Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants.

NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.

Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants.

WRKY-type transcription factors are involved in multiple aspects of plant growth, development and stress response. WRKY genes have been found to be responsive to abiotic stresses; however, their roles in abiotic stress tolerance are largely unknown especially in crops. Here, we identified stress-responsive WRKY genes from wheat (Triticum aestivum L.) and studied their functions in stress tolerance. Forty-three putative TaWRKY genes were identified and two multiple stress-induced genes, TaWRKY2 and TaWRKY19, were further characterized. TaWRKY2 and TaWRKY19 are nuclear proteins, and displayed specific binding to typical cis-element W box. Transgenic Arabidopsis plants overexpressing TaWRKY2 exhibited salt and drought tolerance compared with controls. Overexpression of TaWRKY19 conferred tolerance to salt, drought and freezing stresses in transgenic plants. TaWRKY2 enhanced expressions of STZ and RD29B, and bound to their promoters. TaWRKY19 activated expressions of DREB2A, RD29A, RD29B and Cor6.6, and bound to DREB2A and Cor6.6 promoters. The two TaWRKY proteins may regulate the downstream genes through direct binding to the gene promoter or via indirect mechanism. Manipulation of TaWRKY2 and TaWRKY19 in wheat or other crops should improve their performance under various abiotic stress conditions.

Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

Effects of tobacco ethylene receptor mutations on receptor kinase activity, plant growth and stress responses.

Ethylene receptor is the first component of ethylene signaling that regulates plant growth, development and stress responses. Previously, we have demonstrated that tobacco subfamily 2 ethylene receptor NTHK1 had Ser/Thr kinase activity, and overexpression of NTHK1 caused large rosette, reduced ethylene sensitivity, and increased salt sensitivity in transgenic Arabidopsis plants. Here we found that N-box mutation in the NTHK1 kinase domain abolished the kinase activity and led to disruption of NTHK1 roles in conferring reduced ethylene sensitivity and salt sensitive response in transgenic Arabidopsis plants. However, N-box mutation had partial effects on NTHK1 regulation of rosette growth and expression of salt- and ethylene-responsive genes AtNAC2, AtERF1 and AtCor6.6. Mutation of conserved residues in the H box did not affect kinase activity, seedling growth, ethylene sensitivity or salt-induced epinasty in transgenic plants but did influence NTHK1 function in control of specific salt- and ethylene-responsive gene expression. Compared with NTHK1, the tobacco subfamily 1 ethylene receptor NtETR1 had His kinase activity and played a weak role in regulation of rosette growth, triple response and salt response. Mutation of the conserved His residue in the NtETR1 H box eliminated phosphorylation and altered the effect of Ntetr1-1 on reporter gene activity. These results imply that the Ser/Thr kinase activity of NTHK1 is differentially required for various responses, and NTHK1 plays a larger role than NtETR1.

Roles of ethylene receptor NTHK1 domains in plant growth, stress response and protein phosphorylation.

Ethylene receptors sense ethylene and regulate downstream signaling events. Tobacco ethylene receptor NTHK1, possessing Ser/Thr kinase activity, has been found to function in plant growth and salt-stress responses. NTHK1 contains transmembrane domains, a GAF domain, a kinase domain and a receiver domain. We examined roles of these domains in regulation of plant leaf growth, salt-stress responses and salt-responsive gene expressions using an overexpression approach. We found that the transgenic Arabidopsis plants harboring the transmembrane domain plus kinase domain exhibited large rosettes, had reduction in ethylene sensitivity, and showed enhanced salt sensitivity. The transgenic plants harboring the transmembrane domain plus GAF domain also showed larger rosettes. Truncations of NTHK1 affected salt-induced gene expressions. Transmembrane domain plus kinase domain promoted RD21A and VSP2 expression but decreased salt-induction of AtNAC2. The kinase domain itself promoted AtERF4 gene expression. The GAF domain itself enhanced Cor6.6 induction. Moreover, the NTHK1 functional kinase domain phosphorylated the HIS and ATP subdomains, and five putative phosphorylation sites were identified in these two subdomains. In addition, the salt-responsive element of the NTHK1 gene was in the transmembrane-coding region but not in the promoter region. These results indicate that NTHK1 domains or combination of them have specific functions in plant leaf growth, salt-stress response, gene expression and protein phosphorylation.

Isolation of trehalose-6-phosphate phosphatase gene from tobacco and its functional analysis in yeast cells.

Trehalose is a nonreducing disaccharide composed of two glucose units joined by an alpha, alpha-1, 1 linkage, and has been found in bacteria, yeast, fungi, invertebrates and plants. Accumulation of trehalose in organisms plays a role in enhancing the stress tolerance. A trehalose-6-phosphate phosphatase gene, NtTPPL, was isolated from tobacco in this report. The predicted NtTPPL protein has a putative trehalose_PPase domain. The transcription of the NtTPPL gene was significantly induced by heat stress, and was only slightly induced by NaCl, PEG and low-temperature treatments. When expressing in yeast tps2 mutant, NtTPPL rescued the mutant phenotype under high temperature. This result indicated that NtTPPL functioned as a trehalose-6-phosphate phosphatase in yeast and may play similar roles in plants.

Treatment of intracranial hydatid cysts.

BACKGROUND: Echinococcosis is still endemic in many countries, including China, where it is especially prevalent in the northwest. The aim of this study was to enrich the international literature about the treatment of intracranial hydatid cysts. METHODS: We retrospectively reviewed the clinical features, radiological manifestations, and surgical outcome of 97 patients with intracranial hydatid cysts, who received surgical treatment at the Neurosurgical Department of First Affiliated Hospital of Xinjiang Medical University from 1985 to 2010 and followed up the patient via sending a questionnaire or telephone contact. Clinical outcome was evaluated using the Karnofsky Performance Scale Index. RESULTS: Headache and vomiting were the most common initial symptoms in our patients. Neurological deficits caused by the mass effect of the cysts were seen in 82 cases. On the X-ray, significant bone erosion was seen in only two cases with epidural hydatid cysts. Round-shaped and thin-walled homogeneous low-density cystic lesions without surrounding edema and enhancement were the main findings on computerized tomography (CT) in 95 patients with intraparenchymal hydatid cysts, while two cases with epidural hydatid cysts presented as a heterodensity lesions. On magnetic resonance imaging (MRI), hydatid cyst presented as a round-shaped low signal lesion in T1-weighted images and high signal lesion in T2-weighted images, without enhancement after contrast media injection, while the two cases with epidural cysts presented as mixed signal masses. Surgical removal of cyst was performed in all cases. Total removal was achieved in 93 cases without rupturing the cyst wall. Only two cysts ruptured during the dissection, resulting in two surgery-related mortalities. There was no other additional neurological deficit caused directly by surgery. In 97.2% of the patients, the Karnofsky Performance Scale score was 80 to 90 at the last follow-up. CONCLUSIONS: Intracranial hydatid cyst is still a main cause of increased intracranial pressure among the patients in endemic areas for echinococcosis. CT and MRI are the best diagnostic methods and surgery is the treatment of choice for intracranial hydatid cysts.

Soybean GmPHD-type transcription regulators improve stress tolerance in transgenic Arabidopsis plants.

BACKGROUND: Soybean [Glycine max (L.) Merr.] is one of the most important crops for oil and protein resource. Improvement of stress tolerance will be beneficial for soybean seed production. PRINCIPAL FINDINGS: Six GmPHD genes encoding Alfin1-type PHD finger protein were identified and their expressions differentially responded to drought, salt, cold and ABA treatments. The six GmPHDs were nuclear proteins and showed ability to bind the cis-element "GTGGAG". The N-terminal domain of GmPHD played a major role in DNA binding. Using a protoplast assay system, we find that GmPHD1 to GmPHD5 had transcriptional suppression activity whereas GmPHD6 did not have. In yeast assay, the GmPHD6 can form homodimer and heterodimer with the other GmPHDs except GmPHD2. The N-terminal plus the variable regions but not the PHD-finger is required for the dimerization. Transgenic Arabidopsis plants overexpressing the GmPHD2 showed salt tolerance when compared with the wild type plants. This tolerance was likely achieved by diminishing the oxidative stress through regulation of downstream genes. SIGNIFICANCE: These results provide important clues for soybean stress tolerance through manipulation of PHD-type transcription regulator.

Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis.

From soybean plant, 131 bZIP genes were identified and named as GmbZIPs. The GmbZIPs can be classified into ten groups and more than one third of these GmbZIPs are responsive to at least one of the four treatments including ABA, salt, drought and cold stresses. Previous studies have shown that group A bZIP proteins are involved in ABA and stress signaling. We now chose four non-group A genes to study their features. The four proteins GmbZIP44, GmbZIP46, GmbZIP62 and GmbZIP78 belong to the group S, I, C and G, respectively, and can bind to GLM (GTGAGTCAT), ABRE (CCACGTGG) and PB-like (TGAAAA) elements with differential affinity in both the yeast one-hybrid assay and in vitro gel-shift analysis. GmbZIP46 can form homodimer or heterodimer with GmbZIP62 or GmMYB76. Transgenic Arabidopsis plants overexpressing the GmbZIP44, GmbZIP62 or GmbZIP78 showed reduced ABA sensitivity. However, all the transgenic plants were more tolerant to salt and freezing stresses when compared with the Col plants. The GmbZIP44, GmbZIP62 and GmbZIP78 may function in ABA signaling through upregulation of ABI1 and ABI2 and play roles in stress tolerance through regulation of various stress-responsive genes. These results indicate that GmbZIP44, GmbZIP62 and GmbZIP78 are negative regulators of ABA signaling and function in salt and freezing tolerance.

The soybean Dof-type transcription factor genes, GmDof4 and GmDof11, enhance lipid content in the seeds of transgenic Arabidopsis plants.

Soybean is one of the most important leguminous seed crops among the oil crops. Although the pathways for lipid biosynthesis have been identified, the factors that regulate the biosynthetic pathways at the transcriptional level are largely unknown. Here, we report our findings on the involvement of soybean Dof-type transcription factor genes in the regulation of the lipid content in soybean seeds. We identified 28 Dof-type transcription factor genes in soybean plants, and these genes displayed diverse patterns of expression in various organs. Seven flower/pod-specific genes and one constitutively expressed gene were further investigated. The proteins encoded by these seven genes were localized in the nucleus, and exhibited different abilities for transcriptional activation and DNA binding. Two genes, GmDof4 and GmDof11, were found to increase the content of total fatty acids and lipids in GmDof4 and GmDof11 transgenic Arabidopsis seeds. We also found that the 1000-seed weight was increased in the GmDof4 and GmDof11 transgenic plants. Using microarray and DNA binding analysis, we found that the two Dof-like proteins, GmDof4 and GmDof11, activated the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene, respectively, by direct binding to the cis-DNA elements in their promoter regions. In addition, both proteins downregulated the storage protein gene, CRA1, through direct binding. These results suggest that the two GmDof genes may augment the lipid content of soybean seeds by upregulating genes that are associated with the biosynthesis of fatty acids.

Functional analysis of a putative Ca2+ channel gene TaTPC1 from wheat.

The cytosolic free-calcium concentration [Ca2+](cyt) transiently increases under abiotic stresses and the proteins that control this process are gradually disclosed. The Ca2+-permeable channel is one type of these proteins in plants. In the present study, a novel Ca2+-permeable channel gene TaTPC1 encoding a putative membrane protein was cloned from wheat. It was induced under high salinity, polyethylene glycol, low temperature (4 degrees C), and abscisic acid. Expression of TaTPC1 in the yeast mutant lacking CCH1 can recover its growth under lithium stress through functional complementation. TaTPC1 transgenic plants exhibited more stomatal closing in the presence of Ca2+ than the control, supporting a role for the calcium channel in regulating plant responses to environmental change.