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.     

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.     

Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.     

Expression and functional analysis of <Emphasis Type="Italic">ZmDWF4</Emphasis>, an ortholog of <Emphasis Type="Italic">Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Expression of Indica rice <Emphasis Type="Italic">OsBADH1</Emphasis> gene under salinity stress in transgenic tobacco

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T₂ progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.     

Molecular cloning and functional characterization of MdSOS2 reveals its involvement in salt tolerance in apple callus and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Plants respond to various environmental stresses by activating “stress genes”. CIPKs (CBL-interacting protein kinases) family genes play an important role in the process of stress response. In this study, a CIPK gene MdSOS2 was isolated from apple (Malus × Domestica). Sequence alignment and phylogenetic analysis showed that it is highly similar with Arabidopsis AtSOS2 and contained the conserved domains and motifs. Expression analysis demonstrated that MdSOS2 expressed in all tested organs at different levels, and positively in response to salt stress. Furthermore, the ectopic expression of MdSOS2 complemented the function of Arabidopsis sos2 mutant, and conferred enhanced salt tolerance to the transgenic Arabidopsis. Yeast two-hybrid assay indicated that the N-terminal of MdSOS2 protein physically interacted with MdSOS3 and AtSOS3, respectively, suggesting that SOS pathway operates in apple tree. Finally, MdSOS2 overexpression enhanced, while its suppression reduced the tolerance to salt in transgenic apple calluses, indicating that MdSOS2 acts as a positive regulator in response to salt stress in apple.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants overexpressing <Emphasis Type="Italic">Ramosa1</Emphasis> maize gene show an increase in organ size due to cell expansion

The structure of the plant inflorescence and flower is an important agronomic and ornamental trait studied for its potential economic applications. In particular, the capacity to modify flower size has always been a breeder’s goal. Genetic and molecular studies have shown that the Zea mays gene Ramosa1 (Ra1) is involved in inflorescence branching regulation. In fact the ra1 loss of function mutation causes extra branching of the inflorescence. In this work we suggest a possible utilization of the Ramosa1 maize gene as a tool to modify inflorescence architecture and flower size in transgenic plants. In fact overexpression of this gene in Arabidopsis plants promotes an increase in reproductive organ size. Pollen, seeds, cotyledons, leaves and roots are also larger than those of the wild type. Analysis of organs from transformants showed that cell expansion was increased without apparently affecting cell division. These results suggest that the RA1 protein is able to up-regulate cell expansion in all organs of Arabidopsis plants.     

Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress

Mitogen-activated protein kinase (MAPK) cascades play a remarkably crucial role in plants. It has been studied intensively in model plants Arabidopsis, tobacco and rice. However, the function of MAPKs in maize (Zea mays L.) has not been well documented. ZmSIMK1 (Zea mays salt-induced mitogen-activated protein kinase 1) is a previously identified MAPK gene in maize. In this research, we charactered ZmSIMK1 and showed that ZmSIMK1 was involved in Arabidopsis salt stress. The genomic organization of ZmSIMK1 gene and its expression in maize have been analyzed. In order to investigate the function of ZmSIMK1, we generated transgenic Arabidopsis constitutively overexpressing ZmSIMK1. Ectopic expression of ZmSIMK1 in Arabidopsis resulted in increased resistance against salt stress. Importantly, ZmSIMK1-overexpressing Arabidopsis exhibited constitutive expression of stress-responsive marker genes, RD29A and P5CS1. Furthermore, RD29A and P5CS1 were upregulated under salt stress. These results suggest that ZmSIMK1 may play an important role in plant salt stress.     

Functional study of a salt‐inducible TaSR gene in Triticum aestivum

The gene expression chip of a salt‐tolerant wheat mutant under salt stress was used to clone a salt‐induced gene with unknown functions. This gene was designated as TaSR (Triticum aestivum salt‐response gene) and submitted to GenBank under accession number EF580107. Quantitative polymerase chain reaction (PCR) analysis showed that gene expression was induced by salt stress. Arabidopsis and rice (Oryza sativa) plants expressing TaSR presented higher salt tolerance than the controls, whereas AtSR mutant and RNA interference rice plants were more sensitive to salt. Under salt stress, TaSR reduced Na⁺ concentration and improved cellular K⁺ and Ca²⁺ concentrations; this gene was also localized on the cell membrane. β‐Glucuronidase (GUS) staining and GUS fluorescence quantitative determination were conducted through fragmentation cloning of the TaSR promoter. Salt stress‐responsive elements were detected at 588–1074 bp upstream of the start codon. GUS quantitative tests of the full‐length promoter in different tissues indicated that promoter activity was highest in the leaf under salt stress. Bimolecular fluorescence complementation and yeast two‐hybrid screening further showed the correlation of TaSR with TaPRK and TaKPP. In vitro phosphorylation of TaSR and TaPRK2697 showed that TaPRK2697 did not phosphorylate TaSR. This study revealed that the novel TaSR may be used to improve plant tolerance to salt stress.     

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.     

Cloning and characterization of a novel CBL-interacting protein kinase from maize

A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfeng Zhao and Zhenfei Sun are contributed equally to this work.     

A new tip homolog, <Emphasis Type="Italic">ShTIP</Emphasis>, from <Emphasis Type="Italic">Salicornia</Emphasis> shows a different involvement in salt stress compared to that of TIP from <Emphasis Type="Italic">Arabidopsis</Emphasis>

To obtain an insight into the comprehensive molecular characteristics of the salt tolerance mechanism, we performed a screening for salt inducible genes in a halophytic plant, Salicornia herbacea, using mRNA differential display. A comparative analysis of gene expression in Salicornia grown in control and salt-stressed conditions led to the detection of a gene that was induced by salt. Both sequence analysis and a subsequent database search revealed that this gene was highly homologous to tonoplast intrinsic proteins (TIPs) from a variety of plant species. This gene, designated as ShTIP, is 1014 bp in size and contains a coding region of 762 nucleotides, which encodes a protein of 254 amino acids. Northern blot analysis revealed that ShTIP was predominantly expressed in shoots under normal conditions. However, salt stress induced high expression of ShTIP in both the shoots and roots. The expression of ShTIP in a salt-sensitive calcineurin-deficient yeast mutant (cnbΔ) resulted in a resistance to the high salt conditions. In addition, we compared the expression of a TIP gene in Arabidopsis with that of ShTIP under different conditions and found that the Salicornia TIP has a different regulatory mechanism for adapting to salt stress conditions compared with the glycophyte Arabidopsis TIP. These results indicate that ShTIP plays an important role in salt tolerance.     

Structural analysis of 83-kb genomic DNA from Thellungiella halophila: Sequence features and microcolinearity between salt cress and Arabidopsis thaliana

Salt cress (Thellungiella halophila) has become a desirable plant model for molecular analysis of the mechanisms of salt tolerance. Analysis of its physiological action and expressed EST has resulted in better understanding. However, less is known about its genomic features. Here we determined a continuous sequence approximately 83 kb from a salt cress BAC clone, providing the first insight into the genomic feature for this species. The gene density is approximately one gene per 3.6 kb in this sequence. Many types of repetitive sequences are present in this salt cress sequence, including LTR retroelements, DNA transposons and a number of simple sequence repeats. Comparison of sequence similarity indicated that salt cress shares a close relationship with Arabidopsis. Extensive conservation and high-level microcolinearity were uncovered for both genomes. Our study also indicated that genomic DNA alternations (involving chromosome inversion, sequence loss and gene translocation) contributed to the genomic discrepancies between salt cress and Arabidopsis.