Promoter variations of ClERF1 gene determines flesh firmness in watermelon.

BACKGROUND: Flesh firmness is a critical factor that influences fruit storability, shelf-life and consumer's preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon. RESULTS: In this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxin-responsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis. CONCLUSIONS: Our results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding.

Allelic variations of ClACO gene improve nitrogen uptake via ethylene-mediated root architecture in watermelon.

KEY MESSAGE: The ClACO gene encoding 1-aminocyclopropane-1-carboxylate oxidase enabled highly efficient 15N uptake in watermelon. Nitrogen is one of the most essential nutrient elements that play a pivotal role in regulating plant growth and development for crop productivity. Elucidating the genetic basis of high nitrogen uptake is the key to improve nitrogen use efficiency for sustainable agricultural productivity. Whereas previous researches on nitrogen absorption process are mainly focused on a few model plants or crops. To date, the causal genes that determine the efficient nitrogen uptake of watermelon have not been mapped and remains largely unknown. Here, we fine-mapped the 1-aminocyclopropane-1-carboxylate oxidase (ClACO) gene associated with nitrogen uptake efficiency in watermelon via bulked segregant analysis (BSA). The variations in the ClACO gene led to the changes of gene expression levels between two watermelon accessions with different nitrogen uptake efficiencies. Intriguingly, in terms of the transcript abundance of ClACO, it was concomitant with significant differences in ethylene evolutions in roots and root architectures between the two accessions and among the different genotypic offsprings of the recombinant BC2F1(ZJU132)-18. These findings suggest that ethylene as a negative regulator altered nitrogen uptake efficiency in watermelon by controlling root development. In conclusion, our current study will provide valuable target gene for precise breeding of 'green' watermelon varieties with high-nitrogen uptake efficiencies.

An allelic variant in the ACS7 gene promotes primary root growth in watermelon.

KEY MESSAGE: Gene mining in a C. lanatus × C. amarus population revealed one gene, ACS7, linked to primary root elongation in watermelon. Watermelon is a xerophytic crop characterized by a long primary root and robust lateral roots. Therefore, watermelon serves as an excellent model for studying root elongation and development. However, the genetic mechanism underlying the primary root elongation in watermelon remains unknown. Herein, through bulk segregant analysis we identified a genetic locus, qPRL.Chr03, controlling primary root length (PRL) using two different watermelon species (Citrullus lanatus and Citrullus amarus) that differ in their root architecture. Fine mapping revealed that xaa-Pro dipeptidase and 1-aminocyclopropane-1-carboxylate synthase 7 (ACS7) are candidate regulators of the primary root growth. Allelic variation in the delimited region among 193 watermelon accessions indicated that the long-root alleles might only exist in C. amarus. Interestingly, the discrepancy in PRL among the C. amarus accessions was clearly associated with a nonsynonymous single nucleotide polymorphism variant within the ACS7 gene. The ACS7 expression and ethylene levels in the primary root tips suggested that ethylene is a negative regulator of root elongation in watermelon, as supported by the application of 1-aminocyclopropane-1-carboxylate (ACC, the ethylene precursor) or 2-aminoethoxyvinyl glycine (AVG, an ACS inhibitor). To the best of our knowledge, these findings provide the first description of the genetic basis of root elongation in watermelon. The detected markers of the ACS7 gene will facilitate marker-assisted selection for the PRL trait to improve water and nutrient use efficacy in watermelon and beyond.

The Novel Cucurbitaceae miRNA ClmiR86 Is Involved in Grafting-Enhanced Phosphate Utilization and Phosphate Starvation Tolerance in Watermelon.

Watermelon (Citrullus lanatus) is a globally important Cucurbitaceae crop in which grafting is commonly used to improve stress tolerance and enhance nutrient utilization. However, the mechanism underlying grafting-enhanced nutrient assimilation remains unclear. Here, we demonstrate the possible involvement of a novel Cucurbitaceae miRNA, ClmiR86, in grafting-enhanced phosphate-starvation tolerance via CALCINEURIN B-LIKE INTERACTING PROTEIN KINASE 5 (ClCIPK5) suppression in watermelon. Transcript analyses revealed that the induction of ClmiR86 expression was correlated with the downregulation of ClCIPK5 in squash-grafted watermelon under phosphate starvation. In addition, the differential expression of ClmiR86 in various watermelon genotypes was consistent with their phosphate utilization efficiency. Furthermore, ClmiR86 overexpression in Arabidopsis enhanced root growth and phosphate uptake under phosphate starvation and promoted inflorescence elongation under normal conditions. These results suggest that the ClmiR86-ClCIPK5 axis is involved in phosphate starvation response as well as grafting-enhanced growth vigor and phosphate assimilation. The present study provides valuable insights for investigating long-distance signaling and nutrient utilization in plants.