OsbZIP09, a Unique OsbZIP Transcription Factor of Rice, Promotes Rather Than Suppresses Seed Germination by Attenuating Abscisic Acid Pathway

doi:10.1016/j.rsci.2021.05.006

Abstract

Abstract:

We successfully identified a novel and unique OsbZIP transcription factor, OsbZIP09, whose mutants exhibited longer seeds and less severe pre-harvest sprouting than the wild type, but shared similar germination rate as the wild type under normal germination conditions. The expression of OsbZIP09 was induced by abscisic acid (ABA) and declined as the germination process. As a nucleus-localized transcription factor, the conserved binding motif of OsbZIP09 was identified via DNA affinity purification sequencing technique. Further evidences indicated that OsbZIP09 directly enhanced the expression of ABA catabolism gene ABA8ox1, thus reducing ABA accumulation. In addition, OsbZIP09 also directly bound to the promoter of LEA3 gene to inhibit its expression, thus further alleviating the suppressive effect of ABA on seed germination. These results demonstrated that OsbZIP09 likely functions as a brake of the ABA pathway to attenuate the inhibitory effect of ABA on rice seed germination via dual strategies.

Key words: OsbZIP09, pre-harvest sprouting, abscisic acid, seed germination, ABA8ox1 gene, LEA3 gene, rice, transcription factor

Chuxin Wang, Chengchao Zhu, Yu Zhou, Min Xiong, Jindong Wang, Huang Bai, Chenya Lu, Changquan Zhang, Qiaoquan Liu, Qianfeng Li. OsbZIP09, a Unique OsbZIP Transcription Factor of Rice, Promotes Rather Than Suppresses Seed Germination by Attenuating Abscisic Acid Pathway[J]. Rice Science, 2021, 28(4): 358-367.

Figures/Tables 11

Fig. 1. Knock-out of OsbZIP09 alleviates rice pre-harvest sprouting (PHS).A, Schematic diagram of the two targets in OsbZIP09 gene and the mutation information of the related mutants. The protospacer adjacent motif (PAM) is highlighted in blue, insert nucleotide is highlighted in red, and deleted nucleotides are shown with minus sign ‘-’. B, Germination phenotype of mature rice panicles after 6 d imbibition in water. Scale bar, 2 cm. ZH11, Zhonghua 11.C, Time-course analysis of germination rates of seeds in the panicles. Data are Mean ± SD (n = 30).

Fig. 2. Agronomic traits and normal seed germination assay of osbzip09 mutants.A, Morphology of osbzip09 mutants and its wild type Zhonghua 11 (ZH11). Scale bar, 10 cm. B?J, Plant height (B), heading date (C), tiller number per plant (D), panicle length (E), grain number per panicle (F), 1000-grain weight (G), grain shape (scale bar, 1 cm) (H), grain length (I) and grain width (J) of osbzip09 mutants and wild type. Data are Mean ± SD (n = 10 in B?G, and n = 30 in I and J). **, P < 0.01 (t-test).

Fig. S1. Time-course germination analysis of rice seeds.HAI, Hours after imbibition. Data are Mean ± SD (n = 3).

Fig. 3. Expression and sub-cellular localization analysis of OsbZIP09.A, Expression profiling of OsbZIP09 transcript abundance in developing seeds of rice. B, Time-course expression analysis of OsbZIP09 after imbibition. C, Expression analysis of OsbZIP09 in response to abscisic acid (ABA, 5 μmol/L) and gibberellins (GA, 10 μmol/L) treatments. D, Sub-cellular localization of OsbZIP09 (35S::OsbZIP09-GFP) in tobacco epidermal cells. Scale bars, 20 μm. Actin served as an internal reference gene for normalization in A?C. Data are Mean ± SD (n = 3). **, P < 0.01 (t-test).

Fig. S2. Expression analysis of ABA or GA responsive marker genes.A, Expression analysis of RAB16A, ABA8ox1 and ABA8ox2 in response to ABA (5 μmol/L) treatment.B, Expression analysis of GA20ox1 and GA2ox1 in response to GA (10 μmol/L). Actin served as an internal reference gene for normalisation in A and B. Data are Mean ± SD (n = 3). ** indicates significant difference (P < 0.01, t-test).

Fig. 4. Identification of OsbZIP09 binding motif and its targets involved in regulation of rice seed germination.A, Conserved motif identified from the DNA affinity purification sequencing (DAP-seq) data. B, Distribution of OsbZIP09 binding motifs in the 2-kb region preceding the initial codon (ATG) of ABA8ox1 and LEA3. C and D, Time-course expression analysis of ABA8ox1 and LEA3 after imbibition. Data are Mean ± SD (n = 3).E, Schematic program of the reporter and effector constructs used in the dual-luciferase reporter assay. F and G, OsbZIP09 activates the promoter activity of ABA8ox1 but suppresses the promoter activity of LEA3. Data are Mean ± SD (n = 5). ** indicates significant difference (P < 0.01, t-test).

Table S1. DAP-seq data related to ABA8ox1, LEA3 and GA3ox1.

Chromosome	Start	End	-log10(P-value)'	Strand	Distance_ to_TSS	Category	Gene ID	Gene name
5	26927280	26927615	7.60524	+	3	Promoter	Os05g0542500	OsLEA3; Similar to isoform 2 of late embryogenesis abundant protein 2C group 3.
2	28994350	28994666	8.95194	+	1052	Promoter	Os02g0703600	OsABA8ox1; Similar to abscisic acid 8'-hydroxylase 1. (Os02t0703600-01)
5	4663664	4663921	6.50438	-	-1100	Promoter	Os05g0178100	OsGA3ox1;3beta-hydroxylase GA metabolism (Os05t0178100-01)

Fig. 5. Expression analysis of ABA8ox1 and LEA3 in rice seeds before and after imbibition of abscisic acid (ABA).A and B, Expression analysis of ABA8ox1 (A) and LEA3 (B) in osbzip09 mutants and wild type Zhonghua 11 (ZH11) with or without ABA treatment. Actin served as an internal reference gene for normalization. Different lowercase letters indicate significant differences at P < 0.05 level based on one-way analysis of variance. C, ABA content in osbzip09 mutants and ZH11 before or at 12 h after imbibition (HAI).

Fig. S3. Expression analysis of GA3ox1 and quantification of GA3 content in rice seeds before and after imbibition. A, Expression analysis of GA3ox1 in osbzip09 mutants and ZH11. Actin served as an internal reference gene for normalisation. B, GA3 content in osbzip09 mutants and ZH11 before or at 12 h after imbibition (HAI). Data are Mean ± SD (n = 3). ** indicates significant difference (P < 0.01, t-test). NS, Not significant.

Fig. 6. Simplified model showing OsbZIP09 promotes rather than suppresses seed germination via reducing abscisic acid (ABA) accumulation and attenuating ABA-induction of downstream responsive genes in rice.

Table S2. Oligonucleotide primer sequences used.

Primer name	Sequence ( 5' → 3' )	Usage
OsbZIP09a-SG-seq	GTACAGCCTCACCCTCGACG	SG sequence-1 for editing OsbZIP09 SG sequence-2 for editing OsbZIP09
OsbZIP09b-SG-seq	AAGAGGAATGCCGAAACGGG
OsbZIP09seq-F	ATCTCGTCTCGATCATGGGG	Primers for sequencing and confirming mutation of OsbZIP09
OsbZIP09seq-R	CCCCAGCTTTGACCAGGAAA	Primers for sequencing and confirming mutation of OsbZIP09
bZIP09qRT-F	GAGGCATGTCAGGGGATGTG	For qRT-PCR analysis of OsbZIP09
bZIP09qRT-R	ACTTGGGTTCTGGTACAGGC	For qRT-PCR analysis of OsbZIP09
RAB16AqRT-F	ATGAGGGAGGAGCACAAGAC	For qRT-PCR analysis of RAB16A
RAB16AqRT-R	TTGATCCCCTTCTTCCTCCT
ABA8ox1qRT-F	AAGCTGGCAAAACCAACATC	For qRT-PCR analysis of ABA8ox1
ABA8ox1qRT-R	TGGATTCCGTATTAGCACGG
ABA8ox2qRT-F	GCGAGACGCTCCAGCTCT	For qRT-PCR analysis of ABA8ox2
ABA8ox2qRT-R	GGGCACCCCAGCAGATT
GA20ox1qRT-F	GCCACTACAGGGCCGACAT	For qRT-PCR analysis of GA20ox1
GA20ox1qRT-R	TGGTTGCAGGTGACGATGAT
GA2ox1qRT-F	TGACGATGATGACAGCGACAA	For qRT-PCR analysis of GA2ox1
GA2ox1qRT-R	CCATAGGCATCGTCTGCAATT	For qRT-PCR analysis of GA2ox1
LEA3qRT-F	TCACTTCAAATTCGGTGCAA	For qRT-PCR analysis of LEA3
LEA3qRT-R	CACACCCGTCAGAAATCCTC	For qRT-PCR analysis of LEA3
Actin1qRT-F	CCAAGGCCAATCGTGAGAAGA	Reference gene for qRT-PCR assay
Actin1qRT-R	AATCAGTGAGATCACGCCCAG
pZIP09-Smal-F	TCCCCCGGGATGGCGTCGAAGGCCGGAG	For subcellular assay of OsbZIP09
pZIP09-XbaI-R	GCTCTAGAGAAATCTGCGGAGCTTGTTC	For subcellular assay of OsbZIP09
pZIP09-EcoRI-F	CGGAATTCATGGCGTCGAAGGCCGGAG	For construction of OsbZIP09-62-SK
pZIP09-KpnI-R	GGGGTACCTCAGAAATCTGCGGAGCTTG	For construction of OsbZIP09-62-SK
proLEA3Kpn-F	GGGGTACCGTCGGCCACCCTCCGCAATA	For construction of LEA3pro-pGreenII
proLEA3PstI-R	AACTGCAGACCTGCCCCGCCTTCTCCTG	For construction of LEA3pro-pGreenII
proABA8ox1Kpn-F	GGGGTACCGTAGCCTGTTGGTAGCTTGG	For construction of ABA8ox1pro-pGreenII
proABA8ox1PstI-R	AACTGCAGCGCCTGCTCACTTCACTTAT	For construction of ABA8ox1pro-pGreenII
DAP-seq Adapter A	CACGACGCTCTTCCGATCT	For DAP-seq sampling
DAP-seq Adapter B	GATCGGAAGAGCACACGTCTG

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