SDF5 Encoding P450 Protein Is Required for Internode Elongation in Rice

doi:10.1016/j.rsci.2021.05.001

Abstract

Yachun Yang, Juan Li, Hao Li, Zuntao Xu, Ruiying Qin, Wenge Wu, Pengcheng Wei, Yong Ding, Jianbo Yang. SDF5 Encoding P450 Protein Is Required for Internode Elongation in Rice[J]. Rice Science, 2021, 28(4): 313-316.

Figures/Tables 11

Fig. 1. Characterization of sdf5A, Phenotypes of the wild type (WT) and the sdf5 mutant. B, Internodes of WT and the sdf5 mutant. C, Grain size of WT and the sdf5 mutant. D, Cell length of the fifth internode in WT and the sdf5 mutant. E, Diagram of T-DNA insertion in the sdf5 mutant. FP1, FP2, RP1, and RP2 indicate the primers used for genotyping. F, Transcripts of LOC_ Os05g34320 and LOC_Os05g34325. G, Phenotypes of wild type, sdf5 mutant and LOC_Os05g34325 overexpression lines. T11, T12 and T13 indicate different overexpression plants. H, Transcripts of SDF5 in wild type, sdf5, T11, T12 and T13. Data are Mean ± SD (n = 3). * and ** indicate significant differences at the 0.05 and 0.01 levels by the Student’s t-test, respectively.

Fig. S1. Agronomic traits of wild type (WT) and sdf5 mutant. A, Tiller number. B, Total grain number per panicle. C, Empty grain number per panicle. D, Full grain number per panicle. E, Grain fertility rate. F, 1000-grain weight; G, Grain length. H, Grain width. I, Internode length. All agronomic character surveys were conducted after the rice harvest. Data are Mean ± SD (n = 3). * and ** indicate significant differences at the 0.05 and 0.01 levels by the Student’s t-test, resppectively.

Table S1. Genetic analysis of SDF5.

Generation	Total number of plants	Number of normal plants	Number of semi-dwarf	χ²
T₁	21	7	14	0.38
T₂	420	178	242	0.07

Fig. S2. Segregation of SDF5 in F2 population with self-pollilation. FP1 and RP1 indicate the left primer and right primer on T-DNA border; FP2 is a primer of the Psuper primer; RP2 is a primer in the HPT sequence. Lanes 1 and 2 are wild type. Lanes 3 to 26 are SDF5. ‘-’ indicates the blank. M, Marker.

Table S2. Primers used for gene over-expression, PCR and RT-PCR analysis.

Primer name	Primer sequence (5'-3')	Primer name	Primer sequence (5'-3')
FP1	CAATAAGGAGCAGGGCCCAGT	SDF5-qRT-F	ATTGCATCGCTCTCCTAGGGC
FP2	AGGACGACCAATGTATGTTATG	SDF5-qRT-R	GCCGGATTGCTGAAGGCGACG
RP1	ACAAGTATGGAAACTACTAGA	LRT2-qRT-F1	CTACAAGGGGAGCACCTTCCA
RP2	CATGTCATATAGTTTCCATAGC	LRT2-qRT-F2	TGTACGCGAAGGACGTGCCG
34325F CDS 1300 KpnI	GGTACCATGGAGTTAACTAGTAGCAGTGCCA	LRT2-qRT-R1/2	GAAGAACTGGGACCCGTTAGT
34325R CDS 1300 XbaI	CTCTAGAAGAGGTAGGAAGCAATTGCCTTCT	OsActin2-qRT-F	TACAGTGTCTGGATAGGAGGGTC
34320F CDS 1300 BamHI	GGATCCATGGCGCCAGCGATGGCTCTGAGGT	OsActin2-qRT-R	ACCAACAATCCCAAACAGAGTAG
34320R CDS 1300 XbaI	CTCTAGCTGCTTTATACAGGAGCTTGGCTCT	OsIAA1-qRT-F	CGAGGCCTACTCCGGCTACGAC
SDF5F CDS GFP XbaI	CTCTAGATGGAGTTAACTAGTAGCAGTGCCA	OsIAA1-qRT-R	CGCTGGCAAGTTTCCACAAACA
SDF5R CDS GFP KpnI	GGTACCAAGAGGTAGGAAGCAATTGCCTTCT	OsIAA5-qRT-F	GAACTACCGAAAGAACACGCTG
OsKO2-qRT-F	TGCTACCAGCGACTATTGTGATTT	OsIAA5-qRT-R	TTTGATGATTTACGCAAACCGC
OsKO2-qRT-R	GTGCAGAAGTACCCAACATGCTT	OsIAA10-qRT-F	ATGAGAGGAGGAGTAGCTGGGC
OsKO-qRT-F	CTTCCTCCATCATTTTCTCC	OsIAA10-qRT-R	TCAGGATCTGCCTCTTGTTG
OsKO-qRT-R	AAGCAGTTGTCCACAGGC	OsIAA11-qRT-F1	TGTTCACCTCCTTCACCAT
OsGA20ox2-qRT-F	CCAATTTTGGACCCTACCGC	OsIAA11-qRT-F2	CCGAGACCATCGACCTCAA
OsGA20ox2-qRT-R	GAGAGAAGCCCAACCCAACC	OsIAA11-qRT-R1	GCATTTCTCCACTGCCCTT
OsGA2ox1-qRT-F	CGTCTTGTAGATGGTGGTGC	OsIAA11-qRT-R2	CGGCCACCCCACCACCTGC
OsGA2ox1-qRT-R	CCTGCCTGATGAGTTAGAAAAG	OsIAA12-qRT-F	GCAACAACAAAGACGAGCCCAT
OsGA2ox3-qRT-F	TGGTGGCCAACAGCCTAAAG	OsIAA12-qRT-R	CCTTGTCCTGGTAGGTGATGGC
OsGA2ox3-qRT-R	TGGTGCAATCCTCTGTGCTAAC	OsIAA13-qRT-F	AGCATGGACGGGGCACCCTACCT
GID1-qRT-F	GCCCATCCTTGAGTTCCTGAC	OsIAA13-qRT-R	TCTGCCACGGGACATCTCCAACA
GID1-qRT-F	GTCCTGGAGCGTCACGAAGTA	OsIAA14-qRT-F	GAGGAGGGCTGTACGTGAAGGTG
GA3OX2-qRT-F	CGGACTCGGGCTTCTTCACC	OsIAA14-qRT-R	AGAGATGAACATGTCCCAGGGCA
GA3OX2-qRT-R	AGGAAGTAGCCGAGCGAGACCC	OsIAA16-qRT-F	GACGACGACGACGAGGCGAGGT
GA2OX1-qRT-F	TACCTGCTCCTCCACGCCAACC	OsIAA16-qRT-R	GCCGCCGGAGAAGCAGAGGAACAT
GA2OX1-qRT-R	CACTGTCAGCGTTTGTGGTAAG	OsIAA20-qRT-F	CGGTGGGTTCAAAACCGACGA
KO1-qRT-F	AAGCAGCAGAAGGAAAGG	OsIAA20-qRT-R	GCTCAGCGTATGAGCCGAGGA
KO1-qRT-R	TGGTAAAGCCGTTCCTGT	OsIAA22-qRT-F	TCATCAGCTCAGCTGGTGGGATG
OsGID2-qRT-F	GGGAAATGACAGTGATAAGAATGGA	OsIAA22-qRT-R	AGAGCCTGGGACAGCGAGTCGTA
OsGID2-qRT-R	TGATGCTTGTTGGAGTGAAGCT	OsIAA30-qRT-F	AAGATGTACAAGAGCTACCTTGA
OsSLR1-qRT-F	GACGTCAACGAACGCTCAATT	OsIAA30-qRT-R	TTCTGATCCTTTCATTATCCTAA
OsSLR1-qRT-R	CGGAGTCCAGTCGTCGATCT	OsIAA31-qRT-F	CGGAGACGCAGCAGAAGGAGGATG
OsActin-F	GCCTTGGCAATCCACATC	OsIAA31-qRT-R	GTAGGTGACGGCGAAGTCGGACGG
OsActin-R	AGCATGAAGATCAAGGTGGTC

Table S2. Primers used for gene over-expression, PCR and RT-PCR analysis.

Primer name	Primer sequence (5'-3')	Primer name	Primer sequence (5'-3')
FP1	CAATAAGGAGCAGGGCCCAGT	SDF5-qRT-F	ATTGCATCGCTCTCCTAGGGC
FP2	AGGACGACCAATGTATGTTATG	SDF5-qRT-R	GCCGGATTGCTGAAGGCGACG
RP1	ACAAGTATGGAAACTACTAGA	LRT2-qRT-F1	CTACAAGGGGAGCACCTTCCA
RP2	CATGTCATATAGTTTCCATAGC	LRT2-qRT-F2	TGTACGCGAAGGACGTGCCG
34325F CDS 1300 KpnI	GGTACCATGGAGTTAACTAGTAGCAGTGCCA	LRT2-qRT-R1/2	GAAGAACTGGGACCCGTTAGT
34325R CDS 1300 XbaI	CTCTAGAAGAGGTAGGAAGCAATTGCCTTCT	OsActin2-qRT-F	TACAGTGTCTGGATAGGAGGGTC
34320F CDS 1300 BamHI	GGATCCATGGCGCCAGCGATGGCTCTGAGGT	OsActin2-qRT-R	ACCAACAATCCCAAACAGAGTAG
34320R CDS 1300 XbaI	CTCTAGCTGCTTTATACAGGAGCTTGGCTCT	OsIAA1-qRT-F	CGAGGCCTACTCCGGCTACGAC
SDF5F CDS GFP XbaI	CTCTAGATGGAGTTAACTAGTAGCAGTGCCA	OsIAA1-qRT-R	CGCTGGCAAGTTTCCACAAACA
SDF5R CDS GFP KpnI	GGTACCAAGAGGTAGGAAGCAATTGCCTTCT	OsIAA5-qRT-F	GAACTACCGAAAGAACACGCTG
OsKO2-qRT-F	TGCTACCAGCGACTATTGTGATTT	OsIAA5-qRT-R	TTTGATGATTTACGCAAACCGC
OsKO2-qRT-R	GTGCAGAAGTACCCAACATGCTT	OsIAA10-qRT-F	ATGAGAGGAGGAGTAGCTGGGC
OsKO-qRT-F	CTTCCTCCATCATTTTCTCC	OsIAA10-qRT-R	TCAGGATCTGCCTCTTGTTG
OsKO-qRT-R	AAGCAGTTGTCCACAGGC	OsIAA11-qRT-F1	TGTTCACCTCCTTCACCAT
OsGA20ox2-qRT-F	CCAATTTTGGACCCTACCGC	OsIAA11-qRT-F2	CCGAGACCATCGACCTCAA
OsGA20ox2-qRT-R	GAGAGAAGCCCAACCCAACC	OsIAA11-qRT-R1	GCATTTCTCCACTGCCCTT
OsGA2ox1-qRT-F	CGTCTTGTAGATGGTGGTGC	OsIAA11-qRT-R2	CGGCCACCCCACCACCTGC
OsGA2ox1-qRT-R	CCTGCCTGATGAGTTAGAAAAG	OsIAA12-qRT-F	GCAACAACAAAGACGAGCCCAT
OsGA2ox3-qRT-F	TGGTGGCCAACAGCCTAAAG	OsIAA12-qRT-R	CCTTGTCCTGGTAGGTGATGGC
OsGA2ox3-qRT-R	TGGTGCAATCCTCTGTGCTAAC	OsIAA13-qRT-F	AGCATGGACGGGGCACCCTACCT
GID1-qRT-F	GCCCATCCTTGAGTTCCTGAC	OsIAA13-qRT-R	TCTGCCACGGGACATCTCCAACA
GID1-qRT-F	GTCCTGGAGCGTCACGAAGTA	OsIAA14-qRT-F	GAGGAGGGCTGTACGTGAAGGTG
GA3OX2-qRT-F	CGGACTCGGGCTTCTTCACC	OsIAA14-qRT-R	AGAGATGAACATGTCCCAGGGCA
GA3OX2-qRT-R	AGGAAGTAGCCGAGCGAGACCC	OsIAA16-qRT-F	GACGACGACGACGAGGCGAGGT
GA2OX1-qRT-F	TACCTGCTCCTCCACGCCAACC	OsIAA16-qRT-R	GCCGCCGGAGAAGCAGAGGAACAT
GA2OX1-qRT-R	CACTGTCAGCGTTTGTGGTAAG	OsIAA20-qRT-F	CGGTGGGTTCAAAACCGACGA
KO1-qRT-F	AAGCAGCAGAAGGAAAGG	OsIAA20-qRT-R	GCTCAGCGTATGAGCCGAGGA
KO1-qRT-R	TGGTAAAGCCGTTCCTGT	OsIAA22-qRT-F	TCATCAGCTCAGCTGGTGGGATG
OsGID2-qRT-F	GGGAAATGACAGTGATAAGAATGGA	OsIAA22-qRT-R	AGAGCCTGGGACAGCGAGTCGTA
OsGID2-qRT-R	TGATGCTTGTTGGAGTGAAGCT	OsIAA30-qRT-F	AAGATGTACAAGAGCTACCTTGA
OsSLR1-qRT-F	GACGTCAACGAACGCTCAATT	OsIAA30-qRT-R	TTCTGATCCTTTCATTATCCTAA
OsSLR1-qRT-R	CGGAGTCCAGTCGTCGATCT	OsIAA31-qRT-F	CGGAGACGCAGCAGAAGGAGGATG
OsActin-F	GCCTTGGCAATCCACATC	OsIAA31-qRT-R	GTAGGTGACGGCGAAGTCGGACGG
OsActin-R	AGCATGAAGATCAAGGTGGTC

Fig. S3. Phylogenetic tree of CYP51 proteins. Cluster analysis using the protein sequences of the CYP51 proteins of rice, yeast, human and Arabidopsis thaliana aligned by ClustalW. The relationships of the sequences were examined by MEGA-X.

Fig. S4. Expression pattern of SDF5. Total RNA was isolated from 8-week-old seedlings. Data are Mean ± SD (n = 3).** indicate significant difference by Student’s t-test. P < 0.01.

Fig. S5. Wild type (WT) and sdf5 mutants treated with gibberellic acid (GA3), paclobutrazol (PAC) and brassinosteriod (BR). A and C, Plant height with GA3 treatment. B and D, Plant height with PAC treatment. Red frame marks seedling death. E, Phonetype of wild type and sdf5 treated with BR and benzoyl-CoA ligase (BZL). F, Plant height of WT and sdf5 mutant with BR and BZL treatments. 15-day-old seedlings were subjected to 10 d. Data are Mean ± SD (n = 3). * and ** indicate significant differences at the 0.05 and 0.01 levels by the Student’s t-test, resppectively. CK, Control.

Fig. 2. Gibberellin (GA) compounds were tested in wild type (WT) and sdf5 mutant. A, Phenotypes of WT and sdf5 mutant treated with different concentrations of GA4. Fifteen-day-old seedlings were subjected to 10 d of homone culture and their plant heights were measured. CK, Control. B, Height of WT and sdf5 seedlings in different concentrations of GA4. C, Endogenous levels of GAs were measured in WT and sdf5 mutant. ND indicates not detectable. Data are Mean ± SD (n = 3). * and ** indicate significant differences at the 0.05 and 0.01 levels by the Student’s t-test, respectively.

Fig. S6. Transcriptional analysis of GA genes in wild type (WT) and sdf5 mutant. Total RNA was isolated from 2-week-old seedlings. Data are Mean ± SD (n = 3). * indicate significant differences at the 0.05 level by the Student’s t-test.

Fig. S7. Indoleacetic acid (IAA) analysis in wild type (WT) and sdf5 mutant. A, IAA content in WT and sdf5 mutant. IAA content was tested with 2-week-old seedlings. B, Transcript level of IAA genes in sdf5 mutant. Total RNA was isolated from 2-week-old seedlings. Data are Mean ± SD (n = 3).

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