Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Comparative Functional Analysis of Wheat (Triticum aestivum) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.     

Expression of the <Emphasis Type="Italic">Vicia faba VfPIP1</Emphasis> gene in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants improves their drought resistance

Plant aquaporins are believed to facilitate water transport across cell membranes. However, the relationship between aquaporins and drought resistance in plants remains unclear. VfPIP1, a putative aquaporin gene, was isolated from Vicia faba leaf epidermis, and its expression was induced by abscisic acid (ABA). Our results indicated that the VfPIP1 protein was localized in the plasma membrane, and its expression in V. faba was induced by 20% polyethylene glycol 6000. To further understand the function of VfPIP1, we obtained VfPIP1-expressing transgenic Arabidopsis thaliana plants under the control of the CaMV35S promoter. As compared to the wild-type control plants, the transgenic plants exhibited a faster growth rate, a lower transpiration rate, and greater drought tolerance. In addition, the stomata of the transgenic plants closed significantly faster than those of the control plants under ABA or dark treatment. These results suggest that VfPIP1 expression may improve drought resistance of the transgenic plants by promoting stomatal closure under drought stress.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

The wheat gene <Emphasis Type="Italic">TaST</Emphasis> can increase the salt tolerance of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

On the basis of the results of gene chip analysis of the salt-tolerant wheat mutant RH8706-49 under conditions of salt stress, we identified and cloned an unknown salt-induced gene TaST (Triticum aestivum salt-tolerant). Real-time quantitative PCR analysis showed that the expression of the gene was induced by salt stress. Transgenic Arabidopsis plants overexpressing the TaST gene showed higher salt tolerance than the wild-type controls. Subcellular localization studies revealed that the protein encoded by this gene was in the nucleus. In comparison with wild-type controls, transgenic Arabidopsis plants accumulated more Ca²⁺, soluble sugar, and proline and less Na⁺ under salt stress. Real-time quantitative PCR analysis showed that Arabidopsis plants overexpressing TaST also showed increased expression of many stress-related genes. All these findings indicated that TaST can enhance the salt tolerance of transgenic Arabidopsis plants.     

Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses

Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause–effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse‐maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress‐inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.     

The cotton endocycle‐involved protein SPO11‐3 functions in salt stress via integrating leaf stomatal response,ROS scavenging and root growth

The SPORULATION 11 (SPO11) proteins are among eukaryotic the topoisomerase VIA (Topo VIA) homologs involved in modulating various important biological processes, such as growth, development and stress response via endoreduplication in plants, but the underlying mechanism response to stress remains largely unknown under salt treatment. Here, we attempted to characterize a homolog of TOP VIA in upland cotton (Gossypium hirsutum L.), designated as GhSPO11‐3. The silencing of GhSPO11‐3 in cotton plants resulted in a dwarf phenotype with a failure of cell endoreduplication and a phase shift in the ploidy levels. The GhSPO11‐3‐silenced plants also showed substantial changes including accumulated malondialdehyde, significantly reduced chlorophyll and proline contents and decreased antioxidative enzyme activity after salt treatment. In addition, transgenic Arabidopsis lines overexpressing GhSPO11‐3 accelerated both leaf and root growth with cell expansion and endopolyploidy. Both leaf stomatal density and aperture were markedly decreased, and the transgenic Arabidopsis lines were more tolerant with expression of stress‐responsive genes under salinity stress. Furthermore, consistent with the reduced reactive oxygen species (ROS), the expression of ROS scavenging‐related genes was largely reinforced, and antioxidant enzyme activities were accordingly significantly enhanced in transgenic Arabidopsis lines under salt stress. In general, these results indicated that GhSPO11‐3 likely respond to salt stress by positively regulating root growth, stomatal response, ROS production and the expression of stress‐related genes to cope with adverse conditions in plants.     

Over-expression of PIP2;5 aquaporin alleviates gas exchange and growth inhibition in poplars exposed to mild osmotic stress with polyethylene glycol

The effects of mild osmotic stress conditions on aquaporin-mediated water transport are not well understood. In the present study, mild osmotic stress treatments with 20 and 50 g L^?1 polyethylene glycol 6000 (PEG) in Hoagland’s mineral solution were applied for 3 weeks under controlled environmental conditions to transgenic Populus tremula × Populus alba plants constitutively over-expressing a Populus PIP2;5 aquaporin and compared with the wild-type plants. The PEG treatments resulted in growth reductions and triggered changes in net photosynthesis, transpiration, stomatal conductance and root hydraulic conductivity in the wild-type plants. However, height growth, leaf area, gas exchange, and root hydraulic conductivity were less affected by the PEG treatments in PIP2;5-over-expressing poplar lines. These results suggest that water transport across the PIP2;5 aquaporin is an important process contributing to tolerance of mild osmotic stress in poplar. Greater membrane abundance of PIP2;5 was most likely the factor that was responsible for higher root hydraulic conductivity leading to improved plant water flux and, consequently, greater gas exchange and growth rates under mild osmotic stress conditions. The results also provide evidence for the functional significance of PIP2;5 aquaporin in water transport and its strong link to growth processes in poplar.     

ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

Arabidopsis thaliana Atrab28: a nuclear targeted protein related to germination and toxic cation tolerance

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues.Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.     

Overexpression of microRNA395c or 395e affects differently the seed germination of Arabidopsis thaliana under stress conditions

The Arabidopsis genome encodes six members of microRNA395 (miR395) family previously determined to regulate the expression of ATP sulfurylase (APS) and the sulfate transporter SULTR2;1. However, the mRNA targets for the individual miR395 family members and the biological consequences produced by target gene regulation of each miR395 remain to be identified. In this study, a transgenic approach was employed to determine the mRNA targets for each miR395 family member as well as the role each member plays in plant growth under abiotic stress conditions. Overexpression of miR395c or miR395e retarded and accelerated, respectively, the seed germination of Arabidopsis under high salt or dehydration stress conditions. Despite a single nucleotide difference between miR395c and miR395e, the cleavage of mRNA targets, APS1, APS3, APS4 and SULTR2;1, was not same in miR395c- and miR395e-overexpressing plants. These results demonstrate that a given miRNA family containing a single nucleotide difference can guide the cleavage of various mRNA targets, thereby acting as a positive or negative regulator of seed germination under stress.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils.     

Expression and inhibition of aquaporins in germinating Arabidopsis seeds

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.