Comparative transcriptome analysis of high- and low-embryogenic Hevea brasiliensis genotypes reveals involvement of phytohormones in somatic embryogenesis.

BACKGROUND: Rubber plant (Hevea brasiliensis) is one of the major sources of latex. Somatic embryogenesis (SE) is a promising alterative to its propagation by grafting and seed. Phytohormones have been shown to influence SE in different plant species. However, limited knowledge is available on the role of phytohormones in SE in Hevea. The anther cultures of two Hevea genotypes (Yunyan 73477-YT and Reken 628-RT) with contrasting SE rate were established and four stages i.e., anthers (h), anther induced callus (y), callus differentiation state (f), and somatic embryos (p) were studied. UPLC-ESI-MS/MS and transcriptome analyses were used to study phytohormone accumulation and related expression changes in biosynthesis and signaling genes. RESULTS: YT showed higher callus induction rate than RT. Of the two genotypes, only YT exhibited successful SE. Auxins, cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), gibberellins (GAs), and ethylene (ETH) were detected in the two genotypes. Indole-3-acetic acid (IAA), CKs, ABA, and ETH had notable differences in the studied stages of the two genotypes. The differentially expressed genes identified in treatment comparisons were majorly enriched in MAPK and phytohormone signaling, biosynthesis of secondary metabolites, and metabolic pathways. The expression changes in IAA, CK, ABA, and ETH biosynthesis and signaling genes confirmed the differential accumulation of respective phytohormones in the two genotypes. CONCLUSION: These results suggest potential roles of phytohormones in SE in Hevea.

VaSUS2 confers cold tolerance in transgenic tomato and Arabidopsis by regulation of sucrose metabolism and ROS homeostasis.

KEY MESSAGE: VaSUS2 enhances cold tolerance of transgenic tomato and Arabidopsis by regulating sucrose metabolism and improving antioxidant enzymes activity. Sucrose synthetase (SUS) is a key enzyme of sugar metabolism, and plays an important role in response to abiotic stress in plant. However, the function of VaSUS2 remains unknown in cold tolerance. Here, the cloning and functional characterization of the plasma membrane-localized VaSUS2 gene isolated from Vitis amurensis was studied. The transcript level of VaSUS2 was up-regulated under cold stress in Vitis amurensis. Heterologous expression of VaSUS2 in tomato increased SUS activity, which promoted the accumulation of glucose and fructose under cold treatment. The transgenic tomato and Arabidopsis exhibited higher levels of antioxidant enzymes activity, lower relative electrolyte leakage (REL), malondialdehyde (MDA) and hydrogen peroxide (H2O2) content compared to wild type under cold stress. Importantly, the ability of scavenging reactive oxygen species (ROS) in transgenic plants was significantly improved. Moreover, yeast two-hybrid (Y2H) indicated that VaSnRK1 might be a potential interaction protein of VaSUS2. qRT-PCR showed that sucrose metabolism-related genes SlSUS, SlSPS and SlINV were significantly up-regulated in transgenic tomatoes. Meanwhile, the expression levels of antioxidant enzyme genes and cold-related genes CBF1, COR47 and ICE1 were up-regulated in transgenic plants. Taken together, these results suggested that VaSUS2 was involved in cold tolerance by increasing the levels of soluble sugars, improving the activity of antioxidant enzymes, and up-regulating the expression of cold-related genes in transgenic tomatoes and Arabidopsis.

VaBAM1 weakens cold tolerance by interacting with the negative regulator VaSR1 to suppress ß-amylase expression.

Cold stress is a key climatic factor that limits grape productivity and quality. Although ß-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1-overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1-overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1-overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance.

Identification and expression analysis of the grape pentatricopeptide repeat (PPR) gene family in abiotic stress.

The pentatricopeptide repeat (PPR) is one of the largest gene family in plants, and play important role in regulating plant growth, development and abiotic stress response. However, PPR genes have been poorly studied in grapes. In this study, based on the grape genome database, bioinformatics methods and quantitative real-time PCR (qRT-PCR) were used to identify the VvPPR family and the response to abiotic stress. A total of 181 PPR genes were identified in grape and divided into two subfamilies. Subcellular localization predicted that this gene family mainly functions in chloroplasts, nucleus, and mitochondria. Protein-protein interaction prediction indicated that there may be interaction between VvPPR44,53 and VvPPR44. The promoter region of VvPPR gene family contained various cis-acting elements, which were related to light and hormone. Expression pattern analysis showed that the VvPPR gene family was highly expressed in grape leaves, buds and carpel tissues. qRT-PCR results showed that the expression of VvPPR genes in roots was higher than stems and leaves under NAA, SA, ABA, MeJA and GA3 treatments. VvPPR8 was significantly up-regulated after GA3 and MeJA treatment for 24 h, VvPPR53 was significantly up-regulated after SA, NAA, ABA and MeJA treatment. In addition, In grape leaves, VvPPR53 was up-regulated under PEG, Nacl and 4 â„ƒ treatments. These data indicate that VvPPR gene family members are responsive to hormones and abiotic stresses, and that there are some differences in the degree of response and expression in different grape tissues. This study provides a certain theoretical basis for grape resistance breeding.

Changes and response mechanism of sugar and organic acids in fruits under water deficit stress.

The content and the ratio of soluble sugars and organic acids in fruits are significant indicators for fruit quality. They are affected by multiple environmental factors, in which water-deficient is the most concern. Previous studies found that the content of soluble sugars and organic acids in fruit displayed great differences under varied water stress. It is important to clarify the mechanism of such difference and to provide researchers with systematic knowledge about the response to drought stress and the mechanism of sugar and acid changes in fruits, so that they can better carry out the study of fruit quality under drought stress. Therefore, the researchers studied dozens of research articles about the content of soluble sugar and organic acid, the activity of related metabolic enzymes, and the expression of related metabolic genes in fruits under water stress, and the stress response of plants to water stress. We found that after plants perceived and transmitted the signal of water deficit, the expression of genes related to the metabolism of soluble sugars and organic acids changed. It was then affected the synthesis of metabolic enzymes and changed their metabolic rate, ultimately leading to changes in soluble sugar and organic acid content. Based on the literature review, we described the pathway diagrams of sugar metabolism, organic acid metabolism, mainly malic acid, tartaric acid, and citric acid metabolism, and of the response to drought stress. From many aspects including plants' perception of water stress signal, signal conversion and transmission, induced gene expression, the changes in soluble sugar and the enzyme activities of organic acids, as well as the final sugar and acid content in fruits, this thesis summarized previous studies on the influence of water stress on soluble sugars and the metabolism of organic acids in fruits.

Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme.

BACKGROUND: Abscisic acid (ABA) has been reported in controlling plant growth and development, and particularly dominates a role in resistance to abiotic stress. The Pyrabactin Resistance1/PYR1-Like /Regulatory Components of ABA receptors (PYR1/PYL/RCAR) gene family, of which the PYL9 is a positive regulator related to stress response in ABA signaling transduction. Although the family has been identified in grape, detailed VaPYL9 function in cold stress remains unknown. RESULTS: In order to explore the cold tolerance mechanism in grape, VaPYL9 was cloned from Vitis amurensis. The subcellular localization showed that VaPYL9 was mainly expressed in the plasma membrane. Yeast two-hybrid (Y2H) showed VaPCMT might be a potential interaction protein of VaPYL9. Through the overexpression of VaPYL9 in tomatoes, results indicated transgenic plants had higher antioxidant enzyme activities and proline content, lower malondialdehyde (MDA) and H2O2 content, and improving the ability to scavenge reactive oxygen species than wild-type (WT). Additionally, ABA content and the ratio of ABA/IAA kept a higher level than WT. Quantitative real-time PCR (qRT-PCR) showed that VaPYL9, SlNCED3, SlABI5, and antioxidant enzyme genes (POD, SOD, CAT) were up-regulated in transgenic tomatoes. Transcriptome sequencing (RNA-seq) found that VaPYL9 overexpression caused the upregulation of key genes PYR/PYL, PYL4, MAPK17/18, and WRKY in transgenic tomatoes under cold stress. CONCLUSION: Overexpression VaPYL9 enhances cold resistance of transgenic tomatoes mediated by improving antioxidant enzymes activity, reducing membrane damages, and regulating key genes in plant hormones signaling and antioxidant enzymes.

VaAPL1 Promotes Starch Synthesis to Constantly Contribute to Soluble Sugar Accumulation, Improving Low Temperature Tolerance in Arabidopsis and Tomato.

ADP-glucose pyrophosphorylase (AGPase) is a key rate-limiting enzyme involved in starch synthesis. APL1, an AGPase large subunit, plays an important role in the growth and development of grapes; however, its function in withstanding low temperature (LT) remains elusive. Hence, VaAPL1 was cloned from Vitis amurensis (Zuoshan I), and its function was characterized. The gene was highly expressed in the phloem of V. amurensis during winter dormancy (0, -5, and - 10°C). Phylogenetic relationships demonstrated that VaAPL1 was closely genetic related to SlAPL1 (from Solanum lycopersicum), and clustered into I group. Further, VaAPL1 was ectopically expressed in Arabidopsis thaliana (ecotype Columbia, Col) and tomato ("Micro-Tom" tomato) to characterize its function under LT. Compared with Col, the average survival rate of VaAPL1-overexpressing A. thaliana exceeded 75.47% after freezing treatment. Moreover, reactive oxygen species (ROS) content decreased in VaAPL1-overexpressing A. thaliana and tomato plants under LT stress. The activities of AGPase, and starch contents in VaAPL1-overexpressing A. thaliana were higher than in Col after LT stress. The contents of sucrose and glucose were accumulated in overexpressing plants compared with wild-type at 0 h and 24 h after LT stress. Transcriptome sequencing of overexpressing tomato plants revealed involvement in sugar metabolism and the hormone signal pathway, and Ca2+ signaling pathway-related genes were up-regulated. Hence, these results suggest that overexpression of VaAPL1 not only ensured sufficient starch converting into soluble sugars to maintain cell osmotic potential and provided energy, but also indirectly activated signal pathways involved in LT to enhance plant tolerance.

Temperature-phase transcriptomics reveals that hormones and sugars in the phloem of grape participate in tolerance during cold acclimation.

KEY MESSAGE: Most of the upregulated genes contributed to the accumulation of soluble sugars and ABA in the phloem of 'Vitis amurensis' compared to 'Merlot' during cold acclimation. Extreme cold is one of the dominant abiotic factors affecting grape yield and quality. However, the changes in sugars, phytohormones, and gene expression in the branch phloem of different tolerant grape varieties during cold acclimation remain elusive. The data supported that with decreasing temperature, the contents of fructose, sucrose, and ABA in the phloem of Vitis amurensis (cold-tolerant, T) and 'Merlot' (cold-sensitive, S) increased during cold acclimation, and these indicators were higher in T than in S. Furthermore, the activities of sucrose synthetase, sucrose phosphate synthetase, and acid invertase peaked in the early phase of cold acclimation (approximately 5 °C) compared to other phases (approximately 28 °C, 0 °C, - 5 °C and - 10 °C). Moreover, the RNA sequencing results helped identify a total of 11,343 differentially expressed genes in the phloem of T and S, among which 4912 were upregulated and 6431 were downregulated. In the abscisic acid pathway, CRTISO, PSPY1-1, CYCP707A4-2, PYL4-1, PYL4-2, P2C08, SAPK2, TARAB1, and DBF3 were more highly expressed in T than in S. In the starch and sucrose metabolism pathway, HXK1, PGMP, GLGL1, SUS6, VCINV, BGL11, SSY1, GPS, BAM1 and BAM3 were also more highly expressed in T than in S. Moreover, the genes related to oxidative phosphorylation, such as NDHF, ND4, ND1, NAD7, NAD2, ATPB, YMF19, ATP9, PMA1 and AHA8, were upregulated in T. These results will be beneficial for understanding the potential differences in tolerance across two different cold-tolerant grapes with respect to sugar metabolism and gene expression.

Insight into VvGH3 genes evolutional relationship from monocotyledons and dicotyledons reveals that VvGH3-9 negatively regulates the drought tolerance in transgenic Arabidopsis.

The Gretchen Hagen3 (GH3) gene family is necessary for growth and development in plants and is regulated by osmotic stress and various hormones. Although it has been reported in many plants, the evolutionary relationship of GH3 in grape has not been systematically analyzed from the perspective of monocotyledonous and dicotyledonous. This study identified and analyzed 188 GH3 genes, which were distinctly divided into 9 subgroups, and found these subgroups have obviously been clustered between monocotyledonous and dicotyledonous. VvGH3-x genes had higher synteny with apple and Arabidopsis than that of rice, and the average Ka/Ks value in monocotyledons was higher than that of dicotyledons. The codon usage index showed that monocotyledons preferred to use G3s, C3s, and GC3s, while dicotyledons preferred to use A3s and T3s. The GH3 genes of grape exhibited different expression patterns in various tissues, different abiotic stresses, and hormonal treatments. The subcellular localization showed that VvGH3-9 was expressed in the nucleus and cytoplasm. Additionally, under 20% PEG treatment, the IAA and ABA contents, relative expression levels of VvGH3-9, relative electrical conductivity (REC), as well as MDA were obviously increased in VvGH3-9 overexpression lines at 72 h. In contrast, compared to WT, the contents of proline and H2O2, the activities of POD, SOD, and CAT, and the relative expression levels of drought responsive genes were significantly decreased in overexpressing lines. Collectively, this study provided helpful insight for the evolution of GH3 genes and presented some possibilities to study the functions of GH3 genes in monocotyledons and dicotyledons.

Quantification of temperature dependence of vehicle evaporative volatile organic compound emissions from different fuel types in China.

The evaporative emissions of volatile organic compounds (VOCs) from motor vehicles are dependent upon the ambient temperature. However, the quantitative relationship between evaporative VOC emissions and ambient temperature has rarely been reported, and it is not reflected in the Chinese VOCs emission inventory (EI). In this study, a series of evaporative tests were conducted on a parked gasoline-fueled vehicle in a Variable Temperature Sealed Housing Evaporative Determination chamber under seven temperatures from 298 K to 313 K at intervals of 2.5 K. Results showed that total hydrocarbon emissions at 313 K were 25.7, 12.3, and 26.7 times those at 298 K for China V, China VI, and ethanol-blended E10 fuels, respectively. China V consistently exhibited the lowest evaporative VOC emissions at all temperatures, while those of E10 surpassed even those of China VI and became the highest at 308 K and higher. Along with increasing temperature, the proportions of alkanes and alkenes gradually increased whereas those of aromatics and oxygenated VOCs decreased. Alkenes accounted for less than 20% of the evaporative VOC emissions but contributed to approximately 60% of the total OH loss (LOH) at 298 K and to over 70% at 313 K. cis-2-Butene and trans-2-butene were responsible for the greatest increase in LOH from China V, due to their higher OH reactivity. Our results clearly demonstrated the exponential increases of evaporative VOC emissions and the associated atmospheric reactivity with temperature, and also highlighted that upgrading the emission standard from China V to China IV and promoting the E10 fuel would not contribute to the reduction of evaporative VOC emissions. The strong temperature dependence of evaporative VOC emissions underscores the importance of developing a temperature-driven dynamic EI in China, and the functional relationships retrieved from this study form an essential step in developing such a dynamic EI.

Genome-wide identification of BAM genes in grapevine (Vitis vinifera L.) and ectopic expression of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato.

BACKGROUND: Low temperature (LT) is one of the main limiting factors that affect growth and development in grape. Increasing soluble sugar and scavenging reactive oxygen species (ROS) play critical roles in grapevine resistance to cold stress. However, the mechanism of ß-amylase (BAM) involved in the regulation of sugar levels and antioxidant enzyme activities in response to cold stress is unclear. RESULTS: In this study, six BAM genes were identified and clustered into four groups. Multiple sequence alignment and gene structure analysis showed that VvBAM6 lacked the Glu380 residue and contained only an exon. The transcript abundance of VvBAM1 and VvBAM3 significantly increased as temperature decreased. After LT stress, VvBAM1 was highly expressed in the leaves, petioles, stems, and roots of overexpressing tomato lines. The total amylase and BAM activities increased by 6.5- and 6.01-fold in transgenic plants compared with those in wild-type tomato plants (WT) subjected to LT, respectively. The glucose and sucrose contents in transgenic plants were significantly higher than those in WT plants, whereas the starch contents in the former decreased by 1.5-fold compared with those in the latter under LT stress. The analysis of transcriptome sequencing data revealed that 541 genes were upregulated, and 663 genes were downregulated in transgenic plants. One sugar transporter protein gene (SlSTP10), two peroxidase (POD)-related genes (SlPER7 and SlPER5), and one catalase (CAT)-related gene (SlCAT1) were upregulated by 8.6-, 3.6-, 3.0-, and 2.3-fold in transgenic plants after LT stress, respectively. CONCLUSIONS: Our results suggest that VvBAM1 overexpression promotes ROS scavenging and improves cold tolerance ability by modulating starch hydrolysis to affect soluble sugar levels in tomato plants.

Alleviating damage of photosystem and oxidative stress from chilling stress with exogenous zeaxanthin in pepper (Capsicum annuum L.) seedlings.

As a typical thermophilous vegetable, the growth and yield of peppers are easily limited by chilling conditions. Zeaxanthin, a crucial carotenoid, positively regulates plant abiotic stress responses. Therefore, this study investigated the regulatory mechanisms of zeaxanthin-induced chilling tolerance in peppers. The results indicated that the pretreatment with zeaxanthin effectively alleviated chilling damage in pepper leaves and increased the plant fresh weight and photosynthetic pigment content under chilling stress. Additionally, alterations in photosynthetic chlorophyll fluorescence parameters and chlorophyll fluorescence induction curves after zeaxanthin treatment highlighted the participation of zeaxanthin in improving the photosystem response to chilling stress by heightening the quenching of excess excitation energy and protection of the photosynthetic electron transport system. In chill-stressed plants, zeaxanthin treatment also enhanced antioxidant enzyme activity and transcript expression, and reduced hydrogen peroxide (H2O2) and superoxide anion (O2•-) content, resulting in a decrease in biological membrane damage. Additionally, exogenous zeaxanthin upregulated the expression levels of key genes encoding ß-carotene hydroxylase (CaCA1, CaCA2), zeaxanthin epoxidase (CaZEP) and violaxanthin de-epoxidase (CaVDE), and promoted the synthesis of endogenous zeaxanthin during chilling stress. Collectively, exogenous zeaxanthin pretreatment enhances plant tolerance to chilling by improving the photosystem process, increasing oxidation resistance, and inducing alterations in endogenous zeaxanthin metabolism.

Genome-Wide Identification and Expression Analysis of GA2ox, GA3ox, and GA20ox Are Related to Gibberellin Oxidase Genes in Grape (VitisVinifera L.).

Gibberellin (GAs) plays the important role in the regulation of grape developmental and growth processes. The bioinformatics analysis confirmed the differential expression of GA2, GA3, and GA20 gibberellin oxidase genes (VvGA2oxs, VvGA3oxs, and VvGA20oxs) in the grape genome, and laid a theoretical basis for exploring its role in grape. Based on the Arabidopsis GA2oxs, GA3oxs, and GA20oxs genes already reported, the VvGA2oxs, VvGA3oxs, and VvGA20oxs genes in the grape genome were identified using the BLAST software in the grape genome database. Bioinformatics analysis was performed using software such as DNAMAN v.5.0, Clustalx, MapGene2Chrom, MEME, GSDS v.2.0, ExPASy, DNAsp v.5.0, and MEGA v.7.0. Chip expression profiles were generated using grape Affymetrix GeneChip 16K and Grape eFP Browser gene chip data in PLEXdb. The expression of VvGA2oxs, VvGA3oxs, and VvGA20oxs gene families in stress was examined by qRT-PCR (Quantitative real-time-PCR). There are 24 GAoxs genes identified with the grape genome that can be classified into seven subgroups based on a phylogenetic tree, gene structures, and conserved Motifs in our research. The gene family has higher codon preference, while selectivity is negative selection of codon bias and selective stress was analyzed. The expression profiles indicated that the most of VvGAox genes were highly expressed under different time lengths of ABA (Abscisic Acid) treatment, NaCl, PEG and 5 °C. Tissue expression analysis showed that the expression levels of VvGA2oxs and VvGA20oxs in different tissues at different developmental stages of grapes were relatively higher than that of VvGA3oxs. Last but not least, qRT-PCR (Real-time fluorescent quantitative PCR) was used to determine the relative expression of the GAoxs gene family under the treatment of GA3 (gibberellin 3) and uniconazole, which can find that some VvGA2oxs was upregulated under GA3 treatment. Simultaneously, some VvGA3oxs and VvGA20oxs were upregulated under uniconazole treatment. In a nutshell, the GA2ox gene mainly functions to inactivate biologically active GAs, while GA20ox mainly degrades C20 gibberellins, and GA3ox is mainly composed of biologically active GAs. The comprehensive analysis of the three classes of VvGAoxs would provide a basis for understanding the evolution and function of the VvGAox gene family in a grape plant.