Genetic tapestry of Capsicum fruit colors: a comparative analysis of four cultivated species.

KEY MESSAGE: Genome-wide association study of color spaces across the four cultivated Capsicum spp. revealed a shared set of genes influencing fruit color, suggesting mechanisms and pathways across Capsicum species are conserved during the speciation. Notably, Cytochrome P450 of the carotenoid pathway, MYB transcription factor, and pentatricopeptide repeat-containing protein are the major genes responsible for fruit color variation across the Capsicum species. Peppers (Capsicum spp.) rank among the most widely consumed spices globally. Fruit color, serving as a determinant for use in food colorants and cosmeceuticals and an indicator of nutritional contents, significantly influences market quality and price. Cultivated Capsicum species display extensive phenotypic diversity, especially in fruit coloration. Our study leveraged the genetic variance within four Capsicum species (Capsicum baccatum, Capsicum chinense, Capsicum frutescens, and Capsicum annuum) to elucidate the genetic mechanisms driving color variation in peppers and related Solanaceae species. We analyzed color metrics and chromatic attributes (Red, Green, Blue, L*, a*, b*, Luminosity, Hue, and Chroma) on samples cultivated over six years (2015-2021). We resolved genomic regions associated with fruit color diversity through the sets of SNPs obtained from Genotyping by Sequencing (GBS) and genome-wide association study (GWAS) with a Multi-Locus Mixed Linear Model (MLMM). Significant SNPs with FDR correction were identified, within the Cytochrome P450, MYB-related genes, Pentatricopeptide repeat proteins, and ABC transporter family were the most common among the four species, indicating comparative evolution of fruit colors. We further validated the role of a pentatricopeptide repeat-containing protein (Chr01:31,205,460) and a cytochrome P450 enzyme (Chr08:45,351,919) via competitive allele-specific PCR (KASP) genotyping. Our findings advance the understanding of the genetic underpinnings of Capsicum fruit coloration, with developed KASP assays holding potential for applications in crop breeding and aligning with consumer preferences. This study provides a cornerstone for future research into exploiting Capsicum's diverse fruit color variation.

Regulatory mechanisms of the LBD40 transcription factor in Arabidopsis thaliana somatic embryogenesis.

Somatic embryogenesis (SE) is a process by which an embryo is derived from somatic tissue. Transcription factors (TFs) have been identified that control this process. One such TF that promotes SE is AGAMOUS-like 15 (AGL15). Prior work has shown that AGL15 can both induce and repress gene expression. One way this type of dual function TF works is via protein interactions, so a yeast 2-hybrid (Y2H) screen was undertaken. One intriguing protein with which AGL15 interacted in Y2H was LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)-domain TF that is unique to plants and is primarily expressed during seed development. Here, we confirm the AGL15-LBD40 interaction by quantitative assays and in planta co-immunoprecipation. We also document a role for LBD40, and the closely related protein LBD41, in supporting SE. To determine downstream genes potentially controlled by LBD40, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) was used. More than 400 binding regions for LBD40 were consistently found genome-wide. To determine genes responsive to LBD40/41 accumulation, RNA-seq analysis of transcriptomes of wild-type control and loss-of-function lbd40/lbd41 was performed. Combining these datasets provides insight into genes directly and indirectly controlled by these LOB domain TFs. The gene ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE. This work provides insight into SE, a poorly understood, but essential process to generate transgenic plants to meet agricultural demands or test gene function. This manuscript reports on experiments to understand the role that LDB40, a TF, plays in support of SE by investigating genes directly and indirectly controlled by LBD40 and examining physical and genetic interactions with other TFs active in SE. We uncover targets of LBD40 and an interacting TF of the MADS family and investigate targets involvement in SE.

Role of EIN2-mediated ethylene signaling in regulating petal senescence, abscission, reproductive development, and hormonal crosstalk in tobacco.

Ethylene plays a pivotal role in a wide range of developmental, physiological, and defense processes in plants. EIN2 (ETHYLENE INSENSITIVE2) is a key player in the ethylene signaling pathway. To characterize the role of EIN2 in processes, such as petal senescence, where it has been found to play important roles along with various other developmental and physiological processes, the tobacco (Nicotiana tabacum) ortholog of EIN2 (NtEIN2) was isolated and NtEIN2 silenced transgenic lines were generated using RNA interference (RNAi). Silencing of NtEIN2 compromised plant defense against pathogens. NtEIN2 silenced lines displayed significant delays in petal senescence, and pod maturation, and adversely affected pod and seed development. This study further dissected the petal senescence in ethylene insensitive lines, that displayed alteration in the pattern of petal senescence and floral organ abscission. Delay in petal senescence was possibly because of delayed aging processes within petal tissues. Possible crosstalk between EIN2 and AUXIN RESPONSE FACTOR 2 (ARF2) in regulating the petal senescence process was also investigated. Overall, these experiments indicated a crucial role for NtEIN2 in controlling diverse developmental and physiological processes, especially in petal senescence.

Effects of Mars Global Simulant (MGS-1) on Growth and Physiology of Sweet Potato: A Space Model Plant.

Growing food autonomously on Mars is challenging due to the Martian soil's low nutrient content and high salinity. Understanding how plants adapt and evaluating their nutritional attributes are pivotal for sustained Mars missions. This research delves into the regeneration, stress tolerance, and dietary metrics of sweet potato (Ipomoea batatas) across different Mars Global Simulant (MGS-1) concentrations (0, 25, 50, and 75%). In our greenhouse experiment, 75% MGS-1 concentration significantly inhibited sweet potato growth, storage root biomass, and chlorophyll content. This concentration also elevated the plant tissues' H2O2, proline, and ascorbic acid levels. Higher MGS-1 exposures (50 and 75%) notably boosted the vital amino acids and sugar groups in the plant's storage roots. However, increased MGS-1 concentrations notably diminished the total C:N ratio and elemental composition in both the vines and storage roots. In summary, sweet potato exhibited optimal growth, antioxidant properties, yield, and nutrient profiles at 25% MGS-1 exposure as compared to higher concentrations. This study underscores the need for future interventions, like nutrient enhancements and controlled metal accessibility, to render sweet potato a suitable plant for space-based studies.

Differential gene expression in tall fescue tissues in response to water deficit.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool-season grasses. Also, its seed-borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water-deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown.

The MADS-domain factor AGAMOUS-Like18 promotes somatic embryogenesis.

AGAMOUS-Like 18 (AGL18) is a MADS domain transcription factor (TF) that is structurally related to AGL15. Here we show that, like AGL15, AGL18 can promote somatic embryogenesis (SE) when ectopically expressed in Arabidopsis (Arabidopsis thaliana). Based on loss-of-function mutants, AGL15 and AGL18 have redundant functions in developmental processes such as SE. To understand the nature of this redundancy, we undertook a number of studies to look at the interaction between these factors. We studied the genome-wide direct targets of AGL18 to characterize its roles at the molecular level using chromatin immunoprecipitation (ChIP)-SEQ combined with RNA-SEQ. The results demonstrated that AGL18 binds to thousands of sites in the genome. Comparison of ChIP-SEQ data for AGL15 and AGL18 revealed substantial numbers of genes bound by both AGL15 and AGL18, but there were also differences. Gene ontology analysis revealed that target genes were enriched for seed, embryo, and reproductive development as well as hormone and stress responses. The results also demonstrated that AGL15 and AGL18 interact in a complex regulatory loop, where AGL15 inhibited transcript accumulation of AGL18, while AGL18 increased AGL15 transcript accumulation. Co-immunoprecipitation revealed an interaction between AGL18 and AGL15 in somatic embryo tissue. The binding and expression analyses revealed a complex crosstalk and interactions among embryo TFs and their target genes. In addition, our study also revealed that phosphorylation of AGL18 and AGL15 was crucial for the promotion of SE.

Differential expression of SlKLUH controlling fruit and seed weight is associated with changes in lipid metabolism and photosynthesis-related genes.

The sizes of plant organs such as fruit and seed are crucial yield components. Tomato KLUH underlies the locus fw3.2, an important regulator of fruit and seed weight. However, the mechanism by which the expression levels of KLUH affect organ size is poorly understood. We found that higher expression of SlKLUH increased cell proliferation in the pericarp within 5 d post-anthesis in tomato near-isogenic lines. Differential gene expression analyses showed that lower expression of SlKLUH was associated with increased expression of genes involved in lipid metabolism. Lipidomic analysis revealed that repression of SlKLUH mainly increased the contents of certain non-phosphorus glycerolipids and phospholipids and decreased the contents of four unknown lipids. Co-expression network analyses revealed that lipid metabolism was possibly associated with but not directly controlled by SlKLUH, and that this gene instead controls photosynthesis-related processes. In addition, many transcription factors putatively involved in the KLUH pathway were identified. Collectively, we show that SlKLUH regulates fruit and seed weight which is associated with altered lipid metabolism. The results expand our understanding of fruit and seed weight regulation and offer a valuable resource for functional studies of candidate genes putatively involved in regulation of organ size in tomato and other crops.

Wide-ranging transcriptome remodelling mediated by alternative polyadenylation in response to abiotic stresses in Sorghum.

Alternative polyadenylation (APA) regulates diverse developmental and physiological processes through its effects on gene expression, mRNA stability, translatability, and transport. Sorghum is a major cereal crop in the world and, despite its importance, not much is known about the role of post-transcriptional regulation in mediating responses to abiotic stresses in Sorghum. A genome-wide APA analysis unveiled widespread occurrence of APA in Sorghum in response to drought, heat, and salt stress. Abiotic stress treatments incited changes in poly(A) site choice in a large number of genes. Interestingly, abiotic stresses led to the re-directing of transcriptional output into non-productive pathways defined by the class of poly(A) site utilized. This result revealed APA to be part of a larger global response of Sorghum to abiotic stresses that involves the re-direction of transcriptional output into non-productive transcriptional and translational pathways. Large numbers of stress-inducible poly(A) sites could not be linked with known, annotated genes, suggestive of the existence of numerous unidentified genes whose expression is strongly regulated by abiotic stresses. Furthermore, we uncovered a novel stress-specific cis-element in intronic poly(A) sites used in drought- and heat-stressed plants that might play an important role in non-canonical poly(A) site choice in response to abiotic stresses.

Transcriptome Landscape Variation in the Genus Thymus.

Among the Lamiaceae family, the genus Thymus is an economically important genera due to its medicinal and aromatic properties. Most Thymus molecular research has focused on the determining the phylogenetic relationships between different species, but no published work has focused on the evolution of the transcriptome across the genus to elucidate genes involved in terpenoid biosynthesis. Hence, in this study, the transcriptomes of five different Thymus species were generated and analyzed to mine putative genes involved in thymol and carvacrol biosynthesis. High-throughput sequencing produced ~43 million high-quality reads per sample, which were assembled de novo using several tools, then further subjected to a quality evaluation. The best assembly for each species was used as queries to search within the UniProt, KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups) and TF (Transcription Factors) databases. Mining the transcriptomes resulted in the identification of 592 single-copy orthogroups used for phylogenetic analysis. The data showed strongly support a close genetic relationship between Thymus vulgaris and Thymus daenensis. Additionally, this study dates the speciation events between 1.5-2.1 and 9-10.2 MYA according to different methodologies. Our study provides a global overview of genes related to the terpenoid pathway in Thymus, and can help establish an understanding of the relationship that exists among Thymus species.

A common genetic mechanism underlies morphological diversity in fruits and other plant organs.

Shapes of edible plant organs vary dramatically among and within crop plants. To explain and ultimately employ this variation towards crop improvement, we determined the genetic, molecular and cellular bases of fruit shape diversity in tomato. Through positional cloning, protein interaction studies, and genome editing, we report that OVATE Family Proteins and TONNEAU1 Recruiting Motif proteins regulate cell division patterns in ovary development to alter final fruit shape. The physical interactions between the members of these two families are necessary for dynamic relocalization of the protein complexes to different cellular compartments when expressed in tobacco leaf cells. Together with data from other domesticated crops and model plant species, the protein interaction studies provide possible mechanistic insights into the regulation of morphological variation in plants and a framework that may apply to organ growth in all plant species.

Genome-wide atlas of alternative polyadenylation in the forage legume red clover.

Studies on prevalence and significance of alternative polyadenylation (APA) in plants have been so far limited mostly to the model plants. Here, a genome-wide analysis of APA was carried out in different tissue types in the non-model forage legume red clover (Trifolium pratense L). A profile of poly(A) sites in different tissue types was generated using so-called 'poly(A)-tag sequencing' (PATseq) approach. Our analysis revealed tissue-wise dynamics of usage of poly(A) sites located at different genomic locations. We also identified poly(A) sites and underlying genes displaying APA in different tissues. Functional categories enriched in groups of genes manifesting APA between tissue types were determined. Analysis of spatial expression of genes encoding different poly(A) factors showed significant differential expression of genes encoding orthologs of FIP1(V) and PCFS4, suggesting that these two factors may play a role in regulating spatial APA in red clover. Our analysis also revealed a high degree of conservation in diverse plant species of APA events in mRNAs encoding two key polyadenylation factors, CPSF30 and FIP1(V). Together with our previously reported study of spatial gene expression in red clover, this study will provide a comprehensive account of transcriptome dynamics in this non-model forage legume.

Fruit weight is controlled by Cell Size Regulator encoding a novel protein that is expressed in maturing tomato fruits.

Increases in fruit weight of cultivated vegetables and fruits accompanied the domestication of these crops. Here we report on the positional cloning of a quantitative trait locus (QTL) controlling fruit weight in tomato. The derived allele of Cell Size Regulator (CSR-D) increases fruit weight predominantly through enlargement of the pericarp areas. The expanded pericarp tissues result from increased mesocarp cell size and not from increased number of cell layers. The effect of CSR on fruit weight and cell size is found across different genetic backgrounds implying a consistent impact of the locus on the trait. In fruits, CSR expression is undetectable early in development from floral meristems to the rapid cell proliferation stage after anthesis. Expression is low but detectable in growing fruit tissues and in or around vascular bundles coinciding with the cell enlargement stage of the fruit maturation process. CSR encodes an uncharacterized protein whose clade has expanded in the Solanaceae family. The mutant allele is predicted to encode a shorter protein due to a 1.4 kb deletion resulting in a 194 amino-acid truncation. Co-expression analyses and GO term enrichment analyses suggest association of CSR with cell differentiation in fruit tissues and vascular bundles. The derived allele arose in Solanum lycopersicum var cerasiforme and appears completely fixed in many cultivated tomato's market classes. This finding suggests that the selection of this allele was critical to the full domestication of tomato from its intermediate ancestors.

De novo Transcriptome Assembly and Dynamic Spatial Gene Expression Analysis in Red Clover.

Red clover ( L.) is a cool-season forage legume grown throughout the northeastern United States and is the most widely planted forage legume after alfalfa ( L.). Red clover provides high-value feed to the livestock because of high protein content and easy digestibility. To date, genomic resources for red clover are scarce. In the current study, a de novo transcriptome assembly of red clover was constructed representing different tissue types. The draft assembly consists of 37,565 contigs with N50 and average contig length of 1707 and 1262 bp, respectively. A comparative study with three other legume species displayed a high degree of sequence conservation between red clover and other legumes. The assembled transcriptome was annotated to allow identification of desirable genes. In particular, a genome-wide identification of red clover transcripts encoding putative transcription factors was performed. A comparative gene expression analysis between different tissue types was performed using the assembled transcriptome as the reference, which revealed dynamic gene expression patterns across different tissue types and also identified spatially dynamic gene coexpression clusters. Genes representing tissue-enriched clusters were subjected to gene ontology (GO) enrichment analysis to identify over-represented functional groups. Identification of these tissue-enriched gene coexpression clusters can help in future research focusing on developmental studies across tissues or in biotechnological improvement of red clover.

CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants.

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules.

What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape.

Domestication of fruit and vegetables resulted in a huge diversity of shapes and sizes of the produce. Selections that took place over thousands of years of alleles that increased fruit weight and altered shape for specific culinary uses provide a wealth of resources to study the molecular bases of this diversity. Tomato (Solanum lycopersicum) evolved from a wild ancestor (S. pimpinellifolium) bearing small and round edible fruit. Molecular genetic studies led to the identification of two genes selected for fruit weight: FW2.2 encoding a member of the Cell Number Regulator family; and FW3.2 encoding a P450 enzyme and the ortholog of KLUH. Four genes were identified that were selected for fruit shape: SUN encoding a member of the IQD family of calmodulin-binding proteins leading to fruit elongation; OVATE encoding a member of the OVATE family proteins involved in transcriptional repression leading to fruit elongation; LC encoding most likely the ortholog of WUSCHEL controlling meristem size and locule number; FAS encoding a member in the YABBY family controlling locule number leading to flat or oxheart shape. For this article, we will provide an overview of the putative function of the known genes, when during floral and fruit development they are hypothesized to act and their potential importance in regulating morphological diversity in other fruit and vegetable crops.

A cytochrome P450 regulates a domestication trait in cultivated tomato.

Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SlKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SlKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SlKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SlKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops.

CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway.

Nicotine to nornicotine conversion in tobacco (Nicotiana tabacum L.) is regulated by an unstable converter locus which in its activated state gives rise to a high nornicotine, low nicotine phenotype in the senescing leaves. In plants that carry the high nornicotine trait, nicotine conversion is primarily catalyzed by a cytochrome P450 protein, designated CYP82E4 whose transcription is strongly upregulated during leaf senescence. To further investigate the regulation of CYP82E4 expression, we examined the spatiotemporal distribution and the stress- and signaling molecule-elicited expression patterns of CYP82E4 using alkaloid analysis and a fusion construct between the 2.2 kb upstream regulatory region of CYP82E4 and the beta-glucurodinase (GUS) gene. Histochemical and fluorometric analyses of GUS expression revealed that the CYP82E4 promoter confers high levels of expression in the senescing leaves and flowers, and in the green stems of young and mature plants, but only very low activity was detected in the roots. In the leaves, GUS activity was strongly correlated with the progression of senescence. Treatments of leaf tissue with various signaling molecules including abscisic acid, ethylene, jasmonic acid, salicylic acid and yeast extract; and stresses, such as drought, wounding and tobacco mosaic virus infection did not enhance nicotine conversion or GUS activity in the green leaves, but an increase in CYP82E4 expression was observed in response to ethylene- or tobacco mosaic virus-induced senescence. These results suggest that the expression of CYP82E4 is senescence-specific in the leaves and the use of the CYP82E4 promoter could provide a valuable tool for regulating gene expression in the senescing leaves.

Inactivation of the cytochrome P450 gene CYP82E2 by degenerative mutations was a key event in the evolution of the alkaloid profile of modern tobacco.

The alkaloid profile of cultivated tobacco (Nicotiana tabacum) is different from that of its two progenitors, Nicotiana sylvestris and Nicotiana tomentosiformis, in that tobacco accumulates nicotine as the most abundant alkaloid, while its ancestors convert nicotine to nornicotine in the senescing leaf. The nicotine-retaining phenotype of tobacco is thought to have evolved through the inactivation of the conversion loci inherited from its two progenitors. Here, the genetic changes associated with the inactivation of the conversion locus derived from N. sylvestris were investigated. Candidate genes were isolated from a N. sylvestris senescing leaf cDNA library and characterized by heterologous gene expression in yeast, site-directed mutagenesis and quantitative real-time polymerase chain reaction. A cytochrome P450 gene, designated NsylCYP82E2, was isolated from N. sylvestris. Located on the chromosomal fragment defined by the N. sylvestris conversion locus, NsylCYP82E2 confers high nicotine N-demethylase (NND) activity in the senescing leaves of N. sylvestris, but the gene is inactivated by two degenerative mutations in tobacco. Collectively with previously published data, these results show that inactivation of NND genes by degenerative mutations and/or transcriptional suppression played a key role in the evolution of the alkaloid profile of modern tobacco.