A quick and effective method for thermostability differentiation in cucumber (Cucumis sativus L.).

High temperature affects the growth and production of cucumber. Selecting thermotolerant cucumber cultivars is conducive to coping with high temperatures and improving production. Thus, a quick and effective method for screening thermotolerant cucumber cultivars is needed. In this study, four cucumber cultivars were used to identify heat resistance indexes. The morphological, physiological and biochemical indexes were measured. When exposed to high temperatures, thermotolerant cucumber had a more stable photosystem, membrane, and oxidation-reduction systems. The impact of high temperatures on plants is multifaceted, and the accurate discrimination of heat resistance cannot be achieved solely based on a single or multiple indicators. Therefore, principal component analysis (PCA) was employed to comprehensively evaluate the heat resistance of cucumber plants. The results showed that the heat resistance obtained by PCA was significantly correlated with the heat injury index. In addition, the stepwise regression equation identified two heat-related indices, hydrogen peroxide content (H2 O2 ) and photosynthetic operating efficiency (Fq'/Fm'), and they can quickly distinguish the heat resistance of the other 8 cucumber cultivars. These results will help to accelerate the selection of thermotolerant resources and assist in cucumber breeding.

A putative E3 ubiquitin ligase substrate receptor degrades transcription factor SmNAC to enhance bacterial wilt resistance in eggplant.

Bacterial wilt caused by Ralstonia solanacearum is a severe soil-borne disease globally, limiting the production in Solanaceae plants. SmNAC negatively regulated eggplant resistance to Bacterial wilt (BW) though restraining salicylic acid (SA) biosynthesis. However, other mechanisms through which SmNAC regulates BW resistance remain unknown. Here, we identified an interaction factor, SmDDA1b, encoding a substrate receptor for E3 ubiquitin ligase, from the eggplant cDNA library using SmNAC as bait. SmDDA1b expression was promoted by R. solanacearum inoculation and exogenous SA treatment. The virus-induced gene silencing of the SmDDA1b suppressed the BW resistance of eggplants; SmDDA1b overexpression enhanced the BW resistance of tomato plants. SmDDA1b positively regulates BW resistance by inhibiting the spread of R. solanacearum within plants. The SA content and the SA biosynthesis gene ICS1 and signaling pathway genes decreased in the SmDDA1b-silenced plants but increased in SmDDA1b-overexpression plants. Moreover, SmDDB1 protein showed interaction with SmCUL4 and SmDDA1b and protein degradation experiments indicated that SmDDA1b reduced SmNAC protein levels through proteasome degradation. Furthermore, SmNAC could directly bind the SmDDA1b promoter and repress its transcription. Thus, SmDDA1b is a novel regulator functioning in BW resistance of solanaceous crops via the SmNAC-mediated SA pathway. Those results also revealed a negative feedback loop between SmDDA1b and SmNAC controlling BW resistance.

Systematic analysis of the UDP-glucosyltransferase family: discovery of a member involved in rutin biosynthesis in Solanum melongena.

Eggplant (Solanum melongena) is an economically important crop and rich in various nutrients, among which rutin that has positive effects on human health is found in eggplant. Glycosylation mediated by UDP-glycosyltransferases (UGTs) is a key step in rutin biosynthesis. However, the UGT gene has not been reported in eggplant to date. Herein, 195 putative UGT genes were identified in eggplant by genome-wide analysis, and they were divided into 17 subgroups (Group A-P and Group R) according to the phylogenetic evolutionary tree. The members of Groups A, B, D, E and L were related to flavonol biosynthesis, and rutin was the typical flavonol. The expression profile showed that the transcriptional levels of SmUGT genes in Clusters 7-10 were closely related to those of rutin biosynthetic pathway genes. Notably, SmUGT89B2 was classified into Cluster 7 and Group B; its expression was consistent with rutin accumulation in different tissues and different leaf stages of eggplant. SmUGT89B2 was located in the nucleus and cell membrane. Virus-induced gene silencing (VIGS) and transient overexpression assays showed that SmUGT89B2 can promote rutin accumulation in eggplant. These findings provide new insights into the UGT genes in eggplant, indicating that SmUGT89B2 is likely to encode the final enzyme in rutin biosynthesis.

Genome-Wide Analysis and Characterization of Eggplant F-Box Gene Superfamily: Gene Evolution and Expression Analysis under Stress.

F-box genes play an important role in plant growth and resistance to abiotic and biotic stresses. To date, systematic analysis of F-box genes and functional annotation in eggplant (Solanum melongena) is still limited. Here, we identified 389 F-box candidate genes in eggplant. The domain study of F-box candidate genes showed that the F-box domain is conserved, whereas the C-terminal domain is diverse. There are 376 SmFBX candidate genes distributed on 12 chromosomes. A collinearity analysis within the eggplant genome suggested that tandem duplication is the dominant form of F-box gene replication in eggplant. The collinearity analysis between eggplant and the three other species (Arabidopsis thaliana, rice and tomato) provides insight into the evolutionary characteristics of F-box candidate genes. In addition, we analyzed the expression of SmFBX candidate genes in different tissues under high temperature and bacterial wilt stress. The results identified several F-box candidate genes that potentially participate in eggplant heat tolerance and bacterial wilt resistance. Moreover, the yeast two-hybrid assay showed that several representative F-box candidate proteins interacted with representative Skp1 proteins. Overexpression of SmFBX131 and SmFBX230 in tobacco increased resistance to bacterial wilt. Overall, these results provide critical insights into the functional analysis of the F-box gene superfamily in eggplant and provide potentially valuable targets for heat and bacterial resistance.

Ethylene Inhibits Anthocyanin Biosynthesis by Repressing the R2R3-MYB Regulator SlAN2-like in Tomato.

Fruit ripening is usually accompanied by anthocyanin accumulation. Ethylene is key in ripening-induced anthocyanin production in many fruits. However, the effects of fruit ripening and ethylene on anthocyanin biosynthesis in purple tomato fruits are unclear. This study shows that bagged fruits of the purple tomato cultivar 'Indigo Rose' failed to produce anthocyanins at the red ripening stage after bag removal. In contrast, the bagged immature fruits accumulated a significant amount of anthocyanins after removing the bags. The transcriptomic analyses between immature and red ripening fruit before and after bag removal revealed that anthocyanin-related genes, including the key positive R2R3-MYB regulator SlAN2-like, were repressed in the red ripening fruit. The 86 identified transcription factors, including 13 AP2/ERF, 7 bZIP, 8 bHLH and 6 MYB, showed significantly different expressions between immature and red ripening fruits. Moreover, subjecting bagged immature fruits to exogenous ethylene treatment significantly inhibited anthocyanin accumulation and the expression of anthocyanin-related genes, including the anthocyanin structure genes and SlAN2-like. Thus, ethylene inhibits anthocyanin biosynthesis by repressing the transcription of SlAN2-like and other anthocyanin-related genes. These findings provide new insights into anthocyanin regulation in purple tomato fruit.

CsIVP Modulates Low Nitrogen and High-Temperature Resistance in Cucumber.

Crop plants experience various abiotic stresses that reduce yield and quality. Although several adaptative physiological and defense responses to single stress have been identified, the behavior and mechanisms of plant response to multiple stresses remain underexamined. Herein, we determined that the leaf and vascular changes in Cucumis sativus Irregular Vasculature Patterning (CsIVP)-RNAi cucumber plants can enhance resistance to nitrogen deficiency and high-temperature stress. CsIVP negatively regulated high nitrate affinity transporters (NRT2.1, NRT2.5) and reallocation transporters (NRT1.7, NRT1.9, NRT1.12) under low nitrogen stress. Furthermore, CsIVP-RNAi plants have high survival rate with low heat injury level under high-temperature condition. Several key high-temperature regulators, including Hsfs, Hsps, DREB2C, MBF1b and WRKY33 have significant expression in CsIVP-RNAi plants. CsIVP negatively mediated high-temperature responses by physically interacting with CsDREB2C. Altogether, these results indicated that CsIVP integrates innate programming of plant development, nutrient transport and high-temperature resistance, providing a potentially valuable target for breeding nutrient-efficient and heat-resistant crops.

Heat Stress Resistance Mechanisms of Two Cucumber Varieties from Different Regions.

High temperatures affect the yield and quality of vegetable crops. Unlike thermosensitive plants, thermotolerant plants have excellent systems for withstanding heat stress. This study evaluated various heat resistance indexes of the thermotolerant cucumber (TT) and thermosensitive cucumber (TS) plants at the seedling stage. The similarities and differences between the regulatory genes were assessed through transcriptome analysis to understand the mechanisms for heat stress resistance in cucumber. The TT plants exhibited enhanced leaf status, photosystem, root viability, and ROS scavenging under high temperature compared to the TS plants. Additionally, transcriptome analysis showed that the genes involved in photosynthesis, the chlorophyll metabolism, and defense responses were upregulated in TT plants but downregulated in TS plants. Zeatin riboside (ZR), brassinosteroid (BR), and jasmonic acid (JA) levels were higher in TT plants than in TS. The heat stress increased gibberellic acid (GA) and indoleacetic acid (IAA) levels in both plant lines; however, the level of GA was higher in TT. Correlation and interaction analyses revealed that heat cucumber heat resistance is regulated by a few transcription factor family genes and metabolic pathways. Our study revealed different phenotypic and physiological mechanisms of the heat response by the thermotolerant and thermosensitive cucumber plants. The plants were also shown to exhibit different expression profiles and metabolic pathways. The heat resistant pathways and genes of two cucumber varieties were also identified. These results enhance our understanding of the molecular mechanisms of cucumber response to high-temperature stress.

SlGID1a Is a Putative Candidate Gene for qtph1.1, a Major-Effect Quantitative Trait Locus Controlling Tomato Plant Height.

Plant height is an important agronomic trait in crops. Several genes underlying tomato (Solanum lycopersicum) plant height mutants have been cloned. However, few quantitative trait genes for plant height have been identified in tomato. In this study, seven quantitative trait loci (QTLs) controlling plant height were identified in tomato. Of which, qtph1.1 (QTL for tomato plant height 1.1), qtph3.1 and qtph12.1 were major QTLs and explained 15, 16, and 12% of phenotypic variation (R2), respectively. The qtph1.1 was further mapped to an 18.9-kb interval on chromosome 1. Based on the annotated tomato genome (version SL2.50, annotation ITAG2.40), Solyc01g098390 encoding GA receptor SlGID1a was the putative candidate gene. The SlGID1a gene underlying the qtph1.1 locus contained a single nucleotide polymorphism (SNP) that resulted in an amino acid alteration in protein sequence. The near-isogenic line containing the qtph1.1 locus (NIL-qtph1.1) exhibited shorter internode length and cell length than the wild type (NIL-WT). The dwarf phenotype of NIL-qtph1.1 could not be rescued by exogenous GA3 treatment. Transcriptome analysis and real-time quantitative reverse transcription PCR (qPCR) showed that several genes related to biosynthesis and signaling of GA and auxin were differentially expressed in stems between NIL-qtph1.1 and NIL-WT. These findings might pave the road for understanding the molecular regulation mechanism of tomato plant height.

Rapid analysis of anthocyanin and its structural modifications in fresh tomato fruit.

Anthocyanin is derived from a flavylium cation structure, and it promotes health in humans and functions in plants as protection against environmental stress. The rapid analysis of anthocyanin structure and content is a critical challenge for improving fruit quality. In this study, the tomato cultivar Indigo Rose, which is a popular purple cultivated tomato used for breeding, was taken as an example for anthocyanin analysis. A rapid analysis method was developed to minimize anthocyanin loss from the fresh fruit. Four new anthocyanins were discovered in the tomato, and the structures of a total of 12 anthocyanins were determined. Among these, petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside and malvidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside were the main anthocyanins in Indigo Rose. The structural modifications of these anthocyanins were mainly glycosylation and acylation, and there were also hydroxylation and methylation. Our findings provide new insight into the biosynthesis pathway in tomato fruit.

Comparative transcriptome analysis of differentially expressed genes between the fruit peel and flesh of the purple tomato cultivar 'Indigo Rose'.

Anthocyanins are considered health-promoting phytonutrients; however, anthocyanins strictly occurr in the fruit peel of purple tomato cultivars, making the total anthocyanin content limited per tomato fruit. In this study, we performed a transcriptome analysis between the fruit peel and flesh of a purple tomato cultivar 'Indigo Rose' at both the mature green stage and breaking stage. In total, 1,945 differently expressed genes, including 165 transcription factors, were detected between the fruit peel and flesh, both at and after the mature green stage. We further analyzed the transcription of anthocyanin biosynthesis genes and the regulatory genes composing the MYB-bHLH-WD40 (MBW) complex between the fruit peel and flesh at both development stages. In addition, several light-sensing genes and other transcription factor genes, including BBX family genes and WRKY genes, showed different expression patterns between the fruit peel and flesh. These findings deepen our understanding of anthocyanin biosynthesis in tomato fruit peels and facilitate the identification of genes limiting the anthocyanin biosynthesis in tomato fruit flesh.

CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant.

KEY MESSAGE: Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar 'Indigo Rose.' Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar 'Indigo Rose' using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. 'Indigo Rose,' while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. 'Indigo Rose.' Our findings further our understanding of anthocyanin production in tomato and other plant species.

Anthocyanin Fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit.

Anthocyanin fruit (Aft) and atroviolacea (atv) were characterized in wild tomato and can enhance anthocyanin content in tomato fruit. However, the gene underlying the Aft locus and the mechanism by which Aft and atv act remain largely unknown. In this study, the Aft locus was fine-mapped to an approximately 145-kb interval on chromosome 10, excluding SlAN2 (Solyc10g086250), SlANT1 (Solyc10g086260) and SlANT1-like (Solyc10g086270), which have previously been suggested as candidates. Thus, the R2R3-MYB transcription factor SlAN2-like (Solyc10g086290) was considered the best candidate gene for Aft. The CRISPR/Cas9-mediated SlAN2-like mutants show a much lower accumulation of anthocyanins associated with the downregulation of multiple anthocyanin-related genes compared to the wild-type tomato, indicating that SlAN2-like is responsible for the Aft phenotype. The repressive function of SlMYBATV also was confirmed through the CRISPR/Cas9 approach. A yeast-two-hybrid assay revealed that SlMYBATV interacts with the bHLH protein SlJAF13. Furthermore, yeast-one-hybrid and dual-luciferase transient expression assays showed that Aft directly binds to the SlMYBATV promoter and activates its expression. The results herein provide candidate genes to enhance anthocyanin content in tomato fruit. This research also provides insight into a mechanism involving the Aft-SlMYBATV pathway that fine-tunes anthocyanin accumulation in tomato fruit.

The eggplant transcription factor MYB44 enhances resistance to bacterial wilt by activating the expression of spermidine synthase.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious disease affecting the production of Solanaceae species, including eggplant (Solanum melongena). However, few resistance genes have been identified in eggplant, and therefore the underlying mechanism of BW resistance remains unclear. Hence, we investigated a spermidine synthase (SPDS) gene from eggplant and created knock-down lines with virus-induced gene silencing. After eggplant was infected with R. solanacearum, the SmSPDS gene was induced, concurrent with increased spermidine (Spd) content, especially in the resistant line. We speculated that Spd plays a significant role in the defense response of eggplant to BW. Moreover, using the yeast one-hybrid approach and dual luciferase-based transactivation assay, an R2R3-MYB transcription factor, SmMYB44, was identified as directly binding to the SmSPDS promoter, activating its expression. Overexpression of SmMYB44 in eggplant induced the expression of SmSPDS and Spd content, increasing the resistance to BW. In contrast, the SmMYB44-RNAi transgenic plants showed more susceptibility to BW compared with the control plants. Our results provide insight into the SmMYB44-SmSPDS-Spd module involved in the regulation of resistance to R. solanacearum. This research also provides candidates to enhance resistance to BW in eggplant.

Identification of Candidate HY5-Dependent and -Independent Regulators of Anthocyanin Biosynthesis in Tomato.

High quantities of anthocyanins in plants confer potential protective benefits against biotic and abiotic stressors. Studies have shown that the bZIP transcription factor HY5 plays a key role in controlling anthocyanin accumulation in response to light. However, in hy5 mutants, residual anthocyanins have been detected, indicating that other regulators exist to regulate anthocyanin biosynthesis in an HY5-independent manner. Here, we employed the CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9) system specifically to induce targeted mutagenesis of SlHY5 in the purple tomato cultivar 'Indigo Rose'. The T2 generation of tomato plants homozygous for the null allele of the SlHY5 frameshift mutated by a 1 bp insertion contained a lower anthocyanin content. Transcriptional analysis showed that most of the anthocyanin biosynthesis structural genes and several regulatory genes were down-regulated in the hy5 mutant lines. With transcriptome analyses of the various tissues from hy5 mutant lines, eight candidate transcription factors were identified that may regulate anthocyanin biosynthesis in an HY5-independent manner. These findings deepen our understanding of how light controls anthocyanin accumulation and facilitate the identification of the regulators of anthocyanin biosynthesis in an HY5-independent manner.

A putative R3 MYB repressor is the candidate gene underlying atroviolacium, a locus for anthocyanin pigmentation in tomato fruit.

Anthocyanins are potential health-promoting compounds in the human diet. The atv (atroviolacium) locus, derived from the wild tomato species Solanum cheesmaniae, has been shown to enhance anthocyanin pigmentation in tomato fruit when it co-exists with either the Aft (Anthocyanin fruit) or the Abg (Aubergine) locus. In the present study, the atv locus was fine-mapped to an approximately 5.0-kb interval on chromosome 7. A putative R3 MYB repressor was identified in this interval and is hereby designated as SlMYBATV. The allele of SlMYBATV underlying the atv locus harbored a 4-bp insertion in its coding region, which is predicted to result in a frame-shift and premature protein truncation. The other candidate R3 MYB and R2R3 MYB repressors of anthocyanin biosynthesis were also identified in tomato via a genome-wide search. Transcriptional analysis showed that most of the structural genes and several regulatory genes of anthocyanin biosynthesis were up-regulated in the tomato SlMYBATV mutant lines. These findings may facilitate the elucidation of the molecular mechanisms underlying anthocyanin pigmentation in tomato fruit and help in the marker-assisted selection of anthocyanin-enriched tomato cultivars.

Fine Mapping of a Gene (ER4.1) that Causes Epidermal Reticulation of Tomato Fruit and Characterization of the Associated Transcriptome.

The hydrophobic cuticle that covers the surface of tomato (Solanum lycopersicum) fruit plays key roles in development and protection against biotic and abiotic stresses, including water loss, mechanical damage, UV radiation, pathogens, and pests. However, many details of the genes and regulatory mechanisms involved in cuticle biosynthesis in fleshy fruits are not well understood. In this study, we describe a novel tomato fruit phenotype, characterized by epidermal reticulation (ER) of green fruit and a higher water loss rate than wild type (WT) fruit. The ER phenotype is controlled by a single gene, ER4.1, derived from an introgressed chromosomal segment from the wild tomato species S. pennellii (LA0716). We performed fine mapping of the single dominant gene to an ~300 kb region and identified Solyc04g082540, Solyc04g082950, Solyc04g082630, and Solyc04g082910as potential candidate genes for the ER4.1 locus, based on comparative RNA-seq analysis of ER and WT fruit peels. In addition, the transcriptome analysis revealed that the expression levels of genes involved in cutin, wax and flavonoid biosynthesis were altered in the ER fruit compared with WT. This study provides new insights into the regulatory mechanisms and metabolism of the fruit cuticle.

Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development.

Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which depends on the orchestrated accessibility of regulatory proteins to promoters and other cis-regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the developmental process. To understand the regulatory mechanism and functional elements modulating morphological and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites (DHSs) from the fruit tissues of the tomato cultivar "Moneymaker" at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening.

Spatiotemporal transcriptome provides insights into early fruit development of tomato (Solanum lycopersicum).

Early fruit development is crucial for crop production in tomato. After fertilization, the ovary undergoes cell division and cell expansion before maturation. Although the roles of regulatory signals such as hormone and carbohydrate during early fruit development have been studied, the spatial distribution and the sequential initiation of these regulatory signals still need to be explored. Using the tomato cultivar 'Moneymaker', we analyzed the transcriptome of the ovule and the ovary wall/pericarp dissected from four different stages of the early developing fruits by stereoscope. These datasets give us the whole picture about the spatial and temporal signal distribution in early development of ovule and pericarp. Our results indicate that the hormone signal was initiated in both ovule and pericarp after fertilization. After that, different signals were activated in ovule and pericarp due to their distinct developmental processes. Our study provides spatiotemporal regulatory landscape of gene expression with sequential information which was not studied by previous work and further strengthens the comprehension of the regulatory and metabolic events controlling early fruit development.

The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures.

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.