Exogenous spraying of IAA improved the efficiency of microspore embryogenesis in Wucai (Brassica campestris L.) by affecting the balance of endogenous hormones, energy metabolism, and cell wall degradation.

BACKGROUND: Microspore embryogenesis is an extraordinarily complicated process, comprehensively regulated by a composite network of physiological and molecular factors, among which hormone is one of the most crucial factors. Auxin is required for stress-induced microspore reprogramming, however, the mechanism of its regulation of microspore embryogenesis is still unclear. RESULTS: In this study, we found exogenously spraying 100 mg·L- 1 IAA on the buds of Wucai significantly increased the rate of microspore embryogenesis, and moreover accelerated the process of embryogenesis. Physiological and biochemical tests showed that the contents of amino acids, soluble total sugar, soluble protein, and starch were significantly increased after IAA treatment. Furthermore, exogenously spraying 100 mg·L- 1 IAA significantly enhanced IAA, GA4, and GA9 content, increased catalase (CAT) and malondialdehyde (MDA) activity, and reduced abscisic acid (ABA), MDA and soluble protopectin content, H2O2 and O2·- production rate in the bud with the largest population of late-uninucleate-stage microspores. Transcriptome sequencing was performed on buds respectively treated with 100 mg·L- 1 IAA and fresh water. A total of 2004 DEGs were identified, of which 79 were involved in micropores development, embryonic development and cell wall formation and modification, most of which were upregulated. KEGG and GO analysis revealed that 9.52% of DEGs were enriched in plant hormone synthesis and signal transduction pathways, pentose and glucuronic acid exchange pathways, and oxidative phosphorylation pathways. CONCLUSIONS: These findings indicated that exogenous IAA altered the contents of endogenous hormone content, total soluble sugar, amino acid, starch, soluble protein, MDA and protopectin, the activities of CAT and peroxidase (POD), and the production rate of H2O2 and O2·-. Combined with transcriptome analysis, it was found that most genes related to gibberellin (GA) and Auxin (IAA) synthesis and signal transduction, pectin methylase (PME) and polygalacturonase (PGs) genes and genes related to ATP synthesis and electron transport chain were upregulated, and genes related to ABA synthesis and signal transduction were downregulated. These results indicated that exogenous IAA treatment could change the balance of endogenous hormones, accelerate cell wall degradation, promote ATP synthesis and nutrient accumulation, inhibit ROS accumulation, which ultimately promote microspore embryogenesis.

Auxin participates in regulating the leaf curl development of Wucai (Brassica campestris L.).

Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) belongs to the Brassica genus of the Cruciferae family, and its leaf curl is a typical feature that distinguishes Wucai from other nonheading cabbage subspecies. Our previous research found that plant hormones were involved in the development of the leaf curl in Wucai. However, the molecular mechanisms and the hormones regulating the formation of leaf curl in Wucai have not yet been reported. This study aimed to understand the molecular functions related to hormone metabolism during the formation of leaf curl in Wucai. A total of 386 differentially expressed genes (DEGs) were identified by transcriptome sequencing of two different morphological parts of the same leaf of Wucai germplasm W7-2, and 50 DEGs were found to be related to plant hormones, which were mainly involved in the auxin signal transduction pathway. Then, we measured the content of endogenous hormones in two different forms of the same leaf of Wucai germplasm W7-2. A total of 17 hormones with differential content were identified, including auxin, cytokinins, jasmonic acids, salicylic acids, and abscisic acid. And we found that treatment with auxin transport inhibitor N-1-naphthylphthalamic acid can affect the leaf curl phenotype of Wucai and pak choi (Brassica rapa L. subsp. Chinensis). These results indicated that plant hormones, especially auxin, are involved in developing the leaf curl of Wucai. Our findings provide a potentially valuable reference for future research on the development of leaf curls.

Combined Metabolome and Transcriptome Analysis Elucidates Sugar Accumulation in Wucai (Brassica campestris L.).

Wucai (Brassica campestris L.) is a leafy vegetable that originated in China, its soluble sugars accumulate significantly to improve taste quality during maturation, and it is widely accepted by consumers. In this study, we investigated the soluble sugar content at different developmental stages. Two periods including 34 days after planting (DAP) and 46 DAP, which represent the period prior to and after sugar accumulation, respectively, were selected for metabolomic and transcriptomic profiling. Differentially accumulated metabolites (DAMs) were mainly enriched in the pentose phosphate pathway, galactose metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and fructose and mannose metabolism. By orthogonal projection to latent structures-discriminant s-plot (OPLS-DA S-plot) and MetaboAnalyst analyses, D-galactose and ß-D-glucose were identified as the major components of sugar accumulation in wucai. Combined with the transcriptome, the pathway of sugar accumulation and the interact network between 26 DEGs and the two sugars were mapped. CWINV4, CEL1, BGLU16, and BraA03g023380.3C had positive correlations with the accumulation of sugar accumulation in wucai. The lower expression of BraA06g003260.3C, BraA08g002960.3C, BraA05g019040.3C, and BraA05g027230.3C promoted sugar accumulation during the ripening of wucai. These findings provide insights into the mechanisms underlying sugar accumulation during commodity maturity, providing a basis for the breeding of sugar-rich wucai cultivars.

Transcriptome analysis and differential gene expression profiling of wucai (Brassica campestris L.) in response to cold stress.

BACKGROUND: Wucai suffers from low temperature during the growth period, resulting in a decline in yield and poor quality. But the molecular mechanisms of cold tolerance in wucai are still unclear. RESULTS: According to the phenotypes and physiological indexes, we screened out the cold-tolerant genotype "W18" (named CT) and cold-sensitive genotype "Sw-1" (named CS) in six wucai genotypes. We performed transcriptomic analysis using seedling leaves after 24 h of cold treatment. A total of 3536 and 3887 differentially expressed genes (DEGs) were identified between the low temperature (LT) and control (NT) comparative transcriptome in CT and CS, respectively, with 1690 DEGs specific to CT. The gene ontology (GO) analysis showed that the response to cadmium ion (GO:0,046,686), response to jasmonic acid (GO:0,009,753), and response to wounding (GO:0,009,611) were enriched in CT (LT vs NT). The DEGs were enriched in starch and sucrose metabolism and glutathione metabolism in both groups, and α-linolenic acid metabolism was enriched only in CT (LT vs NT). DEGs in these processes, including glutathione S-transferases (GSTs), 13S lipoxygenase (LOX), and jasmonate ZIM-domain (JAZ), as well as transcription factors (TFs), such as the ethylene-responsive transcription factor 53 (ERF53), basic helix-loop-helix 92 (bHLH92), WRKY53, and WRKY54.We hypothesize that these genes play important roles in the response to cold stress in this species. CONCLUSIONS: Our data for wucai is consistent with previous studies that suggest starch and sucrose metabolism increased the content of osmotic substances, and the glutathione metabolism pathway enhance the active oxygen scavenging. These two pathways may participated in response to cold stress. In addition, the activation of α-linolenic acid metabolism may promote the synthesis of methyl jasmonate (MeJA), which might also play a role in the cold tolerance of wucai.

Genome-wide analysis of the DREB family genes and functional identification of the involvement of BrDREB2B in abiotic stress in wucai (Brassica campestris L.).

Dehydration responsive element binding protein (DREB) is a significant transcription factor class known to be implicated in abiotic stresses. In this study, we systematically conducted a genome-wide identification and expression analysis of the DREB gene family, including gene structures, evolutionary relationships, chromosome distribution, conserved domains, and expression patterns. A total of 65 DREB family gene members were identified in Chinese cabbage (Brassica rapa L.) and were classified into five subgroups based on phylogenetic analysis. Through analysis of the conserved domains of BrDREB family genes, only one exon existed in the gene structure. Through the analysis of cis-acting elements, these genes were mainly involved in hormone regulation and adversity stress. In order to identify the function of BrDREB2B, overexpressed transgenic Arabidopsis was constructed. After different stress treatments, the germination rate, root growth, survival rate, and various plant physiological indicators were measured. The results showed that transgenic Arabidopsis thaliana plants overexpressing BrDREB2B exhibited enhanced tolerance to salt, heat and drought stresses. Taken together, our results are the first to report the BrDREB2B gene response to drought and heat stresses in Chinese cabbage and provide a basis for further studies to determine the function of BrDREBs in response to abiotic stresses.

Mapping and Validation of BrGOLDEN: A Dominant Gene Regulating Carotenoid Accumulation in Brassica rapa.

In plants, the accumulation of carotenoids can maintain the balance of the photosystem and improve crop nutritional quality. Therefore, the molecular mechanisms underlying carotenoid synthesis and accumulation should be further explored. In this study, carotenoid accumulation differed significantly among parental Brassica rapa. Genetic analysis was carried out using the golden inner leaf '1900264' line and the light-yellow inner leaf '1900262' line, showing that the golden inner leaf phenotype was controlled by a single dominant gene. Using bulked-segregant analysis sequencing, BraA09g007080.3C encoding the ORANGE protein was selected as a candidate gene. Sequence alignment revealed that a 4.67 kb long terminal repeat insertion in the third exon of the BrGOLDEN resulted in three alternatively spliced transcripts. The spatiotemporal expression results indicated that BrGOLDEN might regulate the expression levels of carotenoid-synthesis-related genes. After transforming BrGOLDEN into Arabidopsis thaliana, the seed-derived callus showed that BrGOLDENIns and BrGOLDENDel lines presented a yellow color and the BrGOLDENLdel line presented a transparent phenotype. In addition, using the yeast two-hybrid assay, BrGOLDENIns, BrGOLDENLdel, and Brgoldenwt exhibited strong interactions with BrPSY1, but BrGOLDENDel did not interact with BrPSY1 in the split-ubiquitin membrane system. In the secondary and 3D structure analysis, BrGOLDENDel was shown to have lost the PNFPSFIPFLPPL sequences at the 125 amino acid position, which resulted in the α-helices of BrGOLDENDel being disrupted, restricting the formation of the 3D structure and affecting the functions of the protein. These findings may provide new insights into the regulation of carotenoid synthesis in B. rapa.

Transcriptome Analysis Reveals the Symbiotic Mechanism of Ustilago esculenta-Induced Gall Formation of Zizania latifolia.

Zizania latifolia is a perennial aquatic vegetable, whose symbiosis with the fungus Ustilago esculenta (member of Basidiomycota, class Ustilaginaceae) results in the establishment of swollen gall formations. Here, we analyzed symbiotic relations of Z. latifolia and U. esculenta, using a triadimefon (TDF) treatment and transcriptome sequencing (RNA-seq). Specifically, accurately identify the whole growth cycle of Z. latifolia. Microstructure observations showed that the presence of U. esculenta could be clearly observed after gall formation but was absent after the TDF treatment. A total of 17,541 differentially expressed genes (DEGs) were identified, based on the transcriptome. According to gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway results, plant hormone signal transduction, and cell wall-loosening factors were all significantly enriched due to U. esculenta infecting Z. latifolia; relative expression levels of hormone-related genes were identified, of which downregulation of indole 3-acetic acid (IAA)-related DEGs was most pronounced in JB_D versus JB_B. The ultra-high performance liquid chromatography analysis revealed that IAA, zeatin+trans zeatin riboside, and gibberellin 3 were increased under U. esculenta infection. Based on our results, we proposed a hormone-cell wall loosening model to study the symbiotic mechanism of gall formation after U. esculenta infects Z. latifolia. Our study thus provides a new perspective for studying the physiological and molecular mechanisms of U. esculenta infection of Z. latifolia causing swollen gall formations as well as a theoretical basis for enhancing future yields of cultivated Z. latifolia.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

Morphological characteristics and transcriptome analysis at different anther development stages of the male sterile mutant MS7-2 in Wucai (Brassica campestris L.).

BACKGROUND: The discovery of male sterile materials is of great significance for the development of plant fertility research. Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variety of non-heading Chinese cabbage. There are few studies on the male sterility of wucai, and the mechanism of male sterility is not clear. In this study, the male sterile mutant MS7-2 and the wild-type fertile plant MF7-2 were studied. RESULTS: Phenotypic characteristics and cytological analysis showed that MS7-2 abortion occurred at the tetrad period. The content of related sugars in the flower buds of MS7-2 was significantly lower than that of MF7-2, and a large amount of reactive oxygen species (ROS) was accumulated. Through transcriptome sequencing of MS7-2 and MF7-2 flower buds at three different developmental stages (a-c), 2865, 3847, and 4981 differentially expressed genes were identified in MS7-2 at the flower bud development stage, stage c, and stage e, respectively, compared with MF7-2. Many of these genes were enriched in carbohydrate metabolism, phenylpropanoid metabolism, and oxidative phosphorylation, and most of them were down-regulated in MS7-2. The down-regulation of genes involved in carbohydrate and secondary metabolite synthesis as well as the accumulation of ROS in MS7-2 led to pollen abortion in MS7-2. CONCLUSIONS: This study helps elucidate the mechanism of anther abortion in wucai, providing a basis for further research on the molecular regulatory mechanisms of male sterility and the screening and cloning of key genes in wucai.

Characterization and transcriptomic analysis of a novel yellow-green leaf wucai (Brassica campestris L.) germplasm.

BACKGROUND: Leaf color mutants are the ideal materials to explore the pathways of chlorophyll (Chl) metabolism, chloroplast development, and photosynthesis system. In this study, a spontaneous yellow-green leaf wucai (Brassica campestris L.) mutant "WY16-13" was identified, which exhibited yellow-green leaf color during its entire growth period. However, current understanding of the molecular mechanism underlying Chl metabolism and chloroplast development of "WY16-13" is limited. RESULTS: Total Chl and carotenoid content in WY16-13 was reduced by 60.92 and 58.82%, respectively, as compared with its wild type parental line W16-13. Electron microscopic investigation revealed fewer chloroplasts per cell and looser stroma lamellae in WY16-13 than in W16-13. A comparative transcriptome profiling was performed using leaves from the yellow-green leaf type (WY16-13) and normal green-leaf type (W16-13). A total of 54.12 million (M) (WY16-13) and 56.17 M (W16-13) reads were generated. A total of 40,578 genes were identified from the mapped libraries. We identified 3882 differentially expressed genes (DEGs) in WY16-13 compared with W16-13 (i.e., 1603 upregulated genes and 2279 downregulated genes). According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, these DEGs are involved in porphyrin and Chl metabolism [i.e., chlorophyllase (CLH), heme oxygenase (HO), chlorophyll (ide) b reductase (NYC), and protochlorophyllide oxidoreductase (POR) genes], carbohydrate metabolism, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, deficiency in Chl biosynthetic intermediates in WY16-13 revealed that the formation of the yellow-green phenotype was related to the disorder of heme metabolism. CONCLUSIONS: Our results provide valuable insights into Chl deficiency in the yellow-green leaf mutant and a bioinformatics resource for further functional identification of key allelic genes responsible for differences in Chl content.

Gene co-expression network analysis reveals key pathways and hub genes in Chinese cabbage (Brassica rapa L.) during vernalization.

BACKGROUND: Vernalization is a type of low temperature stress used to promote rapid bolting and flowering in plants. Although rapid bolting and flowering promote the reproduction of Chinese cabbages (Brassica rapa L. ssp. pekinensis), this process causes their commercial value to decline. Clarifying the mechanisms of vernalization is essential for its further application. We performed RNA sequencing of gradient-vernalization in order to explore the reasons for the different bolting process of two Chinese cabbage accessions during vernalization. RESULTS: There was considerable variation in gene expression between different-bolting Chinese cabbage accessions during vernalization. Comparative transcriptome analysis and weighted gene co-expression network analysis (WGCNA) were performed for different-bolting Chinese cabbage during different vernalization periods. The biological function analysis and hub gene annotation of highly relevant modules revealed that shoot system morphogenesis and polysaccharide and sugar metabolism caused early-bolting 'XBJ' to bolt and flower faster; chitin, ABA and ethylene-activated signaling pathways were enriched in late-bolting 'JWW'; and leaf senescence and carbohydrate metabolism enrichment were found in the two Chinese cabbage-related modules, indicating that these pathways may be related to bolting and flowering. The high connectivity of hub genes regulated vernalization, including MTHFR2, CPRD49, AAP8, endoglucanase 10, BXLs, GATLs, and WRKYs. Additionally, five genes related to flower development, BBX32 (binds to the FT promoter), SUS1 (increases FT expression), TSF (the closest homologue of FT), PAO and NAC029 (plays a role in leaf senescence), were expressed in the two Chinese cabbage accessions. CONCLUSION: The present work provides a comprehensive overview of vernalization-related gene networks in two different-bolting Chinese cabbages during vernalization. In addition, the candidate pathways and hub genes related to vernalization identified here will serve as a reference for breeders in the regulation of Chinese cabbage production.

Transcriptional profiling reveals changes in gene regulation and signaling transduction pathways during temperature stress in wucai (Brassica campestris L.).

BACKGROUND: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a cold-tolerant plant that is vulnerable to high temperature. This study explored the response mechanism of wucai to low temperature. In this study, wucai seedlings were treated with different temperatures, including low temperature (LT), high temperature (HT), and a control. RESULTS: According to transcriptomics analysis, the number of differentially expressed genes (DEGs) in HT and LT was 10,702 and 7267, respectively, compared with the control. The key genes associated with the physiological response of wucai to the treatments were analyzed. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology annotations indicated the importance of the photosynthesis and photosynthetic-antenna protein pathways. We found that a high-temperature environment greatly inhibited the expression of important genes in the photosynthetic pathway (BrLhc superfamily members, PsaD, PsaE, PsaD, PsaD, PsbO, PsbP, PsbQ, PsbR, PsbS, PsbW, PsbY, Psb27, and Psb28), whereas low temperature resulted in the expression of certain key genes (BrLhc superfamily members, Psa F, Psa H, Psb S, Psb H, Psb 28). In addition, the wucai seedlings exhibited better photosynthetic performance under low-temperature conditions than high-temperature conditions. CONCLUSIONS: Based on the above results, we speculate that upon exposure to low temperature, the plants developed higher cold tolerance by upregulating the expression of genes related to photosynthesis. Conversely, high-temperature stress inhibited the expression of pivotal genes and weakened the self-regulating ability of the plants.

Comparative transcriptome analysis reveals that chlorophyll metabolism contributes to leaf color changes in wucai (Brassica campestris L.) in response to cold.

BACKGROUND: Chlorophyll (Chl) is a vital photosynthetic pigment involved in capturing light energy and energy conversion. In this study, the color conversion of inner-leaves from green to yellow in the new wucai (Brassica campestris L.) cultivar W7-2 was detected under low temperature. The W7-2 displayed a normal green leaf phenotype at the seedling stage, but the inner leaves gradually turned yellow when the temperature was decreased to 10 °C/2 °C (day/night), This study facilitates us to understand the physiological and molecular mechanisms underlying leaf color changes in response to low temperature. RESULTS: A comparative leaf transcriptome analysis of W7-2 under low temperature treatment was performed on three stages (before, during and after leaf color change) with leaves that did not change color under normal temperature at the same period as a control. A total of 67,826 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis revealed that the DEGs were mainly enriched in porphyrin and Chl metabolism, carotenoids metabolism, photosynthesis, and circadian rhythm. In the porphyrin and chlorophyll metabolic pathways, the expression of several genes was reduced [i.e. magnesium chelatase subunit H (CHLH)] under low temperature. Almost all genes [i.e. phytoene synthase (PSY)] in the carotenoids (Car) biosynthesis pathway were downregulated under low temperature. The genes associated with photosynthesis [i.e. photosystem II oxygen-evolving enhancer protein 1 (PsbO)] were also downregulated under LT. Our study also showed that elongated hypocotyl5 (HY5), which participates in circadian rhythm, and the metabolism of Chl and Car, is responsible for the regulation of leaf color change and cold tolerance in W7-2. CONCLUSIONS: The color of inner-leaves was changed from green to yellow under low temperature in temperature-sensitive mutant W7-2. Physiological, biochemical and transcriptomic studies showed that HY5 transcription factor and the downstream genes such as CHLH and PSY, which regulate the accumulation of different pigments, are required for the modulation of leaf color change in wucai under low temperature.

Genome-wide analysis of proline-rich extension-like receptor protein kinase (PERK) in Brassica rapa and its association with the pollen development.

BACKGROUND: Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development. RESULTS: Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERKs were investigated. Gene duplication and synteny analyses and the calculation of Ka/Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana, B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERKs exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds. CONCLUSION: Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.

Response of photosynthetic capacity and antioxidative system of chloroplast in two wucai (Brassica campestris L.) genotypes against chilling stress.

Chilling stress during the growing season could cause a series of changes in wucai (Brassica campestris L.). WS-1 (chilling-tolerant genotype) and Ta2 (chilling-sensitive genotype) were sampled in present study to explore the chilling tolerance mechanisms. Our results indicated that photosynthetic parameters exhibited lower level in Ta2 than in WS-1 under chilling stress. The rapid chlorophyll fluorescence dynamics curve showed that chilling resulted in a greater inactivation of photosystem II reaction center in Ta2. Reactive oxygen species and malondialdehyde content of chloroplast in Ta2 were higher than WS-1. The ascorbate-glutathione cycle in chloroplast of WS-1 played a more crucial role than Ta2, which was confirmed by higher activities of antioxidant enzymes including Ascorbate peroxidase, Glutathione reductase, Monodehydroascorbate reductase and Dehydroascorbate reductase and higher content of AsA and GSH. In addition, the ultrastructure of chloroplasts in Ta2 was more severely damaged. After low temperature stress, the shape of starch granules in Ta2 changed from elliptical to round and the volume became larger than that of WS-1. The thylakoid structure of Ta2 also became dispersed from the original tight arrangement. Combined with our previous study under heat stress, WS-1 can tolerant both chilling stress and heat stress, which was partly due to a stable photosynthetic system and the higher active antioxidant system in plants, in comparison to Ta2.

Comparative Proteomic Analysis Reveals That Chlorophyll Metabolism Contributes to Leaf Color Changes in Wucai ( Brassica campestris L.) Responding to Cold Acclimation.

Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai ( Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7-2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7-1 was selected as the control to evaluate the chlorophyll metabolism changes of W7-2. Compared to W7-1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7-2 were significantly reduced at five developmental stages (13, 21, 29, 37, and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7-2 and W7-1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W7-2 compared with W7-1, respectively. The expression analysis of genes of three subunits of Mg-chelatase ( CHLI1, CHLD, and CHLH), Genomes Uncoupled 4 ( GUN4), and Thioredoxin ( TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7-2. In particular, PORB showed significant up-regulation in W7-2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7-2 and W7-1 were in terms of altered photosynthesis, carbohydrate, and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7-2 than in W7-1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation.

Transcriptome analysis reveals a positive effect of brassinosteroids on the photosynthetic capacity of wucai under low temperature.

BACKGROUND: Brassinosteroids (BRs) have a positive effect on many processes during plant growth and development, and in response to various abiotic stressors. Low-temperature (LT) stress constricts the geographic distribution, growth, and development of wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). However, there is little information on the global gene expression of BRs under LT stress in wucai. In this study, the molecular roles of 24-epibrassinolide (EBR) after exogenously application, were explored by RNA sequencing under LT conditions. RESULTS: According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, photosynthesis was significantly enriched after spraying EBR under LT. The transcripts encoding the photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin were up-regulated after the application of EBR. Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. Notably, transcriptional and physiological analyses revealed that under LT stress, plant responses to EBR involved a major reorientation of photosynthesis, as well as porphyrin and chlorophyll metabolism. CONCLUSION: This study explored the role of EBR as an LT stress tolerance mechanism in wucai. At the transcription level, LT tolerance manifests as an enhancement of photosynthesis, and the amelioration of porphyrin and chlorophyll metabolism.

Transgenic Wucai (Brassica campestris L.) produced via Agrobacterium-mediated anther transformation in planta.

KEY MESSAGE: We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target. Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59-1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T2 transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.

Comparative Proteomics Indicates That Redox Homeostasis Is Involved in High- and Low-Temperature Stress Tolerance in a Novel Wucai (Brassica campestris L.) Genotype.

The genotype WS-1, previously identified from novel wucai germplasm, is tolerant to both low-temperature (LT) and high-temperature (HT) stress. However, it is unclear which signal transduction pathway or acclimation mechanisms are involved in the temperature-stress response. In this study, we used the proteomic method of tandem mass tag (TMT) coupled with liquid chromatography-mass spectrometry (LC-MS/MS) to identify 1022 differentially expressed proteins (DEPs) common to WS-1, treated with either LT or HT. Among these 1022 DEPs, 172 were upregulated in response to both LT and HT, 324 were downregulated in response to both LT and HT, and 526 were upregulated in response to one temperature stress and downregulated in response to the other. To illustrate the common regulatory pathway in WS-1, 172 upregulated DEPs were further analyzed. The redox homeostasis, photosynthesis, carbohydrate metabolism, heat-shockprotein, and chaperones and signal transduction pathways were identified to be associated with temperature stress tolerance in wucai. In addition, 35S:BcccrGLU1 overexpressed in Arabidopsis, exhibited higher reduced glutathione (GSH) content and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and less oxidative damage under temperature stress. This result is consistent with the dynamic regulation of the relevant proteins involved in redox homeostasis. These data demonstrate that maintaining redox homeostasis is an important common regulatory pathway for tolerance to temperature stress in novel wucai germplasm.

Physiological and Transcriptomic Analyses Elucidate That Exogenous Calcium Can Relieve Injuries to Potato Plants (Solanum tuberosum L.) under Weak Light.

Light is one of the most important abiotic factors for most plants, which affects almost all growth and development stages. In this study, physiological indicators suggest that the application of exogenous Ca2+ improves photosynthesis and changes phytohormone levels. Under weak light, photosynthetic parameters of the net photosynthetic rate (PN), stomatal conductance (Gs), and transpiration rate (Tr) decreased; the antioxidation systems peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) reduced; the degrees of malondialdehyde (MDA), H2O2, and superoxide anion (O2-) free radical damage increased; while exogenous Ca2+ treatment was significantly improved. RNA-seq analysis indicated that a total of 13,640 differently expressed genes (DEGs) were identified and 97 key DEGs related to hormone, photosynthesis, and calcium regulation were differently transcribed. Gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, plant hormone signal transduction, photosynthesis, carbon metabolism, and phenylpropanoid biosynthesis were significantly enriched. Additionally, quantitative real-time PCR (qRT-PCR) analysis confirmed some of the key gene functions in response to Ca2+. Overall, these results provide novel insights into the complexity of Ca2+ to relieve injuries under weak light, and they are helpful for potato cultivation under weak light stress.

Comparative Transcriptome Analysis between Fertile and CMS Flower Buds in Wucai (Brassica campestris L.).

BACKGROUND: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq). RESULTS: Microscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai. CONCLUSION: The present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.