Chengguo Jia

AcPIP2 , a plasma membrane intrinsic protein from halophyte Atriplex canescens , enhances plant growth rate and abiotic stress tolerance when overexpressed in Arabidopsis thaliana

Key messageAn aquaporin protein AcPIP2 fromAtriplex canescenswas involved in plant growth rate, abiotic stress tolerance in Arabidopsis. Under limited water condition, AcPIP2 leaded to the sensitivity to drought stress.AbstractAn aquaporin protein (AcPIP2) was obtained from the saltbush Atriplex canescens, which was in PIP2 subgroup belonging to the PIP subfamily, MIP superfamily. The subcellular localization of AcPIP2 showed the fusion protein AcPIP2-eGFP located at the plasma membrane in Nicotiana benthamiana. Overexpression of AcPIP2 in Arabidopsis fully proved that AcPIP2 was involved in plant growth rate, transpiration rate and abiotic stress tolerance (NaCl, drought and NaHCO3) in Arabidopsis, which is mostly in correspondence to gene expression pattern characterized by qRT-PCR performed in A. canescens. And under limited water condition, AcPIP2 overexpression leaded to the sensitivity to drought stress. In the view of the resistant effect in transgenic Arabidopsis overexpressing AcPIP2, the AcPIP2 may throw some light into understanding how the A. canescens plants cope with abiotic stress, and could be used in the genetic engineering to improve plant growth or selective tolerance to the abiotic stress.

A Heavy Metal-Associated Protein (AcHMA1) from the Halophyte, Atriplex canescens (Pursh) Nutt., Confers Tolerance to Iron and Other Abiotic Stresses When Expressed in Saccharomyces cerevisiae

Many heavy metals are essential for metabolic processes, but are toxic at elevated levels. Metal tolerance proteins provide resistance to this toxicity. In this study, we identified and characterized a heavy metal-associated protein, AcHMA1, from the halophyte, Atriplex canescens. Sequence analysis has revealed that AcHMA1 contains two heavy metal binding domains. Treatments with metals (Fe, Cu, Ni, Cd or Pb), PEG6000 and NaHCO3 highly induced AcHMA1 expression in A. canescens, whereas NaCl and low temperature decreased its expression. The role of AcHMA1 in metal stress tolerance was examined using a yeast expression system. Expression of the AcHMA1 gene significantly increased the ability of yeast cells to adapt to and recover from exposure to excess iron. AcHMA1 expression also provided salt, alkaline, osmotic and oxidant stress tolerance in yeast cells. Finally, subcellular localization of an AcHMA1/GFP fusion protein expressed in tobacco cells showed that AcHMA1 was localized in the plasma membrane. Thus, our results suggest that AcHMA1 encodes a membrane-localized metal tolerance protein that mediates the detoxification of iron in eukaryotes. Furthermore, AcHMA1 also participates in the response to abiotic stress.

Generation and Analysis of Expressed Sequence Tags (ESTs) from Halophyte Atriplex canescens to Explore Salt-Responsive Related Genes

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources.

Functional Analysis of the Maize C-Repeat/DRE Motif-Binding Transcription Factor CBF3 Promoter in Response to Abiotic Stress

The ZmCBF3 gene is a member of AP2/ERF transcription factor family, which is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. To understand the regulatory mechanism of ZmCBF3 gene expression, we isolated and characterized the ZmCBF3 promoter (PZmCBF3). Three deletion fragments of PZmCBF3 were generated, C1–C3, from the translation start codon at position −1079, −638, and −234, and fused to the GUS reporter gene. Each deletion construct was analyzed by Agrobacterium-mediated stable transformation and expression in Arabidopsis thaliana. GUS expression assays indicated that the PZmCBF3 exhibited root-specific expression activity. A 234-bp fragment upstream of the ZmCBF3 gene conferred a high level of GUS activity in Arabidopsis. Some cis-acting elements involved in the down-regulation of gene expression were detected in the promoter, encompassing positions −1079 to −234. PZmCBF3 was activated by cold stress. The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress. The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

ZmDBF3, a Novel Transcription Factor from Maize (Zea mays L.), Is Involved in Multiple Abiotic Stress Tolerance

The dehydration-responsive element-binding (DREB) transcription factors play important roles in regulation of plant responses to abiotic stresses. In the present study, ZmDBF3, a novel DREB transcription factor gene from maize (Zea mays L.), was cloned and characterized. Sequence analyses revealed that ZmDBF3 is classified into A-4 group. It was demonstrated that ZmDBF3 was induced in by salt, drought, cold, and high temperature, as well as by signaling molecules abscisic acid (ABA), but no significant changes were observed under salicylic acid (SA) and methyl jasmonate (MeJA) conditions. The results of transient expression assays and transcriptional activity analysis revealed that ZmDBF3 is a nuclear protein with transcriptional activity. Overexpression of ZmDBF3 in yeast (Saccharomyces cerevisiae) exhibited increased survival rate under NaCl, KCl, Na2CO3, NaHCO3, PEG6000, freezing, and sorbitol treatment, compared with the control. Furthermore, ectopic expression of ZmDBF3 in Arabidopsis significantly enhanced tolerance to salt, drought, and freezing tolerance. Taken together, the findings indicated that the ZmDBF3 is a novel member of DREB transcription factor which may act as a regulatory factor involved in multiple stress response pathways.

Cloning and Functional Analysis of the Promoter of a Stress-inducible Gene (ZmRXO1) in Maize

The ZmRXO1 gene is a nucleotide-binding site leucine-rich repeat (NBS–LRR) type of R gene in maize (Zea mays). To understand the regulatory mechanism of ZmRXO1 gene expression, we isolated and characterized the ZmRXO1 promoter (PZmRXO1)—the 5′ flanking region of ZmRXO1. A series of PZmRXO1 deletion derivatives, R1–R4, from the translation start code (−1,576, −934, −829, and −582) were fused to the GUS reporter gene, and each deletion construct was analyzed by Agrobacterium-mediated transformation into tobacco. Sequence analysis showed that several cis-acting elements (MBS, Box-I, TGA-element and CCAAT-box) were located within the promoter. Deletion analysis of the promoter suggested that the 1,576-bp fragment upstream of ZmRXO1 gene showed a high level of GUS expression in tobacco. The promoter sequence (−582 to −1) was sufficient to improve transcription of GUS gene under hormones (MeJA, GA, ABA), drought and low temperature. Moreover, there might be repressor elements in the region (−1,576 to −934xa0bp) to repress ZmRXO1 gene expression under treatment with salicylic acid.