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. 2017 Jul;15(7):850-864.
doi: 10.1111/pbi.12681. Epub 2017 Mar 23.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice

Shuen-Fang Lo  1   2 , Tuan-Hua David Ho  2   3   4 , Yi-Lun Liu  1   2 , Mirng-Jier Jiang  1   2 , Kun-Ting Hsieh  5 , Ku-Ting Chen  1 , Lin-Chih Yu  1 , Miin-Huey Lee  6 , Chi-Yu Chen  6 , Tzu-Pi Huang  6 , Mikiko Kojima  7 , Hitoshi Sakakibara  7 , Liang-Jwu Chen  2   5 , Su-May Yu  1   2   4
Affiliations

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice

Shuen-Fang Lo et al. Plant Biotechnol J. 2017 Jul.

Abstract

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

Keywords: GA 2 oxidase 6; gibberellin; photosynthesis rate; plant architecture; rice; stress tolerance; yield.

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Figures

Figure 1
Figure 1
Three conserved motifs essential for function of C20 GA2oxs in controlling plant height. (a) Amino acid sequence alignment of C20 GA2oxs from different plant species. Roman numerals above the sequences indicate the three unique and conserved motifs present in C20 GA2oxs. Identical and conserved amino acid residues are highlighted in yellow and blue, respectively. Red underlines denote the conserved 30 amino acids in motifs I, II and III of C20 GA2oxs. Point mutations were introduced into the rice GA2ox6 (OsGA2ox6). Mutations that reduced or enhanced GA2ox6 impacts in transgenic rice are marked by red or blue squares, respectively. (b) Three‐month‐old T0 plants of nontransformed control (NT) and transgenic lines overexpressing various GA2ox6 mutants. The impact of GA2ox6‐WT on plant height in transgenic lines was set as 100%, and the impact of other GA2ox6 mutants was calculated relative to this value.
Figure 2
Figure 2
Ectopic expression of GA2ox6 and its mutants reduces plant height but increases tiller numbers in transgenic rice. Ninety‐five‐day‐old T2 transgenic plants were used in this study. (a) Morphology of NT and transgenic plants. Scale bar = 10 cm. Plant heights were quantified. (b) Tiller number. n = 18 for each line.
Figure 3
Figure 3
Ectopic expression of GA2ox6 and its mutants reduces the accumulation of GA precursors in transgenic lines. (a) Concentrations of GA precursors in NT and transgenic rice over‐expressing wild‐type GA2ox6 and its mutants. ND: nondetectable. (b) GA2ox6 is known to inactivate GA 53 in the GA biosynthesis pathway. n = 7, 8, 8, 8 for NT and lines A141E, G343A, and GA2ox6‐WT, respectively.
Figure 4
Figure 4
Root growth and WUE are significantly enhanced in GA‐deficient transgenic rice. (a) Morphology of 25‐day‐old plants, scale bar = 5 cm. (b) Shoot/root ratio. (c) WUE. n = 21 for each line.
Figure 5
Figure 5
Grain yield is significantly enhanced in GA‐deficient transgenic rice. (a) Morphology of rice plants near harvest and grain yield in ton per hectare in spring and fall, 2011, and in fall, 2013. (b) Harvest index in fall, 2013; 2011‐Spring: n = 10 for each line; 2011‐Fall: n = 18, 24, 20, 27 for NT and lines A141E, G343A and GA2ox6‐WT, respectively; 2013‐Fall: n = 32, 55, 55, 30 for NT and lines A141E, G343A and GA2ox6‐WT, respectively. ND: not‐determined.
Figure 6
Figure 6
Moderate GA deficiencies improve multiple agronomic traits. Measured parameters: total chlorophyll content in 25‐day‐old plants, maximal photosynthesis rate, number of productive tillers and numbers of fertile (filled) seeds per plant before harvest. n = 21 for each line.
Figure 7
Figure 7
Abiotic stress tolerance, proline contents and ROS scavenging enzymes are enhanced in GA‐deficient transgenic rice plants. Fourteen‐day‐old plants were used in following experiments. (a) Survival rates after recovery from various stress treatments, n = 120, 49, 64 and 73 for drought treatment; n = 124, 74, 58 and 85 for salt treatment; n = 117, 44, 58 and 85 for cold treatment; and n = 134, 74, 69 and 56 for heat treatment, for NT and lines A141E, G343A and GA2ox6‐WT, respectively. (b) Proline content, catalase (CAT) and ascorbate peroxidase (APX) activities and total peroxide contents in plants treated with or without dehydration. n = 6 for each line.
Figure 8
Figure 8
The elevated abiotic stress tolerance is associated with enhanced bulliform cell volume and water contents in GA‐deficient plants. Fourteen‐day‐old plants were used in following experiments. (a) Leaf morphology before and after 3.5‐h dehydration. Scale bar = 3 cm. Live images are also shown in Video S1. (b) Cross section of first fully expanded leaf under normal growth conditions. Scale bar = 100 μm. Arrow indicates bulliform cells. More leaf sections are also shown in Fig. S7. (c) Quantification of bulliform cell number, depth and area, n = 18, 14, 22, 14 for NT, A141E, G343A and GA2ox6‐WT, respectively. (d) Plant dry weight after dehydration and water contents before and after 3.5‐h dehydration. n = 21 for each line.
Figure 9
Figure 9
GA‐deficient transgenic rice is more resistant to pathogens. (a) Lesion expansion on leaves after infection by Xanthomonas oryze pv. oryzae. (b) Seedling weight after infection by Pythium arrhenomanes. (c) Upward migration of F. fujikuoi from shoot base. n = 10 for each line in each treatment.
Figure 10
Figure 10
Genes that are significantly up‐regulated contribute to increase in grain yield and abiotic and biotic stress tolerance in GA‐deficient transgenic rice. (a) Expression profiles of six groups of genes (number in parenthesis labelled in blue) related to grain yield and abiotic and biotic stress tolerance were compared in roots and shoots between A141E and G343A mutants and NT. Clustered genes up‐ and down‐regulated are marked in red and green, respectively. For detailed lists of genes in each cluster and extent of changes, see Tables S2 and S3. (b) Coordinated events and pathways leading to increase in grain yield and abiotic and biotic stress tolerance. Numbers denote up‐regulated genes corresponding to gene clusters in A. Red and green fonts indicate representative up‐ and down‐regulated genes, respectively. For abbreviation of gene names, see Materials and Methods. Small upward and downward arrowheads indicate up‐ and down‐regulation of genes. Open arrowheads indicate suggested sequences of events.
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