CONSTANS (CO) and CONSTANS-LIKE (COL) transcription factors are known to regulate a series of cellular processes, including the transition from vegetative growth to flower development in plants. However, their role in regulating the fruit chlorophyll content is poorly understood. In this study, SlCOL1, the tomato (Solanum lycopersicum) ortholog of Arabidopsis CONSTANS, was shown to play key roles in controlling fruit chlorophyll. The suppression of SlCOL1 expression led to a reduction in the chlorophyll content of immature green fruit, while the overexpression of SlCOL1 increased it. An analysis of protein–protein interactions indicated that SlCOL1 forms a complex with GOLDEN2-LIKE (GLK2), which promotes the stability of its protein. The overexpression of SlCOL1 in the glk2 null mutation background of tomato failed to promote chlorophyll accumulation in the immature green fruit, which suggests that GLK2 is required for the function of SlCOL1 in regulating chlorophyll content. These results shed new light on the mechanisms used by COL1 and GLK2 to regulate fruit development and chlorophyll accumulation in tomato.

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

Leaf-color mutations have been studied extensively in plants. However, to better understand the complex mechanisms underlying the formation of leaf color, it is essential to continue discover novel genes involved in the process of leaf color development. In this study, we identified a variegated-leaf (vg) mutant in tomato that exhibited defective phenotypes in thylakoids and photosynthesis. To clone the vg locus, an F₂ population was constructed from the cross between the vg mutant (Solanum lycopersicum) and the wild tomato LA1589 (S. pimpinellifolium). Using the map-based cloning approach, the vg locus was mapped on chromosome 7 and narrowed down to a 128 kb region that contained 21 open reading frames (ORFs). The expression levels of ORF9, ORF10, and ORF13 were significantly lower in vg than in the wild-type plants, while the ORF11 transcript level was elevated in vg. We then mutagenized ORF9, ORF10, and ORF13 by the CRISPR/Cas9 system in the wild-type tomato background and found that only the ORF10 mutation reproduced the phenotype of variegated leaves, indicating that ORF10 represents VG and its down-regulated expression was responsible for the variegated leaf phenotype. ORF10 encodes a thylakoid formation protein and its mutant lines showed reduced levels of chlorophyll synthesis and photosynthesis. Taken together, these results suggest that VG is necessary for chloroplast development, chlorophyll synthesis, and photosynthesis in tomato.

Cell cycle regulation plays a critical role in plant growth and development. In this study, the role of a tomato cell cycle gene SlCycB1 has been characterized. Expression analysis revealed that SlCycB1 was mostly expressed in stem, root, and leaves, with relative lower expression in flower and fruit. Tomato plants overexpressing SlCycB1 exhibited a reduction in cell number and increased cell size leading to the growth retardation. Furthermore, yeast two-hybrid analysis and bimolecular fluorescence complementation revealed that SlCycB1 interacted with histone H3.2, an essential component of the nucleosome. Histone H3.2 was transcriptionally up-regulated in the SlCycB1 overexpressing tomato lines. Furthermore, the overexpression of histone H3.2 in transgenic plants showed similar phenotypes to SlCycB1 overexpressing lines. Based on these findings, we concluded that SlCycB1 overexpression altered tomato architecture in association with histone H3.2.