Watermelon (Citrullus lanatus) is the world\u2019s third-largest fruit crop, and commercial cultivation relies heavily on F1 hybrids because of their superior yield, quality, and adaptability. Male-sterile lines can simplify hybrid seed production by preventing self-pollination. The tapetum, the innermost cell layer of the anther, supplies nutrients and materials required for pollen-wall formation before undergoing programmed cell death. Although regulatory pathways controlling these processes have been characterized in Arabidopsis, rice, and maize, their operation in insect-pollinated cucurbit crops remains poorly understood. In particular, researchers have lacked a clear molecular explanation of how watermelon coordinates tapetal development, tetrad-wall degradation, pollen-wall construction, and formation of the lipid-rich adhesive pollen coat required for insect-mediated pollination.<\/p>\n
A study (DOI: 10.48130\/seedbio-0026-0020<\/a>) published in Seed Biology <\/a>on 09 September 2026 by Li Yuan’s team, Northwest A&F University, demonstrates that the ClMS188\u2013ClMS1L1 regulatory module is indispensable for male fertility.<\/p>\n The researchers first examined the expression and cellular localization of ClMS188 and ClMS1L1 using quantitative real-time PCR, RNA in situ hybridization, and fluorescence microscopy. Both transcription factors localized to the nucleus and were expressed predominantly in developing anthers: ClMS188 expression peaked in tapetal cells at the tetrad stage, or stage 8b, whereas ClMS1L1 peaked at stage 9, when microspores are released. The team then used CRISPR\/Cas9 editing to generate three independent knockout lines for each gene. All clms188 and clms1l1 lines were completely male-sterile, producing indehiscent anthers without viable pollen. Semi-thin anther sections revealed that abnormalities in clms188 began at stage 8b, including malformed tetrads, shrinking microspores, and enlarged tapetal cells. Defects in clms1l1 appeared later, at stage 9, when microspores became deformed and the tapetum expanded abnormally. Dual-luciferase assays showed that ClMS188 activated the ClMS1L1 promoter. Yeast one-hybrid and electrophoretic mobility shift assays further demonstrated that ClMS188 binds specifically to a TAACCA motif within that promoter, establishing ClMS1L1 as a direct downstream target. Comparative RNA sequencing of wild-type and mutant anthers identified 1,875 differentially expressed genes in clms188 and 2,266 in clms1l1, with 710 shared between the mutants. Both mutations altered hormone-signaling and phenylpropanoid-biosynthesis pathways, but they also produced distinct molecular effects. ClMS188 influenced genes responsible for tetrad-wall degradation and the synthesis and transport of sporopollenin precursors, which form the durable pollen exine. ClMS1L1 exerted a stronger influence on lipid metabolism and genes involved in producing pollen-coat precursors. These results indicate that ClMS188 operates through two connected routes: one controls tetrad separation and sporopollenin production independently of ClMS1L1, while the other activates ClMS1L1 to promote formation of the pollen coat.<\/p>\n Together, the findings extend a conserved anther-development pathway into watermelon while revealing how it has been adapted to support the specialized pollen coat of an insect-pollinated crop. The clean male-sterile phenotypes of the edited lines make ClMS188 and ClMS1L1 promising targets for breeding systems designed to maintain and propagate male sterility. With further development, targeted editing of this module could support scalable hybrid seed production, lower breeding costs, and accelerate the introduction of desirable traits into commercial watermelon varieties.<\/p>\n ###<\/p>\n References<\/p>\n DOI<\/p>\n 10.48130\/seedbio-0026-0020<\/a><\/p>\n Original Source URL<\/p>\n https:\/\/doi.org\/10.48130\/seedbio-0026-0020<\/a><\/p>\n Funding information<\/p>\n This work was supported by the Key Program of the National Natural Science Foundation of China (U23A20208), the National Natural Science Foundation of China (32573035), the Young Scientists Fund of the National Natural Science Foundation of China (3250181085), the National Key Research and Development Program of China (2023YFE0206900), the Central Guidance on Local Science and Technology Development Fund of Xinjiang Province (ZYYD2024ZY02), and the Earmarked Fund for China Agriculture Research System (CARS-25).<\/p>\n About Seed Biology<\/a><\/p>\n