The Regulation of Sporopollenin Biosynthesis Genes for Rapid Pollen Wall Formation

Abstract

Sporopollenin is the major component of the outer pollen wall (sexine). It is synthesized using a pathway of approximately eight genes in Arabidopsis (Arabidopsis thaliana). MALE STERILITY188 (MS188) and its direct upstream regulator ABORTED MICROSPORES (AMS) are two transcription factors essential for tapetum development. Here, we show that all the sporopollenin biosynthesis proteins are specifically expressed in the tapetum and are secreted into anther locules. MS188, a MYB transcription factor expressed in the tapetum, directly regulates the expression of POLYKETIDE SYNTHASE A (PKSA), PKSB, MALE STERILE2 (MS2), and a CYTOCHROME P450 gene (CYP703A2). By contrast, the expression of CYP704B1, ACYL-COA SYNTHETASE5 (ACOS5), TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 are significantly reduced in ams mutants but not affected in ms188 mutants. However, MS188 but not AMS can activate the expression of CYP704B1, ACOS5, and TKPR1 In ms188, dominant suppression of MS188 homologs reduced the expression of these genes, suggesting that MS188 and other MYB family members play redundant roles in activating their expression. The expression of some sporopollenin synthesis genes (PKSA, PKSB, TKPR2, CYP704B1, and ACOS5) was rescued when MS188 was expressed in ams Therefore, MS188 is a key regulator for activation of sporopollenin synthesis, and AMS and MS188 may form a feed-forward loop that activates the expression of the sporopollenin biosynthesis pathway for rapid pollen wall formation.

Figure 1.

Protein localization of AMS-GFP, MS188-GFP, and sporopollenin synthesis protein-GFPs. Confocal images of the fluorescence of the AMS-GFP, MS188-GFP, CYP704B1-GFP, ACOS5-GFP, TKPR1-GFP, TKPR2-GFP, PKSB-GFP, and PKSA-GFP fusion proteins from stages 6 through 10. GFP expression (530 nm) is shown in the green channel, while chlorophyll autofluorescence (>560 nm) is shown in the red channel. A to C and F to H, The fluorescence of AMS-GFP and MS188-GFP at stages 6 through 8. Right, high-magnification images of GFP localization. D, E, I, and J, The fluorescence of AMS-GFP and MS188-GFP at stages 9 and 10. K, P, U, Z, E1, and J1, The fluorescence of CYP704B1-GFP, ACOS5-GFP, TKPR1-GFP, TKPR2-GFP, PKSB-GFP, and PKSA-GFP at stage 6. L to N, Q to S, V to X, A1 to C1, F1 to H1, and K1 to M1, The fluorescence of CYP704B1-GFP, ACOS5-GFP, TKPR1-GFP, TKPR2-GFP, PKSB-GFP, and PKSA-GFP at stages 7 to 9. Right, high-magnification images. O, T, Y, D1, I1, and N1, The fluorescence of CYP704B1-GFP, ACOS5-GFP, TKPR1-GFP, TKPR2-GFP, PKSB-GFP, and PKSA-GFP at stage 10. Scale bars, 50 μm.

Figure 2.

Expression of the sporopollenin synthesis genes in tapetal transcription factor mutants. A, The proposed hierarchical relationship between the tapetal transcription factors required for exine development. B, Results from microarray analysis of PKSA, PKSB, CYP703A2, MS2, TKPR1, TKPR2, CYP704B1, and ACOS5 expression in the inflorescences of wild type (WT), dyt1, tdf1, ams, and ms188 mutants. C, RT-qPCR analysis of the above sporopollenin synthesis genes in the inflorescences of wild-type, dyt1, tdf1, ams, ms188, and ms1 plants. x axis represents the genes, and y axis is shown as relative expression. sd is indicated as error bar.

Figure 3.

Transcription factor MS188 directly regulates the expression of PKSA, PKSB, and MS2. A to C, The designed probes of PKSB, PKSA, and MS2, respectively. Probe 1 (P1) and P2 contain the binding sites (black dots) of MS188, and P3 was designed in the coding region as a control. The black lines show the fragments of the probes for the ChIP-qPCR assay. D to F, The fragments of the above probes in PKSB, PKSA, and MS2 promoters specifically amplified by ChIP-qPCR using 4xMYC-MS188 transgenic plant samples. The fold of enrichment is the average of three replicates, and sd is indicated as error bar. −AB, absence of antibody; +AB, presence of antibody. G to I, EMSA assay showing MS188 binding to probes in vitro. The first panel represents free probe, and the last panel indicates the mixture of free probe and MBP tag; both are used as negative controls. The shift band is indicated by the arrowhead, which is highlighted by the positive control of mixture of biotin-tagged probe and non-biotin-tagged probe and MBP-MS188. J to L, p35s::MS188 was cotransformed with pPKSB::LUC, pPKSA::LUC, and pMS2::LUC, respectively, and a transient dual-luciferase assay was conducted in the Arabidopsis protoplasts. Three replicates were performed, and the y axis is shown as the ratio of Luciferase/Renilla. sd is indicated as error bar.

Figure 4.

MS188 but not AMS activates the expression of ACOS5, CYP704B1, and TKPR1. A to C, The designed probes in the promoter and genomic regions of TKPR1, ACOS5, and CYP704B1, respectively. D to F, The fragments of the above probes in TKPR1, ACOS5, and CYP704B1 promoters were specifically amplified by ChIP-qPCR using 4xMYC-MS188 transgenic plant samples. The fold enrichment is the average of three replicates (error bar is shown by sd). −AB, absence of antibody; +AB, presence of antibody. G to I, Transient dual-luciferase assays were performed in Arabidopsis leaf protoplasts. The p35S::NOS vector was used as negative control. The p35S::MS188 and p35S::AMS were transformed into the protoplasts with reporter plasmids separately. The last column of three panels was cotransformation of p35S::MS188, p35S::AMS, and each reporter plasmid. sd is indicated as error bar.

Figure 5.

Redundant genes of the MYB family are involved in regulating the expression of sporopollenin synthesis genes. A, pMS188::MS188-SRDX and pAMS::MS188-SRDX constructs. B to E, Fertility comparison between wild-type (WT), ms188, pMS188::MS188-SRDX (ms188^−/−), and pAMS::MS188-SRDX (ms188^−/−) plants, respectively. F, Reverse transcription quantitative PCR (RT-qPCR) analysis of TKPR1, TKPR2, CYP704B1, and ACOS5 expression in the inflorescences of WT, ams, ms188, pMS188::MS188-SRDX (ms188^−/−), and pAMS::MS188-SRDX (ms188^−/−) plants. y axis is shown as relative expression, and error bar is indicated as sd.

Figure 6.

Expression of MS188 in ams rescues the expression of sporopollenin synthesis-related genes. A, Reverse transcription quantitative PCR (RT-qPCR) analysis of PKSB, PKSA, MS2, TKPR1, TKPR2, CYP704B1, and ACOS5 in the inflorescences of wild-type (WT), ams, ms188, and pAMS::MS188 (ams^−/−) plants. y axis is shown as relative expression, and error bar is indicated as sd. B, A proposed model of MS188 acting as the main regulator together with AMS and other MYB family and bHLH family members to modulate sexine formation. For the genetic pathway (DYT1-TDF1-AMS-MS188-MS1) in the tapetal layer, MS188 is directly regulated by AMS and MS188 interacts with AMS to form a feed-forward loop to regulate the expression of sporopollenin synthesis genes. Some other MYB family and bHLH family members participate in the regulation process. The colorful ellipses indicate the transcription factors, and the black ones indicate the sporopollenin enzymes. The black circles represent the sporopollenin precursors.