Download TaCYP78A5 regulates seed size in wheat (Triticum aestivum)

Journal of Experimental Botany, Vol. 67, No. 5 pp. 1397–1410, 2016
doi:10.1093/jxb/erv542 Advance Access publication 27 December 2015
RESEARCH PAPER
TaCYP78A5 regulates seed size in wheat (Triticum aestivum)
Meng Ma1, Huixian Zhao1,2,*, Zhaojie Li1, Shengwu Hu2,3, Weining Song2,3 and Xiangli Liu1,*
1 College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China
State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A & F University, Yangling, Shaanxi 712100, China
3 College of Agronomy, Northwest A & F University, Yangling, Shaanxi 712100, China
2 * Correspondence: [email protected]; [email protected]
Received 3 August 2015; Accepted 25 November 2015
Editor: Gerhard Leubner, Royal Holloway, University of London
Abstract
Seed size is an important agronomic trait and a major component of seed yield in wheat. However, little is known about
the genes and mechanisms that determine the final seed size in wheat. Here, we isolated TaCYP78A5, the orthologous
gene of Arabidopsis CYP78A5/KLUH in wheat, from wheat cv. Shaan 512 and demonstrated that the expression of
TaCYP78A5 affects seed size. TaCYP78A5 encodes the cytochrome P450 (CYP) 78A5 protein in wheat and rescued the
phenotype of the Arabidopsis deletion mutant cyp78a5. By affecting the extent of integument cell proliferation in the
developing ovule and seed, TaCYP78A5 influenced the growth of the seed coat, which appears to limit seed growth.
TaCYP78A5 silencing caused a 10% reduction in cell numbers in the seed coat, resulting in a 10% reduction in seed
size in wheat cv. Shaan 512. By contrast, the overexpression of TaCYP78A5 increased the number of cells in the seed
coat, resulting in seed enlargement of ~11–35% in Arabidopsis. TaCYP78A5 activity was positively correlated with
the final seed size. However, TaCYP78A5 overexpression significantly reduced seed set in Arabidopsis, possibly due
to an ovule development defect. TaCYP78A5 also influenced embryo development by promoting embryo integument
cell proliferation during seed development. Accordingly, a working model of the influence of TaCYP7A5 on seed size
was proposed. This study provides direct evidence that TaCYP78A5 affects seed size and is a potential target for crop
improvement.
Key words: BSMV-VIGS, cell proliferation, CYP78A, seed size, Triticum aestivum L., TaCYP78A5.
Introduction
Wheat (Triticum aestivum L.) is a major food crop. The agronomic characteristics of wheat, particularly seed size, have been
studied extensively (Smidansky et al., 2002; Khalid et al., 2004;
Shomura et al., 2008; Wang et al., 2012). Seed size is a major
component of seed yield and an important trait selected during
domestication and modern crop breeding (Shomura et al., 2008).
Small-seeded species produce more seeds for a given amount
of energy than large-seeded species (Aarssen and Jordan, 2001;
Henery and Westoby, 2001), and studies suggest that small seeds
have a greater tendency to accumulate as persistent seed banks
in the soil (Thompson et al., 2001). However, larger seeds, by
offering a greater supply of nutrients, have favorable effects on
seeding vigor to tolerate many of the stresses encountered during seedling establishment, thus promoting and stabilizing yield
(Leishman et al., 2000; Moles et al., 2005). Therefore, increasing
seed size is a major goal of crop breeding globally.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
1398 | Ma et al.
Seed size is determined by the growth and development of
the embryo, endosperm and maternal integument (Ohto et al.,
2005; Sundaresan, 2005; Berger et al., 2006). For example,
ABSCISIC ACID DEFICIENT2 (ABA2) regulates embryo
and endosperm size by promoting cell proliferation and early
cellularization of the endosperm during early seed development, respectively, ultimately affecting seed size (Cheng
et al., 2014). Gain-of-function mutants of HAIKU1 (IKU1),
HAIKU2 (IKU2) and MINISEED3 (MINI3) produce larger
seeds due to enhanced endosperm proliferation and delayed
endosperm cellularization (Garcia et al., 2003; Luo et al.,
2005; Zhou et al., 2009; Wang et al., 2010). TRANSPARENT
TESTA GLABRA 2 (TTG2) and APETALA 2 (AP2) promote
seed growth by increasing cell expansion in the integuments;
the integuments limit the size of the seed coat (Johnson, 2002;
Garcia et al., 2005; Jofuku et al., 2005); AUXINRESPONSE
FACTOR2 (ARF2) and DA1 (DA means ‘large’ in Chinese)
act on seed size by restricting cell proliferation in the integuments (Jofuku et al., 2005; Ohto et al., 2005; Schruff et al.,
2006; Li et al., 2008).
CYP78A is a plant-specific gene family that is highly
conserved in land plants from bryophytes to angiosperms
(Mizutani and Ohta, 2010; Nelson, 1999, 2006). In Arabidopsis
thaliana and rice, several CYP78As have been implicated in
the control of seed development. For example, CYP78A6,
CYP78A8 and CYP78A9 exhibit redundant functions in
controlling floral organ growth and integument development
by promoting cell proliferation in Arabidopsis (Fang et al.,
2012; Sotelo-Silveira et al., 2013). Overexpression of these
genes increases Arabidopsis seed size. In rice, CYP78A13
affects the proper size balance between the embryo and
endosperm by controlling cell size in the embryo and cell
death in the endosperm (Nagasawa et al., 2013). Loss-offunction mutants of CYP78A13 exhibit a large embryo and
small endosperm; by contrast, CYP78A13 overexpression
results in a small embryo and enlarged endosperm, resulting
in a large seed (Nagasawa et al., 2013; Yang et al., 2013; Xu
et al., 2015). CYP78A5/KLUH acts independently of other
tested maternal factors that influence integument cell proliferation; the expression of CYP78A5/KLUH is limiting for
seed growth in Arabidopsis (Adamski et al., 2009). Similarly,
SlKLUH, an orthologous gene of Arabidopsis CYP78A5 in
tomato (Solanum lycopersicum), controls fruit size by promoting integument cell proliferation (Chakrabarti et al., 2013;
Monforte et al., 2014; van der Knaap et al., 2014). However,
the influence of potential wheat homologs of CYP78A5 on
seed development has not been reported.
Because of the importance of seed size, quantitative trait
loci (QTLs) related to seed size characteristics have been
identified in wheat (Nakamura et al., 2007; Somyong et al.,
2011; Williams and Sorrells, 2014). However, only a few genes
associated with seed size have been isolated (Ma et al., 2012a;
Yang et al., 2012), and little is known about the mechanisms
that determine final seed size in wheat. Here, we isolated
TaCYP78A5, an ortholog of Arabidopsis CYP78A5/KLUH
in wheat that encodes wheat CYP78A5 and can rescue the
phenotype of the Arabidopsis deletion mutant cyp78a5.
We characterized the features of TaCYP78A5 including
gene structure, genome localization and expression pattern.
TaCYP78A5 silencing caused a reduction in cell numbers
in the wheat seed coat, leading to a 10% decrease in wheat
seed size. By contrast, TaCYP78A5 overexpression resulted
in the production of more cells in the seed coat, resulting
in seed enlargement of ~11–35% in Arabidopsis. Moreover,
TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation. These results,
which demonstrate that TaCYP78A5 affects embryo development, provide new insights on the possible function of
CYP78A5 gene in seed development. In summary, our results
indicate that TaCYP78A5 affects wheat seed size by promoting integument cell proliferation in the developing ovule and
seed and that seed size is positively associated with the expression level of TaCYP78A5.
Materials and methods
Plant materials and growth
Wheat (Triticum aestivum L.) cultivars ‘Shaan 512’ and ‘Shaanmai
159’, which exhibit large-grain and small-grain phenotypes, respectively, were utilized to clone the target gene TaCYP78A5 and to
knockdown the target in this study. To investigate the genomic origin of the target gene, different wheat species, including T. urartu
(AA), Aegilops tauschii (DD), T. turgidum ssp. durum (AABB) and
T. aestivum (AABBDD), were selected for gene-specific PCR using
their genomic DNA as templates. To further confirm the chromosome location of the target gene, genomic DNA from a set of
nulli-tetrasomic (NT) and double ditelosomic (DT) lines of wheat
accession ‘Chinese Spring’ was used as the template for gene-specific
PCR. The wheat growth conditions for target gene silencing by the
barley stripe mosaic virus-induced gene silencing (BSMV-VIGS)
technique were as described previously (Ma et al., 2012b).
The wild-type (WT) Arabidopsis thaliana ecotype Columbia-0
(Col) and mutant cyp78a5 (Salk_024697C) in the Columbia-0 background obtained from the Arabidopsis Biological Resource Center
(ABRC, https://abrc.osu.edu/) were used to transform Arabidopsis
with the target gene TaCYP78A5. Its seeds were germinated and
then grown under suitable conditions with a 16-h-light/8-h-dark
cycle and 70% relative humidity (light intensity 170–200 mmol m−2
s−1) at 20–22°C for 70 d until the last silique on the inflorescences
were dry.
Cloning and sequence analysis of TaCYP78A5
To clone wheat TaCYP78A5, the Arabidopsis CYP78A5 (GenBank
accession no. EFH69027.1) protein was used as a query probe
to search the wheat and barley (Hordeum vulgare L.) expressed
sequence tag (EST) databases in GenBank (http://www.ncbi.nlm.
nih.gov/). A wheat EST (GenBank accession no. BE424262.1) and
a barley full-length cDNA (GenBank accession no. BAK05332.1)
were retrieved and assembled as a putative TaCYP78A5 mRNA.
The predicted mRNA sequence was used to design the gene-specific
PCR primers TaCYP78A5-R and -F (Supplementary Table S1 available at JXB online) to amplify the genomic DNA of TaCYP78A5
in wheat.
Details of TaCYP78A5 and its promoter sequence analysis are
provided in Supplementary Method S1.
Total RNA extraction and cDNA synthesis
To clone TaCYP78A5 cDNA from wheat, total RNA was extracted
from immature seeds one week after anthesis using RNAiso-mate
(TaKaRa, Dalian, China). The details of the total RNA extraction
TaCYP78A5 genes influence wheat seed size | 1399
are provided in Supplementary Method S1. cDNA was recovered
from total RNA using Oligotex-dT30 (TaKaRa, Dalian, China).
TaCYP78A5 cDNA was cloned using the primers TaCYP78A5-R
and TaCYP78A5–F (Supplementary Table S1) and the PrimeScript™
II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China).
To analyze TaCYP78A5 silencing in wheat spikes or seeds, spikelets or seeds were collected from the middle part of BSMV-infected
spikes, and total RNA and TaCYP78A5 cDNA were prepared from
the spikelets or seeds as described above.
BSMV-mediated TaCYP78A5 gene silencing
To knockdown the expression of TaCYP78A5 in wheat, the
BSMV-VIGS technique was used as previously reported (Ma
et al., 2012b). The construction of the barley stripe mosaic virus
(BSMV)-derived vector for target gene silencing in wheat seeds is
described in Supplementary Method S1. The BSMV construct
BSMV:TaCYP78A5 (carrying a fragment of the TaCYP78A5
cDNA sequence) was developed as previously described (Ma et al.,
2012b). Ten plants of each wheat cultivar were infected with the
BSMV:TaCYP78A5, BSMV:PDS (BSMV vector carrying a 185 bp
fragment of a barley phytoene desaturase gene PDS) or BSMV:00
(empty vector) constructs on the spikes at the heading stage;
BSMV:00 was used as a negative control, and BSMV:PDS was used
as a positive control (Ma et al., 2012b). The spikelets or seeds from
the middle part of the BSMV-infected spikes were collected to quantify the transcript abundance of the target gene. This experiment was
repeated five times, and the data were averaged.
Construction of the TaCYP78A5 expression vector and
Arabidopsis transformation
To investigate the effect of TaCYP78A5 overexpression on seed
development, three expression vectors with TaCYP78A5 under
the control of three promoters with different expression patterns
in developing Arabidopsis ovules or/and seeds were constructed,
respectively. The CYP78A5 promoter (pCYP78A5 or pKLU) has
high activity specifically in developing ovules and seeds (Zondlo
and Irish, 1999; Adamski et al., 2009). The CaMV 35S promoter
(p35S) has relatively low activity in the ovules but high activity in
developing seeds (Jenik and Irish, 2000; Hraška et al., 2008), and the
INNER NO OUTER promoter (pINO) is highly expressed specifically in the ovule integument (Villanueva et al., 1999).
To determine the subcellular localization of TaCYP78A5, the
TaCYP78A5 coding sequence without a stop codon was inserted
upstream of the GUS coding sequence in the pCAMBIA3301
expression vector to obtain the fusion protein TaCYP78A5-GUS
upon target gene expression. The details of the construction of the
three TaCYP78A5 expression vectors, p35S::TaCYP78A5:GUS,
pKUL::TaCYP78A5:GUS and pINO::TaCYP78A5:GUS are
described in Supplementary Method S1.
Arabidopsis transformation was performed according
to Zhang et al. (Zhang et al., 2006). Transgenic plants were
selected by spraying 0.1% glufosinate (BASTA) on seedling
leaves. At least three independent transgenic lines bearing
each construct were obtained.
Reverse transcriptase-polymerase chain reaction and
quantitative real-time RT-PCR
To determine the efficacy of TaCYP78A5 silencing at the
RNA level in wheat, seeds on the middle of wheat spikes from
BSMV:TaCYP78A5-, BSMV:PDS- or BSMV:00-infected plants
were collected at 5, 10, 15, 20 and 25 days post inoculation (dpi)
for reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis. The amount of RNA in each reaction was normalized
using primers specific for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), which is constitutively expressed in wheat. To determine
the expression level of TaCYP78A5 in different transgenic T3 lines
of Arabidopsis, RT-PCR analysis was performed with total RNA
isolated from the top 2 cm of the inflorescence or siliques from each
transgenic line, and ubiquitin-conjugating 21 (UBC) was used as a reference. All primers are listed in Supplementary Table S1. Details of
the quantitative real-time RT-PCR are provided in Supplementary
Method S1.
PCR-based genotyping
The cyp78a5 mutant allele was identified by PCR analysis using
the LBa1 primer on the T-DNA left border. The CYP78A5 wildtype allele was identified using the primers SALK_024697-LP and
SALK_024697-RP. The molecular identification of TaCYP78A5transgenic plants was performed by PCR analysis using the primers
TaCYP78A5-RT-F and TaCYP78A5-RT-R. Detailed information
on the primers is provided in Supplementary Table S1.
Morphological and cytological characterization and GUS
staining of transgenic plants
Morphological differences in the transgenic plants obtained above
were characterized as previously described (Disch et al., 2006;
Sotelo-Silveira et al., 2013). To measure ovule size, the dissected
pistil was stained with a 0.01% solution of Fluorescent Brightener
28 (SIGMA, China) as previously described (Adamski et al., 2009).
The cell number and cell size in the integument of the seed coat and
cotyledons were measured as previously described (Alonso-Blanco
et al., 1999). To measure the biomass and seed yield, transgenic
plants and five controls were planted in identical pots and cultured
under well-watered conditions. Detailed protocols for the measurements of floral organs, main inflorescence stems, seeds, embryos,
cells, histochemical staining for TaCYP78A5 activity and microscopy are described in Supplementary Method S1.
Results
Isolation and characterization of TaCYP78A5 in wheat
To clone TaCYP78A5 in wheat, a putative TaCYP78A5
mRNA sequence was obtained by assembling wheat
ESTs encoding proteins with similarity to Arabidopsis
CYP78A5. Based on the putative mRNA sequence, primers
TaCYP78A5-F and TaCYP78A5-R were designed to amplify
the complete coding sequence from genomic DNA of wheat
cv. Shaan 512 as the template, and the PCR products were
subcloned into a T clone vector. Three TaCYP78A5 cDNA
sequences were obtained from 20 monoclones, and each of
these three sequences contained a complete open reading
frame (ORF) encoding a polypeptide. These three sequences
were temporarily named TaCYP78A5-1, TaCYP78A5-2 and
TaCYP78A5-3 (Supplementary Fig. S1A). To determine the
chromosomal locations of the three TaCYP78A5 genes, the
PCR mapping method was applied using primers A5-P1,
A5-P2 and A5-P3, and genomic DNA from the nulli-tetrasomic (NT) and double ditelosomic (DT) lines of the wheat
accession Chinese Spring and wheat species with AA, DD,
AABB or AABBDD genomes as templates (Supplementary
Fig. S1B, C). The three TaCYP78A5 sequences were also
aligned with the chromosome-based draft sequence of hexaploid bread wheat (T. aestivum L.) (International Wheat
Genome Sequencing Consortium, http://www.wheatgenome.
1400 | Ma et al.
org/) for further confirmation of their chromosome localization. TaCYP78A5-1, -2 and -3 were located on chromosomes
2BS, 2AS and 2DS, respectively, and consequently designated
TaCYP78A5-A, TaCYP78A5-B and TaCYP78A5-D, respectively. Their GenBank accession numbers are KP768392,
KP768393 and KP768394, respectively.
The coding regions of TaCYP78A5-A, -B and -D are 1651,
1663 and 1650 bp, respectively, and contain two exons and
one intron (Fig. 1A), identical to the coding regions of other
CYP78A family members. The deduced proteins all contained
a hydrophobic region in the N terminus and putative oxygenand heme-binding domains that are typical characteristics of
CYP78A family members (Fig. 1A; Supplementary Fig. S2A)
(Nebert and Gonzalez, 1987). The deduced proteins also exhibited high similarity with each other (Supplementary Fig. S2B).
A phylogenetic tree was constructed with the TaCYP78A5
(putative amino acid sequences of TaCYP78A5-B) and other
reported CYP78A family members from various plant species (Fig. 1B). TaCYP78A5 exhibits very high similarity to
Arabidopsis CYP78A5 (55% identity) and CYP78A10 (53%
identity; GenBank accession no. AEE35551) and is most
similar to Oryza sativa CYP78A13 (76% identity; GenBank
accession no. AB780362.1). CYP78A13 in O. sativa is the
ortholog of Arabidopsis CYP78A5, and CYP78A13 can rescue the cyp78a5 mutant phenotype (Xu et al., 2015). Thus,
we concluded that TaCYP78A5 encodes a cytochrome P450
protein and is the ortholog of CYP78A5.
Most CYP78A family members are involved in seed development (Ito and Meyerowitz, 2000; Anastasiou et al., 2007;
Adamski et al., 2009; Fang et al., 2012; Sotelo-Silveira et al.,
2013; Xu et al., 2015). TaCYP78A5 expression was mainly
detected in immature seeds (0–20 days post flowering)
and young panicles (at 1 and 8 cm) in wheat cv. Shaan512
(Supplementary Fig. S3), consistent with the information for
the EST (GenBank accession no. BG416626.2, 94% identity
with TaCYP78A5) from a H. vulgare seed coat EST library in
the NCBI database. These findings suggest that TaCYP78A5
is involved in seed development.
Silencing of TaCYP78A5 reduces seed size in wheat
To investigate the effect of TaCYP78A5 on seed development,
we knocked down the expression of all three TaCYP78A5
genes in wheat cv. Shaan 512 (large-grain) and Shaanmai
159 (small-grain) by the BSMV-VIGS technique. A 291-bp
fragment of the TaCYP78A5-A cDNA (TaCYP78A5-VIGS)
sequence (corresponding to 45 bp to 335 bp after the translation start codon) was chosen to develop the recombinant
BSMV construct BSMV:TaCYP78A5 (Supplementary
Method S1; Supplementary Figs S4, S5) to specifically and
efficiently silence TaCYP78A5. The infectivity and efficacy
of BSMV-VIGS in both wheat cultivars were investigated as
described previously (Ma et al., 2012b).
We inoculated ten plants of each wheat cultivar by
rubbing each spike with the BSMV:00, BSMV:PDS or
BSMV:TaCYP78A5 construct at the heading stage. Seed
photobleaching was observed in all spikes infected with the
BSMV:PDS construct at 20 dpi, whereas the seeds from
BSMV:00- and BSMV:TaCYP78A5-infected plants remained
green throughout; the representative phenotype of wheat cultivar Shaan 512 is shown in Fig. 2A. RT-PCR analysis indicated that the transcript levels of PDS and TaCYP78A5 were
reduced in BSMV:PDS- and BSMV:TaCYP78A5-infected
plants, respectively, compared to BSMV:00-infected plants
(Fig. 2A). These results indicate that VIGS manipulation of
wheat seeds was effective in this experiment. Furthermore,
mature seeds from the BSMV:TaCYP78A5-infected spikes
were shorter and narrower than those from BSMV:00- and
BSMV:PDS-infected spikes (Fig. 2B). Statistical analysis revealed that seed length, width and seed size of the
BSMV:TaCYP78A5-infected plants were significantly
reduced by ~8%, ~7% and 10% (Fig. 2C; Supplementary Fig.
Fig. 1. TaCYP78A5 structures and phylogenetic analysis of the CYP78A family. (A) Exon and intron structures of the TaCYP78A5-A, -B and -D
genes. Two black boxes indicate exons; black lines in the middle indicate introns; the split indicates the regions in TaCYP78A5-B that are absent in
TaCYP78A5-A and -D; regions encoding hydrophobic domains, oxygen-binding motifs and heme-binding motifs are indicated by oval, circular and
rectangular boxes, respectively. (B) The phylogenetic tree of TaCYP78A5 (putative amino acid sequences of TaCYP78A5-B) and other reported CYP78A
family proteins from Triticum urartu, Arabidopsis thaliana, Oryza sativa, Zea mays, Pinusradiata, Phalaenopsis and Glycine max, constructed by the
neighbor-joining method. (This figure is available in colour at JXB online.)
TaCYP78A5 genes influence wheat seed size | 1401
Fig. 2. TaCYP78A5 silencing leads to a reduction in seed size in wheat cv. Shaan 512. (A) RT-PCR analysis of total RNA isolated from the seeds of
BSMV:00-, BSMV:PDS (phytoenedesaturase)- and BSMV:TaCYP78A5-infected plants at 20 dpi. (B) Detection of target gene silencing in mature seeds
from BSMV:00-, BSMV:PDS- and BSMV:TaCYP78A5-infected plants. (C) Comparison of the length and width of mature seeds from the middle spikelets
of the BSMV:00-, BSMV:PDS- and BSMV:TaCYP78A5-infected plants in which the target gene was silenced. Measurements were obtained from three
plants from each of five replicates and two seeds from each plant (n=30). (This figure is available in colour at JXB online.)
S6C, E), respectively, compared to those of the control group
(BSMV:00- and BSMV:PDS-infected plants). Similar results
were obtained for wheat cv. Shaanmai 159 (Supplementary
Fig. S6B, D, F) and have been reported for cyp78a5 mutants
(Adamski et al., 2009). These results suggest that TaCYP78A5
plays an important role in wheat seed development.
Cell number in the wheat seed coat is reduced in
TaCYP78A5-silenced plants
The effect of CYP78A5 on final seed size is mediated by controlling cell proliferation in ovule integuments. The integument is the future seed coat that ultimately provides an
upper limit to the final seed size (Adamski et al., 2009). To
determine whether the reduced seed size in TaCYP78A5silenced spikes was due to a decreased cell number in the
seed coat, we investigated the cell number in the seed coats
(by measuring the number of outer layer cells in the seed
coat) of BSMV:00- and BSMV:TaCYP78A5-infected plants.
The cell numbers of the seed coat were reduced by ~10% in
seeds of BSMV:TaCYP78A5-infected plants compared with
BSMV:00-infected plants at 20 dpi (Fig. 3A–E). However,
no significant change in the cell length of the seed coat
was observed (Fig. 3C, F). Therefore, the reduced seed size
(Fig. 3D) is most likely attributable to the reduced size of the
seed coat (the seed coat size was represented by the seed coat
perimeter, which was calculated by multiplying the seed coat
cell number by the cell length, n=10), which was due to a 10%
reduction in cell number (Fig. 3E).
CYP78A5 controls seed size by affecting cell proliferation in the ovule integument (Adamski et al., 2009). Whether
TaCYP78A5 also affects cell proliferation in the ovule integument in wheat is unclear. Moreover, CYP78A5 is expressed
not only in ovules but also in developing seeds of Arabidopsis
(Zondlo and Irish, 1999), identical to the expression pattern
observed for TaCYP78A5 in wheat (Supplementary Fig. S3).
To characterize the role of TaCYP78A5 during seed development in wheat, we investigated the integument cell numbers of the developing ovule and seed in plants infected
with the BSMV:TaCYP78A5 or BSMV:00 construct on the
wheat spikes at ~7 days prior to anthesis as described previously (Ma, et al. 2012b). Because the effective silencing of
BSMV-VIGS can last up to 25 days as described previously
(Ma et al., 2012b), the silencing covered nearly all stages of
ovule (0–7 dpi) and seed (8–25 dpi) development. In contrast
to CYP78A5, which only affects ovule integument cell proliferation (Adamski et al., 2009), TaCYP78A5 silencing affected
both ovule and seed integument cell development (Fig. 3H),
resulting in a reduction of seed size (Fig. 3D, G). These results
suggest that the expression of TaCYP78A5 may determine
the growth of the seed coat by affecting the extent of integument cell proliferation in the developing ovule and seed.
Overexpression of TaCYP78A5 increases seed size in
Arabidopsis
To confirm the effects of TaCYP78A5 on the developing
ovule and seed, we characterized TaCYP78A5-overexpressing
transgenic T3 lines bearing different expression vectors
p35S::TaCYP78A5:GUS, pKLU::TaCYP78A5:GUS and
pINO::TaCYP78A5:GUS that exhibit different expression
patterns in the developing Arabidopsis ovule or/and seed. At
1402 | Ma et al.
Fig. 3. TaCYP78A5 silencing causes a reduction in integument cell proliferation wheat ovules and seeds. (A) Seeds from BSMV:00- and
BSMV:TaCYP78A5-infected plants at 20 dpi. (B) Cross-sections (at position of dashed lines in panel A) of seeds stained by Fluorescent Brightener.
(C) Magnified view of the cross-section box in panel B. (D–F) Comparison of seed size (D, represented by the projected area of a seed), cell number
(E) and cell length (F) of the outer seed coat between BSMV:00- and BSMV:TaCYP78A5-infected plants at 20 dpi. (G–H) Comparison of ovule or seed
size (G, represented by the projected area of an ovule or a seed) and cell number of a cross-section of the ovule or outer seed integument (H) between
BSMV:00- and BSMV:TaCYP78A5-infected plants from 5 to 25 dpi. The data represent means ±SE of at least ten independently collected seeds in
which the target gene was silenced; measurements were obtained from two plants from each of five replicates and one seed from each plant. Asterisks
(**) indicate a significant difference from WT at P<0.01 (t-test). Bars: 2 mm (A); 200 μm (B); 100 μm (C). (This figure is available in colour at JXB online.)
least three independent Arabidopsis transgenic lines bearing
each construct were evaluated in this experiment.
The final seed sizes of the pKLU::TaCYP78A5,
35S::TaCYP78A5 and pINO::TaCYP78A5 transgenic lines
were all significantly increased compared to WT (Fig. 4A,
B, J). Moreover, TaCYP78A5 overexpression caused obvious overgrowth of the reproductive organs, as evidenced by
larger and more abundant flowers (Fig. 4C, D), longer and
thicker stems (Fig. 4E) and wider, shorter and more plentiful
siliques (Fig. 4F, L, M). To conduct histological localization
of TaCYP78A5, all constructs expressed TaCYP78A5-GUS
fusion proteins. GUS staining indicated that TaCYP78A5 was
highly expressed in the seed (Fig. 4G) and/or ovule (Fig. 4H).
RT-PCR analysis further confirmed that TaCYP78A5 was
significantly expressed in the inflorescence of transgenic T3
lines bearing each construct (Fig. 4I). A moderate increase
in TaCYP78A5 expression could increase the growth-promoting effects of each genotype in the transgenic lines. Thus,
together with the effect of TaCYP78A5 silencing on seed size
and plant height (Fig. 3A, Supplementary Fig. S7), these
results indicate that TaCYP78A5 plays an important role in
promoting organ and seed growth and that the altered phenotypes are attributable to changes in TaCYP78A5 activity.
Similar observations have been reported for CYP78A5- or
CYP78A9-overexpressing plants (Zondlo and Irish, 1999;
Anastasiou et al., 2007; Adamski et al., 2009; Eriksson et al.,
2010).
The overexpression of TaCYP78A5 also caused defects
in reproductive development, which manifested as shorter
siliques in all transgenic lines containing fewer seeds than WT
(Fig. 4K). These similar phenotypes are normally observed in
cyp78a9 mutants (Ito and Meyerowitz, 2000; Sotelo-Silveira
et al., 2013) or 35S::CYP78A5 transgenic plants (Zondlo
and Irish, 1999). Pollinating WT flowers with pollen from
the TaCYP78A5-overexpressing plants yielded normal numbers of seeds per silique compared to WT, suggesting that
TaCYP78A5 genes influence wheat seed size | 1403
Fig. 4. The overexpression of TaCYP78A5 affects the growth of reproductive organs and seeds in Arabidopsis. (A) Open ethanol-decolorized silique from
the Col and pINO::TaCYP78A5-4 transgenic lines. Images of mature seeds (B), flowers at stage 14 (C), inflorescences (D), main inflorescence stems (E)
and siliques (F) from different genotypes. For panels B, D–F, from left to right are: WT Col, transgenic lines pKLU::TaCYP78A5-5, 35S::TaCYP78A5-3 and
pINO::TaCYP78A5-4 as representative transgenic Arabidopsis bearing each construct and mutant cyp78a5. PKLU/CYP78A5, Arabidopsis CYP78A5
promoter is active in the developing ovule and seed; pINO, INNER NO OUTER promoter is specifically active in ovules. (G) Developing seeds exhibiting
TaCYP78A5 expression in the seed coat and embryo at 2 days after anthesis as determined by transgenic line p35S::TaCYP78A5::GUS analysis (arrow
heads). (H) A developing flower with sepals and stamens removed at stage 13, displaying TaCYP78A5 expression in the developing ovule inside the
gynoecium as determined by transgenic line pINO::TaCYP78A5::GUS analysis (arrow heads). (I) RT-PCR analysis of total RNA isolated from a mixture
of top inflorescences from ten lines per genotype. (J–M) Quantification of mature seed and silique (at stage 14) characteristics in response to altered
TaCYP78A5 expression levels. Thirty plants of the transgenic lines per genotype were grown to maturity without any assisted pollination and harvested
for measurements: (J) Seed size as represented by the projected area of a seed; (K) Seed number per silique; (L) Silique number per main inflorescence
stem; (M) Silique length. The values represent means ±SE (n=20). Asterisks (*) and (**) indicate significant differences from WT (Col) at P<0.05 and
P<0.01 (t-test), respectively. Bars: 2 mm (A–D); 1 mm (E); 200 μm (F, G); and 500 μm (H). Stages are in accordance with those described by Smyth et al.
(1990). (This figure is available in colour at JXB online.)
the reduced seed set in TaCYP78A5-overexpressing plants is
attributable to a defect on the maternal side. Further detailed
analysis of the ovules of pINO::TaCYP78A5-4 transgenic
plants revealed that the overexpression of TaCYP78A5
caused an over-proliferation of ovule integument cells and
tremendous individual differences during ovule development
(Supplementary Fig. S8A–D). Some ovules (31/50) exhibited various degrees of wrinkling, asymmetry and decreased
size and failed to further develop, resulting in female sterility; by contrast, other ovules (19/50) exhibited a full shape
and further developed into mature seeds that were larger than
those of WT (Fig. 4E; Supplementary Fig. S8A, B). In general, the reduction in seed set per silique in the TaCYP78A5overexpressing transgenic line was most likely related to a
developmental defect of the ovule, which was likely due to the
ovule integument cell over-proliferation by the overexpression
of TaCYP78A5. However, the increased seed size was not due
to reduced seed set in the TaCYP78A5-overexpressing transgenic line because pINO::TaCYP78A5-4 plants produced
larger seeds than WT plants with a similar seed set per plant
or per silique (Supplementary Fig. S9A, B, D).
Overexpression of TaCYP78A5 promotes integument
cell proliferation in Arabidopsis
In the above gene silencing experiments, we speculated that
the effect of TaCYP78A5 on seed size was mediated by promoting integument cell proliferation in the developing wheat
ovule and seed. To further confirm this relationship, we analyzed the cell numbers in the seed coat in different TaCYP78A5
transgenic lines. Indeed, TaCYP78A5 overexpression resulted
in an increased cell number in the seed coat, and the increased
cell number was positively associated with the TaCYP78A5
activity level (Fig. 5A, B). However, no obvious difference
in cell area was observed between WT and TaCYP78A5overexpressing plants, except pINO::TaCYP78A5 transgenic
lines, which exhibited reduced cell area (Fig. 5C). Thus, the
formation of large seeds by TaCYP78A5 transgenic plants
1404 | Ma et al.
appears to be the result of increased cell numbers in the
seed coat.
The cell number of the final seed coat is determined by
integument cell proliferation during ovule and seed development (Haughn and Chaudhury, 2005). Therefore, we further
investigated the integument cell number in the developing ovule and seed of TaCYP78A5-overexpressing plants.
TaCYP78A5 overexpression led to increased cell numbers
in the ovule integument of the pKLU::TaCYP78A5-5 transgenic plants compared with WT plants (Supplementary Fig.
S8A, B). Similar effects have been observed in CYP78A5overexpressing plants (Adamski et al., 2009). Moreover, we
investigated the seed integument cells of cyp78a5 mutants and
pKLU::TaCYP78A5-5 transgenic plants during seed development at 1, 2, 4, 6 and 8 dap. The stages were in accordance
with those described by Cheng et al. (2014). At the initial seed
development stage (from 1–4 dap), the rate of cell proliferation was faster in pKLU::TaCYP78A5-5 transgenic plants
than in WT plants but was decreased to the same level as in
WT plants after 6 dap (Fig. 5D–F). By contrast, relatively slow
cell proliferation was observed in cyp78a5 mutants (Fig. 5F).
These results suggest that expression of TaCYP78A5 affects
seed coat development by promoting integument cell proliferation in the developing ovule and seed. Similar effects of
Da1, TTG2, ARF2 and AP2 on seed size via restriction of
cell proliferation or cell expansion in the seed integuments
Fig. 5. TaCYP78A5 overexpression results in increased cell numbers in the Arabidopsis seed coat. (A) Fluorescence micrographs of the mature seed
coat stained by Fluorescent Brightener. Bar, 100 μm. Quantification of the cell number (B) and cell area (C) in the outer integument of mature seeds from
each genotype. Light micrographs of alcohol-discolored seeds from (D) WT Col and (E) pKLU::TaCYP78A5-5 transgenic lines at 1–8 dap. (F) The cell
numbers in the outer integument of developing seeds in response to TaCYP78A5 activity from 1 dap to 8 dap. The values represent means ±SE (n=20).
Asterisks (*) and (**) indicate significant differences from the wild type (Col) at P<0.05 and P<0.01 (t-test), respectively. (This figure is available in colour at
JXB online.)
TaCYP78A5 genes influence wheat seed size | 1405
have been reported (Garcia et al., 2005; Schruff et al., 2006;
Li et al., 2008; Ohto et al., 2009). However, TaCYP78A5 may
act independently of these genes because the expression levels
of these genes in the transgenic lines were similar to those in
WT plants (Supplementary Fig. S10).
Abnormally developed embryos were observed in the double-knockout cyp78a5 cyp78a7 mutant (Wang et al., 2008),
and enlarged embryos were also observed in seeds from
TaCYP78A5-overexpressing plants compared to seeds from
WT plants (Fig. 6A–C, G). To examine the potential role of
TaCYP78A5 in embryo growth, we measured the cell numbers in the cotyledon integument of pKLU::TaCYP78A5,
pINO::TaCYP78A5 and cyp78a5 mutant and WT plants.
The enlarged cotyledons in pKLU::TaCYP78A5 transgenic plants exhibited significantly increased cell numbers
(~11%) compared to WT (Fig. 6A, B, G, I) but similar cell
size (Fig. 6D, E, H). These findings suggest that the enlarged
embryo of pKLU::TaCYP78A5 transgenic plants resulted
from enhanced cell proliferation rather than cell elongation.
By contrast, the reduced cotyledons of the cyp78a5 mutant
exhibited a smaller number of integument cells compared to
WT (Fig. 6C, F, I). Compared to WT, pINO::TaCYP78A5
transgenic plants with TaCYP78A5 expression only in the
ovule exhibited similar cell numbers in the embryo integument
(Fig. 6I) but significantly enlarged cell size (~35%) (Fig. 6H),
suggesting that the significantly enlarged cotyledon of
pINO::TaCYP78A5 transgenic plants was due to the effect
of TaCYP78A5 not on embryo growth but on seed size. The
effects of TaCYP78A5 on seed size were attributed to the promotion of integument cell proliferation by TaCYP78A5 in
the ovule only. These results suggest that TaCYP78A5 affects
embryo development by promoting embryo integument cell
proliferation.
Together with the effects of the silencing and overexpression of TaCYP78A5 in the ovule or/and seed on seed size,
these results indicate that the alteration in seed size represents
the combined effects of TaCYP78A5 on seed coat size and
embryo size. These effects are mediated by the modulation of
the extent of integument cell proliferation by the TaCYP78A5
expression level. By contrast, previous studies have demonstrated that CYP78A5 controls seed size by affecting cell proliferation in the ovule integument only (Adamski et al., 2009).
TaCYP78A5 can rescue the phenotype of the cyp78a5
deletion mutant of Arabidopsis
To further verify the involvement of TaCYP78A5 in promoting organ and seed growth, we transformed three constructs,
Fig. 6. TaCYP78A5 promotes cell proliferation in the embryo integument. (A–C) Light micrographs of alcohol-discolored mature embryos from the
WT Col, transgenic pKLU::TaCYP78A5-5 and cyp78a5 mutant plants. (D–F) Magnified views of the cross-section boxed areas in panels A, B and C,
respectively. (G–I) Quantification of the characteristics of mature cotyledon integument cells in response to altered TaCYP78A5 activity. Asterisks (*) and
(**) indicate significant differences from wild type (Col) at P<0.05 and P<0.01 (t-test), respectively. The values represent means ±SE (n=20). Bars: 100 μm
(A–C); 10 μm (D–F). (This figure is available in colour at JXB online.)
1406 | Ma et al.
Fig. 7. TaCYP78A5 can rescue the phenotype of mutant cyp78a5. For panels A–F, from left to right: WT Col, transgenic pKLU(CYP78A5)::TaCYP78A52;cyp78a5, p35S::TaCYP78A5-5;cyp78a5 and pINO::TaCYP78A5-3;cyp78a5 as representative Arabidopsis transgenic lines bearing each construct
and mutant cyp78a5 plants. Phenotypes of WT, cyp78a5 mutant and TaCYP78A5 transgenic plants at: (A) the seedling stage (Arabidopsis plants at
12 days post germination, with leaf numbers indicated by the numbers in white); (B) stage 6.00; and (C) stage 6.50. In panels B, C the multi-branched
phenotype of the cyp78a5 mutant was rescued by TaCYP78A5 overexpression. Elongated main inflorescence stems in response to the overexpression of
TaCYP78A5 are also visible. (D) Silique phenotypes. (E) The expression level of TaCYP78A5 transgenic lines against a cyp78a5 mutant background was
detected by semi-quantitative RT-PCR analysis of total RNA isolated from a mixture of several top inflorescences per genotype. (F) Phenotypes of mature
seeds. (G, H) Quantification characteristics of mature seeds and siliques (at stage 14) in response to altered TaCYP78A5 expression levels. Thirty plants
from each transgenic line per genotype were grown to maturity without any assisted pollination and harvested for measurements. (G) Silique length. (H)
Seed size. The values represent means ±SE (n=20). Asterisks (*) and (**) indicate significant differences from WT (Col) at P<0.05 and P<0.01 (t-test),
respectively. Bars: 1 cm (A); 2 cm (B, C); 2 mm (D, F). The stages are in accordance with those described by Boyes et al. (2001). (This figure is available in
colour at JXB online.)
pKLU/CYP78A5::TaCYP78A5, 35S::TaCYP78A5 and
pINO::TaCYP78A5, into homozygous cyp78a5 mutants
exhibiting a phenotype of multi-branched, fast leaf initiation rate and small seed size (Supplementary Fig. S11A)
(Anastasiou et al., 2007). A homozygous mutant of cyp78a5
(Salk_024697C) in the Columbia-0 background obtained
from the ABRC was identified (Supplementary Fig. S11B,
C).
The fast leaf initiation rate and multi-branching phenotype of the cyp78a5 mutants was rescued to normal WT levels by transferring pKLU::TaCYP78A5 and
35S::TaCYP78A5 constructs (Fig. 7A–D, G). However,
the phenotype of cyp78a5 mutants at the seedling stage was
not rescued by pINO::TaCYP78A5 because TaCYP78A5
was expressed only in the ovules of pINO::TaCYP78A5transgenic plants. Furthermore, the overexpression of
TaCYP78A5 in the cyp78a5 mutant rescued the slightly
reduced apical dominance phenotype of the cyp78a5
mutant (Adamski et al., 2009) (Fig. 7C). RT-PCR analysis further confirmed that TaCYP78A5 was significantly
expressed in the inflorescence of transgenic T3 lines
bearing each construct (Fig. 7E). The overexpression of
TaCYP78A5 in the cyp78a5 mutant resulted in the production of larger seeds compared to WT plants (Fig. 7F, H).
These results suggest that TaCYP78A5 can rescue the phenotype of cyp78a5 mutants and has a conserved function
in promoting organ and seed growth in both monocots and
dicots.
TaCYP78A5 genes influence wheat seed size | 1407
Overexpression of TaCYP78A5 increases growth
duration but not seed yield
First flower formation, bolting and growth stoppage occur a
few days (2–5 d) earlier in the cyp78a5 mutant compared to
WT plants (Anastasiou et al., 2007). These results suggest that
the functional deletion of CYP78A5 might induce a shortened
growth duration. To determine if TaCYP78A5 also affects
growth duration, we investigated the effects of TaCYP78A5
activity on reproductive organ growth duration by measuring the main inflorescence length and the silique number of
WT, cyp78a5 mutant and transgenic pINO::TaCYP78A5-4
plants. The cyp78a5 mutant and WT plants stopped growing
at 5 and 6 weeks of growth, respectively. However, the main
inflorescences of transgenic line pINO::TaCYP78A5-4 continued growing at 8 weeks of growth to produce more flowers
and siliques (Supplementary Fig. S12A–D). Moreover, these
overgrowth effects were positively associated with the level of
TaCYP78A5 expression in transgenic plants. Thus, the main
reason pINO::TaCYP78A5-4 transgenic plants produced a
longer main inflorescence and more siliques may be not only
the overgrowth of the floral organ but also extension of growth
duration (~2 more weeks) in the transgenic plants induced by
TaCYP78A5 overexpression. Similar effects were observed in
CYP78A9-overexpressing plants (Sotelo-Silveira et al., 2013).
Overexpression of TaCYP78A5 increases longevity in
Arabidopsis, but the effects on seed yield remain unclear. We
therefore investigated the effect of TaCYP78A5 activity on
overall seed yield. TaCYP78A5 silencing caused a 10% reduction in seed size (Fig. 2) and did not significantly affect seed
set. Both the 100-seed weight and total seed weight per panicle were 10% lower in BSMV::TaCYP78A5-silenced plants
compared to the control groups (BSMV:00 and BSMV:PDS
infected plants) (Supplementary Fig. S13A, C). By contrast, the
overexpression of TaCYP78A5 in the WT background significantly increased seed size and the 100-seed weight of transgenic
plants (Fig. 4J; Supplementary Fig. S13B) but did not lead to an
increased total seed yield per plant (Supplementary Fig. S13D).
The increased seed size at the whole-plant level may have been
offset by a reduced seed number per silique and per plant; the
reduced effects of TaCYP78A5 on seed set were even stronger
than the increased effects on seed size (Fig. 4K; Supplementary
Fig. S13B). Moreover, we further investigated the harvest index
(the proportion of total seed yield to total aerial biomass; n=20)
of TaCYP78A5-silencing or -overexpressing plants. The alteration of TaCYP78A5 activity in both wheat and Arabidopsis
led to a reduced harvest index (Supplementary Fig. S13E, F).
These results indicate that either silencing or overexpression of
TaCYP78A5 leads to a reduced seed yield due to reduced seed
size or reduced seed set.
Discussion
CYP78A family members have similar functions in
influencing reproductive development in plants
Cytochrome P450 is one of the largest families of plant proteins (http://drnelson.uthsc.edu/cytochromeP450.html). The
CYP78A class of the CYP family appears to be involved in
plant-specific reactions (Nelson 1999). Several CYP78A family members, such as CYP78A5, CYP78A6 and CYP78A9 in
Arabidopsis (Ito and Meyerowitz, 2000; Adamski et al., 2009;
Fang et al., 2012; Sotelo-Silveira et al., 2013), CYP78A13 in
rice (Nagasawa et al., 2013; Yang et al., 2013) TaCYP78A3
(Ma et al., 2015) and TaCYP78A5 in wheat, which was characterized in the present study, exhibit growth-promoting
effects during reproductive organ and seed development.
The overexpression of these genes results in overgrowth of
seeds and reproductive organs; by contrast, the homozygous
cyp78a5, cyp78a6 cyp78a9, cyp78a8 and cyp78a9 mutants
exhibit smaller reproductive organs and seeds compared to
WT plants (Ito and Meyerowitz, 2000; Anastasiou et al., 2007;
Fang et al., 2012; Sotelo-Silveira et al., 2013). In the present
experiment, TaCYP78A5-silenced wheat plants produced
smaller seeds than the control plants (Fig. 2). However, some
members of the CYP78A family perform overlapping functions in controlling reproductive organ and seed development.
For example, the seed size and weight phenotype of cyp78a6
or cyp78a8 was synergistically enhanced by cyp78a9, suggesting that CYP78A9 functions redundantly with CYP78A6
and CYP78A8 to control seed growth (Fang et al., 2012;
Sotelo-Silveira et al., 2013). Embryos of the double-knockout mutant cyp78a5 cyp78a7 do not develop correctly, yet no
loss-of-function phenotype has been described for cyp78a7
mutants. CYP78A5 and CYP78A7 appear to play redundant
roles in regulating embryo growth in Arabidopsis (Wang
et al., 2008). In addition, two CYP78A members, CYP78A27
and CYP78A28 in Physcomitrella patens, act redundantly
during reproductive organ development (Katsumata et al.,
2011). Thus, CYP78A family members have similar functions
in the regulation of reproductive development.
Expression of CYP78A family members affects female
fertility in plants
Many studies have indicated that CYP78As are involved in
female fertility. Either loss-of-function or gain-of-function of
CYP78A members lead to reduced seed set in Arabidopsis.
These alterations of fertility are all related to the female reproductive organ ovule (Ito and Meyerowitz, 2000; Adamski
et al., 2009; Fang et al., 2012; Sotelo-Silveira et al., 2013).
For example, in a reciprocal cross experiment, Adamski and
co-workers observed that the reduced seed set per silique in
pINO::CYP78A5 transgenic plants appears to be attributable to a defect on the maternal side (Adamski et al., 2009).
In the present study, overexpression of TaCYP78A5 also
caused a decrease in fertility due to a defect in ovule development that was probably due to the promotion of integument cell over-proliferation during ovule development
(Supplementary Fig. S8). Similarly, the overexpression or
functional deletion of CYP78A9 also induced female sterility in Arabidopsis. Scanning electron microscopy analysis
of the ovules revealed that most of the ovules are shriveled
in CYP78A9-overexpressing mutants (Ito and Meyerowitz,
2000). By contrast, the double homozygous mutant cyp78a8cyp78a9 exhibits a short integument of the ovule that cannot
1408 | Ma et al.
Fig. 8. Working model for the influence of TaCYP7A5 on seed size. During ovule and seed development, TaCYP78A5 affects ovule, seed coat and
embryo development by promoting integument cell proliferation, ultimately increasing the mature seed size. (This figure is available in colour at JXB
online.)
accommodate the developing embryo sac (Sotelo-Silveira
et al., 2013). Thus, the expression of CYP78As not only significantly affects seed size but also impacts female plant fertility, which requires a normal expression pattern of CYP78A
members during ovule development.
The effect of TaCYP78A5 on seed size in wheat and
the underlying mechanisms
In wheat, a large number of QTLs have been reported to
control seed size and yield components (Nakamura et al.,
2007; Somyong et al., 2011; Williams and Sorrells, 2014).
Thirty QTLs for seed yield have been detected on ten chromosomes (Su et al., 2006). Most (24/30) of these QTLs were
mapped to the chromosomes of homologous groups 2, 5
and 7. Interestingly, the three TaCYP78A5 genes were also
located on the short arms of the homologous group 2 chromosomes. TaGW2, like rice grain width-associated gene 2
(OsGW2) (Song et al., 2007), is involved in grain development in wheat. Its haplotype, Hap-6A-A, has a significantly
positive effect on grain size and is considered a potentially
superior allele for the improvement of grain yield in wheat
(Su et al., 2011). TaCwi-A1, which is highly homologous to
the rice cell wall invertase (CWI) gene (Hirose et al., 2002),
has been characterized by allelic variations and QTL analysis. QTL analysis indicated that TaCwi-A1 is associated with
kernel weight in Chinese commercial wheat varieties and
landraces (Ma et al., 2012a). However, little is known about
the mechanisms underlying final seed size in wheat. Here, we
isolated a seed size-related gene, TaCYP78A5, and provided
direct evidence for a role of TaCYP78A5 in determining seed
size. Seeds from TaCYP78A5-silenced wheat plants were
10% smaller than those from control plants (Supplementary
Fig. S6C), whereas TaCYP78A5-overexpressing Arabidopsis
plants produced seeds that were larger than the seeds of WT
plants by ~11–35% (Fig. 4J). The effects of TaCYP78A5 on
final seed size are mediated by reducing or increasing integument cell numbers during ovule and seed development.
In summary, TaCYP78A5, the ortholog of Arabidopsis
CYP78A5 in wheat, affects seed size by influencing integument cell proliferation during ovule and seed development.
TaCYP78A5 also influences embryo development by affecting embryo integument cell proliferation during seed development. The expression level of TaCYP78A5 is positively
correlated with the final seed size but also affects female fertility. Accordingly, we have proposed a working model for the
effects of TaCYP7A5 on seed size (Fig. 8). This study provides direct evidence for control of seed size by TaCYP78A5.
These results suggest that TaCYP78A5 may be a target gene
for crop improvement.
Supplementary Data
Supplementary data are available at JXB online.Figure S1.
Chromosome location of TaCYP78A5.
Figure S2. Alignment of three TaCYP78A5 proteins.
Figure S3. TaCYP78A5 expression pattern in wheat
organs or tissues as detected by quantitative real time-PCR
(qRT-PCR).
Figure S4. Multiple sequence alignment of TaCYP78A5VIGS and three TaCYP78A5 cDNA sequences.
Figure S5. Schematic organization of the Barley stripe
mosaic virus (BSMV) genomes and the inserts used for
BSMV-induced gene silencing.
Figure S6. Comparison of seed size, seed length and seed
width of BSMV:00-, BSMV:PDS- and BSMV:TaCYP5infected plants of the wheat cultivars Shaan 512 (Large-seed)
and Shaanmai 159 (Small-seed).
Figure S7. The effect of TaCYP78A5 silencing by barley
stripe mosaic virus (BSMV)-induced gene silencing at the
seedling stage.
Figure S8. TaCYP78A5 promotes cell proliferation in the
ovule integument of Arabidopsis.
Figure S9. Additional seed measurements of Arabidopsis
in response to increased TaCYP78A5 activity.
TaCYP78A5 genes influence wheat seed size | 1409
Figure S10. Expression levels of Da1, TTG2, ARF2 and
AP2 in wild type Col and pINO::TaCYP78A5-4, -8 transgenic lines as detected by quantitative real-time PCR.
Figure S11. Identification of the homozygous mutant
cyp78a5 in Arabidopsis.
Figure S12. Overexpression of TaCYP78A5 increases the
reproductive period in Arabidopsis.
Figure S13. The TaCYP78A5 expression level affects seed
yield.
Method S1. Further details of methods used in this
study.
Table S1. Sequences of the primers used in this study.
Acknowledgements
This work was financially supported by grants from the National Nature
Science Foundation of China (30871578 and 31471482). We thank Dr Li
Huang, Department of Plant Sciences & Plant Pathology, Montana State
University, for providing the BSMV vector. We also thank Dr Xianchun Xia
for providing the NT- and DT-lines of Chinese Spring for target gene chromosome localization.
References
Aarssen LW, Jordan CY. 2001. Between-species patterns of covariation
in plant size, seed size and fecundity in monocarpic herbs. Ecoscience 8,
471–477.
Adamski NM, Anastasiou E, Eriksson S, O’Neill CM, Lenhard M.
2009. Local maternal control of seed size by KLUH/CYP78A5-dependent
growth signaling. Proceedings of the National Academy of Sciences, USA
106, 20115–20120.
Alonso-Blanco C, Blankestijn-de Vries H, Hanhart CJ, Koornneef M.
1999. Natural allelic variation at seed size loci in relation to other life history
traits of Arabidopsis thaliana. Proceedings of the National Academy of
Sciences, USA 96, 4710–4717.
Anastasiou E, Kenz S, Gerstung M, MacLean D, Timmer J, Fleck
C, Lenhard M. 2007. Control of plant organ size by KLUH/CYP78A5dependent intercellular signaling. Developmental Cell 13, 843–856.
Berger F, Grini PE, Schnittger A. 2006. Endosperm: an integrator
of seed growth and development. Current Opinion in Plant Biology 9,
664–670.
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE,
Davis KR, Görlach J. 2001. Growth stage–based phenotypic analysis
of Arabidopsis a model for high throughput functional genomics in plants.
The Plant Cell 13, 1499–1510.
Chakrabarti M, Zhang N, Sauvage C, Muños S, Blanca J,
Cañizares J, Diez MJ, Schneider R, Mazourek M, McClead J.
2013. A cytochrome P450 regulates a domestication trait in cultivated
tomato. Proceedings of the National Academy of Sciences, USA 110,
17125–17130.
Cheng ZJ, Zhao XY, Shao XX, Wang F, Zhou C, Liu YG, Zhang Y,
Zhang XS. 2014. Abscisic Acid Regulates Early Seed Development in
Arabidopsis by ABI5-Mediated Transcription of SHORT HYPOCOTYL
UNDER BLUE1. The Plant Cell 26, 1053–1068.
Disch S, Anastasiou E, Sharma VK, Laux T, Fletcher JC, Lenhard M.
2006. The E3 ubiquitin ligase BIG BROTHER controls Arabidopsis organ
size in a dosage-dependent manner. Current Biology 16, 272–279.
Eriksson S, Stransfeld L, Adamski NM, Breuninger H, Lenhard M.
2010. KLUH/CYP78A5-dependent growth signaling coordinates floral
organ growth in Arabidopsis. Current Biology 20, 527–532.
Fang W, Wang Z, Cui R, Li J, Li Y. 2012. Maternal control of seed
size by EOD3/CYP78A6 in Arabidopsis thaliana. The Plant Journal 70,
929–939.
Garcia D, Fitz Gerald JN, Berger F. 2005. Maternal control of integument
cell elongation and zygotic control of endosperm growth are coordinated to
determine seed size in Arabidopsis. The Plant Cell 17, 52–60.
Garcia D, Saingery V, Chambrier P, Mayer U, Jürgens G, Berger
F. 2003. Arabidopsis haiku mutants reveal new controls of seed size by
endosperm. Plant Physiology 131, 1661–1670.
Haughn G, Chaudhury A. 2005. Genetic analysis of seed coat
development in Arabidopsis. Trends in Plant Science 10, 472–477.
Henery ML, Westoby M. 2001. Seed mass and seed nutrient content as
predictors of seed output variation between species. Oikos 92, 479–490.
Hirose T, Takano M, Terao T. 2002. Cell wall invertase in developing rice
caryopsis: molecular cloning of OsCIN1 and analysis of its expression in
relation to its role in grain filling. Plant and Cell Physiology 43, 452–459.
Hraška M, Rakouský S, Čurn V. 2008. Tracking of the CaMV-35S
promoter performance in GFP transgenic tobacco, with a special
emphasis on flowers and reproductive organs, confirmed its predominant
activity in vascular tissues. Plant Cell, Tissue and Organ Culture 94,
239–251.
Ito T, Meyerowitz EM. 2000. Overexpression of a gene encoding
a cytochrome P450, CYP78A9, induces large and seedless fruit in
Arabidopsis. The Plant Cell 12, 1541–1550.
Jenik PD, Irish VF. 2000. Regulation of cell proliferation patterns by homeotic
genes during Arabidopsis floral development. Development 127, 1267–1276.
Jofuku KD, Omidyar PK, Gee Z, Okamuro JK. 2005. Control of seed
mass and seed yield by the floral homeotic gene APETALA2. Proceedings
of the National Academy of Sciences, USA 102, 3117–3122.
Johnson CS. 2002. TRANSPARENT TESTA GLABRA2, a trichome
and seed coat development gene of Arabidopsis, encodes a WRKY
transcription factor. The Plant Cell 14, 1359–1375.
Katsumata T, Fukazawa J, Magome H, Jikumaru Y, Kamiya Y,
Natsume M, Kawaide H, Yamaguchi S. 2011. Involvement of the
CYP78A subfamily of cytochrome P450 monooxygenases in protonema
growth and gametophore formation in the moss Physcomitrella patens.
Bioscience, Biotechnology, and Biochemistry 75, 331–336.
Khalid A, Arshad M, Zahir Z. 2004. Screening plant growth-promoting
rhizobacteria for improving growth and yield of wheat. Journal of Applied
Microbiology 96, 473–480.
Leishman MR, Wright IJ, Moles AT, Westoby M. 2000. The
evolutionary ecology of seed size. Seeds: the Ecology of Regeneration in
Plant Communities 2, 31–57.
Li Y, Zheng L, Corke F, Smith C, Bevan MW. 2008. Control of final seed
and organ size by the DA1 gene family in Arabidopsis thaliana. Genes &
Development 22, 1331–1336.
Luo M, Dennis ES, Berger F, Peacock WJ, Chaudhury A. 2005.
MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a
leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in
Arabidopsis. Proceedings of the National Academy of Sciences, USA 102,
17531–17536.
Ma D, Yan J, He Z, Wu L, Xia X. 2012a. Characterization of a cell
wall invertase gene TaCwi-A1 on common wheat chromosome 2A and
development of functional markers. Molecular Breeding 29, 43–52.
Ma M, Yan Y, Huang L, Chen M, Zhao H. 2012b. Virus-induced
gene-silencing in wheat spikes and grains and its application in functional
analysis of HMW-GS-encoding genes. BMC Plant Biology 12, 141.
Ma M, Wang Q, Li Z, Cheng H, Li Z, Liu X, Song W, Appels R, Zhao
H. 2015. Expression of TaCYP78A3, a gene encoding cytochrome P450
CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size. The
Plant Journal 83, 312–325.
Mizutani M, Ohta D. 2010. Diversification of P450 genes during land
plant evolution. Annual Review of Plant Biology 61, 291–315.
Moles AT, Ackerly DD, Webb CO, Tweddle JC, Dickie JB, Westoby
M. 2005. A brief history of seed size. Science 307, 576–580.
Monforte AJ, Diaz AI, Caño-Delgado A, van der Knaap E. 2014.
The genetic basis of fruit morphology in horticultural crops: lessons from
tomato and melon. Journal of Experimental Botany 65, 4625–4637.
Nagasawa N, Hibara KI, Heppard EP, Vander Velden KA, Luck
S, Beatty M, Nagato Y, Sakai H. 2013. GIANT EMBRYO encodes
CYP78A13, required for proper size balance between embryo and
endosperm in rice. The Plant Journal 75, 592–605.
Nakamura S, Komatsuda T, Miura H. 2007. Mapping diploid wheat
homologues of Arabidopsis seed ABA signaling genes and QTLs for seed
dormancy. Theoretical and Applied Genetics 114, 1129–1139.
1410 | Ma et al.
Nebert DW, Gonzalez F. 1987. P450 genes: structure, evolution, and
regulation. Annual Review of Biochemistry 56, 945–993.
Nelson DR. 1999. Cytochrome P450 and the individuality of species.
Archives of Biochemistry and Biophysics 369, 1–10.
Nelson DR. 2006. Plant cytochrome P450s from moss to poplar.
Phytochemistry Reviews 5, 193–204.
Ohto MA, Fischer RL, Goldberg RB, Nakamura K, Harada JJ.
2005. Control of seed mass by APETALA2. Proceedings of the National
Academy of Sciences, USA 102, 3123–3128.
Ohto MA, Floyd SK, Fischer RL, Goldberg RB, Harada JJ. 2009.
Effects of APETALA2 on embryo, endosperm, and seed coat development
determine seed size in Arabidopsis. Sexual Plant Reproduction 22,
277–289.
Schruff MC, Spielman M, Tiwari S, Adams S, Fenby N, Scott RJ.
2006. The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links
auxin signalling, cell division, and the size of seeds and other organs.
Development 133, 251–261.
Shomura A, Izawa T, Ebana K, Ebitani T, Kanegae H, Konishi S,
Yano M. 2008. Deletion in a gene associated with grain size increased
yields during rice domestication. Nature Genetics 40, 1023–1028.
Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK, Talbert
LE, Giroux MJ. 2002. Enhanced ADP-glucose pyrophosphorylase activity
in wheat endosperm increases seed yield. Proceedings of the National
Academy of Sciences, USA 99, 1724–1729.
Smyth DR, Bowman JL, Meyerowitz EM. 1990. Early flower
development in Arabidopsis. The Plant Cell 2, 755–767.
Somyong S, Munkvold JD, Tanaka J, Benscher D, Sorrells ME.
2011. Comparative genetic analysis of a wheat seed dormancy QTL with
rice and Brachypodium identifies candidate genes for ABA perception and
calcium signaling. Functional & Integrative Genomics 11, 479–490.
Song XJ, Huang W, Shi M, Zhu MZ, Lin HX. 2007. A QTL for rice grain
width and weight encodes a previously unknown RING-type E3 ubiquitin
ligase. Nature Genetics 39, 623–630.
Sotelo-Silveira M, Cucinotta M, Chauvin AL, Chavez Montes RA,
Colombo L, Marsch-Martinez N, de Folter S. 2013. Cytochrome P450
CYP78A9 is involved in Arabidopsis reproductive development. Plant
Physiology 162, 779–799.
Su JY, Tong YP, Liu QY, Li B, Jing RL, Li JY, Li ZS. 2006. Mapping
quantitative trait loci for post‐anthesis dry matter accumulation in wheat.
Journal of Integrative Plant Biology 48, 938–944.
Su Z, Hao C, Wang L, Dong Y, Zhang X. 2011. Identification and
development of a functional marker of TaGW2 associated with grain weight
in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics
122, 211–223.
Sundaresan V. 2005. Control of seed size in plants. Proceedings of the
National Academy of Sciences, USA 102, 17887–17888.
Thompson K, Jalili A, Hodgson JG, Hamzeh’ee B, Asri Y, Shaw S,
Shirvany A, Yazdani S, Khoshnevis M, Zarrinkamar F. 2001. Seed
size, shape and persistence in the soil in an Iranian flora. Seed Science
Research 11, 345–355.
van der Knaap E, Chakrabarti M, Chu YH, Clevenger JP, Illa-Berenguer
E, Huang Z, Keyhaninejad N, Mu Q, Sun L, Wang Y. 2014. What lies
beyond the eye: the molecular mechanisms regulating tomato fruit weight and
shape. Frontiers in Plant Science 5, doi: 10.3389/fpls.2014.00227
Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz
K, Gasser CS. 1999. INNER NO OUTER regulates abaxial- adaxial
patterning in Arabidopsis ovules. Genes & Development 13,
3160–3169.
Wang A, Garcia D, Zhang H, Feng K, Chaudhury A, Berger F,
Peacock WJ, Dennis ES, Luo M. 2010. The VQ motif protein IKU1
regulates endosperm growth and seed size in Arabidopsis. The Plant
Journal 63, 670–679.
Wang J, Schwab R, Czech B, Mica E, Weigel D. 2008. Dual effects of
miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length
and organ size in Arabidopsis thaliana. Plant Cell 20, 1231.
Wang S, Wu K, Yuan Q, Liu X, Liu Z, Lin X, Zeng R, Zhu H, Dong G,
Qian Q, Zhang G, Fu X. 2012. Control of grain size, shape and quality by
OsSPL16 in rice. Nature Genetics 44, 950–954.
Williams K, Sorrells ME. 2014. Three-dimensional seed size and shape
QTL in hexaploid wheat (Triticum aestivum L.) populations. Crop Science
54, 98–110.
Xu F, Fang J, Ou S et. al. 2015. Variations in CYP78A13 coding
region influence grain size and yield in rice. Plant, cell & environment 38,
800–811.
Yang W, Gao M, Yin X, Liu J, Xu Y, Zeng L, Li Q, Zhang S, Wang
J, Zhang X. 2013. Control of rice embryo development, shoot apical
meristem maintenance, and grain yield by a novel cytochrome P450.
Molecular plant 6, 1945–1960.
Yang Z, Bai Z, Li X, Wang P, Wu Q, Yang L, Li L. 2012. SNP
identification and allelic-specific PCR markers development for TaGW2, a
gene linked to wheat kernel weight. Theoretical and Applied Genetics 125,
1057–1068.
Zhang X, Henriques R, Lin SS, Niu QW, Chua NH. 2006.
Agrobacterium-mediated transformation of Arabidopsis thaliana using the
floral dip method. Nature Protocols 1, 641–646.
Zhou Y, Zhang X, Kang X, Zhao X, Zhang X, Ni M. 2009. SHORT
HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2
promoters in vivo to regulate Arabidopsis seed development. Plant Cell 21,
106–117.
Zondlo SC, Irish VF. 1999. CYP78A5 encodes a cytochrome P450 that
marks the shoot apical meristem boundary in Arabidopsis. The Plant
Journal 19, 259–268.