Water-deficit tolerance in citrus is mediated by the down regulation of PIP gene expression in the roots

Water deficit (WD) is a growing problem in agriculture. In citrus crops, genetically-determined rootstock characteristics are important factors influencing plant responses to WD. Aquaporins are involved in regulating the water supply to the plant by mediating water flow through the cell membranes. Recent studies support a direct role for aquaporins in plant water relations and demonstrate their involvement in WD tolerance. This study investigates the relationship between photosynthetic and water-balance parameters with aquaporin expression levels and hydraulic conductance of roots (Kr) in conditions of moderate WD in citrus rootstocks. The plant materials used were the rootstocks Poncirus trifoliata (L.) Raf. (PT), Cleopatra mandarin (Citrus reshni Hort ex Tan.) (CM) and 030115 (a hybrid of the two former rootstocks), all grafted with the citrus variety ??Valencia Late?? (C. sinensis (L.) Osb). Plants were irrigated with two differents irrigation doses (normal irrigation and moderate WD) during 70 days and leaf water potential (??s), net CO₂ assimilation (A_CO2), transpiration, stomatal conductance (gs) and substomatal CO₂ concentration (Ci) were measured periodically under both irrigation conditions. Kr and PIP1 and PIP2 gene expression levels in fine roots of control plants and plants subjected to WD on day 43 of the experiment were determined. Under WD conditions, the hybrid 030115 drastically reduced aquaporin expression and Kr, accompanied by a loss of plant vigour but without reducing the net CO₂ assimilation (A_CO2). PT maintained the same aquaporin expression level and similar Kr under WD as under normal irrigation conditions, but suffered a sharp reduction in A_CO2. CM, which has lower Kr and aquaporin expression than PT under both normal irrigation conditions and WD, responded better to water stress conditions than PT. Low aquaporin levels, or down-regulated aquaporin expression, accompanied by decreased plant vigour led to decreased plasma membrane permeability, thereby facilitating water retention in the cells under water stress conditions. This may induce water stress tolerance in citrus rootstocks.     

Omega-3 fatty acid desaturase (FAD3, FAD7, FAD8) gene expression and linolenic acid content in cowpea leaves submitted to drought and after rehydration

Membrane polyunsaturated fatty acids (PUFA) and particularly linolenic acid (18:3, LA) are known to be implicated in plant tolerance to low temperature. Their role in resistance to drought is much less investigated. In this work, three full-length cDNAs corresponding to omega-3 fatty acid desaturases: fad3 (endoplasmic reticulum), fad7 and fad8 (chloroplastic) were isolated from Vigna unguiculata leaves. Two cowpea cultivars, one drought-tolerant, EPACE-1, and one drought-susceptible, 1183, were compared in terms of fad isoform gene expression and leaf LA contents in plants submitted to water stress followed by rehydration. In EPACE-1, LA content in the main leaf polar lipids increased in response to mild water deficit. Severe water deficits induced a decrease in MGDG LA content while those of PC and DGDG continued to increase. Variations in FAD gene expression, matched those in LA contents. In 1183, LA contents decreased in all lipid classes in response to water stress, as did FAD3 and FAD8 gene expression levels. Rehydration after a moderate water stress induced stimulation mostly in FAD3 gene expression in both cvs. LA contents were equivalent to control levels in EPACE-1. In 1183, they were back to control levels in PC shortly after rehydration but remained low in galactolipids. These results suggested that omega-3 FAD activities were involved in the increase in leaf membrane unsaturation, in the drought tolerant plants whereas the sensitive plants lost PUFAs in response to the treatment. The significance of this discrepancy between the two cvs. in terms of adaptation to drought is discussed.     

Phenology of glucosinolate concentrations in roots,stems and leaves of Cardamine cordifolia

Total concentrations of isothiocyanate-yielding glucosinolates (IYG) were measured in roots, stems, basal leaves and cauline leaves of the herbaceous perennial Cardamine cordifolia (bittercress, Cruciferae), sampled at three sites in the Colorado Rockies during 1981. Significant variation in quantity was partitioned among plant parts, among sampling dates throughout the growing season, and among the three sites. Roots and basal leaves maintained high and similar concentrations of IYG through the season, while cauline leaves and stems showed seasonal declines, associated partly with flowering. Roots also consistently produced oxazolidinethione-yielding glucosinolates (hydroxylated analogues of IYG), whereas above-ground parts were variable for the presence of these compounds. Seasonal and plant-part variability in glucosinolate content and spatial patchiness of glucosinolate phenotypes contribute to the variation in herbivore occurrence and damage documented in previous studies of this native crucifer.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs

A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain

DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N‐terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi‐methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N‐terminal regulatory domain. By deletion analysis and co‐immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N‐terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex has a bipartite interaction interface and is formed in a head‐to‐head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi‐methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co‐ordinated. J. Cell. Biochem. 106: 521–528, 2009. © 2009 Wiley‐Liss, Inc.     

Chloramphenicol-inducible gene expression in Bacillus subtilis is independent of the chloramphenicol acetyltransferase structural gene and its promoter

Lactobacillus plantarum has an unusually high Mn(II) requirement for growth and accumulated over 30 mM intracellular Mn(II). The acquisition of Mn(II) by L. plantarum occurred via a specific active transport system powered by the transmembrane proton gradient. The Mn(II) uptake system has a Km of 0.2 microM and a Vmax of 24 nmol mg-1 of protein min-1. Above a medium Mn(II) concentration of 200 microM, the intracellular Mn(II) level was independent of the medium Mn(II) and unresponsive to oxygen stresses but was reduced by phosphate limitation. At a pH of 5.5, citrate, isocitrate, and cis-aconitate effectively promoted MN(II) uptake, although measurable levels of 1,5-[14C]citrate were not accumulated. When cells were presented with equimolar Mn(II) and Cd(II), Cd(II) was preferentially taken up by the Mn(II) transport system. Both Mn(II) and Cd(II) uptake were greatly increased by Mn(II) starvation. Mn(II) uptake by Mn(II)-starved cells was subject to a negative feedback regulatory mechanism functioning less than 1 min after exposure of the cells to Mn(II) and independent of protein synthesis. When presented with a relatively large amount of exogenous Mn(II), Mn(II)-starved cells exhibited a measurable efflux of their internal Mn(II), but the rate was only a small fraction of the maximal Mn(II) uptake rate.     

Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes

Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.     

Seasonal flux of isothiocyanate-yielding glucosinolates in roots, stems and leaves of Cardamine cordifolia

Concentrations of constituent isothiocyanate-yielding glucosinolates (IYG) declined seasonally in cauline leaves and stems of Cardamine cordifolia, sampled in montane Colorado. All five constituents (isopropyl, 2-butyl, isobutyl, benzyl and 2-phenyl-ethyl glucosinolates) were correlated positively with total concentration over the growing season. In contrast, no seasonal decline was discerned for total and constitutent IYG concentrations in roots and basal leaves, and constituents showed a mixed pattern of positive and negative correlations with total amount. Vegetative organs thus constitute two compartments relative to glucosinolate metabolism. In above-ground stems and associated cauline leaves, IYG concentrations decline individually and in concert over the growing season. In below-ground rhizomes and associated roots and basal leaves, individual compounds fluctuate in concentration, but in the aggregate do not decline seasonally. Metabolic fluxes of individual glucosinolates within and between vegetative organs, as well as changes in total concentrations, may influence feeding by herbivores.     

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5 non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (–623 to –471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (–341 to –306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C+D+E, B+C+D, B+C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B+C+E is active only in vascular meristematic cells. Domain C (–216 to –158) seems to have a double regulatory role as construct B+E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.