Evolution of cytosolic and organellar invertases empowered the colonization and thriving of land plants.

The molecular innovation underpinning efficient carbon and energy metabolism during evolution of land plants remains largely unknown. Invertase-mediated sucrose cleavage into hexoses is central to fuel growth. Why some cytoplasmic invertases (CINs) function in the cytosol, whereas others operate in chloroplasts and mitochondria, is puzzling. We attempted to shed light on this question from an evolutionary perspective. Our analyses indicated that plant CINs originated from a putatively orthologous ancestral gene in cyanobacteria and formed the plastidic CIN (α1 clade) through endosymbiotic gene transfer, while its duplication in algae with a loss of its signal peptide produced the ß clade CINs in the cytosol. The mitochondrial CINs (α2) were derived from duplication of the plastidic CINs and coevolved with vascular plants. Importantly, the copy number of mitochondrial and plastidic CINs increased upon the emergence of seed plants, corresponding with the rise of respiratory, photosynthetic, and growth rates. The cytosolic CIN (ß subfamily) kept expanding from algae to gymnosperm, indicating its role in supporting the increase in carbon use efficiency during evolution. Affinity purification mass spectrometry identified a cohort of proteins interacting with α1 and 2 CINs, which points to their roles in plastid and mitochondrial glycolysis, oxidative stress tolerance, and the maintenance of subcellular sugar homeostasis. Collectively, the findings indicate evolutionary roles of α1 and α2 CINs in chloroplasts and mitochondria for achieving high photosynthetic and respiratory rates, respectively, which, together with the expanding of cytosolic CINs, likely underpin the colonization of land plants through fueling rapid growth and biomass production.

Fine mapping and candidate gene analysis of CaFCD1 affecting cuticle biosynthesis in Capsicum annuum L.

KEY MESSAGE: CaFCD1 gene regulates pepper cuticle biosynthesis. Pepper (Capsicum annuum L.) is an economically important vegetable crop that easily loses water after harvesting, which seriously affects the quality of its product. The cuticle is the lipid water-retaining layer on the outside of the fruit epidermis, which regulates the biological properties and reduces the rate of water-loss. However, the key genes involved in pepper fruit cuticle development are poorly understood. In this study, a pepper fruit cuticle development mutant fcd1 (fruit cuticle deficiency 1) was obtained by ethyl methanesulfonate mutagenesis. The mutant has great defects in fruit cuticle development, and the fruit water-loss rate of fcd1is significantly higher than that of the wild-type '8214' line. Genetic analysis suggested that the phenotype of the mutant fcd1 cuticle development defect was controlled by a recessive candidate gene CaFCD1 (Capsicum annuum fruit cuticle deficiency 1) on chromosome 12, which is mainly transcribed during fruit development. In fcd1, a base substitution within the CaFCD1 domain resulted in the premature termination of transcription, which affected cutin and wax biosynthesis in pepper fruit, as revealed by the GC-MS and RNA-seq analysis. Furthermore, the yeast one-hybrid and dual-luciferase reporter assays verified that the cutin synthesis protein CaCD2 was directly bound to the promoter of CaFCD1, suggesting that CaFCD1 may be a hub node in the cutin and wax biosynthetic regulatory network in pepper. This study provides a reference for candidate genes of cuticle synthesis and lays a foundation for breeding excellent pepper varieties.

Analysis of the response regulatory network of pepper genes under hydrogen peroxide stress.

Hydrogen peroxide (H2O2) is a regulatory component related to plant signal transduction. To better understand the genome-wide gene expression response to H2O2 stress in pepper plants, a regulatory network of H2O2 stress-gene expression in pepper leaves and roots was constructed in the present study. We collected the normal tissues of leaves and roots of pepper plants after 40 days of H2O2 treatment and obtained the RNA-seq data of leaves and roots exposed to H2O2 for 0.5-24 h. By comparing the gene responses of pepper leaves and roots exposed to H2O2 stress for different time periods, we found that the response in roots reached the peak at 3 h, whereas the response in leaves reached the peak at 24 h after treatment, and the response degree in the roots was higher than that in the leaves. We used all datasets for K-means analysis and network analysis identified the clusters related to stress response and related genes. In addition, CaEBS1, CaRAP2, and CabHLH029 were identified through a co-expression analysis and were found to be strongly related to several reactive oxygen species-scavenging enzyme genes; their homologous genes in Arabidopsis showed important functions in response to hypoxia or iron uptake. This study provides a theoretical basis for determining the dynamic response process of pepper plants to H2O2 stress in leaves and roots, as well as for determining the critical time and the molecular mechanism of H2O2 stress response in leaves and roots. The candidate transcription factors identified in this study can be used as a reference for further experimental verification.

Integrated analysis of multi-omics and fine-mapping reveals a candidate gene regulating pericarp color and flavonoids accumulation in wax gourd (Benincasa hispida).

Wax gourd (Benincasa hispida), a popular fruit of the Cucurbitaceae (cucurbits) family, contains many nutrients with health benefits and is widely grown in China and other tropical areas. In this study, a wax gourd mutant hfc12 with light-color pericarp was obtained through ethane methylsulfonate (EMS) mutagenesis. Integrative analysis of the metabolome and transcriptome identified 31 differentially accumulated flavonoids (DAFs; flavonoids or flavonoid glycosides) and 828 differentially expressed genes (DEGs) between the hfc12 mutant and wild-type 'BWT'. Furthermore, BSA-seq and kompetitive allele specific PCR (KASP) analysis suggested that the light-color pericarp and higher flavonoid content was controlled by a single gene BhiPRR6 (Bhi12M000742), a typical two-component system (TCS) pseudo-response regulator (PRR). Genetic analysis detected only one nonsynonymous mutation (C-T) in the second exon region of the BhiPRR6. Weighted correlation network analysis (WGCNA) identified the downstream target genes of BhiPRR6, probably regulated by light and were intermediated in the regulatory enzyme reaction of flavonoid biosynthetic pathway. Thus, these results speculated that the transcription factor BhiPRR6, interacting with multiple genes, regulates the absorption of light signals and thereby changes the pericarp color and synthesis of flavonoids in wax gourd.

Evolution and functional diversification of catalase genes in the green lineage.

BACKGROUND: Catalases (CATs) break down hydrogen peroxide into water and oxygen to prevent cellular oxidative damage, and play key roles in the development, biotic and abiotic stresses of plants. However, the evolutionary relationships of the plant CAT gene family have not been systematically reported. RESULTS: Here, we conducted genome-wide comparative, phylogenetic, and structural analyses of CAT orthologs from 29 out of 31 representative green lineage species to characterize the evolution and functional diversity of CATs. We found that CAT genes in land plants were derived from core chlorophytes and detected a lineage-specific loss of CAT genes in Fabaceae, suggesting that the CAT genes in this group possess divergent functions. All CAT genes were split into three major groups (group α, ß1, and ß2) based on the phylogeny. CAT genes were transferred from bacteria to core chlorophytes and charophytes by lateral gene transfer, and this led to the independent evolution of two types of CAT genes: α and ß types. Ten common motifs were detected in both α and ß groups, and ß CAT genes had five unique motifs, respectively. The findings of our study are inconsistent with two previous hypotheses proposing that (i) new CAT genes are acquired through intron loss and that (ii) the Cys-343 residue is highly conserved in plants. We found that new CAT genes in most higher plants were produced through intron acquisition and that the Cys-343 residue was only present in monocots, Brassicaceae and Pp_CatX7 in P. patens, which indicates the functional specificity of the CATs in these three lineages. Finally, our finding that CAT genes show high overall sequence identity but that individual CAT genes showed developmental stage and organ-specific expression patterns suggests that CAT genes have functionally diverged independently. CONCLUSIONS: Overall, our analyses of the CAT gene family provide new insights into their evolution and functional diversification in green lineage species.

Genome-Wide Characterization and Anthocyanin-Related Expression Analysis of the B-BOX Gene Family in Capsicum annuum L.

The transcription factors, B-box (BBX), belong to a subfamily of the zinc finger family of proteins and exhibit multiple biological functions in plant growth, development, and abiotic stress response pathways. In this study, a total of 23 CaBBX members were identified using the pepper reference genome database. According to the gene structure, conserved domains, and the phylogenetic tree, 23 CaBBX genes were divided into four groups, wherein the analysis of the promoter region indicated the presence of cis-acting elements related to plant development, hormones, and stress response. Interspecies collinearity analysis showed that the CaBBXs had three duplicated gene pairs, and the highest gene density was found on chromosomes 2 and 7. Transcriptome RNA-seq data and quantitative polymerase chain reaction (qRT-PCR) analysis of pepper plants spanning the entire period showed that more than half of the CaBBX genes were widely expressed in diversity tissues of pepper. Co-expression network analysis indicated that the CaBBXs and the anthocyanin structural genes had a close co-expression relationship. Thus, it was reasonably speculated that the CaBBX genes may be involved in the regulation of anthocyanin biosynthesis. Overall, this study involved the genome-wide characterization of the CaBBX family and may serve as a solid foundation for further investigations on CaBBX genes involved in the anthocyanin synthesis mechanisms and development in pepper.

Genomic Organization and Comparative Phylogenic Analysis of NBS-LRR Resistance Gene Family in Solanum pimpinellifolium and Arabidopsis thaliana.

NBS-LRR (nucleotide-binding site and leucine-rich repeat) is one of the largest resistance gene families in plants. The completion of the genome sequencing of wild tomato Solanum pimpinellifolium provided an opportunity to conduct a comprehensive analysis of the NBS-LRR gene superfamily at the genome-wide level. In this study, gene identification, chromosome mapping, and phylogenetic analysis of the NBS-LRR gene family were analyzed using the bioinformatics methods. The results revealed 245 NBS-LRRs in total, similar to that in the cultivated tomato. These genes are unevenly distributed on 12 chromosomes, and ~59.6% of them form gene clusters, most of which are tandem duplications. Phylogenetic analysis divided the NBS-LRRs into 2 subfamilies (CNL-coiled-coil NBS-LRR and TNL-TIR NBS-LRR), and the expansion of the CNL subfamily was more extensive than the TNL subfamily. Novel conserved structures were identified through conserved motif analysis between the CNL and TNL subfamilies. Compared with the NBS-LRR sequences from the model plant Arabidopsis thaliana, wide genetic variation occurred after the divergence of S. pimpinellifolium and A thaliana. Species-specific expansion was also found in the CNL subfamily in S. pimpinellifolium. The results of this study provide the basis for the deeper analysis of NBS-LRR resistance genes and contribute to mapping and isolation of candidate resistance genes in S. pimpinellifolium.

Evolutionary Conservation and Expression Patterns of Neutral/Alkaline Invertases in Solanum.

The invertase gene family in plants is composed of two subfamilies of enzymes, namely, acid- and neutral/alkaline invertases (cytosolic invertase, CIN). Both can irreversibly cleave sucrose into fructose and glucose, which are thought to play key roles in carbon metabolism and plant growth. CINs are widely found in plants, but little is reported about this family. In this paper, a comparative genomic approach was used to analyze the CIN gene family in Solanum, including Solanumtuberosum, Solanumlycopersicum, Solanumpennellii, Solanumpimpinellifolium, and Solanummelongena. A total of 40 CINs were identified in five Solanum plants, and sequence features, phylogenetic relationships, motif compositions, gene structure, collinear relationship, and expression profile were further analyzed. Sequence analysis revealed a remarkable conservation of CINs in sequence length, gene number, and molecular weight. The previously verified four amino acid residues (D188, E414, Arg430, and Ser547) were also observed in 39 out of 40 CINs in our study, showing to be deeply conserved. The CIN gene family could be distinguished into groups α and ß, and α is further subdivided into subgroups α1 and α2 in our phylogenetic tree. More remarkably, each species has an average of four CINs in the α and ß groups. Marked interspecies conservation and collinearity of CINs were also further revealed by chromosome mapping. Exon-intron configuration and conserved motifs were consistent in each of these α and ß groups on the basis of in silico analysis. Expression analysis indicated that CINs were constitutively expressed and share similar expression profiles in all tested samples from S. tuberosum and S.lycopersicum. In addition, in CIN genes of the tomato and potato in response to abiotic and biotic stresses, phytohormones also performed. Overall, CINs in Solanum were encoded by a small and highly conserved gene family, possibly reflecting structural and functional conservation in Solanum. These results lay the foundation for further expounding the functional characterization of CIN genes and are also significant for understanding the evolutionary profiling of the CIN gene family in Solanum.

Analysis of the ASMT Gene Family in Pepper (Capsicum annuum L.): Identification, Phylogeny, and Expression Profiles.

Acetylserotonin methyltransferase (ASMT) in plant species, one of the most important enzymes in melatonin biosynthesis, plays a rate-limiting role in the melatonin production. In this study, based on the whole genome sequence, we performed a systematic analysis for the ASMT gene family in pepper (Capsicum annuum L.) and analyzed their expression profiles during growth and development, as well as abiotic stresses. The results showed that at least 16 CaASMT genes were identified in the pepper genome. Phylogenetic analyses of all the CaASMTs were divided into three groups (group I, group II, and group III) with a high bootstrap value. Through the online MEME tool, six distinct motifs (motif 1 to motif 6) were identified. Chromosome location found that most CaASMT genes were mapped in the distal ends of the pepper chromosomes. In addition, RNA-seq analysis revealed that, during the vegetative and reproductive development, the difference in abundance and distinct expression patterns of these CaASMT genes suggests different functions. The qRT-PCR analysis showed that high abundance of CaASMT03, CaASMT04, and CaASMT06 occurred in mature green fruit and mature red fruit. Finally, using RNA-seq and qRT-PCR technology, we also found that several CaASMT genes were induced under abiotic stress conditions. The results will not only contribute to elucidate the evolutionary relationship of ASMT genes but also ascertain the biological function in pepper plant response to abiotic stresses.

The Tryptophan Decarboxylase in Solanum lycopersicum.

Melatonin plays an important role in plant growth, development, and environmental stress. In this study, a systematic analysis of tomato tryptophan decarboxylase (SlTrpDC), which is the first enzyme of melatonin biosynthesis, was conducted by integrating structural features, phylogenetic relationships, an exon/intron feature, and a divergent expression profile. The results determined that the tomato genome encoded five members (SlTrpDC1-SlTrpDC5). The phylogenetic relationships indicated that gene expansion was proposed as the major mode of evolution of the TrpDC genes from the different plant algae species to the higher plants species. The analyses of the exon/intron configurations revealed that the intron loss events occurred during the structural evolution of the TrpDCs in plants. Additionally, the RNA-seq and qRT-PCR analysis revealed that the expression of the SlTrpDC3 was high in all of the tested tissues, while the SlTrpDC4 and SlTrpDC5 were not expressed. The expression patterns of the remaining two (SlTrpDC1 and SlTrpDC2) were tissue-specific, which indicated that these genes may play important roles within the different tissues. No expression difference was observed in the tomato plants in response to the biotic stresses. This study will expand the current knowledge of the roles of the TrpDC genes in tomato growth and development.