Isolation of carrot basic leucine zipper transcription factor using yeast one-hybrid screening

Abscisic acid (ABA) is involved in various physiological and developmental processes, including stress responses and seed maturation. Many ABA-regulated genes associated with these processes have been identified and analyzed. Previously, we identified 2 important elements in the promoter of the carrotDcECP31 gene: motif X (CACACGTGGG), which is like an ABA-responsive element (ABRE), and motif Y (CACACGTATC). Together, these are sufficient for embryo-specific ABA-inducible promoter activity. We also showed that motif X functions is an enhancerlike element and that motif Y participates in ABA responsiveness. In this study, we isolated the nuclear protein that interacts with motif Y of theDcECP31 promoter. We performed yeast one-hybrid screening using integrated motif Y as bait and isolated clones. Sequence analysis revealed that clone 22 included the carboxyl-terminal half of bZIP, which contains the basic and leucine zipper domains and binds to G-boxes containing the sequence ACGT. This result supports the hypothesis that carrot C-ABI3, a homologue of theArabidopsis ABI3 protein, functions as a coactivator that interacts with the G-box via protein-protein contacts and suggests that the complex controls the expression of theDcECP31 gene.     

Isolation and Functional Analysis of the Poplar RbcS Gene Promoter

A tissue-specific promoter, Pt-RbcS, from Populus was isolated and cloned based on alignment of AtRBCS-2B cDNA with genomic Populus sequences. Sequence analysis of Pt-RbcS revealed cis-acting regulatory elements in the promoter region, including an ATCT-motif, BoxI, GAG-motif, I-box, G-box, BoxII, GATA-motif, and TCT-motif, which are involved in light responses. In transgenic tobacco lines carrying the β-glucuronidase (GUS) gene driven by the Pt-RbcS promoter, GUS expression was detected in leaves and stems, but not in roots. Transgenic poplar lines harboring constructs carrying the GUS gene driven by truncated Pt-RbcS promoters revealed distinctive expression patterns for five different promoter constructs. The Pt-RbcS promoter was expressed preferentially in photosynthetic tissues such as leaves and stems. Moreover, deletion analysis of the 1,547 bp Pt-RbcS promoter region revealed that a 927-bp DNA segment is critical for expression of Pt-RbcS in green tissues. Overall, our study suggests that the Pt-RbcS promoter from Populus could be applied to genetically improve the photosynthetic efficiency of woody plants.     

A double SORLIP1 element is required for high light induction of ELIP genes in Arabidopsis thaliana

Promoter elements that contribute to high light (HL) induction of the Arabidopsis ELIP1 gene were defined using a transgenic promoter-reporter system. Two adjacent SORLIP1 elements (double SORLIP1, dSL) were found to be essential for HL induction of a GUS reporter gene. The dSL element was also found to be essential for HL induction conferred by the ELIP2 promoter. SORLIP1 elements were enriched in ELIP promoters throughout the plant kingdom, and showed a clade-specific pattern of gain or loss that suggested functionality. In addition, two G-box elements were found to redundantly contribute to HL induction conferred by the ELIP1 promoter.     

A promoter analysis of MOTHER OF FT AND TFL1 1 (JcMFT1), a seed-preferential gene from the biofuel plant Jatropha curcas

MOTHER OF FT AND TFL1 (MFT)-like genes belong to the phosphatidylethanoamine-binding protein (PEBP) gene family in plants. In contrast to their homologs FLOWERING LOCUS T (FT)-like and TERMINAL FLOWER 1 (TFL1)-like genes, which are involved in the regulation of the flowering time pathway, MFT-like genes function mainly during seed development and germination. In this study, a full-length cDNA of the MFT-like gene JcMFT1 from the biodiesel plant Jatropha curcas (L.) was isolated and found to be highly expressed in seeds. The promoter of JcMFT1 was cloned and characterized in transgenic Arabidopsis. A histochemical β-glucuronidase (GUS) assay indicated that the JcMFT1 promoter was predominantly expressed in both embryos and endosperms of transgenic Arabidopsis seeds. Fluorometric GUS analysis revealed that the JcMFT1 promoter was highly active at the mid to late stages of seed development. After seed germination, the JcMFT1 promoter activity decreased gradually. In addition, both the JcMFT1 expression in germinating Jatropha embryos and its promoter activity in germinating Arabidopsis embryos were induced by abscisic acid (ABA), possibly due to two ABA-responsive elements, a G-box and an RY repeat, in the JcMFT1 promoter region. These results show that the JcMFT1 promoter is seed-preferential and can be used to control transgene expression in the seeds of Jatropha and other transgenic plants.     

Construction of Stress Responsive Synthetic Promoters and Analysis of Their Activity in Transgenic Arabidopsis thaliana

To obtain strong inducible promoters to drive abiotic stress-inducible transgene expression with minimal negative effects, we constructed three artificial synthetic promoters (EKCM, EKCRM, and ECCRM) comprising multiple cis-acting stress-response elements. Each promoter was fused independently to the β-glucuronidase (GUS) reporter gene, and GUS expression was analyzed in stable expression systems in Arabidopsis thaliana. T2 transgenic progenies showed integration of the promoter-GUS construct in their genome. RT-PCR assays and histochemical staining analysis showed that GUS expression driven by each promoter increased under desiccation, cold, and high salt conditions. The activity of synthetic promoters, assessed by fluorometric quantitative analysis of GUS enzyme activity, was significantly higher than that of the rd29A promoter under various stress treatments. The most powerful promoter, EKCM, allowed about 1.29-fold in GUS activity relative to the rd29A promoter, on average, under dehydration conditions. All three synthetic promoters could drive stress-inducible GUS expression in different organs of transgenic Arabidopsis. These synthetic promoters represent valuable tools for improving the stress tolerance of crops.