Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development

Abstract

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

Figure 1.

Characterization of tga9 and tga10 mutant alleles, and tissue expression of TGA9 and TGA10. A, Scale diagrams of TGA9 and TGA10 genomic sequences showing the positions of features as indicated in the key at bottom left. Black boxes indicate exons, and horizontal arrows represent annealing positions of primers used for transcript analysis. B, RT-PCR analysis of TGA9 transcripts in the wild type (WT) and tga9 mutants (40 cycles). C, RT-PCR analysis of TGA10 transcripts in the wild type and tga10 mutants (40 cycles). Full, Product obtained using P1/P3 primer combination; Partial, product obtained using P1/P2 primer combination. D and E, RT-PCR analysis of TGA9 and TGA10 transcripts in wild-type plant tissues (35 and 45 cycles). In B to E, GAPC control transcript used 23 cycles.

Figure 2.

Sterility phenotype in tga9 tga10 double mutants. A and B, Comparison of wild-type (WT) and tga9 tga10 inflorescences. Arrows indicate empty siliques. C, Wild-type and tga9 tga10 siliques. D and E, Wild-type and tga9 tga10 dissected siliques. Bars = 1 mm. [See online article for color version of this figure.]

Figure 3.

Flower and anther morphology in the wild type and tga9 tga10 mutants. Floral stages are as indicated. A, Wild-type (WT) flower. B, Wild-type anther, adaxial view. C, Wild-type dissected flower. D, tga9 tga10 flower. E, tga9 tga10 anther, adaxial view. F, tga9 tga10 dissected flower; anthers are nondehiscent. Bars = 1 mm, except 100 μm in B and E. [See online article for color version of this figure.]

Figure 4.

Comparison of wild-type and tga9 tga10 anther development. Cross-sections of wild-type (WT; A–G) and tga9 tga10 (H–N) anthers were stained with toluidine blue. A, Stage 4. Sporogenous cells in all four lobes. B, Stage 5. Characteristic layered structure of anther lobes is resolved. C, Stage 6. Meiosis of microspore mother cells. D, Stage 7. Tetrads form, and the middle layer is crushed. E, Stage 8. Developing microspores; tapetum degeneration is under way. F, Stage 11. Developing microspores; septum degeneration begins and endothecium expands. G, Stage 13. Anthers are dehisced; ruptured stomium. H, Stage 4. Sporogenous cells are often missing in adaxial lobes. I, Stage 5. Sporogenous cells proliferate in abaxial lobes; adaxial lobes are underdeveloped. J, Stage 6. Microspore mother cells in abaxial lobes undergo meiosis, and tapetal cells are abnormally vacuolated; development is variable in adaxial lobes. K, Stage 7. Tetrads appear in abaxial lobes. L, Stage 8. Microspores are released in abaxial lobes; the middle layer fails to degrade. M, Stage 11. Microspore development is delayed; breakdown of the middle layer is incomplete, but endothecium cells are expanded. N, Stage 13. Anthers are indehiscent; pollen is clumped or degraded. En, Endothecium; MC, meiotic cells; ML, middle layer; MMC, microspore mother cells; MSp, microspores; PG, pollen grains; Sm, septum; Sp, sporogenous cells; St, stomium; StR, stomium region; T, tapetum; Tds, tetrads. Bars = 50 μm. [See online article for color version of this figure.]

Figure 5.

Expression of TGA9 in wild-type inflorescences and flowers. Expression was monitored using a TGA9::GUS reporter gene (A–E) or by in situ hybridization (F–L). A to E, GUS activity is first detected in stage 7 flowers, peaks at stage 8, and declines during stages 9 to 10. E, Stage 10 flower cross section; expression persists in the locule wall (arrowheads). TGA9 in situ probe hybridized to anther cross-sections. F, Stage 2 to 3 anthers. Expression laterally and along the adaxial face of primordia (arrows) is shown. G, Stage 2. Anther primordia. H, Stage 4. Expression in all cell layers. I and J, Stages 5 to 6. Expression is restricted to the tapetum and middle layer. K and L, Stages 8 to 11. Expression declines. Bars = 100 μm except in A (0.5 mm) and F to L (25 μm). LW, locule wall; ML, middle layer; T, tapetum. [See online article for color version of this figure.]

Figure 6.

In situ analysis of SPL/NZZ expression in wild-type (WT) and tga9 tga10 anthers. Anther stages are as indicated. A to C, The wild type. SPL is uniformly expressed in adaxial versus abaxial anther lobes. D to F, The tga9 tga10 mutant. SPL expression is reduced in adaxial anther lobes (asterisks). Sp, Sporogenous cells. Bars = 25 μm. [See online article for color version of this figure.]

Figure 7.

Double mutant analysis and RT-PCR analysis of anther transcripts in the wild type and tga9 tga10 mutants. A to F, Cross-sections of stage 11 anthers stained with toluidine blue for the indicated genotypes. Bars = 50 μm. G, RT-PCR analysis of anther transcripts (35 cycles) in wild-type (WT) and mutant inflorescence apices. Numerical values for transcript down-regulation in roxy1 roxy2 mutants (Xing and Zachgo, 2008) are shown. H, Genetic framework for control of anther development (modified from Wilson and Zhang, 2009) showing overlap for tapetal genes down-regulated in roxy1 roxy2 and tga9 tga10 mutants. Shaded ovals, Down-regulated in roxy1 roxy2; dashed perimeter, down-regulated in tga9 tga10; shaded ovals with dashed perimeter; down-regulated in both. Genes not discussed in the text are as follows: TAPETAL DETERMINANT1 (TPD1; Yang et al., 2003), EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS (EMS1/EXS; Canales et al., 2002; Zhao et al., 2002), MYB103/MYB80 (Higginson et al., 2003; Zhang et al., 2007), MYB99 (Ito et al., 2007), ABORTED MICROSPORES (AMS; Sorensen et al., 2003), DEFECTIVE IN EXINE FORMATION1 (DEX1; Paxson-Sowders et al., 2001), NO EXINE FORMATION1 (NEF1; Ariizumi et al., 2004), and CALLOSE SYNTHASE5 (CalS5; Dong et al., 2005). [See online article for color version of this figure.]

Figure 8.

TGA9 and TGA10 directly interact with ROXY1/2. A, Yeast two-hybrid assay. ROXY proteins served as bait. NPR1 and CRUCIFERIN were negative controls. Error bars indicate se (three replicates). B, ROXY proteins interact with TGA9/10 in the nuclei of transiently transformed tobacco leaves. Images show reconstitution of YFP fluorescence 3 to 5 d after coexpression of protein pairs. The N terminus of YFP (YN) was fused in-frame upstream of ROXY1 and ROXY2. The C terminus of YFP (YC) was fused in-frame upstream of TGA9 and TGA10. As a negative control, coexpression of YN-ROXY1 and YC alone failed to reconstitute a fluorescent YFP chromophore (data not shown). Bars = 50 μm. [See online article for color version of this figure.]

Figure 9.

Model for redox control of TGA9/10 activity by ROXY1/2. In their inactive form, one or more oxidized C-terminal Cys residues block TGA9/10 transcription factor activity (a dithiol bridge is shown) by an unknown mechanism. In response to redox changes triggered by a developmental signal, ROXY1 and ROXY2 reduce TGA9 and TGA10, converting them into a transcriptionally active form.