The plant-specific WRKY transcription factor family members have diverse regulatory effects on the genes associated with many plant processes. Although the WRKY proteins in Arabidopsis thaliana and other species have been thoroughly investigated, there has been relatively little research on the WRKY family in Luffa cylindrica, which is one of the most widely grown vegetables in China. In this study, we performed a genome-wide analysis to identify L. cylindrica WRKY genes, which were subsequently classified and examined in terms of their gene structures, chromosomal locations, promoter cis-acting elements, and responses to abiotic stress. A total of 62 LcWRKY genes (471–2238 bp) were identified and divided into three phylogenetic groups (I, II, and III), with group II further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) in accordance with the classification in other plants. The LcWRKY genes were unevenly distributed across 13 chromosomes. The gene structure analysis indicated that the LcWRKY genes contained 0–11 introns (average of 4.4). Moreover, 20 motifs were detected in the LcWRKY proteins with conserved motifs among the different phylogenetic groups. Two subgroup IIc members (LcWRKY16 and LcWRKY31) contained the WRKY sequence variant WRKYGKK. Additionally, nine cis-acting elements related to diverse responses to environmental stimuli were identified in the LcWRKY promoters. The subcellular localization analysis indicated that three LcWRKY proteins (LcWRKY43, LcWRKY7, and LcWRKY23) are localized in the nucleus. The tissue-specific LcWRKY expression profiles reflected the diversity in LcWRKY expression. The RNA-seq data revealed the effects of low-temperature stress on LcWRKY expression. The cold-induced changes in expression were verified via a qRT-PCR analysis of 24 differentially expressed WRKY genes. Both LcWRKY7 and LcWRKY12 were highly responsive to the low-temperature treatment (approximately 110-fold increase in expression). Furthermore, the LcWRKY8, LcWRKY12, and LcWRKY59 expression levels increased by more than 25-fold under cold conditions. Our findings will help clarify the evolution of the luffa WRKY family while also providing valuable insights for future studies on WRKY functions.

Plant-specific APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factors are involved in the regulation of genes associated with the growth and developmental processes of numerous plants. Although AP2/ERF proteins from other species have been intensively studied, no studies have been reported on the AP2/ERF family of Luffa cylindrica, an important vegetable of the cucurbit family, and one of the most popular vegetables in the world. In this study, 133 genes (315–6696 bp) encoding LcAP2/ERF proteins with complete AP2/ERF domains were identified according to the luffa P93075 genome. These LcAP2/ERF genes were subsequently classified and analyzed for their gene structures, chromosomal distribution locations, promoter cis-acting elements, conserved structural domains of encoded proteins, and responses to abiotic stresses. The LcAP2/ERF genes were identified and divided into five phylogenetic groups (AP2, DREBs, ERFs, RAV, and soloists). These genes were unevenly distributed across 13 chromosomes. An analysis of gene structures indicated the LcAP2/ERF genes contained 0–11 introns (average of 4.4). Additionally, 16 motifs were identified in the LcAP2/ERF proteins that were conserved across different phylogenetic groups. Moreover, 11 cis-acting elements associated with response to the environment were analyzed in a 2000 bp region upstream of the LcAP2/ERF gene promoters. A transcriptome analysis involving RNA-seq data revealed tissue-specific LcAP2/ERF expression profiles and the diversity in LcAP2/ERF expression. The effects of low-temperature stress on LcAP2/ERF expression were determined. Furthermore, fruit-development-related and low-temperature-induced expressional changes were verified by RT-qPCR analyses of 14 differentially expressed LcAP2/ERF genes in luffa. Our findings will help clarify the evolution of the luffa AP2/ERF family, while also providing valuable insights for future studies on AP2/ERF functions.

Cyclic nucleotide-gated ion channels (CNGCs) are cell membrane channel proteins for calcium ions. They have been reported to play important roles in survival and in the responses to environmental factors in various plants. However, little is known about the CNGC family and its functions in luffa (Luffa cylindrica L.). In this study, a bioinformatics-based method was used to identify members of the CNGC gene family in L. cylindrica. In total, 20 LcCNGCs were detected, and they were grouped into five subfamilies (I, II, Ⅲ, IV-a, and IV-b) in a phylogenetic analysis with CNGCs from Arabidopsis thaliana (20 AtCNGCs) and Momordica charantia (17 McCNGCs). The 20 LcCNGC genes were unevenly distributed on 11 of the 13 chromosomes in luffa, with none on Chromosomes 1 and 5. The members of each subfamily encoded proteins with highly conserved functional domains. An evolutionary analysis of CNGCs in luffa revealed three gene losses and a motif deletion. An examination of gene replication events during evolution indicated that two tandemly duplicated gene pairs were the primary driving force behind the evolution of the LcCNGC gene family. PlantCARE analyses of the LcCNGC promoter regions revealed various cis-regulatory elements, including those responsive to plant hormones (abscisic acid, methyl jasmonate, and salicylic acid) and abiotic stresses (light, drought, and low temperature). The presence of these cis-acting elements suggested that the encoded CNGC proteins may be involved in stress responses, as well as growth and development. Transcriptome sequencing (RNA-seq) analyses revealed tissue-specific expression patterns of LcCNGCs in various plant parts (roots, stems, leaves, flowers, and fruit) and the upregulation of some LcCNGCs under low-temperature stress. To confirm the accuracy of the RNA-seq data, 10 cold-responsive LcCNGC genes were selected for verification by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Under cold conditions, LcCNGC4 was highly upregulated (>50-fold increase in its transcript levels), and LcCNGC3, LcCNGC6, and LcCNGC13 were upregulated approximately 10-fold. Our findings provide new information about the evolution of the CNGC family in L. cylindrica and provide insights into the functions of the encoded CNGC proteins.

Floret color is a crucial phenotypic trait in broccoli, serving as an indicator of maturity and determining its market value. However, the mechanisms underlying color variation remain unclear. In this study, six broccoli varieties with different floret colors at harvest were chosen as materials. The color difference and pigment content of florets were measured, and a combined analysis of anthocyanin-targeted metabolome and transcriptome was conducted. Our findings revealed that chlorophyll a primarily influences green, yellow-green, and light green coloration, while the wax content may contribute to gray-green coloration. The blue-green and dark blue-green coloration are regulated by both chlorophyll a and anthocyanins. Targeted metabolomics identified five anthocyanin compounds, with peonidin-3-O-glucoside as a key metabolite for blue-green coloration and delphinidin-3-O-glucoside-5-O-galactoside and peonidin-3,5-O-diglucoside for dark blue-green coloration. Transcriptomic analysis identified CHLG as a potential key regulator for yellow-green and light-green floret coloration. The blue-green coloration appears to be coregulated by a combination of genes, including the chlorophyll biosynthesis gene HEMF; anthocyanin biosynthesis genes (PAL, FLS, and UGT); and chlorophyll degradation genes (SGR, PPD, and NYC). Furthermore, upstream genes involved in both chlorophyll metabolism (CHLI, CHLD, CHLM, DVR, and CLH) and anthocyanin biosynthesis (PAL, 4CL, CHS, F3′H, and FLS) play crucial roles in determining the dark blue-green coloration of florets. Meanwhile, transcription factors of the WRKY, NAC, and TCP families are involved in chlorophyll metabolism, while those of the bHLH and MYB families participate in anthocyanin synthesis. The WGCNA identified one Hub gene for chlorophyll metabolism and two for anthocyanin synthesis. In conclusion, 35 candidate genes were identified, including 21 involved in chlorophyll metabolism and 14 in anthocyanin biosynthesis. This study provides novel insights into the molecular mechanisms of floret coloration and establishes a foundation for molecular breeding in broccoli.

Luffa (Luffa acutangula) serves as an important edible, medicinal, and industrial crop. Sucrose synthase (SUS, EC 2.4.1.13) catalyzes sucrose metabolism and facilitates the entry of photosynthetically derived sucrose into metabolic pathways, playing crucial roles in plant growth, development, and stress responses. However, systematic investigations on the SUS gene family in luffa remain relatively scarce. In this study, we identified nine LaSUS family members distributed unevenly across six chromosomes. Their physicochemical properties and evolutionary relationships were systematically elucidated using bioinformatics tools. RNA-seq analysis revealed distinct expression patterns of LaSUS genes during luffa fruit aging, with most genes showing significant down-regulation during this process. Notably, several genes exhibited significant correlations with sucrose content during fruit aging. RT-qPCR analysis demonstrated the drought stress responsiveness of LaSUS genes, with LaSUS1 showing marked up-regulation under drought conditions. Furthermore, overexpression experiments in tobacco confirmed that LaSUS1 contributed to sugar accumulation, increased antioxidant enzyme activities, and positively regulated drought tolerance in luffa. This comprehensive study not only characterizes the LaSUS gene family and bridges the research gap of SUS genes in luffa but also provides theoretical support for investigating the roles of SUS genes in fruit ripening and abiotic stress responses in luffa.

The pyrabactin resistance 1-like (PYR/PYL) proteins are abscisic acid receptors that perform multiple functions in various plant growth and development processes. However, the PYR/PYL gene family in luffa (Luffa cylindrica L.) has not been well-explored. In this study, we analysed the effects of whole-genome member identification, endogenous soluble sugars (SS), soluble proteins (SP), abscisic acid (ABA), indole-3-acetic acid (IAA, auxin) and the gene expression pattern of PYR/PYL influenced by exogenous abscisic acid (ABA) during the fruit development of luffa through the use of physiological and biochemical analyses, bioinformatics, and RT-qPCR techniques. We conducted a comprehensive genome-wide identification and characterisation of the PYR/PYL gene family in luffa fruit development. Four LcPYR and 10 LcPYL genes were identified in the luffa reference genome via bioinformatics analyses. A chromosomal mapping of the identified LcPYR/PYL genes showed that they were distributed on 9 of the 13 chromosomes in the luffa genome. Conserved structural domain analyses of the 14 proteins encoded by the LcPYR/PYL genes identified the PYR_PYL_RCAR_like structural domains typical of this family; however, no regulatory component of abscisic acid receptor (RCAR)-type genes was found. At six luffa fruit development stages (i.e., 0, 3, 6, 9, 12, and 15 days after pollination), the contents of soluble sugars, soluble proteins, and endogenous hormones ABA and IAA in the fruit significantly increased. Under the exogenous ABA treatments, the contents of these four endogenous substances in the fruits were significantly higher than they were in the control group at the same time period, and ABA and IAA seemed to be synergistically involved in the luffa fruit-ripening process. An analysis of the luffa transcriptome data and real-time fluorescence quantitative PCR (RT-qPCR) experiments showed that multiple LcPYR/PYLs (e.g., LcPYL10 and LcPYR4) had differential expression levels in the seven different tissues and exogenous ABA-treated fruits that were analysed, suggesting their roles in ABA hormone-mediated ripening of luffa fruit. Together, the results provide basic information about the LcPYR/PYL family in L. cylindrica and their involvement in fruit development.