Genetic Mechanisms of Cotton Seed Size

On April 29, 2024, Tianzhen Zhang's team from Zhejiang University published a research paper titled "UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development" in Plant Communications. This study found that UDP-glucosyltransferase 71C4 (UGT71C4) participates in cotton seed development by changing the metabolic flux between lignin and flavonoid metabolic pathways, thereby affecting cotton seed yield.

Seeds are unique propagules of gymnosperms and angiosperms. They play a decisive role in the continuation of species and are also the basis of agricultural production. Therefore, studying the functional mechanism of genes regulating seed development has important theoretical and practical significance for people to understand the mechanism of seed development. However, in recent years, research on cotton has still mostly focused on cotton fiber-related research. Cotton seeds have long been considered a by-product of cotton fiber, and little attention has been paid to research on cotton seed size. The genetic mechanism of cotton seed development still needs to be further studied.

This study found a UDP-glucosyltransferase gene UGT71C4, which is specifically expressed in cotton ovules, especially during the critical stages of ovule formation at 10DPA, 15DPA and 20DPA. It is speculated that UGT71C4 plays a role in cotton seed development. In upland cotton, after overexpression of UGT71C4, the length and width of cotton seeds increased, the seed index of mature seeds significantly increased from 10.66g to 11.91g, and the lint percentage decreased from 41.42% to 37.55%. CRISPR/Cas9 gene editing technology was used to obtain different types of loss-of-function mutant lines in the UGT71C4 coding region sequence. It was found that the length and width of cotton seeds decreased, the seed index decreased from 10.66g to 8.60g, and the lint percentage increased from 41.42% to 43.40%. Observing the growth and development of ovules in cotton overexpressing and gene editing UGT71C4, it was found that the size of ovules at 15DPA has changed significantly, and the number of cells in the internal cross section of cotton ovules overexpressing UGT71C4 increased and the ovules were larger. However, the number of cells in the internal cross-section of the seeds of the UGT71C4 gene-edited strain is relatively reduced, and the ovules are smaller.

Combined transcriptomic and metabolomic analysis was performed to explore the changes in metabolite content and related gene expression in the 15DPA ovules of the overexpressed and gene-edited UGT71C4 strain. The results show that in overexpressing UGT71C4 transgenic ovules, flavonoid metabolites such as naringenin, chalcone, dihydroquercetin and dihydrokaempferol accumulated significantly in the phenylpropanoid metabolism pathway, and the key catalytic enzymes CHS, CHI, F3H and F3'H in the synthesis pathway were significantly up-regulated at the transcription level. Using in vitro protein purification technology and in vitro enzyme activity reactions to screen the metabolites that UGT71C4 protein may catalyze, it was found that UGT71C4 specifically catalyzes naringenin in vitro, which combines with the donor UDP-glucose to catalyze the formation of naringenin-7-O-glucoside. In addition, flavonoid glycoside metabolites such as naringenin-4'-O-glucoside, quercetin-4'-O-glucoside, kaempferol-3-O-arabinoside, scopolamine-7-O-glucoside, and quercetin-3'-O-glucoside also accumulated significantly. Genes related to cell proliferation are also significantly activated by the enlargement of seeds, and their expression levels are increased, promoting the expansion of ovule cells and forming larger seeds.

In the cotton ovules of the gene-edited UGT71C4 line, the expression levels of key catalytic enzymes in the lignin metabolism pathway, such as shikimate/quinic acid hydroxycinnamoyl transferase, caffeic acid/5-hydroxyferulic acid-3-O-methyltransferase, and cinnamoyl-CoA reductase, were significantly increased, and the expression of key transcription factors affecting lignin synthesis such as NST1/2, MYB46, MYB83 and other genes increased significantly, and the expression level of the portal enzyme shikimate/quinic acid hydroxycinnamoyl transferase of the lignin pathway increased significantly, and the corresponding lignin synthesis precursors such as p-coumarol, ferulic acid, etc. accumulated significantly, causing ectopic accumulation of lignin in the ovules, restricting the growth and development of the ovules, and causing the seeds to become smaller.

The above results show that overexpression of UGT71C4 can introduce phenylpropanoid metabolic flux into flavonoid metabolism, activate related gene expression, promote cell proliferation in seeds, and increase seed size. In the gene-edited UGT71C4 ovules, the phenylpropanoid metabolism pathway flows more metabolic flux into the lignin metabolism pathway, and the expression of enzymes related to lignin synthesis and key transcription factors for lignin synthesis is activated, increasing the ectopic deposition of lignin in the ovules, causing premature lignification of the ovules and limiting the growth and development of the ovules, resulting in smaller seeds after gene editing UGT71C4.

This study found that UDP-glucosyltransferase can regulate the internal metabolic flux distribution of phenylpropanoid metabolism, affect cotton ovule development, and change seed size. UGT71C4 may be a candidate gene for future research aimed at improving yield, and has important potential value in cotton seed development, cotton yield improvement, and comprehensive utilization of cotton seeds. This study provides new insights into seed size in cotton and other plants, laying the foundation for improving crop yields.

Reference

1. Cao, Y., et al. UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development. Plant Commun. 2024 Apr 30: 100938.

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