A β-ketoacyl-CoA Synthase OsCUT1 Confers Increased Drought Tolerance in Rice

doi:10.1016/j.rsci.2021.12.009

Abstract

Abstract:

Drought stress is one of the major environmental factors affecting crop growth and productivity. Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on leaf epidermis. In this study, we analyzed nine members (OsCUT1?OsCUT9) of β-ketoacyl-CoA synthase, the rate-limiting key enzyme for cuticular wax synthesis in rice by homology search and domain prediction. The expression levels of OsCUT genes under different abiotic stresses were investigated and OsCUT1 down-regulated by abiotic stress was selected for further function validation. Compared to the wild type, overexpression of OsCUT1 (OX-OsCUT1) exhibited significantly increased drought resistance. Epicuticular wax was increased on the leaf surface of OX-OsCUT1 and the chlorophyll leaching experiment showed that the cuticular permeability was decreased in the OX-OsCUT1 plants. Moreover, overexpression of OsCUT1 didn’t result in the significant changes of major agronomic traits. In total, these results suggested that OsCUT1 is a promising gene for engineering rice plants with enhanced drought tolerance.

Key words: rice, β-ketoacyl-CoA synthase, drought tolerance, cuticular wax, OsCUT1

Gao Xiuying, Zhang Ye, Zhang Hongsheng, Huang Ji. A β-ketoacyl-CoA Synthase OsCUT1 Confers Increased Drought Tolerance in Rice[J]. Rice Science, 2022, 29(4): 353-362.

Figures/Tables 7

Table 1. Details of nine CUT proteins in rice.

Gene name	MSU ID	RAP ID	ORF length (bp)	Protein length (aa)	Molecular weight (kDa)
OsCUT1	LOC_Os01g34560	Os01g0529800	1 437	479	52.2
OsCUT2	LOC_Os02g49920	Os02g0731900	1 458	486	53.3
OsCUT3	LOC_Os03g06705	Os03g0162800	1 143	308	33.8
OsCUT4	LOC_Os03g08360	Os03g0181500	1 629	543	60.1
OsCUT5	LOC_Os06g14810	Os06g0260500	1 479	493	54.3
OsCUT6	LOC_Os06g15020	Os06g0262200	1 476	492	54.0
OsCUT7	LOC_Os06g15170	Os06g0262800	1 485	495	54.3
OsCUT8	LOC_Os06g15250	Os06g0263400	1 515	505	54.8
OsCUT9	LOC_Os10g28060	Os10g0416200	822	274	30.3

Fig. 1. Phylogenetic tree of CUT family. The phylogenetic tree was constructed by MEGAX using the maximum likelihood method. Different subfamilies are highlighted with different colors.

Fig. 2. Gene structure, conserved motif and predicted cis-regulatory element in OsCUT genes. A, Distributions of conserved motifs in OsCUT genes. Ten putative motifs are indicated in different colored boxes. The sequence information of the 10 motifs was in Table S1. The right figure shows exon/intron organization of OsCUT genes. Green boxes represent exons and black lines with the same length represent introns. The upstream/downstream regions of OsCUT genes are indicated with yellow boxes. The length of exons can be inferred by the scale at the bottom. UTR, Untranslated region; CDS, Coding sequence. B, Predicted cis-regulatory element in OsCUT promoters. The sequence information of the 11 motifs was in Table S2. ARE, Elements for the anaerobic induction; LTR, Low-temperature-responsive element; ABRE, ABA-responsive element; MYB, MYB-binding site.

Fig. 3. Expression patterns of OsCUT1 after 20% PEG6000, 100 mmol/L NaCl, 0.1 mmol/L H2O2, 0.1 mmol/L abscisic acid (ABA), 4 ºC and 42 ºC treatments. The relative expression of OsCUT1 in different periods was verified by qRT-PCR. Total RNAs were extracted from rice seedlings of two-week-old plants. OsActin gene was used as a control. The transcript level at 0 h was defined as ‘1’. Data were Mean ± SE (n = 3), and compared by the Student’s t-test. *, P < 0.05; **, P < 0.01.

Fig. 4. Spatial expression pattern and subcellular location of OsCUT1. A, OsCUT1 expression in different tissues by reverse transcription semi-quantitative PCR. RAC1 gene was used as a control. B, OsCUT1 expression in different tissues by qRT-PCR. OsActin gene was used as a control. Data were Mean ± SE (n = 3), and compared by the Student’s t-test. **, P < 0.01. C, GUS expression patterns of OsCUT1:GUS transgenic rice plants. GUS activity was detected in different tissues: glume (a), root (b), lamina joint (c), stem (d), sheath (e) and leaf (f). D, Co-expression of OsCUT1-GFP fusion protein and ER-mCherry fusion protein in rice protoplasts was imaged by a confocal microscopy with a Zeiss LSM780 fitted with green (OsCUT1-GFP) and red filters (ER-mCherry). Scale bars, 5 μm. R, Root; S, Stem; L, Leaf; LS, Leaf sheath; P1, Panicle (~3 cm); P2, Panicle (~8 cm); P3, Panicle (~12 cm); GUS, β-Glucuronidase; GFP, Green fluorescent protein; ER, Endoplasmic reticulum.

Fig. 5. Phenotypes of OsCUT1 over- expression OX-OsCUT1 plants. A, Morphology of OX-OsCUT1 (OX3) and wild type Zhonghua 11 (ZH11) plants at the reproductive stage. Scale bars are 20 cm and 5 cm for the left and right figures, respectively. B?E, Quantification of plant height (B), panicle length (C), 1000-grain weight (D) and spikelet fertility (E) of the OX- OsCUT1 plants (OX3) compared with wild type ZH11. For quantification of plant height, panicle length and spikelet fertility, data were Mean ± SE (n = 10). For quantification of 1000-grain weight, data were Mean ± SE (n = 3). All data were compared by the Student’s t-test. F, Scanning electron microscopy of epicuticular wax on leaf surface of OX-OsCUT1 and wild type ZH11. Scale bars, 10 μm. G, OX-OsCUT1 transgenic plants in ZH11 background show significantly increased wax crystals on leaf surface compared to ZH11. Data were Mean ± SE (n = 3), and compared by the Student’s t-test. **, P < 0.01.

Fig. 6. Effects of drought stress and altered cuticular permeability in OsCUT1 overexpression plants (OX-OsCUT1). A, Two-month-old wild type (WT) Zhonghua 11 (ZH11) and OX-OsCUT1 transgenic plants before drought stress and after re-watering for 14 d. Scale bars, 10 cm. B, Three-week-old WT and OX-OsCUT1 transgenic plants before drought stress and after re-watering for 14 d. C, Survival rates of WT and OX-OsCUT1 transgenic plants for drought stress and re-watering for 14 d. D, Ratios of total chlorophyll extracted from WT and OX-OsCUT1. E, Water loss rates in WT and OX-OsCUT1 transgenic leaves. Data were Mean ± SE (n = 3), and compared by the Student’s t-test. *, P < 0.05; **, P < 0.01.

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