Self-incompatibility (SI) is an intraspecific reproductive barrier widely present in angiosperms. The SI system with the broadest occurrence in angiosperms is based on an S-RNase linked to a cluster of multiple S-locus F-box (SLF) genes found in the Solanaceae, Plantaginaceae, Rosaceae, and Rutaceae. Recent studies reveal that non-self S-RNase is degraded by the Skip Cullin F-box (SCF)^SLF-mediated ubiquitin–proteasome system in a collaborative manner in Petunia, but how self-RNase functions largely remains mysterious. Here, we show that S-RNases form S-RNase condensates (SRCs) in the self-pollen tube cytoplasm through phase separation and the disruption of SRC formation breaks SI in self-incompatible Petunia hybrida. We further find that the pistil SI factors of a small asparagine-rich protein HT-B and thioredoxin h together with a reduced state of the pollen tube all promote the expansion of SRCs, which then sequester several actin-binding proteins, including the actin polymerization factor PhABRACL, the actin polymerization activity of which is reduced by S-RNase in vitro. Meanwhile, we find that S-RNase variants lacking condensation ability fail to recruit PhABRACL and are unable to induce actin foci formation required for pollen tube growth inhibition. Taken together, our results demonstrate that phase separation of S-RNase promotes SI response in P. hybrida, revealing a new mode of S-RNase action.

Pollen hydration, germination, and tube growth are vital processes for the successful fertilization of flowering plants. These processes involve complex signaling pathways. Reactive oxygen species (ROS) generated in apoplast involves signaling for the cell wall expansion during tube growth, however molecular regulators are less known. We identified pollen-specific receptor-like kinase (OsPRK) family genes from rice (Oryza sativa), which have conserved leucine-rich repeat (LRR) and kinase domains. To understand the function of these genes, we produced single and triple mutations for OsPRK1, OsPRK2, and OsPRK3 using the clustered regularly interspaced palindromic repeats (CRISPR/Cas9) system. Among these mutants, triple knockout (KO) lines (osprk1/2/3) exhibited the male-sterile phenotype with normal vegetative growth and floret formation. Through cytological analysis, we confirmed that the reduced seed fertility was due to defects in pollen hydration and germination with low ROS accumulation. This defect of pollen germination was partially recovered by treatment with exogenous H₂O₂. We also confirmed that OsPRKs could interact with the LRR extension protein. Our results suggest that rice PRKs redundantly play a role in ROS signaling for pollen hydration and germination, and fertility can be controlled by exogenous application.

Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.

Sucrose functions as a signaling molecule in several metabolic pathways as well as in various developmental processes. However, the molecular mechanisms by which sucrose regulates these processes remain largely unknown. In the present study, we demonstrate that sucrose promotes flowering by mediating the stability of a regulatory protein that represses flowering in rice. Exogenous application of sucrose promoted flowering by inducing florigen gene expression. Reduction of sucrose levels in the phloem through genetic modifications, such as the overexpression of the vacuolar invertase OsVIN2 or the mutation of OsSUT2, a sucrose transporter, delayed flowering. Analysis of relative transcript levels of floral regulatory genes showed that sucrose activated Ehd1 upstream of the florigen, with no significant effect on the expression of other upstream genes. Examination of protein stability after sucrose treatment of major floral repressors revealed that the Ghd7 protein was specifically degraded. The Ghd7 protein interacted with the E3 ligase IPA INTERACTING PROTEIN1 (IPI1), and sucrose-induced K48-linked polyubiquitination of Ghd7 via IPI1, leading to protein degradation. Mutants defective in IPI1 delayed flowering, confirming its role in modulating proteins involved in flowering. We conclude that sucrose acts as a signaling molecule to induce flowering by promoting Ghd7 degradation via IPI1.

Flowering time and growth period are key agronomic traits which directly affect soybean (Glycine max (L.) Merr.) adaptation to diverse latitudes and farming systems. The FLOWERING LOCUS T (FT) homologs GmFT2a and GmFT5a integrate multiple flowering regulation pathways and significantly advance flowering and maturity in soybean. Pinpointing the genes responsible for regulating GmFT2a and GmFT5a will improve our understanding of the molecular mechanisms governing growth period in soybean. In this study, we identified the Nuclear Factor Y-C (NFY-C) protein GmNF-YC4 as a novel flowering suppressor in soybean under long-day (LD) conditions. GmNF-YC4 delays flowering and maturation by directly repressing the expression of GmFT2a and GmFT5a. In addition, we found that a strong selective sweep event occurred in the chromosomal region harboring the GmNF-YC4 gene during soybean domestication. The GmNF-YC4^Hap3 allele was mainly found in wild soybean (Glycine soja Siebold & Zucc.) and has been eliminated from G. max landraces and improved cultivars, which predominantly contain the GmNF-YC4^Hap1 allele. Furthermore, the Gmnf-yc4 mutants displayed notably accelerated flowering and maturation under LD conditions. These alleles may prove to be valuable genetic resources for enhancing soybean adaptability to higher latitudes.

Maize (Zea mays subspecies mays) is an important commercial crop across the world, and its flowering time is closely related to grain yield, plant cycle and latitude adaptation. FKF1 is an essential clock-regulated blue-light receptor with distinct functions on flowering time in plants, and its function in maize remains unclear. In this study, we identified two FKF1 homologs in the maize genome, named ZmFKF1a and ZmFKF1b, and indicated that ZmFKF1a and ZmFKF1b independently regulate reproductive transition through interacting with ZmCONZ1 and ZmGI1 to increase the transcription levels of ZmCONZ1 and ZCN8. We demonstrated that ZmFKF1b underwent artificial selection during modern breeding in China probably due to its role in geographical adaptation. Furthermore, our data suggested that ZmFKF1b^Hap_C7 may be an elite allele, which increases the abundance of ZmCONZ1 mRNA more efficiently and adapt to a wider range of temperature zone than that of ZmFKF1b^Hap_Z58 to promote maize floral transition. It extends our understanding of the genetic diversity of maize flowering. This allele is expected to be introduced into tropical maize germplasm to enrich breeding resources and may improve the adaptability of maize at different climate zones, especially at temperate region.

Plants monitor daylength to synchronize their flowering time with their surroundings and thus maximize reproductive fitness. In Arabidopsis (Arabidopsis thaliana), CONSTANS (CO) activates the expression of FLOWERING LOCUS T (FT); this activation is a crucial aspect of the daylength-dependent regulation of flowering time. Here, we demonstrate that the basic leucine zipper 3 (bZIP3) transcription factor is important for CO-induced FT expression under long photoperiod conditions in Arabidopsis. We isolated bZIP3 as a CO-interacting protein by yeast two-hybrid screening and verified bZIP3–CO complex formation in Arabidopsis through co-immunoprecipitation assays. The temporal and spatial expression patterns of bZIP3 are very similar to those of CO, and bZIP3 protein levels fluctuate throughout the day, with high abundance in the late afternoon. The bzip3 mutant displayed delayed flowering under long photoperiods, whereas bZIP3 overexpression accelerated flowering regardless of daylength. bZIP3 directly binds to the FT promoter region containing CO-responsive elements in vivo. FT messenger RNA (mRNA) levels in the bzip3 mutant and bZIP3 overexpression lines correlated with their flowering times and changed only during the daytime. bZIP3 overexpression resulted in significantly lower FT transcript levels in the co mutant background than in the wild type. Furthermore, bZIP3 forms a complex with ASYMMETRIC LEAVES1 (AS1), a CO partner that helps CO induce FT expression. The bzip3 as1 double mutant flowered later than the two single mutants under longer daylengths, and FT mRNA levels were much lower in the double mutant than in the bzip3 single mutant. Collectively, our findings uncover a new layer of photoperiod-dependent FT regulation in which bZIP3 facilitates CO to activate FT transcription by forming a complex with AS1.

Flowering time is a crucial rice trait that influences its adaptation to various environments, cropping schedules, and agronomic characteristics. Rice breeders have exploited spontaneous mutations in heading date genes to regulate the flowering time. In the present study, we investigated how breeders in Fukui Prefecture regulated days to heading while developing promising rice varieties. Genome-wide association studies (GWAS) identified Hd1, Hd16, and Hd18 as the major genes controlling days to heading in the population. However, we suspected that this highly bred population might exhibit genomic stratification, which could lead to spurious or false correlations in the GWAS. Thus, we also conducted correlation and partial correlation analyses, which uncovered another key heading date gene, Hd17, that GWAS failed to detect because of its linkage disequilibrium with the major effect gene Hd16. Examination of haplotype frequencies across different breeding periods revealed that the early-heading Hd16 (Hd16(E)) and late-heading Hd17 (Hd17(L)) were increasingly co-selected in the Hd1 functional population. Varieties carrying this Hd16(E)/Hd17(L) combination exhibited days to heading in the range of 70–80, which corresponds to the peak temperature and sunshine period and is also optimal for grain quality and yield components in the Fukui environment. The present study highlights that it is imperative to remain vigilant for Type I (false positives) and Type II (false negatives) errors when performing GWAS on highly bred populations and to implement appropriate countermeasures by accounting for gene-by-gene interactions established through the breeding process. We also discuss the effectiveness of Hd16(E), which is not used outside Japan for subtle days to heading control but is widely used in Japan at certain latitudes.