Investigation of heat stress responses and adaptation mechanisms by integrative metabolome and transcriptome analysis in tea plants (Camellia sinensis).

Extreme high temperature has deleterious impact on the yield and quality of tea production, which has aroused the attention of growers and breeders. However, the mechanisms by which tea plant varieties respond to extreme environmental heat is not clear. In this study, we analyzed physiological indices, metabolites and transcriptome differences in three different heat-tolerant tea plant F1 hybrid progenies. Results showed that the antioxidant enzyme activity, proline, and malondialdehyde were significantly decreased in heat-sensitive 'FWS' variety, and the accumulation of reactive oxygen molecules such as H2O2 and O2- was remarkably increased during heat stress. Metabolomic analysis was used to investigate the metabolite accumulation pattern of different varieties in response to heat stress. The result showed that a total of 810 metabolites were identified and more than 300 metabolites were differentially accumulated. Transcriptional profiling of three tea varieties found that such genes encoding proteins with chaperon domains were preferentially expressed in heat-tolerant varieties under heat stress, including universal stress protein (USP32, USP-like), chaperonin-like protein 2 (CLP2), small heat shock protein (HSP18.1), and late embryogenesis abundant protein (LEA5). Combining metabolomic with transcriptomic analyses discovered that the flavonoids biosynthesis pathway was affected by heat stress and most flavonols were up-regulated in heat-tolerant varieties, which owe to the preferential expression of key FLS genes controlling flavonol biosynthesis. Take together, molecular chaperons, or chaperon-like proteins, flavonols accumulation collaboratively contributed to the heat stress adaptation in tea plant. The present study elucidated the differences in metabolite accumulation and gene expression patterns among three different heat-tolerant tea varieties under extreme ambient high temperatures, which helps to reveal the regulatory mechanisms of tea plant adaptation to heat stress, and provides a reference for the breeding of heat-tolerant tea plant varieties.

Soil Microbial Community Characteristics and Their Effect on Tea Quality under Different Fertilization Treatments in Two Tea Plantations.

Fertilization is an essential aspect of tea plantation management that supports a sustainable tea production and drastically influences soil microbial communities. However, few research studies have focused on the differences of microbial communities and the variation in tea quality in response to different fertilization treatments. In this work, the soil fertility, tea quality, and soil microbial communities were investigated in two domestic tea plantations following the application of chemical and organic fertilizers. We determined the content of mineral elements in the soil, including nitrogen, phosphorus, and potassium, and found that the supplementation of chemical fertilizer directly increased the content of mineral elements. However, the application of organic fertilizer significantly improved the accumulation of tea polyphenols and reduced the content of caffeine. Furthermore, amplicon sequencing results showed that the different ways of applying fertilizer have limited effect on the alpha diversity of the microbial community in the soil while the beta diversity was remarkably influenced. This work also suggests that the bacterial community structure and abundance were also relatively constant while the fungal community structure and abundance were dramatically influenced; for example, Chaetomiaceae at the family level, Hypocreaceae at the order level, Trichoderma at the genus level, and Fusarium oxysporum at the species level were predominantly enriched in the tea plantation applying organic fertilizer. Moreover, the bacterial and fungal biomarkers were also analyzed and it was found that Proteobacteria and Gammaproteobacteria (bacteria) and Tremellomycetes (fungi) were potentially characterized as biomarkers in the plantation under organic fertilization. These results provide a valuable basis for the application of organic fertilizer to improve the soil of tea plantations in the future.

Anatomical Mechanisms of Leaf Blade Morphogenesis in Sasaella kogasensis 'Aureostriatus'.

There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and growth patterns of Sasaella kogasensis 'Aureostriatus' and provides a three-dimensional anatomical analysis of cell division, expansion, and degradation. Leaves on the same branch develop bottom-up, while individual leaves develop the other way around. Like bamboo shoots and culms, the leaves follow a "slow-fast-slow" growth pattern, with longitudinal growth being predominant during their development. The growth zones of individual leaves included division, elongation, and maturation zones based on the distribution of growth space. By measuring 13,303 epidermal long cells and 3293 mesophyll cells in longitudinal sections of rapidly elongating leaves, we observed that in the rapid elongation phase (S4-S5), the division zone was located in the 1-2 cm segment at the bottom of the leaf blade and maintained a constant size, continuously providing new cells for leaf elongation, whereas in the late rapid elongation phase (S6), when the length of the leaf blade was approaching that of a mature leaf, its cells at the bottom of the blade no longer divided and were replaced by the ability to elongate. Furthermore, to gain an insight into the dynamic changes in the growth of the S. kogasensis 'Aureostriatus' leaves in the lateral and periclinal directions, the width and thickness of 1459 epidermal and 2719 mesophyll cells were counted in the mid-cross section of leaves at different developmental stages. The results showed that during the early stages of development (S1-S3), young leaves maintained vigorous division in the lateral direction, while periplasmic division gradually expanded from the bottom to the top of the leaf blade and the number of cell layers stabilized at S4. The meristematic tissues on both sides of the leaf were still able to divide at S4 but the frequency of the division gradually decreased, while cell division and expansion occurred simultaneously between the veins. At S6, the cells at the leaf margins and between the veins were completely differentiated and the width of the leaf blade no longer expanded. These findings revealed changes in cell growth anisotropically during the leaf development of S. kogasensis 'Aureostriatus' and demonstrated that leaf elongation was closely related to the longitudinal expansion of epidermal cells and proliferative growth of mesophyll cells, whereas the cell division of meristematic tissues and expansion of post-divisional cells contributed to the increases in blade width and thickness. The presented framework will facilitate a further exploration of the molecular regulatory mechanisms of leaf development in S. kogasensis 'Aureostriatus' and provide relevant information for developmental and taxonomic studies of bamboo plants.

miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis.

Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.

Integrated Transcriptome and Metabolome Analyses Reveal Bamboo Culm Color Formation Mechanisms Involved in Anthocyanin Biosynthetic in Phyllostachys nigra.

Phyllostachys nigra has green young culms (S1) and purple black mature culms (S4). Anthocyanins are the principal pigment responsible for color presentation in ornamental plants. We employ a multi-omics approach to investigate the regulatory mechanisms of anthocyanins in Ph. nigra. Firstly, we found that the pigments of the culm of Ph. nigra accumulated only in one to four layers of cells below the epidermis. The levels of total anthocyanins and total flavonoids gradually increased during the process of bamboo culm color formation. Metabolomics analysis indicated that the predominant pigment metabolites observed were petunidin 3-O-glucoside and malvidin O-hexoside, exhibiting a significant increase of up to 9.36-fold and 13.23-fold, respectively, during pigmentation of Ph. nigra culm. Transcriptomics sequencing has revealed that genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and starch and sucrose metabolism pathways were significantly enriched, leading to color formation. A total of 62 differentially expressed structural genes associated with anthocyanin synthesis were identified. Notably, PnANS2, PnUFGT2, PnCHI2, and PnCHS1 showed significant correlations with anthocyanin metabolites. Additionally, certain transcription factors such as PnMYB6 and PnMYB1 showed significant positive or negative correlations with anthocyanins. With the accumulation of sucrose, the expression of PnMYB6 is enhanced, which in turn triggers the expression of anthocyanin biosynthesis genes. Based on these findings, we propose that these key genes primarily regulate the anthocyanin synthesis pathway in the culm and contribute to the accumulation of anthocyanin, ultimately resulting in the purple-black coloration of Ph. nigra.

Genome-Wide Identification and Analysis of ZF-HD Gene Family in Moso Bamboo (Phyllostachys edulis).

Zinc finger-homeodomain (ZF-HD) proteins play essential roles in plant growth, development and stress responses. However, knowledge of the expression and evolutionary history of ZF-HD genes in moso bamboo remains limited. In this study, a total of 24 ZF-HD genes were found unevenly distributed on 12 chromosomes in moso bamboo (Phyllostachys edulis). Phylogenetic analysis indicated that PeZF-HDs were divided into two subfamilies: ZHD and MIF. The ZHD subfamily genes were further classified into seven groups according to their orthologous relationships among the rice and Arabidopsis ZF-HD gene family. The gene structures and conserved motifs of PeZF-HDs were analyzed. Whole-genome duplication (WGD) or segmental duplication promoted the evolution and expansion of the moso bamboo ZF-HD gene family. Ka/Ks ratios suggested that the twenty-four duplication pairs had undergone purifying selection. Promoter analysis showed that most PeZF-HDs contained cis-elements associated with stress responses and hormones. Expression analysis demonstrated that many PeZF-HDs were responsive to abiotic stress treatment. Overall, this work investigated PeZF-HD genes in moso bamboo using bioinformatic approaches. The evolutionary research on gene structure, motif distribution and cis-regulatory elements indicated that PeZF-HDs play distinct roles in biological processes, which provides a theoretical basis for exploring the physiological functions of ZF-HDs and selecting candidate stress-related genes in moso bamboo.

Metabolomic and transcriptomic analyses reveal a MYB gene, CsAN1, involved in anthocyanins accumulation separation in F1 between 'Zijuan' (Camellia sinensis var. assamica) and 'Fudingdabaicha' (C. sinensis var. sinensis) tea plants.

'Zijuan' (Camellia sinensis var. assamica), a somatic mutant with purple foliage and stem selected from the Yunnan Daye cultivar, has been well developed owing to abnormal pattern of anthocyanin accumulation. However, the genetic basis for the specific accumulation of phloem glycosides is not clear. Tea plants are self-incompatible, so parents with large differences in foliage color were used for crosses to investigate the genetic mechanism of anthocyanins. In this study, 'Zijuan' and green foliage cultivar 'Fudingdabaicha' (C. sinensis var. sinensis) were used as female and male parents, respectively, to generated F1 hybrid progenies with various anthocyanin contents. In order to decipher the genetic rules of anthocyanins accumulation, we performed widely targeted metabolic and transcriptomic profiling. The results showed that cyanidin-3-O-galactoside, delphinidin-3-O-galactoside and petunidin-3-O-galactoside were the major types of anthocyanins and factors directly led to the color variation between parents and F1 plants. Transcriptomic analyses suggested the significant up-regulation of anthocyanidin synthase gene (CsANS1) and CsAN1, a MYB family gene positively regulated the expression of CsANS1, in anthocyanin-rich tea plants. Furthermore, the deletion mutation of CsAN1 was found by cloning and alignment in anthocyanin-lacking cultivars. Taken together, the function deficiency of CsAN1 is predominantly responsible for the inability of anthocyanins accumulation, and this trait is heritable in progenies through hybridization. The present study elucidated the molecular basis of leaf purple trait formation in 'zijuan' and 'Fudingdabaicha' and their F1 plants, which helps to elucidate the genetic mechanism of leaf anthocyanin accumulation regulation in tea plants, and the results provide a research reference for the selection and breeding of high anthocyanin type tea varieties.

Genome-Wide Identification and Characterization of TCP Family Genes in Pak-Choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis var. communis].

The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family, a kind of plant specific transcription factor, is essential for stress response, cell growth, and cell proliferation. However, the characterization of TCP family is still not clear in Pak-choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis var. communis]. In this study, genome-wide analysis of TCP gene family was performed and 26 TCP genes were identified in Pak-choi. Phylogenetic analysis demonstrated that the 26 BcTCPs were divided into two classes: Class I and Class II. Class II was further classified into two subclasses, CIN and CYC/TB1. The qPCR results suggested that most BcTCPs respond to abiotic stresses. The expressions of BcTCP3, BcTCP12, BcTCP21, and BcTCP22 were significantly changed under ABA and cold treatment. BcTCP3 and BcTCP12 were also up-regulated under osmotic treatment. Subcellular localization showed that BcTCP3 and BcTCP21 were located in the nucleus. Our results will facilitate revealing the functions and regulatory mechanisms of BcTCPs.

BcGR1.1, a Cytoplasmic Localized Glutathione Reductase, Enhanced Tolerance to Copper Stress in Arabidopsis thaliana.

Copper is a mineral element, which is necessary for the normal growth and development of plants, but high levels of copper will seriously damage plants. Studies have shown that AtGR1 improves the tolerance of Arabidopsis to aluminum and cadmium stress. However, the role of GR in the copper stress response of plants is still unclear. Here, we identified four genes (named BcGR1.1, BcGR1.2, BcGR2.1 and BcGR2.2, respectively) encoding glutathione reductase (GR) in non-heading Chinese cabbage (Brassica campestris (syn. Brassica rapa) ssp. chinensis), which could be divided into two types based on the subcellular localization. Among them, BcGR1.1, which belonged to the cytoplasmic localization type, was significantly upregulated under copper stress. Compared to WT (the wild type), Arabidopsis thaliana heterologously overexpressed BcGR1.1 had longer roots, higher fresh weight, higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and accumulated more superoxide dismutase and peroxidase under copper stress. However, in the AsA-GSH cycle under copper stress, the contents of AsA and AsA/DHA were significantly downregulated, and the contents of DHA and T-AsA (total AsA) were upregulated, in the BcGR1.1-overexpressing Arabidopsis. Therefore, BcGR1.1 could improve the scavenging ability of reactive oxygen species (ROS) by increasing the activity of GR, antioxidant enzymes and the utilization of AsA, and then enhance the copper stress tolerance of plants.

BcHTT4 Inhibits Branching of Non-Heading Chinese Cabbage at the Vegetative Stage.

Branching is speculated to contribute to the plant architecture and crop yield. As a quantitative trait, branching is regulated by multiple genes in non-heading Chinese cabbage (NHCC). Several related candidate genes have been discovered in previous studies on the branching of NHCC, but their specific functions and regulatory mechanisms still need to be verified and explored. In this study, we found that the expression of BcHTT4, the ortholog to HEAT-INDUCED TAS1 TARGET4 (HTT4) in Arabidopsis, was significantly different between 'Suzhouqing' (common type) and 'Maertou' (multiple shoot branching type) in NHCC, which was consistent with the previous transcriptome sequencing results. The silencing of BcHTT4 expression in non-heading Chinese cabbage promotes axillary bud growth at the vegetative stage. When BcHTT4 is overexpressed in Arabidopsis, branching will decrease. In further study, we found that BcHTT4 interacts with immunophilin BcFKBP13 in vivo and in vitro through yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays. Moreover, quantitative real-time PCR analysis showed that when the expression of BcHTT4 was silenced in 'Suzhouqing', the expression of BcFKBP13 also decreased significantly. Our findings reveal that BcHTT4 is involved in the branching mechanism and interacts with immunophilin BcFKBP13 in NHCC.

Identification and Expression Analysis of Cold Shock Protein 3 (BcCSP3) in Non-Heading Chinese Cabbage (Brassica rapa ssp. chinensis).

A cold-related protein, cold shock protein 3 (BcCSP3), was isolated from non-heading Chinese cabbage in this study. BcCSP3 can encode 205 amino acids (aa) with an open reading frame (ORF) of 618 base pairs (bp). Multiple sequence alignment and phylogenetic tree analyses showed that BcCSP3 contains an N-terminal cold shock domain and is highly similar to AtCSP2, their kinship is recent. Real-time quantitative polymerase chain reaction (RT-qPCR) showed that the expression level of BcCSP3 in stems and leaves is higher than that in roots. Compared with other stress treatments, the change in BcCSP3 expression level was most pronounced under cold stress. In addition, a BcCSP3-GFP fusion protein was localized to the nucleus and cytoplasm. These results indicated that BcCSP3 may play an important role in response to cold stress in non-heading Chinese cabbage. This work may provide a reference for the identification and expression analysis of other CSP genes in non-heading Chinese cabbage.

BcAP3, a MADS box gene, controls stamen development and male sterility in Pak-choi (Brassica rapa ssp. chinensis).

Stamen development is an important developmental process controlled by multiple internal and external factors. Developmental abnormalities of stamens can disrupt the structure and function of anthers, and then result in male sterility. As well known, APETELA 3 (AP3) has a clear function in regulating stamen development, which may impact in male sterility. However, the mechanisms of stamen development and male sterility controlled by AP3 are still not very clear, particular in Pak-choi (Brassica rapa ssp. chinensis). In this work, BcAP3 encoded a protein containing a MADS-box domain, which was a homolog of AtAP3, was identified in Pak-choi. Sequence alignments and phylogenetic analysis indicated that BcAP3 was highly similar to AtAP3. BcAP3 was shown to be localized to the nucleus and exhibited the potential of transcription factor. The transcript of BcAP3 was only expressed in flowers of Pak-choi, indicating that it may act in flower development. Overexpression of BcAP3 in Arabidopsis resulted in developmental abnormalities of anther wall and low vigor pollen, which were associated with the phenotype of male sterility. Expression levels of NST1 and NST2, involved in secondary wall thickening in anther walls, were significantly higher in the BcAP3-transgenic plants than in control plants, suggesting that BcAP3 may affect anther wall development by regulating NST1 and NST2. Taken together, our study demonstrated that BcAP3 could play an essential role in stamen development and male sterility.

Megasporogenesis, microsporogenesis, and female and male gametophyte development in Ziziphus jujuba Mill.

Jujube (Ziziphus jujuba Mill.) is an important fruit tree species in China. In this study, we studied the megasporogenesis, microsporogenesis, and female and male gametophyte development of two major jujube cultivars, "Dongzao" and "Mayazao," using the squash technique, improved paraffin section technology, and optical microscopy. Our investigation revealed that both "Dongzao" and "Mayazao" have bilocular ovaries, basal placenta, and anatropous, bitegmic, crassinucellate ovules. The tetrads formed by meiosis of megaspore mother cells are arranged in a straight line or a tetrahedron. Embryo sac development is of the Polygonum type. The flower buds contain five anthers, each having four pollen sacs. The anther wall, which is of the fundamental form, is composed of epidermis, endothecium, one or two middle layers, and glandular tapetum. Mature pollen grains are two-celled and three-colporate. Both "Dongzao" and "Mayazao" can form normal mature pollen grains. Our study, which has revealed the basic phenomena and progression of megasporogenesis, microsporogenesis, and female and male gametophyte development in jujube, has generated important data for further research on jujube cytology and reproductive biology. Finally, our explorations of the cytological mechanism of male sterility in "Dongzao" also have provided a cytological basis for crossbreeding.

BcMAF2 activates BcTEM1 and represses flowering in Pak-choi (Brassica rapa ssp. chinensis).

KEY MESSAGE: BcMAF2 plays a key role in flowering regulation by controlling BcTEM1, BcSOC1 and BCSPL15 in Pak-choi. Flowering is a key event in the life cycle of plants. Flowering time shows an extensive variation from different Pak-choi (Brassica rapa ssp. chinensis) cultivars. However, the regulation mechanism of flowering in Pak-choi remains rarely known. In this study, a systematic identification and functional analysis of a Pak-choi MADS Affecting Flowering (MAF) gene, BcMAF2, was carried out. BcMAF2 encoded a protein containing a conserved MADS-box domain, which was localized in the nucleus. QPCR analysis indicated that the expression of BcMAF2 was higher in the leaves and flowers. Overexpression of BcMAF2 in Arabidopsis showed that BcMAF2 repressed flowering, which was further confirmed by silencing endogenous BcMAF2 in Pak-choi. In addition, Tempranillo 1 (TEM1) expression was up-regulated and MAF2 expression was down-regulated in the BcMAF2-overexpressing Arabidopsis. The expression of BcMAF2 and BcTEM1 was down-regulated in BcMAF2-silencing Pak-choi plants. The yeast one-hybrid, dual luciferase and qPCR results revealed that BcMAF2 protein could directly bind to BcTEM1 promoter and activate its expression, which was not reported in Arabidopsis. Meanwhile, a self-inhibition was found in BcMAF2. Taken together, this work suggested that BcMAF2 could repress flowering by directly activating BcTEM1.

Identification and Functional Characterization of a Cold-Related Protein, BcHHP5, in Pak-Choi (Brassica rapa ssp. chinensis).

In plants, heptahelical proteins (HHPs) have been shown to respond to a variety of abiotic stresses, including cold stress. Up to the present, the regulation mechanism of HHP5 under low temperature stress remains unclear. In this study, BcHHP5 was isolated from Pak-choi (Brassica rapa ssp. chinensis cv. Suzhouqing). Sequence analysis and phylogenetic analysis indicated that BcHHP5 in Pak-choi is similar to AtHHP5 in Arabidopsis thaliana. Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative. Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that BcHHP5 was induced to express by cold and other abiotic stresses. In Pak-choi, BcHHP5-silenced assay, inhibiting the action of endogenous BcHHP5, indicated that BcHHP5-silenced might have a negative effect on cold tolerance, which was further confirmed. All of these results indicate that BcHHP5 might play a role in abiotic response. This work can serve as a reference for the functional analysis of other cold-related proteins from Pak-choi in the future.

Isolation and functional characterization of a floral repressor, BcFLC2, from Pak-choi (Brassica rapa ssp. chinensis).

MAIN CONCLUSION: BcFLC2 functioned as a repressor of flowering by directly regulating BcTEM1, BcMAF2, BcSOC1 and BcSPL15 in Pak-choi. FLOWERING LOCUS C (FLC) plays an important role in regulating flowering time. Here, we functionally described an FLC homologous gene, BcFLC2, that negatively regulated flowering in Pak-choi (Brassica rapa ssp. chinensis). The sequence comparison to Arabidopsis FLC showed that BcFLC2 also had a MADS-box domain at the N terminus. BcFLC2 was highly expressed in the leaves, roots, stems and stamens, and its expression was repressed by vernalization in Pak-choi. Interestingly, BcFLC2 expression exhibited a small peak at 2 weeks of vernalization treatment, suggesting that BcFLC2 may be involved in preventing premature flowering under short-term cold exposure in Pak-choi, which is different from the AtFLC expression pattern. Overexpression of BcFLC2 in Arabidopsis caused late flowering, while silencing of BcFLC2 in Pak-choi caused early flowering. BcFLC2 localized to the cell nucleus and functioned as a transcription factor. Yeast one-hybrid analysis revealed that BcFLC2 could bind to the promoters of Pak-choi Tempranillo 1 (BcTEM1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BcSOC1), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (BcSPL15) and MADS AFFECTING FLOWERING 2 (BcMAF2). Taken together, the present results suggested that BcFLC2 played a key role in flowering regulation as a negative regulator by controlling BcTEM1, BcMAF2, BcSOC1 and BcSPL15 expression.

Isolation and Functional Characterization of a Floral Repressor, BcMAF1, From Pak-choi (Brassica rapa ssp. Chinensis).

MADS-box genes form a large gene family in plants and are involved in multiple biological processes, such as flowering. However, the regulation mechanism of MADS-box genes in flowering remains unresolved, especially under short-term cold conditions. In the present study, we isolated BcMAF1, a Pak-choi (Brassica rapa ssp. Chinensis) MADS AFFECTING FLOWERING (MAF), as a floral repressor and functionally characterized BcMAF1 in Arabidopsis and Pak-choi. Subcellular localization and sequence analysis indicated that BcMAF1 was a nuclear protein and contained a conserved MADS-box domain. Expression analysis revealed that BcMAF1 had higher expression levels in leaves, stems, and petals, and could be induced by short-term cold conditions in Pak-choi. Overexpressing BcMAF1 in Arabidopsis showed that BcMAF1 had a negative function in regulating flowering, which was further confirmed by silencing endogenous BcMAF1 in Pak-choi. In addition, qPCR results showed that AtAP3 expression was reduced and AtMAF2 expression was induced in BcMAF1-overexpressing Arabidopsis. Meanwhile, BcAP3 transcript was up-regulated and BcMAF2 transcript was down-regulated in BcMAF1-silencing Pak-choi. Yeast one-hybrid and dual luciferase transient assays showed that BcMAF1 could bind to the promoters of BcAP3 and BcMAF2. These results indicated that BcAP3 and BcMAF2 might be the targets of BcMAF1. Taken together, our results suggested that BcMAF1 could negatively regulate flowering by directly activating BcMAF2 and repressing BcAP3.

Vernalization can regulate flowering time through microRNA mechanism in Brassica rapa.

Vernalization is an important process that regulates the floral transition in plants. MicroRNAs (miRNAs) are endogenous non-coding small RNA (sRNA) molecules that function in plant growth and development. Despite that miRNAs related to flowering have previously been characterized, their roles in response to vernalization in pak-choi (Brassica rapa ssp. chinensis) has never been studied. Here, two sRNA libraries from B. rapa leaves (vernalized and non-vernalized plants) were constructed and sequenced. Two hundred eight known and 535 novel miRNAs were obtained, of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization-related miRNAs. The corresponding targets were predicted on the basic of sequence homology search. In addition, 11 miRNAs and eight targets were selected for real-time quantitative PCR to confirm their expression profiles. Functional annotation of targets using gene ontology and Kyoto encyclopedia of genes and genomes results suggested that most targets were significantly enriched in the hormone signaling pathway. Moreover, a decreased indole-3-acetic acid (IAA) and an increased GA3 hormone were detected after vernalization, indicating that the IAA and GA3 might response to vernalization. These results indicated that vernalization regulates flowering through microRNA mechanism by affecting endogenous hormone level in B. rapa. This study provides useful insights of promising miRNAs candidates involved in vernalization in B. rapa, and facilitates further investigation of the miRNA-mediated molecular mechanisms of vernalization in B. rapa.

Cold acclimation alters DNA methylation patterns and confers tolerance to heat and increases growth rate in Brassica rapa.

Epigenetic modifications are implicated in plant adaptations to abiotic stresses. Exposure of plants to one stress can induce resistance to other stresses, a process termed cross-adaptation, which is not well understood. In this study, we aimed to unravel the epigenetic basis of elevated heat-tolerance in cold-acclimated Brassica rapa by conducting a genome-wide DNA methylation analysis of leaves from control (CK) and cold-acclimated (CA) plants. We found that both methylation and demethylation occurred during cold acclimation. Two significantly altered pathways, malate dehydrogenase activity and carbon fixation, and 1562 differentially methylated genes, including BramMDH1, BraKAT2, BraSHM4, and Bra4CL2, were identified in CA plants. Genetic validation and treatment of B. rapa with 5-aza-2-deoxycytidine (Aza) suggested that promoter demethylation of four candidate genes increased their transcriptional activities. Physiological analysis suggested that elevated heat-tolerance and high growth rate were closely related to increases in organic acids and photosynthesis, respectively. Functional analyses demonstrated that the candidate gene BramMDH1 (mMDH: mitochondrial malate dehydrogenase) directly enhances organic acids and photosynthesis to increase heat-tolerance and growth rate in Arabidopsis. However, Aza-treated B. rapa, which also has elevated BramMDH1 levels, did not exhibit enhanced heat-tolerance. We therefore suggest that DNA demethylation alone is not sufficient to increase heat-tolerance. This study demonstrates that altered DNA methylation contributes to cross-adaptation.

Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.

Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.