OsABT, a Rice WD40 Domain-Containing Protein, Is Involved in Abiotic Stress Tolerance

doi:10.1016/j.rsci.2021.07.012

Abstract

Abstract:

Plant growth and crop productivity are severely affected by abiotic stress on a global scale. WD40 repeat-containing proteins play a significant role in the development and environmental adaptation of eukaryotes. In this study, OsABT, a stress response gene, was cloned from rice (Oryza sativa L. cv. Nipponbare). OsABT encodes a protein containing seven WD40 domains. Expression analysis revealed that the OsABT gene was first up-regulated and then down-regulated following treatment with abscisic acid (ABA) and NaCl, but was down-regulated when treated with PEG8000. Subcellular localization results showed that OsABT was located in the cytoplasm and nucleus of Arabidopsis roots. OsABT transgenic Arabidopsis showed significantly increased tolerance to ABA and salt stress during plant seedling development. However, the transgenic lines were more sensitive to drought stress. Moreover, OsABT can interact with OsABI2, a component of ABA signaling pathway. These results showed that OsABT plays a positive regulatory role in response to salt stress and a negative role in response to drought stress in Arabidopsis.

Key words: abscisic acid, Arabidopsis, drought stress, rice, salt stress, WD40 domain-containing protein

Chen Eryong, Shen Bo. OsABT, a Rice WD40 Domain-Containing Protein, Is Involved in Abiotic Stress Tolerance[J]. Rice Science, 2022, 29(3): 247-256.

Figures/Tables 8

Fig. 1. Protein sequence analysis of OsABT and phylogenetic relationships of OsABT and its homologous proteins in plants. A, Multiple sequence alignment of OsABT and AtABT. WD-1 to WD-7 show the amino acid sequences of seven tryptophan-aspartate (WD) domains. B, Domain analysis of OsABT protein. The pink solid rectangle represents low compositional complexity, and the ruler represents the number of amino acids. C, Phylogenetic relationships of OsABT and its homologs in other plant species. The phylogenetic tree was generated in MEGA 6.0. Bootstrap values were 1 000 replications.

Fig. 2. Gene structure of OsABT and subcellular localization of OsABT protein. A, Comparison of gene structure between OsABT and AtABT. B, Protein location analysis of OsABT in Arabidoposis roots using confocal laser scanning microscopy. The three top images show free green fluorescence protein (GFP) signal, and the three subsequent images show GFP-OsABT fusion proteins. Scale bars, 20 μm.

Table 1. cis-elements that respond to abscisic acid (ABA) and stress in OsABT promoter.

cis-element	Sequence	Position from ATG	Biological function
ABRELATERD1	ACGTG	-1851, -1321	ABA responsive element
ABRERATCAL	MACGYGB	-1694	ABA responsive element
EBOXBNNAPA	CANNTG	-1264, -1089, -781, -762, -590	ABA responsive element
DRE2COREZMRAB17	ACCGAC	-232	ABA responsive element, dehydration-responsive element
PROXBBNNAPA	CAAACACC	-846	ABA responsive element
GT1CONSENSUS	GRWAAW	-515	Salt responsive element
MYB1AT	WAACCA	-459, -401	Dehydration-responsive element
MYB2AT	TAACTG	-353	Dehydration-responsive element
MYB2CONSENSUSAT	YAACKG	-1669, -353, -169	Dehydration-responsive element
MYCCONSENSUSAT	CANNTG	-1827, -1264, -1089, -781, -762, -590	Dehydration-responsive element
DRECRTCOREAT	RCCGAC	-1581, -880, -232	Dehydration-responsive element
CBFHV	RYCGAC	-1581, -880, -232	Dehydration-responsive element
ACGTATERD1	ACGT	-1858, -1852, -1322, -995	Required for etiolation-induced expression of erd1 (early responsive to dehydration)
LTRECOREATCOR15	CCGAC	-1581, -1566, -1035, -880, -840, -232	Low-temperature-responsive element
LTRE1HVBLT49	CCGAAA	-517	Low-temperature-responsive element
CCAATBOX1	CCAAT	-876, -377	Heat stress response element
BIHD1OS	TGTCA	-1556, -653, -336	Binding site of OsBIHD1 in disease resistance response

Fig. 3. Expression profiles of OsABT under stress. A?C, Expression patterns of OsABT in response to abscisic acid (ABA) (A), NaCl stress (B) and PEG8000 treatment (drought simulation) (C). RNA was extracted from rice roots at 0 (control), 1, 3, 6, 12 and 24 h after treatments with ABA (50 μmol/L), NaCl (150 mmol/L) and PEG8000 (10 g/mL). OseEF-1α was used as an internal reference. Data are Mean ± SD (n = 3).

Fig. 4. Response of transgenic OsABT Arabidopsis lines (1-5 and 4-5) to abscisic acid (ABA). A, OsABT was detected in Col-0 and transgenic lines of Arabidopsis, with AtUBQ10 as an internal reference. B, Analysis of the greening rate of transgenic OsABT Arabidopsis on Murashige and Skoog (MS) medium and MS medium containing ABA (1 μmol/L). C, Statistic analysis of greening rate of transgenic lines at 14 d after germination. Data are Mean ± SD (n = 3). Asterisks represent significant differences between transgenic Arabidopsis lines and Col-0 plants (Student’s t-test, *, P < 0.05; **, P < 0.01).

Fig. 5. Response of transgenic OsABT Arabidopsis to salt stress. A, Greening rate analysis of transgenic lines 1-5 and 4-5 on Murashige and Skoog (MS) medium and MS medium containing NaCl (125 mmol/L). B, Statistical analysis of greening rate of transgenic lines 1-5 and 4-5 at 5, 6, 7 and 8 d after germination, respectively. Data are Mean ± SD (n = 3). Significant differences compared with Col-0 (Student’s t-test): *, P < 0.05; **, P < 0.01.

Fig. 6. Response of transgenic Arabidopsis overexpression OsABT lines (1-5 and 4-5) to drought stress. A, Eleven-day-old seedlings of Col-0 and transgenic Arabidopsis overexpression lines were transplanted into different pots and grew for 14 d (before drought stress). Then, water was withheld for 2 weeks (after drought stress), after which the plants were rewatered for 1 week (after water recovery). The photos were taken at the time of before drought stress, after drought stress and after water recovery, respectively. B, Survival rates of transgenic lines 1-5 and 4-5 after drought stress compared with Col-0, respectively. Three independent biological replicates were applied. Data are Mean ± SD (n = 48, Student’s t-test, *, P < 0.05).

Fig. 7. Interaction between OsABT and OsABI2 as measured via bimolecular fluorescent complimentary assay. OsABT-YFPN and OsABI2-YFPC were co-localized in Nicotiana benthamiana leaf cells. Scale bars, 20 μm.

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