Specific expression of the gibberellin 3.BETA.-hydroxylase gene, HvGA3ox2, in the epithelium is important for Amy1 expression in germinating barley seeds

Abstract

We have cloned gibberellin (GA) 3b-hydroxylase cDNA, (HvGA3ox), a homolog of the rice gene OsGA3ox from germinating barley seeds. HvGA3ox cDNA was expressed in Escherichia coli as a fusion protein capable of oxidizing GA 9 to GA 4 and GA 20 to GA 1 . Deduced amino acid sequence analysis showed that HvGA3ox was identical to HvGA3ox2, which shares a high homology with the OsGA3ox2 isoform that plays an important role for induction of a-amylase in germinating rice seeds. Northern blot analysis showed that HvGA3ox2 gene was expressed only in germinating seeds, not in other organs. Typical feedback regulation was not observed in seedling treated with either biologically active GA (GA 3 ) or uniconazole, an inhibitor of GA biosynthesis. HvGA3ox2 mRNA was detected from 12 h after imbibition just before expression of a-amylase gene, Amy1. In situ hybridization of germinating seeds revealed that HvGA3ox2 mRNA was localized in the epithelium up to at least 3 days after imbibition. Our results suggest that GA biosynthesis in epithelium is important for expression of a-amylase in germinating barley seeds, and that HvGA3ox2 encodes the key enzyme of this event.Key words: a-amylase, epithelium, gibberellin, Hordeum vulgare. Original Paper Copyright © 2005 The Japanese Society for Plant Cell and Molecular BiologyAbbreviations: DIG, dioxigenin; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA end. The nucleotide sequence reported in this paper has been submitted to DDBJ/GenBank/EMBL nucleotide sequence database under the accession number AB189152. a TEPresent address: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan 1998; Yamaguchi et al. 1998;Yamaguchi et al. 2001;Itoh et al. 2001). Itoh et al. (2001) cloned and analyzed two GA3ox genes (OsGA3ox1 and OsGA3ox2) from rice. Their study indicated that OsGA3ox1 was preferentially expressed in unopened flower and OsGA3ox2 in elongating leaves. Kaneko et al. (2002) reported that OsGA3ox2 production in epithelium cells was important for a-amylase induction in early stages of germinating rice seeds. On the other hand, little is known about the molecular mechanism of GA biosynthesis in germinating barley seeds. Recently, Spielmeyer et al. (2004) reported isolation and chromosome mapping of two genes encoding GA 3b-hydroxylase, HvGA3ox1 and HvGA3ox2, from barley. However, there is no information about the expression pattern of these genes, and their role in a-amylase expression during germination remains to be clarified. The research described here predicts that HvGA3ox2 plays an important role in GA biosynthesis for the induction of hydrolytic enzymes, including a-amylase, in germinating barley seeds. Materials and methods Plant materialsBarley seeds (Hordeum vulgare cv. Harrington) were surface-sterilized in 1% NaClO for 10 min and rinsed in distilled water. The seeds were imbibed in distilled water for 8 h, plated on petri dishes with wetted filter paper, and grown in a culture chamber at 20°C in the dark. Seedling s...

Molecular Mechanisms Underlying Abscisic Acid/Gibberellin Balance in the Control of Seed Dormancy and Germination in Cereals

“…It has been shown previously that vivipary phenotype in maize kernels due to ABA deficiency can be reversed through inhibition of GA synthesis, and this demonstrates the role of GA in antagonizing the action of ABA and thereby seed transition from dormancy to germination ( White et al, 2000 ). Changes in the balance between ABA and GA levels, which modulate seed dormancy status, are associated with alterations in the expression patterns of their metabolic genes; for example, imbibition of non-dormant barley seeds induces the expression of ABA catabolic gene HvABA8′OH1 and GA biosynthetic gene HvGA3ox2 ( Suzuki et al, 2005 ; Millar et al, 2006 ), which leads to reduced ABA and increased GA levels. Furthermore, imbibition of non-dormant wheat seeds has been shown to lead to transcriptional induction of TaGA3ox2 and HvABA8′OH1 genes ( Liu et al, 2013 ; Son et al, 2016 ).…”

Cortical microtubule disorganized related to an endogenous gibberellin increase plays an important role in rice mesocotyl elongation

“…GA2oxs are involved in GAs catabolism and inactivate GAs through 2β-hydroxylation (Lo et al 2008). GA20ox2 and GA3ox2 play an important role in GA biosynthesis and GID1 is involved in sensing and transducing GA signal (Hedden and Phillips 2000;Itoh et al 2001;Spielmeyer et al 2002;Suzuki et al 2005;Wu et al, 2011). Our results demonstrated that the expression of Figure 5.…”

Manipulating Gibberellin Control Over Growth and Fertility as a Possible Target for Managing Wild Radish Weed Populations in Cropping Systems

“…There was no detectable GID1-DELLA interaction when the yeast cultures were supplemented with bio-inactive GA 9 (Figures 5Aiii, B). However, when we introduced a third transgene into the yeast that encodes for a GA3OX protein from either Arabidopsis or barley, which have confirmed GA 3b-hydroxylase activity (Williams et al, 1998;Spielmeyer et al, 2004;Suzuki et al, 2005;Pearce et al, 2015), the GA 9 supplemented yeast cultures grew. This implies the yeast were synthesizing GA 4 from the GA 9 which in turn enabled the GID1-DELLA interaction, leading to activation of the HIS3 reporter gene and allowing the HIS deficient yeast to grow (Figures 5Aiv, C).…”