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. 2023 Nov 13;4(6):100682.
doi: 10.1016/j.xplc.2023.100682. Epub 2023 Sep 9.

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin

Wenqi Yang  1 , Dongdong Yao  1 , Haiyang Duan  2 , Junli Zhang  3 , Yaling Cai  1 , Chen Lan  1 , Bing Zhao  1 , Yong Mei  1 , Yan Zheng  1 , Erbing Yang  4 , Xiaoduo Lu  5 , Xuehai Zhang  2 , Jihua Tang  6 , Ke Yu  7 , Xuebin Zhang  8
Affiliations

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin

Wenqi Yang et al. Plant Commun. .

Abstract

Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid derivatives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The NMR and GC-MS results confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these species. We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot plants. Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance in pollen.

Keywords: UV radiation; heat stress; lignin monomers; pollen cell wall; sporopollenin.

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Figures

Figure 1
Figure 1
H, G, and S lignin units are structural components of maize sporopollenin. (A) Electron micrographs of maize pollen grains (top), the fine powder after ball milling (middle), and the destarched extractive-free cell-wall residues of maize pollen (bottom). Scale bar, 500 μm. (B) 2D-NMR spectra of thioacidolysis-released products of maize sporopollenin and cell walls extracted from a mixture of lignified maize tissues (stem, leaf, and leaf sheath), showing the aromatic signals of H, G, and S lignin units, as well as tricin, p-BA, p-CA, and FA. (C) Selected ion chromatograms and MS2 spectra of the indicated lignin monomers in thioacidolysis-released products of maize sporopollenin or maize stems from GC–MS. The structures of characteristic base peak fragment ions are shown. Results obtained from the maize inbred line MN are used here for representation.
Figure 2
Figure 2
H units comprise the majority of lignin monomers in maize sporopollenin. (A) Quantification of lignin monomers in thioacidolysis-released products of sporopollenin from 117 maize inbred lines. One-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 117, P < 0.05). Error bars represent SD. Letters indicate significant differences. (B) Descriptive statistics for lignin monomer composition in sporopollenin of 117 maize inbred lines. (C) Quantification of lignin monomers in thioacidolysis-released products of sporopollenin from B73. One-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 4, P < 0.05). Error bars represent SD. Letters indicate significant differences. (D) Quantification of lignin monomers in thioacidolysis-released products of leaf, stem, tassel, glume, and leaf sheath tissues of B73. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 4, P < 0.05). Error bars represent SD. Letters indicate significant differences in the abundance of individual monomers within each tissue.
Figure 3
Figure 3
Monolignol biosynthesis is essential for pollen resistance to stresses in Arabidopsis. (A) Comparison of total lignin monomers in thioacidolysis-released products of sporopollenin from maize inbred lines originating from tropical/subtropical and temperate areas. Student’s t-test was used for statistical analysis (tropical/subtropical, n = 65; temperate, n = 52; P < 0.05). Error bars represent SD. Asterisk indicates a significant difference. (B) Heatmap showing the percentage of defective pollen in inbred lines with the highest and lowest levels of total lignin monomers in sporopollenin before and after heat treatment. Five biological replicates (shown in five rows) were analyzed for each inbred line. (C) Pollen viability of Arabidopsis before and after heat treatment. Fresh pollen grains were exposed to 37°C for the indicated times and then tested for in vitro germination. Scale bar represents 200 μm. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 10, P < 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each time point.
Figure 4
Figure 4
Natural variation in ZmVAMP726 is associated with lignin monomer composition in maize sporopollenin. (A) GWAS results for total lignin monomer content in thioacidolysis-released products of maize sporopollenin. The lead SNP is highlighted in red. The window (middle) represents the 60-kb genomic region surrounding the most significant SNP. The LD heatmap (bottom) shows pairwise R2 values between polymorphisms in the 60-kb region centered on the most significant SNP. (B) Protein sequence alignment of AtVAMP726 and GRMZM2G075588. Identical residues are highlighted in gray, and similar residues are highlighted in yellow and cyan. (C) Developmental expression pattern of ZmVAMP726. Relative expression was calculated using the 2−ΔΔCt method. Zm00001d015327 (UBQ) was used as the reference. Error bars represent SD (n = 3). (D) Quantification of H, G, and S units and total lignin monomers in thioacidolysis-released products of sporopollenin from B73 and Zmvamp726. Student’s t-test was used for statistical analysis (n = 10, P < 0.05). Error bars represent SD. Asterisks indicate significant differences. n.s., not significant.
Figure 5
Figure 5
ZmVAMP726 is involved in maize pollen resistance to heat and UV radiation. (A) Pollen viability of B73 and Zmvamp726 after heat treatment. Fresh pollen grains were exposed to 37°C for the indicated times and then stained with TTC. Student’s t-test was used for statistical analysis (n = 5, P < 0.05). Error bars represent SD. Asterisks indicate significant differences. Scale bar, 500 μm. (B) Pollen viability of B73 and Zmvamp726 after UV radiation treatment. Fresh pollen grains were exposed to UV for the indicated times and then stained with TTC. Student’s t-test was used for statistical analysis (n = 5, P < 0.05). Error bars represent SD. Asterisks indicate significant differences. Scale bar, 500 μm.
Figure 6
Figure 6
Both AtVAMP726 and ZmVAMP726 are involved in resistance of Arabidopsis pollen to heat and UV radiation. (A) AtVAMP726 is involved in resistance of Arabidopsis pollen to heat and UV radiation. Fresh pollen grains of the indicated Arabidopsis genotypes were exposed to 37°C or UV radiation for the indicated times and then tested for in vitro germination. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 5, P < 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each time point. Scale bar, 200 μm. (B)ZmVAMP726 restored pollen stress resistance in Atvamp726. Fresh pollen grains were exposed to 37°C or UV radiation for the indicated times and then tested for in vitro germination. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n = 5, P < 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each time point. Scale bar, 200 μm.
Figure 7
Figure 7
Overexpression of AtVAMP726 alters the proteome of the apoplastic space. (A) GO enrichment analysis of differentially accumulated proteins in apoplastic washing fluids obtained from Arabidopsis plants overexpressing VAMP726. The top ten most significantly enriched GO terms are listed. (B) Relative abundance of proteins associated with the GO term “peroxidase activity.” The protein abundances were normalized by Z score and plotted. Proteins were organized according to hierarchical clustering. Three biological replicates per genotype are represented by three columns. Color scale represents the Z-score value. (C) Quantification of p-coumaryl alcohol in apoplastic washing fluids obtained from Arabidopsis plants. Student’s t-test was used for statistical analysis (n = 3, P < 0.05). Error bars represent SD. Asterisks indicate a significant difference.
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