Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

Characterization and primary functional analysis of a bamboo NAC gene targeted by miR164b

Key message

PeSNAC1 , a stress-related NAC1 from Phyllostachys edulis , was characterized. Ectopic expression in Arabidopsis indicated that PeSNAC1 together with ped -miR164b participated in the regulation of organ boundaries and stress tolerance.

Abstract

NAC (NAM, ATAF1/2 and CUC2) participates in many different processes regulating plant growth, development, and stress response. A total of 125 NAC genes have been predicted in moso bamboo (Phyllostachys edulis), but their roles are poorly understood. PeSNAC1 targeted by ped-miR164b was focused for further study. The cleavage of PeSNAC1 mRNA guided by ped-miR164b was validated using RLM-5′ RACE. Tissue-specific expression analysis demonstrated that ped-miR164b had a declining trend from root, sheath, leaf, to that of stem, which was opposite to that of PeSNAC1. Transgenic Arabidopsis plants overexpressing either PeSNAC1 (OX-PeSNAC1) or, ped-miR164b (OX-ped-miR164b) driven by the CaMV35S promoter were generated. OX-ped-miR164b plants showed similar phenotype of cuc2 mutants whose growth was seriously suppressed. Compared with Col-0, sense OX-PeSNAC1 plants grew rapidly and flowered earlier, whereas antisense plants grew slowly and exhibited delayed flowering. Sense OX-PeSNAC1 plants had the greatest number of lateral roots, while antisense OX-PeSNAC1 and OX-ped-miR164b plants had fewer lateral roots than Col-0. Under NaCl and PEG6000 stresses, survival rates were higher and F _v/F _m values declined more slowly in sense OX-PeSNAC1 plants than in Col-0, with lower survival rates and a more rapid decrease in F _v/F _m values conversely observed in antisense OX-PeSNAC1 and OX-ped-miR164b plants. These findings indicated that ped-miR164b-targeted PeSNAC1 may play key roles in plant development and tolerance to salinity and drought stresses.

     

AM1 is a potential ABA substitute for drought tolerance as revealed by physiological and ultra-structural responses of oilseed rape

Abscisic acid (ABA) is an important signaling molecule for plants under drought tolerance. However, ABA itself has many limitations to be used in agriculture practically. Recently, AM1 (ABA-mimicking ligand) has been found to replace ABA. In this study, we have investigated AM1’s potential role for drought tolerance by growing two contrasting rapeseed (Brassica napus L.) genotypes: Qinyou 8 (drought sensitive) and Q2 (drought resistant) with exogenous ABA or AM1 application under well-watered and drought-stressed conditions. Results demonstrate that drought stress has hampered plant growth (relative height growth rate, plant biomass, leaf area), plant water status (leaf relative water content, root moisture content, leaf water potential), photosynthetic gas exchange attributes like net photosynthesis rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), transpiration rate (E); chlorophyll fluorescence parameters like photosynthetic efficiency (Fv/Fm), effective quantum yield of PSII (Φ _PSII ), photochemical quenching coefficient (qL), electron transport rate (ETR) and chlorophyll content, especially for Qinyou 8 significantly compared to well-watered plants. Whereas increased root/shoot ratio (R/S), water use efficiency (WUE) and non-photochemical quenching (NPQ) was recorded in both genotypes under drought stress. On the other hand, exogenous ABA or AM1 treatment has regulated all the above parameters in a rational way to avoid drought stress. Chloroplast transmission electron microscope images, especially for Qinyou8, have revealed that oxidative stress induced by drought has blurred the grana thylakoids, increased the size or number of plastoglobules due to lipid peroxidation, and the presence of starch granules depict weak capacity to convert them into simple sugars for osmotic adjustment. However, intact grana thylakoid, few plastoglobules with no or very few starch granules were observed in the chloroplast from ABA- or AM1-treated plants under drought. More importantly, AM1-treated plants under drought stress have responded in an extremely similar way like ABA-treated ones. Finally, it is suggested that AM1 is a potential ABA substitute for plant drought tolerance.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

Over-expression of <Emphasis Type="Italic">CsGSTU</Emphasis> promotes tolerance to the herbicide alachlor and resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> in transgenic tobacco

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm^-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with enzyme properties, recombinant CsGSTs were in vitro expressed and tested for GST activity using alachlor as substrate. The recombinant GSTU2 enzyme was twice more active than GSTU1 in conjugating alachlor to GSH thus indicating that CsGSTU2 might play a crucial role in the plant defense against the herbicide. Moreover, as a consequence of the infiltration with a bacterial suspension of the P. syringae pv. tabaci, transgenic tobacco plants but not wild type plants bestowed the capability to limit toxic metabolite diffusion through plant tissues as indicated by the absence of chlorotic halos formation. Consequently, the transgenic tobacco plants described in the present study might be utilized for phytoremediation of residual xenobiotics in the environment and might represent a model for engineering plants that resist to pathogen attack.     

Cloning <Emphasis Type="Italic">PIP</Emphasis> genes in drought-tolerant vetiver grass and responses of transgenic <Emphasis Type="Italic">VzPIP2;1</Emphasis> soybean plants to water stress

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.     

Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.