Genomewide computational analysis of nitrate response elements in rice and Arabidopsis

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualifies to be the cis-acting NRE for all the responsive genes. We searched for the specific occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-specific bias. This is the first comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any specificity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-specific and shoot-specific response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolutionary conservation of an atypical glucocorticoid‐responsive element in the human tyrosine hydroxylase gene

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at ?7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein‐1 (AP‐1)‐like motif in the rat TH gene. We cloned this hTH‐CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH‐CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5′‐TGACTAA at ?7243 bp completely abolished the Dex‐stimulated Luc activity of hTH‐CRII construct. The AP‐1 agonist, tetradeconoyl‐12,13‐phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP‐1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid‐responsive element in a 7 bp AP‐1‐like motif in the promoter region at ?7.24 kb of the human TH gene.     

OsASR2 regulates the expression of a defence‐related gene,Os2H16, by targeting the GT‐1 cis‐element

The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.     

The NIN‐like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize

Maize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN‐like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild‐type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate‐responsive cis‐element at the 5′ UTR of the gene. Interestingly, a natural loss‐of‐function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.     

RPW8 promoter is involved in pathogen‐And stress‐inducible expression from Vitis pseudoreticulata

Based on previous cloning of VpRPW8‐e, we obtained a 1,126 bp VpRPW8‐e promoter sequence in this study. A large number of TATA‐boxes, CAAT‐boxes, and other cis‐acting elements were predicted including light‐responsive elements, hormone‐responsive elements, stress‐responsive elements, and growth‐ and development‐associated elements within the promoter sequence. To further investigate the function of this promoter, we examined its activity in response to biotic and abiotic stress. The VpRPW8‐e promoter was strongly activated by Plasmopara viticola infection, and activation also occurred when the orientation of the promoter was reversed, although to a lesser extent. Deletion analysis showed that the ?1,126 to ?475 bp region of VpRPW8‐e promoter had high activity. A promoter fragment 5′ deleted to ?475 bp (P_?475) was activated in response to heat and cold stress, and even more strongly in response to Phytophthora capsici and salicylic acid (SA). Furthermore, Transgenic Nicotiana benthamiana were generated, VpRPW8‐e driven by P_?475 enhanced resistance to Ph. capsici in N. benthamiana. Based on these results, the ?475 bp region was deduced to be an indispensable part of the VpRPW8‐e promoter. VpRPW8‐e promoter is involved in pathogen‐ and stress‐inducible expression.     

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis‐regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)‐inducible motif discovery among promoters of 18 co‐expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean–SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co‐localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN‐inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN‐inducible, leading to the discovery of 23 core motifs of 5‐ to 7‐bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W‐AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high‐throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.     

Quercetin‐induced upregulation of human GCLC gene is mediated by cis‐regulatory element for early growth response protein‐1 (EGR1) in INS‐1 beta‐cells

The catalytic subunit of γ‐glutamylcysteine ligase (GCLC) catalyses the rate‐limiting step in the de novo synthesis of glutathione (GSH), which is involved in maintaining intracellular redox balance. GSH is especially important for antioxidant defense system since beta‐cells show intrinsically low expression of antioxidant enzymes. In the present study, we investigated the regulatory mechanisms by which quercetin, a flavonoid, induces the expression of the GCLC gene in rat pancreatic beta‐cell line INS‐1. Promoter study found that the proximal GC‐rich region (from ?90 to ?34) of the GCLC promoter contained the quercetin‐responsive cis‐element(s). The quercetin‐responsive region contains consensus DNA binding site for early growth response 1 (EGR1) at ‐67 (5′‐CGCCTCCGC‐3′) which overlaps with a putative Sp1 binding site. Electrophoretic mobility shift assay showed that an oligonucleotide containing the EGR1 site was bound to nuclear factors EGR1, Sp1, and Sp3. In the promoter analysis, mutation of EGR1 site significantly reduced the quercetin response, whereas mutation of Sp1 site decreased only the basal activity of the GCLC promoter. Additionally, the transient overexpression of EGR1 significantly increased basal activity of the GCLC promoter. Finally, we showed that quercetin potently induced both EGR1 mRNA and its protein levels without affecting the expression of Sp1 and Sp3 proteins. Therefore, we concluded that EGR1 was bound to GC‐rich region of the GCLC gene promoter, which was prerequisite for the transactivation of the GCLC gene by quercetin. J. Cell. Biochem. 108: 1346–1355, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.