MULTI-FLORET SPIKELET 4 (MFS4) Regulates Spikelet Development and Grain Size in Rice

doi:10.1016/j.rsci.2021.05.005

Abstract

Abstract:

In rice, the spikelet is the basic unit of inflorescence, and its development is important for determining the grain yield and quality. We reported a rice spikelet mutant multi-floret spikelet 4 (mfs4) which resulted in the production of extra floral organs or a whole extra floret, and elongated sterile lemmas. The results suggested that the mutation of the MFS4 gene interfered with spikelet meristem determinacy and floral organ identity. In addition, the plant height and the grain length and width in the mfs4 mutant were all less than those in the wild type. Using the bulked segregant analysis method, the MFS4 gene was localized in a 557-kb region on the long arm of chromosome 1. Sequence analysis showed that there was a C-base deletion at the open reading frame of LOC_Os01g67430. Further tests indicated that a wild type copy of LOC_Os01g67430 was able to reverse the mfs4 defects, which indicated that LOC_Os01g67430 was the MFS4 gene. The MFS4 gene encodes a lipase located in the mitochondria and is expressed strongly in the young inflorescence. qRT-PCR results showed that the expression of some genes that were known to regulate spikelet meristem determinacy and grain size were decreased in the mfs4 mutant, which indicated that the MFS4 gene regulates spikelet meristem determinacy and grain size by modulating the expression of these genes.

Key words: rice, sterile lemma, multi-floret spikelet, meristem, grain size

Yan Wang, Xiaoqin Zeng, Lu Lu, Qinglan Cheng, Fayu Yang, Mingjiang Huang, Mao Xiong, Yunfeng Li. MULTI-FLORET SPIKELET 4 (MFS4) Regulates Spikelet Development and Grain Size in Rice[J]. Rice Science, 2021, 28(4): 344-357.

Figures/Tables 11

Fig. 1. Phenotypes of spikelet in wild type (WT) and mfs4 mutants. A, Spikelet of the WT. A1, WT spikelet; A2, The lemma and palea were removed in A1; A3 to A6, The WT spikelet surface characters of sl, pa and le; A7, Transverse section of WT spikelet. B?E, Spikelets of the mfs4 mutants. B1 and B2, The Type I mfs4 mutant spikelet; B3?B5, The Type I mfs4 mutant spikelet surface characters of spikelet, le and ele; B6, Transverse section of the Type I mfs4 mutant. C1 and C2, The Type II mfs4 mutant spikelet; C2, The lemma and extra lemma were removed; C3?C6, The Type II mfs4 mutant spikelet surface characters of sl, pa, le and ele; C7, Transverse section of the Type II mfs4 mutant. D1 and D2, The Type II mfs4 mutant spikelet; D2, The lemma, palea and elongate sterile lemma were removed; D3?D6, The Type II mfs4 mutant spikelet surface characters of sl, pa, le and mrp; D7, Transverse section of the Type II mfs4 mutant. E1, The Type III mfs4 mutant spikelet; E2?E4, The Type III mfs4 mutant spikelet surface characters of sl, pa and le; E5, Transverse section of the Type III mfs4 mutant. F, Percentage of mutant organs in mfs4 spikelets.

Fig. 2. Histological and qRT-PCR analysis of lateral organs in spikelets of wild type (WT) and mfs4 mutants. A?C, Transverse sections of lateral organs in the WT spikelet. A and B, The lemma and palea of WT. C, The sterile lemma of WT. Red box indicates the vascular bundle. Scale bars are 500 μm. D?H, Transverse sections of lateral organs in the mfs4 mutant spikelets. D?G, The lemma, palea and extra lemma of the mfs4 mutant spikelets. H, The elongate sterile lemma of the mfs4 mutant spikelets. Red boxes indicate the vascular bundles. Scale bars are 500 μm. I?P, qRT-PCR analysis of OsMADS1, OsMADS6, DL, G1, OsMADS14 and OsMADS15 expression. Actin was used as a control. RNA was isolated from the ?ower organs of the WT and mfs4 mutant spikelets. Data are Mean ± SE (n = 3). *, P? ≤ 0.05 and **, P? ≤ 0.01 by the Student’s t-test. bop, Body of palea; ele, Extra lemma; le, Lemma; lo, Lodicule; lsl, Lemma- like sterile lemma; mrp, Marginal region of palea; Pa, Palea; bs, Residual bop-like structure; sl, Sterile lemma. bop, Body of palea; ele, Extra lemma; le, Lemma; lo, Lodicule; mrp, Marginal region of palea; lsl, Lemma-like sterile lemma; ov, Ovule; pa, Palea; pi, Pistil; rbs, Residual bop-like structure; rg, Rudimentary glume; sl, Sterile lemma; st, Stamen. Scale bars are 1 000 μm in A1, A2, B1, B2, C1, C2, D1, D2 and E1; Scale bars are 500 μm in A3?A6, B3?B5, C3?C6, D3?D6 and E2?E4; Scale bars are 100 μm in A7, B6, C7, D7 and E5.

Fig. 3. Scanning electron micrographs of spikelets at early developmental stages in wild type (WT) and mfs4 mutant. A-D, WT. E-H, mfs4 with ele. Asterisks indicate the stamens. Scale bars are 100 μm. ele, Extra lemma; fm, Flower meristem; le, Lemma; pa, Palea; rg, Rudimentary glume; sl, Sterile lemma.

Fig. 4. Comparison of morphological characters between wild type (WT) and mfs4 mutant. A, Plant morphology of WT and mfs4 mutant at the heading stage. B, Internodes and panicles of WT and mfs4 mutant. C and D, Comparisons of grain length (C) and width (D) in the WT and mfs4 mutant.E?K, Comparisons of internode length (E), panicle length (F), plant height (G), grain number per panicle (H), grain width (I), grain length (J) and 1000-grain weight (K) between the WT and mfs4 mutant. Scale bars are 10 cm in A and B, and 1 cm in C and D. *, P ≤ 0.05 and **, P ≤ 0.01 by the Student’s t-test.

Fig. 5. Fine mapping and sequencing analysis of MFS4 gene. A, Gene mapping of MFS4 on rice chromosome 1. The red triangles indicate the target gene. The red line in the exon indicates the mutational site of candidate gene. WT, Wild type. B, Schematic structure of the complementary vector pCAMBIA1300-MFS4-GFP and MFS4 genome. MFS4-COM-F/MFS4-COM-R are complementary vector primers, F1/R2 are primers for amplifying endogenous MFS4, and F1/R1 are primers for amplifying exogenous MFS4-GFP. C, Defect in mfs4 flowers were completely rescued by introduction of pCAMBIA1300-MFS4. mfs4-com, mfs4 complementary flower. Scale bars are 1 mm. D, Identification of transgenic plants. M, DNA marker; Lanes 1?3, Detection of positive transgenic plants by F1/R2 primers; Lanes 4?6, Detection of positive transgenic plants by F1/R1 primers.

Fig. 6. Spatiotemporal expression pattern of MFS4 gene. A and B, qRT-PCR of MFS4. Actin was used as a control. RNA was isolated from young panicles < 0.5 cm, 0.5?1.0 cm, 1.0?2.0 cm, 2.0?5.0 cm and ≥ 5.0 cm, as well as vegetative organ and floral organ of wild type plants. Data are Mean ± SD (n = 3). C?H, In situ hybridization expression in panicles of wild type (C and D), and spikelets at Sp2?Sp3 (E), Sp4?Sp5 (F), Sp5?Sp6 (G) and Sp7?Sp8 (H). bm, Branch meristem; fm, Floral meristem; fo, Floral organ; le, Lemma; lo, Lodicule; pa, Palea; rg, Rudimentary glume; sl, Sterile lemma; sm, Spikelet meristem; st, Stamen. Scale bars are 500 μm in C and D, and 100 μm in E?H.

Fig. 7. Subcellular localization of MFS4 protein. A and D, GFP illuminant. B and E, mCherry illuminant which indicates the mitochondria marker. C and F, Merge illuminant. Scale bars are 50 μm.

Fig. 8. Expression levels of related genes in wild type (WT) and mfs4 mutant. A, Expression analysis of related genes influencing spikelet meristem determination in young panicles. B, Expression analysis of related genes influencing grain size by cell cycle-related genes in panicles. C, Expression analysis of related genes influencing grain size by hull cell expansion-related genes in panicles. D, Expression analysis of related genes influencing grain size by hull cell proliferation-related genes in panicles. Actin was used as a control. RNA was isolated from panicles of WT and mfs4 mutant. At least three replicates were performed and the mean value was used. Error bars mean standard error. *, P?≤ 0.05 and **, P?≤ 0.01 by the Student’s t-test.

Table S1. Sequences of primers for gene mapping.

Primer	Forward sequence (5′→3′)	Reverse sequence (3′→5′)
CHR1-WY-4	GGATGAACCATACACGCCCATT	GCATGAGAATGCCTGACTGGATG
CHR1-TJ-4	CATGAACAAATCTCAAGTTGTTGAG	TCTGCTTTCTTGGTTACGAGGT
CHR1-WY-5	CGGAGAACTCTGGTTGTGCTTTG	TGCCGTTGAGCCAAAGACATCT
CHR1-WY-6	ACTGAGCTAGCTATGCGGGCTG	AGCAGAGTCATCGACGAGTCGA
CHR1-WY-7	TCAGACCCCATTCTTCACTCCG	GTCGACTTGTTGCCCTCCTCTT
CHR1-WY-8	GCTCTCTCGTCGATCTTAGCAGC	CGTTCGTTGCATGAAGAGGAG

Table S2. Sequences of primers for RT-qPCR.

Primer	Forward sequence (5′→3′)	Reverse sequence (3′→5′)
OsMADS1	ATCACCATCAGGGTCTTCTC	CAACCATGTCTGCTGCTTCA
OsMADS6	CCAACAATGCACTTTCTGAAAC	GGAGGCTTGCTGCATGGC
DL	CCCATCTGCTTACAACCGCTT	GTTGGAGGTGGAAACCGTCG
MFS4	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
G1	GGCGTCTACTTGCCATTTCTG	TCGATCAGCATCAAAGCACAG
OsMADS14	CCATTAACGAGCTTCAACGG	TGGTATGGATCTGAAGCCTCC
OsMADS15	AGTACGCCACTGACTCCAGG	TGCTGGCCCCTCACATTC
MFS1	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
MFS2	GGAGGGCCTCAGATGGCAGAGG	GCTTCAACACGGGACCAAGATGG
MFS3	AGCGAGCCGTGCATGGTGTA	GCTGCTGCTTAATTGCAGTGATG
SNB	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
SHAT1	GCGATGGGAATCGCACATATGG	AAACCCGTCCATGCTGCTCTTC
OsIDS1	GATGGGTGGCTCTCAGCATTCT	CAGCTTTGCAAGCCAACACGTC
RSR1	AGCTGCCTACTGATGCTGCTGCT	GGTTCAGATCCACATCGGCTCCT
Os06g43220	GTGGAGTGGAGGCAGACATCA	CCGGAGAACCTGAACGAACTC
CYCT1	AGACAGCAGGTGATCAGGCCAC	CCTTGCTGGGCAAACAGTTGA
CYCD3-1	TTGCTGGTTTACCTGTCATGCC	AGGAACCGGTCCAGGTAGTTCA
CYCD3-2	GATGCCCCCAAGGTCAGTAGTA	TCGGAGATGATCTTGGCGCA
CYCD2-1	GATGGAGGAAACCCTAGTTCCC	CTACCTGGCAACGAACAATGCA
CYCD2-2	TCAGTGATCGCGCGATGATCT	ATGGGGTCACAGCTTGCAGC
CYCD2-3	AGCACCATGAAAGGTACCATCG	ACACCGCCAAAGTCAAGAACG
GS2	GTTGGGTTCTCAGCTGCACATG	ACTGCATCCACCTGACACCAAT
SRS3	ACCAGGCGACAAAGAGATCGAG	GCATTCCTTCAGAGCGAGCAG
SRS5	CTCGACATTGAGCGCCCAAC	TTCAGAGCACCATCGAACCTCA
PGL1	GCTCGACTCCGGAGCAATGATA	CCGATCATGTTCTACCCCCAT
PGL2	CGAGCTCATCTCCAAGCTCCAG	ACCACCACCCACACACACGACT
APG	CAAGAGCTCATCCCAAACTGCA	CGTCTTGAGGTACTCGATGGCT
SLG	TGGAGCACATCACATCCACCTC	AGGTGATGTAGGGGAGGTCGAAT
WTG1	TAGTGCATGCTTTGTCATGGCA	TGCCAGTGCCAGGTTATTCACA
SMOS1	ATCCTGCCAGCAAGTCAACCTG	AAGCTCGTACGGTCACTCTTGA
GSA1	TGCAAGTCCCCAAGCTGATCC	GCATCGTGGGTCTCAATTGGAT
FZP	ACACAAGTGCAGTGCATGCATG	GCCTCGAGTGTTCATGACCAAT
GS3	GTTCTACCGAAATGGCCGGAA	AGGAGAGGTAGCTGAGGCAGCA
GS5	TGCTTGGCCATCTACAGGTGAT	GCATAGCTCTCCCCTGAGATGT
OsCCS5	TGGAACGTGTTTCCATCTCCC	ATGATGTGGCCCCGATGCTA
GW2	CAGGTCTGCACAGGCTGCAA	TGCTAGTCTGGGCAGAGCATGA
GW5	CGTTGTGTGTTGGCGATGGAT	ATCTTGGGGCTCCGGTCGTA
GW8	GGTGCAAGGAGGACCTGAGCAAG	AATTTGGCGGGAAGGAAGGAGAC
TGW2	GGTAGGTGCAAGCACTGAAGCA	TTCGTCTACCTCATCATGCCGA
TGW3	TGTGTGGCTGGAAATCCGGAT	AATGCCGTTTCCTCCAGCATC
TGW6	AACGAGAGCACCAGCACGAGA	GAGAGCAGCACGAGGAAGACAA
OsMKKK10	GATGCCGAAAATGCAACACTTG	GACACCCCTTGGTGATGTGGAT
OsMKK4	ACGGCTTCACTTGGCCTCGT	GCGTGTGCGTTTTCTGGTGG
OsLG3	ACACTTCGTGTTCGCGTCCAA	ACGACTGCACGAACAAAACCAA
BSG1	AGATCGGCTGCTACCTGACCTAG	GTGGTGATGGTGACGATCCATC

Table S2. Sequences of primers for RT-qPCR.

Primer	Forward sequence (5′→3′)	Reverse sequence (3′→5′)
OsMADS1	ATCACCATCAGGGTCTTCTC	CAACCATGTCTGCTGCTTCA
OsMADS6	CCAACAATGCACTTTCTGAAAC	GGAGGCTTGCTGCATGGC
DL	CCCATCTGCTTACAACCGCTT	GTTGGAGGTGGAAACCGTCG
MFS4	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
G1	GGCGTCTACTTGCCATTTCTG	TCGATCAGCATCAAAGCACAG
OsMADS14	CCATTAACGAGCTTCAACGG	TGGTATGGATCTGAAGCCTCC
OsMADS15	AGTACGCCACTGACTCCAGG	TGCTGGCCCCTCACATTC
MFS1	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
MFS2	GGAGGGCCTCAGATGGCAGAGG	GCTTCAACACGGGACCAAGATGG
MFS3	AGCGAGCCGTGCATGGTGTA	GCTGCTGCTTAATTGCAGTGATG
SNB	AACGTACACGACCCGATCAC	CCGACCTTGAAGAAGTCGAG
SHAT1	GCGATGGGAATCGCACATATGG	AAACCCGTCCATGCTGCTCTTC
OsIDS1	GATGGGTGGCTCTCAGCATTCT	CAGCTTTGCAAGCCAACACGTC
RSR1	AGCTGCCTACTGATGCTGCTGCT	GGTTCAGATCCACATCGGCTCCT
Os06g43220	GTGGAGTGGAGGCAGACATCA	CCGGAGAACCTGAACGAACTC
CYCT1	AGACAGCAGGTGATCAGGCCAC	CCTTGCTGGGCAAACAGTTGA
CYCD3-1	TTGCTGGTTTACCTGTCATGCC	AGGAACCGGTCCAGGTAGTTCA
CYCD3-2	GATGCCCCCAAGGTCAGTAGTA	TCGGAGATGATCTTGGCGCA
CYCD2-1	GATGGAGGAAACCCTAGTTCCC	CTACCTGGCAACGAACAATGCA
CYCD2-2	TCAGTGATCGCGCGATGATCT	ATGGGGTCACAGCTTGCAGC
CYCD2-3	AGCACCATGAAAGGTACCATCG	ACACCGCCAAAGTCAAGAACG
GS2	GTTGGGTTCTCAGCTGCACATG	ACTGCATCCACCTGACACCAAT
SRS3	ACCAGGCGACAAAGAGATCGAG	GCATTCCTTCAGAGCGAGCAG
SRS5	CTCGACATTGAGCGCCCAAC	TTCAGAGCACCATCGAACCTCA
PGL1	GCTCGACTCCGGAGCAATGATA	CCGATCATGTTCTACCCCCAT
PGL2	CGAGCTCATCTCCAAGCTCCAG	ACCACCACCCACACACACGACT
APG	CAAGAGCTCATCCCAAACTGCA	CGTCTTGAGGTACTCGATGGCT
SLG	TGGAGCACATCACATCCACCTC	AGGTGATGTAGGGGAGGTCGAAT
WTG1	TAGTGCATGCTTTGTCATGGCA	TGCCAGTGCCAGGTTATTCACA
SMOS1	ATCCTGCCAGCAAGTCAACCTG	AAGCTCGTACGGTCACTCTTGA
GSA1	TGCAAGTCCCCAAGCTGATCC	GCATCGTGGGTCTCAATTGGAT
FZP	ACACAAGTGCAGTGCATGCATG	GCCTCGAGTGTTCATGACCAAT
GS3	GTTCTACCGAAATGGCCGGAA	AGGAGAGGTAGCTGAGGCAGCA
GS5	TGCTTGGCCATCTACAGGTGAT	GCATAGCTCTCCCCTGAGATGT
OsCCS5	TGGAACGTGTTTCCATCTCCC	ATGATGTGGCCCCGATGCTA
GW2	CAGGTCTGCACAGGCTGCAA	TGCTAGTCTGGGCAGAGCATGA
GW5	CGTTGTGTGTTGGCGATGGAT	ATCTTGGGGCTCCGGTCGTA
GW8	GGTGCAAGGAGGACCTGAGCAAG	AATTTGGCGGGAAGGAAGGAGAC
TGW2	GGTAGGTGCAAGCACTGAAGCA	TTCGTCTACCTCATCATGCCGA
TGW3	TGTGTGGCTGGAAATCCGGAT	AATGCCGTTTCCTCCAGCATC
TGW6	AACGAGAGCACCAGCACGAGA	GAGAGCAGCACGAGGAAGACAA
OsMKKK10	GATGCCGAAAATGCAACACTTG	GACACCCCTTGGTGATGTGGAT
OsMKK4	ACGGCTTCACTTGGCCTCGT	GCGTGTGCGTTTTCTGGTGG
OsLG3	ACACTTCGTGTTCGCGTCCAA	ACGACTGCACGAACAAAACCAA
BSG1	AGATCGGCTGCTACCTGACCTAG	GTGGTGATGGTGACGATCCATC

Table S3. Primers used in this study.

Primer	Sequence	Purpose
MFS4-com-GFP-F-EcoRI	CTATGACCATGATTACGAAGATAGCTCGATCAGCCACAACCAT	Complementary test
MFS4-com-GFP-R-BamHI	CATGTCGACTCTAGAGGATCCGATTAGCCCGAGCGTCTGCAC	Complementary test
580-MFS4-XbalI-F	AAGTCCGGAGCTAGCTCTAGAATGACGCTCCCGAGGCAATG	Subcellular localization
580-MFS4-BamHI-R	GCCCTTGCTCACCATGGATCCGATTAGCCCGAGCGTCTGCA	Subcellular localization
MFS4-F	GCTCAAGATGGATTGGGAAGGTC	Gene test
MFS4-R	TGCCAGATCGTCCAACTGGTGT	Gene test
MFS4-COM-F1	AAGGTGATGGACTTCGTGGGTC	Complementary plant test
MFS4-COM-R1	CGGTGGTGCAGATGAACTTCAG	Complementary plant test
MFS4-COM-R2	CTGCTCAGATTTGGGCGTTCTG	Complementary plant test

References 54

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