Genome-wide identification of GH9 gene family and the assessment of its role during fruit abscission zone formation in Vaccinium ashei.

KEY MESSAGE: The genomic location and stage-specific expression pattern of GH9 genes reveal their critical roles during fruit abscission zone formation in Vaccinium ashei. Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into 3 subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit.

Transcriptomic Analysis of Heat Stress Response in Brassica rapa L. ssp. pekinensis with Improved Thermotolerance through Exogenous Glycine Betaine.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is sensitive to high temperature, which will cause the B. rapa to remain in a semi-dormancy state. Foliar spray of GB prior to heat stress was proven to enhance B. rapa thermotolerance. In order to understand the molecular mechanisms of GB-primed resistance or adaptation towards heat stress, we investigated the transcriptomes of GB-primed and non-primed heat-sensitive B. rapa 'Beijing No. 3' variety by RNA-Seq analysis. A total of 582 differentially expressed genes (DEGs) were identified from GB-primed plants exposed to heat stress relative to non-primed plants under heat stress and were assigned to 350 gene ontology (GO) pathways and 69 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The analysis of the KEGG enrichment pathways revealed that the most abundantly up-regulated pathways were protein processing in endoplasmic reticulum (14 genes), followed by plant hormone signal transduction (12 genes), ribosome (8 genes), MAPK signaling pathway (8 genes), homologous recombination (7 genes), nucleotide excision repair metabolism (5 genes), glutathione metabolism (4 genes), and ascorbate and aldarate metabolism (4 genes). The most abundantly down-regulated pathways were plant-pathogen interaction (14 genes), followed by phenylpropanoid biosynthesis (7 genes); arginine and proline metabolism (6 genes); cutin, suberine, and wax biosynthesis (4 genes); and tryptophan metabolism (4 genes). Several calcium sensing/transducing proteins, as well as transcription factors associated with abscisic acid (ABA), salicylic acid (SA), auxin, and cytokinin hormones were either up- or down-regulated in GB-primed B. rapa plants under heat stress. In particular, expression of the genes for antioxidant defense, heat shock response, and DNA damage repair systems were highly increased by GB priming. On the other hand, many of the genes involved in the calcium sensors and cell surface receptors involved in plant innate immunity and the biosynthesis of secondary metabolites were down-regulated in the absence of pathogen elicitors in GB-primed B. rapa seedlings. Overall GB priming activated ABA and SA signaling pathways but deactivated auxin and cytokinin signaling pathways while suppressing the innate immunity in B. rapa seedlings exposed to heat stress. The present study provides a preliminary understanding of the thermotolerance mechanisms in GB-primed plants and is of great importance in developing thermotolerant B. rapa cultivars by using the identified DEGs through genetic modification.

The GPCR-ß-arrestin complex allosterically activates C-Raf by binding its amino terminus.

G protein-coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and ß-arrestins (ßarrs). In a ßarr-dependent signaling pathway, ßarrs link GPCRs to various downstream signaling partners, such as the Raf-mitogen-activated protein kinase extracellular signal-regulated kinase-extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR-ßarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, ßarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR-ßarr1 complexes in vitro. Furthermore, we show that ßarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates ßarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and ßarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between ßarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and ßarr1 bind to the same region on C-Raf. Furthermore, we found that ßarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR-ßarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how ßarrs regulate effector molecules to activate downstream signaling pathways.

An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity.

TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.

Enhanced Resistance to Sclerotinia sclerotiorum in Brassica rapa by Activating Host Immunity through Exogenous Verticillium dahliae Aspf2-like Protein (VDAL) Treatment.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most destructive diseases in Brassica rapa. Verticillium dahliae Aspf2-like protein (VDAL) is a secretory protein of V. dahliae which has been shown to enhance the resistance against fungal infections in several plants. Nonetheless, the molecular mechanisms of VDAL-primed disease resistance are still poorly understood. In this study, we performed physiological, biochemical, and transcriptomic analyses of Brassica rapa in order to understand how VDAL confers resistance to S. sclerotiorumn infections in plants. The results showed that foliar application of VDAL significantly reduced the plaque area on leaves inoculated with S. sclerotiorum. It also enhanced antioxidant capacity by increasing activities of superoxide dismutase (SOD), peroxidase (POD), peroxidase (APX), glutathione reductase (GR), protoporphyrinogen oxidase (PPO), and defense-related enzymes ß-1,3-glucanase and chitinase during the infection periods. This occurred in parallel with significantly reduced relative conductivity at different periods and lower malondialdehyde (MDA) content as compared to sole S. sclerotiorum inoculation. Transcriptomic analysis showed a total of 146 (81 up-regulated and 65 down-regulated) differentially expressed genes (DEGs) in VDAL-treated leaves compared to the control. The most enriched three Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were the mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, and plant-pathogen interaction, all of which were associated with plant immunity. DEGs associated with MAPK and hormone signal transduction pathways were ethylene response sensor ERS2, EIN3 (Ethylene Insensitive3)-binding F-box protein 2 (EBF2), ethylene-responsive transcription factor ERF94, MAPK 9 (MKK9), protein phosphatase 2C (PP2C37), auxin-responsive proteins (AUX/IAA1 and 19), serine/threonine-protein kinase CTR1, and abscisic acid receptors (PLY 4 and 5). Among the DEGs linked with the plant-pathogen interaction pathway were calmodulin-like proteins (CML5, 24, 27), PTI1-like tyrosine protein kinase 3 (Pti13) and transcription factor MYB30, all of which are known to play key roles in pathogen-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI) for hypersensitive response (HR), cell wall reinforcement, and stomatal closure in plants. Overall, VDLA treatment triggered repression of the auxin and ABA signaling pathways and de-repression of the ethylene signaling pathways in young B. rapa seedlings to increase plant innate immunity. Our results showed that VDAL holds great potential to enhance fungal disease resistance in B. rapa crop.

Glutamic Acid and Poly-γ-glutamic Acid Enhanced the Heat Resistance of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) by Improving Carotenoid Biosynthesis, Photosynthesis, and ROS Signaling.

Heat stress is one of the most common agrometeorological risks in crop production in the middle and lower reaches of the Yangtze River in China. This study aimed to investigate whether glutamic acid (Glu) or poly-γ-glutamic acid (γ-PGA) biostimulants can improve the thermotolerance of a cool-season Chinese cabbage (Brassica rapa L. ssp. pekinensis) crop. Priming with Glu (2.0 mM) or γ-PGA (20 mg·L-1) was conducted at the third leaf stage by applying as daily foliar sprays for 5 days before 5 days of heat stress (45 °C in 16-h light/35 °C in 8-h dark). Coupled with morpho-physiological and biochemical analyses, transcriptomes of Glu or γ-PGA-primed Chinese cabbage under heat stress were examined by RNA-seq analysis. The results showed that the thermotolerance conferred by Glu and γ-PGA priming was associated with the increased parameters of vegetative growth, gas exchange, and chlorophyll fluorescence. Compared with the control, the dry weights of plants treated with Glu and γ-PGA increased by 51.52% and 39.39%, respectively. Glu and γ-PGA application also significantly increased the contents of total chlorophyll by 42.21% and 23.12%, and carotenoid by 32.00% and 24.00%, respectively. In addition, Glu- and γ-PGA-primed plants markedly inhibited the levels of malondialdehyde, electrolyte leakage, and super-oxide anion radical, which was accompanied by enhanced activity levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD). Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) categories within the differentially expressed genes (DEGs) functional clusters of RNA-seq data indicated that the expression levels of the genes for DNA replication, DNA repair system, linoleic acid metabolism, cysteine and methionine metabolism, glutathione metabolism, purine and pyrimidine metabolism, carotenoid biosynthesis, and plant-pathogen interaction were commonly up-regulated by both Glu and γ-PGA priming. Glu treatment enhanced the expression levels of the genes involved in aliphatic glucosinolate and 2-oxocarboxylic acid, while γ-PGA treatment activated carotenoid cleavage reaction to synthesize abscisic acid. Taken together, both Glu and γ-PGA have great potential for the preadaptation of Chinese cabbage seedlings to heat stress, with Glu being more effective than γ-PGA.

Genome-Wide Identification and Analysis of Polygalacturonase Genes in Solanum lycopersicum.

Polygalacturonase (PG), a large hydrolase family in plants, is involved in pectin disassembly of the cell wall in plants. The present study aims to characterize PG genes and investigate their expression patterns in Solanum lycopersicum. We identified 54 PG genes in the tomato genome and compared their amino acid sequences with their Arabidopsis counterpart. Subsequently, we renamed these PG genes according to their Arabidopsis homologs. Phylogenetic and evolutionary analysis revealed that these tomato PG genes could be classified into seven clades, and within each clade the exon/intron structures were conserved. Expression profiles analysis through quantitive real-time polymerase chain reaction (qRT-PCR) revealed that most SlPGs had specific or high expression patterns in at least one organ, and particularly five PG genes (SlPG14, SlPG15, SlPG49, SlPG70, and SlPG71) associated with fruit development. Promoter analysis showed that more than three cis-elements associated with plant hormone response, environmental stress response or specific organ/tissue development exhibited in each SlPG promoter regions. In conclusion, our results may provide new insights for the further study of PG gene function during plant development.

Melatonin improves the storage quality of rabbiteye blueberry (Vaccinium ashei) by affecting cuticular wax profile.

Cuticular wax is the first line of structural defense for plants against external stresses. This study investigated the effects of melatonin (MT) on chemical composition and accumulation profile of wax, as well as fruit quality of rabbiteye blueberry during storage. The results indicated a significant reduction in the overall wax content during storage. Nevertheless, MT effectively delayed the decline, with a higher amount of 9.8% and 15.17% in the treated 'Baldwin' and 'Garden Blue' compared to their respective controls at 21st day of storage. The wax composition significantly varied depending on storage time, MT treatment, and cultivars. Additionally, MT markedly improved the fruit quality of rabbiteye blueberries. Correlation analysis revealed water loss and decay rates were negatively correlated with triterpenoids and fatty acids. Taken together, this study highlights the positive effects of post-harvest MT application on shelf life and fruit quality of blueberry by modifying the wax profile during storage.

A BrLINE1-RUP insertion in BrCER2 alters cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Glossiness is an important quality-related trait of Chinese cabbage, which is a leafy vegetable crop in the family Brassicaceae. The glossy trait is caused by abnormal cuticular wax accumulation. In this study, on the basis of a bulked segregant analysis coupled with next-generation sequencing (BSA-seq) and fine-mapping, the most likely candidate gene responsible for the glossy phenotype of Chinese cabbage was identified. It was subsequently named Brcer2 because it is homologous to AtCER2 (At4g24510). A bioinformatics analysis indicated a long interspersed nuclear element 1 (LINE-1) transposable element (named BrLINE1-RUP) was inserted into the first exon of Brcer2 in HN19-G via an insertion-mediated deletion mechanism, which introduced a premature termination codon. Gene expression analysis showed that the InDel mutation of BrCER2 reduced the transcriptional expression levels of Brcer2 in HN19-G. An analysis of cuticular waxes suggested that a loss-of-function mutation to BrCER2 in Chinese cabbage leads to a severe decrease in the abundance of very-long-chain-fatty-acids (> C28), resulting in the production of a cauline leaf, inflorescence stem, flower, and pistil with a glossy phenotype. These findings imply the insertion of the LINE-1 transposable element BrLINE1-RUP into BrCER2 can modulate the waxy traits of Chinese cabbage plants.

Optimization of near-infrared reflectance models in determining flavonoid composition of okra (Abelmoschus esculentus L.) pods.

Okra pods have been utilized as a functional food due to their rich active ingredient composition, especially the high content of flavonoid compounds. This study conducted near-infrared spectroscopy (NIRS) modeling optimization and external validation based on the flavonoid components of 219 pod samples. Spectral correlation analyses identified two types of spectral response patterns classified as quercetin-3-O-xylose (1-2) glucoside (QOXG) and total flavonoid content (TFC), consisting of six different spectral regions. Different modeling effects were observed for QOXG and TFC with various spectral region combination analyses, where the lower wave-number region contributed more to both flavonoids calibration models. The combination of standard normal variate / "1, 9, 3" / partial least squares was found to be the most effective for developing calibration models for both flavonoids. The resulting models had small root mean square errors of prediction for external validation and high determination coefficients, indicating their usefulness for rapid prediction of flavonoid composition in okra pods.

Genome-Wide Identification and Expression Analysis of ESPs and NSPs Involved in Glucosinolate Hydrolysis and Insect Attack Defense in Chinese Cabbage (Brassica rapa subsp. pekinensis).

Glucosinolates are secondary plant metabolites that are part of the plant's defense system against pathogens and pests and are activated via enzymatic degradation by thioglucoside glucohydrolases (myrosinases). Epithiospecifier proteins (ESPs) and nitrile-specifier proteins (NSPs) divert the myrosinase-catalyzed hydrolysis of a given glucosinolate to form epithionitrile and nitrile rather than isothiocyanate. However, the associated gene families have not been explored in Chinese cabbage. We identified three ESP and fifteen NSP genes randomly distributed on six chromosomes in Chinese cabbage. Based on a phylogenetic tree, the ESP and NSP gene family members were divided into four clades and had similar gene structure and motif composition of Brassica rapa epithiospecifier proteins (BrESPs) and B. rapa nitrile-specifier proteins (BrNSPs) in the same clade. We identified seven tandem duplicated events and eight pairs of segmentally duplicated genes. Synteny analysis showed that Chinese cabbage and Arabidopsis thaliana are closely related. We detected the proportion of various glucosinolate hydrolysates in Chinese cabbage and verified the function of BrESPs and BrNSPs in glucosinolate hydrolysis. Furthermore, we used quantitative RT-PCR to analyze the expression of BrESPs and BrNSPs and demonstrated that these genes responded to insect attack. Our findings provide novel insights into BrESPs and BrNSPs that can help further promote the regulation of glucosinolate hydrolysates by ESP and NSP to resist insect attack in Chinese cabbage.

Role of melatonin in promoting plant growth by regulating carbon assimilation and ATP accumulation.

Melatonin (MT) is a phytohormone important in mediating diverse plant growth processes. In this study, we performed transcriptomic, qRT-PCR, physiological and biochemical analyses of Brassica rapa seedlings in order to understand how MT promotes plant growth. The results showed that exogenous MT increased the rate of cyclic electron flow around photosystem (PS) I, fluorescence quantum yield, and electron transport efficiency between PSII and PSI to promote the vegetative growth of B. rapa seedlings without affecting oxidative stress level, as compared to control. However, MT treatment significantly reduced photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) by 2.25-, 1.23- and 3.50-fold at 0.05 level, respectively. This occurred in parallel with the down-regulation of the genes for carbon fixation in photosynthetic organisms in a KEGG pathway enrichment. More accelerated plant growth despite the reduced photosynthesis rate and the enhanced electron transport rate suggested that NADPH and adenosine triphosphate (ATP) were preferentially diverted into other anabolic reactions than the Calvin cycle upon MT application. MT treatment increased ATP level and facilitated carbon assimilation into primary metabolism that led to a significant enhancement of soluble protein, sucrose, and fructose, but a significant decrease in glucose content. MT-induced carbon assimilation into primary metabolism was driven by up-regulation of the genes for glutathione metabolism, Krebs cycle, ribosome, and DNA replication in a KEGG pathway enrichment, as well as down-regulation of the genes for secondary metabolites. Our results provide an insight into MT-mediated metabolic adjustments triggered by coordinate changes in a wide range of gene expression profiles to help improve the plant functionality.

Identification and Quantification of Flavonoids in Okra (Abelmoschus esculentus L. Moench) and Antiproliferative Activity In Vitro of Four Main Components Identified.

Okra is a kind of flavonoid-rich food which was reported to have a variety of health functions. Flavonoids are the major polyphenolic compounds in okra and are thought to play a role in reducing the risk of disease. The aim of this study was to isolate and identify the flavonoids composition in okra pods and explore the activity of the main flavonoids components identified on inhibiting tumor cell proliferation in vitro. Six individual flavonoids were identified by HPLC-MS/MS: quercetin-3-gentiobioside (Q3G), quercetin-3-sambubioside (Q3S), rutin, quercetin-7-glucoside (Q7G), isoquercitrin (ISO) and quercetin-3-malonylglucoside (Q3M), which were all separated well within 30 min. The analytical method was validated by the recovery of spiked samples and so on. Moreover, four main flavonoids components, namely Q3G, Q3S, ISO and Q3M, exhibited significant (p < 0.05) inhibition of NCI-N87, A375, A549 cells proliferation (25−100 µmol/L) and of HFLS-RA (200−300 µmol/L) in different levels, according to MTT method, respectively. It is demonstrated that the flavonoids components of okra exhibited a noteworthy development prospect as a possible nutraceutical dietary supplement.

Glycine Betaine and ß-Aminobutyric Acid Mitigate the Detrimental Effects of Heat Stress on Chinese Cabbage (Brassica rapa L. ssp. pekinensis) Seedlings with Improved Photosynthetic Performance and Antioxidant System.

Heat stress is one of the major abiotic factors that limit the growth, development, and productivity of plants. Both glycine betaine (GB) and ß-aminobutyric acid (BABA) have received considerable attention due to their roles in stimulating tolerance to diverse abiotic stresses. In order to understand how GB and BABA biostimulants alleviate heat stress in a cool-weather Chinese cabbage (Brassica rapa L. ssp. pekinensis) plant, we investigated the GB- and BABA-primed heat-stressed plants in terms of their morpho-physiological and biochemical traits. Priming with GB (15 mM) and BABA (0.2 mM) was conducted at the third leaf stage by applying foliar sprays daily for 5 days before 5 days of heat stress (45 °C in 16 h light/35 °C in 8 h dark) on Chinese cabbage seedlings. The results indicate that GB and BABA significantly increased chlorophyll content, and the parameters of both gas exchange and chlorophyll fluorescence, of Chinese cabbage under heat stress. Compared with the unprimed heat-stressed control, the dry weights of GB- and BABA-primed plants were significantly increased by 36.36% and 45.45%, respectively. GB and BABA priming also greatly mitigated membrane damage, as indicated by the reduction in malondialdehyde (MDA) and electrolyte leakage through the elevation of proline content, and increased activity levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). Taken together, GB and BABA have great potential to enhance the thermotolerance of Chinese cabbage through higher photosynthesis performance, osmoprotection, and antioxidant enzyme activity.

Melatonin affects cuticular wax profile in rabbiteye blueberry (Vaccinium ashei) during fruit development.

Cuticular wax has been implicated in the first line of plant defense mechanism against external stresses. In this study, cuticular wax on the fruits of two rabbiteye blueberry cultivars cv. Baldwin and Brightwell were examined in terms of the morphology and chemical composition during fruit development in the presence and absence of pre-harvest melatonin (MT) treatment. It revealed that the crystal wax appeared as numerous tubules, and its morphology was not affected by MT treatment. A dominant constituent of triterpenoid at veraison stage was ß-amyrin (45.62% of total wax) in Baldwin and oleanolic acid (22.79% of total wax) in Brightwell. Exogenous MT application significantly promoted fruit quality and increased total content of cuticular wax, however, the effect of MT on individual wax components greatly varied depending on cultivars and fruit developmental stage. Consequently, MT application may enhance sustainability of post-harvest fruit production systems by facilitating accumulation of cuticular wax.

Transcriptomic analysis of succulent stem development of Chinese kale (Brassica oleracea var. alboglabra Bailey) and its synthetic allotetraploid via RNA sequencing.

Chinese kale (Brassica oleracea var. alboglabra Bailey, CC) is a succulent stem vegetable in the Brassica family. Its allotetraploid (AACC) vegetable germplasm, which was synthesized via distant hybridization with the colloquially named 'yellow turnip' (B. rapa L. ssp. rapifera Matzg., AA), has a swelling stem similar to CC. To address the molecular mechanism of stem development for CC and AACC, RNA sequencing (RNA-seq) was used to investigate transcriptional regulation of their stem development at three key stages including 28 days, 42 days and the bolting stage (BS) after sowing. As a result, 32,642, 32,665, 33,816, 32,147, 32,293 and 32,275 genes were identified in six corresponding cDNA libraries. Among them, 25,459 genes were co-expressed, while 7,183, 7,206, 8,357, 6,688, 6,834 and 6,814 genes were specifically expressed. Additionally, a total of 29,222 differentially expressed genes (DEGs) were found for functional enrichment as well as many genes involved in plant hormones including gibberellin (GA), abscisic acid (ABA), cytokinin (CTK) and auxin (AUX). Based on gene expression consistency between CC and AACC, the gene families including DELLA, GID, PYR/PYL, PP2C, A-ARR and AUX/IAA might be related to stem development. Among these, eight genes including Bo00834s040, Bo5g093140, Bo6g086770, Bo9g070200, Bo7g116570, Bo3g054410, Bo7g093470 and Bo5g136600 may play important roles in stem development based on their remarkable expression levels as confirmed by qRT-PCR. These findings provide a new theoretical basis for understanding the molecular mechanism of stem development in Brassica vegetable stem breeding.

Melatonin elevated Sclerotinia sclerotiorum resistance via modulation of ATP and glucosinolate biosynthesis in Brassica rapa ssp. pekinensis.

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SIGNIFICANCE: Melatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

Effects of BrMYC2/3/4 on Plant Development, Glucosinolate Metabolism, and Sclerotinia sclerotiorum Resistance in Transgenic Arabidopsis thaliana.

MYC2/3/4, known as a basic helix-loop-helix (bHLH) transcription factor, directly activate the genes involved in diverse plant development and secondary metabolites biosynthesis. In this study, we identified and cloned five MYC paralogs (BrMYC2/3-1/3-2/4-1/4-2) from Chinese cabbage (Brassica rapa ssp. pekinensis). In-silico analyses for the physicochemical properties suggested that BrMYC2/3-1/3-2/4-2/4-3 are unstable hydrophobic and acidic proteins, while BrMYC4-1 is an unstable hydrophobic and basic protein. BrMYC2/3/4 belong to the bHLH superfamily and are closely related to AthMYC2/3/4 orthologs that mediate the regulation of various secondary metabolites. It was demonstrated that BrMYC2/3/4-GFP fusion protein localized in the nucleus and expression levels of five BrMYC2/3/4 homologous genes all elevated relative to control (Ctrl). When expressed in Arabidopsis under the control of 35S promoter, each of the BrMYC2/3-1/3-2/4-1/4-2 transgenes differentially influenced root and shoot elongation, vegetative phase change, flowering time, plant height and tiller number after flowering, and seed production. Despite the variation of phenotypes between the transgenic lines, all the lines except for BrMYC4-2 exhibited shorter seed length, less seed weight, higher accumulation of glucosinolates (GSs), and resistance to Sclerotinia sclerotiorum than Ctrl. Notably, BrMYC2 overexpression (OE) line significantly reduced the lengths of root and hypocotyl, seed length, and weight, along with faster bolting time and strikingly higher accumulation of total GSs. Accumulation of GSs at the highest levels in the BrMYC2 OE line conferred the highest resistance to S. sclerotiorum. Unlike BrMYC3 OE and BrMYC4 OE , BrMYC2 OE stimulated the growth of plant height after fluorescence. The results of this study point to the BrMYC2 overexpression that may provide a beneficial effect on plant growth and development via plant resistance to the fungal pathogen.

Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1.

Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glu-cobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.

Development of a yeast internal-subunit eGFP labeling strategy and its application in subunit identification in eukaryotic group II chaperonin TRiC/CCT.

Unambiguous subunit assignment in a multicomponent complex is critical for thorough understanding of the machinery and its functionality. The eukaryotic group II chaperonin TRiC/CCT folds approximately 10% of cytosolic proteins and is important for the maintenance of cellular homeostasis. TRiC consists of two rings and each ring has eight homologous but distinct subunits. Unambiguous subunit identification of a macromolecular machine such as TRiC through intermediate or low-resolution cryo-EM map remains challenging. Here we present a yeast internal-subunit eGFP labeling strategy termed YISEL, which can quickly introduce an eGFP tag in the internal position of a target subunit by homologous recombination, and the tag labeled protein can be expressed in endogenous level. Through this method, the labeling efficiency and tag-occupancy is ensured, and the inserted tag is usually less mobile compared to that fused to the terminus. It can also be used to bio-engineer other tag in the internal position of a protein in yeast. By applying our YISEL strategy and combined with cryo-EM 3D reconstruction, we unambiguously identified all the subunits in the cryo-EM map of TRiC, demonstrating the potential for broad application of this strategy in accurate and efficient subunit identification in other challenging complexes.