Summary: Plants produce countless specialized metabolites crucial for their development and fitness, and many are useful bioactive compounds. Capsaicinoids are intriguing genus‐specialized metabolites that confer a pungent flavor to Capsicum fruits, and they are widely applied in different areas. Among the five domesticated Capsicum species, Capsicum chinense has a high content of capsaicinoids, which results in an extremely hot flavor. However, the species‐specific upregulation of capsaicinoid‐biosynthetic genes (CBGs) and the evolution of extremely pungent peppers are not well understood. We conducted genetic and functional analyses demonstrating that the quantitative trait locus Capsaicinoid1 (Cap1), which is identical to Pun3 contributes to the level of pungency. The Cap1/Pun3 locus encodes the Solanaceae‐specific MYB transcription factor MYB31. Capsicum species have evolved placenta‐specific expression of MYB31, which directly activates expression of CBGs and results in genus‐specialized metabolite production. The capsaicinoid content depends on MYB31 expression. Natural variations in the MYB31 promoter increase MYB31 expression in C. chinense via the binding of the placenta‐specific expression of transcriptional activator WRKY9 and augmentation of CBG expression, which promotes capsaicinoid biosynthesis. Our findings provide insights into the evolution of extremely pungent C. chinense, which is due to natural variations in the master regulator, and offers targets for engineering or selecting flavor in Capsicum.

The organization of chromatin into self-interacting domains is universal among eukaryotic genomes, though how and why they form varies considerably. Here we report a chromosome-scale reference genome assembly of pepper (Capsicum annuum) and explore its 3D organization through integrating high-resolution Hi-C maps with epigenomic, transcriptomic, and genetic variation data. Chromatin folding domains in pepper are as prominent as TADs in mammals but exhibit unique characteristics. They tend to coincide with heterochromatic regions enriched with retrotransposons and are frequently embedded in loops, which may correlate with transcription factories. Their boundaries are hotspots for chromosome rearrangements but are otherwise depleted for genetic variation. While chromatin conformation broadly affects transcription variance, it does not predict differential gene expression between tissues. Our results suggest that pepper genome organization is explained by a model of heterochromatin-driven folding promoted by transcription factories and that such spatial architecture is under structural and functional constraints.

Background: The basic helix–loop–helix (bHLH) transcription factors (TFs) serve crucial roles in regulating plant growth and development and typically participate in biological processes by interacting with other TFs. Capsorubin and capsaicinoids are found only in Capsicum, which has high nutritional and economic value. However, whether bHLH family genes regulate capsorubin and capsaicinoid biosynthesis and participate in these processes by interacting with other TFs remains unknown. Results: In this study, a total of 107 CabHLHs were identified from the Capsicum annuum genome. Phylogenetic tree analysis revealed that these CabHLH proteins were classified into 15 groups by comparing the CabHLH proteins with Arabidopsis thaliana bHLH proteins. The analysis showed that the expression profiles of CabHLH009, CabHLH032, CabHLH048, CabHLH095 and CabHLH100 found in clusters C1, C2, and C3 were similar to the profile of carotenoid biosynthesis in pericarp, including zeaxanthin, lutein and capsorubin, whereas the expression profiles of CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 found in clusters L5, L6 and L9 were consistent with the profile of capsaicinoid accumulation in the placenta. Moreover, CabHLH007, CabHLH009, CabHLH026 and CabHLH086 also might be involved in temperature-mediated capsaicinoid biosynthesis. Yeast two-hybrid (Y2H) assays demonstrated that CabHLH007, CabHLH009, CabHLH026, CabHLH063 and CabHLH086 could interact with MYB31, a master regulator of capsaicinoid biosynthesis. Conclusions: The comprehensive and systematic analysis of CabHLH TFs provides useful information that contributes to further investigation of CabHLHs in carotenoid and capsaicinoid biosynthesis.

Plants are constantly challenged by environmental stresses, including drought and high salinity. Improvement of drought and osmotic stress tolerance without yield decrease has been a great challenge in crop improvement. The Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a protein of the class IV HD-Zip family, has been demonstrated to significantly improve drought tolerance in Arabidopsis, rice and pepper. Here, we report that AtEDT1/HDG11 confers drought and osmotic stress tolerance in the Chinese kale. AtEDT1/HDG11-overexpression lines exhibit auxin-overproduction phenotypes, such as long hypocotyls, tall stems, more root hairs and a larger root system architecture. Compared with the untransformed control, transgenic lines have significantly reduced stomatal density. In the leaves of transgenic Chinese kale plants, proline (Pro) content and reactive oxygen species-scavenging enzyme activity was significantly increased after drought and osmotic stress, particularly compared to wild kale. More importantly, AtEDT1/HDG11-overexpression leads to abscisic acid (ABA) hypersensitivity, resulting in ABA inhibitor germination and induced stomatal closure. Consistent with observed phenotypes, the expression levels of auxin, ABA and stress-related genes were also altered under both normal and/or stress conditions. Further analysis showed that AtEDT1/HDG11, as a transcription factor, can target the auxin biosynthesis gene YUCC6 and ABA response genes ABI3 and ABI5. Collectively, our results provide a new insight into the role of AtEDT1/HDG11 in enhancing abiotic stress resistance through auxin- and ABA- mediated signaling response in Chinese kale.

Background: ERF transcription factors (TFs) belong to the Apetala2/Ethylene responsive Factor (AP2/ERF) TF family and play a vital role in plant growth and development processes. Capsorubin and capsaicinoids have relatively high economic and nutritional value, and they are specifically found in Capsicum. However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis. Results: In this study, a total of 142 ERFs were identified in the Capsicum annuum genome. Subsequent phylogenetic analysis allowed us to divide ERFs into DREB (dehydration responsive element binding proteins) and ERF subfamilies, and further classify them into 11 groups with several subgroups. Expression analysis of biosynthetic pathway genes and CaERFs facilitated the identification of candidate genes related to the regulation of capsorubin and capsaicinoids biosynthesis; the candidates were focused in cluster C9 and cluster C10, as well as cluster L3 and cluster L4, respectively. The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids (β-carotene, zeaxanthin and capsorubin) in the pericarp. In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids. Furthermore, CaERF92, CaERF102 and CaERF111 were found to be potentially involved in temperature-mediated capsaicinoids biosynthesis. Conclusion: This study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers.

Bitter gourd (Momordica charantia L.) is a significant vegetable. Although it has a special bitter taste, it is still popular with the public. The industrialization of bitter gourd could be hampered by a lack of genetic resources. The bitter gourd’s mitochondrial and chloroplast genomes have not been extensively studied. In the present study, the mitochondrial genome of bitter gourd was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of bitter gourd is 331,440 bp with 24 unique core genes, 16 variable genes, 3 rRNAs, and 23 tRNAs. We identified 134 SSRs and 15 tandem repeats in the entire mitochondrial genome of bitter gourd. Moreover, 402 pairs of repeats with a length greater than or equal to 30 were observed in total. The longest palindromic repeat was 523 bp, and the longest forward repeat was 342 bp. We found 20 homologous DNA fragments in bitter gourd, and the summary insert length was 19,427 bp, accounting for 5.86% of the mitochondrial genome. We predicted a total of 447 potential RNA editing sites in 39 unique PCGs and also discovered that the ccmFN gene has been edited the most often, at 38 times. This study provides a basis for a better understanding and analysis of differences in the evolution and inheritance patterns of cucurbit mitochondrial genomes.