Fei Wang

Citric acid (CA) plays a crucial role as a fruit flavor enhancer and serves as a mediator in multiple metabolic pathways in tomato fruit development. Understanding factors influencing CA metabolism is essential for enhancing fruit flavor and CA-mediated biological processes. The accumulation of CA, however, is influenced by a complex interplay of genetic and environmental factors, leading to challenges in accurately predicting and regulating its levels. In this study, we conducted a genome-wide association study (GWAS) on CA, employing six landmark models based on genome-wide variations including structural variants, insertions and deletions, and single nucleotide polymorphisms. The identification of 11 high-confidence candidate genes was further facilitated by leveraging linkage disequilibrium and causal variants associated with CA. The transcriptome data from candidate genes were examined, revealing higher correlations between the expression of certain candidate genes and changes in CA metabolism. Three CA-associated genes exerted a positive regulatory effect on CA accumulation, while the remaining genes exhibited negative impacts based on gene cluster and correlation analyses. The CA content of tomatoes is primarily influenced by improvement sweeps with minimal influence from domestication sweeps in the long-term breeding history, as evidenced by population differentiation and variants distribution. The presence of various causal variants within candidate genes is implicated in the heterogeneity of CA content observed among the tomato accessions. This observation suggests a potential correlation between the number of alternative alleles and CA content. This study offers significant function-based markers that can be utilized in marker-assisted breeding, thereby enhancing their value and applicability.

TIFY [TIF(F/Y)XG] proteins are a plant particular transcription factor family that regulates plant stress responses. Therefore, to fill this gap, we investigated CaTIFY genes in pepper. Gene structure and conserved motifs of the pepper TIFY gene family were systematically analyzed using sequence alignment analysis, Cis-acting element analysis, transcriptomic data, and RT-qPCR analysis, and their expression patterns were further analyzed using Virus-Induced Gene Silencing (VIGS) and cold stress reactive oxygen species (ROS) response. We identified 16 CaTIFY genes in pepper, which were dispersed among seven subgroups (JAZI, JAZII, JAZIII, PPD, TIFY, and ZIM/ZML). Several CaTIFY members had stress-related harmonic-responsive elements, and four (CaTIFY7, CaTIFY10b, CaTIFY1b, and CaTIFY6b) had low-temperature-responsive elements. Transcriptomic data and RT-qPCR analysis revealed that the TIFY genes in pepper displayed different expression patterns in the roots, stems, leaves, flower fruits, and seeds. In particular, CaTIFY7 was highly expressed in young leaves, and CaTIFY10b was highly expressed in roots. CaTIFYs participated in the regulation of several different abiotic stresses and CaTIFY7 and CaTIFY10b were significantly induced by cold stress. Additionally, Virus-Induced Gene Silencing (targeting CaTIFY7 and CaTIFY10b) resulted in plants that were sensitive to cold stress. Conversely, overexpression of CaTIFY7 and CaTIFY10b enhanced plant cold tolerance by promoting the expression of genes related to cold stress and the ROS response. CaTIFY7 and CaTIFY10b interacted with themselves and CaTIFY7 also interacted with CaTIFY10b in the yeast two-hybrid (Y2H) system. Our data provide a basis for further analysis of the role of pepper TIFY genes in cold-stress responses in the future.

The purple color of unripe pepper fruit is attributed to the accumulation of anthocyanins. Only a few genes controlling the biosynthesis and regulation of anthocyanins have been cloned in Capsicum. In this study, we performed a bulked segregant analysis of the purple striped trait using an F² population derived from a cross between the immature purple striped fruit line Chen12-4-1-1-1-1 and the normal green fruit line Zhongxian101-M-F⁹. We mapped the CaPs locus to an 841.39 kb region between markers M-CA690-Xba and MCA710-03 on chromosome 10. CA10g11690 encodes an R2R3-MYB transcription factor that is involved in the biosynthesis of anthocyanins as the best candidate gene. Overexpression and silencing in transformed tobacco (Nicotiana tabacum) lines indicated that CA10g11690 is involved in the formation of purple stripes in the exocarp. A comparison of parental sequences identified an insertion fragment of 1,926 bp in the second intron region of Chen12-4, and eight SNPs were detected between the two parents. Additionally, there were 49 single nucleotide polymorphic variations, two sequence deletions, and four sequence insertions in the promoter region. We found that CA10g11690 undergoes alternative splicing and generates different transcripts. Thus, the functional transcript of CA10g11690 appeared to be primarily involved in the development of purple phenotype in the exocarp. Our data provide new insight into the mechanism of anthocyanin biosynthesis and a theoretical basis for the future breeding of purple striped pepper varieties.