Locus TUTOU2 determines the panicle apical abortion phenotype of rice (Oryza sativa L.) in tutou2 mutant

ZHU Zi-chao,LUO ShengLEI BinLI Xian-yong,CHENG Zhi-jun

1 Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,P.R.China

2 Rice Research Institute,Chongqing Academy of Agricultural Sciences,Chongqing 400060,P.R.China

Abstract Rice panicle apical abortion (PAA) is a detrimental agronomic trait resulting in spikelet number reduction and yield loss. To understand its underlying molecular mechanism,we identified one recessive PAA mutant tutou2 from the offspring of tissue cultures. The mutation locus was finely mapped to a 75-kb interval on the long arm of chromosome 10. Sequence analysis revealed a single nucleotide substitution of A to T at the 941 position of LOC_Os10g31910 in tutou2,resulting in an amino acid change from isoleucine to phenylalanine. Complementation analysis showed that the degenerated panicle phenotype in tutou2 was rescued in the transgenic lines. A phenotype similar to tutou2 can also be obtained by LOC_Os10g31910 knockout in wild-type rice. These results suggested that LOC_Os10g31910 is the causative locus TUTOU2 responsible for the tutou2 PAA phenotype and probably also the locus of DEL1,previously documented as a leaf senescence gene. The significant phenotypic differences between del1 and tutou2 suggest that the locus DEL1/TUTOU2 plays roles in both leaf and panicle development which were not considered fully in previous studies.

Keywords:Oryza sativa L.,panicle apical abortion,pectate lyase-like,gene cloning,DEL1

1.Introduction

Rice (Oryza sativaL.) is a staple food crop for more than half of the world’s population. Grain yield in rice is mainly determined by three key components,namely number of panicles,number of grains per panicle and grain weight(Xing and Zhang 2010). Rice panicle development is initiated following the switch from vegetative to reproductive growth,during which the shoot apical meristem is converted into an inflorescence meristem,and then to main axis and primary branch meristems (BMs). Primary BMs produce secondary BMs,and both can initiate spikelet meristems and finally form spikelets (Ikedaet al.2004;Tanakaet al.2013).The number of grains per panicle is determined mainly by the length of the main axis and the length and number of primary and secondary branches.

Genes related to grain number per panicle in rice were first reported in the transgenic analysis ofREDUCED CULM NUMBER1(RCN1) andRCN2(Nakagawaet al.2002). Their overexpression delays the transformation of shoot-tip meristem from vegetative to reproductive growth,thereby increasing the number of secondary branches.Several genes related to panicle development,such as panicle erectness genesDENSE AND ERECT PANICLE1(DEP1)/qPE9-1,ERECT PANICLE2(EP2) andDEP3,increase the number of grains per panicle (Huanget al.2009;Zhouet al.2009;Zhuet al.2010;Qiaoet al.2011).When the expression ofOsCKX2encoding cytokinin (CK)oxidase/dehydrogenase is decreased,CK accumulates in the inflorescence meristem and consequently promotes axillary bud growth and increases the numbers of branches and florets (Ashikariet al.2005). Loss of function ofLONELY GUY,a CK-activating enzyme encoding gene,prematurely terminates the shoot meristem (Kurakawaet al.2007).The mutation of transcription factorDROUGHT AND SALT TOLERANCE,which regulates the expression ofOsCKX2,can lead to CK accumulation in the shoot apical meristem,thus increasing the activity of the meristem and subsequently the number of grains per spike (Liet al.2013).

Other genes,such asSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14(OsSPL14),increase the number of grains per panicle by promoting the formation of panicle branches (Jiaoet al.2010;Miuraet al.2010).EnhancingGhd7expression can increase the grain number per panicle under long days (Xueet al.2008).LAX PANICLE1(LAX1) encodes a bHLH domain,andLAX2encodes a plant-specific domain;mutations in both genes can reduce the numbers of branches and grains(Komatsuet al.2003;Oikawa and Kyozuka 2009;Tabuchiet al.2011). The overexpression ofABERRANT PANICLE ORGANIZATION1(APO1),which encodes the F-box protein,increases the number of grains per panicle (Ikedaet al.2007).APO1andAPO2/RFLcan coordinate panicle development (Ikeda-Kawakatsuet al.2012),and theFRIZZY PANICLEgene is involved in its regulation (Baiet al.2017;Huoet al.2017). Overexpression of a gene encoding an S-domain receptor kinase can alternate plant type and increase grain number per panicle (Zouet al.2015). A panicle-grain-number incremental gene was isolated,which is an allele of the narrow leaf gene (NAL1,Fujitaet al.2013). MutationofTAW1,the panicle development regulator gene from a dominant mutanttawawa1-D,can prolong the differentiation time of branches and increase the number of grains per panicle (Yoshidaet al.2013).

Spikelet degeneration is an important element causing yield loss (Senanayakeet al.1991). Both the length of the main axis and the number of primary and secondary branches are down-regulated significantly due to early-stage degeneration of the spikelet,which occurs either at the apex or basal portion of the panicle (Yamagishiet al.2004;Chenget al.2011). Mutation at theSP1locus encoding the PT leads to panicle apical abortion (PAA) in mutantPAA-Hwa(Akteret al.2014).TUTOU1participates in panicle development and encodes a suppressor of cAMP receptor-like protein(Baiet al.2015),while loss of function inSPL6can cause rice panicle cell death (Wanget al.2018). The mutation ofOsALMT7,a putative aluminum-activated malate transporter encoding gene,causes the degeneration of spikelet on the apical portion at the late stage of panicle development (Henget al.2018). The rice calcineurin B-like protein-interacting protein kinase 31 (OsCIPK31) is involved in the development of the panicle apical spikelet (Penget al.2018). Recently,it was found that the protein containing CYSTATHIONINE β-SYNTHASE DOMAIN is required for panicle development in rice,and the mutation in the geneDEGENERATED PANICLE AND PARTIAL STERILITY1resulted in the degenerated panicles and partial sterility (Zafaret al.2020).

Given that PAA is a complex trait resulting from the interactions of multiple genes and the external environment,the molecular basis for this phenotype is still elusive.Cloning related genes will be helpful for understanding the genetic mechanism underlying the PAA phenotype.Previously,five top spikelet degeneration mutants have been identified,namelytutou1,paa1,spl6,paa1019,anddps1(Baiet al.2015;Henget al.2018;Penget al.2018;Zafaret al.2020). However,four mutants (excepttutou1) only showed PAA phenotype with no effect on leaf senescence.tutou1,in addition to the top panicle abortion,also showed a degenerated leaf phenotype. In the process of cloning a novel PAA-like mutanttutou2,we found it is an allelic mutant of geneDEL1,which was considered a determinant of leaf senescence in mutantdel1.Our results provide novel evidence that both leaf senescence and PAA share one pathway,probably programmed cell death (PCD).

2.Materials and methods

2.1.Plant materials and genetic mapping population construction

The PAA mutanttutou2was isolated from the tissue culture ofjaponicaricecv.Kitaake. In 2017,tutou2was crossed with thejaponicaricecv.IRAT129 to construct a genetic mapping population at the Beijing Experimental Station(summer season,39°54´N). After the reproduction of F1at the Sanya Experimental Station (in winter,18°16´N,tropical climate),the F2population plants were planted at the Beijing Experimental Station together with the two parents on 5 May,2018. Ten plants were grown in one row with 20 cm between rows and 15 cm between plants. After the emergence of the earliest panicle on most individuals,leaf samples were collected and grouped from typical PAA plants.

2.2.DNA extraction,linkage analysis and map-based cloning

The DNA of the parents and single plants selected from the mapping population was extracted from fresh leaf tissueviathe cetyl trimethylammonium bromide method. DNA from 10 single plants with typical PAA in the F2population was extracted and mixed equally to construct a mixed pool. For linkage analysis,108 polymorphic insertion/deletion (InDel)and simple sequence repeat (SSR) markers between the parents of the mutant and IRAT129,and distributed evenly over the rice genome,were used to genotype the mixed DNA pool. PCR was performed and the products were separated on 8% non-denaturing polyacrylamide gels and then visualized by silver staining. For fine mapping,770 F2single plants with the PAA phenotype were used. The newly developed CAPS markers for fine mapping were designed by comparing the genomic sequences fromjaponicacv.IRAT129 and Kitaake by using the Software dCAPS Finder 2.0 and Primer Premier 5.0 (Table 1).

2.3.Vector construction for genetic transformation

For the complementation test,a 7.3-kb genomic fragment including the entire coding region ofLOC_Os10g31910(5 315 bp),and 1 500-bpupstream and 500-bp downstream sequences,was isolated from the wild type (WT) with the primer pair p2300-TUTOU2-F and p2300-TUTOU2-R(Table 2). The fragment was introduced into a pCAMBIA2300 vector,andtutou2was subsequently transformed by theAgrobacterium-mediated method.

Table 1 The molecular markers used in TUTOU2 mapping

Table 2 The primers used in vector constructions

To create the knockout line,an 18-bp sgRNA CTCGTGCAGCGGATGCCT targeting the second exon of Kitaake was cloned into the CRISPR-Cas9 expression vector according to a previously described method (Miaoet al.2013). The resulting construct was transformed into Kitaake by theAgrobacterium-mediated method.All complementation and knockout lines were planted in the Hainan Experimental Station.

3.Results

3.1.Phenotype of tutou2 mutant

The stable rice PAA mutanttutou2(Fig.1-A–D,following the designation of the mutanttutou1(Baiet al.2015)) was identified from the generation of tissue culture asjaponica cv.Kitaake. The agronomic traits surveyed (Fig.1-I–N)demonstrated that in addition to top panicle degeneration,the panicle length of the mutant was 27.9% shorter than in WT. The seed-setting rate and 1 000-grain weight decreased by 44.3 and 41.1%,respectively,compared with WT. Meanwhile,the plant height oftutou2was about 79.3%of the WT. Furthermore,the lengths of both the first and forth internodes from the top were clearly shorter than WT(P<0.05;Fig.1-E). In addition,the mutant displayed a late heading date phenotype of 7 days later than that of WT in 2018 at the Beijing Experimental Station. However,tutou2tiller number was increased by 33.7% compared with WT.

The mutant also displayed an early leaf senescence phenotype at the grain filling stage. After heading 7 days,the apex of the flag leaf exhibited a pale yellow color,and the leaf margin became withered intutou2. Meanwhile,the top second and third leaves displayed more serious premature senescence with larger areas turning into reddish brown color and becoming withered. Especially in the top third leaf,the withered area nearly accounted for 20% of the whole leaf (Fig.1-F–H). This phenotype was more obvious in the lower part leaves of the mutant.

Fig.1 Gross morphology and statistics on the agronomic traits of wild type (WT) and tutou2. A and B,phenotype comparison of plants and panicles between WT (left) and mutant (right). C and D,magnified views of the top panicles of WT and mutant. E,internode length comparison of WT (left) and tutou2 (right) at the filling stage. I–IV,1st–4th internodes from top,respectively.F–H,leaf senescence in flag leaf,top second leaf and top third leaf between WT (left) and mutant (right) after heading 7 days.I–N,phenotype comparisons of plant height,days to flowering,number of tillers per plant,seed-setting rate,panicle length,and 1 000-grain weight between WT (left) and mutant (right),respectively. Values are mean±SE (n=10). Asterisks above the bars indicate statistically significant differences from the WT analyzed by Student’s t-test (n=10;＊＊,P<0.01). Bars=15,2,0.6,3,and 2.5 cm in A,B,C–D,E,and F–H,respectively.

To reveal when the top spikelet starts to abort,we divided the panicle development course into 1-,3-,7-,11-,and 13-cm stages according to panicle length (Henget al.2018),and13 cm is the maximum length of panicle development.Except the smaller spikelet size found intutou2,no apparent panicle degeneration was observed ahead of the 7-cm stage between WT and mutant(Fig.2). From the 11-and 13-cm stages on,tutou2displayed a phenotype of combined apical spikelet degeneration,short panicle length and small sized spikelets. Therefore,the mutant apical panicle degeneration may occur after the 7-cm stage.

Fig.2 Top spikelet abortion occurs at a late stage of panicle development in tutou2. A–E,developing panicles of wild type(WT) (left) and tutou2 (right) at different stages indicated by panicle length:1 cm (A),3 cm (B),7 cm (C),11 cm (D),and 13 cm (E),respectively,with 13 cm being the final panicle size.In D and E,white arrows indicate the abortion spikelets. F–J,tip spikelet from the corresponding panicles in A–E. Bars=1 cm in A–E and 5 mm in F–J.

3.2.Genetic analysis and map-based cloning of the candidate gene for TUTOU2

The mutant was crossed withcv.IRAT129 to determine thetutou2genetic model. In the F1generation,all individuals displayed non-PAA phenotype.Within the F2population,tutou2exhibited typical single-gene Mendelian segregation(2 356 normal:770tutou2,χ2=0.2256,P=0.6348),indicating that the PAA occurring intutou2is determined by a single recessive nuclear gene (Appendix A).

A total of 162 pairs of SSR and InDel markers evenly distributed in the rice genome were employed to detect polymorphism between IRAT129 andtutou2. Finally,108 pairs of polymorphic markers were screen out.Linkage analysis revealed that theTUTOU2locus was delimited by the markers RJ10-1 and M178 on the long arm of chromosome 10. A total of 770 F2mutant individuals were further evaluated,and 91 and 27 recombinants were identified by the two polymorphic markers. For fine mapping of the candidate gene,six InDel markers (M16616,M16765,M161,M164,M174,and M178),one SSR marker RJ10-1,and one CAPS marker S6 were newly developed. The candidate gene forTUTOU2was finally narrowed into a 75-kb interval flanked by the markers S6 and M16765(Fig.3-A).

Fig.3 Map-based cloning of TUTOU2. A,the TUTOU2 locus was broadly mapped to the region between RJ10-1 and M178 on the long arm of chromosome 10 and finally narrowed to a 75-kb interval flanked by the primers S6 and M16765 using 770 F2 mutant individuals. The positions of the markers,number of recombinant plants,and relative positions of 10 open reading frames (ORFs)are presented. The candidate open reading frame is highlighted in grey. B,diagram of the mutation site in gene LOC_Os10g31910.According to sequence analysis,an A to T mutation occurred at 941 bp from the start codon,causing an amino acid change from Ile to Phe. The filled and white boxes represent exons and untranslated regions,respectively,and black lines indicate the introns of LOC_Os10g31910.

This 75 kb interval includes 10 open reading frames(ORFs). Sequence comparisons between WT andtutou2revealed that only ORF10,a putative pectate lyase-like protein encoding the geneLOC_Os10g31910,harbors an A to T mutation at the position of 941 on exon 2,resulting in encoding an amino acid alternation from isoleucine (Ile)to phenylalanine (Phe).

3.3.Complementation and gene knockout tests

To verify whether the mutation of a single nucleotide in geneLOC_Os10g31910is responsible for the PAA phenotype oftutou2,the 7.3 kb fragment covering the wholeLOC_Os10g31910sequence together with its 1 500 bp upstream and 500 bp downstreamwas amplified from the WTcv.Kitaake,and then cloned into the pCAMBIA2300 vector to create a complementary construct. This construct was subsequently transformed intotutou2mutant by theAgrobacterium-mediated method. As a result,the five complementation lines obtained were fully rescued and displayed a normal panicle without degenerated apical panicle and normal leaves. Other traits such as plant height,seed-setting rate and 1 000-grain weight,etc.,were also restored similar to the WT (Fig.4-A and B).The determination of their sequences confirmed that the transgenic plants were successfully complemented in the T0and subsequent T1generations (Fig.4-C).

The CRISPR-Cas9 technique was employed to provide further evidence forLOC_Os10g31910asthe candidate gene ofTUTOU2. A CRISPR-Cas9 construct targeting the second exon ofLOC_Os10g31910was transformed into WT and 13 plants displaying the top spikelet degeneration and premature leaf senescence phenotypes. Phenotypes such as decreased seed-setting rate,dwarfism and reduced 1 000-seed weight,etc.,also occurred. Among these 13 knockout plants,we detected one line with a typical apical spikelet abortion and leaf senescence phenotype,and confirmed that this line carried one base deletion in the target region (Fig.4-A,B and D). Collectively,complementation and gene knockout experiments demonstrated that the mutation in the geneLOC_Os10g31910is responsible for thetutou2PAA and leaf senescence phenotype.

Fig.4 Complementation and knockout experiments of TUTOU2. A and B,in complementation individuals,the degenerated panicle phenotype is rescued to normal;in TUTOU2 knockout individuals,obvious panicle apical abortion (PAA) appeared on the tip of transgenic panicle. A,gross morphology of wild type (WT),tutou2 and transgenic plants. B,panicles of WT,mutant and transgenic plants. Com and crispr represent the complementation and gene-knockout plant,respectively. C,at the position of 941 bp,the complemented individuals with nucleotides A/T compared with A/A and T/T at the same position of WT and tutou2. D–F,one base pair deletion occurred in the target area of crispr-plant (G),compared with WT (E) and mutant (F). The sequence in blue color represents the target region,and the black rectangle indicates the deleted base pair. Bars=15 cm in (A) and 2 cm in (B).

4.Discussion

We isolated a PAA mutant from the tissue culture population.The mutanttutou2displayed reduced plant height,leaf senescence,late heading date,shortened panicle length,top spikelet degeneration,less seed setting,and light 1 000-grain weight. Using map-based cloning,we narrowed the locus ofTUTOU2to a 75-kb interval on chromosome 10.Sequence analysis revealed that an A→T mutation occurred at the 941 positon of the geneLOC_Os10g31910and putatively caused a one amino acid substitution from Ile to Phe. Both transgenic complementation and knockout experiments verified that the mutation occurring on geneTUTOU2is a causative factor for the phenotype of the mutanttutou2.

Gene cloning and characterization suggest thatTUTOU2encodes a putative pectate lyase-like protein. In plants,thepectate lyase-likegene is frequently present in multiple copies. Genome sequencing has identified 12,22,26,46,53,42,and 83pectate lyase-likegenes in rice (Senechalet al.2014),tomato (Yanget al.2017),Arabidopsis thaliana(Shinet al.2014),Brassica rapa(Senechalet al.2014),Gossypium raimondii,Gossypium arboretum,andGossypium hirsutum(Sunet al.2018),respectively. InArabidopsis(Shinet al.2014),cotton (Sunet al.2018),tomato (Kulikauskas and McCormick 1997),alfalfa (Wuet al.1996),tobacco(Rogerset al.2001),Brassica rapa(Jianget al.2013),andBrassica campestris(Jianget al.2014),thepectate lyase-likegene members play a variety of roles in plant development,such as in pollen development,and fruit maturation of tomato (Yanget al.2017),strawberry(Benitez-Burracoet al.2003),grape (Nunanet al.2001),and banana (Marin-Rodriguezet al.2003),as well as in cotton fiber elongation (Sunet al.2018),latex release inHevea brasiliensis(Chotigeatet al.2009),Arabidopsispowdery mildew (Vogelet al.2002),and tomato grey mold resistance (Yanget al.2017).

In rice,somepectate lyase-likefamily members regulate leaf senescence.ospse1is a premature leaf senescence mutant that exhibits a leaf senescence phenotype from the booting stage to ripening. Its plant height,grain weight,and seed-setting rate are decreased significantly.Theospse1locus was mapped on a 38-kb region of chromosome 1 and the candidate gene isOs01g0546800,putatively encoding a pectate lyase protein (Wuet al.2013). Mutantdel1displayed dwarfism and early senescent leaf phenotypes.It was found thatLOC_Os10g31910is responsible for this mutant locus. TheDEL1encodes a pectate lyase precursor and is involved in the maintenance of normal cell division and leaf senescence (Lenget al.2017).

TUTOU2is the allele of the geneDEL1,which is mutated at the position 1 940 bp and was previously shown to determine leaf senescence performance (Lenget al.2017).Although bothdel1andtutou2display dwarfism and premature leaf senescence,there are many obvious differences specifically in top spikelet abortion.tutou2exhibits a degenerated spikelet,whiledel1floret appears normal. These different phenotypes caused by different mutation positions have also been found in mutants of bothtutou1andes1-1on the same gene ofLOC_Os01g11040. Thetutou1displayed a typical top spikelet abortion accompanied with significantly reduced 1 000-kernal weight,seed-setting rate,plant height,tiller number,root length,chlorophyll content and whitened and senescent leaves (Baiet al.2015). However,es1-1mainly shows a phenotype of premature of leaves rather than any apical panicle degeneration (Raoet al.2015).The results presented here suggest thatDEL1/TUTOU2may have a wider role in both leaf and panicle development than previously considered.

Indel1plants,accumulation of reactive oxygen species(ROS) triggered PCD and eventually resulted in premature leaf senescence (Lenget al.2017). Additionally,PCD might cause rice top panicle degeneration (Henget al.2018;Penget al.2018;Wanget al.2018;Zafaret al.2020). Based on these findings,we presumed that thetutou2premature leaf senescence and PAA phenotypes might be related to PCD triggered by ROS accumulation.

5.Conclusion

In this study,we identified a top spikelet degenerated mutanttutou2,which is accompanied by premature leaf senescence,reduced plant height,late heading date,short panicle length,and decreased seed-setting rate and 1 000-grain weight phenotypes. The panicle growth process analysis revealed that the mutant top spikelet degeneration started after the 7-cm stage. With a mapbased strategy,the candidate gene was delimited to an approximate 75 kb physical distance by markers S6 and M16765 on chromosome 10. Sequence alignment found a single nucleotide substitution of A to T at the 941 position ofLOC_Os10g31910in the mutant,leading to an amino acid change from isoleucine to phenylalanine. Complementation and knockout tests further confirmed thatLOC_Os10g31910is the causative geneTUTOU2responsible fortutou2,which putatively encodes a pectate lyase-like protein. Phenotypes and sequence analysis suggested thatTUTOU2is probably the alleleDEL1,previously reported as a leaf senescence and dwarf gene. Although typical phenotypes oftutou2anddel1were different,both of them might be caused by PCD triggered by ROS accumulation. This study may provide insight into the roles thatpectate lyase-likegene plays in both leaf and panicle development which were not considered fully in previous studies.

Acknowledgements

This work was supported by grants from the National Transgenic Science and Technology Program,China(2016ZX08009003-003),the National Key Research and Development Program of China (2016YFD0101100),the Youth Innovation Team Program of Chongqing Academy of Agricultural Sciences,China (NKY-2018QC03) and the National Natural Science Foundation of China (31960401).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm