Emerging crosstalk between two signaling pathways coordinates K+ and Na+ homeostasis in the halophyte Hordeum brevisubulatum.

K+/Na+ homeostasis is the primary core response for plant to tolerate salinity. Halophytes have evolved novel regulatory mechanisms to maintain a suitable K+/Na+ ratio during long-term adaptation. The wild halophyte Hordeum brevisubulatum can adopt efficient strategies to achieve synergistic levels of K+ and Na+ under high salt stress. However, little is known about its molecular mechanism. Our previous study indicated that HbCIPK2 contributed to prevention of Na+ accumulation and K+ reduction. Here, we further identified the HbCIPK2-interacting proteins including upstream Ca2+ sensors, HbCBL1, HbCBL4, and HbCBL10, and downstream phosphorylated targets, the voltage-gated K+ channel HbVGKC1 and SOS1-like transporter HbSOS1L. HbCBL1 combined with HbCIPK2 could activate HbVGKC1 to absorb K+, while the HbCBL4/10-HbCIPK2 complex modulated HbSOS1L to exclude Na+. This discovery suggested that crosstalk between the sodium response and the potassium uptake signaling pathways indeed exists for HbCIPK2 as the signal hub, and paved the way for understanding the novel mechanism of K+/Na+ homeostasis which has evolved in the halophytic grass.

Genome-wide identification and characterization of FAD family genes in barley.

Fatty acid desaturases (FADs) play pivotal roles in determining plant stress tolerance. Barley is the most salt-tolerant cereal crop. In this study, we performed genome-wide identification and characterization analysis of the FAD gene family in barley (Hordeum vulgare). A total of 24 HvFADs were identified and divided into four subfamilies based on their amino acid sequence similarity. HvFADs unevenly distributed on six of seven barley chromosomes, and three clusters of HvFADs mainly occurred on the chromosome 2, 3 and 6. Segmental duplication events were found to be a main cause for the HvFAD gene family expansion. The same HvFAD subfamily showed the relatively consistent exon-intron composition and conserved motifs of HvFADs. Cis-element analysis in HvFAD promoters indicated that the expression of HvFADs may be subject to complex regulation, especially stress-responsive elements that may involve in saline-alkaline stress response. Combined transcriptomic data with quantitative experiments, at least five HvFADs highly expressed in roots under salt or alkali treatment, suggesting they may participate in saline or alkaline tolerance in barley. This study provides novel and valuable insights for underlying salt/alkali-tolerant mechanisms in barley.

Overexpressing a putative aquaporin gene from wheat, TaNIP, enhances salt tolerance in transgenic Arabidopsis.

High soil salinity is a major abiotic stress in plant agriculture worldwide. Here, we report the characterization of a novel aquaporin gene TaNIP (Triticum asetivum L. nodulin 26-like intrinsic protein), which was involved in salt tolerance pathways in plants. TaNIP was identified and cloned through the gene chip expression analysis of a salt-tolerant wheat mutant RH8706-49 under salt stress. Quantitative reverse transcription-PCR (Q-RT-PCR) was used to detect TaNIP expression under salt, drought, cold and ABA treatment. The overexpression of TaNIP in transgenic Arabidopsis produced higher salt tolerance than wild-type plants. Localization analysis showed that TaNIP proteins tagged with green fluorescent protein (GFP) were localized to the cell plasma membrane. Under salt stress treatment, TaNIP-overexpressing Arabidopsis accumulated higher K(+), Ca(2+) and proline contents and lower Na(+) level than the wild-type plants. The overexpression of TaNIP in transgenic Arabidopsis also up-regulated the expression of a number of stress-associated genes. Our results suggest that TaNIP plays an important role in salt tolerance in Arabidopsis and can also enhance plants' tolerance to other abiotic stresses.

Cloning and function analysis of BAG family genes in wheat.

By analysing the cDNA microarray of the salt tolerant mutant of wheat RH8706-49 under salinity stress, our results showed an expressed sequence tag fragment and acquired an unknown gene (designated as TaBAG) with a BAG conserved domain through electronic cloning and RT-PCR technology. The gene was registered into GenBank (No. FJ599765). After homologous alignment analysis, electronic cloning, and amplifying with RT-PCR, the other gene with a BAG conserved domain, TaBAG2, was obtained and registered into GenBank (No. GU471210). Quantitative PCR analysis demonstrated that TaBAG2 expression was induced by saline and heat stress. TaBAG gene expression under salinity stress increased remarkably but showed an insignificant response to heat stress. The adversity stress detection results showed that Arabidopsis overexpressing TaBAG and TaBAG2 exhibited an obvious salt tolerance increase. Under heat stress, Arabidopsis overexpressing TaBAG2 showed increased heat tolerance; however, the heat tolerance of Arabidopsis overexpressing TaBAG did not vary significantly under heat stress. Subcellular localisation results demonstrated that TaBAGs were mainly located in the cytoplasm and the cell nucleus. We applied fluorescence complementation and yeast two-hybrid technique to prove that TaBAG2 can obviously bond with TaHsp70 and TaCaMs. After the respective mutation of aspartic acid (D) and arginine (R) at high conservation in BAG domain of TaBAG2, the bonding interaction between TaBAG2 and TaHsp70 disappeared, indicating that the two amino acids were the key loci for the interaction between TaBAG2 and TaHsp70. Heat tolerance detection results demonstrated that the heat tolerance of Arabidopsis overexpressing and cotransfected with TaBAG2 and TaHsp70 was much higher than that of Arabidopsis overexpressing TaBAG2 and Arabidopsis overexpressing TaHSP70. This finding implies that the synergistic use of TaBAG2 and TaHSP70 can improve heat tolerance of plants.

Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum.

Ferredoxin is a small iron-sulfer protein involved in various one-eletron transfer pathways. Little is known about how ferredoxin is regulated to distribute electron under abiotic stress. Our previous study has showed that HbCIPK2 conferred salinity and drought tolerance. Thus, we hypothesized that HbCIPK2 could mediate the activities of interacting partners as a signal transducer. In this report, we identified a novel HbCIPK2-interacting ferredoxin (HbFd1) from halophyte Hordeum brevisubulatum by yeast two-hybrid screens, confirmed this interaction by BiFC in vivo and CoIP in vitro, and presented the expression pattern of HbFd1. HbFd1 was down-regulated under salinity and cold stress but up-regulated under PEG stress, its expression showed tissue-specific, mainly in shoot chloroplast, belonging to leaf-type subgroup. Moreover, HbCIPK2 could recruit HbFd1 to the nucleus for their interaction. The C-terminal segment in HbFd1 protein was involved in the interaction with HbCIPK2. These results provided insight into the connection between CBL-CIPK signaling network and Fd-dependent metabolic pathways.

[Proteomic analysis of the salt tolerance mutant of wheat under salt stress].

Two dimensional electrophoresis was used to analyse the proteome of the salt-tolerant mutant of wheat (RH8706-49) and the salt-sensitive mutant of wheat (H8706-34) which had been treated by 1% NaCl for 72 hours. After being analysed by MALDI-TOF-MS and Mascot software, the qualitative and quantitative differences were identified between the two materials for five candidate proteins: H+-transporting two-sector ATPase, glutamine synthetase 2 precursor, putative 33 kD oxygen evolving protein of photosystem II and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit. These five proteins are all belong to chloroplast proteins. They are likely to play a crucial role in keeping the function of the chloroplast and the whole cells when the plant was under salt-stress.

Overexpression of the receptor-like protein kinase genes AtRPK1 and OsRPK1 reduces the salt tolerance of Arabidopsis thaliana.

AtRPK1 (AT1G69270) is a leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene in Arabidopsis thaliana. The rice gene Os07g0602700 (OsRPK1) is the homolog of AtRPK1. AtRPK1 and OsRPK1 were overexpressed and the expression of AtRPK1 was inhibited by RNAi in A. thaliana. The functional results showed that the degrees of salt tolerance of the 35S:RPK1 A. thaliana plants were significantly lower than that of the control plants. The AtRPK1-RNAi A. thaliana plants exhibited higher salt tolerance than the wild-type plants (Col). The subcellular localisation results showed that the RPK1 proteins were mainly distributed on the cell membrane and that the overexpressed AtRPK1 proteins exhibited a significantly clustered distribution. The physiological analyses revealed that the overexpression of the RPK1 genes increased the membrane permeability in the transgenic A. thaliana plants. In response to salt stress, these plants exhibited an increased Na(+) flux into the cell, which caused greater damage to the cell. The real-time quantitative PCR analysis showed that the expression of the P5CS1 gene was inhibited and the SOS signalling pathway was blocked in the 35S:AtRPK1 A. thaliana plants. These effects at least partially contribute to the salt-sensitive phenotype of the 35S:RPK1 plants.

Cloning and functional analysis of wheat V-H+-ATPase subunit genes.

The root microsomal proteomes of salt-tolerant and salt-sensitive wheat lines under salt stress were analyzed by two-dimensional electrophoresis and mass spectrum. A wheat V-H(+)-ATPase E subunit protein was obtained whose expression was enhanced by salt stress. In silicon cloning identified the full-length cDNA sequences of nine subunits and partial cDNA sequences of two subunits of wheat V-H(+)-ATPase. The expression profiles of these V-H(+)-ATPase subunits in roots and leaves of both salt-tolerant and salt-sensitive wheat lines under salt and abscisic acid (ABA) stress were analyzed. The results indicate that the coordinated enhancement of the expression of V-H(+)-ATPase subunits under salt and ABA stress is an important factor determining improved salt tolerance in wheat. The expression of these subunits was tissue-specific. Overexpression of the E subunit by transgenic Arabidopsis thaliana was able to enhance seed germination, root growth and adult seedling growth under salt stress.

Overexpression of TaSTRG gene improves salt and drought tolerance in rice.

High salt and drought are the main factors affecting agricultural production. Thus, cloning stress-tolerance-related genes and identifying their functions are essential to enhancing crop tolerance to stresses. In this study, a salt-induced unknown wheat (Triticum aestivum L.) gene was identified and cloned according to microarray analysis of salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt tolerance-related gene (TaSTRG) and submitted to Genbank (Accession number: EF599631). TaSTRG expression in wheat is induced by multiple stresses including salt, polyethylene glycol (PEG), abscisic acid (ABA), and cold. Transgenic rice plants overexpressing TaSTRG gene showed higher salt and drought tolerance than the control. Under salt stress, the transgenic rice had a lower intracellular Na(+)/K(+) ratio than the control. Under salt and PEG treatments, these TaSTRG overexpressing rice plants had higher survival rate, fresh weight and chlorophyll content, accumulated higher proline and soluble sugar contents, and had significantly higher expression levels of putative proline synthetase and transporter genes than the control plants. These results indicate that the wheat TaSTRG gene could enhance plant tolerance to multiple types of stresses.

[A modified TAIL-PCR and its application in isolating gene promoter of wheat].

Using a modified TAIL-PCR technique, the 5' -flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29 degrees C instead of 44 degrees C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5' -flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemical analysis of the transgenic plants showed the report gene was driven by isolated 5'-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.

[Introduce Tagsk1 into salt-sensitive callus to improve the capacity of salt-tolerance by micropartical bombardment].

The Tagsk1 (Triticum asetium L. glycogen synthase kinase 1) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was cloned by PCR. The special primers designed according to full length cDNA sequence of Tagsk1 (AF525086). A binary expression vector pBI121-gsk1 containing Gus and Tagsk1 was constructed. And pBI121-gsk1 was introduced into the callus induced from mature embryos of salt-sensitive wheat H8706-34 and cv. China Spring by particle bombardment. The transformed callus were screened by Kanamycin and 0.5% NaCl. The salt-tolerance callus were obtained, which showed higher ability of salt-tolerance and could diffirentiate roots and buds on the medium containing 0.5% NaCl.