Evaluation of the Use of the Tobacco PR-1a Promoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

A comparison of plants regenerated from a variegated <Emphasis Type="Italic">Epipremnum aureum</Emphasis>

In order to study chloroplast biogenesis, we chose natural variegated Epipremnum aureum (golden pothos) and regenerated pale yellow, variegated and green plants from all three types of tissue explants. The percentage of three types of regenerated shoots from three different explants was very close. Regenerated plants have been maintained for a year and show no sign of a colour switch. By comparing their protein profiles, two major differences between pale yellow and green plants were observed at the 15 and 40 to 50 kDa proteins. Moreover, pale yellow plants had unexpected high molecular mass proteins (greater than 60 kDa). Both variegated and green plants had more chlorophyll (Chl) a than Chl b, the ratios were about 1.46 and 1.93, respectively. In contrast, the pale yellow plants not only had less total Chl, but also the reduction of Chl a was much greater than Chl b, resulting in a higher content of Chl b than Chl a. Microscopic analysis revealed that pale yellow plants contained predominantly undeveloped chloroplasts with low Chl contents, even though their mesophyll cells were similar to green and variegated plants. PCR amplification of chloroplast DNA with 14 universal chloroplast primers did not reveal any difference among these regenerated plants.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

ZmDREB2A regulates ZmGH3.2 and ZmRAFS,shifting metabolism towards seed aging tolerance over seedling growth

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION‐RESPONSIVE ELEMENT‐BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.2, encoding indole‐3‐acetic acid [IAA] deactivating enzyme), and increased IAA in their embryo, produced longer seedling shoots and roots, than the null segregant (NS) controls. However, the zmdreb2a seeds, with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryo, exhibit decreased seed aging tolerance, than the NS controls. Overexpression of ZmDREB2A in maize protoplasts increased the expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2‐ or ZmRAFS‐promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS. Exogenous supplementation of IAA to the unaged, germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2a seedlings from unaged seeds. These findings provide evidence that ZmDREB2A regulates the longevity of maize seed by stimulating the production of raffinose while simultaneously acting to limit auxin‐mediated cell expansion.     

Expression analysis and functional characterization of a novel cold-responsive gene <Emphasis Type="Italic">CbCOR15a</Emphasis> from <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd’s purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA₃ accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA₃ regulation and confers enhanced cold acclimation in transgenic plants.     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

A heat-shock promoter fusion to the Ac transposase gene drives inducible transposition of a Ds element during Arabidopsis embryo development

A heat-shock promoter fusion to the Ac transposase gene (hs::TPase) was constructed and introduced into Arabidopsis. In five transformants containing the fusion the abundance of transposase mRNA increased approximately 120-fold on exposure to high temperatures. Hybrid plants containing hs::TPase and a Ds element inserted in a streptomycin resistance gene (Ds::SPT) were made and these plants were self-fertilized either after heat shocking at different stages in development or without exposure to high temperature. The progeny of these plants were sown on streptomycin-containing medium and the frequency with which variegated or streptomycin-resistant (strep^R) seedlings occurred was used as an indication of the frequency of Ds excision. Very few of the progeny of plants not exposed to heat shock or of those heat shocked only during vegetative development were variegated or strep^R. However, plants that were heat shocked after the appearance of flower buds and during seed development produced high frequencies (approaching 100%) of variegated, but very few strep^R, progeny. Furthermore, when variegated seedlings were grown to maturity and self-fertilized without further exposure to heat shock then strep^R seedlings often occurred at high frequency among their progeny. Southern analysis indicated that the majority of these strep^R plants contained a transposed Ds at a new location. These data indicate that in response to heat shock Ds excision frequently occurs in embryonic cells which ultimately give rise to the gametes, as well as in cells of the developing cotyledons. The importance of an inducible transposon system for transposon tagging is discussed.