A Fragment Substitution in Promoter of MS92/PTC1 Causes Male Sterility in Rice

doi:10.1016/j.rsci.2020.03.004

Abstract

Abstract:

Persistent tapetal cell1 (PTC1) plays a curial role in pollen development, and is thought to function as a transcriptional activator in rice. However, the molecular mechanism of PTC1 in regulating pollen development and its cis-elements are not well understood. We identified a novel weak male sterility mutant (ms92) which exhibited expanded tapetum and shrink pollen grains. Map-based cloning and allelic analysis suggested that the male sterility of ms92 was caused by a DNA fragment substitution in the promoter of PTC1. The decreased expression of MS92/PTC1 in ms92 and cis-element analysis indicated that the substituted sequence contained several potential binding cis-element of negative feedback. MS92/PTC1 was specifically expressed in tapetum and microspores at the young microspore stage, and its protein was localized in nucleus. We further found that MS92/PTC1 functions as a transcription activator by recognizing H3K4me3. Transcriptomic analysis revealed that a number of genes involved in tapetum degeneration and pollen wall formation were down-regulated in ms92, which are the potential targets of MS92/PTC1. The substitution fragment in MS92/PTC1 promoter was essential for pollen development, and we provided a novel mutant for further identifying the cis-elements in promoter and the molecular network of MS92/PTC1.

Key words: rice, MS92/PTC1, plant homeodomain finger, cis-element, male sterility, anther

Peng Qin, Luchang Deng, Weilan Chen, Juan Huang, Shijun Fan, Bin Tu, Jun Tan, Hua Yuan, Yuping Wang, Bingtian Ma, Shigui Li. A Fragment Substitution in Promoter of MS92/PTC1 Causes Male Sterility in Rice[J]. Rice Science, 2020, 27(5): 396-404.

Figures/Tables 15

Supplemental Table 1 Primers used in this study.

Name	Primer sequence	Purpose	Gene symbol
RM24431	CTTACTTAGCACGTGCCTGTTGC	Map-based cloning
RM24431	CATTGGGATCATGCATCTCTGC
RM3600	TGCCCACACATGATGAGC
RM3600	AACGGGCAAGAGATCTTCTG
RM7424	CAGATCAAGCTAGCCACACAGC
RM7424	GAAGGCAGAGCAGGAGAGAAGC
RM7048	CGTGAAAGTGACGAGTTTCAGTCC
RM7048	CGAAGTGAACATGGCACAAACC
Promoter-lost	GTGGGGGACACTCTTGGCTGACA
Promoter-lost	GGGTAGCCGGGCTGGCAGAA
MS92-GFP F	TCCCCCCGGGATGGCGCCTAAGATGGTGATCAG	Vector construction
MS92-GFP R	GGACTAGTACAGTTGAAGGACGGGAACG
MS92-BD F	CGGAATTCATGGCGCCTAAGATGGTGATCAG
MS92-BD R	TTTGGATCCACAGTTGAAGGACGGGAACG
MS92ΔN1-BD F	CGGAATTCATGGCGCCTAAGATGGTGATCAG
MS92ΔN1-BD R	TTTGGATCCGCAGAGCGGCATGGAGCCGAG
MS92ΔN2-BD F	CACCGAATTCATGGCGCCTAAGATGGTGA
MS92ΔN2-BD R	TTTGGATCCGCGCTCGCCGCCGCCGCTCCCCT
MS92ΔN3-BD F	CACCGAATTCGTGCTGGTGCCGCACCTGTCGTGCT
MS92ΔN3-BD R	TTTGGATCCACAGTTGAAGGACGGGAACG
MS92ΔC1-BD F	CACCGAATTCGTCGTGGACTGCGCGTGCGG
MS92ΔC1-BD R	TTTGGATCCACAGTTGAAGGACGGGAACGACACGACG
MS92ΔC2-BD F	ACGCCATGCGCTTGCCGTCGTCGTC
MS92ΔC2-BD R	GACGACGACGGCAAGCGCATGGCGT
MS92ΔC3-BD F	GTGCTGCCACGCCTCCGCGATGTCGCAGCACGC
MS92ΔC3-BD R	GCGTGCTGCGACATCGCGGAGGCGTGGCAGCAC
MS92ΔC4-BD F	CCCGGCGCCCCGCGTGTTCTGCCACG
MS92ΔC4-BD R	CGTGGCAGAACACGCGGGGCGCCGGG
MS92 PHD GST F	TTTGGATCCGTCGTGGACTGCGCGTGCGG
MS92 PHD GST R	CCGGAATTCCGGACAGTTGAAGGACGGGAACG
RNA in situ F	CGTTGATTGGCAGCAACTAGC
RNA in situ R	CATATCAGATGCCTCCCCCAC
MS92 qPCR-F	GGCATCCCCACATCAACCT	qRT-PCR analysis
MS92 qPCR-R	ATCTCGCTGAACACCCTCT
UBQ qPCR-F	GGAAGTAAGGAAGGAGGAG
UBQ qPCR-R	CAGAGGTGATGCTAAGGT
LOC_Os04g57490 qPCR-F	CAAGGAGTATCCCGTCTGCAA		OsCP1
LOC_Os04g57490 qPCR-R	AGAGATTGAATCAGGTTGTTCCG
LOC_Os07g05150 qPCR-F	AAGGAGGTTGCCGACGACAC
LOC_Os07g05150 qPCR-R	CGAAGCTCTTCAGCTCCTTCTC
LOC_Os06g02019 qPCR-F	CTGACCCCAAAATGTTCAACCC		OsKAO
LOC_Os06g02019 qPCR-R	CTCACCCTGCACTTTGGATTTG
LOC_Os06g16300 qPCR-F	CTGATCGAGATCAAGAACCTGCT
LOC_Os06g16300 qPCR-R	AGAATAGTCTCAAGATTGGTCAGGT
LOC_Os04g51070 qPCR-F	TGAGGACGAACGAGATGTGATT		EAT1
LOC_Os04g51070 qPCR-R	TCACCTACTATTGAGGCCCTGT
LOC_Os09g27940 qPCR-F	CCACAGCTGCGTGAGGCCCGACACC
LOC_Os09g27940 qPCR-R	TCATCGTGCCGCAGCCGAACCCGAG
LOC_Os08g43290 qPCR-F	GAACAGGCTGCTGGCGTGCCGCGCGTA		OsC4
LOC_Os08g43290 qPCR-R	AGCAGTTGATCTGGCTGAGGTGGCAT
LOC_Os09g10260 qPCR-F	ATGTCGAAGCTAGTGAGCAACA
LOC_Os09g10260 qPCR-R	TTGTCGACATCGATGAAGCTGG
LOC_Os07g05180 qPCR-F	CGACACAATCCTCCTAATCAGC
LOC_Os07g05180 qPCR-R	GTTCTTGTCCACATCGATGAAC
LOC_Os07g23570 qPCR-F	CTCCACCTTCCTCACTGTTCAA
LOC_Os07g23570 qPCR-R	CAACCCAAGTAGGTCATTCCCA
LOC_Os10g20470 qPCR-F	TGTTCTTGCTTCGCAATGTCTG
LOC_Os10g20470 qPCR-R	ATGCCTACAAGGTAGAATGCCC
LOC_Os01g34970 qPCR-F	AACATTGCTGAACAGACTATTG
LOC_Os01g34970 qPCR-R	AACACCTTCTTGTAGAGTAGATT
LOC_Os07g42490 qPCR-F	GCTGGAAACAAGGAAACTGTGG
LOC_Os07g42490 qPCR-R	GAGGTGAGCCTCTTCTGTGATT

Supplemental Table 1 Primers used in this study.

Name	Primer sequence	Purpose	Gene symbol
RM24431	CTTACTTAGCACGTGCCTGTTGC	Map-based cloning
RM24431	CATTGGGATCATGCATCTCTGC
RM3600	TGCCCACACATGATGAGC
RM3600	AACGGGCAAGAGATCTTCTG
RM7424	CAGATCAAGCTAGCCACACAGC
RM7424	GAAGGCAGAGCAGGAGAGAAGC
RM7048	CGTGAAAGTGACGAGTTTCAGTCC
RM7048	CGAAGTGAACATGGCACAAACC
Promoter-lost	GTGGGGGACACTCTTGGCTGACA
Promoter-lost	GGGTAGCCGGGCTGGCAGAA
MS92-GFP F	TCCCCCCGGGATGGCGCCTAAGATGGTGATCAG	Vector construction
MS92-GFP R	GGACTAGTACAGTTGAAGGACGGGAACG
MS92-BD F	CGGAATTCATGGCGCCTAAGATGGTGATCAG
MS92-BD R	TTTGGATCCACAGTTGAAGGACGGGAACG
MS92ΔN1-BD F	CGGAATTCATGGCGCCTAAGATGGTGATCAG
MS92ΔN1-BD R	TTTGGATCCGCAGAGCGGCATGGAGCCGAG
MS92ΔN2-BD F	CACCGAATTCATGGCGCCTAAGATGGTGA
MS92ΔN2-BD R	TTTGGATCCGCGCTCGCCGCCGCCGCTCCCCT
MS92ΔN3-BD F	CACCGAATTCGTGCTGGTGCCGCACCTGTCGTGCT
MS92ΔN3-BD R	TTTGGATCCACAGTTGAAGGACGGGAACG
MS92ΔC1-BD F	CACCGAATTCGTCGTGGACTGCGCGTGCGG
MS92ΔC1-BD R	TTTGGATCCACAGTTGAAGGACGGGAACGACACGACG
MS92ΔC2-BD F	ACGCCATGCGCTTGCCGTCGTCGTC
MS92ΔC2-BD R	GACGACGACGGCAAGCGCATGGCGT
MS92ΔC3-BD F	GTGCTGCCACGCCTCCGCGATGTCGCAGCACGC
MS92ΔC3-BD R	GCGTGCTGCGACATCGCGGAGGCGTGGCAGCAC
MS92ΔC4-BD F	CCCGGCGCCCCGCGTGTTCTGCCACG
MS92ΔC4-BD R	CGTGGCAGAACACGCGGGGCGCCGGG
MS92 PHD GST F	TTTGGATCCGTCGTGGACTGCGCGTGCGG
MS92 PHD GST R	CCGGAATTCCGGACAGTTGAAGGACGGGAACG
RNA in situ F	CGTTGATTGGCAGCAACTAGC
RNA in situ R	CATATCAGATGCCTCCCCCAC
MS92 qPCR-F	GGCATCCCCACATCAACCT	qRT-PCR analysis
MS92 qPCR-R	ATCTCGCTGAACACCCTCT
UBQ qPCR-F	GGAAGTAAGGAAGGAGGAG
UBQ qPCR-R	CAGAGGTGATGCTAAGGT
LOC_Os04g57490 qPCR-F	CAAGGAGTATCCCGTCTGCAA		OsCP1
LOC_Os04g57490 qPCR-R	AGAGATTGAATCAGGTTGTTCCG
LOC_Os07g05150 qPCR-F	AAGGAGGTTGCCGACGACAC
LOC_Os07g05150 qPCR-R	CGAAGCTCTTCAGCTCCTTCTC
LOC_Os06g02019 qPCR-F	CTGACCCCAAAATGTTCAACCC		OsKAO
LOC_Os06g02019 qPCR-R	CTCACCCTGCACTTTGGATTTG
LOC_Os06g16300 qPCR-F	CTGATCGAGATCAAGAACCTGCT
LOC_Os06g16300 qPCR-R	AGAATAGTCTCAAGATTGGTCAGGT
LOC_Os04g51070 qPCR-F	TGAGGACGAACGAGATGTGATT		EAT1
LOC_Os04g51070 qPCR-R	TCACCTACTATTGAGGCCCTGT
LOC_Os09g27940 qPCR-F	CCACAGCTGCGTGAGGCCCGACACC
LOC_Os09g27940 qPCR-R	TCATCGTGCCGCAGCCGAACCCGAG
LOC_Os08g43290 qPCR-F	GAACAGGCTGCTGGCGTGCCGCGCGTA		OsC4
LOC_Os08g43290 qPCR-R	AGCAGTTGATCTGGCTGAGGTGGCAT
LOC_Os09g10260 qPCR-F	ATGTCGAAGCTAGTGAGCAACA
LOC_Os09g10260 qPCR-R	TTGTCGACATCGATGAAGCTGG
LOC_Os07g05180 qPCR-F	CGACACAATCCTCCTAATCAGC
LOC_Os07g05180 qPCR-R	GTTCTTGTCCACATCGATGAAC
LOC_Os07g23570 qPCR-F	CTCCACCTTCCTCACTGTTCAA
LOC_Os07g23570 qPCR-R	CAACCCAAGTAGGTCATTCCCA
LOC_Os10g20470 qPCR-F	TGTTCTTGCTTCGCAATGTCTG
LOC_Os10g20470 qPCR-R	ATGCCTACAAGGTAGAATGCCC
LOC_Os01g34970 qPCR-F	AACATTGCTGAACAGACTATTG
LOC_Os01g34970 qPCR-R	AACACCTTCTTGTAGAGTAGATT
LOC_Os07g42490 qPCR-F	GCTGGAAACAAGGAAACTGTGG
LOC_Os07g42490 qPCR-R	GAGGTGAGCCTCTTCTGTGATT

Fig. 1. Phenotypic comparisons of wild type (WT) and ms92 mutant.A, Comparison of WT and ms92 plants at the heading stage. Scale bar, 10 cm. B, WT floret at the mature pollen stage. Scale bar, 0.5 mm. C, ms92 floret at the mature pollen stage. Scale bar, 0.5 mm. D, Pollen of WT. Scale bar, 30 μm. E, Pollen of ms92. Scale bar, 30 μm.

Fig. 2. Comparison of anther semi-thin section between wild type (WT) and ms92.A, WT at the microspore mother cell stage. B, WT at the meiosis stage. C, WT at the tetrad stage. D, WT at the young microspore stage. E, WT at the vacuolated pollen stage. F, WT at the mature pollen stage. G, ms92 at the microspore mother cell stage. H, ms92 at the meiosis stage. I, ms92 at the tetrad stage. J, ms92 at the young microspore stage. K, ms92 at the vacuolated pollen stage. L, ms92 at the mature pollen stage. Scale bars, 15 μm.E, Epidermis; En, Endothecium; M, Middle layer; MP, Mature pollen; Ms, Microsporocyte; Msp, Microspore; T, Tapetum; Tds, Tetrads.

Fig. 3. Defective pollen wall of ms92.A and B, Microspore of wild type (A) and ms92 (B) at the young microspore stage treated with water. C and D, Microspore of wild type (C) and ms92 (D) at the young microspore stage treated with acetolysis mixture for 10 min. E and F, Primexine of wild type (E) and ms92 (F) at the young microspore stage. G and H, Exine of wild type (G) and ms92 (H) at the mature pollen stage. I and G, Pollen of wild type (I) and ms92 (J) at the mature pollen stage by scanning electron microscopy (SEM). K and L, Magnification of wild type (K) and ms92 (L) pollen.Scale bars are 20 μm in A to D, 0.5 μm in E to J, 2 μm in K and L.Ba, Baculum; Em, Electron-dense material; Lb, Lipid body; Ms, Microspore; Ne, Nexine; Te, Tectum.

Supplemental Fig. 1. SEM analysis of anther surface of WT (A to D) and ms92 (E to H).AA and E, The meiosis stage. B and F, The young microspore stage. C and G, The vacuolated pollen stage; D and H, The mature pollen stage. Bars, 5 μm.

Fig. 4. Transmission electron microscopy analysis of wild type (WT) and ms92 anthers.A, Anthers of WT at the young microspore stage. Scale bar, 1 μm. B, Magnification of WT tapetum in A. Scale bar, 0.5 μm. C, Anthers of WT at the mature pollen stage. Scale bar, 1 μm. D, Magnification of WT tapetum in C. Scale bar, 0.5 μm. E, Anthers of ms92 at the young microspore stage. Scale bar, 1 μm. F, Magnification of ms92 tapetum in E. Scale bar, 0.5 μm. G, Anthers of ms92 at the mature pollen stage. Scale bar, 1 μm. H, Magnification of ms92 tapetum in G. Scale bar, 0.5 μm.E, Epidermis; En, Endothecium; M, Middle layer; Ms, Microsporocyte; Nu, Nucleus; ER, Endoplasmic reticulum; T, Tapetum; U, Ubisch bodies.

Fig. 5. Map based cloning of MS92, its expression pattern and RNA in situ hybridization.A, Schematic represents the exon (solid black box), intron (black lines) and UTR (gray lines) of MS92, 1 036 bp DNA fragment (black arrow) was replaced by 25 bp DNA fragment (white box) in the promoter. B, qRT-PCR analysis of MS92 expression in wild type (WT) and ms92 spikelet. M, Meiosis stage; Y, Young microspore stage; V, Vacuolated pollen stage. Values are Mean ± SD (n = 3). C, RNA in situ hybridization analysis of MS92 using WT anther sections at the young microspore stage with sense probe. Scale bar, 50 μm. D, RNA in situ hybridization analysis of MS92 using WT anther sections at the young microspore stage with anti-sense probe. Scale bar, 50 μm.

Supplemental Fig. 2. Sequence alignment of the WT and ms92 promoter.The sequences were aligned using ClustalW and displayed using BOXSHADE.

Supplemental Table 2 Allelic analysis between ms92 and ptc1.

	Fertile	sterile	χ2（1:1）
ptc1×MS92 B	17	12	0.209
ms92×PTC1 B	22	26	0.606

Supplemental Table 3 Summary of 31 known genes with changed expression in both ms92 and ptc1.

Gene ID	Gene symble	Expression tissue a	Function	LOGms92/WT	Isolation	Reference
LOC_Os04g48400	HTH1	Epidermis-expressed	Anther cuticle and Pollen development	-3.07	KD and M	Xu et al, 2017
LOC_Os03g18480	OsTDF1	Tapetum-specific	Tapetum degeneration	1.15	M	Cai et al, 2015
LOC_Os04g37570	OsAP37	Tapetum-specific	Tapetum degeneration	-2.76	None	Niu et al, 2013
LOC_Os04g51070	EAT1/DTD1	Tapetum-specific	Tapetum degeneration	-1.93	M	Niu et al, 2013
LOC_Os08g43290	OsC4	Tapetum-specific	Pollen exine development	-1.81	None	Tsuchiya et al, 1994
LOC_Os01g02190	OsNIP4;2	RPMCT	Pollen development and pollination	-4.99	None	Pérez et al, 2016
LOC_Os10g38050	OsNP1	RPMCT	Anther cuticle and Pollen exine patterning	-2.64	M	Liu et al, 2017
LOC_Os12g13930	KAR	RPMCT	Pollen exine formation	-3.65	None	Aya et al, 2009
LOC_Os01g62430	OsERG1	RPMCT	Plant defense	-2.23	None	Kim et al, 2003
LOC_Os04g45960	OsSub42	RPMCT	Panicle development	-3.03	None	Sharma et al, 2012
LOC_Os03g07140	OsMS2/DPW	Tapetum and microspore	Pollen wall development	-2.56	M	Shi et al, 2011
LOC_Os04g57490	OsCP1	Tapetum and microspore	Pollen development	-1.87	KD	Lee et al, 2004
LOC_Os05g49900	OsCHS	Tapetum and microspore	Pollen exine formation(decreased)	-3.13	None	Zhang et al, 2008
LOC_Os06g47880	OsCDKG1	Tapetum and microspore	Pollen wall formation	-2.78	None	Huang et al, 2013
LOC_Os04g52320	OsQRT3	Tapetum and microspore	Pollen mother cell wall degradation	-2.11	None	Rhee et al, 2003
LOC_Os04g02640	OsKCS6	Tapetum and microspore	Pollen wall development	-1.33	None	A et al, 2013
LOC_Os01g50622	OsBBD1	Tapetum and microspore	Callose deposition in leaf	-1.29	None	You et al, 2010
LOC_Os12g04980	DMC1A/DMC1	Tapetum and microspore	Homologous paring in rice meiosis	1.82	KD and M	Deng and Wang, 2007
LOC_Os01g50940	OsMYL1	Tapetum and microspore	Anther cell identity	1.51	None	Yang et al, 2016
LOC_Os08g06110	OsLHY	Tapetum and microspore	Flowering time regulation	1.11	None	Ogiso et al, 2010
LOC_Os01g15900	RDD1	Tapetum and microspore	Phytochrome regulation	1.09	KD	Iwamoto et al, 2009
LOC_Os05g50930	OsSIG5	Tapetum and microspore	Light-responsive gene	1.17	None	Kubota et al, 2007
LOC_Os07g48830	WSI76	Tapetum and microspore	Stress-induced gene	1.45	None	Rabbani et al, 2003
LOC_Os04g51150	OsAOX1a	Tapetum and microspore	Cold tolerance	1.25	OX	Li et al, 2013
LOC_Os11g32650	CHS	Tapetum and microspore	Plant defense	-2.38	None	Qiu et al, 2007
LOC_Os05g51090	OsSWEET5	Tapetum and microspore	Crosstalk between sugar and auxin	-6.79	OX	Zhou et al, 2014
LOC_Os01g64890	OsMGT1	Tapetum and microspore	Salt or aluminum tolerance	-1.52	M	Chen et al, 2017
LOC_Os07g37090	OSRIP18	Tapetum and microspore	Salt and drought tolerance	-3.46	OX	Jiang et al, 2012
LOC_Os05g35400	OsBiP4	Tapetum and microspore	Endoplasmic reticulum stress response	-1.81	KD	Wakasa et al, 2012
LOC_Os04g48350	OsDREB1-1	Tapetum and microspore	Stress response	-1.29	None	Tian et al, 2005
LOC_Os04g46940	OsHMA5	Tapetum and microspore	Xylem loading of copper	-1.35	M	Deng et al, 2013

Supplemental Table 3 Summary of 31 known genes with changed expression in both ms92 and ptc1.

Gene ID	Gene symble	Expression tissue a	Function	LOGms92/WT	Isolation	Reference
LOC_Os04g48400	HTH1	Epidermis-expressed	Anther cuticle and Pollen development	-3.07	KD and M	Xu et al, 2017
LOC_Os03g18480	OsTDF1	Tapetum-specific	Tapetum degeneration	1.15	M	Cai et al, 2015
LOC_Os04g37570	OsAP37	Tapetum-specific	Tapetum degeneration	-2.76	None	Niu et al, 2013
LOC_Os04g51070	EAT1/DTD1	Tapetum-specific	Tapetum degeneration	-1.93	M	Niu et al, 2013
LOC_Os08g43290	OsC4	Tapetum-specific	Pollen exine development	-1.81	None	Tsuchiya et al, 1994
LOC_Os01g02190	OsNIP4;2	RPMCT	Pollen development and pollination	-4.99	None	Pérez et al, 2016
LOC_Os10g38050	OsNP1	RPMCT	Anther cuticle and Pollen exine patterning	-2.64	M	Liu et al, 2017
LOC_Os12g13930	KAR	RPMCT	Pollen exine formation	-3.65	None	Aya et al, 2009
LOC_Os01g62430	OsERG1	RPMCT	Plant defense	-2.23	None	Kim et al, 2003
LOC_Os04g45960	OsSub42	RPMCT	Panicle development	-3.03	None	Sharma et al, 2012
LOC_Os03g07140	OsMS2/DPW	Tapetum and microspore	Pollen wall development	-2.56	M	Shi et al, 2011
LOC_Os04g57490	OsCP1	Tapetum and microspore	Pollen development	-1.87	KD	Lee et al, 2004
LOC_Os05g49900	OsCHS	Tapetum and microspore	Pollen exine formation(decreased)	-3.13	None	Zhang et al, 2008
LOC_Os06g47880	OsCDKG1	Tapetum and microspore	Pollen wall formation	-2.78	None	Huang et al, 2013
LOC_Os04g52320	OsQRT3	Tapetum and microspore	Pollen mother cell wall degradation	-2.11	None	Rhee et al, 2003
LOC_Os04g02640	OsKCS6	Tapetum and microspore	Pollen wall development	-1.33	None	A et al, 2013
LOC_Os01g50622	OsBBD1	Tapetum and microspore	Callose deposition in leaf	-1.29	None	You et al, 2010
LOC_Os12g04980	DMC1A/DMC1	Tapetum and microspore	Homologous paring in rice meiosis	1.82	KD and M	Deng and Wang, 2007
LOC_Os01g50940	OsMYL1	Tapetum and microspore	Anther cell identity	1.51	None	Yang et al, 2016
LOC_Os08g06110	OsLHY	Tapetum and microspore	Flowering time regulation	1.11	None	Ogiso et al, 2010
LOC_Os01g15900	RDD1	Tapetum and microspore	Phytochrome regulation	1.09	KD	Iwamoto et al, 2009
LOC_Os05g50930	OsSIG5	Tapetum and microspore	Light-responsive gene	1.17	None	Kubota et al, 2007
LOC_Os07g48830	WSI76	Tapetum and microspore	Stress-induced gene	1.45	None	Rabbani et al, 2003
LOC_Os04g51150	OsAOX1a	Tapetum and microspore	Cold tolerance	1.25	OX	Li et al, 2013
LOC_Os11g32650	CHS	Tapetum and microspore	Plant defense	-2.38	None	Qiu et al, 2007
LOC_Os05g51090	OsSWEET5	Tapetum and microspore	Crosstalk between sugar and auxin	-6.79	OX	Zhou et al, 2014
LOC_Os01g64890	OsMGT1	Tapetum and microspore	Salt or aluminum tolerance	-1.52	M	Chen et al, 2017
LOC_Os07g37090	OSRIP18	Tapetum and microspore	Salt and drought tolerance	-3.46	OX	Jiang et al, 2012
LOC_Os05g35400	OsBiP4	Tapetum and microspore	Endoplasmic reticulum stress response	-1.81	KD	Wakasa et al, 2012
LOC_Os04g48350	OsDREB1-1	Tapetum and microspore	Stress response	-1.29	None	Tian et al, 2005
LOC_Os04g46940	OsHMA5	Tapetum and microspore	Xylem loading of copper	-1.35	M	Deng et al, 2013

Fig. 3. Conservation plots and cis-elements analyses of the substitution sequence in the promoter of MS92. A, Comparison of conservation plots relative to individual plant species. The promoter of the MS92 was compared with that of orthologous genes from 30 plant species using a 60-bp window and a step width of 1 bp. The substitution sequence was denoted with a red dotted box, including conserved core region (red box) and responsive region (green box).B, Graphical representation of 1.5-kb upstream regulatory region of MS92 and presence of key cis-elements. Square box represents the presence of important cis-regulatory motif on the sequence with respect to its TSS.

Fig. 6. Subcellular localization and transcriptional activation assay of MS92 protein.A, Subcellular localization of MS92 protein in onion epidermis cells. (a) Bright-filed image of 35S::GFP; (b) Green fluorescence signal of 35S::GFP; (c) Merged image of 35S::GFP; (d) Bright-filed image of MS92::GFP; (e) Green fluorescence signal of MS92::GFP; (f) Merged image of MS92::GFP. Scale bars, 20 μm. B, Subcellular localization of MS92 protein in Nicotiana benthamiana leaf cells. (a) Bright-filed image of 35S::GFP; (b) Green fluorescence signal of 35S::GFP; (c) Merged image of 35S::GFP; (d) Bright-filed image of MS92::GFP; (e) Green fluorescence signal of MS92::GFP; (f) Merged image of MS92::GFP. Scale bars, 20 μm. C, Transcriptional activation assay of MS92 protein and truncated proteins. PHD, Plant homeodomain. D, Pull-down assay of calf histones with MS92 PHD and analyzed by western blots using the antibodies indicated on the left. GST, Only glutathione-S-transferases tagged protein. GST-MS92PHD, PHD tagged with GST protein.

Supplemental Fig. 4. Neighbor-joining tree using PHD (Plant Homeodomain) finger protein sequences.Hs, Homo sapiens; Sc, Saccharomyces cerevisiae, Ath, Arabidopsis thaliana; Os, Oryza sativa.The number for each interior branch shows the percentage of the bootstrap value (1000 replicates). The scale bar indicates the estimated number of amino acid substitutions per site.

Supplemental Fig. 5. Validation of transcriptome data by qRT-PCR.Values are Mean ± SD (n = 3).

Fig. 7. Differential expression gene analysis between using RNA-seq of wild type (WT) and ms92 and microarray data of WT and ptc1.A, Venn diagram depicting the numbers of DEG between WT and ms92 or ptc1. B, Cluster analysis of 31 genes altered in both ms92 and ptc1 which had been reported. Genes were classified into Clusters I, II, III and IV based on their expression patterns. Other details are showed in Supplemental Table 2. Red and green represents higher and lower expression levels, respectively. Two independent biological samples were analyzed in heatmap.

References 33

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