Isolation and Functional Analysis of Two Gibberellin 20-Oxidase Genes from Satsuma Mandarin (<i>Citrus unshiu</i> Marc.)

Abstract

Satsuma mandarin (Citrus unshiu Marc.) has two GA 20-oxidase genes, CuGA20ox1 and CuGA20ox2, and the genomic sequence for CuGA20ox1 is shorter than that for CuGA20ox2, although the coding region of cDNA for CuGA20ox1 is slightly longer than that for CuGA20ox2. Southern blot analysis revealed that 12 Citrus cultivars examined and trifoliate orange [Poncirus trifoliata (L.) Raf.] have at least two types of GA 20-oxidase genes, such as CuGA20ox1 and CuGA20ox2 genes. CuGA20ox1 and CuGA20ox2 were differentially expressed in various tissues. CuGA20ox1 was expressed in almost all of the tissues investigated with relatively higher expression in vegetative than in reproductive tissues, whereas CuGA20ox2 was specifically expressed in flower buds just before anthesis. These distinct expression patterns of CuGA20ox1 and CuGA20ox2 imply that function of these two genes diverged in the process of evolution. The specific and relatively higher expression of CuGA20ox2 in flower buds would explain why GA-like activity was higher in Satsuma mandarin's ovaries at anthesis. Transgenic Arabidopsis [Arabidopsis thaliana (L.) Heynh] plants ectopically expressing CuGA20ox1 or CuGA20ox2 were examined to elucidate the function of these two Satsuma mandarin genes. Phenotypic analysis revealed that both CuGA20ox1 and CuGA20ox2 caused elongated inflorescence but did not affect the timing of flowering in transgenic Arabidopsis as compared with wild-type controls. Ectopic expression of CuGA20ox1 and CuGA20ox2 significantly affected the levels of GA 24 and GA 34 on the non-13-hydroxylation pathway; GA 24 decreased and GA 34 increased. This observation indicates that both CuGA20ox1 and CuGA20ox2 accelerated the conversion of GA 24 , a substrate of a GA 20-oxidase, to GA 9 , a precursor of an active form of GA 4 . Likewise, on the early-13-hydroxylation pathway, ectopic expression of CuGA20ox1 significantly decreased GA 19 and increased GA 29 and GA 8 , inactive metabolites of 2-hydroxylation of GA 20 and GA 1 , respectively, suggesting the activation of this biosynthetic pathway. CuGA20ox2 also had a tendency to activate the early-13-hydroxylation pathway although it increased only GA 20 with a statistically significant difference. Taken together, we concluded that CuGA20ox1 and/or CuGA20ox2 activated both the early-13-and non-13-hydroxylation pathways for increasing active GAs, resulting in elongated inflorescences in transgenic Arabidopsis.

Gibberellin 2-Oxidase Genes from Satsuma Mandarin (<i>Citrus unshiu</i> Marc.) Caused Late Flowering and Dwarfism in Transgenic <i>Arabidopsis</i>

“…A previous study identified two GA 20-oxidase genes from Satsuma mandarin and confirmed their function using transgenic Arabidopsis. Consistent with the accumulation of active GAs in the ovaries at around anthesis, CuGA20ox2 was specifically expressed in the flower bud just before anthesis in Satsuma mandarin (Kotoda et al, 2016). In this study, Satsuma mandarin GA 2oxidase genes encoding enzymes with an inactivation activity of GAs were characterized because the accumulation of active GAs is regulated by the balance between their synthesis and inactivation.…”

“…The expression pattern of CuGA2ox4 resembled that of CuGA20ox1 in the reproductive tissues of the adult trees in that both genes were expressed in juice sacs in November and December and in peel in December (Fig. 3A; Kotoda et al, 2016), suggesting that CuGA2ox4 and CuGA20ox1 act in concert to regulate the accumulation of active GAs in fruit at the maturing stage. Because these genes are expressed in both juice sacs and peel, they might be involved in the occurrence of peel puffins to some extent.…”

“…These results imply a relationship between the degree of seedlessness and the endogenous GAs. In recent years, GA biosynthetic genes have been isolated and analyzed to clarify their functions in citrus (Fagoaga et al, 2007;Huerta et al, 2009;Kotoda et al, 2016;Vidal et al, 2001Vidal et al, , 2003. A previous study identified two GA 20-oxidase genes from Satsuma mandarin and confirmed their function using transgenic Arabidopsis.…”

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes

“…Likewise, Kishu shows no parthenocarpy (Shimizu, T., unpublished) but Satsuma is an exceptionally high and stable parthenocarpic variety [117]. Kotoda et al isolated two gibberellin 20-oxidase genes from Satsuma thought to be involved in parthenocarpy, and demonstrated their different biological functions [71]. Very recently, Kotoda et al isolated three gibberellin 2-oxidase genes of Satsuma involved in the degradation of bioactive gibberellic acid [132].…”

“…Citrus genome sequence resources in public databases, including BAC end sequences of Clementine [69] and Satsuma [70,71], and whole genome shotgun sequences of sweet orange ‘Ridge Pineapple’ in the trace file repository of Sanger reads (ftp://ftp.ncbi.nlm.nih.gov/pub/TraceDB/citrus_sinensis/), were also used for DNA marker design. Preliminary evaluation of the quality and length of each of these data sets was carried out using pregap4 [72], then a consensus sequence set was obtained for each set with Mira assembler [73] to reduce redundancy.…”