Transgenic tobacco plants expressing grass <Emphasis Type="Italic">AstEXPA1</Emphasis> gene show improved performance to several stresses

Expansins are cell wall-loosening proteins and now widely accepted to associate with the plant resistance against various abiotic stresses. In this study, we cloned an expansin gene of AstEXPA1 from Agrostis stolonifera, a heat-resistant creeping bentgrass cultivar, and transformed it into tobacco plants. Physiological index test showed that the transgenic lines were resistant to various abiotic stresses of drought, heat, cold, and salt in comparison to non-transgenic plants. Comprehensive analysis of four physiological response indices showed that the transgenic plants performed much better resistance to drought, following to heat, cold and salt stress, respectively. Meanwhile soluble sugar content displayed more weight to plant resistance by over-expressing AstEXPA1 gene, followed as proline content, REL, and MDA content. The results here would expand our understanding of the expansin roles and drive better insights into plant molecular breeding against stress.     

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.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

<Emphasis Type="Italic">MsZEP</Emphasis>, a novel zeaxanthin epoxidase gene from alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>), confers drought and salt tolerance in transgenic tobacco

Key message

The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum.

Abstract

Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

The opposite roles of <Emphasis Type="Italic">OsmiR408</Emphasis> in cold and drought stress responses in <Emphasis Type="Italic">Oryza sativa</Emphasis>

MiR408 is a conserved miRNA family in plants. Although AtmiR408 is generally regarded as participating in stress responses, it still remains obscure whether OsmiR408 modulates tolerance to environmental stress. In the current study, expression of Pre-OsmiR408 and OsmiR408 was found to be induced by cold stress, but repressed by drought stress in the rice cultivar “Kongyu 131”. By comparing the wild type and OsmiR408 transgenic lines, we found that OsmiR408 overexpression conferred enhanced cold tolerance at both the early seedling stage and the young seedling stage. On the other hand, the OsmiR408 transgenic lines exhibited decreased drought tolerance, which is further verified by greater water loss. We also predicted the putative target genes of OsmiR408 and verified the decreased expression of seven targets in OsmiR408 transgenic lines, including four phytocyanins and three atypical target genes. Among them, Os09g29390, a phytocyanin gene, and Os01g53880, an auxin responsive Aux/IAA gene, were down-regulated by cold treatment, which is opposite to the cold-induced expression of OsmiR408. Taken together, our results suggest opposite roles of OsmiR408 in plant responses to cold and drought stresses.     

Overexpression of sugarcane gene <Emphasis Type="Italic">SoSnRK2.1</Emphasis> confers drought tolerance in transgenic tobacco

Key message

Overexpression of SoSnRK2.1 improved drought tolerance and growth of tobacco plants.

Abstract

Sucrose non-fermenting1-related protein kinase 2 (SnRK2) is a key enzyme in regulating ABA signal transduction in plants, and it plays a significant role in response to multiple abiotic stresses. In this research, SoSnRK2.1 gene was cloned from sugarcane variety GT21 and characterized under various stresses. The cloned SoSnRK2.1 gene has a complete open reading frame of 1002 bp, encoding a peptide of 333 amino acids. The amino acid sequence of SoSnRK2.1 has high homology with those of Zea mays and Oryza sativa, which belongs to SnRK2 s families. The expression of SoSnRK2.1 under stresses of drought, PEG, and ABA indicated that this gene is involved in stress responses in sugarcane. To investigate the gene function, fusional SoSnRK2.1-GFP-pBI121 under control of CaMV 35S was transformed into tobacco plants. Growth and morphology of transgenic plants demonstrated that overexpression of SoSnRK2.1 enhanced drought tolerance in tobacco. Transgenic tobacco plants had lower levels of ion leakage (IL), and contents of maleic dialdehyde (MDA) and H₂O₂, with higher activities of three antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and chlorophyll and relative water content (RWC) than those in wide type (WT) tobacco. SoSnRK2.1 was stably transmitted to the next generation via sexual reproduction. Though the data presented here are from a heterologous system, it is highly likely that SoSnRK2.1 is involved in the abiotic stress response in sugarcane and may be playing an important role in regulation of its growth.

     

Overexpression of <Emphasis Type="Italic">AlTMP2</Emphasis> gene from the halophyte grass <Emphasis Type="Italic">Aeluropus littoralis</Emphasis> in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H₂O₂, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na⁺ and higher K⁺ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.