Genome-Wide Analysis of the ZRT,IRT-Like Protein (ZIP) Family and Their Responses to Metal Stress in <Emphasis Type="Italic">Populus trichocarpa</Emphasis>

The ZRT, IRT-like protein (ZIP) family plays an important role in the transport of zinc (Zn) and iron (Fe) across the cell membrane in many different species. However, studies on ZIP family are mainly limited in herbaceous species; hence, we investigated functional divergence of ZIP family in Populus trichocarpa. We identified 21 ZIP genes in P. trichocarpa and classified them into four groups based on phylogenetic analysis. Structural analyses revealed that most of the PtrZIP transporters have eight transmembrane domains (TMDs). PtrZIP members were unequally positioned in 19 P. trichocarpa linkage groups (LGs), with six tandem duplications and four segmental duplications. The promoter regions of PtrZIP genes contain Zn, Fe, copper (Cu), and other metal stress-related cis-elements. Additionally, tissue-specific expression of PtrZIP genes showed that most of them had relatively high expression levels in the root. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of PtrZIP genes were induced not only under deficiency or excess condition of Zn, Fe, Cu and manganese (Mn) but also under excess condition of cadmium (Cd) and lead (Pb) stress. These findings indicated that PtrZIP genes may have played potential roles in metal transporters. Genome-wide analysis of PtrZIP genes in P. trichocarpa provided more comprehensive insights on the structure and function of this gene family.     

Characterization and expression analysis of cytokinin biosynthesis genes in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Strawberry is one of the most economically important fruit crops in the world. Cytokinins (CKs) play a critical role in plant growth and development, as well as the stress response, and the level of CKs in plants is regulated by synthesis and degradation pathways. The key synthetic enzymes of CKs are isopentenyl transferases (IPTs) and LONELY GUYS (LOGs). We surveyed the strawberry genome and identified seven FvIPT genes and nine FvLOG genes. We analyzed gene structures, conserved domains, and their phylogenetic relationships with rice and Arabidopsis. The isoelectric points and glycosylation sites of the proteins were predicted. We also analyzed tissue- or organ-specific expression patterns of the FvIPT and FvLOG genes. The FvIPT and FvLOG genes showed different expression profiles in different organs. Most FvIPT and FvLOG genes were down-regulated in response to osmotic stress, high-temperature treatment, and exogenous abscisic acid (ABA) application, suggesting possible roles of these genes in the plants’ resistance to abiotic stresses. In addition, we found that the results of bioinformatics analyses to identify cis-regulatory elements may not be consistent with experimental expression data; thus, computer-predicted putative cis-elements need to be confirmed by experiments. Our systematic analyses of the FvIPT and FvLOG families provide a foundation for characterizing the function of these genes in the regulation of growth, development, and stress tolerance in Fragaria vesca, as well as a reference for improving stress tolerance by manipulating CK content.     

Characterization of genes coding for galacturonosyltransferase-like (GATL) proteins in rice

In the present study, seven galacturonosyltransferase-like (GATL) genes (OsGATLs) in rice (Oryza sativa L.) were genome-widely identified and the chromosomal locations and the gene structures of which were characterized. Under normal condition, OsGATL2 and OsGATL3 are highly expressed in root, while OsGATL4 is highly expressed in stem and leaf. Many cis-elements related to stress response and plant hormone were found in the promoter sequence of each OsGATL. The expression patterns of these OsGATL genes under treatment with abscisic acid (ABA), drought and low temperature were assessed by qRT-PCR. The expression levels of most OsGATLs significantly increased following the treatments with drought or low temperature. In addition, physicochemical properties of OsGATLs and phylogenetic analysis with GATL from rice and several other species were performed. 3D structures and protein–protein interaction (PPI) network of OsGATLs were further predicted by Swiss-model and STRING 9.0 database, respectively. The identification and bioinformatic analysis of GATL family in rice could provide reference data for further study on their biological functions, especially in the responsiveness to hormones and stress signaling.     

<Emphasis Type="Italic">Wsi18</Emphasis> promoter from wild rice genotype, <Emphasis Type="Italic">Oryza nivara</Emphasis>, shows enhanced expression under soil water stress in contrast to elite cultivar, <Emphasis Type="Italic">IR20</Emphasis>

Identification and characterization of plant promoters from wild rice genotypes showing inducible expression under soil water stress (SWS) is desirable for transgene expression to generate stress tolerant rice cultivars. A comparative expression profiling of Wsi18, a group 3 LEA gene, revealed differential response under SWS conditions between modern cultivated rice (IR20) and its wild progenitor (Oryza nivara). Wsi18 promoter from O. nivara showed enhanced inducible expression of the reporter gusA gene, encoding β-glucuronidase, in transgenic rice plants in comparison to similar promoter from IR20. Deletion analysis unravelled the cis-acting regulatory elements minimally required for optimal expression of Wsi18 promoter from O. nivara under SWS condition. This is the first report of characterization of an inducible promoter from a wild rice genotype to drive the gene expression under water stress conditions. The Wsi18 promoter element from the wild rice genotype can be used in future genetic manipulation strategies for the generation of SWS tolerant rice cultivars with improved yield characteristics.     

Regulatory <Emphasis Type="Italic">cis</Emphasis>-elements on citrus peel-specific expressed gene, <Emphasis Type="Italic">CuCRTISO-like</Emphasis>, promoter respond to hormones and abiotic stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.     

Isolation and characterization of the 4-coumarate:coenzyme A ligase (4CL1) promoter from <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

The most important enzyme of the phenylpropanoid pathway, 4-coumarate:coenzyme A ligase (4CL), is encoded by several homologous genes including 4CL1. The 4CL1 promoter is a tissue-specific gene expression element, particularly active in the secondary xylem or older stems. In this study, the 1127 bp 5′- upstream region of the 4CL1 coding sequence from Eucalyptus camaldulensis, Euc4CL1, was isolated and characterized. Essential putative cis-elements in the Euc4CL1 promoter included: a TATA-box at ?22/?28 position, two CCAAT-boxes at ?256/?260 and ?277/?281 positions, respectively, an AC-element at ?328/?336 and A-boxes at ?115/?120 and ?990/?995 positions. To investigate the effect of the Euc4CL1 promoter on gene expression, a plant transformation vector, pEuc4CL1p, containing the reporter gene for β-glucuronidase (GUS) under the control of Euc4CL1 promoter was constructed based on the pBI101 backbone and introduced in tobacco plants. Stable expression of the GUS gene in transgenic lines was analysed by a histochemical GUS assay. The results indicated the specific expression of the GUS gene in the stem xylem cells of transgenic tobacco lines was controlled by the Euc4CL1 promoter. The observations suggest the isolated Euc4CL1 promoter is a potential candidate for driving the expression of a foreign gene in plant xylem tissues.     

Ectopic expression of phloem motor protein pea forisome PsSEO-F1 enhances salinity stress tolerance in tobacco

Key message

PsSEOF-1 binds to calcium and its expression is upregulated by salinity treatment. PsSEOF - 1 -overexpressing transgenic tobacco showed enhanced salinity stress tolerance by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway.

Abstract

Calcium (Ca²⁺) plays important role in growth, development and stress tolerance in plants. Cellular Ca²⁺ homeostasis is achieved by the collective action of channels, pumps, antiporters and by Ca²⁺ chelators present in the cell like calcium-binding proteins. Forisomes are ATP-independent mechanically active motor proteins known to function in wound sealing of injured sieve elements of phloem tissue. The Ca²⁺-binding activity of forisome and its role in abiotic stress signaling were largely unknown. Here we report the Ca²⁺-binding activity of pea forisome (PsSEO-F1) and its novel function in promoting salinity tolerance in transgenic tobacco. Native PsSEO-F1 promoter positively responded in salinity stress as confirmed using GUS reporter. Overexpression of PsSEO-F1 tobacco plants confers salinity tolerance by alleviating ionic toxicity and increased ROS scavenging activity which probably results in reduced membrane damage and improved yield under salinity stress. Evaluation of several physiological indices shows an increase in relative water content, electrolyte leakage, proline accumulation and chlorophyll content in transgenic lines as compared with null-segregant control. Expression of several genes involved in cellular homeostasis is perturbed by PsSEO-F1 overexpression. These findings suggest that PsSEO-F1 provides salinity tolerance through cellular Ca²⁺ homeostasis which in turn modulates ROS machinery providing indirect link between Ca²⁺ and ROS signaling under salinity-induced perturbation. PsSEO-F1 most likely functions in salinity stress tolerance by improving antioxidant machinery and mitigating ion toxicity in transgenic lines. This finding should make an important contribution in our better understanding of the significance of calcium signaling in phloem tissue leading to salinity stress tolerance.