Abstract
BackgroundGA 2-oxidases (GA2oxs) are involved in regulating GA homeostasis in plants by inactivating bioactive GAs through 2β-hydroxylation. Rice GA2oxs are encoded by a family of 10 genes; some of them have been characterized, but no comprehensive comparisons for all these genes have been conducted.ResultsRice plants with nine functional GA2oxs were demonstrated in the present study, and these genes not only were differentially expressed but also revealed various capabilities for GA deactivation based on their height-reducing effects in transgenic plants. Compared to that of wild-type plants, the relative plant height (RPH) of transgenic plants was scored to estimate their reducing effects, and 8.3% to 59.5% RPH was observed. Phylogenetic analysis of class I GA2ox genes revealed two functionally distinct clades in the Poaceae. The OsGA2ox3, 4, and 8 genes belonging to clade A showed the most severe effect (8.3% to 8.7% RPH) on plant height reduction, whereas the OsGA2ox7 gene belonging to clade B showed the least severe effect (59.5% RPH). The clade A OsGA2ox3 gene contained two conserved C186/C194 amino acids that were crucial for enzymatic activity. In the present study, these amino acids were replaced with OsGA2ox7-conserved arginine (C186R) and proline (C194P), respectively, or simultaneously (C186R/C194P) to demonstrate their importance in planta. Another two amino acids, Q220 and Y274, conserved in OsGA2ox3 were substituted with glutamic acid (E) and phenylalanine (F), respectively, or simultaneously to show their significance in planta. In addition, through sequence divergence, RNA expression profile and GA deactivation capability analyses, we proposed that OsGA2ox1, OsGA2ox3 and OsGA2ox6 function as the predominant paralogs in each of their respective classes.ConclusionsThis study demonstrates rice has nine functional GA2oxs and the class I GA2ox genes are divided into two functionally distinct clades. Among them, the OsGA2ox7 of clade B is a functional attenuated gene and the OsGA2ox1, OsGA2ox3 and OsGA2ox6 are the three predominant paralogs in the family.
Highlights
GA 2-oxidases (GA2oxs) are involved in regulating GA homeostasis in plants by inactivating bioactive GAs through 2β-hydroxylation
Functional Screening of Rice GA2ox1, GA2ox2, GA2ox4, GA2ox7 and GA2ox8 Using T-DNA Activation-Tagged Mutants Our previous study showed that enhanced expression of OsGA2ox3 (M77777), OsGA2ox5 (M27337), OsGA2ox6 (M47191) and OsGA2ox9 (M58817) in their respective T-DNA activation-tagged mutants reduced plant height, which provided a useful tool for functional screening of GA2ox genes (Lo et al 2008)
The present study demonstrated at least four amino acid variants that are distinct between clade A (C186, C194, Q220 and Y274 in OsGA2ox3) and clade B (R191, P201, E227 and F283 in OsGA2ox7); these variants are associated with strong clade A and weak clade B GA deactivation capability
Summary
GA 2-oxidases (GA2oxs) are involved in regulating GA homeostasis in plants by inactivating bioactive GAs through 2β-hydroxylation. The availability of bioactive GAs in plant tissues must be homeostatically maintained for proper plant growth, which is precisely regulated by both GA biosynthetic and catabolic enzymes. The GA 20oxidase (GA20ox), GA 3-oxidase (GA3ox) and GA 2oxidase (GA2ox) members of the 2OGD family are thought to be the main enzymes involved in regulating GA homeostasis in plants during growth and in response to environmental signals (Yamaguchi 2008). GA20oxs and GA3oxs are GA biosynthetic enzymes (Toyomasu et al 1997; Itoh et al 2001), and GA2oxs are recognized as GA deactivation enzymes that convert GA precursors or bioactive GAs into inactive GAs by 2β-hydroxylation (Thomas et al 1999). Phylogenetic analysis based on protein sequences classified GA2oxs into three classes (Additional file: Fig. S1, Lee and Zeevaart 2005): class I and class II, whose members catalyze C19-GAs, are referred to as C19-type GA2oxs, and class III, whose members catalyze C20-GAs, are referred to as C20-type GA2oxs (Schomburg et al 2003; Lo et al 2008)
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