Auxin controls Arabidopsis anther dehiscence by regulatingendothecium lignification and jasmonic acid biosynthesis
Valentina Cecchetti1,2, Maria Maddalena Altamura3, Patrizia Brunetti1, Valentina Petrocelli2,†, Giuseppina Falasca3, Karin
Ljung4, Paolo Costantino1 and Maura Cardarelli2,*1Dipartimento di Biologia e Biotecnologie, Sapienza Universit!a di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy,2Istituto di Biologia e Patologia Molecolari Consiglio Nazionale delle Ricerche, Sapienza Universit!a di Roma, Piazzale AldoMoro 5, 00185 Rome, Italy,3Dipartimento di Biologia Ambientale, Sapienza Universit!a di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy, and4Ume"a Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sci-ences, SE-90183 Ume"a, Sweden
Received 20 November 2012; revised 24 January 2013; accepted 27 January 2013; published online 14 February 2013.*For correspondence (e-mail [email protected]).† Present address: Istituto FIRC di Oncologia Molecolare, Via Adamello 16,20139 Milano, Italy.
SUMMARY
It has been suggested that, in Arabidopsis, auxin controls the timing of anther dehiscence, possibly by pre-
venting premature endothecium lignification. We show here that auxin content in anthers peaks before the
beginning of dehiscence and decreases when endothecium lignification occurs. We show that, in the auxin-
perception mutants afb1-3 and tir1 afb2 afb3, endothecium lignification and anther dehiscence occur earlier
than wild-type, and the gene encoding the transcription factor MYB26, which is required for endothecium
lignification, is over-expressed specifically at early stages; in agreement, MYB26 expression is reduced in
naphthalene acetic acid-treated anthers, and afb1 myb26 double mutants show no endothecial lignification,
suggesting that auxin acts through MYB26. As jasmonic acid (JA) controls anther dehiscence, we analysed
how auxin and JA interact. In the JA-defective opr3 mutant, indehiscent anthers show normal timing of
endothecium lignification, suggesting that JA does not control this event. We show that expression of the
OPR3 and DAD1 JA biosynthetic genes is enhanced in afb1-3 and tir1 afb2 afb3 flower buds, but is reduced
in naphthalene acetic acid-treated flower buds, suggesting that auxin negatively regulates JA biosynthesis.
The doublemutant afb1 opr3 shows premature endothecium lignification, as in afb1-3, and indehiscent anthers
due to lack of JA, which is required for stomium opening. By treating afb1 opr3 and opr3 inflorescences with
JA, we show that a high JA content and precocious endothecium lignification both contribute to induction of
early anther dehiscence. We propose that auxin controls anther dehiscence timing by negatively regulating
two key events: endothecium lignification viaMYB26, and stomiumopening via the control of JA biosynthesis.
In Arabidopsis, late stamen development consists of threedevelopmental programs: anther dehiscence, pollen matu-ration and filament elongation. Coordination of these threeprocesses ensures the release of mature pollen grains ontothe stigma when filament elongation is completed.
Anther dehiscence and pollen maturation begin whenthe epidermis, endothecium, middle layer and tapetuminside the anther have differentiated, and meiosis has beencompleted. Anther dehiscence is characterized by threemain events: development of lignified secondary walls in
endothecial cells, followed by degradation of septum cellsleading to a bi-locular anther, and finally breakage of thestomium, a group of specialized epidermal cells, whichallows the release of pollen grains at anthesis (Goldberget al., 1993). The first step in the anther dehiscence processis expansion of endothecial cells and the start of degenera-tion of the tapetum and middle layer. Development of lig-nified secondary walls occurs after endothecial cells haveexpanded when the degeneration of tapetum and middlelayer is observed, and this correlates with the first pollen
The Plant Journal (2013) 74, 411–422 doi: 10.1111/tpj.12130
mitotic division. Endothecium lignification is essential foranther opening, as it is responsible for the tension thatleads to stomium breakage (Keijzer, 1987; Bonner and Dick-inson, 1989; Mitsuda et al., 2005; Yang et al., 2007). It hasbeen demonstrated that the transcription factor MYB26plays a regulatory role in endothecium lignification, as wallthickening is not observed in the endothecial cells of thems35/myb26 mutant that is defective in MYB26, which dis-plays indehiscent anthers. MYB26 acts upstream of the lig-nin biosynthesis pathway, and MYB26 expression inanthers is mainly observed from the start of pollen mitosisI to the end of the bicellular pollen stage (Dawson et al.,1999; Yang et al., 2007).
In a previous study (Cecchetti et al., 2008), we showedthat auxin regulates the start of anther dehiscence by con-trolling the timing of endothecium lignification, as the tri-ple and quadruple auxin receptor mutants tir1 afb2 afb3and tir1 afb1 afb2 afb3 exhibited precocious endotheciumlignification and early anther dehiscence; pollen matura-tion was also precocious in these mutants, and stamen fila-ment growth was reduced, leading to shorter filamentscompared to wild-type. Auxin is synthesized in anthers atpre-meiotic and meiotic stages by YUC2 and YUC6, andaccumulation of auxin, as indicated by the activity of theauxin-inducible promoter DR5 (Ulmasov et al., 1997), wasobserved at the end of meiosis. DR5 activity decreaseswhen endothecium lignification occurs, and is no longerdetectable at the bi-locular stage, suggesting that auxinacts at the start of late stamen development (Cecchettiet al., 2008).
It has been shown by various authors that jasmonic acid(JA) is also involved in the control of anther dehiscence, asmutants defective in JA biosynthesis (Stintzi and Browse,2000; Ishiguro et al., 2001) or perception (Xie et al., 1998;Devoto et al., 2002) show indehiscent anthers and are con-sequently male-sterile. Five biosynthetic genes areinvolved in JA biosynthesis: DAD1, LOX, AOS, AOC andOPR3. It has been shown that DAD1 and OPR3 areexpressed in stamen filaments and anthers, respectively,during late stamen development. JA is also involved inother processes occurring during late stamen develop-ment, as both dad1 and opr3 mutants show reduced pollenviability, and the opr3 mutant has shortened filaments(Sanders et al., 2000; Stintzi and Browse, 2000).
The effect of JA on anther dehiscence was analysed indad1mutants by Ishiguro et al. (2001), who proposed that JAcauses anther dehydration, which is responsible for stomiumbreakage and opening at the final stages of anther develop-ment. Accordingly, Sanders et al. (2000) have shown that sto-mium breakage is delayed in the dde1 and opr3mutant.
Indirect evidence suggesting that auxin may activate thebiosynthesis of JA in late stamen development comesfrom the analysis of double mutants defective in the auxinresponse factors (ARFs) ARF6 and ARF8, which have inde-
hiscent anthers and decreased jasmonate production inflower buds during late development (Nagpal et al., 2005).Furthermore, it has been shown that, in arf6 arf8 flowerbuds, the expression of DAD1 is severely decreased (Taba-ta et al., 2010). Very recently, an auxin-dependent regula-tory role against JA has been proposed for ARF5/MONOPTEROS (Garrett et al., 2012), as the gain-of-func-tion mutant mpabn shows indehiscent anthers that openupon JA treatment.
The present study was designed to extend our knowl-edge on how auxin controls the timing of endothecium lig-nification, and to clarify the interaction between auxin andJA in determining anther dehiscence. Using auxin and JAconcentration measurements, auxin perception single andmultiple mutants, naphthalene acetic acid (NAA) and JAtreatments, JA biosynthetic mutants and analysis of thelevels of relevant transcripts, we show that changes inauxin concentration control the timing of endothecium lig-nification and modulate the JA biosynthesis that is respon-sible for stomium opening.
RESULTS
Changes in IAA concentration control late anther
developmental stages
In a previous paper (Cecchetti et al., 2008), we showed thatthe activity of the DR5 promoter in anthers is undetectableat stages 8 and 9 [pre-meiotic and meiotic stages of flowerdevelopment respectively (Bowman, 1994)], and peaks atstage 10 before tapetum degeneration and endotheciumlignification; DR5 activity decreases at stage 11 when thetapetum degenerates and endothecium lignificationoccurs, and is no longer detectable at stage 12 after sep-tum lysis when tapetum degeneration is completed.
To verify whether these changes in DR5 activitycorresponded to actual changes in auxin concentrationcontrolling and coordinating the events leading to antherdehiscence, we measured the free indole-3-acetic acid(IAA) content in anthers at various developmental stages(see Methods S1). As shown in Table 1, free IAA content
Table 1 Free IAA content in wild-type anthers at various develop-mental stages
Floral stage IAA concentration (pg/mg)*
8 52.33 ! 3.5a,†
9 52.37 ! 8.6a,†
10 93.57 ! 9.911 47.61 ! 11.25b,†
12 21.36 ! 1.01c
*In each case, values are means of three measurements ! SE.†These values are not significantly different.aP < 0.01 compared with stage 10.bP < 0.05 compared with stage 10.cP < 0.01 compared with stages 8, 9, 10 and 11.
was quite low during stages 8 and 9 (pre-meiotic and mei-otic stages), peaked at stage 10 (microspore stage), i.e.before endothecium lignification, decreased at stage 11(pollen mitosis II stage) when lignification has occurred,and further decreased at stage 12 (tricellular pollen stage)when the anther is bi-locular. These data show thatchanges in auxin concentration do indeed take place dur-ing anther development, and are consistent with thereported changes in DR5 activity and the proposed role ofauxin in stamen development (Cecchetti et al., 2008).
The afb1 auxin perception mutant is defective only
in anther dehiscence
Here, we aim to better define the role of auxin and shedlight on its interaction with JA in controlling specificallyanther dehiscence. We therefore looked for tir1 or afbauxin receptor mutants (Dharmasiri et al., 2005) that werealtered only in anther dehiscence by analysing the pheno-type of tir1-1, afb1-3, afb2-3 and afb3-4 mutants (Cecchettiet al., 2008).
We first measured the percentage of dehiscent anthersin mutant and wild-type flowers at various developmentalstages. We also analysed this percentage in the triplemutant tir1 afb2 afb3, which is defective in anther dehis-cence (Cecchetti et al., 2008). We observed early antherdehiscence at stage 12 initial (12i) before rapid filamentelongation (compared to stage 13 in the wild-type) inapproximately 10% of afb1-3 and afb2-3 (Figure 1a and Fig-ure S1a) and in 35% of tir1 afb2 afb3 flowers (Figure 1a)but not in tir1-1 and afb3-4 (Figure S1a). To assess whetherpollen maturation is also premature in afb1-3 and afb2-3,we performed in vitro germination assays on pollen grainsfrom stage 12 and 13 flowers. Wild-type and afb1-3 stage12 pollen grains did not germinate after 24 h of culture,whereas 5% of afb2-3 pollen grains at the same stagedeveloped pollen tubes (Figure S1b,c). At stage 13, wild-type and afb1-3 pollen grains showed comparable percent-ages of germination, but afb2-3 pollen grains showed asignificantly higher one (Table S1). Filament length in afb1-3 stamens was comparable to that of wild-type, but afb2-3filaments were slightly but significantly shorter than thoseof wild-type (Table S1). These results suggest afb1-3 as themost suitable mutant for the present study as it is onlydefective in anther dehiscence.
To assess whether expression of AFB1 in wild-typeplants is compatible with the early anther dehiscence ofthe afb1-3 mutant, we performed an in situ hybridizationanalysis on flower buds at various developmental stages.As shown in Figure 1(b–g), the AFB1 mRNA signal is faintat pre-meiotic stage 8 (Figure 1b) but strong at meioticstage 9, localized in the tapetum, endothecium and tetrads(Figure 1c). At stage 10, before endothecium lignification,the AFB1 mRNA signal is still present in tissues surround-ing the theca (Figure 1d) but is undetectable at stage 11
during endothecium lignification, while tapetum degenera-tion occurs (Figure 1e). The signal is absent at all stages inprocambial cells (Figure 1b–e) and in negative controls(Figure 1f,g). These data indicate that AFB1 mRNA isdetectable specifically in tissues surrounding the thecabefore endothecium lignification.
Thus afb1-3 mutant lines were used for subsequentanalysis. The triple mutant tir1 afb2 afb3 was also used,although it is also defective in pollen maturation and fila-ment elongation (Cecchetti et al., 2008), because it showsa higher percentage of early dehiscent anthers (seeabove) compared to afb1-3 (Figure 1a), but expressesAFB1.
Auxin controls the timing of endothecium lignification
via the MYB26 gene
Endothecium lignification is the main event at the startof the dehiscence process, and occurs at stage 11 offlower development in wild-type anthers, during tapetum
(a)
(b) (c) (d)
(e) (f) (g)
Figure 1. Early anther dehiscence in afb1-3 and tir1 afb2 afb3 flowers andexpression profile of AFB1.(a) Wild-type, afb1-3 and tir1 afb2 afb3 flowers at stage 12: non-dehiscentanthers are visible in wild-type flowers, whereas partially dehiscent anthersare visible in afb1-3 and tir1 afb2 afb3 flowers (arrows). The insets showmagnifications of an indehiscent anther from a wild-type flower and par-tially dehiscent anthers from afb1-3 and tir1 afb2 afb3 flowers.(b–g) RNA in situ hybridization of AFB1. Transverse sections of anthers. (b)Stage 8: a weak signal is present in the theca archesporium. (c) Stage 9: astrong signal is present in the endothecium, tapetum and tetrads. (d) Stage10: a weak signal is present in tissues surrounding the theca and in mi-crospores. (e) Stage 11: no signal in tissues surrounding the theca or in pol-len grains. (f,g) Control hybridization experiments for AFB1. (f) Stage 9: nospecific signal with AFB1 sense probe in tissues surrounding the theca or intetrads. (g) Transverse section of an afb1-3 mutant anther at stage 10: nospecific signal with antisense AFB1 probe in tissues surrounding the thecaor in microspores. Scale bars = 10 lm. En, endothecium; Ms, microspores;PG, pollen grains; T, tapetum; Tds, tetrads; Th, theca.
degeneration. In a previous study (Cecchetti et al., 2008),we showed that early anther dehiscence occurs in tir1 afb1afb2 afb3 quadruple mutants due to precocious endothe-cium lignification at the end of stage 10, before tapetumdegeneration. To verify whether early anther dehiscence inafb1-3 and tir1 afb2 afb3 mutants is also due to precociousendothecium lignification, we performed a comparativehistological analysis at various developmental stages (seeMethods S2). As shown in Figure 2(a), at stage 10, noendothecium lignification is observed in wild-type anthers,which show lignified thickenings at stage 11 (Figure 2b). Incontrast, endothecium lignification is clearly visible inafb1-3 and tir1 afb2 afb3 anthers from stage 10 (Figure 2a,b), but not earlier than stage 10 (Figure S2).
It has been shown by Yang et al. (2007) that the tran-scription factor MYB26 is required for endothecium lignifi-cation. To assess whether MYB26 expression in afb1-3 andtir1 afb2 afb3 is consistent with the precocious endothe-cium lignification in these mutants, we analysed the levelof MYB26 transcript in flower buds at various developmen-tal stages by quantitative RT-PCR.
As shown in Figure 2(c), the expression of MYB26 wasnegligible in wild-type flower buds until stage 10 when it
peaked before tapetum degeneration, and then decreasedat stage 11 when endothecium lignification occurs. In con-trast, in both afb1-3 and tir1 afb2 afb3 flower buds, thelevel of MYB26 mRNA was significantly higher than in thewild-type before stage 10, i.e. just before the precociousendothecium lignification, but the peak of MYB26 expres-sion at stage 10 was lower in both afb1-3 and tir1 afb2afb3.
To assess whether MYB26 is expressed early specificallyin anthers of afb1-3 and tir1 afb2 afb3 mutants, we gener-ated and analysed wild-type (Col-0 and Ws), afb1-3 andtir1 afb2 afb3 plants harbouring the GUS reporter gene dri-ven by the MYB26 promoter (see Methods S3). As shownin Figure 3(a–c), MYB26:GUS activity was undetectablebefore stage 10 in wild-type anthers. At stage 10, stainingwas observed in the tapetum, middle layer and endothe-cium (Figure 3a,d), and also in stamen filaments and inthe pistil.
The same GUS staining pattern, but more intense, wasobserved at early stage 11, before endothecium lignifica-tion (Figure 3a,e), whereas GUS staining was mainlydetectable in the stamen filament from late stage 11 (Fig-ure 3a). In agreement with the above described quantitative
(a)
(c)
(b)
Figure 2. Endothecium lignification and MYB26 expression in afb1-3, tir1 afb2 afb3 and opr3 flower buds.(a, b) Transverse sections of wild-type, afb1-3, tir1 afb2 afb3 and opr3 anthers visualized by fluorescence microscopy. (a) Stage 10: endothecium lignification isabsent in Col-0, Ws and opr3 anthers, whereas abundant autofluorescence of fibrous bands, perpendicular to the epidermis surface, is visible in afb1-3 and tir1afb2 afb3 endothecium (arrowheads). A non-specific diffuse fluorescence signal is observed in microspores and tapetum cells mainly in Col-0, Ws and opr3anthers. (b) Stage 11: very abundant autofluorescence of fibrous bands is visible in Col-0, Ws, afb1-3, tir1 afb2 afb3 and opr3 endothecium (arrowheads). A non-specific diffuse fluorescence signal is observed in pollen grains and tapetum cells of all sections. Scale bars = 20 lm (a, b). En, endothecium; Ms, microspores;PG, pollen grains; T, tapetum.(c) Quantitative RT-PCR analysis of MYB26 transcripts in wild-type and afb1-3 (left), wild-type and tir1 afb2 afb3 (middle), and wild-type and opr3 (right) flowerbuds at developmental stages up to 10 (indicated as <10), 10 and 11. Values are means ! SEM for MYB26 cDNA levels relative to actin cDNA. Asterisks indicatesignificant differences from the wild-type value (*P < 0.05; **P < 0.01).
RT-PCR data, MYB26:GUS activity was detectable at stage9 (meiosis) in the afb1-3 background, mainly in theanther, where it was localized in tapetum, middle layer,endothecium, tetrads and procambium (Figure 3a,c). Atstage 10, MYB26:GUS activity was still visible in the tape-tum and middle layer, and less in endothecium (Fig-ure 3d), while no GUS activity was detectable in theprocambium. In addition, GUS staining is almost unde-tectable in the afb1-3 pistil from stages 10–12 (Figure 3a),
possibly explaining why the transcript level of MYB26was higher in whole flower buds from the wild-type linecompared to those of afb1-3 at stages 10 and 11 of devel-opment. In tir1 afb2 afb3 anthers, MYB26:GUS activitywas detectable at pre-meiotic and meiotic stages: at pre-meiotic stage 8, it was quite diffuse in the theca (Fig-ure 3b), while at meiosis (stage 9), it was localized mainlyin the tapetum and to a lesser extent in the middle layer,endothecium and procambium (as in the afb1-3 back-ground) (Figure 3a,c); MYB26:GUS activity in the tir1 afb2afb3 background at stage 10 was still very high in tissuessurrounding the theca, mainly in tapetum, but was low inthe filament and the pistil (Figure 3a). In contrast to whatwas observed in MYB26:GUS afb1 anthers, GUS stainingwas still detectable at stage 11 in the anther tissues, asshown in Figure 3(a,e).
Thus, in afb1-3 and tir1 afb2 afb3 mutants, whichshowed early endothecium lignification, there was anenhanced activity of the MYB26 promoter specifically atpre-meiotic and meiotic stages in the endothecium andin tissues surrounding the theca, suggesting that auxincontrols the timing of endothecium lignification viaMYB26.
(a)
(b)
(c)
(d)
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Figure 3. Expression of MYB26:GUS is regulated by auxin.(a–e) Early expression of MYB26:GUS is higher in afb1-3 and tir1 afb2 afb3mutant anthers. (a) MYB26:GUS expression in inflorescences of wild-type(Col-0 and Ws ecotype, upper panel) and of the afb1-3 and tir1 afb2 afb3mutants (lower panel). In the wild-type inflorescences, GUS staining (blue)is observed in anthers absent at late stage 10 and stage 11 (more in Col-0than in the Ws background) and is absent at stage 12. In the mutant inflo-rescences, GUS staining is observed at stages 9 and 10 in anthers, and thisis more intense in the tir1 afb2 afb3 than in the afb1-3 background. GUSstaining is weak in afb1-3 anthers at stage 11 and absent at stage 12, whereasit is strong in tir1 afb2 afb3 anthers at stage 11 and absent at stage 12.(b–e) Histochemical analysis of anthers of MYB26:GUS Col, MYB26:GUSWs, MYB26:GUS afb1 and MYB26:GUS tir1 afb2 afb3 inflorescences. (b)Stage 8: GUS staining is absent in MYB26:GUS Col, MYB26:GUS Ws andMYB26:GUS afb1 anthers at pre-meiotic stage whereas is visible in MYB26:GUS tir1 afb2 afb3 anthers.(c) Stage 9: GUS staining is absent in MYB26:GUS Col and MYB26:GUS Wsanthers at tetrad stage, but is observed in MYB26:GUS afb1 anthers and isintense in MYB26:GUS tir1 afb2 afb3 anthers, mainly localized in the tape-tum, middle layer, endothecium and procambium.(d) Late stage 10: GUS staining is observed in MYB26:GUS Col, MYB26:GUS Ws, MYB26:GUS afb1 and MYB26:GUS tir1 afb2 afb3 anthers, localizedin the tapetum, middle layer and endothecium.(e) Stage 11: GUS staining is intense in MYB26:GUS Col, MYB26:GUS Wsand MYB26:GUS tir1 afb2 afb3 anthers, mainly localized in the tapetum, butis weak in MYB26:GUS afb1 anthers. Strong GUS staining was observed inMYB26:GUS Ws stamen filaments. Scale bars = 10 lm (b–d) and 30 lm (e).(f–h) Exogenous NAA treatments reduce MYB26 promoter activity. (f) Quan-titative RT-PCR analysis of MYB26 transcript in mock-treated and NAA-trea-ted flower buds at developmental stages up to 9, 10 and 11. Values aremeans ! SEM for MYB26 cDNA levels relative to actin cDNA. Asterisks indi-cate a significant difference from the mock-treated (**P < 0.01).(g,h) Histochemical analysis of anthers of mock-treated (g) and NAA-treated(h) MYB26:GUS Col inflorescences. (g) GUS staining is absent in anthers atstage 9 (left) but is observed in anthers at stages 10 and 11. (h) GUS stainingis absent in anthers at stage 9 (left), but is faint in anthers at stages 10 (mid-dle) and 11 (right). Scale bars = 10 lm (g,h). En, endothecium; ML, middlelayer; Ms, microspores; P, procambium; T, tapetum; Tds, tetrads; Th, theca.
To confirm this hypothesis, we treated wild-type inflo-rescences with 50 lM NAA in planta (Cecchetti et al., 2008),and analysed the MYB26 transcript level in flower buds atvarious developmental stages. As shown in Figure 3(f),MYB26 mRNA levels were comparably low in NAA- andmock-treated flower buds up to stage 10. In contrast,MYB26 mRNA levels were severely reduced in NAA-treatedcompared to mock-treated flower buds at stages 10 and11, confirming that auxin exerts negative control overMYB26 expression. To verify the effects of NAA treatmentson the activity of the MYB26 promoter in anthers, we trea-ted MYB26:GUS inflorescences with NAA and analysedGUS staining at various develpmental stages. In agree-ment with quantitative RT-PCR data, no GUS staining wasobserved in NAA- and mock-treated anthers prior to stage10, whereas only faint GUS staining was observed in allNAA-treated anthers at stages 10 and 11 (Figure 3g,h). Incontrast, at the same stages, MYB26 promoter activity wasclearly detectable inmock-treated (Figure 3g,h)MYB26:GUSanthers similar to untreated anthers shown in Figure 3a,d.
To provide further evidence that auxin acts via MYB26,we crossed afb1-3 and ms35/myb26 single mutants andanalysed three independent double knockout afb1 myb26lines (Figure S3a) phenotypically and histologically toassess their fertility and the timing of endothecium lignifi-cation (see Methods S3). As shown in Figure 4(a), afb1myb26 flowers had indehiscent anthers like ms35/myb26flowers (Dawson et al., 1999). Histological analysisrevealed that, at stage 11, endothecium expansion hadalready taken place in wild-type anthers, but only partiallyin afb1 myb26 anthers (Figure 4b); the lignified thickeningsthat were observed at late stage 11 in wild-type (Figure 4b,c) were absent in afb1 myb26 anthers at this and subse-quent stages (Figure 4b–d).
Thus, endothecium lignification does not occur in theafb1 myb26 double mutant, confirming that auxin actsthrough MYB26 to regulate this event.
Jasmonic acid controls stomium opening, but is not
involved in the timing of endothecium lignification
Sanders et al. (2000) showed that stomium opening wasdelayed in mutants defective in the JA biosynthetic geneOPR3, but no data on the timing of endothecium lignifica-tion were provided.
To assess whether JA plays a role in endothecium lignifi-cation, we performed a comparative histological analysis ofanthers from opr3 and wild-type flowers at various develop-mental stages. As shown in Figure 2(a), no endotheciumlignification was observed either in wild-type or opr3anthers before tapetum degeneration (stage 10), whereaslignified thickenings were clearly visible at stage 11 inanthers of both genotypes (Figure 2b). We also comparedthe transcript level of MYB26 during stamen developmentin opr3 and wild-type flower buds by quantitative RT-PCR
from stages before 10–12, and did not observe any signifi-cant difference (Figure 2c). These data indicate that JA hasno effect on the timing of endothecium lignification.
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Figure 4. Indehiscent anthers and lack of endothecium lignification in afb1myb26 double mutant flowers.(a) Wild-type and afb1 myb26 flowers at stage 13: fully dehiscent anthersare present in wild-type flowers, whereas non-dehiscent anthers are visiblein afb1 myb26 flowers (arrows). (b–d) Bright-field (b) and fluorescence (c,d)images of wild-type and afb1 myb26 anthers at various developmentalstages. (c) Magnification of a theca from wild-type and afb1 myb26 anthersshown in the bright-field images (b).(b,c) Stage 11: cell expansion and endothecium lignification are observed inwild-type anthers (arrowheads) (left) but are absent in afb1 myb26 endothe-cium (right). A non-specific diffuse fluorescence signal is observed in tape-tum cells and the septum region of the afb1 myb26 anther, and in pollengrains of wild-type and afb1 myb26 anthers.(d) Stage 12: lignification is completed (arrowheads) and initial septum lysisis observed in wild-type anthers (arrow), whereas lignification is absent butinitial septum lysis is observed in afb1 myb26 anthers (arrow). A non-spe-cific diffuse fluorescence signal is observed in pollen grains of wild-typeand afb1 myb26 anthers. Scale bars = 20 lm (b–d). En, endothecium; PG,pollen grains; Sr, septum region; T, tapetum.
We then crossed the afb1-3 auxin-perception mutant andthe opr3 JA-defective mutant. Flowers from three indepen-dent double knockout afb1 opr3 lines (Figure S3b) werephenotypically and histologically analysed to assess stomi-um opening and the timing of endothecium lignification.As shown in Figure 5(a), afb1 opr3 flowers had indehiscentanthers like opr3 flowers and unlike afb1-3 flowers (seeFigure 1a), indicating that stomium opening does notoccur in the absence of JA.
Histological analysis of afb1 opr3 flowers showed ligni-fied thickenings in the endothecium of approximately 10%of double mutant anthers at stage 10 of development (Fig-ure 5b), as previously observed in afb1-3 single mutantanthers, indicating that the early endothecium lignificationcaused by defective auxin perception also occurs in theabsence of JA. In addition, in afb1 opr3 flower buds, thelevel of MYB26 transcript is higher than in wild-type beforestage 10, as described for the afb1-3 single mutant,whereas the peak at stage 10 is slightly higher than that forthe wild-type (Figure 5c). Thus, MYB26 expression isenhanced at early stages in afb1 opr3 flowers showing pre-cocious endothecium lignification, as previously observedin afb1-3 flower buds.
These data indicate that endothecium lignification iscontrolled by auxin and is independent of JA, whereasauxin has no effect on stomium opening in the absence ofJA.
Auxin controls JA production
It has been shown that the JA biosynthetic genes DAD1and OPR3 are expressed in stamens during late develop-ment, and that the JA content peaks in flower buds atstages 11 and 12 (Nagpal et al., 2005) when auxin concen-tration decreases (Cecchetti et al., 2008; this paper). Toanalyse the relationship between auxin and JA biosynthe-sis, we compared the expression of DAD1 and OPR3 inwild-type, afb1-3 and tir1 afb2 afb3 flower buds.
As shown in Figure 6(a–d), OPR3 and DAD1 areexpressed in wild-type flower buds up to stage 10, andtheir transcript levels increase at stage 11 mainly in Col-0;at stage 12, OPR3 and DAD1 transcript levels slightlydecrease in Col-0 flower buds only. In afb1-3 flower buds(Figure 6a,c), the transcript levels of DAD1 and OPR3 arecomparable to those of the wild-type up to stages 10 and11, but are significantly higher at stage 12 (2.5 and 1.3times higher than wild-type for DAD1 and OPR3, respec-tively). In tir1 afb2 afb3 flower buds (Figure 6b,d), DAD1and OPR3 transcript levels are comparable to those of thewild-type up to stage 10 but are significantly higher atstage 11 (3 and 2.3 times higher than wild-type for DAD1and OPR3, respectively); at stage 12, the DAD1 transcriptlevel is slightly but significantly higher than that of thewild-type, whereas the OPR3 transcript level is comparablethat of the wild-type. These data suggest that auxin exertsa negative effect on expression of these JA biosyntheticgenes at specific late stages, i.e. before stomium opening(stages 11 and 12). To verify that the increase in JA biosyn-thetic gene transcripts corresponds to an actual increase inJA concentration in auxin mutants, we measured JA con-tent in afb1-3, tir1 afb2 afb3 and wild-type flower buds atstages 11 and 12 (pooled together), when the peak of JAproduction was observed in wild-type flowers (MethodsS4). As shown in Figure 6(e), JA content is comparable to
(a)
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Figure 5. Indehiscent anthers, precocious endothecium lignification andMYB26 expression in afb1 opr3 double mutants.(a) Wild-type, opr3 and afb1 opr3 flowers at stage13: fully dehiscent anthersare present in wild-type flowers, whereas non-dehiscent anthers are visiblein opr3 and afb1 opr3 flowers (arrows).(b) Wild-type and afb1 opr3 anthers visualized by fluorescence microscopy.Stage 10: lignification is absent in wild-type endothecium but visible in theendothecium of afb1 opr3 anthers (arrowheads). Stage 11: lignification isobserved in the endothecium of wild-type and afb1 opr3 anthers (arrow-heads). Scale bars = 20 lm. En, endothecium; Ms, microspores; PG, pollengrains; T, tapetum.(c) Quantitative RT-PCR analysis of MYB26 transcript in wild-type and afb1opr3 flower buds at developmental stages up to 10 (indicated as <10), 10and 11. Values are means ! SEM for MYB26 cDNA levels relative to actincDNA. Asterisks indicate a significant difference from the wild-type value(**P < 0.01).
that of the wild-type in afb1-3 flower buds, but is approxi-mately two times higher in tir1 afb2 afb3 flower buds.
To confirm the negative effect of auxin on the expres-sion of DAD1 and OPR3 at stages 11 and 12, wild-type infl-orescences were treated with 50 lM NAA or mock-treated,and analysed for DAD1 and OPR3 transcript levels. Asshown in Figure 6(f), mock-treated flower buds similar tountreated ones (Figure 6a,c) had comparable levels ofDAD1 and OPR3 transcripts at all stages. In contrast, when
treated with NAA, flower buds up to stage 10 showedDAD1 and OPR3 transcript levels that were comparable tothose of mock-treated buds, but the transcript levels atstage 11 were significantly lower (1.5 and 1.8 times,respectively) than mock-treated controls; at stage 12, theOPR3 transcript level was significantly lower (1.8 times)than in mock-treated flower buds, but the DAD1 transcriptlevel was not significantly lower.
These data support the notion that auxin negatively reg-ulates the biosynthesis of JA in anthers before stomiumopening at stages 11 and 12 of development, and suggestthat JA concentration has a role in the timing of antherdehiscence.
High JA concentrations induce precocious anther
dehiscence
To assess whether an increase in JA concentration at stages11 and 12 causes precocious anther dehiscence, we treatedopr3 inflorescences with various JA concentrations andmeasured the percentage of dehiscent anthers at variousstages. Mutant opr3 inflorescences were mock-treated ortreated with 1, 3.2 or 6.4 mM JA, and the number of dehis-cent anthers after 48 h was analysed in flower buds atdevelopmental stage 12 initial (12i) before rapid filamentelongation, stage 12 middle (12 m) during rapid filamentelongation, and stage 13, when this elongation phase isover. As shown in Figure 7, mock-treated inflorescencesshowed no dehiscent anthers at stages 12i and 12 m, andvery few dehiscent anthers at stage 13. Dehiscent antherswere observed at stage 12i in inflorescences treated with6.4 mM JA but not those treated with 1 or 3.2 mM JA; atstage 12 m, the percentage of dehiscent anthers increasesas JA concentration increases; at stage 13, nearly all anthers
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Figure 6. JA content is regulated by auxin in flower buds by modulatingDAD1 and OPR3 transcript levels.(a-d) Quantitative RT-PCR of mRNA extracted from wild-type and afb1-3 (a,c) or wild-type and tir1 afb2 afb3 (b,d) flower buds at developmental stagesup to and including 10 (indicated as "10), 11 and 12.(e) JA content in pooled flower buds at developmental stages 11 and 12from wild-type (Col-0 and Ws) afb1-3 and tir1 afb2 afb3 plants. Values aremeans ! SE (n = 3).(f) Quantitative RT-PCR of mRNA extracted from wild-type flower buds atdevelopmental stages " 10, 11 and 12. (a-d,f) Values are means ! SEM forDAD1 and OPR3 cDNA levels relative to actin cDNA. Asterisks indicate a sig-nificant difference from the wild-type value (*P < 0.05; **P < 0.01).
Figure 7. Effects of exogenous JA on anther dehiscence in opr3 and afb1opr3 flower buds at various developmental stages.opr3 and afb1 opr3 inflorescences were treated with 0, 1, 3.2 or 6.4 mM JA.The percentage of dehiscent anthers was measured in flower buds at devel-opmental stages 12i, 12m and 13. Values are means ! SEM from threeexperiments. A single asterisk indicates a significant difference among JAtreatments within the same genotype (P < 0.05). Double asterisks indicate asignificant difference between 6.4 and 1 mM JA treatments within the samegenotype (P < 0.01). Circles (°°) indicate a significant difference betweenopr3 and afb1 opr3 flower buds within the same JA treatment (P < 0.01).
were dehiscent in all JA-treated buds. This data indicatethat precocious anther dehiscence may be caused by highJA concentration, suggesting that auxin controls the timingof anther dehiscence by modulating JA biosynthesis.
As auxin controls the timing of anther dehiscence by mod-ulating JA biosynthesis, we wished to confirm whether, aspreviously suggested (Cecchetti et al., 2008), auxin doesindeed act on the timing of anther dehiscence by control-ling endothecium lignification.
We utilized afb1 opr3 double mutants, which show pre-cocious endothecium lignification but indehiscent anthersdue to the opr3 mutation (Figure 5a,b). afb1 opr3 inflores-cences were treated with 1, 3.2 or 6.4 mM JA, and the per-centage of dehiscent anthers was analysed at stages 12i,12 m, 13 and compared with the results for JA-treatedopr3 inflorescences.
As shown in Figure 7, nearly all anthers were dehiscentat stage 13 in afb1 opr3 JA-treated buds, similar to opr3JA-treated inflorescences; in contrast, at stage 12 m, a sig-nificantly higher percentage of dehiscent anthers wasobserved in JA-treated afb1 opr3 inflorescences comparedto opr3 inflorescences. Similarly, at stage 12i, a high per-centage of dehiscent anthers was observed in all JA-trea-ted afb1 opr3 inflorescences, whereas dehiscent antherswere observed only in opr3 inflorescences treated with6.4 mM JA at stage 12i. These data confirm that the timingof endothecium lignification contributes to regulation ofthe timing of anther dehiscence.
DISCUSSION
Anther dehiscence, pollen maturation and filament elonga-tion are late processes in stamen development. In a previ-ous paper, we showed that auxin negatively controlsanther dehiscence in Arabidopsis, and suggested that adecrease in auxin concentration triggers dehiscence by act-ing on endothecium lignification (Cecchetti et al., 2008).
In this paper, we further clarify the role of auxin inanther dehiscence, and establish a relationship betweenauxin and JA, another hormone that is known to beinvolved in anther dehiscence (Xie et al., 1998; Ishiguroet al., 2001; Nagpal et al., 2005).
By measuring IAA concentration in Arabidopsis anthersat various developmental stages, we found an increase inIAA concentration that begins at meiotic and pre-meioticstages (8 and 9), leading to a peak of IAA at the initial stageof late development (stage 10), followed by a significantdecrease in IAA content when endothecium lignificationhas occurred at stage 11. A further decrease in IAA contentis observed when the anthers become bi-locular, at stage12. These results are in good agreement with data previ-ously obtained by monitoring DR5 activity (Cecchetti et al.,
2008), and support the notion that a local auxin minimumis required to trigger late developmental stages in anthers.Similarly, a decrease in auxin content has been shown tobe necessary during specification of the valve layer whenArabidopsis fruit opening takes place (Sorefan et al., 2009).
To clarify how auxin regulates endothecium lignification,we utilized the auxin receptor mutant afb1-3 because it isaltered only in anther dehiscence, whereas mutants ofother auxin receptors are also altered either in pollen mat-uration and filament elongation (afb2-3), or are not alteredin anther dehiscence (afb3-4 and tir1-1). These diverse phe-notypes are in agreement with data published by Parryet al. (2009), which suggest that auxin receptors have dif-ferent functions in given tissues due to different affinitieswith different AUX/IAA proteins.
In our analysis, we compared the results obtained forafb1-3 with those for the triple mutant tir1 afb2 afb3, whichexpresses AFB1 but not other receptors and shows ahigher percentage of early dehiscing anthers.
By histological fluorescence analysis, we determinedthat endothecium lignification occurs at stage 10 in bothafb1-3 and tir1 afb2 afb3 mutant anthers, i.e. earlier than inwild-type anthers, where lignin deposition is observed atstage 11. These data, together with phenotypic analysis ofmultiple and single auxin-perception mutants (Cecchettiet al., 2008; this paper), indicate that all four auxin recep-tors contribute to regulation of anther dehiscence by actingon the timing of endothecium lignification, with only AFB1acting specifically on anther dehiscence.
We showed that auxin exerts its effect by acting nega-tively on expression of the MYB26 gene, which encodes anMYB transcription factor that is required for endotheciumlignification (Yang et al., 2007). By quantitative RT-PCR andanalysis of the expression of a transcriptional MYB26:GUSfusion, we found that MYB26 is mainly expressed at stage11 in wild-type anthers, and at a low level at late stage 10just before and during endothecium lignification, in agree-ment with results obtained byYang et al. (2007), but is moreactively expressed than in wild-type at early stages in afb1-3 (stage 9) and tir1 afb2 afb3 (stage 8) mutant anthers.
Furthermore, we show that NAA-treated flower budsshow a severe reduction in MYB26 transcript levels atstages 10 and 11, and only faint GUS staining is detectablein anthers from NAA-treated MYB26:GUS inflorescences.These findings indicate that MYB26 transcriptionis induced at late stage 10 and stage 11 in wild-typeanthers, and we propose that this expression is negativelycontrolled by auxin. As auxin content increases inanthers from stage 9 to 10 (this paper), a reduction in auxincontent should occur, at least in endothecial cells, at latestage 10.
However, we cannot rule out the possibility that MYB26expression is under additional transcriptional control inde-pendently of auxin. We obtained further evidence that
auxin controls endothecium lignification through MYB26by generating an afb1 myb26 double mutant. Phenotypicand histological analysis of afb1 myb26 flowers showedindehiscent anthers in this double mutant due to lack ofendothecium lignification, suggesting that MYB26 isrequired for auxin regulation of the timing of endotheciumlignification. The alternative explanation of the geneticdata, i.e. that AFB1 acts downstream of MYB26, is in con-trast with the fact that AFB1 is expressed earlier thanMYB26 in anthers, indicating that AFB1 acts via MYB26 toregulate endothecium lignification.
Interestingly, and in agreement with the results obtainedby Yang et al. (2007), GUS staining in MYB26:GUS antherswas observed not only in endothecium cells but also in thetapetum. It is possible that expression in the tapetum issubsequently repressed by post-transcriptional regulation.Alternatively, MYB26 expression in the tapetum may belinked to exine biosynthesis, which takes place in this tis-sue. Indeed, MYB transcription factors have been linked toregulation of phenylpropanoid metabolism, which isinvolved either in lignin biosynthesis (Yang et al., 2007) orin synthesis of sporopollenin, from which exine is made(Dobritsa et al., 2011).
In this study, we also analysed the relationship betweenauxin and JA, which is also involved in anther dehiscence.We tested the role of JA in endothecium lignification usinga mutant defective in the JA biosynthetic gene OPR3 thatis specifically expressed in anthers (Stintzi and Browse,2000). We found that JA is not involved in controllingendothecium lignification, as the timing of lignin deposi-tion as well as MYB26 transcript levels in opr3 flower budswere comparable to wild-type. Ishiguro et al. (2001) pro-posed that JA controls stomium opening, the final event ofanther dehiscence, and Sanders et al. (2000) showeddelayed and defective stomium opening in opr3 mutants.However, these authors did not assess whether JA controlsstomium opening by controlling endothecium lignificationor whether these two developmental processes are inde-pendently controlled. We show here that the latter is thecase: anthers of afb1 opr3 double mutant flowers showearly endothecium lignification like afb1-3 anthers, but areindehiscent as in the opr3 mutant, which is defective instomium opening. These findings demonstrate that auxinhas no effect on stomium opening in the absence of JA.However, the early dehiscence phenotype of afb1-3 andtir1 afb2 afb3 mutants suggests that auxin controls stomi-um opening through JA.
We show that, during late stages of flower development,auxin negatively controls JA biosynthesis, acting on bothstamen-specific JA biosynthetic genes DAD1 and OPR3. Intir1 afb2 afb3 mutant flower buds, there is an increase inthe level of DAD1 and OPR3 transcripts, resulting inincreased production of JA during late stamen develop-
ment. The increase in JA content in these early-dehiscentmutants is consistent with the phenotype of JA-defectivemutants, which show indehiscent or late dehiscentanthers. The increase in JA production was not detectablein afb1-3 flower buds. However, this mutant showed fewerearly-dehiscing anthers, and a lower level of DAD1 andOPR3 transcripts than tir1 afb2 afb3, possibly due to a lim-ited increase in JA production that may go undetectedunder our experimental conditions. We provide further evi-dence that auxin negatively controls JA biosynthesis bytreating wild-type inflorescences with NAA and showingthat DAD1 and OPR3 transcript levels are reduced in flowerbuds specifically at stages 11 and 12 of development.
Future work is necessary to clarify which gene(s) medi-ate the negative control of auxin on JA biosynthesis in latestages of flower development. Possible candidates may bethe AS1 and AS2 transcription factors, which repress theclass 1 KNOX genes, which in turn repress DAD1 (Tabataet al., 2010). Another candidate may be ARF5/MP: recently,a negative role for ARF5 on JA production has been pro-posed (Garrett et al., 2012), in contrast to the well-knownpositive control of ARF6/ARF8 (Nagpal et al., 2005; Tabataet al., 2010).
We also show here that JA has no effect on auxin syn-thesis and accumulation in anthers. JA production peaks inflower buds when auxin concentration decreases butbefore auxin biosynthetic genes are turned off. To rule outa possible positive feedback effect of JA in negatively regu-lating auxin synthesis, we showed that, in opr3 inflores-cences, the transcript levels of the YUC2 and YUC6 auxinbiosynthetic genes and DR5 activity are comparable to thewild-type (Figure S4 and Data S1).
Thus, our data indicate that auxin negatively regulatesendothecium lignification and JA concentration. We alsoshow that both JA levels and the timing of endotheciumlignification contribute to determining when anther dehis-cence occurs. By treatment of opr3 inflorescences withexogenous JA, we showed that a high JA level causes pre-cocious anther dehiscence, and by comparing the timingof anther dehiscence of JA-treated opr3 and afb1 opr3flower buds, we confirmed our previous suggestion thatprecocious endothecium lignification causes early antherdehiscence (Cecchetti et al., 2008).
In summary, as represented in Figure 8, we suggest that,in Arabidopsis anthers, an auxin maximum at early floralstage 10 blocks premature endothecium lignification viarepression of MYB26. Upon a decrease in auxin concentra-tion at floral stages late 10-11, repression of MYB26 isreleased: this allows MYB26 to trigger endothecium lignifi-cation. Auxin reduction also allows higher expression ofDAD1 and OPR3, which causes an increase in JA concen-tration, leading to stomium breakage and allowing comple-tion of the anther dehiscence process (stage 13).
Arabidopsis wild-type and mutants are described in Methods S5.Anthers collected for IAA measurements were severed from flowerbuds, immediately frozen and divided into five groups according tothe stage: pre-meiotic, meiotic, uninucleate microspore, pollenmitosis II and tricellular pollen grains. The stage was assessed bysqueezing one anther per flower bud in acetic orcein solution todistinguish between pre-meiotic and meiotic stages or stainingwith DAPI (4’,6-diamidino-2-phenylindole) solution (Cecchettiet al., 2004) to distinguish between pollen mitosis I and pollenmitosis II stages. The tricellular pollen grain stage was defined asflower buds with white petals protruding past sepals correspond-ing to stage 12. Flower bud developmental stages were alsochecked by histological analysis of random flower buds.
Flower buds collected for JA measurements and for quantitativeRT-PCR analysis were severed from three plants for each genotypeand immediately frozen. Stages <10 (Figures 2c, 3f, 5c) and " 10(Figure 6a-d,f) were collected after removing all flower buds fromstage 10 onwards and from stage 11 onwards, respectively, fromthe inflorescences. Stages 11 and 12 for JA measurement wereobtained by pooling equal amounts of flower buds at stages 11and 12. For each genotype, 20 mg (dry weight) of pooled flowerbuds in three replicates were collected.
In planta NAA treatment of MYB26 inflorescences was per-formed as described previously (Cecchetti et al., 2008). Twenty-four hours after NAA treatments, wild-type flowers at stages<10, 10, 11 and 12 or "10 and 11 (Figure 3f) and 12 (Figure 6f)were collected for quantitative RT-PCR analysis. JA treatmentswere performed by brushing inflorescences of three plants with1, 3.2 or 6.4 mM methyl jasmonate and water/0.05% Tween(Figure 7).
Quantitative RT-PCR analysis
RNA was extracted from 50 mg flower buds at the indicatedstages of development or from inflorescences, and reverse-tran-scribed as previously described (Cecchetti et al., 2007) (MethodsS6).
SYBR Green-based quantitative assays were performed asdescribed by Savona et al. (2012) using a Bio-Rad iCycler iQ(http://www.bio-rad.com). All quantifications were performed intriplicate.
Statistical analysis
Two-tailed and one-tailed Student’s t tests were used to evaluatestatistical significance. All the statistical analyses were performedusing Graph Pad Prism 5 (Graph Pad Software Inc., http://www.graphpad.com).
ACKNOWLEDGEMENTS
We are grateful to Mark Estelle and Geraint Parry for kindly pro-viding tir1 and afb single mutants, Zoe Wilson for the ms35/myb26 mutant and the proMYB26:GUS construct, and JohnBrowse for the opr3 mutant. We thank Hannah Florence for JAmeasurements performed at the University of Exeter mass spec-trometry facility, and Gun L#ovdahl for excellent technical assis-tance in quantification of IAA.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Analysis of anther dehiscence in tir1-1, afb2-3 and afb3-4 single mutant flowers and in vitro germination assay of pollengrains from tir1-1, afb1-3, afb2-3 and afb3-4 lines.
Figure S2. Analysis of endothecium lignification in afb1-3 and tir1afb2 afb3 anthers.
Figure S3. RT-PCR analysis of inflorescences from wild-type, afb1myb26 and afb1 opr3 double mutants.
Figure S4. DR5:GUS activity and YUC2 and YUC6 transcript levelsare not altered in opr3 flower buds.
Table S1. Stamen filament length at stage 13 of development inafb1-3, afb2-3, tir1-1 and afb3-4 single mutant flowers.
Data S1. Jasmonic acid has no effect on auxin synthesis and accu-mulation.
Methods S1. Quantification of IAA.Methods S2. Morphological, histological and cytological analysis.
Figure 8. Model for auxin control of the anther dehiscence process.Auxin concentration in anthers (red area), anther developmental events and relevant gene activities [DAD1/OPR3 mRNA level in the flower (green area), MYB26mRNA level in the anther (blue area)] as inferred from our data, from stages 9–13 (Bowman, 1994). En, endothecium; St, septum (green circles).
Methods S3. MYB26:GUS plants and crosses.Methods S4. Quantification of JA.Methods S5. Plant material.Methods S6. Primers used for quantitative RT-PCR.PLEASE checkthat all supplemenary methods are referred to in the text innumerical order. You may need to insert a section in Experimentalprocedures entitled ‘Additional methods’. Please check that sub-mitted material is numbered correctly.
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