frameshift in the subsequent coding region creates a premature
stop codon at amino acid position 509 (Figure I-7b). A substitution of C for T was found
in the acl1-3 mutant resulting in a replacement of proline by serine at amino acid
residue 618 (Figure I-7a,b). The expression level of At4g37460 was seemingly not
altered in the acl1-1 and acl1-3 mutants (Figure I-7c). Recently, a new acl1 mutant
allele, 2080 (acl1-4), was isolated from the pool of T-DNA insertional mutants and
kingly gifted from Dr. Goro Horiguchi (Figure I-8). The 2080 mutant also had a
disruption in the coding region of At4g37460 and full-length transcript was not detected
(Figure I-7a,c).
At4g37460 encodes a tetratricopeptide repeat (TPR) domain-containing
protein known as SUPPRESSOR OF rps4-RLD (SRFR1) (Kwon et al., 2009). Loss of
functional SRFR1 in Col background causes increased accumulation of SNC1 and also
other R genes, resulting in the constitutive activation of defense responses (Kim et al.,
2010; Li et al., 2010). Moreover, an acl1-1-like severe stunted phenotype is observed in
all those srfr1 mutants isolated previously in the Col background, among which srfr1-3
has a nonsense mutation near the acl1-1 mutation site (Li et al., 2010). I therefore
concluded that the acl1 phenotype is caused by mutations in At4g37460/SRFR1.
Constitutive activation of defense related genes and the stunted growth were
24
correlated in the acl1 mutants in a temperature-dependent manner
Identification of SNC1 and SRFR1 as the genes involved in the acl1
phenotype suggests that the acl1 phenotype occurs through activation of a defense
response pathway. Thus, I examined the expression of defense related genes. Plants
were grown on sterile media to avoid contact with the pathogens potentially present in
the environment. The acl1-1 mutants had significantly increased expression of defense
related genes: EDS1 and PAD4; SA INDUCTION DEFICIENT2 (SID2), which are
required for SA synthesis (Nawrath and Métraux, 1999); and PR1 and PR2, consistent
with the previous expression analysis using the srfr1 mutants in the Col background
(Figure I-9a) (Kim et al., 2010; Li et al., 2010; Bhattacharjee et al., 2011). Moreover, I
observed a remarkable increase in the expression of NDR1, SID1 (Nawrath and Métraux,
1999), NONEXPRESSOR OF PR GENES1 (NPR1), a positive regulator of SA-mediated
SAR (Cao et al., 1997; Ryals et al., 1997), and another PR gene, PR5, in the acl1-1
mutant (Figure I-9a). These results suggest a constitutive activation of defense
responses through ETI and SA-dependent pathways in the acl-1 mutants at 22˚C. The
acl1-3 mutant also showed higher expression of PR1 and PR2 than wild-type plants.
However, the expression level of other defense related genes in the acl1-3 mutant was
very similar to that in wild type, presumably reflecting a weak activation of defense
responses. By contrast, PLANT DEFENSIN1.2 (PDF1.2), which is repressed by SA
(Spoel et al., 2003), was downregulated in the acl1 mutants (Figure I-9a). I also
observed whether cell death was occurring in the acl1 plants, because cell death is often
accompanied by constitutive activation of defense responses. While no visible cell death
25
was observed in the acl1 leaves when grown on sterile agar plates, cell death was more
frequent in the acl1 leaves than that in wild type when plants were grown in pots and
exposed to outer environment (Figure I-9b,c). This suggests that SAR is induced in the
acl1 mutants and that acl1 plants are in a “primed” state, which enables plants to be
sensitive to pathogen attack (Conrath et al., 2002), although cell death is not essential
for the stunted growth of the acl1 mutants.
Previously, we had observed that the acl1-1 mutant partially restores its plant
growth at 24˚C, which is only 2˚C higher than the usual temperature at which
Arabidopsis plants are grown (Kamata and Komeda, 2008). At temperatures higher than
26˚C, acl1-1 mutant was apparently indistinguishable from wild-type plants (Figure
I-9d). I grew the weak acl1-3 mutant at different temperatures and found that, similar to
the acl1-1 mutant, the acl1-3 mutant showed a partial but considerable restoration of
inflorescence length and leaf size at 24˚C, and was fully restored at 26˚C and 28˚C
(Figure I-9d). Several studies of the mutants with constitutive activation of defense
responses have been shown that both constitutive activation of defense responses and
stunted growth observed at 22˚C were completely suppressed at 28˚C (Yang and Hua,
2004; Gou et al., 2009). I further examined how the expression patterns of the defense
related genes alter at intermediate temperatures between 22˚C and 28˚C. At 24˚C, the
expression of the defense related genes was greatly decreased in the acl1 mutants
compared to that at 22˚C (Figure I-9a). The acl1-1 mutant still showed a faint
expression of PR1 and PR2 genes at 24˚C but it was further repressed at 26˚C. Likewise,
the elevated expression of PR1 and PR2 genes in the acl1-3 mutant was significantly
26
repressed at higher temperatures (Figure I-9a). Pot-grown acl1 plants also showed
gradual repression of defense related genes as growth temperature increased from 22˚C
to 26˚C (Figure I-10). These results show that gradual alteration of the acl1 growth
phenotype and the expression of the defense related genes are correlatively regulated in
a temperature-dependent manner. It was also shown that 26˚C is high enough to
suppress the acl1 phenotype. In the acl1 mutants, the expression level of SNC1 and
RPP5 homologues was also decreased at 28˚C compared from that at 22˚C, but was still
slightly higher than the wild-type level (Figure I-6d).
acl1 phenotype depends on EDS1 and partially on PAD4
To further examine the dependence of the acl1 phenotype on key regulators of
the defense responses, I generated double mutants. Both acl1-1 eds1-2 and acl1-3
eds1-2 double mutants completely restored the mutant phenotype to wild type (Figure
I-11a,b). While the acl1-3 pad4-1 double mutant was almost identical to wild type,
acl1-1 pad4 double mutant had much larger leaves and more elongated inflorescence
stems than the acl1-1 mutant, showing considerable but not complete suppression of the
acl1-1 phenotype (Figure I-11a,b). On the other hand, acl1 ndr1-1 double mutants
showed similar growth to the acl1 single mutants (Figure I-11a). This result suggests
that acl1 growth does not depend on NDR1, although NDR1 expression significantly
increased in the acl1-1 mutant (Figure I-9a).
A number of R proteins further require REQUIRED FOR MLA12
RESISTANCE1 (RAR1), which functions together with SUPPRESSOR OF THE G2
27
ALLELE OF SKP1 (SGT1) and HEAT SHOCK PROTEIN90 (HSP90) for proper
folding and stabilization of R proteins (Austin et al., 2002; Muskett et al., 2002;
Takahashi et al., 2003). I therefore generated acl1 rar1-21 double mutants. The acl1
phenotype was restored to some degree by the rar1-21 mutation: Both acl1-1 rar1-21
and acl1-3 rar1-21 double mutants were larger than the single acl1-1 and acl1-3
mutants, respectively, though they still exhibited short stems and curly leaves (Figure
I-11a,b). This suggests an involvement of RAR1 in the acl1 phenotype.
acl1 sid2-2 double mutants were only slightly larger than the single acl1
mutants (Figure I-11a,b). acl1 npr1-1 double mutants showed no restoration of the acl1
growth and their stunted phenotype was rather enhanced, which was more obvious at
24˚C (Figure I-11a,c). Defense response pathways dependent on SA accumulation and
NPR1 were not essential for the acl1 growth phenotype.
Nitrogen conditions influence the plant growth of acl1 mutants
In the course of this study, I discovered that acl1 mutants grew larger on full
and half-strength Murashige and Skoog (MS) media compared to those grown on the
MGRL medium, which is usually used for growing plants in pots. The total
concentrations of nitrate (NO3-), ammonium (NH4+) and potassium (K+) were 41.3, 3.9
and 6.7 times greater, respectively, for MS than MGRL (Table I-8). I added 10 mM
NH4NO3 or 7 mM KCl to the MGRL medium so that the concentration of these ions
becomes comparable to the half-strength MS medium and grew plants. While the
addition of KCl had no effect on the acl1 mutant growth, the addition of NH4NO3 was
28
effective for growing the acl1 mutants larger (data not shown).
To further examine the effect of nitrate and ammonium on the acl1 plant
growth separately, I developed a nitrogen-free medium by replacing Ca(NO3)2 by CaCl2
and excluding NH4Cl in the MGRL medium. Supplementation of nitrate to the
nitrogen-free medium, by exchanging CaCl2 to Ca(NO3)2, was sufficient for plant
survival, but acl1 plant size remained unchanged even at 10 mM nitrate (Figure I-12a).
Supplementation of ammonium, by adding NH4Cl, enabled the acl1-1 mutants to
increase in size depending on the NH4Cl concentration. The acl1-1 plants reached
almost the same size as that of wild type at 15 mM NH4Cl (Figure I-12b). I further
found that there was an interaction between the presence of ammonium and nitrate
content. For the acl1 and wild-type plants to grow larger in response to NH4Cl, 10 mM
rather than 1 mM nitrate was adequate (Figure I-12b,c). When plants were grown in
pots and watered with media containing different concentrations of ammonium, mature
acl1 plants showed improvement in the elongation of inflorescence stem by increase in
the NH4Cl concentrations and the mature acl1-3 plants grew to similar size to wild-type
plants at higher than 5 mM ammonium (Figure I-12d).
Likewise, growth of the double mutants generated between acl1-1 and
mutants of regulatory genes in defense responses also altered at higher ammonium
concentration (Compare Figures I-11a and I-13a). The acl1-1 pad4-1 mutant became
apparently identical to wild-type plants on the medium containing 10 mM ammonium
(Figure I-13). On the contrary, the acl1-1 rar1-21 double mutant grown on the media
with higher concentration of ammonium was less altered from that on the MGRL
29
medium.
Expression of defense related genes are increased in acl2-1, but the acl2-1
phenotype was not fully suppressed by high temperature and eds1-2 mutation
acl2-1 is a semi-dominant mutation that causes reduction of stem length and a
slight abnormality in leaf morphology (Tsukaya et al., 1993). ACL2 appeared to regulate
stem and petiole length via control of cell length for the most part (Tsukaya et al., 1995;
2002). ACL2 was mapped to an interval of <2 cM on chromosome 1. However, the
precise effect of acl2-1 mutation on the plant growth in a molecular level is not yet been
confirmed (Tsukaya et al., 1995). I observed that the growth defect in the acl2-1 mutant
is alleviated at higher temperatures (Figure I-14a). Although a complete restoration was
not observed even at 28˚C, temperature-dependent growth potential of the acl2-1
mutant suggests a possibility that the acl2-1 phenotype is also related to the constitutive
activation of defense responses similar to the acl1 mutants. By expression analysis, PR1
and PR2 were found to be highly expressed in the acl2-1 mutant, while the key
regulatory genes essential for R gene-mediated defense response, EDS1, PAD4 and
NDR1, were only moderately increased (Figure I-14b). Different from the acl1 mutants,
the PR1 and PR2 expression were not obviously altered by the increases in temperature,
suggesting that activation of defense responses in the acl2-1 mutant is more tolerant to
higher temperatures than those in the acl1 mutants.
The growth defect was most restored in acl2-1 eds1-2 double mutant, though
the restoration was not complete, among the double mutants generated between acl2-1
30
and mutants of key regulatory genes in defense responses (Figure I-14c). Plant growth
of acl2-1 pad4-1 and acl2-1 ndr1-1 double mutants was also restored to some degree,
suggesting that the acl2-1 phenotype is partially dependent on EDS1 and, less on PAD4
and NDR1. acl2-1 sid2-2 double mutant slightly restored the mutant phenotype,
implying a more or less influence of SA on the acl2-1 growth. However, npr1-1
mutation had no effect on the acl2-1 phenotype.
I further examined the acl2-1 mutant growth on different nitrogen conditions.
However, different from the acl1 mutants, the acl2-1 mutants grown on the higher
concentration of ammonium were not so much altered from those grown on low
concentration of ammonium, and nitrogen condition had no striking effects on the
acl2-1 growth (Figure I-14d,e).
Discussions
In the part 1, I isolated the “genuine” acl1-1 plants without the inversion. By
comparing these plants to the original acl1-1 plants with the inversion, the inversion
was likely to be function as an enhancer of the acl1-1 phenotype.
The inversion enhanced the acl1-1 phenotype and altered expression patterns of
various genes
In the previous study, there was a description that the length of the
inflorescence stems of acl1-1 plants varied among plants, even though they were grown
31
side by side under the same conditions at 22˚C (Tsukaya et al., 1993). It is possible that
this unknown factor that influences the growth of the original acl1-1 mutants is the
inversion identified in this study. I observed that the height of plants, the size of rosettes,
and the length of cells were remarkably reduced in the acl1-1 inv/inv plants compared
with the acl1-1 +/+ plants at 22˚C (Tables I-2 and I-3, Figures I-3 and I-4). Moreover,
the inversion functioned more effectively as an enhancer of the acl1-1 phenotype when
plants were grown at 24˚C, inhibiting partial restoration of the acl1-1 phenotype in the
acl1-1 inv/inv plants (Figures I-3b and I-4b,c). When the acl1-1 phenotype was fully
restored to wild-type at 26˚C or 28˚C, the difference between the acl1-1 +/+ plants and
the acl1-1 inv/inv plants disappeared. Considering that the inversion itself was not
sufficient to significantly alter plant morphology in the Col-0 and acl1-3 backgrounds
(Table I-2, Figures I-4 and I-5), it seems that the inversion enhances the defects in the
plant growth only when severe growth defects are already present, such as in acl1-1
plants.
I found that, in addition to loss of full-length transcripts of At4g21960 and
At4g22250, the expression patterns of some other genes within and adjacent to the
inversion and were altered by the inversion (Figure I-2). Although it is not yet
confirmed, it is possible that the genes distant from the inversion are also affected by
the inversion and that organ-specific and/or age-specific expression is altered. I propose
that multiple genes are complicatedly involved in the enhancement of the acl1-1
phenotype.
The original acl1-1 mutant was obtained by X-ray irradiation. In most higher
32
plants, including Arabidopsis, DNA double-strand breaks caused by ionizing irradiation
(fast neutron, X-ray, and γ-ray) are predominantly repaired by non-homologous
end-joining rather than by simple ligation or accurate homologous recombination. In
non-homologous end-joining, any end can fuse with any end. Thus, the repair of
double-strand breaks in plants is suggested to be error-prone (Gorbunova and Levy,
1999). Sometimes, the repair of double-strands breaks has very complex DNA
rearrangements, combining deletions, insertions, inversions and duplications of the
original sequence (Shirley et al., 1992), and that was what was confirmed at the border
regions of the inversion isolated from the original acl1-1 (Figure I-1d).
In the part 2, further studies on the acl1 mutants were carried out using the
acl1-1 +/+ line isolated in the part 1.
5年以内に雑誌等で刊行予定のため非公開。
Growth defect of the acl1 mutants is caused by the loss of the negative regulation
of SNC1, which requires EDS1 for its function
The region containing the RPP5 gene cluster was shown to be co-segregate
with the acl1-1 phenotype, by map-based cloning approach, and it was further
confirmed that the acl1 phenotype require TIR-NBS-LRR R gene SNC1 as an essential
gene for the acl1 growth phenotype. I also identified the acl1 mutations in a negative
regulator of SNC1, the SRFR1 gene, strongly suggesting the loss of negative regulation
of SNC1-induced defense response pathway causes the acl1 stunted growth. Although
RPP4 is another gene that has been reported to cause dwarfism in its gain-of-function
33
mutant (Huang et al., 2010) and I observed that RPP4 expression was increased in the
acl1 mutants, RPP4 was not responsible for the acl1 growth.
The stunted growth and the elevated expression of the defense related genes
correlated positively with rises in temperature (Figures I-9a,d and I-10). Temperature
has been known to influence more than one aspect of plant defense responses, and the
modulation of defense responses by higher temperatures is a general phenomenon
among plants (Whitham et al., 1996; Hwang et al., 2000; Alcázer and Parler, 2011). In
the case of the acl1 mutants, the temperature-dependence of its phenotype could be
explained by the nature of SNC1, which is likely to be a functional sensor of
temperature: Nuclear accumulation of SNC1 is reduced at higher temperature, such as
28˚C, and this is likely to contribute to the inhibition of defense responses (Zhu, Y. et al.,
2010). It has been reported that defense activation and stunted growth in srfr1 mutants
are also suppressed at 28˚C (Kim et al., 2010). However, regarding the acl1 plant
growth, the activity of the SNC1 protein would already be under the threshold level
required to induce defense responses at 26˚C (Figure I-9a,d). I assume that the
accumulation of the SNC1 protein in the nucleus is quite sensitive to temperature and
gradually inhibited with increasing temperature, because a slight change in temperature,
from 22˚C to 24˚C and from 24˚C to 26˚C, apparently modifies the acl1 phenotype.
I also speculate that there is a certain threshold level for the SNC1 gene
product to trigger defense responses and that SNC1 expression in the acl1 snc1-11/+
mutant at 22˚C was under that threshold level, since a heterozygous snc1-11/+ mutation
was enough to suppress the severe stunted growth in the acl1 mutants (Figure I-6e,f).
34
The expression analysis showed that genes related to the defense responses
are constitutively activated in the acl1 mutants (Figure I-9a-c). In addition to the EDS1
and PAD4 that are reported to be activated in the srfr1 mutants (Bhattacharjee et al.,
2011), NDR1 and genes required for SA accumulation and positive regulation of the
SA-mediated defense pathways were also upregulated in the acl1 mutants. Moreover,
frequently induced cell death in the acl1 leaves suggests that the acl1 plants are primed
for stronger activation of SAR and cell death (Figure I-9b,c).
I further examined the involvement of each defense related gene in the acl1
growth by double mutant analysis. It was clearly shown that the acl1 plant growth is
entirely controlled under EDS1 by the complete restoration of the acl1 eds1-2
phenotype to wild type (Figure I-11a,b). According to the plant growth of the double
mutants, the acl1 phenotype partially depends on PAD4, but not on NDR1. These results
are consistent with the dependence of the activity of TIR-NBS-LRR R genes on these
key regulatory genes in inducing defense responses (Glazebrook et al., 1996; Feys et al.,
2001; van der Biezen et al., 2002), indicating that the activity of the TIR-NBS-LRR R
genes mediated by EDS1 and PAD4 controls the plant growth phenotype. Since NDR1 is
dispensable for the acl1 phenotype, increased expression of NDR1 in the acl1 mutants
would not be the primary effect of the acl1 mutation and rather caused by a positive
feedback regulation of the defense response pathway.
Although SA accumulation and the NPR1-dependent signaling pathway play
an important role in plant defense, SID2 and NPR1 were unnecessary for the acl1
phenotype (Figure I-11). Thus, SA and NPR1-dependent pathways are not essential for
35
inducing the growth defects and the acl1 phenotype, and the acl1 growth is regulated
independently of these pathways. However, the sid2-1 mutation slightly restored the
acl1 growth defects and, on the contrary, the npr1-1 mutation enhanced the acl1
phenotype. Assuming that the SA level in the acl1 mutants is increased by the npr1-1
mutation, similarly to that in snc1-1 npr1 mutant (Li et al., 2001), these minor
alterations in the acl1 phenotype can be explained by the SA level.
In my study, the acl1-1 rar1-21 mutant partially repressed the acl1 phenotype
(Figure I-11a,b). Since the predominant R gene regulating the acl1 phenotype is SNC1,
that would mean that some function of SNC1 depends on RAR1. However, the growth of
the snc1-1 rar1-21 plant was less altered from that of the single snc1-1 mutant
(Goritschnig et al., 2007). It is possible that R genes other than SNC1 involved in the
acl1 morphology, though yet not identified, are RAR1-dependent. Another possibility is
that wild-type SNC1 is partially RAR1-dependent and the mutated Snc1-1 is not.
ACL1 encoded SRFR1, which negatively regulates defense responses.
All the acl1 mutant lines possessed a mutation common only in SRFR1
(At4g37460). The acl1-1 phenotype was nearly identical to the srfr1 phenotype, which
has already been described in the Col background (Figures I-6d,e and I-9a) (Kim et al.,
2010). The srfr1 growth phenotype also has been reported to require Col-specific SNC1,
and no growth defect was observed in srfr1 mutants in RLD accession backgrounds nor
in srfr1 plants with the RPP5 gene cluster of Ler (Kwon et al., 2004; Li et al., 2010).
36
SRFR1 is a single-copy gene that encodes tandem repeats of the TPR motif,
which is known to mediate protein–protein interactions and assembly of multiprotein
complexes (D’Andrea and Regan, 2003; Kwon et al., 2009). In Arabidopsis, SRFR1 is
found to interact with EDS1 and SGT1 (Li et al., 2010; Bhattacharjee et al., 2011).
SRFR1-EDS1 complex shows a similar localization pattern to SNC1-EDS1 and other R
protein-EDS1 complexes, suggesting a possibility of the involvement of SRFR1 in
directly affecting the stability of EDS1-resistance protein complexes (Bhattacharjee et
al., 2011). Interaction between SGT1 and SRFR1 through its N-terminal TPR domain is
considered to have a negative role in R protein accumulation, which would prevent
auto-activation of plant defense responses (Azevedo et al., 2006; Li et al., 2010). The
early stop codon caused by a frame shift in the acl1-1 mRNA sequence is located in the
conserved TPR repeat at the middle of the SRFR1 protein (Figure I-7). Since the acl1-1
mutant exhibits a severe phenotype similar to that of a null T-DNA insertion mutant,
srfr1-4, the SRFR1 function is likely to be lost in the acl1-1. On the other hand, the
weak growth defect observed in the acl1-3 mutant suggests that the SRFR1 function is
partially retained in the acl1-3 mutation. The acl1-3 missense mutation is in the
C-terminal region conserved among SRFR1-like proteins. Although the function of this
C-terminal region is still unknown, I presume that it is important for full activity of
SRFR1. All srfr1 mutations identified previously cause a premature stop of SRFR1
protein translation (Kwon et al., 2009; Kim et al., 2010; Li et al, 2010). The acl1-3
mutation would be a unique mutation that enables us to investigate the effects of
alteration in a single amino acid. I expect that further analysis on acl1-3 and, if available,
37
other missense mutations of SRFR1 would help us to understand the molecular basis of
the SRFR1 protein in more detail.
The sequence similarity of TPR domain of SRFR1 to transcriptional
repressors such as Ssn6 in S. cerevisiae and O-linked N-acetylglucosamine transferases
in C. elegans and D. melanogaster implies that the SRFR1 function as transcriptional
co-repressors (Kwon et al., 2009). In the acl1 mutants, expression levels of SNC1 and
some RPP5-homologues were decreased but maintained even at 28˚C, the temperature
that eliminates a positive feedback amplification of SNC1 and R genes mediated by SA
(Figure I-6) (Yang and Hua, 2004; Yi and Richards, 2007). Similarly, expression of
SNC1 in srfr1-4 snc1-11 double mutant was still elevated despite the fact that
self-upregulation of SNC1 is abolished (Kim et al., 2010). From these results, it is
possible to consider that SRFR1 acts in negative regulation of SNC1 transcription,
independent of positive feedback regulation of SNC1.
acl1 plant growth was influenced by the ammonium concentration when an
adequate amount of nitrate was provided
I showed that the nitrogen condition modifies the acl1 phenotype. Although, it
does not completely suppresses the acl1 growth defects like elevated temperature, weak
phenotype such observed in the acl1-3 and the acl1-1 pad4 mutants can be easily
masked on nitrogen-rich media like MS.
The activation of the defense response increases demands on cellular
metabolism. It has been proposed that induction of the defense pathway is costly,
38
consuming the nutrient source for biosynthesis of defense compounds, and therefore
forces the plants to restrict their growth to adjust to a reduced level of nutrient
availability (Tian et al., 2003; Heidel et al., 2004; Smith and Stitt, 2007). Partial
improvement of the acl1 plant growth by an increased supply of nitrogen source (Figure
I-12) might be explained by the increased nutrient allocation to plant growth.
However, considering that two nitrogen forms, ammonium and nitrate, have
distinct effects on acl1 growth, I propose another possibility. Although Arabidopsis has
a pathway for nitrogen reduction from nitrate to ammonium, it has been reported that
Arabidopsis utilizes ammonium preferentially when provided with a mixed nitrogen
source (Gazzarrini et al., 1999) and there would be a specific effect caused only by
ammonium. During pathogenesis, intracellular glutamine (Gln), which is a first major
product of ammonium assimilation, was depleted in plant cells (Liu et al., 2010). This
would inhibit subsequent production of glutaminate (Glu) that consumes reduced
ferredoxin as an electron donor (Suzuki and Knaff, 2005). Moreover, other sinks for
electrons, such as carbon fixation and photorespiration pathways, were also largely
repressed and would perturb redox balance when defense responses are activated (Foyer
et al., 2009; Liu et al., 2010). Redox imbalance triggers the overaccumulation of
reactive oxygen species (ROS) and SA, which enhances the SA-dependent defense
response and PR gene expression (Mach et al., 2001; Liu et al., 2010). I suspect that a
supply of nitrogen source in the form of ammonium can promote production of Gln and
Glu, reduce the redox imbalance, and suppress highly activated defense responses and
associated growth defects in the acl1 mutants. I also observed a decrease in the
39
expression level of defense related gene in the acl1 mutants supplied with higher
concentration of ammonium (Figure I-15). Enhanced ammonium assimilation is enabled
by the presence of nitrate (Britto and Kronzucker, 2002) and this may be why an
adequate concentration of nitrate is required for the acl1 mutants to achieve
ammonium-dependent growth. A low ammonium/nitrate ratio in growth media also
leads accumulation of nitrite and nitric oxide in plants that acts in positive feedback of
EDS1-dependent pathway (Wang et al., 2013). This would be another reason why
higher ammonium concentration alleviates the severe stunted growth in the acl1 mutant.
The genetic dependence of the nitrate-dependent growth of mutants with
constitutive activation of defense responses is poorly understood. Since the eds1-2
mutation completely restored the acl1 mutant phenotype (Figure I-11a,b), it is difficult
to evaluate if there is any EDS1-independent pathway in nitrogen-dependent acl1
growth. The complete restoration of growth phenotype in the acl1-1 pad4-1 mutant only
at higher ammonium concentration (Figure I-13) suggests that PAD4-independent
defense pathway in the acl1 pad4 double mutants can be repressed at high ammonium
concentration. On the other hand, the acl1-1 rar1-21 mutant was less altered from that
grown
on
the
MGRL medium
(Figures
I-12
and
I-13),
suggesting
that
RAR1-independent pathway might be less responsive to the nitrogen.
We have only a little knowledge about the genetic factors involved in the
acl1/srfr1 plant growth. Since it is confirmed that the acl1 phenotype depends on
EDS1-mediated SNC1 activity, investigation of the downstream factors of EDS1 and
SNC1 might help to understand the growth the regulatory mechanism of the acl1
40
mutants. Regarding the SNC1 gene, increasing reports on modifier of snc1-1 (mos)
mutations suggest essential factors for activation of SNC1 and downstream defense
responses (Germain et al., 2010; Monaghan et al., 2010; Zhu, Z. et al., 2010; Xu et al.,
2011; Xia et al., 2013; Xu et al., 2013). However, downstream genes that directly
regulate plant growth are not yet identified. This might be because that multiple plant
growth regulators are involved in plant immunity (Pieterse et al., 2009), and a single
gene suppressor might have little effect on modifying apparent plant growth. In this
report, I propose a possibility that nitrogen is one of those factors that affect some
aspect of plant growth in the acl1 mutants. I expect further detailed genetic and
biochemical analysis using acl1 mutants will help revealing the regulators of plant
growth associated with defense responses.
The acl2 mutation causes constitutive activation of defense response pathways
As shown in Figure I-14b, the acl2-1 mutant showed increased expression of
defense related genes, suggesting the constitutive activation of defense responses.
However, different from the acl1 mutants, the trigger of defense responses in acl12-1
seems to be more tolerant to higher temperatures, since 28˚C was not sufficient to fully
repress the elevated expression of defense related genes in acl2-1 (Figure I-14a,b). Also,
the acl2-1 growth phenotype was not completely restored neither by EDS1 nor NDR1
(Figure I-14c). Considering an early termination of inflorescence stem growth
associated with defense responses that is independent of EDS1 and NDR1, the acl2-1
mutation might have something in common with uni-1d, which caused by a
41
gain-of-function mutation of CC-TIR-LRR type or R gene (Igari et al., 2008). uni-1d
phenotype requires ER receptor kinase family members (Uchida et al., 2011a) and, in
fact, inflorescence elongation of the acl2-1 mutant alleviated in Ler background (Prof.
Atsushi Kato, personal communication). The uni-1D/UNI protein interacts with
regulatory particle triple-ATPase (RPT) subunit 2a of the 19S regulatory particle in the
26S proteasome, which is turned out to be responsible for inducing both defects in
morphology and defense responses (Chung and Tasaka, 2011), although I am not sure
for the involvement of RPT2a in the acl2-1 mutant phenotype at this time. Further
investigations on the genetic factors involved in the acl2-1 phenotype and on the acl2-1
mutation itself are needed to understand the mechanisms regulating the plant growth
and defense responses in the acl2-1 mutant.
Materials and Methods
Plant lines
Arabidopsis thaliana accession Columbia (Col-0) was used as the wild type. acl1-1
(Tsukaya et al., 1993; Kamata and Komeda, 2008), acl1-3, acl2-1 (Tsukaya et al., 1993),
pad4-1 (Glazebrook et al., 1996), ndr1-1 (Century et al., 1995), npr1-1 (Cao et al.,
1994), rar1-21 (Tornero et al., 2002), sid2-2 (Wildermuth et al., 2001), snc1-11
(SALK_047058) and rpp4 (SALK_017521) (Yang and Hua, 2004) mutants were
previously described. The eds1-2 mutation introgressed into Col-0 background was used
as the eds1-2 mutant in this study (Parker et al., 1996). 2080 (acl1-4) was isolated from
42
the pool of T-DNA insertion mutants, by Goro Horiguch, and kindly gifted. 2080 was
backcrossed to Col-0 for more than 5 times. Backcrosses indicated that the 2080
phenotype is caused by a recessive mutation at a single genetic locus, which does not
link with the T-DNA insertion. 2080 was found to be allelic with acl1-1 and acl1-3 by
the phenotype of F1 plants between them. Transgenic line SGT754-5-3 having a T-DNA
insertion in the 3rd intron of At4g21960 (La background), and SALK_018861 and
SALK_044071 lines having T-DNA inserted in the promoter region and 3’UTR of
At4g22250 respectively (Both are Col background) were isolated from Salk T-DNA
lines (Alonso et al., 2003).
To obtain double mutants, acl1 mutants were crossed with each defense
mutant. F2 plants expressing the acl1 phenotype and heterozygous to the defense
mutation were selected and then self-fertilized. PCR primers used for genotyping
defense genes are listed in Table I-9.
Plant growth
Seeds were sown on water-moistened rockwool, which placed on vermiculite in pots,
and placed in darkness at 4˚C for 3 days before they were transferred to growth
chambers. Unless noted, the growth chambers were set at 22˚C under long-day
conditions (16 hours light/ 8 hours darkness) and plants were watered with the modified
MGRL medium (Kamata and Komeda, 2008), which contains 10 mM nitrate and 0.5
mM NH4Cl as nitrogen source. For plants grown on plates, surface-sterilized seeds were
sown on the media containing 2 % (w/v) sucrose and 0.8% (w/v) bacto agar (BD).
43
Growth temperature was altered to 24˚C, 26˚C or 28˚C and MS (Wako), 1/2X MS,
nitrogen-free medium was used if necessary. The nitrogen-free medium contained 5
mM CaCl2 instead of 5 mM Ca(NO3)2 and NH4Cl was excluded from the MGRL
medium. Media were additionally supplemented with nutrients as described in the text.
Map-based cloning of acl1
acl1 mutants were crossed to Ler and F2 to F4 progenies exhibiting the acl1 phenotype
were used for recombination analysis using cleaved-amplified polymorphic sequence
(CAPS) and simple sequence length polymorphism (SSLP) markers based on the
information from the Arabidopsis Information Resource (TAIR, www.arabidopsis.org).
Additional derived CAPS (dCAPS) markers at polymorphisms between Col and Ler
accessions, designed based on information available from TAIR (Table I-7). The 159 Kb
region including RPP5 gene cluster was amplified separately as short overlapping
fragments by PCR (Ex taq, Takara Bio) and sequenced with BigDye® Terminator v.1.1
Cycle Sequencing kit (Applied Biosystem).
Examination of the inversion
Genomic DNA was extracted from rosette leaves as described by Edwards et al. (1991).
Primers used for the examination of the existence of genomic region from At4g22290 to
At4g21690 are designed more than 50 bp outside from the gene regions annotated by
TAIR. Expected PCR fragments were less than 7 kb and amplified with Ex taq (Takara
Bio). PCR fragments were then treated with several restriction enzymes and cleaved
44
into shorter fragments to identify the changes in the length of fragment more precisely
by agarose gel electrophoresis. In the case of genes longer than 7 kb, genes were
separated in two parts to ensure the amplification by PCR. For the amplification of
At4g21960, NK215; 5’-GAGAT CAGTA AAATA GATCG-3’ and NK216; 5’-TTTAA
GGAGC GTGCA TTGC-3’ were used as primers. For At4g22250, NK232; 5’-TATAA
TGTCA TCATC ACTGC-3’ and NK240; 5’-TCGAG TATCT CAATG ATCGG-3’ and
for At4g21920, NK 248; 5’-AAACA TCAAA CTTCA CGGAG-3’ and NK249;
5’-AATAC GTAGT TTTGA CCTGG-3’ were used. PCR fragments of approximately
2.2 Kbp, 0.8 Kbp and 3.3 Kbp in length were obtained by PCR respectively. NK255
5’-AGATC ACATT GAATC TGCAG-3’ and NK256 5’-TAAGT CAGTG TGGAA
CTAAG-3’ were designed at non-gene-coding region between At4g21960 and
At4g21970 to obtain 1.7 Kbp PCR products used just for the positive control of PCR
performed in Figure I-1. For detecting the conjugated fragments of At4g21960 and
At4g22250, NK215 and NK240 were used to obtain approximately 1.3 Kbp PCR
fragment fragment and NK216 and NK232 were used to obtain approximately 1.6 Kbp
PCR fragment only from the inversed DNA. Conjugated fragments were sequenced
with BigDye® Terminator v.1.1 Cycle Sequencing kit (Applied Biosystem).
Expression analysis for genes related to the inversion mutation.
Plants were grown for 10 days on the agar plate at 22˚C under long-day conditions.
RNA was extracted with RNeasy® plant mini kit (QIAGEN). Extracted RNA was
treated with cloned DNaseI (Takara Bio). Reverse-transcriptase reaction was carried out
45
with Random 9 mers to synthesize cDNA from every type of transcripts. Both RT
(reverse transcriptase) reaction and PCR (polymerase chain reaction) were preformed
with RNA PCR kit (AMV) Ver.3.0 (Takara Bio). Gene specific primers used for PCR
were NK244; 5’-ACTGC GCGGT GGAGT CATG-3’, NK856; 5’-GTAGC ATGTG
AGGGA CGTGG-3’, NK398; 5’-AGACG GAGAT TCCAG GTTG-3’, NK239;
5’-TTAGA GTTTC CGTTA CCGAG-3’, NK349; 5’-CTAGG AGGGC GACGA
GGC-3’, NK262; 5’-TTTAT GACAC GTGCA GGG-3, NK350; 5’-TCGTG ACCGC
TCATC TGTC’-3’, NK263; 5’-GTGCT CTAGA GAATT GTGGC-3’. EF1a
(At5g60390, as a control) were amplified with NK24; 5’-ACTTG CAGCT ATGGG
TAAAG-3’ and NK25; 5’-CGAAA GTCTC ATCAT TTGGC-3’. For semi-quantitative
RT-PCR of the genes located near and within the inversion, primers listed on the Table
I-10 were used.
Semi-quantitative RT-PCR for genes related to defense responses
Total RNA from the shoot of 10 day-old seedlings grown on sterile agar plates was
extracted using RNeasy plant mini kit (Qiagen). The first-strand cDNA was synthesized
from 1 µg of total RNA in a 20 µl reaction volume using the PrimeScript RT-PCR kit
(Takara Bio) with Oligo-dT primer. Sequences of gene-specific PCR primers are
provided in Table I-11. For Semi-quantitative RT-PCR analysis, the primers were
designed to span introns to avoid amplification from contaminated genomic DNA,
except for At4g16880. PCR runs consisted of 24-32 cycles, depending on the linear
range of PCR amplification for individual genes. PCR cycle included incubations at
46
94˚C for 30 sec, at 55˚C for 30 sec, and at 72˚C for 90 sec. PCR products were detected
by electrophoresis through 1.2% agarose gels, depending on the length of PCR product
and stained with ethidium bromide. All RT-PCR procedures were repeated at least three
times with similar results.
Observation of cellular structure and measurement of cell length
Inflorescence stems of plants grown for 40 days after germination were fixed overnight
in FAA [70% ethanol: formaldehyde: acetic acid = 18: 1: 1 (v/v)], dehydrated in an
ethanol series and embedded in Technovit resin as described in manual (Kluzer). 5 µm
thick sections were stained with 0.05% (w/v) toluidine blue O dissolved in 1% (w/v)
Na2BO4O7 10H2O aquous solution. Sections were photographed (E-330, Olympus)
under light microscopy (eclipse 80i, Nikon) and cell length was measured using
Photoshop 8.0 (Adobe systems). Three individuals from each strain and each
temperature were used for analysis.
Trypan blue staining
Leaves were stained with lactophenol trypan blue solution [10 ml of lactic acid, 10 ml
of glycerol, 10 g of phenol, and 10 mg of trypan blue dissolved in 10 ml of distilled
water] by boiling for approximately 1 min. Leaves were then cleared in 2.5 g/ml chloral
hydrate solution and examined under a light microscope. Dead cells are stained blue.
Preparation of the genomic DNA library and next-generation sequencing
47
1 g of frozen seedlings of Col, acl1-1 and acl1-3 were individually ground in liquid
nitrogen to a fine powder. Nuclei fraction was enriched using “Semi-pure Preparation of
nuclei procedure” of CelLytic PN Isolation/Extraction Kit (Sigma-Aldrich), and
genomic DNA was isolated using Plant DNeasy mini kit (Qiagen). 0.5 µg of DNA was
sheared using Covaris S2 (Covaris) at 100-bp setting. After being purified using a
QIAquick PCR purification kit (Qiagen), the DNA library was prepared using Genomic
Adaptor Oligo Mix (Illumina) as the DNA adaptor and NEBNext DNA Sample prep
Reagent Set 1 (New England Biolabs) according to manufacturer’s manual. Ligation
products were size-selected by electrophoresis on 2% (w/v) agarose gel. 200-250 bp
DNA fragments were excised from agarose gel and purified using QIAquick gel
extraction kit (Qiagen). Then adopter-modified DNA fragments were enriched by PCR
with PCR Primers 1.1 and 2.1 (Illumina) and KAPA HiFi HotStart ReadyMix (KAPA).
The PCR program was 98˚C for 30 sec, followed by 10 cycles of 98˚C for 10 sec, 65˚C
for 30 sec and 70˚C for 30 sec. PCR products were gel-purified using a QIAquick gel
extraction kit. Sequencing with illumina-GAIIx and informatics were performed as
described in Uchida et al. (2011b).
48
Tables
Table I-1. Segregation of acl1-1 phenotype and the inversion
-------------------------------------------------------------------------------------------------------------------Phenotype
Inversion
--------------------------------------------------------------------------------------Wild type (+/+)
Hemizygote (inv/+)
Homozygote (inv/inv)
-------------------------------------------------------------------------------------------------------------------Wild-type
acl1-1
123
203
45
5
22
119
--------------------------------------------------------------------------------------------------------------------
49
Table I-2. Measurement of plant height at 22˚C
--------------------------------------------------------------------------------------------------------------------Genotype
Average ± standard deviation (cm)
t-value
--------------------------------------------------------------------------------------------------------------------acl1-1 +/+*
6.0
± 1.8
(n = 39)
acl1-1 inv/inv*
4.3
± 1.2
(n = 31)
Col +/+**
179.7
± 17.4
(n = 16)
Col inv/inv**
180.0
± 29.1
(n = 7)
t = 4.52†
t = 0.031††
--------------------------------------------------------------------------------------------------------------------Plants were grown for 40 days.
*Total plant height including the rosette and the inflorescence stem.
**Length of the inflorescence stem was measured as plant height.
†Difference between acl1-1 +/+ and acl1-1 inv/inv is significant (significance level: α = 0.05).
†† Difference between Col +/+ and Col inv/inv is not significant (significance level: α = 0.05).
50
Table I-3. Comparison between cell length of +/+ and inv/inv plants
----------------------------------------------------------------------------------------------------------------------------------------------------------Epidermis
Cortex
Pith
(µm)
(µm)
(µm)
n*
----------------------------------------------------------------------------------------------------------------------------------------------------------22˚C acl1-1 +/+
43.0 ± 14.6
28.5 ± 6.38
78.0 ± 25.0
50
22˚C acl1-1 inv/inv
21.2 ± 9.25
26.1 ± 6.84
43.6 ± 11.1
20
6.19†
1.36
5.91†
22˚C Col +/+ 2met**
266.7 ± 100.4
40.2 ± 8.61
175.3 ± 48.9
20
22˚C Col inv/inv 2met**
243.2 ± 54.0
38.5 ± 11.9
197.2 ± 39.1
30
1.07
0.552
-1.75
22˚C Col +/+ 3met**
232.1 ± 54.4
31.2 ± 9.03
178.1 ± 54.6
30
22˚C Col inv/inv 3met**
245.9 ± 56.5
31.7 ± 7.96
159.8 ± 43.7
20
-0.803
-0.209
1.25
24˚C acl1-1 +/+ 2met**
112.6 ± 62.3
28.6 ± 8.71
92.7 ± 35.5
40
24˚C acl1-1 inv/inv 2met**
25.6 ± 7.24
36.4 ± 10.9
72.1 ± 39.3
20
6.20†
-2.99†
2.04†
24˚C acl1-1 +/+ 3met**
205.0 ± 83.1
31.5 ± 9.56
128.1 ± 31.2
30
24˚C acl1-1 inv/inv 3met**
23.2± 14.4
13.7 ± 6.30
37.4 ± 18.6
30
11.81†
8.53†
13.67†
t-value
t-value
t-value
t-value
t-value
-----------------------------------------------------------------------------------------------------------------------------------------------
† Difference is significant (significance level: α = 0.05).
* Total number of cells assayed for calculate t-value.
** Metameric type of apical meristems: 2met, the type 2 metamer, main inflorescence stem differentiates cauline leaves
with elongating internodes; 3met, the type 3 metamer, main florescence stem bears flowers without bracts formed upon
the type 2 metamer (Schultz and Haughn, 1991).
51
Table I-4. Measurement of plant height at 26˚C
--------------------------------------------------------------------------------------------------------------------Genotype
Average ± standard deviation (cm)
t-value
--------------------------------------------------------------------------------------------------------------------acl1-1 +/+*
227.6
± 27.5
(n = 12)
t = - 0.350†
acl1-1 inv/inv*
227.3
± 52.1
(n = 8)
t = - 0.242 †
Col +/+*
221.4
± 54.5
(n = 12)
Col inv/inv*
231.0
± 58.7
(n = 16)
t = - 0.519†
--------------------------------------------------------------------------------------------------------------------Plants were grown for 40 days.
*Length of the inflorescence stem was measured as plant height.
†Height of the plants is not significantly deviated from Col +/+.
52
Table I-5. Frequency of recombination between acl1-3 and molecular markers on chromosome 4
----------------------------------------------------------------------------------------------------------------------Marker
Kb
Marker segregation in F2
Recombination
Col/Col : Col/Ler : Ler/Ler
frequency (%)
----------------------------------------------------------------------------------------------------------------------LD
1,124
nga8
8:
5:2
30.0
5,629
54 : 25 : 1
20.8
FCA312
8,814
172 : 14 : 3
15.4
SC5
9,165
61 :
4:0
3.1
AG
10,384
296 :
6:0
2.0
CIW7
11,524
216 :
8:0
2.3
CAT2
16,701
89 :
4:0
3.1
DHS1
18,096
14 :
2:0
7.1
-----------------------------------------------------------------------------------------------------------------------
53
Table I-6. Segregation rate of the acl1-1 phenotype in the F2 generation
---------------------------------------------------------------------------------------------------------------Phenotype
---------------------------------------------Cross
Wild type
acl1-1 (percentage)
χ2
---------------------------------------------------------------------------------------------------------------acl1-1 X Col
213
63 (22.8 %)
0.695 (P > 0.1)*
acl1-1 X Ler
376
63 (14.4 %)
26.5 (P < 0.001)*
---------------------------------------------------------------------------------------------------------------* Chi-square value for the expected ratio of 3 wild-type : 1 acl1-1.
54
Table I-7. Additional mapping markers generated for map-based cloning
---------------------------------------------------------------------------------------------------------------------------------------Fragments (bp)
---------------------Marker name (Kb)
Primer sequences (5’-3’)
Restriction enzyme
Col
Ler
---------------------------------------------------------------------------------------------------------------------------------------PERL0770186
(9,253.4)
PERL0771474
(9,357.9)
PERL 0772901
(9,443.2)
PERL0775439
(9,552.2)
PERL0776597
(9,601.7)
PERL0778995
(9,758.4)
GTAGGATATGATCCTCTTTTGG
BamHI
128, 25
153
HinfI
155
125, 30
ClaI
143
118, 25
BglII
125, 25
150
BglII
121, 25
146
EcoRV
125, 25
150
GTTGACAGAAATGTCGAAAAGGGAT
CTATAAATACCCTGACTTGC
TGAGAATGCGAATGGAACTAAGCAAAGAC
TTCACCAGACTCTCTATTC
TTTGTACTCGGTAGTACTCCATCGA
CTTTGTTATACCTCATAATGGG
TTATGTTCCTGGGAAATACAAGATC
CTCTTAGATGTAAGATTGTG
TCGTCCTCTTCAGGTACCTGAGATC
GTACCTCTGTTTAGTGTG
ATTACTACATGCCCCAAGATGATAT
------------------------------------------------------------------------------------------------------------------------------------------
55
Table I-8. Composition of MS and MGRL media used in this study
--------------------------------------------------------------------------------------------------------MS (Wako)
MGRL
------------------------------------------
------------------------------------------
Concentration (mM)
Concentration (mM)
-------------------------------------------------------------------------------------------------------NH4NO3
20.6
Na2HPO4
1.5
KNO3
18.8
NaH2PO4
0.26
CaCl2•2H2O
3.0
KCl
3.0
MgSO4•7H2O
1.5
Ca(NO3)2•4H2O
5.0
KH2PO4
1.25
MgSO4•4H2O
1.5
H3BO3
0.10
NH4Cl
0.50
MnSO4•4H2O
0.10
H3BO3
0.003
ZnSO4•7H2O
0.03
MnSO4•4H2O
0.010
KI
0.005
ZnSO4•7H2O
0.001
Na2MoO4 •2H2O
0.001
MoO3
0.0002
CuSO4•5H2O
0.0001
CuSO4•5H2O
0.0001
CoCl2•6H2O
0.0001
CoCl2•6H2O
0.0001
Na2-EDTA
0.11
Fe(III)-EDTA
0.02
FeSO4•7H2O
0.10
------------------------------------------------------------------------------------------------------
56
Table I-9. Primers and restriction enzymes used for genotyping defense mutants
----------------------------------------------------------------------------------------------------------------------------------------Genotype
Primers (5’-3’)
Restriction enzyme
----------------------------------------------------------------------------------------------------------------------------------------EDS1
ACACATCGGTGATGCGAGACA
GGCTTGTATCATCTTCTATCC
eds1-2
ACACATCGGTGATGCGAGACA
GTGGAAACCAAATTTGACATTAG
PAD4/pad4-1
GAAGCAGCAATGAACAATTC
FinI digests wild-type product
CACTCCTCAGGCACTTTAAC
NDR1
TGGTTTAAGCATGAGAGTCC
TTCGACCACCTTCTGTGTC
ndr1-1
CCAACTAAGCACATTTTGGG
CCCAACATATAATTGTTTCTTG
rar1-21
GGAATGAAAGAGTGGAGCTGCTACTAG SpeI digests mutant product
TTTTGGAACCGATTTGGCCAG
SID2
CAACCACCTGGTGCACCAGC
AAGCAAAATGTTTGAGTCAGCA
sid2-2
TTCTTCATGCAGGGGAGGAG
AAGCAAAATGTTTGAGTCAGCA
npr1-1
GTCTCGAATGTACATAAGGC
NlaIII digests wild-type product
ATCATGAGTGCGGTTCTACC
SNC1
ATGACAAGTTGACATCGG
CCTGAATGAATTGGTGGAGA
snc1-11 (NPTII)
ATTGAACAAGATGGATTGCACG
TCAGAAGAACTCGTCAAGAAGG
RPP4
ATCAATTTGCGTTGGCATCC
GGAGATTTGATTTTAGCCAC
rpp4
ATCAATTTGCGTTGGCATCC
TTAGGCGACTTTTGAACGCG
-------------------------------------------------------------------------------------------------------------------------------------
57
Table I-10. Gene function and sequence of primers used for expression analysis of genes located
near and within the inversion
----------------------------------------------------------------------------------------------------------------------------------------------------------
Locus
Function
Primers (5’-3’), forward and reverse
----------------------------------------------------------------------------------------------------------------------------------------At4g21940 CALCIUM-DEPENDENT PROTEIN KINASE 15
(CPK15)
TGGACAAGAGAACATTGTTG
CTCTGCAATAACCTTTAGAG
At4g21950 Unknown protein similar to AT4G04630.1
CGAAGAATATTCAATTAAAGAG
M13 primer M4 (Takara)
At4g21970 Similar to Os05g0114600
GGAAGGGGGAGAAGAGGTTTC
CTAAAGAGTTAAAAGACCATTG
At4g21980 AUTOPHAGY 8A (APG8A)
CTAAACCTCTCGAGGCAAG
TCAAGCAACGGTAAGAGATC
At4g21990 APS REDUCTASE 3 (APR3)
ATTGTTGCTTCTGAGGTTAC
CAACATTCTCGCTATTGAAG
At4g22000 Hypothetical protein
TGGAAAAGCTGCAGAAGCTG
M13 primer M4 (Takara)
At4g22010 SKU5 SIMILAR 4 (SKS4)
TCGAACTATCCGGAGAAATC
TGGATATTCATCTCGGTACG
At4g22030 F-box family protein
TAGCTTGTCTAGGTTTGATG
AAAGATACAACAGACTTGAG
At4g22050 Aspartyl protease family protein
AATGTTCCAATGGATTCCGC
ACTTAGCGAACCCAACTTTC
At4g22060 F-box family protein
AGCTTCCTTTAGATCTCTTG
M13 primer M4 (Takara)
At4g22070 WRKY DNA-BINDING PROTEIN 31
TGAAGCTGCCATGATAAGCG
ACACATCCGAAACTCAAAAC
At4g22080 Pectate lyase family protein
GAGAATGCCAAGAGTAAGAC
AAGTTTCCCGGAGCTACTG
At4g22090 Pectate lyase family protein
AGAATGCCAAGGGTAAGACG
TCCGGGAGCGACTGTGAATC
At4g22100 Glycosyl hydrolase family 1 protein
TCCTTCACACTCGTAACC
GAAATCTTTGGCTCTTTGAAC
58
At4g22105 SCR-LIKE 26 (SCRL26)
GCTACTTTTTTCTTGGTTTC
TACACCGGCATAAATGTTCCG
At4g22110 Alcohol dehydrogenase
AAAAATTCGAGCTAGGCAAG
AAAGAATGCAGCGGAGAGAC
At4g22115 SCR-LIKE 14 (SCR L14)
GGGCAATGTTAAAGAAGTGG
GCAAGGAACATAACATCTAC
At4g22120 Early-responsive to dehydration protein-related protein
GAATTATGGTGAAGCTTGGC
CGGTTTCTAATGTATCTTTC
At4g22130 STRUBBELIG-RECEPTOR FAMILY 8 (SRF8)
ACTGAGAGACAGGTTTCAAC
CAGAATGAGATATCGACGTG
At4g22140 DNA binding protein
CAAAGTTGTGAGAGCGGGAG
AAACGGACCTTAACATCATC
At4g22150 LANT UBX DOMAIN-CONTAINING PROTEIN 3
(PUX3)
CTTCTTTTCTTGATAGCATTC
TGAATGACTACAGAACTTGC
At4g22160 Unknown protein
AGAATGTCTTGTTGGGTAAAG
M13 primer M4 (Takara)
At4g22165 F-box family protein
AACATAACCCTAATTCCTGG
TACGAACCAATGAGCTCTAG
At4g22170 F-box family protein
AACATAACCCTAATTCCTGG
TACGAACCAATGAGCTCTAG
At4g22180 F-box family protein
AAGCGTCTCAGAGGAGATAC
TAACATATGAAGAAGAAAGTTC
At4g22190 similar to conserved hypothetical protein
GTGTTATGTACCACAAACTC
M13 primer M4 (Takara)
At4g22200 ARABIDOPSIS K+ TRANSPORTER (AKT2/3)
ATTTGGAACGTTTCTTACCC
ACATCCACATAAGAGATGTG
At4g22210 LOW-MOLECULAR-WEIGHT CYSTEINE-RICH 85
(LCR85)
ATGTCTCCTACAGATGGGC
TCACATGCTTTCCATTTCAG*
At4g22212 Defensin-like (DEFL) family protein
CATATCTCCTACAGAAGTAG†
TCACATGCTTTCCATTTCAG*
At4g22214 Defensin-like (DEFL) family protein
ATCTCCTACAGAAGCAGTG
ATGCCTGCTTTTTTATATCC
At4g22217 Defensin-like (DEFL) family protein
CTATCTCCCACCGAAGTGG
59
TATTGAGCACGGATACTATC
At4g22220 IRON-SULFUR CLUSTER ASSEMBLY COMPLEX
PROTEIN (ISU1)
TTCTTCATCTGTTGCCACTG
CACCATTTGTCTTCACACG
At4g22230 Defensin-like (DEFL) family protein
TTTCCTTTTCAGCACCACGC
CATATCTCCTACAGAAGTAG†
At4g22233 Potential natural antisense gene
CTGATCGAAGTCGCTAAC
TATGATCCATACAAAGAGAC
At4g22235 Defensin-like (DEFL) family protein
TCTCCTATAGAAGTGAATGG
ACATGCTTTCCTTTTCAGC
At4g22240 Plastid-lipid associated protein
AAGTAAACCTACAACCACAC
AAATGGATCCTCGCCTACAC
At4g22260 IMMUTANS (IM)
GCTCATAATGGAAGAATTGG
GCAACCACAAAGGCTAGTAG
----------------------------------------------------------------------------------------------------------------------------------------*. †, The same primers were used because these genes shared highly similar sequences.
60
Table I-11. Primers used for semi-quantitative RT-PCR
-------------------------------------------------------------------------------------------------------------------------------Gene
Primer 1 (5’-3’)
Primer 2 (5’-3’)
-------------------------------------------------------------------------------------------------------------------------------EDS1
TGCTC GATCA CCTGA ATAAT C
ACACA TCAAC TGTTG CAAAC
PAD4
CAGTT AAAGA TCAAG GAAGG
TGTAG AAATT CGCAA TGTCG
NDR1
ATGAA GACAC AGAAG GTGG
CGAAT AGCAA AGAAT ACGAG
SID1
CTGGT CGCAG AATCG GTG
GCCGA AACAA TCTGT GAAG
SID2
CCATC TCTCG TAGTT ACTC
CATTA AACTC AACCT GAGGG
NPR1
GATCT TGAAA ATAGA GTTGC AC
ACGAT GAGAG AGTTT ACGG
PR1
GAATT TTACT GGCTA TTCTC TG
TTAGT ATGGC TTCTC GTTC
PR2
TTCAA CCACA CAGCT GGAC
ACTTA GACTG TCGAT CTGG
PR5
CTCGT GTTCA TCACA AGC
AGGGC AGAAA GTGAT TTC
PDF1.2
TGCTT TCGAC GCACC GG
CTCAT AGAGT GACAG AGAC
RPP4
AATAC GTGTG CCACC CAC
CTCGA TCTCA TTTCT ATCTT G
At4g16880
GTATG TTACC AAAGA TTTCA AG
CGTAT GAATT ACCTG GACG
SNC1
CTTCA TAGAT TGGTG AAGTT AG
TCAGT TACCA GAAAC AGGAA AC
At4g16900
CTGTA GGGCA GGTGG AG
TTAAC GTATT CTAGA ATCC
At4g16920
GCGGA TGGGG ATGAC AT
TCAGT TACCA GAATC AGTAG
At4g16930
GATTC TCGAA ACTGG TGTAA TG
CCTGT TCTTC TCGGT TGG
At4g16940
ACGTT TAACA CCGAA TG
CATCA CAGCG TTGAG TCTTC
At4g16950
CTCTT TTTTG CCCCT TCTTC
GATCT TCTGA ACGGG CCTAA TG
At4g16960
ACGTT TAACA CCGAA TG
TTCCC AAGGG ACTGG AC
At4g16990
GATTG CCGGT AATCG TC
CCTTG TTCAC AGTAC TC
SRFR1
ATGGC GACGG CGACG GC
CTGTG TTCGC CTAAT CCATG
EF1a
ACTTG CAGCT ATGGG TAAAG
CGAAA GTCTC ATCAT TTGGC
SRFR1 for cDNA sequencing
ATGGC GACGG CGACG GC
TCAAT CGTTG TAAGT GCTAA G
------------------------------------------------------------------------------------------------------------------------------------
61
Chromosome 4 putative region including acl1-1
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gene model
(a)
CIW7
12,000Kbp
9,000Kbp
本図については、非公開。
oriantation: forward, reverse
Kbp
MASC
G4539 functions
AG
「An inversion identified in acl1-1SC5
mutant
as an enhancer
of500the
acl1-1 phenotype」
04642
pseudogene
Genes and Genetic Systems 誌 83巻、293~300頁
11,550
11,750
11,600
11,650
11,700
Kbp
Fig. 1 参照
At4g21690
At4g22290
amplified PCR
fragment
1*
(d) WT
pNK215
pNK240
645
0
~
~
~
WT
original acl1-1
acl1-3
WT
original acl1-1
acl1-3
0
~
At4g22250
＊
At4g21960 1700
pNK216
pNK232
←4g21950
pNK215
At4g21960＊
＊
pNK240
At4g22250
~
103 1
~
~
~
138
(c)
pNK216
1700 1264
645
1.5
0.95
inversion in acl1-1
At4g22250
1.5
0.95
approx. 120 Kbp
At4g21960
(Kbp)
19.3
6.2
3.5
4g22260→
1
(Kbp)
19.3
6.2
3.5
100 bp
100 bp
1269
WT
original acl1-1
acl1-3
1*
2*
4g21960 4g22250 4g21920
＊
(b)
2*
pNK232
At4g21960
1290
255
256
138
WT
original acl1-1
WT
original acl1-1
WT
original acl1-1
8 bp insertion
103
At4g22250
90
TACTGAAAGAT AC TGACTTT acagacca CGTGACCGCTCATC
1269
1250
At4g21960
(Kbp)
19.3
6.2
3.5
1.5
0.95
1.5
0.95
(e)
v
(Kbp)
19.3
6.2
3.5
At4g21960
At4g22250
C
o
C l +/
o +
ac l inv
l /
ac 1-1 inv
l1 +/
-1 +
in
v/
in
216
232
At4g22250
215
240
160
primer pairs
(NK)
1264
GACCctgcacgtgtcataaaaaaaaaaagtc GTTAGATTCAAATGCATCGGATG
EF1α
Figure I-1. Identification of inversion in the original acl1-1 mutants.
(a) Locations of genes assayed in this study.
(b) PCR performed with DNA from wild-type, the original acl1-1 mutant, and the acl1-3 mutant to detect full-length genomic regions
At4g22250
of At4g21960
At4g21960 and At4g22250.
(c) Detection of novel conjugated fragments in the original acl1-1 mutant.
(d) Structure of wild-type At4g21960 and At4g22250 and conjugated genes caused in the inversion. Filled and open squares: exons.
At4g21960
Bold lines: introns. Arrows with pNK-number: positions of primers used. Within nucleotide sequences, gray shading, At4g21960;
white boxes, At4g22250;
capital
letters,
exons;
small
letters,
introns.
Underlined
6
bp
were
duplicated.
Black
shading
indicates
2 bp
At4g22250
At4g22250
substitution from TT to AC. A 34-bp fragment was deleted from At4g22250.
(e) RT-PCR performed with RNA from 10-day-old plants.
C
o
C l +/
ol +
ac in
l v
ac 1-1 /inv
l1 +
-1 /+
in
v/
in
v
C
o
C l +/
o +
ac l in
l v
ac 1-1 /inv
l1 +
-1 /+
in
v/
in
v
インターネット公表に関する使用承認が雑誌社（出版社）から得られていないため、
本図については、非公開。
(a)
「An inversion identified in acl1-1 mutant functions as an enhancer of the acl1-1 phenotype」
Genes
and Genetic Systems At4g22140
誌 83巻、293~300頁
At4g21940
1 参照
At4g21950Supplementary Fig.
At4g22150
At4g21970
At4g21980
*
At4g21990
At4g22000
*
At4g22010
*
At4g22030
*
At4g22050
At4g22060
*
At4g22070
*
At4g22080
*
At4g22090
*
At4g22100
At4g22105
At4g22110
At4g22115 not detected
At4g22120
At4g22130
At4g21960
*
*
645
0
NK349
NK262
NK244
NK244
NK240
0
NK398
NK350
NK263
NK239
NK239
100 bp
C
o
C l +/
ol +
ac in
l v
ac 1-1 /inv
l1 +
-1 /+
in
v/
in
v
NK856
*
*
*
At4g22250
＊
1700
NK856
*
＊
(b)
At4g22160
At4g22165
At4g22170
At4g22180
At4g22190
At4g22200
At4g22210
At4g22212
At4g22214
At4g22217
At4g22220
At4g22230
At4g22233
At4g22235
At4g22240
At4g22260
EF1α
At4g21960: NK244-NK349
At4g21960: NK856-NK262
At4g22250: NK350-NK239
At4g22250: NK240-NK263
conjugated transcript: NK856-NK398
conjugated transcript: NK244-NK239
appox. length
(Kbp)
(0.2)
(1.0)
(0.5)
EF1α
Figure I-2. Expression analysis of genes within and adjacent to the inversion.
(a) RT-PCR analysis of genes within and adjacent to the inversion. RNA was extracted from 10-days-old plants.
*Genes exhibited different expression patterns among four plant strains.
(b) Partial expressions of At4g21960 and At4g22250 and conjugated transcripts in the inv/inv plants. Gene structure and expected
length of transcripts are indicated by the name of primers. Black and white boxes: exons. Bold lines: introns. Approximate lengths
of the conjugated transcripts are shown in parentheses. Arrows: fusion positions in the inversion.
インターネット公表に関する使用承認が雑誌社（出版社）から得られていないため、
本図については、非公開。
(a) 22˚C
(b) 24˚C
「An inversion
identified in acl1-1 mutant
functions as an enhancer of the acl1-1 phenotype」
acl1-1 +/+
acl1-1 inv/inv
Genes and Genetic Systems 誌 83巻、293~300頁
acl1-1 +/+
Fig. 2 参照
Col +/+
Col inv/inv
acl1-1 inv/inv
Figure I-3. Enhancement of acl1-1 phenotype by the inversion.
(a) Overall development of acl1-1 +/+ plants and acl1-1 inv/inv plants observed at 40 days after
germination (the upper sections) and rosettes of Col +/+ plants and Col inv/inv plants at 35 days after
germination (the lower sections). Plants were grown at 22˚C.
(b) Growth of acl1-1 +/+ and acl1-1 inv/inv plants at 24˚C. Plants were grown for 40 days after
germination.
Bars = 3 cm.
0.8
#
36.7%
†
0.4
17.9%
* 9.4%
0.2
24.6%
†
*
Col +/+ 2met
Col inv/inv 2met
Col +/+ 3met
Col inv/inv 3met
acl1-1 +/+
acl1-1 inv/inv
Col +/+ 2met
Col inv/inv 2met
Col +/+ 3met
Col inv/inv 3met
acl1-1 +/+
acl1-1 inv/inv
0
#
#
epidermis
pith
cortex
(b)
(c)
µm
400
µm
400
type 2 metamer
350
350
300
300
Length of cells in epidermis
Length of cells in epidermis
# 62.3%
0.6
Col +/+ 2met
Col inv/inv 2met
Col +/+ 3met
Col inv/inv 3met
acl1-1 +/+
acl1-1 inv/inv
cell length in ratio to the type 2 metamer of Col +/+
インターネット公表に関する使用承認が雑誌社（出版社）から得られていないため、
(a)
本図については、非公開。
「An inversion identified in acl1-1 mutant functions as an enhancer of the acl1-1 phenotype」
1.2
††
Genes and Genetic Systems 誌 83巻、293~300頁
Fig. 3 参照
1.0
63.1%
250
200
150
100
Col +/+
250
200
150
100
Col +/+
Col inv/inv
50
0
type 3 metamer
acl1-1 +/+
acl1-1 inv/inv
22
24
26
28
growth temperature
Col inv/inv
50
˚C
0
acl1-1 +/+
acl1-1 inv/inv
22
24
26
28
˚C
growth temperature
Figure I-4. Length of cells of inflorescence stems.
(a) Length of cells in epidermis, cortex, and pith. Percentages indicate the ratio of cell length of each
genotype and metamer to the length of type 2 metamer of Col +/+. 2met = type 2 metamer, 3met = type 3
metamer.
(b,c) Length of cells in epidermis of type 2 (b) and type 3 (c) metamer at 22, 24, 26, and 28˚C. At 22˚C, the
type 2 and type 3 metamers of the acl1-1 +/+ plants and the acl1-1 inv/inv plants were assayed together.
Lines: averages of each plant strain.
(a)
(b)
Figure I-5. Comparison of plant growth between Col +/+ and Col inv/inv, and acl1-3
+/+ and acl1-3 inv/inv. (a) Overall appearance of Col +/+, Col inv/inv, acl1-3 +/+ and two plants from distinct
lines of acl1-3 inv/inv. Number in the parenthesis indicate the line number. Bar = 5 cm. (b) Rosette size and plant height of plants of Col +/+, Col inv/inv, acl1-3 +/+ and two
acl1-3 inv/inv lines. Error bars indicate standard deviation.
Plants were grown for 40 days at 22˚C. 5年以内に雑誌等で刊行予定のため、本図については非公開。
(a)
(b)
(c)
(d)
(e)
f
(f)
Figure I-6. Mapping of the locus linked to acl1 phenotype.
(a) 864 plants exhibiting acl1-1 phenotype in F3 population from the cross between acl1-1 and Ler were
examined. Recombination frequency at each marker is shown. (b) Finer mapping was performed using 184 F4 and 240 F3 plants and additional mapping markers. The
numbers indicate the total number of recombinant chromosomes at each marker. The RPP5 gene cluster region
is shown by a yellow box. (c) Structure of the RPP5 gene cluster. Yellow arrows indicate TIR-NBS-LRR R genes. Striped dark-yellowcolored arrows indicate genes that contain partial TIR-NBS-LRR domains and green arrows indicate non-R
genes. (d) Semi-quantitative RT-PCR of the R genes located within and next to the RPP5 gene cluster. PCR products
from At4g16900 and At4g16940 were not detectable. EF1α was used as control. (e,f) Phenotype of the acl1 snc1-11 double mutants. Plants were grown for 25 days (e) or 40 days (f) in pots.
Bars = 1 cm (e) and 5 cm (f). 5年以内に雑誌等で刊行予定のため、本図については非公開。
(a)
(b)
(c)
Figure I-7. Structure of ACL1/SRFR1 (At4g37460). (a) Genetic structure of ACL1/SRFR1. Exons are indicated by red boxes. Introns are
indicated by lines and untranslated regions by gray boxes. Red-colored letters represent
mutations in the acl1 mutants. (b) Protein structure of ACL1/SRFR1. Dark-blue boxes indicate the tetratricopeptide
repeat (TPR). (c) Semi-quantitative RT-PCR of ACL1/SRFR1 in the acl1 mutants at 22˚C. EF1α was
used as control. 5年以内に雑誌等で刊行予定のため、本図については非公開。
Figure I-8. Growth phenotype of a new acl1 mutant allele, 2080. 40 days-old acl1-1 and 2080 plants grown at 22˚C. Bar = 1 cm.
5年以内に雑誌等で刊行予定のため、本図については非公開。
EDS1
T
26˚C
-1 -3
l1 l1
ac ac
W
T
24˚C
-1 -3
l1 l1
ac ac
W
T
W
W
T
(a)
a
22˚C
-1 -3
l1 l1
ac ac
28˚C
-1 -3
l1 l1
ac ac
PAD4
22˚C
(d)d
24˚C
26˚C
28˚C
WT
NDR1
SID1
SID2
NPR1
PR1
PR2
PR5
PDF1.2
acl1-1
EF1α
(b)
(c)
c
WT
acl1-1
acl1-3
WT
acl1-1
acl1-3
acl1-3
Figure I-9. Constitutive activation of defense response genes and temperature
dependence of the acl1 phenotype.
(a) Semi-quantitative expression analysis of SA-mediated defense genes (EDS1, PAD4,
NDR1, SID1, SID2, NPR1, PR1, PR2, and PR5) and a JA-mediated defense gene
(PDF1.2) in 10-days-old seedlings grown on sterile agar plates. EF1 was used as
control.
(b,c) Detection of cell death in the acl1 leaves at 22˚C. Plants were grown on either sterile
agar media (b) or in pots (c) for 25 days and leaves were stained by trypan blue, which
gives blue staining in regions undergoing cell death.
(d) Plant growth of wild type, acl1-1 and acl1-3 at different temperatures. Plants were
grown for 40 days at indicated temperatures in pots.
Bars = 100 µm (b,c) and 5 cm (d).
5年以内に雑誌等で刊行予定のため、本図については非公開。
Figure I-10. Semi-quantitative expression analysis of SA-mediated
defense genes in 14-days-old Wild type (WT) and acl mutant plants
grown at 22˚C in pots. EF1α was used as control. (a)
b
(b)
5年以内に雑誌等で刊行予定のため、本図については非公開。
c
(c)
Figure I-11. Phenotype of double mutants between acl1 and mutants of genes in the
defense response pathways. (a,b) Plants were grown for 25 days (a) or 40 days (b) in pots at 22˚C. (c) acl1-1 npr1-1 and acl1-3 npr1-1 plants were grown for 40 days either at 22˚C or 24˚C.
Bars = 1 cm (a), 5 cm (b) and 2.5 cm (c).
(a)
mm
(d)
Rosette size
25
20
15
0.5
WT
1
NH4Cl (mM)
5
10
10
5
0
(b)
5年以内に雑誌等で刊行予定のため、本図については非公開。
0 1 2.5
5
10
mM
NO 3-
mm
25
Rosette size
20
acl1-1
15
10
5
0
5
10
NH4Cl
with 10 mM NO3-
15
mM
mm
25
Rosette size
(c)
0
20
acl1-3
15
10
5
0
0
5
10
NH4Cl
with 1 mM NO 3-
WT
acl1-1
15
mM
acl1-3
Figure I-12. Effect of nitrate and ammonium on the acl1 plant growth.
(a-c) Plants were grown for 2 weeks on agar plates containing the nitrogen-free medium supplied
with additional nitrogen source as descried. Rosette size indicates average length of long and short
diameter of the rosette.
(d) Mature pots-grown plants watered with nitrogen-free media supplied with 10 mM nitrate and
the indicated concentration of NH4Cl. Plants were grown for 40 days.
Bars = 5 cm.
(a)
5年以内に雑誌等で刊行予定のため、本図については非公開。
(b)
Figure I-13. Growth of the double mutants on media containing higher
concentration of ammonium. Plants were grown for 25 days (a) or 40 days (b) watered with nitrogen-free media
supplemented with 10 mM Nitrate and 10 mM NH4Cl. Bars = 1 cm (a) and 5 cm (b). (a)
(b)
(c)
(d)
(e)
Figure I-14. Phenotype of the acl2-1 mutant
(a) Temperature-dependent growth of the acl2-1 mutant. Plants were grown at indicated
temperature for 40 days, and watered with MGRL medium.
(b) Semi-quantitative RT-PCR of the defense-related genes in the acl2-1 mutant. EF1α
was used as control. (c) Double mutants between acl2-1 and mutants of key regulatory genes in defense
responses. Plants were grown for 40 days at 22˚C, and watered with MGRL medium.
(d) Growth of acl2-1 mutant watered with the nitrogen-free medium supplemented with
10 mM nitrate and indicated concentrations of NH4Cl. (e) Growth of acl2-1 seedlings on the media containing different concentrations of NO3and NH4Cl. Bars = 5 cm. Chapter II: Mutations in epidermis-specific HD-ZIP IV genes affect
floral organ identity in Arabidopsis thaliana.
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Introduction
「Mutations in epidermis-specific HD-ZIP
IV genes affect floral organ identity in Arabidopsis thaliana」
Plant Journal誌 75巻 430~440頁
Homeodomain-leucine zipper (HD-ZIP) proteins are transcription factors
present only in the plant kingdom. They have a homeodomain (HD), which is a
conserved 60-amino acid motif for DNA-binding, and a leucine zipper motif, which
mediates their homo- and hetero-dimerization. The class IV HD-ZIP (HD-ZIP IV)
proteins are characterized by an internal loop in the middle of the leucine zipper motif,
which is thus called a zipper-loop-zipper (ZLZ) domain (Schrick et al., 2004). They also
contain a steroidogenic acute regulatory protein-related lipid transfer (START) domain
and a START-adjacent (SAD) domain in the C-terminus. The HD-ZIP IV family in
Arabidopsis thaliana consists of 16 genes: GLABRA2 (GL2), ARABIDOPSIS
THALIANA
MERISTEM
PROTODERMAL
LAYER1
FACTOR2
(ATML1),
(PDF2)
and
ANTHOCYANINLESS2
HOMEODOMAIN
(ANL2),
GLABROUS1
(HDG1)-HDG12, among which HDG6 is identical to the previously identified FWA
(Nakamura et al., 2006). Expression analyses with promoter-GUS gene fusions have
revealed that ATML1, PDF2, HDG2, HDG5, HDG11 and HDG12 are expressed
predominantly in the epidermal layer of shoot meristems and organs (Nakamura et al.,
2006), suggesting that many members of the HD-ZIP IV family regulate gene
expression in the epidermis. However, only four single knockout mutants are known to
62
show morphological alterations. The gl2 mutant is defective in trichome and seed
mucilage formation, and has ectopic root hairs in non-hair cell files (Rerie et al., 1994;
Di Cristina et al., 1996). In anl2, anthocyanin accumulation is reduced in the shoot and
several extra cells are formed between cortical and epidermal layers of the root (Kubo et
al., 1999; Kubo and Hayashi, 2011). The hdg11 mutant has trichomes with increased
branching and the phenotype is enhanced in hdg11 hdg12 double mutants (Nakamura et
al., 2006), while hdg2 has trichomes with smooth cell walls (Marks et al., 2009).
ATML1 and PDF2, which are closely similar in sequence to each other, are both
expressed specifically in the outermost cell layer (L1) of shoot apical meristems (Lu et
al., 1996; Sessions et al., 1999; Abe et al., 2003). While each T-DNA insertion mutant
of ATML1 and PDF2 shows wild-type phenotype, the atml1-1 pdf2-1 double mutant has
severe defects in the differentiation of shoot epidermal cells, indicating that they play
redundant but critical roles in the formation of the shoot epidermis (Abe et al., 2003).
So far, however, there has been no further information on double mutants within the
family that display abnormal phenotypes except for pdf2-1 hdg3-1 and atml-1 hdg3-1,
which show slight defects in cotyledon development (Nakamura et al., 2006) and
precise functions of HD-ZIP IV members and functional redundancy among them
remain largely to be elucidated.
There are also accumulating reports of epidermis-related functions of HD-ZIP
IV genes in other plants. In maize, OUTER CELL LAYER4 (OCL4) has been suggested
to regulate trichome patterning and anther development (Vernoud et al., 2009), while
OCL1 may be involved in lipid metabolism and cuticle biosynthesis (Javelle et al.,
63
2010). The cotton GbML1 regulates fiber development (Zhang et al., 2010). In tomato,
Woolly (Wo), the closest homolog of Arabidopsis PDF2, is essential for embryo
development and its dominant allele is known to exhibit the woolly trichome phenotype
(Yang et al., 2011). However, these genes represent only a part of the HD-ZIP IV family
and we are still far from comprehensive understanding of the HD-ZIP IV family.
Epidermis is organized in a continuous monolayer of cells that covers plant
body. Epidermal cells in shoot organs are exclusively derived from L1, the outermost
layer of shoot apical meristems, which continues to undergo anticlinal cell division
(Barton and Poethig, 1993). Shoot epidermis plays a critical role in organ separation and
defense responses against drought or pathogens as well as in the integrity of organs. In
flowers, the Arabidopsis cytochrome P450 KLUH (KLU) expressed in the epidermis
stimulates organ growth non-cell-autonomously and may be one of the major players in
the coordination of the final size of floral organs (Anastasiou et al., 2007; Eriksson et
al., 2010). When floral organ identity genes in Arabidopsis such as AGAMOUS (AG),
SEPALLATA3 (SEP3), APETALA3 (AP3) and PISTILLATA (PI) are expressed
specifically in the L1, the flowers show similar modifications to those with constitutive
expression of the respective genes, suggesting that the differentiation and maturation of
floral organs can be partially directed from the L1 cells (Urbanus et al., 2010). Although
AP3 expressed only in the epidermis is not sufficient for full restoration of floral organ
identity in ap3 mutants, it acts non-autonomously to recover overall shape of petals,
confirming the important contribution of the epidermis to organ development (Jenik and
Irish, 2001; Urbanus et al., 2010).
64
To examine further the function of the HD-ZIP IV genes in Arabidopsis, their
double mutants were generated and I found that the combinations of pdf2-1 with mutant
alleles of HDG1, HDG2, HDG5 or HDG12 resulted in abnormal floral organ formation,
suggesting the importance of the interplay in the epidermis between PDF2 and these
HD-ZIP IV proteins during flower development.
Results
A previous study has revealed that double mutants of HD-ZIP IV gene pairs
with high sequence similarity such as HDG2-HDG3 and HDG4-HDG5 (Figure II-1) do
not necessarily show abnormal phenotype (Nakamura et al., 2006). I therefore focused
on the study of double mutant combinations of pdf2 or atml1 alleles with the alleles of
other HD-ZIP IV genes.
pdf2-1 hdg2-3 produces flowers with sepaloid petals and carpelloid stamens
HDG2 is the second closest homologue to ATML1 and PDF2 (Figure II-1).
The mutant alleles, hdg2-2 and hdg2-3, have a T-DNA inserted in the START
domain-coding region and in the HD-coding region of HDG2, respectively, and the
latter may represent a null allele (Figure II-2) (Nakamura et al., 2006). Thus, double
mutant between hdg2-3 with pdf2-1 was first generated. In the course of detailed
phenotypic analysis, one or two of the two short stamens were found to be occasionally
absent in pdf2-1 and hdg2-3 single mutant flowers (Figure II-3a,b). pdf2-1 hdg2-3
flowers had smaller petals and stamens than the wild type, and fertility was severely
65
reduced (Figures II-3c and II-4a-c). The mutant petals were greenish and rather
resembled sepals (Figure II-4d). Tissue sections of the petal revealed that inner cells
were often composed of more than two layers of large cells like sepals (Figure II-4e-g).
I further observed by using scanning electron microscopy (SEM) that the epidermal
cells of the adaxial surface of pdf2-1 hdg2-3 petals were flat and long like sepals rather
than conical like normal petals (Figure II-5a-c). The abaxial surface of these petals was
composed of cells with normal shape and clusters of elongated cells with scattering
guard cells (Figure II-5d-f). Stamens of pdf2-1 hdg2-3 flowers frequently had enlarged
anthers and carpelloid structures such as stigmatic papillae and ovules or filaments
without anthers (Figure II-5g-k). These abnormalities were observed at higher
frequency in the four long stamens than in the two short stamens (Figure II-3b). I also
confirmed that the phenotype of pdf2-1 hdg2-2 flowers was almost identical to that of
pdf2-1 hdg2-3 flowers (Figure II-3).
pdf2-1 hdg1-1, pdf2-1 hdg5-1 and pdf2-1 hdg12-2 also show partial homeotic
phenotype
I found that hdg1-1 also had flowers lacking one or two of the two short
stamens while hdg1-2 had normal flowers (Figure II-3a). The T-DNA insert in hdg1-1 is
located in the intron splitting the START domain-coding region and that in hdg1-2 is in
the exon encoding the ZLZ domain of HDG1 (Figure II-2) (Nakamura et al., 2006).
pdf2-1 hdg1-1 showed partial homeotic conversions of petals into sepals and stamens
into carpels, a phenotype similar to that of pdf2-1 hdg2-3 (Figures II-3b and II-6a,b),
66
while pdf2-1 hdg1-2 showed no homeotic phenotype but instead had flowers lacking in
one or two of the short stamens with higher frequency than pdf2-1 (Figures II-3 and
II-6c,d). Expression of the GUS reporter gene fused to a HDG1 promoter has previously
been detected only in the epidermis of stamen filaments (Nakamura et al., 2006).
However, I confirmed that the longer promoter could direct the GUS expression also in
the epidermis of stems, pedicels, anthers of stamens and the nucellus in flowers (Figure
II-7). On the contrary, I could not detect the GUS expression in flower meristems during
early developmental stages.
The hdg5-1 allele, which carries a T-DNA insertion in the START
domain-coding region of HDG5 (Figure II-2), showed wild-type phenotype but pdf2-1
hdg5-1 also produced flowers with sepaloid petals and carpelloid stamens, resulting in
reduced fertility (Figures II-3 and II-6e,f). On the contrary, double mutants of pdf2-1
with hdg5-2, which has a T-DNA insertion in the SAD domain-coding sequence (Figure
II-2), were indistinguishable from pdf2-1 plants (Figure II-3).
I also found that pdf2-1 hdg12-2 flowers exhibited only weak conversion of
petals and stamens to sepals and carpels, respectively, with low frequency compared to
the double mutants described above (Figures II-3 and II-6g,h). The shape of epidermal
cells of the adaxial surface of pdf2-1 hdg12-2 petals was intermediate between those of
petals and sepals, and guard cells were observed among them (Figure II-8).
Furthermore, we generated double mutants between pdf2-1 and alleles of the
other HD-ZIP IV genes reported in Nakamura et al. (2006) but observed no homeotic
conversions of floral organs in these mutants (Figure II-9 and data not shown). pdf2-1
67
hdg11-1 hdg12-2 triple mutants showed no difference in flower morphology from
pdf2-1 hdg12-2 (Figure II-3).
pdf2-2 and atml1 alleles are not involved in homeotic conversion of floral organs
In addition to pdf2-1, I obtained a new allele of PDF2, which has a T-DNA
insertion in the exon for the more C-terminal part of the START domain than does
pdf2-1, and named pdf2-2 (Figure II-2). Interestingly, pdf2-2 had flowers lacking one of
the two short stamens with higher frequency than pdf2-1 but pdf2-2 hdg2-3 showed no
additional abnormality (Figure II-3). pdf2-2 was also crossed with hdg1-1, hdg5-1, and
hdg12-2 alleles, but these double mutants showed no homeotic conversions of floral
organs and manifested only the pdf2-2 phenotype, namely the lack of a short stamen
(Figure II-3).
Since PDF2 is functionally redundant with ATML1 (Abe et al., 2003), I next
examined double mutants between atml1 alleles and hdg1-1, hdg2-3, hdg5-1, or
hdg12-2. atml1-1 and atml1-3 carry a T-DNA insertion in the exon encoding the SAD
domain and the homeodomain, respectively (Figure II-9) (Abe et al., 2003; Roeder et
al., 2012). But no combination of them showed homeotic changes in floral organs
(Figure II-9).
I further confirmed that hdg1-1 hdg2-3, hdg1-1 hdg5-1, hdg1-1 hdg12-2,
hdg2-3 hdg5-1, hdg2-3 hdg12-2 and hdg5-1 hdg12-2 also showed normal development
of flowers except for an occasional lack of short stamens (data not shown).
68
AP3 expression is reduced in mutant flowers
According to the ABC model, homeotic conversion of petals into sepals and
stamens into carpels is caused by loss-of-function of the class B floral identity genes
(Coen and Meyerowitz, 1991). Thus, I examined expression levels of the two class B
genes, AP3 and PI, in the above-described phenotypic mutants. AP3 expression was
reduced remarkably in pdf2-1 hdg2-3, pdf2-1 hdg1-1, and pdf2-1 hdg5-1 inflorescences,
and moderately in pdf2-1 hdg12-2 (Figure II-10), suggesting the correlation of the
severity of the homeotic phenotype with the level of AP3 expression. On the other hand,
reduction in PI expression was detected only in pdf2-1 hdg1-1 and pdf2-1 hdg2-3
inflorescences (Figure II-10). Expression levels of the class A floral identity gene AP2,
the class C gene AG, and the class E gene SEP3, which are also required for specifying
the organ identity of petals and stamens (Honma and Goto, 2001; Theissen, 2001), were
not reduced in these mutant inflorescences (Figure II-11).
Real-time RT-PCR experiments using RNA prepared from flower buds at
different stages revealed that the level of AP3 expression was much lower in pdf2-1
hdg1-1 and pdf2-1 hdg2-3 than in the wild type throughout different stages of flower
development, while the level of PI expression was gradually decreased in these mutants
at later stages (Figure II-12a). However, in situ hybridization experiments revealed that,
as is the case with wild-type flowers (Jack et al., 1992; Smyth et al., 1990), the AP3
transcript was detected in the mutant flowers at stage 3 (Figure II-12b-e), suggesting
that the onset of the AP3 expression is unaffected in these mutants. After stage 3, AP3
transcript was detected only in the proximal part of petals and stamens (Figure II-12d,
69
arrow heads). PI expression is detected in whorls 2, 3 and 4 of wild-type flowers at
stage 3, then restricted to whorls 2 and 3, and maintained together with AP3 expression
throughout the development of petals and stamens (Goto and Meyerowitz, 1994). PI
expression patterns in pdf2-1 hdg1-1 and pdf2-1 hdg2-3 flowers were similar to those in
wild-type flowers (Figure II-12f-i). Since initiation of AP3 expression involves the
meristem identity genes such as LEAFY (LFY), AP1, and UNUSUAL FLORAL
ORGANS (UFO) (Weigel and Meyerowitz, 1993; Levin and Meyerowitz 1995; Lee et
al., 1997), I examined expression patterns of these genes and confirmed that they were
not altered in pdf2-1 hdg2-3 and pdf2-1 hdg1-1 (Figure II-11).
I also introduced the GFP gene driven by the AP3 promoter into pdf2-1
hdg2-3 and pdf2-1 hdg1-1. Similar to the pattern observed by in situ hybridization, the
GFP fluorescence in wild-type plants transformed with the pAP3::GFP construct was
detected in whorls 2 and 3 at stage 3 and maintained in petals and stamens until later
stages of flower development (Figure II-12). In pdf2-1 hdg1-1 and pdf2-1 hdg2-3, the
GFP signal appeared in the presumptive whorls 2 and 3 of floral meristems at stage 3
and, unlike in the wild type, the signal became restricted to the basal region of petals
and stamens at stages 6 to 9 (Figure II-12k-m). I observed only sporadic signals of GFP
in the middle to upper part of the petal at stage 10 (Figure II-12l), suggesting that AP3 is
not uniformly expressed in these mutants.
Transgenic expression of AP3 or PDF2 rescues the phenotype
70
I examined whether or not transgenic expression of AP3 could restore the
development of petals and stamens in pdf2-1 hdg2-3. When the AP3 full-length cDNA
was fused to the CaMV 35S promoter (35S::AP3) and introduced into pdf2-1 hdg2-3
plants, the gross appearance of petals and stamens were indistinguishable from that of
the wild type (Figure II-13a,b) and the fertility was fully restored. Epidermal cells of the
petals in pdf2-1 hdg2-3 carrying the 35S::AP3 construct were conical in the adaxial
epidermis and uniformly rectangular in the abaxial epidermis (Figure II-13c,d) like
those in the wild-type.
I also performed complementation experiments with the wild-type PDF2. The
PDF2 full-length cDNA was fused to the native PDF2 promoter (pPDF2::PDF2) and
introduced into pdf2-1 hdg2-3. Five transgenic lines were obtained and two of them
showed normal development of flowers as pdf2-1 transformed with the same construct,
but the other three developed flowers with smaller petals than wild-type flowers (Figure
II-13e-g). Even in these flowers, stamens were fully fertile and showed no carpelloid
features (Figure II-13h).
pdf2-1 transcripts are present in the epidermis
T-DNA insertion mutants of PDF2 and other HD-ZIP IV genes could produce
incomplete transcripts from the respective alleles. The presence of the pdf2-1 transcript
was examined by RT-PCR with specific primers designed to amplify the region that is
upstream of the T-DNA insertion and encodes the HD-ZLZ domain. The resulting PCR
products were detected at similar levels in the wild type and pdf2-1 hdg double mutants
71
(Figure II-14a). In situ hybridization using a probe for 5’ UTR of PDF2 showed that the
pdf2-1 transcript was expressed in the L1 cells of inflorescence and floral meristems, a
pattern identical to that of the wild-type PDF2 transcript (Figure II-14b). RT-PCR
experiments were also performed using specific primers for HD-ZLZ domain-coding
sequences of HDG1, HDG2, HDG5 and HDG12, and revealed that the transcripts from
hdg1-1 and hdg5-2 are present but those from hdg1-2, hdg2-3 and hdg12-2 are not in
their respective double mutants with pdf2-1 (Figure II-14a). Since the T-DNA insertion
in hdg1-1 is located in an intron of HDG1, the full-length HDG1 transcript could be
produced but no RT-PCR products were amplified from hdg1-1 by using the primers
encompassing the insertion site (data not shown).
Discussion
HDG2 is involved in determining the identity of petals and stamens
Previous studies have reported that pdf2-1 and hdg2-3 single mutants show no
obvious phenotype (Abe et al., 2003; Nakamura et al., 2006). I found, however, the
short stamens were occasionally absent in these mutants (Figure II-3a). Furthermore,
pdf2-1 hdg2-3 was shown to cause partial homeotic conversions of petals and stamens
into sepals and carpels, respectively. Since hdg2-3 carries a T-DNA insertion in the
HD-coding exon of HDG2 and no transcripts including this exon were detectable
(Figure II-13a), it may represent a loss-of-function mutation, suggesting that HDG2
together with PDF2 is required for the identity of petals and stamens.
72
I note that, while pdf2-2 also sometimes had reduced number of stamens,
pdf2-2 hdg2-3 showed no homeotic phenotype. The site of T-DNA insertion in pdf2-1 is
located in the midst of the START domain-coding sequence of PDF2, while that in
pdf2-2 is in its C-terminal part. Thus, it is conceivable that the pdf2-1 allele, which
could produce PDF2 truncated in the START domain, has some negative effects on the
determination of stamen and petal identity. I also observed that neither atml1-1 nor
atml1-3 in combination with hdg2-3 showed homeotic flower defects. It is possible that,
unlike PDF2, ATML1 does not play a role in determining the floral organ identity.
Alternatively, because these atml1 alleles do not represent the mutant disrupting the
START domain of ATML1 like pdf2-1, their defects might be recovered by PDF2.
HDG1, HDG5, and HDG12 also function in flower development
hdg1-1 has a T-DNA in an intron interrupting the START domain-coding
sequence of HDG1. hdg1-1 also often had reduced number of the short stamens while
hdg1-2, which carries a T-DNA insertion in the ZLZ-coding region and likely represents
a loss-of-function allele, had normal flowers. These results suggest that hdg1-1 also
have some negative effects on floral organ development. The severe phenotype such as
carpelloid stamens and the lack in anthers in pdf2-1 hdg1-1 may reflect synergistic
effects of these alleles. On the other hand, the reduction in the number of the short
stamens in pdf2-1 was enhanced by hdg1-2 rather than by hdg1-1 (Figure II-3a). This
might be due to the only moderate down-regulation of AP3 in pdf2-1 hdg1-2 (Figure
II-10a). Indeed, a very weak mutant allele of AP3, ap3-11, produces fertile stamens but
73
with reduction in number (Yi and Jack, 1998). However, the possibility should not be
excluded that the effect of pdf2 and hdg mutations on the number of stamens is through
the modulation of other factors than AP3. Taken together, I suggest that HDG1 and
PDF2 play a cooperative role in the formation of stamens. Preferential expression of
HDG1 in the epidermis of developing stamens also insists that HDG1 functions in
stamens (Figure II-7). However, given the fact that neither hdg1-1 hdg2-3 nor hdg1-2
hdg2-3 showed any additional phenotype (data not shown), the role of HDG1 might be
subsidiary to that of PDF2 in floral organ development.
I also observed homeotic conversions of petals and stamens in pdf2-1 hdg5-1.
hdg5-1 carries a T-DNA insertion in the middle of the START domain-coding exon of
HDG5, suggesting that the property of hdg5-1 is also similar to that of pdf2-1. However,
the effect of hdg5-1 may be moderate compared to that of pdf2-1 and hdg1-1 because
hdg5-1 single mutants had normal flowers. Furthermore, hdg5-1 had no additional
effect on the phenotype of hdg2-3 and hdg1-1, suggesting again that PDF2 plays a
principal role among the HD-ZIP IV family in flower development. Since hdg5-2, in
which the START domain-coding region is predicted to be intact, showed no homeotic
phenotypes in the double mutant with pdf2-1, it likely represents a weaker allele. It is
also possible again that the START domain has a negative regulatory role in the
activation of HD-ZIP IV proteins.
The T-DNA insertion in hdg12-2 is located in the middle of the ZLZ
domain-coding exon of HDG12, suggesting that hdg12-2 is a loss-of-function allele.
74
Thus relatively weak phenotype observed in pdf2-1 hdg12-2 flowers suggests that
HDG12 is less involved in flower development than is HDG2.
Among other HD-ZIP IV mutants investigated, pdf2-1 hdg3-1 showed
increased frequency of infertile and filamentous stamens (Figure II-9), while the hdg3-1
single mutant was identical to wild type (data not shown). This implies that HDG3 is
also involved in proper development of stamens together with PDF2 and is consistent
with a previous report suggesting that HDG3 functions in anther dehiscence (Li et al.,
2007).
pdf2-1 has non-cell-autonomous effects on AP3 expression
Reduction of AP3 expression was not limited to epidermal cells of the floral
meristem in pdf2-1 hdg2-3, while the pdf2-1 transcript was detected specifically in the
epidermis of the inflorescence meristem (Figures II-12d,l,m and II-14b). This suggests
that pdf2-1 could have non-cell-autonomous effects on the determination of floral organ
identity, although the possibility that the pdf2-1 gene product directly binds to the AP3
promoter and represses its expression cannot be excluded. In maize, the fusion protein
of the ZmOCL1 HD-ZLZ domains with the repressor domain of the Drosophila
Engrailed protein reduces expression of GA20 oxidase and probably the gibberellin
(GA) content (Khaled et al., 2005). Synthesis of functional GAs may be required for
maturation of petals and stamens along with the promotion of AP3 expression (Yu et al.,
2004; Plackett et al., 2011). Although no GA-deficient mutants cause abnormalities in
floral organ identity (Goto and Pharis, 1999), evidence of a link between GA signaling
75
and the floral homeotic genes is mounting (Plackett et al., 2011). It might be possible
that GA synthesis is reduced in the epidermis of pdf2-1 hdg2-3 flowers, thereby leading
to a reduced expression of AP3 non-cell-autonomously. There are some reports
suggesting the existence of epidermis-derived non-cell-autonomous signals that regulate
whole organ growth but the identity of these signals remains elusive (Savaldi-Goldstein
et al., 2007; Savaldi-Goldstein and Chory, 2008). Anastasiou et al. (2007) proposed a
possibility that KLU, a cytochrome P450 monooxygenese, can modify fatty-acid-related
molecules and regulate organ growth non-cell-autonomously. Considering that
epidermis-expressed HD-ZIP IV proteins regulate expression of the genes related to
lipid metabolism such as BODYGUARD (BDG), FIDDELHEAD (FDH), and LIPID
TRANSFER PROTEIN (LTP) (Abe et al., 2003; Wu et al., 2011), it is also possible that
extracellular lipid composition is important for non-autonomous signaling and is greatly
affected in the epidermis of floral organs in the double mutants with the homeotic
phenotype.
AP3 expression is maintained by direct interaction of the AP3/PI heterodimer
to the AP3 promoter region in an autoregulatory fashion (Hill et al. 1998; Tilly et al.
1998; Lamb et al. 2002). PI expression was less affected in the double mutants with
homeotic phenotype than AP3 expression and only gradually decreased as flowers
matured (Figure II-10j). Thus, the inhibitory effect of pdf2-1 and/or hdg mutations may
not be on the autoregulatory mechanism of AP3/PI expression but rather specific to
AP3.
76
In conclusion, my results suggest that cooperative functions of PDF2 with at
least HDG1, HDG2, HDG5, and HDG12 in the epidermis are involved in determining
the identity of petals and stamens. However, because of the limitations of mutant alleles
available for analysis, the reasons why the phenotypes detected in double mutant
combinations remain not completely understood. There might also be additional
HD-ZIP IV members that participate in floral organ formation, although they could have
been missed due to the lack of appropriate mutant alleles. For future work, it will be
necessary to generate further multiple mutant combinations and RNAi-induced
knockdown plants to define precise roles of each gene.
Materials and Methods
Plant material and growth conditions
Arabidopsis thaliana accession Columbia-0 (Col-0) was used as the wild type. Most of
the T-DNA insertion alleles have been described previously (Abe et al., 2003;
Nakamura et al., 2006). atml1-3, pdf2-2, and hdg5-2 alleles were derived from the
SALK insertion line, SALK_033408 (Roeder et al., 2012), the SAIL lines,
SAIL_503_E09 and SAIL_238_A05, respectively, and obtained from ABRC. Plants
were grown under the condition descried in the chapter I, except for the continuous light
and growth temperature, which was at 23˚C. Genotypes of double mutants were
determined by PCR of genomic DNA with gene-specific primers shown in previous
studies (Abe et al., 2003; Nakamura et al., 2006) and Table II-1.
77
Anatomy of floral organs
For anatomy, early-formed (first to tenth) flowers on the primary inflorescence were
examined under stereomicroscopy (n ≥ 15). For cross sections, flowers were embedded
in Technovit 7100 (Kulzer), sectioned and stained as described in the chapter I.
For scanning electron microscopy (SEM), flowers were fixed in FAA for 1 h
at room temperature and dehydrated in an ethanol series. After exchange of ethanol to
isoamyl acetate by a graded ethanol-isoamyl acetate series, samples were critical-point
dried in liquid CO2, coated with platinum, and viewed on a SEM JOEL JSM-6510LV
(JOEL).
Confocal microscopy
Apical part of the primary inflorescence was embedded in 7% agar and sectioned with a
vibratome. Confocal microscopy was performed on an inverted fluorescent microscope
IX70 (Olympus) equipped with a confocal unit. Fluorescence of pAP3::GFP was exited
with a wavelength of 488 nm and detected using a CCD camera through 500-550 nm
band-pass filter. Autofluorescence of chlorophyll was detected at 615-680 nm.
Plasmid construction
For making pAP3::GFP, AP3 promoter of 830-bp length was amplified by PCR using
Ex Taq polymerase (Takara) and cloned into pGEM-T Easy vector (Promega).
Subsequently, the fragment was cut with HindIII and BglII and cloned into the
pBI101-based Ti plasmid vector that carries a GFP reporter gene (Matsuhara et al.,
78
2000). For making 35S::AP3, full-length AP3 cDNA was amplified by RT-PCR with
total RNA extracted from wild-type flowers, cloned into pGEM-T Easy vector, cut with
BamHI, and then cloned into the BamHI site of pBI121 (Clontech). The resulting
35S::AP3 fusion gene was cloned as HindIII/EcoRI fragment into pBarMAP vector
(Breuninger et al., 2008). For pPDF2::PDF2 construction, PDF2 promoter sequence of
1591 bp was amplified from wild-type genomic DNA and the full-length PDF2 cDNA
was amplified by RT-PCR. After the resulting fragments were respectively cloned into
pGEM-T Easy, XbaI/ClaI fragment of the PDF2 promoter and ClaI/BglII fragment of
the PDF2 cDNA were sequentially cloned into the modified pBarMap. Primer pairs
used were listed in Table II-1.
Plant transformation
The Ti plasmids were introduced into Agrobacterium tumefaciens C58C1 by
electroporation. For pAP3::GFP plants, Col-0 plants were transformed by the floral dip
method (Clough and Bent, 1998). The pAP3::GFP transgene was further introduced into
pdf2-1 hdg2-3 and pdf2-1 hdg1-1 mutants by crossing. Transformation with 35S::AP3
and pPDF2::PDF2 constructs was performed by floral spray methods (Chung et al.,
2000) and the transformants were selected by spraying 1:10 dilution of BASTA.
RNA extraction and RT-PCR
Total RNA was prepared with the RNeasy plant mini kit (Qiagen). First-strand cDNA
was synthesized from 2 µg of total RNA with an oligo(dT) primer using the PrimeScript
79
RT-PCR kit (Takara). Real-time PCR was performed using the DNA Engine Opticon2
System (Bio-Rad) with KAPA SYBR Fast qPCR kit (KAPA biosystems). Gene-specific
primers are listed in Table II-1. ACTIN8 (ACT8) gene was used as an internal control to
normalize the expression data for each gene. For detection of the HD-ZIP IV transcripts
in double mutant combinations, each cDNA fragment was amplified by 22 cycles of
PCR for ACT8, 26 cycles for HDG1, HDG2, HDG5, and HDG12, and 28 cycles for
PDF2, respectively. The PCR condition was 94˚C for 2 min, followed by the cycles of
94˚C for 30 s, 53˚C for 30 s, and 72˚C for 60 s.
In situ hybridization
Inflorescences were fixed in FAA, dehydrated in an ethanol series, passed through a
xylene-ethanol series, embedded in Paraplast (Sigma) and sectioned at 7 µm thickness.
Gene-specific RNA probes for AP3, PI, AP1, LFY and UFO were prepared as described
previously (Jack et al., 1992; Weigel et al., 1992; Goto and Meyerowitz, 1994;
Gustafson-Brown et al., 1994; Lee et al., 1997). For the pdf2-1 transcript, PDF2 cDNA
fragment was amplified with the primers used for semi-quantitative RT-PCR (Table
II-1) and cloned into pGEM-T Easy. The fragment was then transferred to the EcoRI
site of pBluescript SK+ (Stratagene) and linearized with BamHI and XhoI to prepare
antisense and sense probes, respectively. Probe labeling, hybridization and
immunological detection were performed as previously described (Abe et al., 1999).
Histochemical GUS staining
80
2.5-kb promoter of HDG1 was amplified with primers 5’-CTGCA GATGA AGGCT
TCCAA TGTTG-3’ and 5’-GGATC CGAGG GAAAT ATTTA ATGAA G-3’ and with
wild-type genomic DNA as template. PCR fragment was cloned into pGEM-T Easy
Vector (Promega) and verified by DNA sequencing. The HDG1 promoter fragment was
cleaved with PstI/BamHI and cloned into the corresponding sites of pBluescript SK+
(Stratagene) and subsequently inserted in front of the GUS gene in the binary vector
pBI101.3 (Clontech) by using the SalI/BamHI restriction sites. The resulting plasmid
pHDG1::GUS was introduced into wild-type plants via Agrobacterium-mediated
transformation and transformants were isolated by kanamycin selection as described in
the Materials and Methods. GUS staining, fixing tissue, and sectioning were performed
as described (Nakamura et al., 2006).
81
Tables
Table II-1. Primers used in this study
(a) Primers used for genotyping mutant alleles.
-----------------------------------------------------------------------------------------------Genotype Primer Sequence (5’ to 3’)
-----------------------------------------------------------------------------------------------PDF2-2 F, ATTGA TAGGA TCTCT GCTAT TGC
R, CTGTT GTCGA CATTG TTGTC
pdf2-2
F, ATTGA TAGGA TCTCT GCTAT TGC
SynLB, GAAAT GGATA AATAG CCTTG CTTCC
HDG5
F, TCCAA GTGAT GTTGA CAATG GGA
R, TAAGT TCTTG AACAC AAGAG ACAGC
hdg5-2
F, TCCAA GTGAT GTTGA CAATG GGA
SynLB, GAAAT GGATA AATAG CCTTG CTTCC
-----------------------------------------------------------------------------------------------(b) Primers used for plasmid constructions for complementation tests.
-----------------------------------------------------------------------------------------------PCR product Primer Sequence (5’ to 3’)
-----------------------------------------------------------------------------------------------AP3 promoter
F, AAGCT TAGTT TTGAA ACAAC ACTAA
R, AGATC TTTTT GTTGA AGAGA TTTGG
AP3 cDNA
F, GGATC CATGG CGAGA GGGAA GATC
R, GGATC CTTCA AGAAG ATGGA AGGTA
PDF2 promoter
F, TCTAG AGAAA AAAAA AACAA GACG
R, ATCGA TGAGA CAATG ATAAA GAGAA G
PDF2 cDNA
F, ATCGA TGTAC CATCC AAACA TG
R, GGGCC CAGAT CTACG CTCCT CCTCC AAC
-----------------------------------------------------------------------------------------------(c) Primers used for RT-PCR.
-----------------------------------------------------------------------------------------------Gene Primer Sequence (5’ to3’)
-----------------------------------------------------------------------------------------------82
AP3
F, GAATA TGGCG AGAGG GAAGA
R, CTAGC CTCTG CTTGA TCTGA
PI
F, TGGGT AGAGG AAAGA TCGAG
R, CTTCA AATGC CTGAG CTCCA
AP2
F, TCCCA ATTCA AACCA CCAAT
R, CGCCG GAAAC AGTGA GAA
AG
F, AGCAC AACCT TACCT TCCAT
R, AACAG AGAGC TCGTA AGCTT
SEP3
F, GAAAG CTGTA CGAGT TTTGC AG
R, TTGAA GGCAC ATTGG GTTCT
ACT8
F, CTCAC GGAGA TCTTC ATCGT C
R, TCTCT TGCTC GTAGT CGACA G
PDF2
F, GCGCT CATCAA TAACT CCTT
R, CAAAC ATGTT TGGAT GGTAC
HDG1
F, TCAAC GGTTT TCTCG ACGAC
R, CGTTC GATTT GAGTC TTCAT C
HDG2
F, AGATC TATGT TCGAG CCAAA TATGC
R, CTCTT CCCTT AATCG AGC
HDG5
F, GGATC CATGT TGACA ATGGG AGAAG
R, GTCTT GTTGT GCCTT CATT
HDG12 F, CGTCA CACAC CTCAC CAGAT
R, CTTCC ATAGC GGTCA CAG
AP1
F, GTGAT GCTGA AGTTG CTCTT G
R, CCACT GCTCC TGTTG AGC
LFY
F, CTCTC CCAAG AAGGG TTATC
R, TATAT CCCAG CCATG ACGAC
UFO
F, TCTCT CTTTT GCTTA TATCC CTTCA
R, CGCTA AAAGG GCTAT AGTTC ATACA-3’
------------------------------------------------------------------------------------------------
83
AT
Figure II-1. Unrooted phylogenetic tree of HD-ZIP IV proteins. The protein sequences were analyzed using CLUSTAL W (Thompson et al.,
1994). The tree was constructed by using the neighbor-joining method (Saitou and
Nei, 1987).
Figure II-2. Locations of T-DNA insertions in mutant alleles of HD-ZIP IV
genes used in this study.
Exons are indicated by boxes and introns are indicated by lines. Black and gray
colored boxes represent HD and the ZLZ domain. Boxes filled with stripes and dots
represent the START domain and SAD, respectively.
a
Figure II- 3. Phenotype of wild type and pdf2 hdg stamens.
(a) Variation of the number of stamens per flower.
(b) Percentage of phenotypes observed in long (left) and short (right) stamens of wild-type and
a series of mutants. Fertile stamens had yellow mature anthers with pollen grains. Infertile
stamens had greenish anthers without any obvious pollen grains. The stamens with carpel-like
structures such as stigmatic papillae and ovule were counted as carpelloid.
(c) Length of filaments of fertile and infertile stamens. Error bars indicate standard deviation.
(a)
(e)
(b)
(d)
(c)
(f)
(g)
Figure II-4. Floral phenotype of pdf2-1 hdg2-3.
(a-c) Mature flowers of wild type (a), hdg2-3 (b) and pdf2-1 hdg2-3 (c). Both sepals and petals were
removed from a flower on the right side (a-c). Only sepals were removed from a flower in the center (c).
(d) From left to right: a wild-type sepal, a wild-type petal, and three petals typically observed in pdf2-1
hdg2-3.
(e-g) Cross-section of a wild-type petal (e), a wild-type sepal (f), and a pdf2-1 hdg2-3 petal (g).
Bars = 1 mm (a-d), 100 µm (e-g).
Figure II-5. SEM images of petals and stamens.
(a-c) Adaxial surface of a wild type petal (a), a pdf2-1 hdg2-3 petal (b), and a wild type sepal (c).
(d-f) Abaxial surface of a wild type petal (d), a pdf2-1 hdg2-3 petal (e), and a wild type sepal (f).
(g) Wild type stamen.
(h-l) a pdf2-1 hdg2-3 stamens with stigmatic papillae (h), with an abnormally enlarged anther
and stigmatic papillae (i), with ectopic ovule-like structures (j, k), and with no anther (l).
Bars = 10 µm (a-f), 100 µm (g-l). Figure II-6. Floral phenotypes of hdg single and pdf2-1 hdg double mutants.
(a,b) hdg1-1(a) and pdf2-1 hdg1-1 (b).
(c,d) hdg1-2 (c) and pdf2-1 hdg1-2 (d).
(e,f) hdg5-1 (e) and pdf2-1 hdg5-1 (f).
(g,h) hdg12-2 (g) and pdf2-1 hdg12-2 (h).
Bars = 1 mm.
Figure II-7. Promoter activity of HDG1 examined by beta-glucuronidase (GUS)
reporter system using 2.5-kbp region upstream of the HDG1.
(a) Inflorescence of pHDG1::GUS introduced Col-0 plants.
(b-e) Epidermis specific GUS staining pattern in floral meristem (b), young stamens
and the center of carpels (c), inflorescence stems (d), and in young ovules (e).
Bars = 1 mm (a) and 100 µm (b-e).
( )
( )
Figure II-8. SEM images of petal surface of pdf2-1 hdg12-2. Adaxial (a) and abaxial surface (b). Bars = 10 µm.
( )
Figure II-9. Stamen phenotype of pdf2-1 hdg and atml1 hdg mutants other
than shown in Figure II-3.
(a) Variation of the number of stamens per flower.
(b) Percentage of phenotypes observed in long and short stamens of wild-type and
mutants.
Figure II-10. Expression analysis of AP3 and PI in mutant inflorescences.
Real-time RT-PCR was performed using inflorescence apices containing floral
buds younger than stage 13. Error bars indicate standard deviations of three
independent samples. Asterisks indicate a significant difference from the wild
type (Student’s t test; *P < 0.05 and **P < 0.1).
Figure II-11. Expression analysis of floral organ identity genes in mutant inflorescences.
(a) Real-time RT-PCR of AP2, AG and, SEP3. RNA was extracted from inflorescence apices
containing floral buds younger than stage 13. Error bars indicate standard deviations of three
independent samples.
(b-l) in situ hybridization patterns of AP1 (b-d), LFY (f-h), and UFO (j-l). Antisense probes for
each gene were hybridized with wild type (b, f, j), pdf2-1 hdg1-1 (c, g, k), and pdf2-1 hdg2-3 (d, h,
l) sections. Sense probes produced no signal in wild type flowers (e, i, k). Bars = 100 µm.
Figure II-12. Temporal and spatial expression patterns of AP3 and PI.
(a) Temporal expression of AP3 and PI during flower development. Flowers were sorted
into four groups according to their size. More than two biological replicates, each
contained flowers from more than 150 independent inflorescences, were analyzed.
(b-e) In situ hybridization of AP3 in wild type (b), pdf2-1 hdg1-1 (c), and pdf2-1 hdg2-3
(d).
Arrows indicate signals at presumptive whorls 2 and 3 at stage 3 (d) and arrowheads
indicate signals at the basal part of petals and stamens at stage 5 (left) and stage 6 (right)
(c). Wild-type sections were also examined with the AP3 sense probe (e).
(f-i) In situ hybridization of PI in wild type (f), pdf2-1 hdg1-1 (g), and pdf2-1 hdg2-3 (h).
Wild-type sections were also examined with the AP3 sense probe (i).
(j-l) Confocal microscopy images of wild-type (j), pdf2-1 hdg1-1 (k), pdf2-1 hdg2-3 (l, m)
inflorescences expressing pAP3::GFP. An arrow indicates the GFP fluorescence in a
developing petal. Numbers indicate floral stages.
Scale bars = 100 µm.
Figure II-13. Floral phenotype of plants transformed with 35S::AP3 or
pPDF2::PDF2.
(a,b) Inflorescences of pdf2-1 carrying 35S::AP3 (a) and pdf2-1 hdg2-3 carrying
35S::AP3 (b).
(c,d) SEM images of adaxial surface (c) and abaxial surface (d) of a petal in pdf2-1
hdg2-3 carrying 35S::AP3.
(e-h) Inflorescences of pdf2-1 carrying pPDF2::PDF2 (e), pdf2-1 hdg2-3 carrying
pPDF2::PDF2 showing wild-type phenotype (f), and pdf2-1 hdg2-3 carrying
pPDF2::PDF2 producing short petals (g, h). Sepals and petals were removed from a
flower on the right side (h).
Bars = 5 mm (a, b, e-g), 10 µm (c, d), and 1 mm (h).
Figure II-14. Detection of HD-ZIP IV transcripts in pdf2 hdg mutants.
(a) Semi-quantitative RT-PCR of HD-ZIP IV genes in pdf2 hdg mutants. PCR was performed
using gene-specific primers designed to amplify the region encompassing the HD-ZIP domain of
each gene.
(b) In situ hybridization of PDF2 in wild type and pdf2-1. The 5’-UTR of PDF2 mRNA was
used as probe. No signal was detected in the wild type with the sense probe. Bar = 100 µm.
General Discussion
I have shown several genetic factors that are involved in the growth and
development of Arabidopsis thaliana. In the first chapter, I reported that the ACL1 gene
was identical to SRFR1, which negatively regulates defense responses against pathogens.
Constitutive activation of defense signaling pathway induced by SNC1 was found to
cause the severe stunted growth such observed in the acl1-1 mutant. And in the next
chapter, HD-ZIP IV genes, whose function in the plant development had been unknown,
are suggested to be involved in the proper development and determination of organ
identity in flowers. As far as I had investigated, it is uncertain that the phenotype of the
HD-ZIP IV mutants is related to the perception of the information from the outer
environment like pathogen infection. However, in the analysis of the acl1 mutants, it
became clear that the ACL1 gene is a key regulator of plant growth correlated with the
resistance pathway against pathogens, whose activity is also influenced by temperature
and nutrient condition in the growth media.
At present, I have only identified the genetic factors or mutations as an input,
and plant growth phenotype as an output of the signaling pathways that regulate plant
growth and development. I expect further studies would reveal the molecular
mechanisms that link these inputs and outputs, and more details in the co-operative
regulation of plant growth and development by genetic and environmental factors. In
molecular aspects, lipid metabolism might be related to both of the signaling pathways
involved in the growth of the acl1 and HD-ZIP IV mutants. EDS1 and PAD4, which are
84
essential for defense response pathways via TIR-NBS-LRR R genes, encode acetyl
hydrolase with homology to eukaryotic lipases (Falk et al., 1999; Jirage et al., 1999). It
was therefore suggested that they might play a role in lipid based signaling by
hydrolysing a lipid substrate. On the other hand, epidermis-expressed HD-ZIP IV
proteins regulate expression of the genes related to lipid metabolism (Abe et al., 2003;
Wu et al., 2011), and it is also possible that extracellular lipid composition is important
for the floral development. Although further investigations on the relationships between
lipid metabolism and the regulation of defense signaling, or floral development are
needed, it would be interesting to examine the involvement of the lipid metabolisms in
the regulatory pathways of plant growth and development in future studies.
85
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