Transcription Factor MYB26 Is Key to Spatial Specificity in Anther Secondary Thickening Formation

Abstract

Successful fertilization relies on the production and effective release of viable pollen. Failure of anther opening (dehiscence), results in male sterility, although the pollen may be fully functional. MYB26 regulates the formation of secondary thickening in the anther endothecium, which is critical for anther dehiscence and fertility. Here, we show that although the MYB26 transcript shows expression in multiple floral organs, the MYB26 protein is localized specifically to the anther endothecium nuclei and that it directly regulates two NAC domain genes, NST1 and NST2, which are critical for the induction of secondary thickening biosynthesis genes. However, there is a complex relationship of regulation between these genes and MYB26. Using DEX-inducible MYB26 lines and overexpression in the various mutant backgrounds, we have shown that MYB26 up-regulates both NST1 and NST2 expression. Surprisingly normal thickening and fertility rescue does not occur in the absence of MYB26, even with constitutively induced NST1 and NST2, suggesting an additional essential role for MYB26 in this regulation. Combined overexpression of NST1 and NST2 in myb26 facilitates limited ectopic thickening in the anther epidermis, but not in the endothecium, and thus fails to rescue dehiscence. Therefore, by a series of regulatory controls through MYB26, NST1, NST2, secondary thickening is formed specifically within the endothecium; this specificity is essential for anther opening.

Figure 1.

Rescue of fertility by DEX induction of MYB26. A, myb26 mutant carrying the MYB26pro:MYB26-GR-YFP transgene line before DEX treatment showing short, sterile siliques due to a lack of self-fertilization as a result of a failure of anther dehiscence. B, myb26 mutant carrying the MYB26pro:MYB26-GR-YFP transgene line after DEX treatment, showing rescued fertility and elongated, filled siliques on the upper region of the inflorescences (arrows); below the rescued fertile siliques were male sterile short, seedless siliques which developed before the DEX treatment. C, Close-up of the inflorescence from the transgene line before DEX treatment, showing short siliques (arrow), which do not contain seeds. D, Close-up of the inflorescence from the transgene line showing rescue of fertility and elongated, filled siliques (arrows) after DEX treatment. E, Wild-type flower showing anther dehiscence and pollen release. F, Flower from the myb26 mutant carrying the MYB26pro:MYB26-GR-YFP transgene before DEX treatment, showing a lack of anther dehiscence and pollen release. G, Flower from the myb26 mutant line carrying the MYB26pro:MYB26-GR-YFP transgene line after DEX treatment, showing rescue of anther dehiscence. H to J, Confocal images of anthers after ethidium bromide/acridine orange staining for secondary thickening. H, Wild-type anther showing lignified endothecium layer (arrow). I, myb26 mutant carrying the MYB26pro::MYB26-YFP-GR transgene before DEX treatment, which lacks endothecium secondary thickening (arrow). J, myb26 MYB26pro:MYB26-GR-YFP transgene line showing restoration of endothecium thickening after DEX treatment (arrow). Scale bars: 100 µm.

Figure 2.

Localization of MYB26 after DEX-induced expression. A and B, Confocal imaging of expression of the functional MYB26pro:MYB26-YFP fusion protein in anthers; expression is only seen in the nuclei of the anther endothecium cells during pollen mitosis I. A, MYB26-YFP fusion protein localized in endothecium nuclei (arrows; excitation 514 nm). B, Overlay of anther chlorophyll autofluorescence (excitation 488 nm) and MYB26-YFP fusion protein. Scale bar represents 75 µm. C, MYB26Pro:GUS expression is seen in many floral tissues, including nectaries, style, filaments, and anthers. D, Time course of MYB26 expression by qRT-PCR in myb26 mutant buds and myb26 mutant carrying the MYB26pro:MYB26-GR-YFP transgene after DEX treatment. Expression levels of the transgene fluctuated slightly but were reduced 1 h post-DEX treatment and strongly reduced by 4 h post-DEX treatment with all samples being at least P < 0.05 after 3 h compared to 0 h control (t test statistical analysis; *P ≤ 0.05; **P ≤ 0.01).

Figure 3.

Induction of NST1 and NST2 expression by MYB26. A, NST1Pro:GUS expression in wild type and (B) increased NST1pro:GUS expression in MYB26 overexpression line, particularly in the peduncle, sepals. and anthers. C, Increased magnification of NST1pro:GUS expression in MYB26 overexpression line showing expression in anthers. D and E, Time course analysis of expression of NST1 and NST2 by qRT-PCR after DEX activation of MYB26 in the transgenic (MYB26pro:MYB26-GR-YFP) myb26 mutant line and in the myb26 mutant control lacking the transgene. D, Induction of NST1 occurred 4 to 6 h after DEX treatment. E, Induction of NST2 was seen 4 to 6 h after DEX treatment. Error bars represent sd (t test statistical analysis compared to 0 h in each line; *P ≤ 0.05; **P ≤ 0.01).

Figure 4.

Ectopic expression of MYB26 is unable to induce secondary thickening in the anther in the absence of NST1 or NST2 expression. A, Wild-type plant showing full fertility as evidenced by silique elongation and full seed set. B, nst1nst2 double mutant showing sterility as indicated by a lack of silique elongation; plants also showed increased bushy growth. C, Expression of 35Spro:MYB26 in nst1nst2 background does not rescue male fertility or bushy growth. D, Expression of 35Spro:MYB26 in nst1nst1NST2nst2 background, which is heterozygous for and thus expressing NST2, is fertile, and growth resembles wild type. NST2 acts redundantly with NST1, and the phenotypes of heterozygous lines are equivalent to wild type, with full fertility and normal growth habit (Mitsuda et al., 2005). Boxed regions show increased magnification of the same lines. E to L, Sections stained with phloroglucinol to detect lignin; scale bar represents 50 µm. E Wild-type anther showing secondary thickening in the endothecium. F The nst1nst2 double mutant fails to develop endothecium secondary thickening (arrow). G, Endothecium secondary thickening is not rescued by expression of 35Spro:MYB26 in the nst1nst2 background (arrow). H, Increased levels of anther endothecium thickening were, however, seen with the 35Spro:MYB26 in the nst1nst1Nst2nst2 heterozygous background (arrow). I to L, Secondary thickening in the inflorescence stems (I) wild type, (J) nst1nst2 double mutant (thickening is slightly reduced), and (K) the nst1nst2 double mutant expressing 35Spro:MYB26. L, Ectopic secondary thickening is seen in the inflorescence stem (arrows) when MYB26 is overexpressed in presence of NST2 (35Spro:MYB26 in the nst1nst1NST2nst2 heterozygous background). M, qRT-PCR expression analysis of MYB26, NST1, NST2, and NST3 in the whole inflorescence of wild type, nst1nst2 mutant, nst1nst2 mutant expressing 35Spro:MYB26, and in the nst1 single mutant (nst1nst1NST2nst2 heterozygous line), and nst1nst1NST2nst2 heterozygous line expressing 35Spro:MYB26. N, qRT-PCR expression of genes involved in secondary thickening pathways in the whole inflorescence of various backgrounds shown in M. Error bars represent sd in M and N (t test statistical analysis compared to its relevant background for each line; *P ≤ 0.05; **P ≤ 0.01).

Figure 5.

ChIP indicates that MYB26 directly binds to upstream regions of NST1 and NST2. A and B, Diagram of upstream region of (A) NST1 (B) NST2; boxes P1 to P10 indicate regions used for ChIP analysis; red/orange boxes are regions that showed positive binding. C and D, ChIP qPCR showing enrichment for (C) P2a in NST1 and P8 for NST2 using anti-GFP, and (D) anti-MYB26 antibodies. E, MYB26-YFP within the nucleus of the endothecium (left; arrows) was detected in the DEX-induced MYB26pro:MYB26-GR-YFP line; no nuclear localized expression was seen in the non-DEX-induced line (right). F, No ChIP qPCR enrichment was seen in the IP-ipG controls. Error bars represent sd (t test statistical analysis compared to control primer [C and F] or anti IpG [D] controls; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Figure 6.

Expression of NST1 or NST2 under the control of the CaMV35S promoter is unable to rescue anther secondary thickening in the myb26 mutant. A to L, Anthers stained for secondary thickening with acridine orange/ethidium bromide and visualized by confocal microscopy. A and B, Wild-type anther showing endothecium thickening (arrow). C and D, myb26 mutant lacking endothecium thickening (arrow). E and I, Overexpression of NST1 (35Spro:NST1) in wild-type background; increased levels of secondary thickening are seen in both the endothecium and epidermal tissues (arrows). F and J, Overexpression of NST1 (35Spro:NST1) in the myb26 mutant background; occasional patches of secondary thickening are seen in the epidermal tissues (arrow), but these are extremely limited, and no endothecium thickening is seen. G and K Overexpression of NST2 (35Spro:NST2) in wild-type background; increased levels of secondary thickening are seen in the endothecium (arrow), but not in the epidermal tissues as seen with NST1 overexpression in wild type. H and L, Overexpression of NST2 (35Spro:NST2) in the myb26 mutant background; occasional patches of secondary thickening are seen in the epidermal cells (arrow); however, these are extremely limited, and the endothecium cells are abnormal and lack the usual expansion seen in these cells prior to secondary thickening deposition. I to L are higher magnifications of the same anther shown in E to H. Scale bars represent 104.85 µm in A, 57.64 µm in B, 80 µm in C, 50 µm in D, 108.69 µm in E, 101.37 µm in F, 108.73 µm in G, 41.67 µm in H, and 50 µm in I to L. M to O, Expression by qRT-PCR analysis in the wild type, myb26 mutant, and overexpression lines of (M) MYB26, (N) NST1, and (O) NST2. Error bars represent sd (t test statistical analysis compared to its relevant background for each line; **P ≤ 0.01).

Figure 7.

Anthers from MYB26myb26 heterozygotes and myb26 mutants that are expressing both NST1 and NST2 under the control of the CaMV35S promoter. A to D, Anthers isolated and stained with phloroglucinol HCl to detect lignified thickening from lines overexpressing both NST1 and NST2. A and B, High levels of native secondary thickening are seen in the endothecium (En) layer (arrows) in the MYB26myb26 heterozygote background with both NST1 and NST2 transgenes. C and D, In the myb26 mutant the anthers appear contorted with ectopic thickening in epidermal tissues (arrows shows ectopic thickening in anther epidermis [Ep] and also in the filament); normal secondary thickening is not seen in the endothecium in the myb26 background, regardless of expression of both NST1 and NST2. Scale bar represents 0.1 mm.

Figure 8.

Rescue of fertility in the myb26 mutant by expression of MYB26 regulated by the NST2 promoter. A, NST2pro:GUS expression in wild type, showing expression in extending filaments prior to dehiscence, and in postmeiotic anthers. B, NST2pro:GUS expression in the myb26 mutant, showing expression in extending filaments prior to dehiscence, and in postmeiotic anthers. C to K, Stamen stained for secondary thickening with acridine orange/ethidium bromide and visualized by confocal microscopy; (C, F, and I) wild-type anthers and filaments; (D, G, and J) NST2pro:MYB26 expression in the myb26 mutant showing high levels of secondary thickening in the endothecium and increased secondary thickening in the filament; (E, H, and K) myb26 mutant with no thickening in the anther endothecium. Scale bars represent 200 µm in C, 150 µm in D, 300 µm in E, 75 µm in F to H, and 150 µm in I to K.

Figure 9.

Model of MYB26 regulation of anther secondary thickening pathway. MYB26 regulation of secondary thickening through downstream the redundant transcription factors NST1/NST2. Arrows represent direct regulation, while bar represents repression, and dotted lines represent predicted regulation/repression. X = unknown factor that enables NST1/2 to initiate secondary thickening. This could be via NST1/2 protein activation/stabilization or removal of an inhibitor involved in NST1/2 degradation/turnover. AHP4, ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSFER FACTOR 4; HDG3, HOMEODOMAIN GLABRA 2-LIKE PROTEIN 3; TCP24 and WRKY2, transcription factor; JA, Jasmonic Acid; DAD1, DEFECTIVE IN ANTHER DEHISCIENCE1; OPR3, 12-OXOPHYTODIENOATE REDUCTASE 3; WAT1, WALLS ARE THIN1.