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.     

Changes in leaf epicuticular wax,gas exchange and biochemistry metabolism between <Emphasis Type="Italic">Jatropha mollissima</Emphasis> and <Emphasis Type="Italic">Jatropha curcas</Emphasis> under semi-arid conditions

Jatropha curcas and Jatropha mollissima plants were evaluated under conditions of high (HSM) and low (LSM) soil moisture in a semi-arid environment, as changes in the content and concentration of epicuticular wax and the leaf metabolism which could have a relationship with drought tolerance. Besides epicuticular wax, gas exchange, antioxidant system and biochemical parameters of the photosynthetic metabolism were measured. The epicuticular wax content increased only in J. mollissima leaves 95 % under LSM, when compared with HSM conditions. Therefore, J. curcas invested less in the production of long-chain n-alkanes than did J. mollissima under LSM conditions. J. mollissima plants showed the highest CO₂ assimilation rate during the HSM period compared to J. curcas. Both species showed high stability in some leaf biochemistry products, highlighting the highest sugar content, free amino acids, total soluble protein, and photosynthetic pigments in the leaves of J. mollissima plants under both of the soil moisture conditions. Moreover, the stability and performance of the different parameters, such as morphologic variables, seem to allow J. mollissima plants to tolerate semi-arid conditions.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Modulation of proline metabolic gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under water-stressed conditions by a drought-mitigating <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ ¹ -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ ¹ -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.     

Green roof <Emphasis Type="Italic">Petunia</Emphasis>, <Emphasis Type="Italic">Ageratum</Emphasis>, and <Emphasis Type="Italic">Mentha</Emphasis> responses to water stress,seaweeds, and trinexapac-ethyl treatments

The availability of sufficient irrigation water and the development of drought-tolerant species are challenging factors in the design and maintenance of green roofs in modern cities. Green roof plants of Petunia hybrid Headliner^® Red Star, Ageratum hybrid Artist^® blue, and Mentha spicata L. grown in a simulated green roof pot system under controlled greenhouse conditions. The plants were watered every 2 or 6 days (2DWI/6DWI) for 8 weeks accompanied by either a 6-day treatment of seaweed extracts of Ascophyllum nodosum as a soil drench or foliar spray, or two concentrations of trinexapac-ethyl (TE) biweekly sprays. Following treatments, leaf number, leaf area, dry weights, plant height, stomatal conductanse, photosynthetic and transpiration rates and leaf water potential and relative water content were determined in two seasons during 2016 and 2017. The prolonged irrigation intervals reduced plant growth as revealed by morphological and physiological parameters. The application of SWE as drench treatment improved Petunia and Ageratum plant vegetative growth, stomatal conductance, photosynthetic and transpiration rates and leaf water potential and relative water content during prolonged irrigation intervals. TE increased the vegetative growth as well as the physiological performance of Ageratum plants. However, neither SWE nor TE treatments improved the performance of Mentha plants under prolonged irrigation intervals. It was suggested that improved photosynthetic rates were stimulated by enhanced stomatal conductance leading to improved leaf water potential as well as increased relative water content during prolonged irrigation conditions.     

Photosynthetic adjustment after rehydration in <Emphasis Type="Italic">Annona emarginata</Emphasis>

Reports indicate that Annona emarginata is tolerant to drought and is also used as an alternative rootstock for atemoya under drought conditions. The photosynthetic process can be adjusted after rehydration, resulting on total or partial recovery. The aim of this study was to determine if A. emarginata shows adjusts in gas exchange and the chlorophyll a fluorescence pattern after rehydration. During water deficits, the gas exchange and water content in the leaf decreased. However, after 5 days of rehydration, the water content in the leaf recovered and rehydrated plants presented the water use efficiency better than irrigated plants. Further remaining gas exchange parameters were lower in relation to irrigated plants. In chlorophyll a fluorescence, the rehydrated plants showed higher dissipation of light energy as heat, maintaining high activity of photoprotection. After rehydration, A. emarginata shows a positive correlation between transpiration and CO₂ assimilation rate, which optimize the water use efficiency. Thus, A. emarginata presents adjustments in gas exchange and photochemical process, resulting on a possible long-term photosynthetic acclimation to water deficiency.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

A glucuronokinase gene in<Emphasis Type="Italic"> Arabidopsis</Emphasis>, <Emphasis Type="Italic">AtGlcAK</Emphasis>, is involved in drought tolerance by modulating sugar metabolism

Arabidopsis glucuronokinase (AtGlcAK), as a member of the GHMP kinases family, is implicated in the de novo synthesis of UDP-glucuronic acid (UDP-GlcA) by the myo-inositol oxygenation pathway. In this study, two T-DNA insertion homozygous mutants of AtGlcAK, atglcak-1 and atglcak-2, were identified. AtGlcAK was highly expressed in roots and flowers. There was reduced primary root elongation and lateral root formation in atglcak mutants under osmotic stress. The atglcak mutants displayed enhanced stomatal opening in response to abscisic acid (ABA), elevated water loss and impaired drought tolerance. Under water stress, the accumulation of reducing and soluble sugars was reduced in atglcak mutants, and the metabolism of glucose and sucrose was affected by the synthetic pathway of UDP-GlcA. Furthermore, a reduced level of starch in atglcak mutants was observed under normal conditions. The phylogenetic analysis suggested that GlcAK was conserved in numerous dicots and monocots plants. In short, AtGlcAK mutants displayed hypersensitivity to ABA and reduced root development under water stress, rendering the plants more susceptible to drought stress.     

Molecular characterization and expression analysis of the <Emphasis Type="Italic">SPL</Emphasis> gene family with <Emphasis Type="Italic">BpSPL9</Emphasis> transgenic lines found to confer tolerance to abiotic stress in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk.

Christolea crassifolia HARDY: gene (CcHRD) belongs to the AP2/ERF-like tanscritpion factor family, and overexpression of HRD gene has been proved to result in improved water use efficiency and enhanced drought resistance in multiple plant species. In the present study, we cloned the CcHRD gene from Christolea crassifolia, which shares 99.1% sequence similarity with the HRD gene from Arabidopsis thaliana. We generated transgenic tomato plants expressing CcHRD gene by agrobacterium-mediated genetic transformation. Our results revealed that the transgenic tomato plants showed a more developed root system and higher fruit yield than the wild-type plants. Furthermore, the leaf relative water content, chlorophyll content and Fv/Fm value in transgenic plants were significantly higher than the wild type, while the relative conductivity and MDA content of transgenic plant leaves were markedly lower than those of wild type under drought stress. We also observed that the major agronomic traits of transgenic tomato plants were improved under natural drought stress compared with those of the wild type. In summary, results in this transgenic study showed that the CcHRD gene could enhance the drought resistance in tomato, and also provided important information for the application of drought-responsive genes in improving crop plant resistance to abiotic stresses.     

Aquaporin gene expression and physiological responses of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L. to the mycorrhizal fungus <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> and drought stress

The influence of arbuscular mycorrhiza (AM) and drought stress on aquaporin (AQP) gene expression, water status, and photosynthesis was investigated in black locust (Robinia pseudoacacia L.). Seedlings were grown in potted soil inoculated without or with the AM fungus Rhizophagus irregularis, under well-watered and drought stress conditions. Six full-length AQP complementary DNAs (cDNAs) were isolated from Robinia pseudoacacia, named RpTIP1;1, RpTIP1;3, RpTIP2;1, RpPIP1;1, RpPIP1;3, and RpPIP2;1. A phylogenetic analysis of deduced amino acid sequences demonstrated that putative proteins coded by these RpAQP genes belong to the water channel protein family. Expression analysis revealed higher RpPIP expression in roots while RpTIP expression was higher in leaves, except for RpTIP1;3. AM symbiosis regulated host plant AQPs, and the expression of RpAQP genes in mycorrhizal plants depended on soil water condition and plant tissue. Positive effects were observed for plant physiological parameters in AM plants, which had higher dry mass and lower water saturation deficit and electrolyte leakage than non-AM plants. Rhizophagus irregularis inoculation also slightly increased leaf net photosynthetic rate and stomatal conductance under well-watered and drought stress conditions. These findings suggest that AM symbiosis can enhance the drought tolerance in Robinia pseudoacacia plants by regulating the expression of RpAQP genes, and by improving plant biomass, tissue water status, and leaf photosynthesis in host seedlings.     

Differentiation of the pollen size in five representatives of <Emphasis Type="Italic">Taraxacum</Emphasis> sect. <Emphasis Type="Italic">Palustria</Emphasis>

Measurements of the pollen size in 5 species of Taraxacum sect. Palustria at three levels of ploidy: 2n = 3x = 24 (T. paucilobum), 2n = 4x = 32 (T. vindobonense, T. trilobifolium), 2n = 5x = 40 (T. mendax) and one taxon of unknown number of chromosomes 2n = ? (T. portentosum) are presented in this paper. Obtained results indicate a lack of distinct positive correlation between the pollen size and ploidy in the studied group of plants. Distinct relationship was, however, found between ploidy and the range of pollen size and shape variability. Most variable were the pollen grains of triploid T. paucilobum and the least — those in pentaploid T. mendax. Ranges of pollen variability in tetraploid T. trilobifolium and T. vindobonense and in T. portentosum of unknown number of chromosomes showed intermediate values.     

Induction of salt tolerance in salicylate-deficient <Emphasis Type="Italic">NahG</Emphasis> Arabidopsis transformants using the nitric oxide donor

The effects of treatment with nitric oxide donor sodium nitroprusside (SNP, 0.5 mM) on salt tolerance of wild type (Col–0) Arabidopsis thaliana plants and Arabidopsis thaliana plants transformed with the bacterial salicylate hydroxylase gene (NahG) were compared. Basic salt tolerance level (200 mM NaCl) was higher in NahG transformants. Under salt stress conditions, these plants showed higher activity levels for antioxidant enzymes as well as higher content of sugars and anthocyanins. The treatment with NO donor induced salt tolerance in the plants of both genotypes, which could be observed as less strong growth inhibition, reduced oxidative damage, and preservation of chlorophyll pool in leaves. After the exposure to salt stress, the activity of both superoxide dismutase and guaiacol peroxidase was higher in SNP-treated wild type plants and NahG transformants than in the nontreated plants. After the imposition of salt stress, proline content in leaves of the wild type plants treated with the nitric oxide donor was lower than in the leaves of the nontreated plants. In contrast, SNP treatment of NahG transformants led to a significant increase in the proline content in leaves under the salt stress conditions. Conclusions have been made that wild type Col-0 plants and NahG transformants differ in how their systems of protection against salt stress are activated and that nitric oxideinduced mobilization of protection systems in A. thaliana may not require the presence of salicylate.