Selecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H₂O₂, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated thatEF-1αwas the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally,UBQexpression was stable following the salt treatment, whereasTUBwas identified as a suitable reference gene for H₂O₂and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

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.

Background: Virus-induced gene silencing (VIGS) is a reverse genetics technology that can efficiently and rapidly identify plant gene functions. Although a variety of VIGS vectors have been successfully used in plants, only a few reports on VIGS technology in Luffa exist. Results: In the present study, a new cucumber green mottle mosaic virus (CGMMV)-based VIGS vector, pV190, was applied to establish the CGMMV-VIGS to investigate the feasibility of the silencing system for Luffa. Phytoene desaturase (PDS) gene was initially selected as a VIGS marker gene to construct a recombinant vector. Plants infected with Agrobacterium harboring pV190-PDS successfully induced effective silencing in Luffa, and an effective gene silencing phenotype with obvious photobleaching was observed. To further validate the efficiency, we selected TEN for gene-silencing, which encodes a CYC/TB1-like transcription factor and is involved in tendril development. Luffa plants inoculated with the pV190-TEN exhibited shorter tendril length and nodal positions where tendrils appear are higher compared to those of non-inoculated plants. RT-qPCR showed that the expression levels of PDS and TEN were significantly reduced in the CGMMV-VIGS plants. Moreover, we evaluated the CGMMV-VIGS efficiency in three cucurbits, including cucumber, ridge gourd, and bottle gourd. Conclusion: We successfully established a CGMMV-based VIGS system on ridge gourd and used marker genes to identify the feasibility of the silencing system in Luffa leaves and stems.

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.

Cauliflower is one of the most important popular vegetables in China. Cauliflower (Brassica oleracea L. var. botrytis) produces glucosinolates, which are secondary metabolites that have anti-cancer properties. The choice of suitable reference ...