Download A Cotton Gene Encoding MYB-Like Transcription Factor is

A Cotton Gene Encoding MYB-Like Transcription Factor is
Specifically Expressed in Pollen and is Involved in Regulation of
Late Anther/Pollen Development
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University,
Wuhan 430079, China
*Corresponding author: E-mail, [email protected]; Fax, +86-27-67862443.
(Received May 8, 2012; Accepted February 21, 2013)
In flowering plants, pollen development is a highly programmed process, in which a lot of genes are involved. In this
study, a gene, designated as GhMYB24, encoding R2R3-MYBlike protein was isolated from cotton. GhMYB24 protein is
localized in the cell nucleus and acts as a transcriptional
activator. Northern blot analysis revealed that GhMYB24
transcripts were predominantly detected in anthers. It was
further found that strong expression of GhMYB24 was
mainly detected in pollen and was regulated during anther
development by in situ hybridization. Overexpression of
GhMYB24 in Arabidopsis caused flower malformation,
shorter filaments, non-dehiscent anthers and fewer viable
pollen grains. Further analysis revealed that the septum
and stomium cells of anthers were not broken, and fewer
fibrous bands were found in the endothecium cells in transgenic plants. A complementation test demonstrated that
GhMYB24 was able to recover partially the male fertility of
the myb21 myb24 double mutant. Expression levels of the
genes involved in the phenylpropanoid biosynthetic pathway and reactive oxygen species homeostasis were altered in
GhMYB24-overexpressing transgenic plants. Furthermore,
the genes involved in jasmonate biosynthesis and its signaling pathway were up-regulated in the transgenic plants.
Yeast two-hybrid assay indicated that GhMYB24 interacted
with GhJAZ1/2 in cells. Taking the data together, our results
suggest that GhMYB24 may play an important role in
normal anther/pollen development.
Keywords: Anther development Cotton (Gossypium hirsutum) Male fertility Pollen viability R2R3-MYB transcription factor Regulation of gene expression.
Abbreviations: ANS, anthocyanidin synthase; AOX, alternative oxidase; AP, altermative pathway; CaMV, Cauliflower
mosaic virus; CCD, charge-coupled device; CP, cytochrome
pathway;
DAPI,
40 ,6-diamidino-2-phenylindole;
DDO
medium, double dropout medium; FAA, formalin–acetic
acid–alcohol; FDA, fluorescein diacetate; GFP, green fluorescent protein; JA, jasmonate; ORF, open reading frame; PAL,
phenylalanine ammonia lyase; PCD, programmed cell death;
PI, propidium iodide; QDO medium, quadruple dropout
medium; ROS, reaction oxygen species; RT–PCR, reverse transcription–PCR.
Regular Paper
Yang Li, Jia Jiang, Man-Li Du, Lan Li, Xiu-Lan Wang and Xue-Bao Li*
Introduction
MYB proteins which comprise a large and multifunctional
family in plants are characterized by a conserved DNA-binding
domain, the MYB domain, consisting of 1–4 amino acid sequence repeats and forming a helix–turn–helix structure.
Depending on the number of adjacent repeats, MYB proteins
are classified into four classes: MYB-related proteins with one
MYB domain, R2R3-MYB with two MYB domains, R1R2R3MYB with three adjacent repeats and 4R-MYB containing
four R1/R2-like repeats. Plant MYB proteins play very important roles in many plant processes (Dubos et al. 2010). Some
MYB proteins are involved in primary and secondary metabolism. For example, AtMYB11, AtMYB12 and AtMYB111 control
flavonol biosynthesis (Stracke et al. 2007), AtMYB75, AtMYB90,
AtMYB113 and AtMYB114 control anthocyanin biosynthesis
(Gonzalez et al. 2008), while AtMYB123 controls the biosynthesis of proanthocyanidins (Lepiniec et al. 2006). It has been
demonstrated that AtMYB20, AtMYB42, AtMYB46, AtMYB52,
AtMYB54, AtMYB58, AtMYB63, AtMYB69, AtMYB83,
AtMYB85 and AtMYB103 activate lignin, xylan and cellulose
biosynthesis (Zhong and Ye 2009), whereas AtMYB68 is a positive regulator of lignin biosynthesis and regulates cellulose and
xylan deposition (Feng et al. 2004). Another important function
of MYB proteins is the control of development and determination of cell fate and identity. For example, GL1 and AtMYB23
control trichome initiation in shoots (Kirik et al. 2005).
AtMYB23 protein is positively regulated by AtMYB66 and provides a positive feedback loop for cell fate specification in controlling root hair patterning (Kang et al. 2009). Moreover, MYB
proteins also regulate plant development. Zhang et al. (2007)
proposed that AtMYB103 facilitates pollen exine formation and
acts downstream of AtMYB35. DUO1 and DUO3 control male
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038, available online at www.pcp.oxfordjournals.org
! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
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germ cell division and differentiation (Twell 2011). In addition,
MYB proteins also participate in mediating hormone actions
and responses to biotic and abiotic stresses. For example,
AtMYB2 is involved in the response to low oxygen by regulating
the AtADH1 promoter (Hoeren et al. 1998).
The stamen, the male reproductive organ in flowering
plants, consists of an anther, a space for pollen development
and a filament providing the anther with structural support
and nutrients. The anther is made up of the anther wall
which has four cell layers (epidermis, endothecium, middle
layer and tapetum) and generation cells (Dawson et al. 1993).
Pollen development can be divided into two sequential stages:
microsporogenesis and microgametogenesis. During the microsporogenesis stage, the archesporial cells go through a series of
mitotic divisions, forming pollen mother cells. Then, the pollen
mother cells undergo meiosis, fashioning tetrads of haploid
microspores. After the microspores have formed, pollen development enters into the microgametogenesis stage. During this
stage, the unicellular microspores undergo mitotic divisions
twice, and become microgametophytes (Goldberg et al. 1993,
McCormick 1993). At same time, the anther wall enters a multiple step process, including the degeneration of the tapetum
and middle layer, weakening of the epidermis, secondary
thickening of the endothecium and retraction of the anther
wall to permit pollen release.
It is believed that a large number of genes play important
roles in this highly programmed process (Chang et al. 2011,
Wilson et al. 2011). Not surprisingly, MYB proteins are involved
in regulation of plant microgamete development. AtMYB103
specifically expressed in the tapetum and middle layer of anthers is important for pollen development. Reducing AtMYB103
expression resulted in earlier degeneration of the tapetum,
and the majority of pollen grains were distorted in shape and
lacked cytoplasm (Higginson et al. 2003). AtMYB26, an
important protein in pollen development, regulates endothecial cell development and acts upstream of the lignin
biosynthesis pathway. In the myb26 null mutant, lignification
does not take place in the endothecium layer. As a result, secondary thickening of the endothecium layer cannot occur and
consequently anthers fail to dehisce (C.Y. Yang et al. 2007). A
previous study showed that AtMBY21and AtMYB24 are
induced by jasmonate (JA) and mediate important aspects of
JA-regulated anther development and filament elongation
(Mandaokar et al. 2006). A further study showed that they
can interact with JAZ1, JAZ8 and JAZ11 to respond to JA signaling and activate expression of some genes essential for the JA
signaling pathway in anther development (Song et al. 2011).
In addition, another study revealed that AtMYB108 and
AtMYB24 have overlapping functions and act downstream of
AtMYB21 in pollen maturation, but AtMYB108 is not involved
in filament elongation of the stamen (Mandaokar and Browse
2009). Interestingly, a subsequent study identified another
JA response gene, AtMYB57, that promotes filament development of the stamen, like AtMYB21 and AtMYB24 (Cheng et al.
2009).
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In the past years, some cotton MYB genes had been identified as being important for fiber development of cotton. Suo
et al. (2003) reported that GhMYB109 is specifically expressed in
cotton fiber initial cells as well as elongating fibers. However,
further investigation in more detail demonstrated that this
protein is not required for cotton fiber initiation but is important for fiber elongation (Pu et al. 2008). Recent studies revealed
that GhMYB25 is required for fiber initiation (Machado et al.
2009), and the GhMYB25-like protein is upstream of GhMYB25
and GhMYB109 (Walford et al. 2011). Although previous studies provided important clues about the roles of MYB genes in
cotton fiber development, little is known about their role in
anther/pollen development of cotton. In this study, we identified a novel MYB gene, GhMYB24, in cotton. The GhMYB24
gene is predominantly expressed in pollen and encodes a
MYB-like transcriptional activator which interacts with
GhJAZ1/2 proteins. Overexpression of GhMYB24 in Arabidopsis
affected the JA signaling pathway, the phenylalanine ammonia
lyase (PAL) pathway and reactive oxygen species (ROS) homeostasis, and consequently caused the male-sterile phenotype,
including non-viable pollen grains, shorter filaments and
non-dehiscent anthers.
Results
Isolation and characterization of GhMYB24
By screening a cotton flower cDNA library, a cDNA encoding
MYB-like protein was identified. The isolated cDNA (designated as GhMYB24; GenBank accession No. JQ923474) encodes
a R2R3-MYB homolog comprising 210 amino acids and shares
relatively high homology (61% and 60% identity, respectively)
with Arabidopsis AtMYB24 and AtMYB21 at the amino acid
level. GhMYB24 protein contains two conserved MYB repeats
in its N-terminal region, and a W-MDDIW motif in its
C-terminal region, like some other MYB proteins (Supplementary Fig. S1A). Subsequently, the genomic DNA sequence of
the GhMYB24 gene was isolated in cotton. Compared with its
cDNA sequence, we found that the GhMYB24 gene contains
two introns in its open reading frame (ORF). The first intron
splits codon 46 (Gly46) and is 184 bp in length, while the second
intron splits codon 89 (Agr89) and is 1,434 bp in length (Supplementary Fig. S1B).
GhMYB24 protein is localized to the nucleus and
acts as a transcriptional activator
To investigate the subcellular localization of GhMYB24, the
ORF of GhMYB24 (without the termination codon) was fused
in-frame to the 50 terminus of a GFP (green fluorescent protein)
reporter gene under the control of the Cauliflower mosaic virus
(CaMV) 35S promoter, and transformed into cotton cells (see
the Materials and Methods). The transformed cotton callus
cells expressing the GhMYB24-eGFP fusion gene were also
stained using 40 ,6-diamidino-2-phenylindole (DAPI). GFP fluorescence and DAPI staining were detected and photographed
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
GhMYB24 is involved in anther/pollen development
under a Leica confocal laser-scanning microscope (Leica Ltd).
As shown in Supplementary Fig. S2A–D, GhMYB24–GFP
fusion proteins accumulated mainly in the nucleus which was
labeled with DAPI. These results suggested that GhMYB24 protein is targeted to the nucleus, consistent with a transcription
regulatory role of GhMYB24 as a transcription factor.
To identify the transcriptional activity of GhMYB24, we
employed the yeast GAL4-responsive reporter system. The
GhMYB24 coding sequence was fused to the GAL4 DNAbinding domain to generate the effector construct and transformed into yeast (Saccharomyces cerevisiae) AH109 and
Y187. The reported gene was then tested, using the constructs
only with the GAL4 DNA-binding domain as negative controls.
As shown in Supplementary Fig. S2E, the transformed yeast
cells containing pGBKT7-GhMYB24 could grow on SD/–Trp/–
Ade medium and turned blue in the flash-freezing filter assay,
indicating that GhMYB24 protein significantly activated LacZ
reporter gene expression in yeast cells. The above data suggested that GhMYB24 is capable of activating transcription
in cells.
GhMYB24 is preferentially expressed in
anthers/pollen
To investigate GhMYB24 expression profiling in cotton tissues,
Northern hybridization was employed. As shown in Fig. 1A,
GhMYB24 transcripts were accumulated at a very high level
in anthers, but very weak signals were found in petals, and no
expression was detected in the other tissues, suggesting that
GhMYB24 is preferentially/specifically expressed in cotton.
To determine further the expression pattern of GhMYB24
during anther development, in situ hybridization was performed, using a fragment (250 bp) of the 30 region of the
GhMYB24 gene as a probe. In early developmental stages of
cotton anthers, GhMYB24 was expressed neither in the pollen
sac nor in microspore mother cells (Fig. 1B). With further
anther development, relatively strong GhMYB24 expression
was detected in microspores (single-nucleus pollen), but few
signals were detected in the other anther cells in cotton anthers
of about 15 days old (Fig. 1C). At late stages of cotton anther
development (>20-day-old anthers), high levels of GhMYB24
expression were still found in male gametophytes (maturing
pollen) (Fig. 1D). In contrast, no signals were observed in any
anther tissues using sense chain transcript probes of the
GhMYB24 gene as controls (Fig. 1E–G). These results indicated
that GhMYB24 is mainly expressed in pollen, implicating that
this gene may function in pollen development.
Overexpression of GhMYB24 in Arabidopsis leads
to male sterility
To analyze the phenotypic effects of GhMYB24 gain of function,
GhMYB24 cDNA with a C-terminal GFP sequence was cloned
into the pCAMBIA1301 vector under the control of the CaMV
35S promoter, and transferred into the wild-type Col-ecotype
Arabidopsis. A total of 23 transgenic plants (T1 generation)
Fig. 1 Analysis of expression of the GhMYB24 gene in cotton tissues.
(A) RNA gel blot analysis of GhMYB24 in cotyledons (c), leaves
(l), roots (r), hypocotyls (h), petals (p), anthers (a), fibers (f) and
ovules (o) (top), and loading of RNA samples (bottom). (B–G) in
situ hybridization of GhMYB24 expression using digoxigenin-labeled
antisense probes (B, C, D) and sense probes as negative controls
(E, F, G). (B) A 9-day-old anther; (C) a 15-day-old anther; (D) pollen
in the 20-day-old anther. (E–G) The same stages of anther/pollen
development are shown as negative controls.
overexpressing the GhMYB24 gene were generated. Among
them, six transgenic plants (L4, L8, L11, L14, L17 and L19)
with a very high level of GhMYB24 were completely sterile,
and 12 transgenic plants (L1, L3, L5, L7, L10, L12, L13, L15,
L20, L21, L22 and L23) with low to moderate levels of
GhMYB24 were partially sterile, whereas the remaining five
transgenic plants (L2, L6, L9, L16 and L18) with very low levels
of GhMYB24 expression were found to be fertile (Supplementary Table S1). Subsequently, five independent homozygous
lines (T2–T4 generations) were selected for further analysis.
Line 3 (L3) represents the line with the highest level of
GhMYB24 overexpression, and the other four lines (L15, L13,
L1 and L9) represent those lines with different levels of
GhMYB24 overexpression (Fig. 2A). The transgenic plants
displayed smaller and narrower cotyledons and leaves in the
vegetal period (Fig. 2C, E) and severely decreased fertility
(Fig. 2G), compared with the control (GFP transgenic plants)
(Fig. 2B, D, F). There were far fewer siliques filled with seed in
GhMYB24-overexpressing transgenic plants than in the wild
type and control (GFP transgenic plants) (Table 1). Furthermore, the transgenic plants with high expression levels of
GhMYB24 had malformed carpels in their flowers (Fig. 2I),
whereas GFP transgenic plants showed normal flowers
(Fig. 2H). When GhMYB24 transgenic flowers were pollinated
with wild-type pollen, seed set was normal (Supplementary
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Table S2). The results suggested that overexpression of
GhMYB24 in Arabidopsis leads to male sterility.
Overexpression of GhMYB24 in Arabidopsis
suppresses filament elongation and anther
dehiscence
Another important phenotype of GhMYB24 overexpression in
Arabidopsis was discovered by measuring the filament elongation and anther dehiscence of the transgenic plants. When
flowering, about 87.3% flowers from controls (GFP transgenic
plants) contained four long stamens which could reach the
stigmas. In contrast, GhMYB24 transgenic flowers displayed a
much lower percentage of stamens of normal length. As shown
in Fig. 3A, only 10% and 12.15% of flowers from GhMYB24
transgenic lines 3 and 13 (L3 and L13), respectively, had four
long stamens, like wild type and control flowers, whereas 50% of
L3 transgenic flowers and 28.04% of L13 transgenic flowers did
not contain any normal length stamens, and the other lines
showed a portion of short filament stamens (i.e. each flower
only had one, two or three normal length stamens).
Furthermore, GhMYB24-overexpressing transgenic flowers displayed non-dehisced anthers. Our data revealed that 96.55% of
opened flowers of the GFP transgenic plants consistently had six
stamens which could release pollen. In contrast, all the anthers
did not dehisce in 76% of L3 transgenic flowers, and 1–6
dehisced anthers were found in the other L3 flowers. A similar
phenotype was also found in the flowers of the GhMYB24 transgenic line 13 (Fig. 3B). Taken together, these results indicated
that stamens of GhMYB24 transgenic plants are inefficient at
transferring pollen to the stigma.
Overexpression of GhMYB24 in Arabidopsis
reduces pollen viability
Fig. 2 Phenotype of GhMYB24-overexpressing transgenic Arabidopsis.
(A) RT–PCR analysis of GhMYB24 expression in transgenic plants. WT,
wild type; GFP, GFP transgenic plants; L1–L15, GhMYB24 transgenic
lines. (B) A 14-day-old wild-type plant. (C) A 14-day-old GhMYB24
transgenic plant. (D) A 24-day-old wild type plant. (E) A 24-day-old
GhMYB24 transgenic plant. (F) Primary inflorescence of a wild-type
plant. (G) Primary inflorescence of a GhMYB24 transgenic plant.
Arrows indicate sterile siliques in transgenic plants. (H) A flower of
the wild type. (I) A flower of a GhMYB24 transgenic plant. Bar = 2 mm.
Analysis of the seed set rate indicated that pollen of the
transgenic Arabidopsis plants expressing GhMYB24 were defective (Supplementary Table S2). To investigate why these
pollen cells are sterile, the pollen viability of several lines of
GhMYB24 transgenic Arabidopsis was examined by double
staining with fluorescein diacetate (FDA) and propidium
iodide (PI), using the GFP transgenic plants and wild type
as controls. After staining, strong green fluorescence in pollen
cells was observed in most of the wild-type and control plants
(Fig. 4A), whereas relatively red fluorescence was detected in
most of the GhMYB24 transgenic pollen (Fig. 4B, C), indicating
that the GhMYB24 transgenic pollen was non-viable. More than
1,000 pollen grains from 10 independent flowers of different
plants for each transgenic line were randomly selected for
Table 1 Seed setting in GhMYB24 transgenic Arabidopsis plants
Line No.
No. of siliques
No. of siliques with seeds
No.of siliques
without seeds
Percentage of
siliques with seeds
P-value
NT
WT
20.27 ± 5.27
19.36 ± 5.66
0.91 ± 0.70
94.62 ± 5.40
GFP
22.83 ± 4.55
21.58 ± 4.46
1.25 ± 1.29
94.49 ± 6.09
0.956
L1
26.08 ± 2.64
15.67 ± 8.38
10.42 ± 8.35
60.14 ± 30.01
0.002
L3
32.25 ± 7.66
1.66 ± 1.50
30.58 ± 6.44
4.46 ± 3.84
<0.001
L9
24.17 ± 4.20
17.92 ± 8.16
6.25 ± 5.83
72.34 ± 28.90
0.023
L13
20.42 ± 4.70
6.42 ± 5.09
14.00 ± 6.78
31.89 ± 27.69
<0.001
L15
30.93 ± 3.15
5.07 ± 4.27
25.86 ± 4.18
16.12 ± 12.81
<0.001
Siliques formed in the primary inflorescences of plants were scored at 50 d. Twelve plants for each line were examined.
Data are means ± SD. Independent t-test demonstrated that there was significant difference in the percentage of siliques with seeds between the transgenic plants and
the wild type.
WT, wild type; GFP, transgenic plants with the pBI-35S-GFP vector as control; L1, L3, L9, L13 and L15, GhMYB24–GFP transgenic lines; NT, not tested.
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GhMYB24 is involved in anther/pollen development
0
1
2
3
4
0
B
100%
100%
90%
90%
80%
80%
% of flower with
each category anthers
% of flower with
each category length of stamens
A
70%
60%
50%
40%
30%
1
2
3
4
5
6
70%
60%
50%
40%
30%
20%
20%
10%
10%
0%
0%
GFP
L3
L13
GFP
L3
L13
Fig. 3 Assay of the reduced filament elongation and non-dehiscent anthers in GhMYB24-overexpressing transgenic Arabidopsis. (A) Percentage
of flowers with normal stamen length. The numbers indicate flowers with zero, one, two, three and four stamens which could touch the stigmas,
respectively. A total of 63 flowers from the control, 100 flowers from transgenic line L3 and 107 flowers from transgenic line L13 were examined.
(B) Percentage of flowers with anthers of each category. The numbers indicate opened flowers with one, two, three, four, five and six dehisced
anthers, respectively. A total of 63 flowers from control, 96 flowers from transgenic line L3 and 108 flowers from transgenic line L13 were
examined. GFP, GFP transgenic plants as control; L3 and L13, GhMYB24 transgenic lines.
measurement of pollen viability. Statistical analysis showed that
>85% of the GFP transgenic pollen was viable. In contrast, only
9.88% (L3) to 27.58% (L13) of the GhMYB24 transgenic pollen
was viable (Fig. 4D). The results indicated that the pollen viability of GhMYB24 transgenic plants was remarkably reduced,
compared with that of wild-type and GFP transgenic plants,
owing to GhMYB24 overexpression.
Overexpression of GhMYB24 in Arabidopsis affects
pollen maturation, septum degeneration and
formation of fibrous bands
To determine the timing of the anther developmental defects
in GhMYB24-overexpressing Arabidopsis plants more precisely,
anthers at different developmental stages were selected for
anatomical observation. The results showed that no significant
differences were detected between transgenic plants and controls until stage 9. Anthers of both transgenic lines and controls
can form four layers of anther wall and a uninucleate microspore (Fig. 5A–F). At stage 12, the septums were degenerated,
and thus the anthers became bilocular, and fibrous bands appeared in the endothecia of wild-type anthers. At the same
time, pollen finished cell division and were filled with cytoplasm
that could be stained by toluidine blue (Fig. 5G). In GhMYB24
transgenic anthers, however, the septums were not degenerated, no or fewer fibrous bands were found in the endothecia,
and pollen were even devoid of cytoplasm and could not be
stained by toluidine blue (Fig. 5H, I). At stage 13, the stomiums
of anthers were broken to release pollen in the wild type (Fig.
5J), whereas most stomiums of anthers were not broken and
consequently no or fewer pollen were released in the transgenic
plants with high levels of GhMYB24 expression (Fig. 5K, L).
These data suggested that overexpression of GhMYB24 in
Arabidopsis resulted in abnormal anther wall structure during
late anther development.
Expression of the genes involved in the
phenylpropanoid biosynthetic pathway and ROS
homeostasis is altered in GhMYB24-overexpression
transgenic Arabidopsis plants
It has been reported that AtMYB24 and AtMYB21 are involved
in regulation of expression of the genes important for plant
fertility (Shin et al. 2002; X.Y. Yang et al. 2007). To investigate
whether GhMYB24 plays a similar role in regulation of male
fertility, we examined the expression of several genes involved
in the phenylpropanoid biosynthetic pathway and AOX1a, a
gene encoding alternative oxidase (AOX), which was reported
to be up-regulated in AtMYB21-overexpressing Arabidopsis. As
shown in Fig. 6, the expression levels of two genes (PAL2 and
ANS) involved in the phenylpropanoid biosynthetic pathway
and AOX1a were up-regulated in flowers of GhMYB24-overexpressing Arabidopsis. However, the expression levels of the
other genes, such as C4H, COMT and 4CL, which also play
roles in the phenylpropanoid biosynthetic pathway, were not
changed in the transgenic plants. In contrast, the expression
level of CCoAOMT, which regulates secondary thickening, was
down-regulated in the transgenic plants.
In agreement with the fact that anthocyanidin synthase
(ANS) and AOX family genes are involved in scavenging ROS
in plants (Mittler et al. 2004, Dong et al. 2005), we also observed
that the expression of the genes related to ROS homeostasis
was altered in flowers of GhMYB24-overexpressing transgenic
plants. In Arabidopsis, ROS are mainly produced by two
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Fig. 4 Assay of pollen viability in GhMYB24-overexpressing transgenic Arabidopsis. (A–C) Pollen from 35S:GFP plants (A), 35S:GhMYB24-GFP line
L3 (B) and 35S:GhMYB24-GFP line L13 (C). Viable pollen displayed green fluorescence and non-viable pollen were red. Bar = 100 mm. (D)
Statistical analysis of the percentage of viable pollen in different transgenic plants. Data are means ± SD for three replicates. **Independent ttest demonstrated that there were very significant differences (P < 0.01) in the percentage of viable pollen between the GhMYB24 transgenic
plants and controls (GFP transgenic plants).
NADPH oxidases (RbohD and RbohF). Semi-quantitative
reverse transcription–PCR (RT–PCR) analysis revealed that
the transcriptional levels of RbohD and RbohF genes were remarkably increased or decreased, respectively, in flowers of
GhMYB24-overexpressing transgenic plants. In addition, the
genes (e.g. GPX4 and CAT1) involved in the ROS-scavenging
network were down-regulated in the transgenic flowers. On
the other hand, little change in expression in the genes encoding superoxide dismutase (CSD1), ascorbate peroxidase (APX1),
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glutathione peroxidase 5 (GPX5) and peroxiredoxin (2-cys PrxR
F and Type 2 PrxR C) was detected in the transgenic Arabidopsis
plants (Fig. 6).
GhMYB24 partially rescues the sterile phenotype
of the myb21 myb24 double mutant
To confirm further whether GhMYB24 has a similar function to
AtMYB24 and AtMYB24, the coding region of GhMYB24 was
constructed into the pCAMBIA1301 vector under the control
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GhMYB24 is involved in anther/pollen development
Fig. 5 Comparative analysis of the histological features of anther development in wild-type and GhMYB24 transgenic plants of Arabidopsis.
(A–C) anthers from the wild type (A), L3 (B) and L13 (C) at developmental stage 7; (D–F) anthers from the wild type (D), L3 (E) and L13
(F) at developmental stage 9; (G–I) anthers from the wild type (G), L3 (G) and L13 (I) at developmental stage 12; (J–L) anthers from the wild
type (J), L3 (K) and L13 (L) at developmental stage 13. The images are of cross-sections through two locules. E, epidermis; En, endothecium; ML,
middle layer; T, tapetum; Tds, tetrads; StR, stomium region; MSp, microspore; PG, pollen grain; Sm, septum; St, stomium; Fb, fibrous bands.
Bar = 50 mm.
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Fig. 6 RT–PCR analysis of expression of the genes associated with
the PAL pathway and ROS in GhMYB24 transgenic Arabidopsis. PAL
(AT3G53260), phenylalanine ammonia lyase gene; C4H (AT2G30490),
cinnamate 4-hydoxylase gene; 4CL (AT1G51680), 4-coumarate-COA
ligase gene; COMT (AT5G54160), caffeic acid O-methyltransferase
gene; CcoAOMT (AT4G34050), caffeoyl-CoA O-methyltransferase
gene; ANS1 (AT2G38240), anthocyanidin synthase 1 gene; ANS2
(AT4G22880), anthocyanidin synthase 2 gene; AOX1a (AT3G22370),
alternative oxidase 1a gene; RbohD (At5G47910), NADPH oxidase D
gene; RbohF (At1G64060), NADPH oxidase F gene; CSD1 (At1G08830),
Cu/Zn superoxide dismutase gene; APX1 (At1G07890), ascorbate peroxidase gene; GPX4 (At2G48150), glutathione peroxidase 4 gene; GPX5
(At3G63080), glutathione peroxidase 5 gene; CAT1 (At1G20630), catalase gene; Type2 PrxR C (At1G65970), type 2 peroxiredoxin C gene;
2-cys PrxRF (At3G06050), peroxiredoxin F gene; ACT2 (AT3G18780),
actin gene 2.
of the AtMYB24 promoter and transferred into MYB21/myb21
myb24/myb24 heterozygous Arabidopsis plants. Subsequently,
GhMYB24 transgenic plants in the myb21 myb24 homozygous
background were identified. The expression levels of AtMYB21,
AtMYB24 and GhMYB24 in different transgenic plants were
determined by RT–PCR analysis (Fig. 7A). Anthers in myb21
myb24 double mutant plants were severely male sterile
(Fig. 7B), but six individual lines with expression of GhMYB24
in the myb21 myb24 double mutant background were found to
be partially fertile (Fig. 7C), compared with the fertile wild-type
plants (Fig. 7D, Table 2). In addition, anthers failed to dehisce,
and filament elongation was arrested in the myb21 myb24
double mutant (Fig. 7E). In contrast, flowers of GhMYB24 transgenic plants (myb21 myb24 background) and the wild type
showed more dehisced anthers and normal filaments
(Fig. 7F, G). These results demonstrated that GhMYB24 has a
similar function to that of AtMYB24 and AtMYB21 in transgenic
Arabidopsis.
GhMYB24 is involved in modulating the JA
signaling pathway
It has been reported that two plant hormones, gibberellins
and JA, act as regulatory molecules to influence stamen
developmental processes. To investigate whether GhMYB24
900
Fig. 7 Test of the complementation of the myb21 myb24 double
mutant by GhMYB24. (A) RT–PCR analysis of expression of
AtMYB21, AtMYB24 and GhMYB24 in the GhMYB24 transgenic
plants in the myb21 myb24 background. WT, wild type; L1–L6,
GhMYB24 transgenic lines. (B) Primary inflorescence of the myb21
myb24 double mutant. (C) Primary inflorescence of a GhMYB24 transgenic plant (myb21 myb24 background). (D) Primary inflorescence of
the wild type. (E) A flower of the myb21 myb24 double mutant. (F) A
flower of a GhMYB24 transgenic plant (myb21 myb24 background).
(G) A flower of the wild type. Arrows indicate sterile siliques in mutant
and transgenic plants.
participates in these signalling processes, the transcript levels
of the genes involved in gibberellin and JA biosynthesis and
their signaling pathways were analyzed in the transgenic Arabidopsis plants and controls. As shown in Fig. 8, the genes
related to JA biosynthesis (DAD1, AOS, LOX1, LOX2, AOC1,
AOC2, AOC3, AOC4 and OPR3) and JA signaling (COI1 and
JAZ1) were up-regulated in flowers of the transgenic plants.
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
GhMYB24 is involved in anther/pollen development
Table 2 Seed setting in GhMYB24 transgenic Arabidopsis plants in the myb21 myb24 background under long-day conditions
Line No.
No. of siliques
No. of siliques
with seeds
No. of siliques
without seeds
Percentage of
siliques with seeds
P-value
myb21 myb24
29.4 ± 4.70
4.73 ± 2.37
24.67 ± 4.79
16.27 ± 8.09
NT
WT
21.27 ± 5.28
19.66 ± 5.12
1.61 ± 0.74
92.43 ± 5.12
NT
L1
25.87 ± 2.72
19.40 ± 2.41
6.47 ± 2.47
75.25 ± 8.07
<0.001
L2
27.60 ± 1.68
15.93 ± 3.20
11.67 ± 2.79
57.61 ± 10.69
<0.001
L3
26.07 ± 2.22
21.40 ± 3.25
4.67 ± 1.80
81.81 ± 7.68
<0.001
L4
26.20 ± 3.21
18.93 ± 4.91
7.27 ± 4.23
72.24 ± 16.31
<0.001
L5
25.13 ± 1.92
20.53 ± 2.61
4.60 ± 2.23
81.73 ± 8.48
<0.001
L6
27.33 ± 4.08
17.20 ± 3.14
10.13 ± 3.64
63.56 ± 11.43
<0.001
Siliques formed in the primary inflorescences of plants were scored at 50 d. Twelve plants for each line were examined.
Data are means ± SD. Independent t-test demonstrated that there was a very significant difference (P < 0.01) in the percentage of siliques with seeds between the
transgenic plants and the wild type. myb21 myb24, myb21 myb24 double mutant; WT, wild type; L1–L6, complementary GhMYB24 (myb21 myb24) transgenic lines;
NT, not tested.
Fig. 8 Real-time quantitative RT–PCR analysis of expression of the genes involved in gibberellin and JA signaling in GhMYB24 transgenic
Arabidopsis. Data were averaged from three batches of independent samples and the ACT2 gene was used as calibrator. The histogram is drawn
based on the log2 scale of the ratio of the expression levels of the genes in the transgenic flowers vs. those in control flowers. AtMYB21
(At3G27810), AtMYB24 (At5G40350), GA20OX1 (At4G25420), GA3OX1 (At1G15550), GA2OX8 (At4G21200), RGA (At2G01570), RGL
(At1G66350), DAD1 (At2G44810), AOS (At5G42650), LOX1 (At1G55020), LOX2 (At3G45140), AOC1 (At3G25760), AOC2 (At3G25770), AOC3
(At3G25780), AOC4 (At1G13280), OPR3 (At2G06050), COI1 (At2G39940), JAZ1 (At1G19180). Data are means ± SD for three replicates.
In addition, AtMYB24 expression was also enhanced in
the transgenic plants. A recent study implied that AtMYB21
and AtMYB24 may be able to promote their own transcription (Song et al. 2011). Similarly, ectopic expression of
GhMYB24 in Arabidopsis enhanced the expression level of
AtMYB24 in the transgenic plants. On the other hand, the
expression levels of the genes which participate in gibberellin biosynthesis (GA20OX1 and GA3OX1), degradation
(GA2OX8) and signaling (RGA and RGL) were not significantly
altered in the transgenic plants, compared with those of the
controls.
To investigate whether GhMYB24 interacts with JA signaling
proteins in cotton, a yeast two-hybrid system was employed to
assay the interactions among GhMYB24 and eight cotton JAZ
proteins (GhJAZ1–GhJAZ8). The ORF of GhMYB24 was cloned
into the pGADT7 vector, and the coding sequences of eight
GhJAZ genes were cloned into the pGBKT7 vector, respectively.
As shown in Supplementary Fig. S3, GhMYB24 protein interacted with two cotton JAZ proteins (GhJAZ1 and GhJAZ2) in
yeast cells. The above data suggested that GhMYB24 protein
may be involved in modulating the JA signaling pathway in
cotton.
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
901
Y. Li et al.
Discussion
The R2R3-MYB class as the activating transcriptional factor is
the largest subfamily of MYB proteins that plays central roles
in plant-specific processes. It has been demonstrated that some
MYB proteins, such as AtMYB21, AtMYB57, AtMYB24,
AtMYB62, AtMYB116, AmMYB305, AmMYB340 and
PsMYB26, show trans-activation activity which depends on
the conversed W-MDDIW motif (Li et al. 2006). In this study,
GhMYB24, encoding R2R3-MYB protein, was identified in
cotton. GhMYB24 also contains the W-MDDIW motif in its
C-terminus and can activate reporter gene expression in yeast
cells. RNA gel blot analysis showed that GhMYB24 was clearly
specifically expressed in anthers, like Arabidopsis MYB21 and
MYB24 genes (Shin et al. 2002, X.Y. Yang et al. 2007, Cheng et al.
2009). A previous study indicated that AtMYB21 is preferentially expressed in anther vascular tissue and in cells at the
junction between the anther and stamen filament, while
AtMYB24 is mainly expressed in microspores from floral
stages 7–12, and the vascular tissue of the stamen filament
(X.Y. Yang et al. 2007, Cheng et al. 2009). Besides, AtMYB21
and AtMYB24 may still be involved in anther wall development.
In our study, however, the expression of GhMYB24 in anthers
was only detected in male gametophytes (maturing pollen),
suggesting that it may play an important role in pollen
development.
The data presented in this study indicated that GhMYB24
transgenic plants displayed greatly reduced fertility, which is
correlated with the levels of GhMYB24 overexpression. Previous
study revealed that the rice TDR gene functions in promoting
tapetum programmed cell death (PCD). Suppression of TDR
expression retarded tapetum degeneration, and consequently
pollen wall formation was significantly altered in the tdr mutant
(Zhang et al. 2008). Likewise, knockout of the GPAT6 gene in
Arabidopsis caused defective tapetum development with
reduced endoplasmic reticulum profiles in the tapetum, leading
to the abortion of pollen grains and defective pollen wall formation in the gpat6 mutant. Furthermore, GPAT6 and GPAT1
have combined effects on the release of microspores from tetrads and stamen filament elongation (Li et al. 2012). In contrast,
our results indicated that the tapetum in GhMYB24 transgenic
plants could develop normally as did the wild type, whereas the
defect in pollen cells was observed, suggesting that formation of
the defective pollen in GhMYB24 transgenic plants may be due
to an effect on pollen development and maturation, instead of
tapetum development.
In self-pollinating plants, filament elongation and anther
dehiscence are also necessary in making flowers fertile. It has
been reported that Arabidopsis MYB21, MYB24 and MYB57 are
gibberellin-dependent stamen-enriched genes. The myb21
myb24 myb57 triple mutant confers a short stamen phenotype
leading to male sterility (Cheng et al. 2009). Also, many genes
are also involved in regulation of anther dehiscence (Wilson
et al. 2011). In our study, large numbers of the abnormal
902
stamens with short filaments and non-dehiscent anthers in
the transgenic plants were generated owing to the ectopic expression of GhMYB24 in anther wall cells. On the other hand,
when pollinated with the transgenic pollen by artificial
cross-pollination, the seed sets were significantly increased,
compared with those of the natural self-pollinated plants
(Table 1; Supplementary Table S2), implying that failure of
pollination also contributes to male sterility of GhMYB24 transgenic plants. It is well known that anther dehiscence and pollen
release depend on PCD of the septum and stomium (Kuriyama
and Fukuda 2002, Sanders et al. 2005). However, this process
was not observed in GhMYB24-overexpressing transgenic
plants. These results suggested that failure of anther dehiscence
is associated with defects in septum and stomium programmed
degradation in GhMYB24 transgenic plants.
As transcription factors, MYB proteins regulate different
branches of flavonoid metabolism by directly mediating the
expression of downstream genes. A previous study indicated
that the MYB domain is required for the MYB protein to recognize and specifically bind to the promoters of their target
genes (Ogata et al. 1995). Sequence analysis demonstrated that
GhMYB24 shares relatively high identity with AtMYB24 and
AtMYB21 in the conserved MYB domain, suggesting that
GhMYB24 may regulate similar target genes to AtMYB21 and
AtMYB24. Further experimental results revealed that the expression levels of PAL2 and ANS2 genes were remarkably
increased in GhMYB24 transgenic Arabidopsis plants, compared with those in the wild type. In contrast, other genes
involved in the PAL pathway and regulated by AtMYB24
(such as C4H and COMT) did not alter their expression levels
in the transgenic plants. Moreover, a complementation test
demonstrated that overexpression of GhMYB24 in the myb21
myb24 double mutant could recover pollen fertility of the
transgenic plants, suggesting that GhMYB24 may play a
similar role to that of AtMYB21 and AtMYB24 in regulating
pollen/anther development.
Arabidopsis overaccumulatng plantacyanin, which has the
ability to produce ROS, displayed indehiscent anthers, and premature PCD occurred in the endothecium during late anther
development (Dong et al. 2005). It has been reported that ANS
catalyzes leucoanthocyanidins to form anthocyanidin which, as
a potent antioxidant, reduces intracellular ROS levels (Scalbert
and Williamson 2000, He and Giusti 2010). AOX is a
mitochondria-located protein and serves a general function
in all plant species by limiting mitochondrial ROS formation
(Maxwell et al. 1999). Similarly, the expression level of ANS was
up-regulated in GhMYB24 transgenic plants, leading to an increase in the content of anthocyanidin and a decrease in ROS
activity. In addition, our results showed that GhMYB24 also
regulates the expression of the AOX gene, and two ROSscavenging genes (GPX4 and CAT1) which are induced by
ROS in transgenic plants. Collectively, we suppose that the
regulation of expression of these genes (such as ANS, AOX,
GPX4 and CAT1) resulted in delaying PCD formation of the
septum and stomium due to the reduced ROS level.
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
GhMYB24 is involved in anther/pollen development
During reproductive development, an abundant energy
supply is necessary for male gamete formation, development
and maturation. The cytochrome pathway (CP) and the alternative pathway (AP) are two major electron transport chains in
higher plants. AP diverges from the main respiratory chain at
ubiquinone, and comprises a single protein, AOX (Vanlerberghe and McIntosh 1997). AOX is non-proton pumping
since it by-passes proton-pumping complexes III and IV, and
electron flow to AOX dramatically reduces the energy yield of
respiration. In our study, superfluous accumulation of AOX in
the transgenic anthers might result in interruption of the
CP–AP balance and, consequently, not enough energy might
be produced for pollen development.
In the plant kingdom, JA as a regulatory molecule participates in many plant developmental processes, including stamen development (Mandaokar et al. 2006). The
JA-deficient mutant opr3 and the JA signaling mutant coi1 displayed shortened filaments, delayed anther dehiscence and
non-viable pollen (Stintzi and Browse 2000). Further study revealed that upon perception of the JA signal, COI1 recruits JAZ
proteins to the SCFCOI1 complex for degradation by the 26S
proteasome. Subsequently, AtMYB21 and AtMYB24 which are
restrained by the interaction with JAZs were released to
activate the downstream genes (Song et al. 2011). Likewise,
yeast two-hybrid assay revealed that GhMYB24 could interact
with two cotton JAZ proteins. Additionally, the expression of
the genes (including AtJAZ1) involved in JA biosynthesis and
signaling was enhanced in GhMYB24 transgenic Arabidopsis.
A previous study indicated that some JAZ genes were repressed
by their own products and by forming a feedback loop of
JA response (Chico et al. 2008). Therefore, we presume
that the ectopic expression of GhMYB24 in Arabidopsis may
directly modulate the expression of AtJAZ1 and subsequently
other genes in the JA signaling pathway. As a result, the transgenic plants displayed the defective phenotype in anther
development, especially in pollen viability. In summary, the
data presented in this study contribute to the understanding of the role of the GhMYB24 gene in plant anther/pollen
development.
Materials and Methods
Plant materials
Seeds of cotton (Gossypium hirsutum cv Coker 312) were germinated on half-strength Murashige and Skoog (MS) medium
in a 16 h light/8 h dark cycle at 28 C for 6 d. Roots, cotyledons
and hypocotyls were collected from these seedlings, and other
tissues such as leaves, anthers, petals, ovules and fibers were
collected from plants grown in the field or the greenhouse.
Seeds of Arabidopsis (Columbia ecotype) were germinated
on MS medium (16 h light/8 h, 22 C) for 10 d, and then transferred to soil in a growth room with a 16 h light/8 h dark photoperiod at 22 C. myb21-3 (CS311165), myb24-t1 (Salk_017221)
and the myb21-3 myb24-t1 double mutant were verified using
primer pairs (myb21-3-LP, myb21-3-RP, myb24-t1-LP, myb24-t1RP and LBb1.3) listed in Supplementary Table S3.
Isolation of GhMYB24 cDNA and genomic DNA
To identify the genes that are functionally expressed in cotton
anthers, >4,000 cDNA clones were randomly selected from the
cotton flower cDNA library constructed previously (Wang and
Li 2009) for sequencing. One clone containing the complete
GhMYB24 sequence was identified from these cDNAs. The corresponding GhMYB24 gene was isolated from cotton genomic
DNA by PCR. The gene-specific primers used in PCRs are
GhMYB24-P1 and GhMYB24-P2 (Supplementary Table S3).
Then, the PCR fragment of the GhMYB24 gene was cloned
into the pGEM-T vector for sequencing.
RNA gel blot analysis
RNA samples (20 mg per lane) from these cotton tissues were
separated on 1.2% agarose–formaldehyde gels, and transferred
onto Hybond-N nylon membranes by capillary blotting. A
250 bp PCR fragment of the 30 -untranslated region was used
as a gene-specific probe for Northern hybridization as described
previously (Li et al. 2002).
In situ hybridization
Cotton flower buds in different development stages were fixed
with FAA (50% ethanol, 5% acetic acid and 3.7% formaldehyde)
overnight and then embedded in paraffin. The samples were
sliced into 7 mm thick sections and mounted on polylysinecoated glass slides. In situ hybridization was carried out as
described previously (Wang and Li 2009). Antisense and sense
mRNA probes transcribed from a GhMYB24 cDNA fragment
(about 250 bp of the 30 -untranslated region) for in situ hybridization were labeled with digoxigenin by in vitro transcription
with T3 or T7 RNA polymerase, respectively. The sections were
photographed under a Nikon microscope (Nikon Co. Ltd.).
RT–PCR analysis
Total RNA was isolated from 10-day-old Arabidopsis seedlings
and inflorescences of matured plants using RNA-Solv Reagent
(Omega Bio-tek). About 2 mg of total RNA was reverse transcribed into cDNAs using reverse transcriptase (TAKARA).
First-strand cDNA was used as the template in
semi-quantitative and quantitative PCR analyses, using
gene-specific primers (Supplementary Table S3). Arabidopsis
ACTIN2 was used as standard control for normalization of RNA
samples. Real-time quantitative RT–PCR analysis was performed by the method described previously (Wang and Li
2009), and the relative values were expressed as the log2 scale
of the ratio of the expression levels of the genes in the transgenic flowers vs. those in control flowers.
Trans-activation activity assays
Trans-activation activity assays were conducted as described
previously (Li et al. 2011). The coding region of GhMYB24 was
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
903
Y. Li et al.
amplified by PCR using Pfu DNA polymerase and gene-specific
primers (GhMYB24-L and GhMYB24-R), and cloned into
pGBKT7 at the Nde† and BamH† sites. The construct was transferred into yeast stains AH109 and Y187. AH109 transformants
were screened on SD/–Trp/–Ade medium (SD minimal
medium lacking tryptophan and adenine), and Y187 transformants were employed to test the b-galactosidase activity by a
flash-freezing filter assay. The yeast cells containing empty
pGBKT7 vector were used as the negative control.
Pollen viability assay
Vector construction and plant transformation
Yeast two-hybrid assay
The coding region (without the termination codon) of
GhMYB24 was amplified using Pfu DNA polymerase and primers (GhMYB24-GL and GhMYB24-GR) (Supplementary
Table S3), and cloned into pBluescript SK-eGFP vector containing the coding sequence of the eGFP gene using the BamH†
and Xba† sites to create the GhMYB24-eGFP fusion construct.
Subsequently, the transcription unit of the CaMV35S
promoter:GUS:NOS-Terminal was digested from the PBI121
vector by Hind††† and EcoR†, and cloned into pCAMBIA1301
vector to obtain a new construct pCAMBIA1301-1. Then, the
fragment of the GhMYB24-eGFP fusion gene was digested from
the pBluescript SK-GhMYB24-eGFP construct and cloned into
PBI121 and pCAMBIA1301-1 by BamH† and Sac†, respectively,
replacing the b-glucuronidase (GUS) gene. On the other hand,
the promoter of AtMYB24 was amplified by PCR using primers
AtMYB24-P1 and AtMYB24-P2 (Supplementary Table S3), and
cloned into pCAMBIA1301-GhMYB24-eGFP by Hind††† and
BamH†, replacing the CaMV35S promoter. The constructs
were transferred into Agrobacterium tumefaciens (strains
LBA4404 and GV3101) for plant transformation. Arabidopsis
transformation was performed by the floral dip method.
The ORF of GhMYB24 was cloned into the pGADT7 vector, and
the coding sequences of eight GhJAZ genes (GhJAZ1–GhJAZ8)
were cloned into the pGBKT7 vector, creating fusions to the
binding domain and activation domain of the yeast transcriptional activator GAL4, respectively, by the method described earlier (Zhang et al. 2010). The constructs were transferred into
AH109 and Y187, respectively. Yeast strains containing
pGADT7-GhMYB24 or pGBKT7-GhJAZ vectors were used in
the mating reaction. The yeast zygotes were selected on double
dropout medium (DDO medium, SD/–Leu–Trp). Then, the
transformants were streaked on quadruple dropout medium
(QDO medium, SD/–Trp/–Leu/-His/–Ade), using transformants
containing pGBKT7 and pGADT7-GhMYB24 vectors as negative
controls and transformants containing pGBKT7-53 and
pGADT7-RecT vectors as positive controls (Zhang et al. 2010).
Subcellular localization
Cotton hypocotyl explants were transformed with Agrobacterium harboring the GhMYB24-eGFP vector by the method
described previously (Li et al. 2002). The transformed cotton
callus cells expressing GhMYB24-eGFP were used for observation of GFP fluorescence. After washing in phosphate-buffered
saline (PBS), cotton cells were collected and incubated in PBS
with 1 mg ml1 DAPI as a counterstain for nuclei. The fluorescence of the GhMYB24–eGFP fusion protein was visualized in
cotton callus cells under a confocal laser scanning microscope
(SP5, Leica) with a filter set of 466 nm for excitation and 506–
538 nm for emission. The fluorescence of DAPI was observed
under the same microscope with a filter set of 358 nm for excitation and 458 nm for emission. SP5 software was employed
to record and process the digital images taken.
Seed setting rate test
The seed setting rates of wild-type and transgenic plants was
tested by examining the percentage of siliques with seeds
formed in primary inflorescences. The data were input into
SPSS software, and the t-test of independent samples was performed for statistical inference.
904
Pollen viability was assayed by double staining with FDA and PI
described previously (Mandaokar and Browse 2009). The
stained pollen grains were viewed under a Nikon microscope
equipped with UV light and a charge-coupled device (CCD)
camera (Nikon Digital Sight DS-5Mc). The data were input
into SPSS software, and the t-test of independent samples
was performed for statistical inference.
Floral sectioning
Whole flowers were collected and fixed in FAA solution overnight, and then dehydrated in a graded ethanol series (10%
increments). The flower samples were embedded in SPI-PON
812 resin, and sliced into semi-thin (about 3 mm) sections by a
Leica microtome (http://www.leica.com). After staining with
0.25% toluidine blue, the sections were photographed under
a Nikon microscope equipped with a CCD camera (Nikon
Digital Sight DS-5Mc).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by National Natural Sciences
Foundation of China [grant No. 31070281]; the Ministry of
Agriculture of China [project for transgenic research (grant
No. 2011ZX08009-003)].
Acknowledgments
The authors thank Professor Daoxin Xie in Tsinghua University
(Beijing, China) for kindly donating seeds of myb21-3
(CS311165), myb24-t1 (Salk_017221) and the myb21-3
myb24-t1 double mutant.
Plant Cell Physiol. 54(6): 893–906 (2013) doi:10.1093/pcp/pct038 ! The Author 2013.
GhMYB24 is involved in anther/pollen development
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