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. 2020 Sep;184(1):478-486.
doi: 10.1104/pp.20.00594. Epub 2020 Jul 13.

The Class III Gibberellin 2-Oxidases AtGA2ox9 and AtGA2ox10 Contribute to Cold Stress Tolerance and Fertility

Theo Lange  1 , Carolin Krämer  1 , Maria João Pimenta Lange  2
Affiliations

The Class III Gibberellin 2-Oxidases AtGA2ox9 and AtGA2ox10 Contribute to Cold Stress Tolerance and Fertility

Theo Lange et al. Plant Physiol. 2020 Sep.

Abstract

Many developmental processes in plants are regulated by GA hormones. GA homeostasis is achieved via complex biosynthetic and catabolic pathways. GA catabolic enzymes include GA 2-oxidases that are classified into three classes. Members of class III GA 2-oxidases typically act on GA precursors containing a C20-skeleton. Here, we identified two further members of this class of GA 2-oxidases, namely AtGA2ox9 and AtGA2ox10, in the Arabidopsis (Arabidopsis thaliana) genome. Both genes encode enzymes that have functional similarities to AtGA2ox7 and AtGA2ox8, which are class III GA 2-oxidases that 2β-hydroxylate C20-GAs. Previously unknown for GA 2-oxidases, AtGA2ox9 performs 2α-hydroxylation of C19-GAs and harbors putative desaturating activity of C20-GAs. Additionally, AtGA2ox9 and AtGA2ox10 exhibit GA 20-oxidase activity. AtGA2ox9 oxidizes carbon-20 to form tricarboxylic acid C20-GAs, whereas AtGA2ox10 produces C19-GA9 AtGA2ox9 transcript levels increase after cold treatment and AtGA2ox10 is expressed mainly in the siliques of Arabidopsis plants. Atga2ox9 loss-of-function mutants are more sensitive to freezing temperatures, whereas Atga2ox10 loss-of-function mutants produce considerably more seeds per silique than wild-type plants. We conclude that in Arabidopsis, AtGA2ox9 contributes to freezing tolerance and AtGA2ox10 regulates seed production.

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Figures

Figure 1.
Figure 1.
Enzymatic properties of class III GA 2-oxidases in Arabidopsis. A, Scheme of reactions catalyzed by the two identified multifunctional enzymes AtGA2ox9 (At5g58660) and AtGA2ox10 (At3g47190), in bold print. C19-GAs are highlighted by the blue background. All others are C20-GAs. B, HPLC radiochromatograms of products from incubation of recombinant AtGA2ox9 and AtGA2ox10 with the C20-GAs 14C-GA12, 14C-GA15, 14C-GA24, and 14C-GA25 and the C19-GAs 14C-GA9 and 14C-GA4 as substrates. Blue traces represent C19-GAs. Products were identified by GC-MS (Supplemental Table S1). Compounds a (putative didehydro-GA110) and b (putative didehydro-GA46) are referred to as “a” and “b,” respectively.
Figure 2.
Figure 2.
Catabolic function and sites of expression of class III GA 2-oxidases. A, Phenotype of transgenic plants overexpressing catabolic AtGA2ox9 and AtGA2ox10. Shown from left to right are representative 32-d-old plants transferred to the soil after 18 d in Murashige and Skoog medium: Col-0 and two overexpressor lines each for AtGA2ox9OE and AtGA2ox10OE. Scale bar = 5 cm. B, Catabolic class III GA2ox9 and GA2ox10 are expressed during seedling and seed development, respectively. Transcript levels of GA2ox7, GA2ox8, GA2ox9, and GA2ox10 genes were determined in different tissues by RT-qPCR (relative to SAND) during Arabidopsis development. Plotted data are means of three biological replicates ± se.
Figure 3.
Figure 3.
Analysis of AtGA2ox9 and AtGA2ox10 loss-of-function mutant plants. Phenotypic analysis of Atga2ox9-1 and Atga2ox10-1 mutants in comparison to wild-type Col-0 A, Plate-grown 7-d-old seedlings. B, Soil-grown 35-d-old plants. Plotted data are means of ≥15 plants, ≥90 siliques, and seeds from 20 siliques per genotype ± se. C, Representative 35-d-old plants (from left to right) Col-0, Atga2ox9-1, and Atga2ox10-1. D, Endogenous GA levels (means ± se) in siliques of 35-d-old Col-0 and Atga2ox10-1 plants. Shown are means of three biological replicates (ng·g−1 fresh weight). Statistical differences relative to Col-0 were determined by Student’s t test; *P < 0.05, **P < 0.01. Scale bar = 5 cm.
Figure 4.
Figure 4.
AtGA2ox9 contributes to freezing tolerance in Arabidopsis. A, Cold regulation of the Arabidopsis class III GA 2-oxidase genes. Transcript levels of GA2ox7, GA2ox8, GA2ox9, and GA2ox10 genes were determined by RT-qPCR (relative to SAND) in Arabidopsis 2-week-old plants subjected to cold treatment (4°C) for the times indicated or kept at 22°C (control). Data plotted are means ± se of three biological replicates. B, Endogenous GA levels (means ± se) in 14-d-old Col-0 and Atga2ox9-1 seedlings without or with cold treatment (12 h at 4°C). Shown are means of three biological replicates (ng·g−1 fresh weight). In some samples, endogenous GA was not detected (n.d.), but the internal standard was recovered. C, Fourteen-day-old Col-0 and Atga2ox9-1 plants were exposed to −5°C for 10 h, as described in “Materials and Methods,” and survival was scored after 14 d of recovery. Top, Representative Col-0 (left two pots) and Atga2ox9-1 (right two pots) plants after freezing treatment. Each pot contained five plants. Bottom, Quantification of the results, with data plotted as means ± se of four biological replicates (n = 36 for each replicate). Statistical differences relative to Col-0 were determined by Student’s t test; *P < 0.05, **P < 0.01. Scale bar = 5 cm.
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