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. 2019 Oct;5(10):1057-1065.
doi: 10.1038/s41477-019-0511-z. Epub 2019 Sep 16.

CYP72A enzymes catalyse 13-hydrolyzation of gibberellins

Juan He  1   2 , Qingwen Chen  1 , Peiyong Xin  3 , Jia Yuan  1 , Yihua Ma  1   2 , Xuemei Wang  1   2 , Meimei Xu  4 , Jinfang Chu  3 , Reuben J Peters  4 , Guodong Wang  5   6   7
Affiliations

CYP72A enzymes catalyse 13-hydrolyzation of gibberellins

Juan He et al. Nat Plants. 2019 Oct.

Erratum in

Abstract

Bioactive gibberellins (GAs or diterpenes) are essential hormones in land plants that control many aspects of plant growth and development. In flowering plants, 13-OH GAs (having low bioactivity-for example, GA1) and 13-H GAs (having high bioactivity-for example, GA4) frequently coexist in the same plant. However, the identity of the native Arabidopsis thaliana 13-hydroxylase GA and its physiological functions remain unknown. Here, we report that cytochrome P450 genes (CYP72A9 and its homologues) encode active GA 13-hydroxylases in Brassicaceae. Plants overexpressing CYP72A9 exhibited semi-dwarfism, which was caused by significant reduction in GA4 levels. Biochemical assays revealed that recombinant CYP72A9 protein catalysed the conversion of 13-H GAs to the corresponding 13-OH GAs. CYP72A9 was expressed predominantly in developing seeds in Arabidopsis. Freshly harvested seeds of cyp72a9 mutants germinated more quickly than the wild type, whereas stratification-treated seeds and seeds from long-term storage did not. The evolutionary origin of GA 13-oxidases from the CYP72A subfamily was also investigated and discussed here.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The GA biosynthesis pathway in Arabidopsis.
All enzymes mapped to the GA biosynthesis pathway were verified with enzymatic assays and chemical profiling of loss-of-function mutants. The dashed line indicates uncharacterized enzymatic step in Arabidopsis. The carbon backbone of GA12 is labeled with numbers, and the bioactive GAs (GA1 and GA4) are marked in red. CPS, ent-copalyl diphosphate synthase; GAMT, GA methyltransferase; GGPP, geranylgeranyl diphosphate; KAO, ent-kaurenoic acid oxidase; KO, ent-kaurene oxidase; KS, ent-kaurenoic acid synthase; MEP, 2-C-methyl-D-erythritol 4-phosphate.
Fig. 2
Fig. 2. Overexpression of CYP72A9 results in a dwarf phenotype and decreases endogenous GA4 levels.
a. GFPPS-sesterTPS-P450 gene cluster and phenotypes of Arabidopsis with increased expression level of each AtCYP72 (CYP72A7, A8, A9, A10, A11, A13, A14, and A15). All plants were grown under the same growth conditions and images were taken at 24 days after germination. This experiment was repeated at least three times with similar results. E.V., empty vector; Scale bar = 5 cm. b. Exogenous applied GA3 rescues the growth of ga1-t mutant and CYP72A9-overexpressing Arabidopsis. All one-week-old seedlings, which grown on ½ MS agar medium, were transferred onto ½ MS agar medium and grown for another week with or without 2 μM bioactive GA3. This experiment was repeated three times with similar results. WT, wild type. Scale bar = 1 cm. c. Profile of endogenous GAs in WT and two independent CYP72A9-overexpressing lines. The GA levels in the rosette leaves of 4-week-old Arabidopsis are presented as the means ± SDs (n = 3 biologically independent samples). **, significant difference from WT (P < 0.01; two-tailed Student’s t-test). N.D., not detectable; N.Q., detected, but not quantifiable due to low abundance.
Fig. 3
Fig. 3. CYP72A9 is a GA 13-hydroxylase.
CYP72A9 converted GA4, GA9, and GA12 to GA1, GA20, and GA53, as verified by comparison to authentic standards. Chromatogram of selected ions of m/z 546 for GA1, m/z 224 for GA4, m/z 270 for GA9, m/z 358 for GA12, m/z 476 for GA20, and m/z 239 for GA53. It is noteworthy that the y axis scale for each reaction is arbitrary for clarity, and the GA1 region has been amplified 20 times to allow easier visualization (inlet window). Control, yeast strain harboring pESC-Leu empty vector. This experiment was repeated at least three times with similar results.
Fig. 4
Fig. 4. Tissue-specific expression and subcellular localization of CYP72A9.
a. qRT-PCR analysis of CYP72A9 transcript levels in different tissues. Error bars represent the SD of three independent experiments. SR, root of 10-day-old seedlings; SL, leaf of 10-day-old seedlings; RO, root of mature plants; RL, rosette leaf; CL, cauline leaf; FL, flowers; GS, germinating seeds; DS, dry seeds; Silique samples were prepared followed the reference by Varbanova et al. At1g13320, At2g28390, and At4g34270 were used as reference genes in this analysis. The lowest level of CYP72A9 transcript in germinating seeds was set as 1.0. b. Histochemical GUS staining of siliques (stages 9 and 10) from Pro72A9:GUS transgenic plants. Scale bar = 0.2 mm. Silique samples had been stained for 24 h before imaging. This experiment was repeated two times with similar results. c. Subcellular localization of CYP72A9 in Arabidopsis leaf-mesophyll protoplasts. The ER was revealed by mCherry marker protein (Nelson et al., 2007). Scale bar = 5 μm. This experiment was repeated two times with similar results. d. Profiles of endogenous GAs in WT and two independent cyp72a9 mutants. The GA levels in the developing seeds/siliques of mature Arabidopsis are presented as the means ± SDs (n = 3 biologically independent samples). **, significant difference from WT (P < 0.01; two-tailed Student’s t-test); *, (P < 0.05; two-tailed Student’s t-test). N.D., not detectable; N.Q., detected, but not quantifiable due to low abundance.
Fig. 5
Fig. 5. Decreased primary seed dormancy of cyp72a9 mutants.
a. Germination of fresh harvested WT and cyp72a9 seeds on water-saturated filter paper without stratification treatment (4 °C for 3 days). Each data point represents the means ± SDs (n = 5 biologically independent experiments). b. Germination of fresh harvested WT and cyp72a9 seeds on water-saturated filter paper with stratification treatment (4 °C for 3 days). Each data point represents the means ± SDs (n = 5 biologically independent experiments). c. Germination of 6-month dry storage WT and cyp72a9 seeds on water-saturated filter paper without stratification treatment. Each data point represents the means ± SDs (n = 5 biologically independent experiments). d. Representative images of precocious germination tests of WT and cyp72a9 siliques. Pictures were taken 14 days after plating on ½MS medium.
Fig. 6
Fig. 6. Phylogenetic and biochemical analysis of CYP72A proteins from three Brassicaceae plants, rice, and soybean.
Gray color means no gene cloned in this study. GmCYP72A61 and GmCYP72A69, previously identified as triterpene oxidase, are indicated by black circles. Relative activity of various CYP72A proteins is expressed as the substrate conversion ratio (%). Values represent means from two independent experiments. It is noteworthy that the product of AtCYP72A15 and GmCYP72A135 using GA9 as the substrate is a mixture of GA20 and one unidentified hydroxylated GA9 (Supplementary Figs. 12 and 13). #, Inactivity with tested GA substrates might just reflect unsuccessful P450 protein expression in WAT11 yeast strain, which not detected by western blot (Supplementary Fig. 16).
Fig. 7
Fig. 7. Updated GA metabolism in developing seeds/silique of Arabidopsis.
CYP72A is highlighted in red. The arrow between GA19 and GA20 (in Arabidopsis pathway) is thinner than the others to show the low catalytic efficiency of this reaction in Arabidopsis, at least in developing seeds/silique. Uncharacterized oxidase(s) catalyzed the conversion from GA34 to GA8.
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