Mechanisms of Salt Tolerance in Transgenic Arabidopsis thaliana Carrying a Peroxisomal Ascorbate Peroxidase Gene from Barley

Ascorbate peroxidases （APX）, localized in the cytosol, peroxisome, mitochondria, and chloroplasts of plant cells, catalyze the reduction of H2O2 to water by using ascorbic acid as the specific electron donor. To determine the role of peroxisomal type ascorbate peroxidase （pAPX）, an antioxidant enzyme, in protection against salt-induced oxidative stress, transgenic Arabidopsis thaliana plant carrying a pAPX gene （HvAPX1） from barley （Hordeum vulgate L.） was analyzed. The transgenic line pAPX3 was found to be more tolerant to salt stress than the wild type. Irrespective of salt stress, there were no significant differences in Na^＋, K^＋, Ca^2＋, and Mg^2＋ contents and the ratio of K^＋ to Na^＋ between pAPX3 and the wild type. Clearly, the salt tolerance in pAPX3 was not due to the maintenance and reestablishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation （measured as the levels of malondialdehyde） accumulation under salt stress was higher in the wild type than in pAPX3. The mechanism of salt tolerance in transgenic pAPX3 can thus be explained by reduction of oxidative stress injury. Under all conditions tested, activities of superoxide, glutathione reductase, and catalase were not significantly different between pAPX3 and the wild type. In contrast, the activity of APX was significantly higher in the transgenic plant than in wild type under salt stress. These results suggested that in higher plants, HvAPX1 played an important role in salt tolerance and was a candidate gene for developing salttolerant crop plants.

Overexpression of GmBIN2,a soybean glycogen synthase kinase 3 gene,enhances tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots

Glycogen synthase kinase 3 （GSK3） is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 （GmBIN2） gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase （SOD） activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.

Maize ABP2 enhances tolerance to drought and salt stress in transgenic Arabidopsis

Abiotic stresses, especially drought and salt, severely affect maize production, which is one of the most important cereal crops in the world. Breeding stress-tolerant maize through biotechnology is urgently needed to maintain maize production. Therefore, it is important to identify new genes that can enhance both drought and salt stress tolerance for molecular breeding. In this study, we identified a maize ABA （abscisic acid）-responsive element （ABRE） binding protein from a 17-day post-pollination （dpp） maize embryo cDNA library by yeast one-hybrid screen using the ABRE2 sequence of the maize Cat1 gene as bait. This protein, designated, ABRE binding protein 2 （ABP2）, belongs to the bZIP transcription factor family. Endogenous expression of ABP2 in maize can be detected in different tissues at various development stages, and can be induced by drought, salt, reactive oxygen species （ROS）-generating agents, and ABA treatment. Constitutive expression of ABP2 in transgenic Arabidopsis plants enhanced tolerance to drought and salt stress, and increased sensitivity to ABA. In exploring the mechanism by which ABP2 can stimulate abiotic stress tolerance, we found that ROS levels were reduced and expression of stress-responsive and carbon metabolism-related genes was enhanced by constitutive ABP2 expression in transgenic plants. In Short, we identified a maize bZIP transcription factor which can enhance both drought and salt tolerance of plants.

Determining the Copy Number of Exogenous Gene in Transgenic Plant by SYBR Green Real-time Quantitative PCR

[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.

Harpin_(xoo) and Its Functional Domains Activate Pathogen-inducible Plant Promoters in Arabidopsis

Harpins are bacterial proteins that can enhance plant growth and defense against pathogens and insects. To elaborate whether harpins perform the diverse functions in coordination with the activation of specific promoters that contain particular elements, we cloned pathogen-inducible plant promoters PPP1, PPP2, and PPP3 from tobacco and investigated their responses to harpinxoo or its truncated fragments DEG, DIR, and DPR (domains for enhancing plant growth, insect resistance and pathogen resistance). PPP1 contains an internal repeat composed of two tandem 111bp fragments; 111bp in the repeat was deleted in PPP2. PPP3 contains a bacteria-inducible element; PPP1 and PPP2 additionally contain TAC-1 and Eli boxes inducible correspondingly by salicylic acid (SA) and elicitors. Function of cloned PPPs was confirmed based on their activation in transgenic Arabidopsis plants by Ralstonia solanacearum (Ralston) or SA. Harpinxoo, DEG, DIR, or DPR activated PPP1 and PPP2 but not PPP3, consistent with the presence of Eli boxes in promoters. PPP1 was ca. 3-fold more active than PPP2, suggesting that the internal repeat affects levels of the promoter activation.

Cloning and Functional Validation of Mung Bean VrPR Gene

For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.

Characterization of NPR1 Genes from Norton and Cabernet Sauvignon Grapevine

Non-expressor of pathogenesis-related genes 1 （NPR1） plays a significant role in the defense responses of plants to pathogens by regulating the expression of defense-related genes. In the present study, we isolated two NPR1 genes from Vitis aestivalis cv. Norton and Vitis vinifera cv. Cabernet Sauvignon, which were referred to as VaNPR1.1 and VvNPR1. 1-CS, respectively. They encode a protein of 584 amino acids with a predicted molecular weight of 64.8 kDa and a theoretical isoelectric point （pI） of 5.74. The predicted amino acid sequences of VaNPR1.1 and VvNPR1.1-CS differ by only one amino acid. Over-expression of VaNPR1.1 gene in Arabidopsis npr1-1 mutant plants restores the transcriptional expression of AtPR-1 gene, though not to the full scale. This result demonstrated that a grapevine VaNPR1.1 possesses a similar function to the Arabidopsis NPR1 in the regulation of defense-related genes. Over-expression of VaNPR1.1 in transgenic Arabidopsis plant increased tolerance to salinity, but had no effect on the drought tolerance. We conclude that VaNPR1.1 is a functional ortholog of AtNPR1 and also involved in grapevine＇s response to the salt stress.

Expression and functional analysis of Fa PHO1;H9 gene of strawberry(Fragaria×ananassa)

Although the phosphate 1（PHO1）gene family has been implicated in inorganic phosphate transport and homeostasis,the underlying mechanism of this gene in the strawberry has not yet been revealed.In the present study,we analyzed the expression of the PHO1;H9 gene in the strawberry（Fragaria×ananassa）,revealing the involvement of this gene in the regulation of phosphorus（P）content.The coding sequence（CDS）of the PHO1;H9 gene,was isolated from the cultivated strawberry‘Sachinoka’and named as Fa PHO1;H9.The full-length CDS of this gene was 2 292 bp,encoding 763 amino acids,and the protein contained both SYG1/Pho81/XPR1（SPX）and ERD1/XPR1/SYG1（EXS）domains,which were involved in phosphate（Pi）signaling.Real-time reverse transcription-polymerase chain reaction（RT-PCR）data suggested that the level of Fa PHO1;H9 expression was consistent with the P content in different organs,except for the petiole.Particularly,its expression level was also correlated with P content in fruits of different developmental stages.The expression of Fa PHO1;H9 was also consistent with P content in leaves under different concentrations of P fertilizer application.Furthermore,transgenic Arabidopsis lines were generated,and the P content in Arabidopsis plants over-expressing Fa PHO1;H9was significantly higher than that in wild-type plants.Therefore,we proposed that Fa PHO1;H9 functions in P transport.

Cloning and functional analysis of the promoter of allergen gene Ara h 1 from peanut

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins or secondary metabolites.Seed-specific promoters(SSPs)can direct the expression of genes specifically in seeds to avoid undesirable effects associated with constitutive expression.However,few SSPs have been identified in peanut.Previous studies have shown that some allergen-encoding genes encode seed storage proteins or exhibit seed-specific/preferential expression.In this study,we characterized allergen-encoding genes from across the genomes of Arachis species to explore seed-specific genes.We found that at least 9 out of 16 identified peanut allergen-encoding genes were expressed specifically in the seeds or were preferentially expressed.A 1493-bp promoter fragment of allergen gene Ara h 1(we named it AHSSP6)was isolated from cultivated peanut genome.cis-element analysis showed that three RY repeat elements which usually exsisted in seed or embryo specific promoter sequence were also present in AHSSP6 sequence.Histochemical analysis showed AHSSP6 could drive the expression of aβ-glucuronidase(GUS)reporter gene specifically in the seeds or cotyledon tissue of transgenic Arabidopsis,while not in other tissues.These findings indicated that these promoters of allergen genes were candidate SSPs,and AHSSP6 was a novel SSP which could be potentially utilized in peanut improvement.

ThERF1 from Tamarix hispida confers decreased tolerance to oxidative and drought stresses and is regulated by a WRKY protein

The ethylene-responsive factor family is one of the largest families of plant-specific transcription factors that are involved in plant development and stress responses. Previously, we demonstrated that the gene Th ERF1,encoding a novel ethylene-responsive factor from Tamarix hispida, negatively modulates abiotic stress tolerance. In the present study, Arabidopsis plants overexpressing Th ERF1 had decreased oxidative tolerance and increased transpirational water loss rate compared with wild-type plants, leading to sensitivity to abiotic stress. Real-time RT-PCR showed that the upstream regulator of Th ERF1,Th WRKY2, is involved in responses to different abiotic stresses. Furthermore, both Th WRKY2 and Th ERF1 shared similar expression patterns in the stems and leaves of T.hispida when exposed to salinity, drought and abscisic acid. Chromatin immunoprecipitation assays further confirmed that Th WRKY2 can directly bind to the promoter of Th ERF1 and regulate its expression. This study revealed the regulatory mechanism of Th ERF1 expression in response to abiotic stresses in T. hispida.

Expression of a High Mobility Group Protein Isolated from Cucumis sativus Affects the Germination of Arabidopsis thaliana under Abiotic Stress Conditions

Although high mobility group B （HMGB） proteins have been identified from a variety of plant species, their importance and functional roles in plant responses to changing environmental conditions are largely unknown. Here, we investigated the functional roles of a CsHMGB isolated from cucumber （Cucumis sativus L.） in plant responses to environmental stimuli. Under normal growth conditions or when subjected to cold stress, no differences in plant growth were found between the wild-type and transgenic Arabidopsis thaliana overexpressing CsHMGB. By contrast, the transgenic Arabidopsis plants displayed retarded germination compared with the wild-type plants when grown under high salt or dehydration stress conditions. Germination of the transgenic plants was delayed by the addition of abscisic acid （ABA）, implying that CsHMGB affects germination through an ABA-dependent way. The expression of CsHMGB had affected only the germination stage, and CsHMGB did not affect the seedling growth of the transgenic plants under the stress conditions. The transcript levels of several germination-responsive genes were modulated by the expression of CsHMGB in Arabidopsis. Taken together, these results suggest that ectopic expression of a CsHMGB in Arabidopsis modulates the expression of several germinationresponsive genes, and thereby affects the germination of Arabidopsis plants under different stress conditions.

ARF2 coordinates with PLETHORAs and PINs to orchestrate ABA-mediated root meristem activity in Arabidopsis

Multiple hormones, including abscisic acid（ABA） and auxin, regulate cell division and differentiation of Arabidopsis root meristems. AUXIN RESPONSE FACTOR2（ARF2） functions as a negative regulator of ABA responses, as seed germination and primary root growth of arf2 mutants are hypersensitive to ABA. In this study, we found that ABA treatment reduced the expression levels of the PIN-FORMEDs（PIN） auxin efflux carriers, PIN1, PIN3,PIN4, and PIN7, to a greater extent in the root meristems of arf2-101 mutant than in the wild type. Also, arf2-101 pin1 and arf2-101 pin4 double mutants show less ABA-induced inhibition of root meristem activity than the arf2-101 mutants. Furthermore, ARF2 positively mediates the transcripts of transcription factor PLETHORA 1（PLT1）gene but negatively mediates PLT2 at protein level in root meristems. Using a dexamethasone（DEX）-inducible transgenic line, Pro35S：PLT2-GR, we showed that PLT2 greatly promotes cell division and completely inhibits cell differentiation in root meristems of the arf2-101 mutant once PLT2 is induced by DEX, which can be partially reversed by ABA treatment, suggesting that ABA regulates root meristem activity in both ARF2-dependent and independent pathways. Our results uncover a complex regulatory architecture in which ARF2 coordinates with PLTs and PINs to orchestrate ABA-mediated regulation of root meristem activity in Arabidopsis.

NPR1-dependent salicylic acid signaling inhibits Agrobacteriummediated transient expression in Arabidopsis

Dear Editor,Arabidopsis thaliana,as a model plant,is the most well-studied plant species.One of the advantages of using Arabidopsis is that obtaining stably transformed plants using flower-dipping method is easy.However,generating transgenic plants is still time-consuming.Therefore,transient expression is frequently used to characterize protein functions.Several transient expression assays have been developed,including protoplast transfection,biolistic bombardment,and Agrobacterium-mediated transient expression.Among these assays,

W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress Responses

Protein kinases play crucial roles in response to external environment stress signals. A putative protein kinase, W55a, belonging to SNFl-related protein kinase 2 （SnRK2） subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5＇ cDNA ends （5＇-RACE） and reverse transcription-polymerase chain reaction（RT-PCR）. It contains a 1 029-bp open reading frame （ORF） encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonate, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexpressing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.

An MYB Transcription Factor from Malus xiaojinensis Has a Potential Role in Iron Nutrition

Regulation of iron uptake and use is critical for plant survival and growth. We isolated an MYB gene from Malus xiaojinensis named MxMYB1, which is induced under Fe-deficient conditions. Expression of MxMYB1 was upregulated by Fe starvation in the roots but not in leaves, suggesting that MxMYB1 might play a role in iron nutrition in roots. Transgenic Arabidopsis plants expressing MxMYB1 exhibited lower iron content as compared with wild type plants under both Fe-normal （40 μM） and Fe-deficient conditions （Fe omitted and Ferrozine 300 μM）. However, the contents of Cu, Zn and Mn were not changed in these transgenic plants. Gene chip and real-time polymerase chain reaction analyses indicated that the expression of two Fe-related genes encoding an iron transporter AtlRT1 and an iron storage protein ferritin AtFER1 might be negatively regulated by MxMYB1 as the expression levels of these genes were lower in MxMYB1 expressing transgenic Arabidopsis plants as compared with wild type plants under both Fe-normal and Fe-deficient conditions. These results suggest that MxMYB1 may function as a negative regulator of iron uptake and storage in plants.