Identification of WRKY Family Members and Characterization of the Low-Temperature-Stress-Responsive WRKY Genes in Luffa (Luffa cylindrica L.).

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.

Transcriptome and Metabolome Provide Insights into Fruit Ripening of Cherry Tomato (Solanum lycopersicum var. cerasiforme).

Insights into flavor formation during fruit ripening can guide the development of breeding strategies that balance consumer and producer needs. Cherry tomatoes possess a distinctive taste, yet research on quality formation is limited. Here, metabolomic and transcriptomic analyses were conducted on different ripening stages. The results revealed differentially accumulated metabolites during fruit ripening, providing candidate metabolites related to flavor. Interestingly, several key flavor-related metabolites already reached a steady level at the mature green stage. Transcriptomic analysis revealed that the expression levels of the majority of genes tended to stabilize after the pink stage. Enrichment analysis demonstrated that changes in metabolic and biosynthetic pathways were evident throughout the entire process of fruit ripening. Compared to disease resistance and fruit color genes, genes related to flavor and firmness may have a broader impact on the accumulation of metabolites. Furthermore, we discovered the interconversion patterns between glutamic acid and glutamine, as well as the biosynthesis patterns of flavonoids. These findings contribute to our understanding of fruit quality formation mechanisms and support breeding programs aimed at improving fruit quality traits.

The reference genome and full-length transcriptome of pakchoi provide insights into cuticle formation and heat adaption.

Brassica rapa includes various vegetables with high economic value. Among them, green petiole type pakchoi (B. rapa ssp. chinensis) is one of the major vegetables grown in southern China. Compared with other B. rapa varieties, green petiole type pakchoi shows a higher level of heat resistance, which is partially derived from the rich epicuticular wax. Here we sequence a high-quality genome of green petiole type pakchoi, which has been widely used as the parent in breeding. Our results reveal that long terminal repeat retrotransposon insertion plays critical roles in promoting the genome expansion and transcriptional diversity of pakchoi genes through preferential insertions, particularly in cuticle biosynthetic genes. After whole-genome triplication, over-retained pakchoi genes escape stringent selection pressure, and among them a set of cuticle-related genes are retained. Using bulked-segregant analysis of a heat-resistant pakchoi cultivar, we identify a frame-shift deletion across the third exon and the subsequent intron of BrcCER1 in candidate regions. Using Nanopore long-read sequencing, we analyze the full-length transcriptome of two pakchoi cultivars with opposite sensitivity to high temperature. We find that the heat-resistant pakchoi cultivar can mitigate heat-caused leaf damage by activating an unfolded protein response, as well as by inhibiting chloroplast development and energy metabolism, which are presumably mediated by both transcriptional regulation and splicing factors. Our study provides valuable resources for Brassica functional genomics and breeding research, and deepens our understanding of plant stress resistance.

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions.

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 that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and 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.

Whole-genome sequencing provides insights into the genetic diversity and domestication of bitter gourd (Momordica spp.).

Bitter gourd (Momordica charantia) is a popular cultivated vegetable in Asian and African countries. To reveal the characteristics of the genomic structure, evolutionary trajectory, and genetic basis underlying the domestication of bitter gourd, we performed whole-genome sequencing of the cultivar Dali-11 and the wild small-fruited line TR and resequencing of 187 bitter gourd germplasms from 16 countries. The major gene clusters (Bi clusters) for the biosynthesis of cucurbitane triterpenoids, which confer a bitter taste, are highly conserved in cucumber, melon, and watermelon. Comparative analysis among cucurbit genomes revealed that the Bi cluster involved in cucurbitane triterpenoid biosynthesis is absent in bitter gourd. Phylogenetic analysis revealed that the TR group, including 21 bitter gourd germplasms, may belong to a new species or subspecies independent from M. charantia. Furthermore, we found that the remaining 166 M. charantia germplasms are geographically differentiated, and we identified 710, 412, and 290 candidate domestication genes in the South Asia, Southeast Asia, and China populations, respectively. This study provides new insights into bitter gourd genetic diversity and domestication and will facilitate the future genomics-enabled improvement of bitter gourd.

A RAD-Based Genetic Map for Anchoring Scaffold Sequences and Identifying QTLs in Bitter Gourd (Momordica charantia).

Genetic mapping is a basic tool necessary for anchoring assembled scaffold sequences and for identifying QTLs controlling important traits. Though bitter gourd (Momordica charantia) is both consumed and used as a medicinal, research on its genomics and genetic mapping is severely limited. Here, we report the construction of a restriction site associated DNA (RAD)-based genetic map for bitter gourd using an F2 mapping population comprising 423 individuals derived from two cultivated inbred lines, the gynoecious line 'K44' and the monoecious line 'Dali-11.' This map comprised 1,009 SNP markers and spanned a total genetic distance of 2,203.95 cM across the 11 linkage groups. It anchored a total of 113 assembled scaffolds that covered about 251.32 Mb (85.48%) of the 294.01 Mb assembled genome. In addition, three horticulturally important traits including sex expression, fruit epidermal structure, and immature fruit color were evaluated using a combination of qualitative and quantitative data. As a result, we identified three QTL/gene loci responsible for these traits in three environments. The QTL/gene gy/fffn/ffn, controlling sex expression involved in gynoecy, first female flower node, and female flower number was detected in the reported region. Particularly, two QTLs/genes, Fwa/Wr and w, were found to be responsible for fruit epidermal structure and white immature fruit color, respectively. This RAD-based genetic map promotes the assembly of the bitter gourd genome and the identified genetic loci will accelerate the cloning of relevant genes in the future.

De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica 'Fusi-3' fruits.

Fresh-cut luffa (Luffa cylindrica) fruits commonly undergo browning. However, little is known about the molecular mechanisms regulating this process. We used the RNA-seq technique to analyze the transcriptomic changes occurring during the browning of fresh-cut fruits from luffa cultivar 'Fusi-3'. Over 90 million high-quality reads were assembled into 58,073 Unigenes, and 60.86% of these were annotated based on sequences in four public databases. We detected 35,282 Unigenes with significant hits to sequences in the NCBInr database, and 24,427 Unigenes encoded proteins with sequences that were similar to those of known proteins in the Swiss-Prot database. Additionally, 20,546 and 13,021 Unigenes were similar to existing sequences in the Eukaryotic Orthologous Groups of proteins and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Furthermore, 27,301 Unigenes were differentially expressed during the browning of fresh-cut luffa fruits (i.e., after 1-6 h). Moreover, 11 genes from five gene families (i.e., PPO, PAL, POD, CAT, and SOD) identified as potentially associated with enzymatic browning as well as four WRKY transcription factors were observed to be differentially regulated in fresh-cut luffa fruits. With the assistance of rapid amplification of cDNA ends technology, we obtained the full-length sequences of the 15 Unigenes. We also confirmed these Unigenes were expressed by quantitative real-time polymerase chain reaction analysis. This study provides a comprehensive transcriptome sequence resource, and may facilitate further studies aimed at identifying genes affecting luffa fruit browning for the exploitation of the underlying mechanism.