The ZmMYB84-ZmPKSB regulatory module controls male fertility through modulating anther cuticle-pollen exine trade-off in maize anthers

Abstract

Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen-stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade-off of lipid-metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male-sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map-based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)-localized polyketide synthase (PKS) with catalytic activities to malonyl-CoA and midchain-fatty acyl-CoA to generate triketide and tetraketide α-pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9-10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84-ZmPKSB regulatory module controlled a trade-off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine-tuning regulation of lipid-metabolic balance to precisely promote anther cuticle and pollen exine formation in plants.

Keywords: ZmMYB84-ZmPKSB regulatory module; anther cuticle; lipid metabolism; pollen exine; polyketide synthase; trade-off.

Figure 1

Phenotypic and cytological comparison of wild type (WT) and pksb mutant. (a) Comparison of tassels, anthers and pollen grains stained with I₂‐KI between WT and pksb mutant. (b) Transverse sections of WT and pksb anthers from stages S8b to S12. (c) SEM analysis of anther cuticle, Ubisch bodies, pollen and pollen surface in WT and pksb mutant at anther stage 13. CMsp, collapsed microspore; E, epidermis; En, endothecium; ML, middle layer; Mp, mature pollen; Msp, microspore; T, tapetum; Tds, tetrads.

Figure 2

TEM and SEM analyses of WT and pksb anthers from stages S9 to S12. (a) TEM observation of anther wall, microspores, Ubisch bodies and exine in WT and pksb mutant. SEM (b) and TEM observations (c) of anther outer surface in WT and pksb mutant. Ba, bacula; Cu, cuticle; Cw, cuticle wall; E, epidermis; En, endothecium; F, foot layer; ML, middle layer; Mp, mature pollen; Msp, microspore; Ta, tapetum; Te, tectum; Ub, Ubisch body.

Figure 3

Map‐based cloning and molecular characterization of ZmPKSB gene. (a) Primary and fine mapping of pksb locus on chromosome 7. n1 and n2, the number of F₂ plants used for gene mapping. (b) Gene structure and DNA sequencing analysis of ZmPKSB in WT and pksb mutant. Black boxes indicate exons, and intervening lines indicate introns. (c) Phenotypes of tassels, anthers and pollen grains (stained with 1% I₂‐KI) of WT, pksb and the three CRISPR/Cas9 knockout lines at the mature pollen stage. (d) Allelic tests between each of two homozygous CRISPR/Cas9 knockout lines (ZmPKSB‐Cas9‐1 and ‐2) and heterozygotic pksb mutant (ZmPKSB/pksb). (e) Phylogenetic analysis of ZmPKSB and its orthologs in 18 plant species. The analysis involved 21 amino acid sequences from Aegilops tauschii (AEGTA), Arabidopsis thaliana (AT), Bradchypodium distachyon (Bd), Brassica napus (Bn), Dichanthelium oligosanthes (Do), Glycine max (Gm), Gossypium raimondii (Gr), Hordeum vulgare (Hv), Hypericum perforatum (Hp), Nicotiana benthamiana (Niben), Oryza sativa (Os), Physcomitrium patens (Pp), Setaria italica (Si), Solanum lycopersicum (Sl), Sorghum bicolor (Sb), Triticum aestivum (Ta), Triticum dicoccoides (Td) and Zea mays (Zm). The numbers on the branches represent the bootstrap values of the phylogenetic tree. (f) Spatiotemporal expression of ZmPKSB by qPCR analysis. (g) qPCR analysis of ZmPKSB in WT and pksb anthers. (h) Subcellular localization of ZmPKSB in maize protoplasts. The ZmPKSB‐GFP was co‐transformed with the ER‐mCherry as an ER marker. The 35S‐GFP vector was used as a negative control. ZmUbi2 served as an internal control, and error bars indicate SD (f and g). Each reaction had three biological replicates with three technical repeats (n = 9).

Figure 4

ZmPKSB is activated by ZmMYB84 and the ZmMYB84‐ZmPKSB regulatory module controls lipid biosynthesis for anther cuticle and pollen exine formation. (a) Expression patterns of the ZmMYB84 and ZmPKSB genes in WT (B73) anthers from stages S5 to S12 based on RNA‐seq data. Data represent mean ± SD, n = 2–4. (b) Expression patterns of the ZmMYB84 and ZmPKSB genes in WT (B73) anthers from stages S5 to S12 by qPCR analysis. Data represent mean ± SD, n = 9. (c) Expression pattern change in ZmPKSB in myb84 anthers compared with WT anthers from stages S6 to S12 based on qPCR analysis. Data represent mean ± SD, n = 9. (d)Transient dual‐luciferase reporter (TDLR) assay of ZmPKSB promoter activity activated by ZmMYB84 in maize protoplasts. Data represent mean ± SD, n = 3. (e) EMSA showing ZmMYB84 binging to the ccaacc box in ZmPKSB promoter in vitro. (f) SEM observation of anther cuticle in WT and myb84 mutant form stages S9 to S13. (g) Analysis of anther cutin and wax contents in WT and myb84 anthers at stage S13. Data represent mean ± SD, n = 3. (h) A proposed model of the ZmMYB84‐ZmPKSB regulatory module, which is required for lipid biosynthesis for maize anther cuticle and pollen exine formation. For (d and g), * and *** indicate the significant levels of P < 0.05 and P < 0.001 by a two‐tailed Student's t‐test, respectively.

Figure 5

Enzyme activity analysis of the recombinant ZmPKSB proteins. (a) ZmPKSB has 417 amino acids and contains chalcone synthases (CHS) N and C domains. The conserved catalytic triad of Cys171, His320 and Asn353 is indicated with arrows. (b) The simulated three‐dimensional structure of ZmPKSB protein and the spatial locations of the conserved catalytic triad consisting of Cys171, His320 and Asn353. (c) Sequence alignment of the three sites and their point mutations in the catalytic triad. (d) SDS‐PAGE analysis of the MBP‐tagged protein ZmPKSB (ZmPKSB‐MBP) and its three single amino acid substitution variants (C171A‐MBP, H320A and N353A) purified from Escherichia coli. (e) High‐performance liquid chromatography (HPLC) analyses of the products of enzymatic reactions catalysed by ZmPKSB‐MBP and its three single amino acid substitution variants (Cys171, His320 or Asn353) with C16‐CoA+malonyl‐CoA and C18‐CoA+malonyl‐CoA as substrates. The cognate triketide and tetraketide α‐pyrone reaction products were detected. The m/z value of the [M‐H]‐ion of each product was indicated. (f) Identification of enzymatic reaction products of ZmPKSB with C16:0‐CoA and malonyl‐CoA as substrates by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) analysis. Structure of the compound and putative fragmentation scheme is shown.

Figure 6

ZmMYB84 and ZmPKSB affect pollen exine and anther cuticle formation by altering expression of sporopollenin‐, cutin‐ and wax‐related genes. (a) Expression pattern changes in nine sporopollenin‐related genes in WT and pksb mutant anthers from stages S7 to S13. (b) Expression pattern changes of three cutin‐related genes (b1) and six wax‐related genes (b2) in WT and pksb mutant anthers from stages S7 to S13. (c) Analysis of anther wax and cutin contents per unit surface area in WT and pksb mutant at stage S13. Anther cutin and wax contents were increased in pksb mutant compared with WT. Data represent mean ± SD of three independent experiments. ** indicates P < 0.01 by a two‐tailed Student's t‐test, respectively. (d) Expression pattern changes of nine sporopollenin‐related genes in WT and myb84 anthers from stages S6 to S12. (e) Expression pattern changes of three cutin‐related genes (e1) and six wax‐related genes (e2) in WT and myb84 anthers from stages S6 to S12. For (a, b, d and e), error bars indicate SD, and each reaction had three biological replicates with three technical repeats. *, **, and *** indicate the significant levels of P < 0.05, 0.01 and 0.001 (Student's t‐test, n = 9), respectively.

Figure 7

ZmMYB84‐ZmPKSB regulatory module controls a trade‐off between anther cuticle and pollen exine formation by regulating genes related to biosynthesis and transport of sporopollenin, cutin and wax in maize anthers.