Genome-wide analysis of respiratory burst oxidase homolog gene family in pea (Pisum sativum L.).

Plant respiratory burst oxidase homologs (RBOHs) are key enzymes regulating superoxide production, which is important for plant development and responses to biotic and abiotic stresses. This study aimed to characterize the RBOH gene family in pea (Pisum sativum L.). Seven PsRBOH genes were identified in the pea genome and were phylogenetically clustered into five groups. Collinearity analyses of the RBOHs identified four pairs of orthologs between pea and soybean. The gene structure analysis showed that the number of exons ranged from 6 to 16. Amino acid sequence alignment, conserved domain, and conserved motif analyses showed that all seven PsRBOHs had typical features of plant RBOHs. The expression patterns of PsRBOH genes in different tissues provided suggested their roles in plant growth and organ development. In addition, the expression levels of PsRBOH genes under different abiotic stresses were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that PsRBOH genes exhibited unique stress-response characteristics, which allowed for functional diversity in response to different abiotic stresses. Furthermore, four PsRBOHs had a high probability of localization in the plasma membrane, and PsRBOH6 was localized to the plasma membrane and endoplasmic reticulum. The results of this study provide valuable information for further functional analysis of pea RBOH genes and their role in plant adaptation to climate-driven environmental constraints.

Candidate regulators of drought stress in tomato revealed by comparative transcriptomic and proteomic analyses.

Drought is among the most common abiotic constraints of crop growth, development, and productivity. Integrating different omics approaches offers a possibility for deciphering the metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. Here, we explored the transcriptional and post-transcriptional changes in drought-stressed tomato plants using transcriptomic and proteomic profiles to determine the molecular dynamics of tomato drought stress responses. We identified 22467 genes and 5507 proteins, among which the expression of 3765 genes and 294 proteins was significantly changed under drought stress. Furthermore, the differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) showed a good correlation (0.743). The results indicated that integrating different omics approaches is promising in exploring the multilayered regulatory mechanisms of plant drought resistance. Gene ontology (GO) and pathway analysis identified several GO terms and pathways related to stress resistance, including response to stress, abiotic stimulus, and oxidative stress. The plant hormone abscisic acid (ABA) plays pivotal roles in response to drought stress, ABA-response element binding factor (AREB) is a key positive regulator of ABA signaling. Moreover, our analysis indicated that drought stress increased the abscisic acid (ABA) content, which activated AREB1 expression to regulate the expression of TAS14, GSH-Px-1, and Hsp, ultimately improving tomato drought resistance. In addition, the yeast one-hybrid assay demonstrated that the AREB1 could bind the Hsp promoter to activate Hsp expression. Thus, this study involved a full-scale analysis of gene and protein expression in drought-stressed tomato, deepening the understanding of the regulatory mechanisms of the essential drought-tolerance genes in tomato.

Comprehensive Analysis to Reveal Amino Acid Metabolism-Associated Genes as a Prognostic Index in Gastric Cancer.

Background: Amino acid metabolism (AAM) is related to tumor growth, prognosis, and therapeutic response. Tumor cells use more amino acids with less synthetic energy than normal cells for rapid proliferation. However, the possible significance of AAM-related genes in the tumor microenvironment (TME) is poorly understood. Methods: Gastric cancer (GC) patients were classified into molecular subtypes by consensus clustering analysis using AAMs genes. AAM pattern, transcriptional patterns, prognosis, and TME in distinct molecular subtypes were systematically investigated. AAM gene score was built by least absolute shrinkage and selection operator (Lasso) regression. Results: The study revealed that copy number variation (CNV) changes were prevalent in selected AAM-related genes, and most of these genes exhibited a high frequency of CNV deletion. Three molecular subtypes (clusters A, B, and C) were developed based on 99 AAM genes, which cluster B had better prognosis outcome. We developed a scoring system (AAM score) based on 4 AAM gene expressions to measure the AAM patterns of each patient. Importantly, we constructed a survival probability prediction nomogram. The AAM score was substantially associated with the index of cancer stem cells and sensitivity to chemotherapy intervention. Conclusion: Overall, we detected prognostic AAM features in GC patients, which may help define TME characteristics and explore more effective treatment approaches.

Metabolic and molecular mechanisms underlying the foliar Zn application induced increase of 2-acetyl-1-pyrroline conferring the 'taro-like' aroma in pumpkin leaves.

Introduction: Fresh pumpkin leaf is popular vegetable for its rich nutrition. The pleasant taro-like odour is important aroma quality of crops, and mostly contributed by 2-acetyl-1-pyrroline in pumpkin. Element Zn can impact metabolite biosynthesis in plants, including aroma formation. However, Zn-induced biochemical responses, especially 2-acetyl-1-pyrroline formation in pumpkin, haven't been elucidated. Methods: This study integrated metabolome and transcriptome to explore molecular fluctuations in pumpkin leaves at different time intervals after foliar Zn treatment. Result and Discussion: We first identified more than one thousand metabolites from pumpkin leaves by integrating different mass spectrometry methods according to the form in which a metabolite exists. Comparative metabolomic analysis revealed there were separately 25 out of 50 and 286 out of 963 metabolites that were respectively identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, differentially regulated by Zn treatment. Our findings revealed that 50mg/L of Zn significantly enhanced 2-acetyl-1-pyrroline production by more than 38%, which was contributed by increased biosynthesis of its precursors, including ornithine and proline. The following transcriptome analysis discovered 30,574 genes, including 953 novel genes. Zn treatment induced the differential expression of 41.6% of identified genes which were supposed to regulate the downstream metabolite changes in a time-dependent manner. Pathway analysis indicated that alternations in primary metabolism, including carbon metabolism and biosynthesis of amino acids, were vital to the fluctuated aromatic compound generation. Phytohormones and transcription factors may regulate the expression of gene P5CS and proline biosynthesis, which, therefore, affect 2-acetyl-1-pyrroline production. This research reveals molecular mechanisms of 2-acetyl-1-pyrroline formation in pumpkin, which will provide the molecular basis for desired aroma compound production through metabolite engineering.

QTL mapping reveals candidate genes for main agronomic traits in Luffa based on a high-resolution genetic map.

Luffa is an important medicinal and edible vegetable crop of Cucurbitaceae. Strong heterosis effects and strikingly complementary characteristics were found between the two domesticated Luffa cultivars, Luffa acutangula and Luffa cylindrica. To explore the genetic basis underlying their important agronomic traits, we constructed the first interspecific high-density genetic linkage map using a BC1 population of 110 lines derived from a cross between S1174 (Luffa acutangula) and P93075 (Luffa cylindrica). The map spanned a total of 2246.74 cM with an average distance of 0.48 cM between adjacent markers. Thereafter, a large-scale field-based quantitative trait loci (QTLs) mapping was conducted for 25 important agronomic traits and 40 significant genetic loci distributed across 11 chromosomes were detected. Notably, a vital QTL (qID2) located on chromosome 9 with a minimum distance of 23 kb was identified to be responsible for the internode diameter and explained 11% of the phenotypic variation. Lac09g006860 (LacCRWN3), encoding a nuclear lamina protein involved in the control of nuclear morphology, was the only gene harbored in qID2. Sequence alignment showed completely different promoter sequences between the two parental alleles of LacCRWN3 except for some nonsynonymous single nucleotide polymorphisms (SNPs) in exons, and the expression level in thick-stem P93075 was distinctively higher than that in thin-stem S1174. According to the natural variation analysis of a population of 183 inbred lines, two main haplotypes were found for LacCRWN3: the P93075-like and S1174-like, with the former haplotype lines exhibiting significantly thicker internode diameters than those of the latter haplotype lines. It showed that LacCRWN3, as the only CRWN3 gene in Cucurbitaceae, was the most likely candidate gene regulating the internode diameter of Luffa. Our findings will be beneficial for deciphering the molecular mechanism of key phenotypic traits and promoting maker-assisted breeding in Luffa.

Metabolome and Transcriptome Analyses of Cucurbitacin Biosynthesis in Luffa (Luffa acutangula).

Cucurbitacins are extremely bitter compounds mainly present in Cucurbitaceae, where Luffa belongs. However, there is no comprehensive analysis of cucurbitacin biosynthesis in Luffa fruit. Therefore, this study analyzed bitter (WM709) and non-bitter (S1174) genotypes of Luffa to reveal the underlying mechanism of cucurbitacin biosynthesis by integrating metabolome and transcriptome analyses. A total of 422 metabolites were detected, including vitamins, essential amino acids, antioxidants, and antitumor substances. Of these, 131 metabolites showed significant differences between bitter (WM709) and non-bitter (S1174) Luffa fruits. The levels of isocucurbitacin B, cucurbitacin D, 23,24-dihydro cucurbitacin E, cucurbitacin F were significantly higher in bitter than in non-bitter Luffa. Transcriptome analysis showed that Bi, cytochromes P450s (CYP450s), and acyltransferase (ACT) of the cucurbitacin biosynthesis pathway, were significantly up-regulated. Moreover, drought stress and abscisic acid (ABA) activated genes of the cucurbitacin biosynthesis pathway. Furthermore, dual-luciferase reporter and yeast one-hybrid assays demonstrated that ABA-response element binding factor 1 (AREB1) binds to the Bi promoter to activate Bi expression. Comparative analysis of the Luffa and cucumber genomes showed that Bi, CYP450s, and ACT are located in the conserved syntenic loci, and formed a cucurbitacin biosynthesis cluster. This study provides important insights into major genes and metabolites of the cucurbitacin biosynthetic pathway, deepening the understanding of regulatory mechanisms of cucurbitacin biosynthesis in Luffa.

Identification of suitable reference genes for quantitative reverse transcription PCR in Luffa (Luffa cylindrica).

Reverse transcription real-time quantitative PCR is widely used to quantify gene expression. Reference genes are usually used as internal controls to measure the target gene expression level. To date, there is no consensus on the use of systematically validated reference genes in different tissues of Luffa. This study evaluated the expression stability of 11 candidate reference genes in different tissues using five algorithms (BestKeeper, comparative delta-Ct method, GeNorm, NormFinder, and RefFinder). Protein phosphatase 2A was the most stable gene, while alpha Tubulin was the least stable. The relative expression of ethylene-related genes in different tissues was also analyzed to reveal their role in sex determination. This study provides the basis for using suitable reference genes to evaluate targeted gene expression. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01182-8.

A mutation in LacDWARF1 results in a GA-deficient dwarf phenotype in sponge gourd (Luffa acutangula).

KEY MESSAGE: A dwarfism gene LacDWARF1 was mapped by combined BSA-Seq and comparative genomics analyses to a 65.4 kb physical genomic region on chromosome 05. Dwarf architecture is one of the most important traits utilized in Cucurbitaceae breeding because it saves labor and increases the harvest index. To our knowledge, there has been no prior research about dwarfism in the sponge gourd. This study reports the first dwarf mutant WJ209 with a decrease in cell size and internodes. A genetic analysis revealed that the mutant phenotype was controlled by a single recessive gene, which is designated Lacdwarf1 (Lacd1). Combined with bulked segregate analysis and next-generation sequencing, we quickly mapped a 65.4 kb region on chromosome 5 using F2 segregation population with InDel and SNP polymorphism markers. Gene annotation revealed that Lac05g019500 encodes a gibberellin 3ß-hydroxylase (GA3ox) that functions as the most likely candidate gene for Lacd1. DNA sequence analysis showed that there is an approximately 4 kb insertion in the first intron of Lac05g019500 in WJ209. Lac05g019500 is transcribed incorrectly in the dwarf mutant owing to the presence of the insertion. Moreover, the bioactive GAs decreased significantly in WJ209, and the dwarf phenotype could be restored by exogenous GA3 treatment, indicating that WJ209 is a GA-deficient mutant. All these results support the conclusion that Lac05g019500 is the Lacd1 gene. In addition, RNA-Seq revealed that many genes, including those related to plant hormones, cellular process, cell wall, membrane and response to stress, were significantly altered in WJ209 compared with the wild type. This study will aid in the use of molecular marker-assisted breeding in the dwarf sponge gourd.

Dysregulated Expression Levels of Circular RNAs Serve as Prognostic and Clinicopathological Markers in Gastric Cancer: a Meta-Analysis.

BACKGROUND: Circular RNAs (circRNAs) are novel biomarkers that are widely investigated in various cancers. There is increasing evidence that the expression levels of circRNAs are upregulated or downregulated in various cancers, but the overall prognostic efficiency of circRNAs in gastric cancer (GC) remains unclear. Therefore, this meta-analysis studies the relationship between circRNA expression and the prognosis of gastric malignancies. METHODS: A systematic search was conducted in the PubMed, Web of Science (WOS), EMBASE, and Cochrane Library databases. Eligible studies reporting on the associations of circRNAs with the clinicopathological characteristics and prognosis of GC patients were included. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were utilized to assess clinicopathological parameters. Hazard ratios (HRs) and 95% CIs were used to evaluate the prognostic value of circRNAs using RevMan 5.3 and Stata 15.1 software. RESULTS: Fifteen eligible studies, including 13 for clinicopathological features and 15 for prognosis, were included in our study. For clinicopathological parameters, the high expression of oncogenic circRNAs was significantly associated with poor clinicopathological features, and the high expression of tumor-suppressor circRNAs was associ-ated with better clinicopathological features. In terms of prognostic value, oncogenic circRNAs had a negative influence on overall survival (OS: HR = 2.27, 95% CI: 1.93 - 2.69), and the high expression of tumor suppressor circRNAs was related to improved survival outcomes (OS: HR = 0.56, 95% Cl: 0.44 - 0.72). CONCLUSIONS: This meta-analysis revealed that the expression of circRNAs might be a useful prognostic biomarker in gastric cancer.

A high-quality sponge gourd (Luffa cylindrica) genome.

Sponge gourd (Luffa cylindrica) is an important cultivated vegetable and medicinal plant in the family Cucurbitaceae. In this study, a draft genome sequence of the sponge gourd inbred line P93075 was analyzed. Using Illumina, PacBio, and 10× Genomics sequencing techniques as well as new assembly techniques such as FALCON and chromatin interaction mapping (Hi-C), a chromosome-scale genome of approximately 656.19 Mb, with an N50 scaffold length of 48.76 Mb, was generated. From this assembly, 25,508 protein-coding gene loci were identified, and 63.81% of the whole-genome consisted of transposable elements, which are major contributors to the expansion of the sponge gourd genome. According to a phylogenetic analysis of conserved genes, the sponge gourd lineage diverged from the bitter gourd lineage approximately 41.6 million years ago. Additionally, many genes that respond to biotic and abiotic stresses were found to be lineage specific or expanded in the sponge gourd genome, as demonstrated by the presence of 462 NBS-LRR genes, a much greater number than are found in the genomes of other cucurbit species; these results are consistent with the high stress resistance of sponge gourd. Collectively, our study provides insights into genome evolution and serves as a valuable reference for the genetic improvement of sponge gourd.

Identification of drought-responsive microRNAs in tomato using high-throughput sequencing.

Drought is a major abiotic stress affecting crop productivity and quality. As a class of noncoding RNA, microRNA (miRNA) plays important roles in plant growth, development, and stress response. However, their response and roles in tomato drought stress is largely unknown. Here, by using high-throughput sequencing, we compared the miRNA profiles before and after drought treatment in two tomato genotypes: M82, a drought-sensitive cultivated tomato (Solanum lycopersicum), and IL2-5, a drought-tolerant introgression line derived from M82 and the tomato wild species S. pennellii (LA0716). A total of 108 conserved and 208 novel miRNAs were identified, among them, 32 and 68 were significantly changed in expression after stress. Further, 1936 putative target genes were predicted for those differentially-expressed miRNAs. Gene ontology and pathway analysis showed that many of the target genes were involved in stress resistance, such as genes in GO terms including response to stress, defense response, response to stimulus, phosphorylation, and signal transduction. Our results suggested that miRNAs play an essential role in the drought response of tomato. This work will help to further characterize specific miRNAs functioning in drought tolerance.

Profiling of drought-responsive microRNA and mRNA in tomato using high-throughput sequencing.

BACKGROUND: Abiotic stresses cause severe loss of crop production. Among them, drought is one of the most frequent environmental stresses, which limits crop growth, development and productivity. Plant drought tolerance is fine-tuned by a complex gene regulatory network. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success to improve plant yield and quality. Recent studies have demonstrated that microRNAs play critical roles in plant drought tolerance. However, little is known about the microRNA in drought response of the model plant tomato. Here, we described the profiling of drought-responsive microRNA and mRNA in tomato using high-throughput next-generation sequencing. RESULTS: Drought stress was applied on the seedlings of M82, a drought-sensitive cultivated tomato genotype, and IL9-1, a drought-tolerant introgression line derived from the stress-resistant wild species Solanum pennellii LA0716 and M82. Under drought, IL9-1 performed superior than M82 regarding survival rate, H2O2 elimination and leaf turgor maintenance. A total of four small RNA and eight mRNA libraries were constructed and sequenced using Illumina sequencing technology. 105 conserved and 179 novel microRNAs were identified, among them, 54 and 98 were differentially expressed upon drought stress, respectively. The majority of the differentially-expressed conserved microRNAs was up-regulated in IL9-1 whereas down-regulated in M82. Under drought stress, 2714 and 1161 genes were found to be differentially expressed in M82 and IL9-1, respectively, and many of their homologues are involved in plant stress, such as genes encoding transcription factor and protein kinase. Various pathways involved in abiotic stress were revealed by Gene Ontology and pathway analysis. The mRNA sequencing results indicated that most of the target genes were regulated by their corresponding microRNAs, which suggested that microRNAs may play essential roles in the drought tolerance of tomato. CONCLUSION: In this study, numerous microRNAs and mRNAs involved in the drought response of tomato were identified using high-throughput sequencing, which will provide new insights into the complex regulatory network of plant adaption to drought stress. This work will also help to exploit new players functioning in plant drought-stress tolerance.