Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing <Emphasis Type="Italic">Datura stramonium</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.     

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.     

Fine mapping of the <Emphasis Type="Italic">qCTS4</Emphasis> locus associated with seedling cold tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Rice seedlings are sensitive to low temperatures (≤15°C) and under prolonged or repeated exposure, yellowing and stunting are commonly observed. Damage to seedlings results in poor stand establishment and delayed maturation, which can cause significant reductions in yield. In general, japonica rice varieties exhibit more cold tolerance than indica varieties. Earlier genetic analysis of the California rice variety M202 revealed several quantitative trait loci (QTL) that contribute to its tolerance to low temperatures in comparison to the indica rice variety IR50. Among these QTL, qCTS4 is associated with tolerance to yellowing and stunting of rice seedlings and accounts for 40% of the phenotypic variation. Here we report on the fine mapping of qCTS4 to a 128 kb region of chromosome 4, which is highly suppressed for recombination in our mapping populations. Our results provide the necessary foundation for identifying the gene(s) underlying qCTS4 and the markers developed here may be used to introgress this region into indica varieties to improve seedling tolerance to low temperatures. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

Systematic analysis of <Emphasis Type="Italic">NPK1</Emphasis>-like genes in rice reveals a stress-inducible gene cluster co-localized with a quantitative trait locus of drought resistance

Phosphorylation by protein kinase is a ubiquitous key mechanism in translating external stimuli such as drought stress. NPK1 is a mitogen-activated protein kinase kinase kinase identified in Nicotiana tabacum and plays important roles in cytokinesis and auxin signaling transduction and responses to multiple stresses. Here we report the evolution, structure, and comprehensive expression profile of 21 NPK1-like genes in rice (Oryza sativa L.). Phylogenetic analysis of NPK1-like sequences in rice (OsNPKL), Arabidopsis, and other plants reveals that NPK1-like genes could be classified into three subgroups. Three OsNPKL gene clusters, located on chromosome 1 (OsNPKL1, 2, 3, and 4), 5 (OsNPKL14 and 15), and 10 (OsNPKL19 and 20), respectively, were identified in the rice genome. These clustered genes, which most likely evolved by tandem gene duplication, belong to the same phylogenetic subgroup, with similar genomic structures and conserved motifs in the kinase domain, which is unique to this subgroup. Expression analysis of OsNPKL genes under abiotic stresses suggests that the stress-responsive genes are mainly from the same subgroup. Especially interesting is that all the clustered genes are induced by drought, salt, or cold stress, and a few members are very strongly induced by drought. Some of the clustered genes are also induced by abscisic acid. The gene cluster on chromosome 1 is co-located with a quantitative trait locus (QTL) related to drought resistance. Although the drought-induced expression levels of the four genes in the cluster show no difference between the two parents used for QTL mapping, sequence variation in coding regions of the genes between the parents has provided some clues for further functional characterization of this gene cluster in abiotic stress tolerance in rice.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> is highly tolerant against drought,salt and osmotic stress

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.     

Function of a novel GDSL-type pepper lipase gene, <Emphasis Type="Italic">CaGLIP1</Emphasis>, in disease susceptibility and abiotic stress tolerance

GDSL-type lipase is a hydrolytic enzyme whose amino acid sequence contains a pentapeptide motif (Gly-X-Ser-X-Gly) with active serine (Ser). Pepper GDSL-type lipase (CaGLIP1) gene was isolated and functionally characterized from pepper leaf tissues infected by Xanthomonas campestris pv. vesicatoria (Xcv). The CaGLIP1 protein was located in the vascular tissues of Arabidopsis root. The CaGLIP1 gene was preferentially expressed in pepper leaves during the compatible interaction with Xcv. Treatment with salicylic acid, ethylene and methyl jasmonate induced CaGLIP1 gene expression in pepper leaves. Sodium nitroprusside, methyl viologen, high salt, mannitol-mediated dehydration and wounding also induced early and transient CaGLIP1 expression in pepper leaf tissues. Virus-induced gene silencing of CaGLIP1 in pepper conferred enhanced resistance to Xcv, accompanied by the suppressed expression of basic PR1 (CaBPR1) and defensin (CaDEF1) genes. The CaGLIP1 lipase produced in Escherichia coli hydrolyzed the substrates of short and long chain nitrophenyl esters. The CaGLIP1-overexpressing Arabidopsis exhibited enhanced hydrolytic activity toward short and long chain nitrophenyl ester, as well as enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato and the biotrophic oomycete Hyaloperonospora parasitica. SA-induced expression of AtPR1 and AtGST1, also was delayed in CaGLIP1-overexpressing plants by SA application. During seed germination and plant growth, the CaGLIP1 transgenic plants showed drought tolerance and differential expression of drought- and abscisic acid (ABA)-inducible genes AtRD29A, AtADH and AtRab18. ABA treatment differentially regulated seed germination and gene expression in wild-type and CaGLIP1 transgenic Arabidopsis. Overexpression of CaGLIP1 also regulated glucose- and oxidative stress signaling. Together, these results indicate that CaGLIP1 modulates disease susceptibility and abiotic stress tolerance. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number AY775336.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

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.     

Overexpression of a putative maize calcineurin B-like protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> confers salt tolerance

The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plants. Although extensive studies and remarkable progress have been made in Arabidopsis (Arabidopsis thaliana) CBLs, their functions in other plant species are still quite limited. Here, we report the cloning and functional characterization of ZmCBL4, a novel CBL gene from maize (Zea mays). ZmCBL4 encodes a putative homolog of the Arabidopsis CBL4/SOS3 protein, with novel properties. ZmCBL4 has one copy in maize genome and harbors seven introns in its coding region. ZmCBL4 expressed differentially in various organs of the maize plants at a low level under normal condition, and its expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of 35S::ZmCBL4 not only complemented the salt hypersensitivity in Arabidopsis sos3 mutant, but also enhanced the salt tolerance in Arabidopsis wild type at the germination and seedling stages. Moreover, the LiCl tolerance in all of the ZmCBL4-expressing lines increased more significantly as compared with the NaCl tolerance, and in consistent with this, it was found that the expression of Arabidopsis AtNHX8, a putative plasma membrane Li⁺/H⁺ antiporter gene identified recently, was induced in these transgenic lines under LiCl stress. The ZmCBL4-expressing Arabidopsis lines accumulated less Na⁺ and Li⁺ as compared with the control plants. This study has identified a putative maize CBL gene which functions in the salt stress-elicited calcium signaling and thus in the tolerance to salinity. Database accession number: EF405963.     

T-DNA tagged knockout mutation of rice <Emphasis Type="Italic">OsGSK1</Emphasis>, an orthologue of <Emphasis Type="Italic">Arabidopsis BIN2</Emphasis>, with enhanced tolerance to various abiotic stresses

T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (O ryza s ativa g lycogen s ynthase k inase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis b rassinosteroid in sensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Serry Koh and Sang-Choon Lee are co-first authors.     

Functional roles of the pepper antimicrobial protein gene, <Emphasis Type="Italic">CaAMP1</Emphasis>, in abscisic acid signaling,and salt and drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Biotic signaling molecules including abscisic acid (ABA) are involved in signal transduction pathways that mediate the defense response of plants to environmental stresses. The antimicrobial protein gene CaAMP1, previously isolated from pepper (Capsicum annuum), was strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. Because transformation is very difficult in pepper, we overexpressed CaAMP1 in Arabidopsis. CaAMP1-overexpressing (OX) transgenic plants exhibited reduced sensitivity to ABA during the seed germination and seedling stages. Overexpression of CaAMP1 conferred enhanced tolerance to high salinity and drought, accompanied by altered expression of the AtRD29A gene, which is correlated with ABA levels and environmental stresses. The transgenic plants were also highly tolerant to osmotic stress caused by high concentrations of mannitol. Together, these results suggest that overexpression of the CaAMP1 transgene modulates salt and drought tolerance in Arabidopsis through ABA-mediated cell signaling. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession number AY548741.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.