Characterization of drought stress-mitigating Rhizobium from faba bean (Vicia faba L.) in the Chinese Qinghai-Tibet Plateau.

Rhizobium-driven symbiotic nitrogen-fixation in legumes not only benefits the growth but also enhances the stress tolerance of plants. Isolating and characterizing efficient, drought-tolerant rhizobia is a central goal for improving crop yields in arid regions. Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium ('QHCD11') sampled from the root nodules of faba beans growing in an arid agricultural area in Qinghai-Tibet. We further tested the drought tolerance of the strain as well as of 'Qingcan 14' faba bean seedlings inoculated with it. Biochemical characterization involved bromothymol blue (BTB) tests, carbon metabolic profiling (Biolog GENIII), DNA-DNA hybridization (dDDH) assays, average nucleotide identity (ANI) analyses, and 16S rRNA sequencing. The result indicated that strain 'QHCD11' likely belongs to the Rhizobium indicum species. Drought stress tolerance was assessed by exposure to polyethylene glycol (PEG-6000) at concentrations of 0, 10, 15, and 20%. Increasing concentrations of PEG-6000 tended to result in decreased growth of 'QHCD11', although the strain performed better at 20% PEG 6000 than at 15%. Inoculation of drought-stressed faba bean seedlings with strain 'QHCD11' improved root vitality, chlorophyll content, antioxidant enzyme activities, and plant height. We suggest that inoculation of faba beans with 'QHCD11' is an environmentally sound strategy for mitigating crop drought stress in arid and semi-arid regions. In addition, the results presents here will benefit future studies into faba bean-rhizobia symbioses under drought stress.

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis.

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR-cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K+ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K+ homeostasis.

Comparative Transcriptome Analysis Reveals Gene Expression Differences in Eggplant (Solanum melongena L.) Fruits with Different Brightness.

Fruit brightness is an important quality trait that affects the market value of eggplant. However, few studies have been conducted on eggplant brightness. In this study, we aimed to identify genes related to this trait in three varieties of eggplant with different fruit brightness between 14 and 22 days after pollination. Using RNA-Seq Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we found that wax- and cutin-related pathways and differentially expressed genes displayed significant differences among different development stages and varieties. Scanning electron microscopy revealed that the wax layer was thinner in '30-1' and 'QPCQ' than in '22-1'. Gas chromatography-mass spectrometry analysis revealed that wax content was significantly lower in '30-1' than in '22-1', which indicated that wax may be an important factor determining fruit brightness. We further identified and analyzed the KCS gene family, which encodes the rate-limiting enzyme of FA elongation in wax synthesis. The results provide an insight into the molecular mechanisms of fruit brightness in eggplants and further eggplant breeding programs.

Metabolomic Analysis, Combined with Enzymatic and Transcriptome Assays, to Reveal the Browning Resistance Mechanism of Fresh-Cut Eggplant.

Browning has been the primary limitation in eggplant processing. This study investigates the molecular mechanism underlying fresh-cut eggplant fruit browning by observing the physicochemical characteristics of browning-resistant ('F') and browning-sensitive ('36') eggplant cultivars. Browning-related enzyme activity and gene expression (PPO, LOX, and PLD) were significantly higher in the '36' eggplant, thereby enhancing the degree of browning, compared to the 'F' eggplant. The MDA content and O2- production rate progressively increased as browning increased, while the antioxidant capacity of the fruit decreased. The cutting injury significantly activated the expression of PAL, thereby inducing the accumulation of phenolic acids, while the PPO gene was significantly upregulated, which activated the activity of polyphenol oxidase. Our results showed that the oxidation of chlorogenic acids to chlorogenic quinones resulted in the occurrence of browning, which suggests chlorogenic acid as the main browning substrate in fresh-cut eggplant.

Small signaling molecules in plant response to cold stress.

Cold stress is one of the harsh environmental stresses that adversely affect plant growth and crop yields in the Qinghai-Tibet Plateau. However, plants have evolved mechanisms to overcome the impact of cold stress. Progress has been made in understanding how plants perceive and transduce low-temperature signals to tolerate cold stress. Small signaling molecules are crucial for cellular signal transduction by initiating the downstream signaling cascade that helps plants to respond to cold stress. These small signaling molecules include calcium, reactive oxygen species, nitric oxide, hydrogen sulfide, cyclic guanosine monophosphate, phosphatidic acid, and sphingolipids. The small signaling molecules are involved in many aspects of cellular and physiological functions, such as inducing gene expression and activating hormone signaling, resulting in upregulation of the antioxidant enzyme activities, osmoprotectant accumulation, malondialdehyde reduction, and photosynthesis improvement. We summarize our current understanding of the roles of the small signaling molecules in cold stress in plants, and highlight their crosstalk in cold signaling transduction. These discoveries help us understand how the plateau plants adapt to the severe alpine environment as well as to develop new crops tolerating cold stress in the Qinghai-Tibet Plateau.

Transcriptome profiling reveals potential genes involved in browning of fresh-cut eggplant (Solanum melongena L.).

Fresh-cut processing promotes enzymatic browning of fresh fruits and vegetables, which negatively affects the product appearance and impacts their nutrition. We used RNA-sequencing to analyze the transcriptomic changes occurring during the browning of fresh-cut eggplant fruit samples from both browning-sensitive and browning-resistant cultivars to investigate the molecular mechanisms involved in browning. A total of 8347 differentially expressed genes were identified, of which 62 genes were from six gene families (i.e., PPO, PAL, POD, CAT, APX, and GST) potentially associated with enzymatic browning. Furthermore, using qRT-PCR, we verified 231 differentially regulated transcription factors in fresh-cut eggplant fruits. The enzyme activities of PPO, POD, PAL, and CAT in '36' were significantly higher than those of 'F' fresh-cut for 15 min. Both PPO and POD play a major role in the browning of eggplant pulp and might therefore act synergistically in the browning process. Meanwhile, qPCR results of 18 browning related genes randomly screened in 15 eggplant materials with different browning tolerance showed variant-specific expression of genes. Lastly, gene regulatory networks were constructed to identify the browning-related genes. This work provides a basis for future molecular studies of eggplants, and lays a theoretical foundation for the development of browning-resistant fresh-cut fruits and vegetables.

Study on browning mechanism of fresh-cut eggplant (Solanum melongena L.) based on metabolomics, enzymatic assays and gene expression.

Enzymatic browning is one of the crucial problems compromising the flavor and texture of fresh-cut fruit and vegetables. In this study, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry (LC-MS) was used to explore the browning mechanism in fresh-cut eggplant. Metabolomics studies showed that with the increase of fresh-cut time, the contents of 946 metabolites changed dynamically. The metabolites having the same trend share common metabolic pathways. As an important browning substrate, the content of chlorogenic acid increased significantly, suggesting that may be more important to fresh-cut eggplant browning; all 119 common differential metabolites in 5 min/CK and 3 min/CK contrastive groups were mapped onto 31 KEGG pathways including phenylpropanol metabolism, glutathione metabolism pathway, et al. In physiological experiments, results showed that the Phenylpropanoid-Metabolism-Related enzymes (PAL, C4H, 4CL) were changed after fresh-cut treatment, the activities of three enzymes increased first and then decreased, and reached the maximum value at 5 min, indicating the accumulation of phenolic substances. At the same time, ROS were accumulated when plant tissue damaged by cutting, the activities of related antioxidant enzymes (SOD, APX and CAT) changed dynamically after oxidative damage. SOD and APX content increased significantly and reached the maximum value at 10 min after cutting, and then showed a downward trend. However, CAT activity increased sharply and reached the maximum value within 3 min after cutting, then maintained the same activity, and showed a downward trend after 30 min. These data fully demonstrated that the activities of browning related enzymes and gene expression increased with the prolonging of fresh cutting time. We explained the browning mechanism of fresh-cut eggplant by combining metabolomics and physiology, which may lay the foundation for better understanding the mechanism of browning during the fruits and vegetables during processing.

The influence of soil drought stress on the leaf transcriptome of faba bean (Vicia faba L.) in the Qinghai-Tibet Plateau.

Water deficit has a significant impact on growth, development and yield of fava bean (Vicia fava L.) in arid and semi-arid climates. The aim of this study was to identify differentially expressed genes in the Qinghai 13 genotype under soil drought through leaf transcriptome analysis. A total of 256.95 M clean reads were obtained and assembled into 176334 unigenes, with an average length of 766 bp. A total of 9126 (4439 upregulated and 4687 downregulated) differentially expressed genes (DEGs) were identified in faba bean leaves under soil drought. In total, 324 putative transcription factors were identified and classified as belonging to different transcription factor families. According to GO and KEGG analysis, the soil drought stress-inducible DEGs encoded proteins mainly involved in regulating photosynthesis, osmotic adjustment, detoxification, autophagy and other functions. In addition, a large portion of DEGs appeared to be novel because they could not be annotated in any functional databases, therefore, suggesting a specific response to soil drought in faba bean. Finally, RNA-seq analysis was validated by quantitative reverse-transcription PCR analysis. This work provides comprehensive and valuable information for understanding the molecular mechanisms which faba bean uses to respond to soil drought.

Transcriptome profiling and gene expression analyses of eggplant (Solanum melongena L.) under heat stress.

Global warming induces heat stress in eggplant, seriously affecting its quality and yield. The response to heat stress is a complex regulatory process; however, the exact mechanism in eggplant is unknown. We analyzed the transcriptome of eggplant under different high-temperature treatments using RNA-Seq technology. Three libraries treated at high temperatures were generated and sequenced. There were 40,733,667, 40,833,852, and 40,301,285 clean reads with 83.98%, 79.69%, and 84.42% of sequences mapped to the eggplant reference genome in groups exposed to 28°C (CK), 38°C (T38), and 43°C (T43), respectively. There were 3,067 and 1,456 DEGs in T38 vs CK and T43 vs CK groups, respectively. In these two DEG groups, 315 and 342 genes were up- and down-regulated, respectively, in common. Differential expression patterns of DEGs in antioxidant enzyme systems, detoxication, phytohormones, and transcription factors under heat stress were investigated. We screened heat stress-related genes for further validation by qRT-PCR. Regulation mechanisms may differ under different temperature treatments, in which heat shock proteins and heat stress transcription factors play vital roles. These results provide insight into the molecular mechanisms of the heat stress response in eggplant and may be useful in crop breeding.

Chalcone synthase (CHS) family members analysis from eggplant (Solanum melongena L.) in the flavonoid biosynthetic pathway and expression patterns in response to heat stress.

Enzymes of the chalcone synthase (CHS) family participate in the synthesis of multiple secondary metabolites in plants, fungi and bacteria. CHS showed a significant correlation with the accumulation patterns of anthocyanin. The peel color, which is primarily determined by the content of anthocyanin, is an economically important trait for eggplants that is affected by heat stress. A total of 7 CHS (SmCHS1-7) putative genes were identified in a genome-wide analysis of eggplants (S. melongena L.). The SmCHS genes were distributed on 7 scaffolds and were classified into 3 clusters. Phylogenetic relationship analysis showed that 73 CHS genes from 7 Solanaceae species were classified into 10 groups. SmCHS5, SmCHS6 and SmCHS7 were continuously down-regulated under 38°C and 45°C treatment, while SmCHS4 was up-regulated under 38°C but showed little change at 45°C in peel. Expression profiles of key anthocyanin biosynthesis gene families showed that the PAL, 4CL and AN11 genes were primarily expressed in all five tissues. The CHI, F3H, F3'5'H, DFR, 3GT and bHLH1 genes were expressed in flower and peel. Under heat stress, the expression level of 52 key genes were reduced. In contrast, the expression patterns of eight key genes similar to SmCHS4 were up-regulated at a treatment of 38°C for 3 hour. Comparative analysis of putative CHS protein evolutionary relationships, cis-regulatory elements, and regulatory networks indicated that SmCHS gene family has a conserved gene structure and functional diversification. SmCHS showed two or more expression patterns, these results of this study may facilitate further research to understand the regulatory mechanism governing peel color in eggplants.

Transcriptome analysis revealed expression of genes related to anthocyanin biosynthesis in eggplant (Solanum melongena L.) under high-temperature stress.

BACKGROUND: Anthocyanin synthesis is affected by many factors, among which temperature is an important environmental factor. Eggplant is usually exposed to high temperatures during the cultivation season in Shanghai, China. Therefore,RNA -seq analysis was used to determine the effects of high-temperature stress on gene expression in the anthocyanin biosynthetic pathway of eggplant (Solanum melongena L.). RESULTS: We tested the heat-resistant cultivar 'Tewangda'. The plants were incubated at 38 °C and 45 °C, and the suitable temperature for eggplant growth was used as a control. The treatment times were 3 h and 6 h. The skin of the eggplant was taken for transcriptome sequencing, qRT-PCR assays and bioinformatic analysis. The results showed that 770 genes were differentially expressed between different treatments. Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses identified 16 genes related to anthocyanin biosynthesis, among which CHSB was upregulated. Other genes, including BHLH62, MYB380, CHI3, CHI, CCOAOMT, AN3, ACT-2, HST, 5MA-T1, CYP75A2, ANT17, RT, PAL2, and anthocyanin 5-aromatic acyltransferase were downregulated. In addition, the Myb family transcription factor PHL11 was upregulated in the CK 3 h vs 45 °C 3 h, CK 3 h vs 38 °C 3 h, and CK 6 h vs 38 °C 6 h comparisons, and the transcription factor bHLH35 was upregulated in the CK 3 h vs 38 °C 3 h and CK 6 h vs 38 °C 6 h comparisons. CONCLUSION: These results indicated that high temperature will downregulate most of the genes in the anthocyanin biosynthetic pathway of eggplant. Our data have a reference value for the heat resistance mechanism of eggplant and can provide directions for molecular breeding of heat-resistant germplasm with anthocyanin content in eggplant.

Drought stress impact on leaf proteome variations of faba bean (Vicia faba L.) in the Qinghai-Tibet Plateau of China.

Water scarcity is a major abiotic stress factor that strongly influences growth, development and yield of grain legumes in arid and semi-arid area of the world. Drought stress frequently occurs during the seedling stage and finally affects yield of faba bean (Vicia faba L.). However, the responses of plant leaf to drought have not been documented very well at the proteomic level. "Ga da dou" of the drought-tolerant faba bean cultivar was exposed to drought to examine the proteome changes of leaves. In this study, 2-week-old seedlings were subjected to water deficit by 7 days drought stress, whereas control plants were regularly irrigated. After withdrawing water, plants exposed to drought for 7 days and control plants at the same developmental stage were included in quantitative proteomic analysis using two-dimensional electrophoresis gels of proteins in combination with mass spectrometry. Over 300 proteins were detected by 2-DE, 50 differentially expressed proteins were detected by t test and 30 proteins were successfully identified by MALDI-TOF/TOF, in which 25 proteins were clearly downregulated and five proteins were upregulated. The quantified proteins were grouped into five functional groups, mainly regulatory proteins (46.7%), energy metabolism (23.3%), cell cytoskeleton (6.7%), other functions (20%) and unknown function (3.3%). Chitinase was upregulated under drought, suggesting that it was an important part of the plant defense system, playing an important role in stress resistance. 50S ribosomal protein was upregulated under drought, suggesting its role in protecting plants against stress by re-establishing normal protein conformations. The abundance of proteins involved in protein synthesis such as chitinase, Bet protein and glutamate-glyoxylate aminotransferase was upregulated under drought stress. These proteins could play important roles in drought tolerance and contribute to the relatively stronger drought tolerance of "Ga da dou".

Structure and function of seed storage proteins in faba bean (Vicia faba L.).

The protein subunit is the most important basic unit of protein, and its study can unravel the structure and function of seed storage proteins in faba bean. In this study, we identified six specific protein subunits in Faba bean (cv. Qinghai 13) combining liquid chromatography (LC), liquid chromatography-electronic spray ionization mass (LC-ESI-MS/MS) and bio-information technology. The results suggested a diversity of seed storage proteins in faba bean, and a total of 16 proteins (four GroEL molecular chaperones and 12 plant-specific proteins) were identified from 97-, 96-, 64-, 47-, 42-, and 38-kD-specific protein subunits in faba bean based on the peptide sequence. We also analyzed the composition and abundance of the amino acids, the physicochemical characteristics, secondary structure, three-dimensional structure, transmembrane domain, and possible subcellular localization of these identified proteins in faba bean seed, and finally predicted function and structure. The three-dimensional structures were generated based on homologous modeling, and the protein function was analyzed based on the annotation from the non-redundant protein database (NR database, NCBI) and function analysis of optimal modeling. The objective of this study was to identify the seed storage proteins in faba bean and confirm the structure and function of these proteins. Our results can be useful for the study of protein nutrition and achieve breeding goals for optimal protein quality in faba bean.

AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis.

The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity.

AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport.

AtNHX5 and AtNHX6, the endosomal Na+,K+/H+ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K+ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K+ and H+ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K+ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K+ at acidic pH while AtCHXs at low K+ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K+ and was sensitive to low K+ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K+ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K+ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K+ transport.

Alleviation of exogenous 6-benzyladenine on two genotypes of eggplant (Solanum melongena Mill.) growth under salt stress.

Cytokinins were recently shown to control plant adaptation to environmental stresses. To characterize the roles of cytokinins in the tolerance of eggplant (Solanum melongena Mill.) to salt stress, the protective effects of 6-benzyladenine (6-BA) on the growth, photosynthesis, and antioxidant capacity in the leaves of two eggplant cultivars Huqie12 (salt-sensitive) and Huqie4 (salt-tolerant) were investigated. Under 90 mM NaCl stress, Huqie4 showed higher biomass accumulation and less oxidative damage compared to the Huqie12. Application of exogenous 10 µM 6-BA significantly alleviated the growth suppression caused by salt stress in two eggplant genotypes. In parallel with the growth, 6-BA application in salt-stressed plants resulted in enhanced chlorophyll contents, as well as photosynthetic parameters such as net CO2 assimilation rate (P n), stomatal conductance (g s), transpiration rate (E), and intercellular CO2 concentration (C i). Furthermore, exogenous 6-BA also significantly reduced the O2 (-) production rate and malondialdehyde content and markedly increased the antioxidant enzymes superoxide dismutase and peroxidase, the antioxidant metabolites ascorbate and reduced glutathione (GSH), and proline in both genotypes under salt stress. The results indicate that exogenous 6-BA is useful to improve the salt resistance of eggplant, which is most likely related to the increase in photosynthesis and antioxidant capacity.