Studies on Ginkgo biloba (E)-4-Hydroxy-3-methylbut-(2)-enyl Diphosphate Reductase Gene and Promoter

Abstract

Isoprenoids, also known as terpenoids, are derived from the five-carbon building units isopentenyl diphophate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Even though they are synthesized in all living organisms, plants have more diverse and abundant isoprenoid compounds compared to others. Plants have two distinct isoprenoid biosynthetic pathways, 2-C-methyl-D-erythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways. In this study, plant IDS, the terminal enzyme in MEP pathway, is focused. Ginkgo biloba, one of the gymnosperm tree known as living fossil, has three copies of IDS genes. They are divided into two classes: GbIDS1 to class 1, and GbIDS2 and 2-1 to class 2. Each enzyme class is known to separately participate in primary and secondary metabolisms. In this research, promoter analysis and overexpression study of GbIDSs were performed respectively in Arabidopsis and poplar. The GbIDS1 and GbIDS2 promoters were fused with GUS protein and then introduced into Arabidopsis. GbIDS1pro::GUS transformant showed GUS expression in most organs except for roots, petals, and stamina, whereas GbIDS2pro::GUS was expressed only in the young leaves, internodes where the flower and shoot branched, and notably in primary root junction. This pattern of GUS expression correlated with high transcript level of GbIDS2 in Ginkgo roots compared to that of GbIDS. Methyl jasmonate (MeJA) treatment resulted in down-regulated GbIDS1pro activity in Arabidopsis leaves and upregulated GbIDS2pro activity in roots. The similar patterns of GUS activity in GbIDS2pro:: GUS Arabidopsis roots were also seen upon treatments of gibberellins (GA), abscisic acid (ABA), and indole butyric acid (IBA). Each of the GbIDS1 and GbIDS2 overexpression construct was introduced into poplars. Ten GbIDS1 overexpression lines were obtained while no transformants were made with GbIDS2. GbIDS1 transgenic poplars were taller than wild-type (WT) BH poplars by 25% and have 2 more leaves in indoor condition 7 weeks after potting in soil. Twenty five weeks after potting in outdoor nursery, GbIDS1 plants in pot gained height by 7% compared to BH, and showed delayed winter bud formation. In addition, overexpression of GbIDS1 gene led increase of chlorophyll and carotenoid contents by approximately 20% in transgenic poplars compared to WT poplars. Chlorophyll-related genes, CHS (chlorophyll synthase) and CAO (chlorophyll a oxidase) transcript levels were higher in transgenic poplars by 30% and 50% respectively. Transcript level analyses of GA biosynthetic genes, KS (kaurene synthase), GA20ox (gibberellin 20 oxidase), and GA2ox (gibberellin 2 oxidase), were performed in poplar. In this analysis, transcript levels of bioactive GA synthesis gene, KS and GA20ox, were up-regulated while GA inactivation gene, GA2ox, was down-regulated in transgenic poplars. In spite of signal peptide deletion, tGbIDS2 (truncated GbIDS2 devoid of signal peptide) targeted to the chloroplast. In the heterozygote Arabidopsis plants, overexpression of tGbIDS2 was previously reported to lead rapid growth and early flowering. However, in homozygote tGbIDS2 overexpression transgenic Arabidopsis in the current research, there were no significant phenotype changes compared to the Col-0 wild type (WT). Besides, little changes were observed in chlorophyll and carotenoids contents in transgenic and Col-0 Arabidopsis plants. On the other hand, transcript levels of floral genes and GA4 displayed differences between WT and transgenic Arabidopsis plants. CO (CONSTANTS) levels were up-regulated by 60% but FLC (FLOWERING LOCUS C), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CO1), and LFY (LEAFY) were down-regulated by 26%, 32%, and 24%, respectively. Also transcript level of GA4 gene increased by 56% in transgenic Arabidopsis, it infers decrease of GA amounts in transgenic Arabidopsis. Through these experiments, physiological differences of each GbIDS were examined, and applicability of GbIDSs for genetic engineering was evaluated.