Over-expression of DREB46 enhances drought tolerance in Populus trichocarpa.

The drought responsive element binding (DREB) gene family has a significant role in plant abiotic stress responses. Here, we cloned a drought-inducible DREB gene, DREB46 (Potri.019G075500), and investigated its function in drought tolerance in Populus trichocarpa. Under treatment with exogenous abscisic acid and 6% PEG6000, DREB46 was rapidly and abundantly expressed. We successfully inserted P. trichocarpa DREB46 constructs into P. trichocarpa. After 11 d of drought stress and 3 d of rehydration treatment, the DREB46 over-expression (OE) lines exhibited significantly increased survival rates relative to the wild type (WT). Histochemical staining showed that the accumulation of reactive oxygen species (ROS) in transgenic plants under drought stress was lower than that in WT plants. Furthermore, OE plants displayed higher superoxide dismutase, peroxidase, and catalase activities and proline content, but lower malondialdehyde content than the WT plants under drought stress. In contrast, DREB46-RNA interference (RNAi) lines exhibited the opposite phenotype. Under PEG-6000 stress, OE plants produced significantly more adventitious roots (ARs) than WT plants. In contrast, RNAi-mediated DREB46-inhibited poplar exhibited fewer ARs. Quantitative real-time PCR indicated that WOX11/12a (Potri.013G066900), a gene related to root growth and development regulation, was significantly increased in OE plants. Additionally, yeast two-hybrid (Y2H) assays showed that DREB46 could interact with protein kinase MPK1 (Potri.002G032100) and protein phosphatase PP2C47 (Potri.007G058700), respectively, and this result was also verified by luciferase complementation assay. Transient co-expression results of leaves showed that PP2C47 and DREB46 Agrobacterium-transformed leaves had strong drought tolerance. These results show that DREB46 plays a key role in drought tolerance by inducing the ROS scavenging system and increasing the number of ARs.

Genome-wide identification of WD40 superfamily in Cerasus humilis and functional characteristics of ChTTG1.

The WD40 superfamily plays an important role in a wide range of developmental and physiological processes. It is a large gene family in eukaryotes. Unfortunately, the research on the WD40 superfamily genes in Cerasus humilis has not been reported. 198 ChWD40s were identified and analyzed in the present study, along with evolutionary relationships, gene structure, chromosome distribution, and collinearity. Then, 5 pairs of tandem duplication and 17 pairs of segmental duplication were found. Based on RNA-Seq data analysis, we screened 31 candidate genes whose expression was up-regulated during the four developmental stages of fruit peel. In addition, we also demonstrated that ChWD40-140, namely ChTTG1, located in the nucleus, cytoplasm, and cytomembrane, has transcriptional activation activity and can form homodimers. ChTTG1 is involved in anthocyanin biosynthesis through heterologous overexpression in Arabidopsis. These research results provide a reference for a comprehensive analysis of the functions of WD40 in the future.

Overexpression of Cerasus humilis ChAOX2 improves the tolerance of Arabidopsis to salt stress.

Alternative oxidase (AOX) has a well-established involvement in plant growth and stress tolerance in many studies. In this study, we isolated and characterized the AOX2 from Cerasus humilis. The ChAOX2 Open Reading Frame (ORF) contains 1029 nucleotides and encodes 342 amino acid residues. The inferred amino acid sequence of ChAOX2 shared the highest sequence similarity with a homolog from Prunus yedoensis. The ChAOX2 transcripts were relatively abundant in the old leaves and significantly up-regulated by salt stress. Subcellular localization analysis showed that ChAOX2 was located in the mitochondria. We transformed ChAOX2 into wild-type Arabidopsis thaliana and found that compared with wild-type and aox mutant lines, heterotopic expression of ChAOX2 increased proline content, and peroxidase and superoxide dismutase activities, while decreasing relative conductivity and the reactive oxygen species level. Further, the ratio of alternate respiration to the total respiration in plants that overexpressed ChAOX2 was significantly higher than that in wild-type and mutant plants under salt stress. These results indicate that ChAOX2 plays a key role in salt tolerance.

Ectopic expression of the transcription factor CUC2 restricts growth by cell cycle inhibition in Arabidopsis leaves.

Plant leaf margins produce small outgrowths or teeth causing serration in a regular arrangement, which is specified by auxin maxima. In Arabidopsis, the spatiotemporal pattern of auxin dependents on both, the transcription factor CUC2 and the signal peptide EPFL2, a ligand of the growth-promoting receptor kinase ERECTA (ER). Ectopic expression of CUC2 can have contrary effects on leaf growth. Ubiquitous expressed CUC2 suppresses growth in the whole leaf, whereas cuc2-1D mutants have enlarged leaves, through ER-dependent cell proliferation in the teeth. Here we investigated the growth dynamics of cuc2-1D leaves and the growth restricting the function of CUC2 using the ubiquitous inducible CUC2-GR transgene. In time courses, we dissected the serration promoting the function of CUC2 in the leaf margin and ectopic growth inhibition by CUC2 in the leaf plate. We found that CUC2 limits growth rather by cell cycle inhibition than by cell size control. Furthermore, endogenous CUC2 was rapidly induced by CUC2-GR indicating a possible auto-inducible feedback. In contrast, EPFL2 was quickly decreased by transient CUC2 induction but increased in cuc2-3 mutant leaves suggesting that CUC2 can also counteract the EPFL2-ER pathway. Therefore, tooth growth promotion and growth inhibition by CUC2 involve partially the same mechanism but in contrary ways.

Overexpression of the ChVDE gene, encoding a violaxanthin de-epoxidase, improves tolerance to drought and salt stress in transgenic Arabidopsis.

To investigate the protective mechanism of violaxanthin de-epoxidase (VDE) zeaxanthin in Cerasus humilis under drought and salt-stress conditions, we cloned the entire cDNA sequence of ChVDE from C. humilis and generated ChVDE-overexpression (OE) and ChVDE-complementation (CE) Arabidopsis plants. The open reading frame of ChVDE contained 1,446 bp nucleotides and encoded 481 amino acids. The ChVDE showed the highest similarity with those of Camellia sinensis and Citrus sinensis. Subcellular localization analysis showed that ChVDE was located in the chloroplasts. OE plants showed stronger root growth and higher levels of total chlorophyll as compared to WT and VDE mutant (npq1-2) plants. Moreover, the relative de-epoxidation state of the xanthophyll cycle pigments (A + Z)/(V + A+Z) was higher in OE plants than in the controls. OE plants had enhanced photosynthetic rates, respiration rates, and transpiration rates compared with the WT or npq1-2 plants after drought or salt treatment. Collectively, our results demonstrate that ChVDE plays a positive role in both drought and salt tolerance.

Drought Tolerance Is Correlated with the Activity of Antioxidant Enzymes in Cerasus humilis Seedlings.

Cerasus humilis, grown in the northern areas of China, may experience water deficit during their life cycle, which induces oxidative stress. Our present study was conducted to evaluate the role of oxidative stress management in the leaves of two C. humilis genotypes, HR (drought resistant) and ND4 (drought susceptible), when subjected to a long-term soil drought (WS). The HR plants maintained lower membrane injury due to low ROS and MDA accumulation compared to ND4 plants during a long-term WS. This is likely attributed to global increase in the activities of superoxide dismutase (SOD) isoenzymes and enzymes of the ascorbate-glutathione (AsA-GSH) cycle and maintenance of ascorbate (AsA) levels. Consistent closely with enzymes activities, the expression of cytosolic ascorbate peroxidase (cAPX) and dehydroascorbate reductase (DHAR) followed a significant upregulation, indicating that they were regulated at the transcriptional level for HR plants exposed to WS. In contrast, ND4 plants exhibited high ROS levels and poor antioxidant enzyme response, leading to enhanced membrane damage during WS conditions. The present study shows that genotypic differences in drought tolerance could be likely attributed to the ability of C. humilis plants to induce antioxidant defense under drought conditions.

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves.

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber (Cucumis sativus L.) and tomato (Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate-glutathione (AsA-GSH) cycle enzymes and the production of H(2)O(2) in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA-GSH cycle enzymes followed by an increased H(2)O(2) accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.

Genotypic variation of Rubisco expression, photosynthetic electron flow and antioxidant metabolism in the chloroplasts of chill-exposed cucumber plants.

Genetic improvement of agronomic crops is necessary to cope with chilling stress. To identify the physiological factors responsible for this genotypic difference in chill-induced inhibition of photosynthesis, leaf CO2 assimilation, the electron flux in the chloroplast and the antioxidant metabolism in isolated chloroplasts were examined in two genotypes of cucumber (Cucumis sativus) plants with distinct chilling tolerance. Cucumber plants were exposed to 100 micromol m(-2) s(-1) at 9/7 degrees C (day/night) for 10 d and were then returned to optimal conditions for 2 d. Chilling resulted in more significant reductions in rbcL and rbcS transcripts, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) content and initial Rubisco activity, leading to higher electron flux to O2 in the chilling-sensitive genotype than in the chilling-tolerant genotype. The chilling-tolerant genotype showed lower H2O2 contents in the chloroplasts by maintaining higher H2O2-scavenging activity in the chloroplasts than in the chilling-sensitive genotype. H2O2 accumulation in chloroplast was negatively correlated with the initial Rubisco activity and photosynthetic rate.

Response of ascorbate peroxidase isoenzymes and ascorbate regeneration system to abiotic stresses in Cucumis sativus L.

Ascorbate peroxidase (APX) isoenzymes, distributing in at least four distinct cell compartments, the chloroplastic stroma (sAPX) and thylakoid membrane (tAPX), microbody (mAPX) and cytosol (cAPX), catalyze the reduction of H(2)O(2) to water by using ascorbic acid (AsA) as specific electron donor. In order to better clarify the response of APX isoenzymes and AsA regeneration enzymes to abiotic stresses, the activities of APX isoenzymes as well as monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were investigated in cucumber plants after heat, methyl viologen (MV) and H(2)O(2) treatments. The activities of cAPX, sAPX, mAPX increased after a slight decline throughout the experiment. Consistent closely with sAPX activity, the expression of sAPX followed a similar change pattern, indicating that sAPX was regulated at the transcriptional level. In contrast, constitutive expression was observed in tAPX activity and no significant changes in tAPX activity were found throughout the experiment. The increases in MDAR and GR were accompanied with enhanced level of AsA/DHA, implying that the AsA regeneration system plays an essential role in compensating AsA degradation.