Identification of New Allele of FLOURY ENDOSPERM2 in White-Core Endosperm Mutant of Rice

Bao Jinsong, Zhang Yu, Zhao Jiajia, Chen Yaling, Wu Weixun, Cao Liyong, Xu Feifei

Letter

Identification of New Allele ofin White-Core Endosperm Mutant of Rice

Bao Jinsong1, 2, Zhang Yu1, Zhao Jiajia1, Chen Yaling3, Wu Weixun4, Cao Liyong4, Xu Feifei1

(Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Rural Affairs and Zhejiang Province / Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; Hainan Institute of Zhejiang University, Yazhou District, Sanya 572025, China; Laboratory of Plant Genetic Improvement and Biotechnology, Jiangxi Normal University, Nanchang 330000, China; State Key Laboratory of Rice Biology / Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China)

Identification of regulatory genes from chalky/floury endosperm mutants is an important approach to understand the mechanism of starch biosynthesis to accelerate rice grain quality improvement. A mutant, identified from60Co γ-irradiation of anrice Guanglu’ai 4 (GLA4), exhibited white-core endosperm and altered starch physicochemical properties. However, the causal gene responsible for the white-core endosperm inhas not been identified. Here, we developed a recombined inbred line (RIL, F7) population derived from a cross betweenand arice Tainong 67 (TN67) with translucent endosperm. Bulked- segregant analysis combined with next generation sequencing revealed five single nucleotide polymorphisms (SNPs) in four candidate genes that were highly associated with the white-core endosperm. Among them, one base pair deletion inresulted in a frame shift mutation after the 983th amino acid (aa), and a premature stop codon occurred after the other 11 aa. Moreover, functional annotation revealed thatwas previously characterized as the() gene. Full-length coding sequence fromdriven by the maize ubiquitin promoter was transformed into, and seeds from these transgenic plants expressingwere largely rescued to translucent, indicating thatwas responsible for the white- core endosperm in.

The most abundant substance stored in rice grain is starch, which offers a primary source of energy for human beings (You et al, 2019; Zhang et al, 2021) and affects rice cooking and eating quality (Bao, 2012). Starch biosynthesis in rice seed endosperm is regulated by many genes involved in various pathways. Loss-of-function of mutants related to starch biosynthesis are usually accompanied by white-core or floury endosperm, where is filled with loosely packed, small and spherical starch granules with large air spaces, while normal grains have translucent endosperm which consists of tightly packed, large and irregularly polyhedral starch granules. In recent years, a lot of genes have been functionally characterized from floury endosperm mutants. Among them, the causal genes for() (Zhou et al, 2016),() (Nishi et al, 2001),() and() (Kawagoe et al, 2005) are starch synthesis-related genes.() (Kang et al, 2005),(Cai et al, 2018),(Lei et al, 2022) and(Long et al, 2018) are involved in lipid biosynthesis.(Wu et al, 2019),(Xue et al, 2019),(Yu et al, 2021) and(Hao et al, 2019), encoding pentatricopeptide repeat (PPR) proteins, play important roles in mitochondrial function and endosperm development.(Matsushima et al, 2010) and(Wang et al, 2021) are involved in compound starch granule (SG) division.encodes a protein with a carbohydrate-binding module 48 (CBM48) domain that binds to SG. FLO6 interacts with ISA1, and recruits ISA1 to SGs during endosperm development (Peng et al, 2014).encodes a NAD-dependent cytosolic malate dehydrogenase, and plays a crucial role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm (Teng et al, 2019). RSR1 and bZIP58 are transcription factors that bind to the promoters of starch synthesis related genes (SSRGs). RSR1 represses the expression of SSRGs (Fu and Xue, 2010), while bZIP58 activates the expression of SSRGs (Wang et al, 2013).encodes a tetratricopeptide repeat motif containing protein, and loss of functions inresults in decreased expression of genes involved in production of storage starch and storage proteins in the endosperm (She et al, 2010). Aspositively regulates the degradation of the nucellus and the nucellar projection during seed development, suppressedexpression causes defective programmed cell death of the nucellus and nucellar projection, resulting in shrunken floury endosperm (Yin and Xue, 2012). NF-YB1, NF-YC12 and bHLH144 can form a heterotrimer complex of NF-YB1- YC12-bHLH144, in which NF-YB1 binds to the G-box motif ingene promoter to regulate amylose synthesis (Bello et al, 2019). Besides, NF-YC12 and bHLH144 enhance the stability of NF-YB1 from 26S proteasome degradation (Bello et al, 2019), and NF-YC12 binds to the promoters ofand(glutamine synthetase1) in developing endosperm (Xiong et al, 2019). Knock-out mutants of,andwere obtained from the CRISPR/Cas9 system,and each mutant exhibits increased percentage of grain with chalkiness (Bello et al, 2019; Xiong et al, 2019). In a previous study, we isolated a white-core endosperm mutant, which has altered amylopectin structure with increased degrees of polymerization of 6 to 9 (DP6–9) and DP22–35, decreased DP10–21, as well as displayed decreased gelatinization temperature, and increased total protein content (Kong et al, 2014). However, the causal gene for the white-core endosperm inhas not been identified.

To isolate the causal gene forphenotype, we constructed two F2populations by crossingwith its parent GLA4 and arice variety TN67 (/GLA4 and/TN67), respectively. Thechalky phenotype in the F2seeds was investigated and the separation ratios of translucent-endosperm to white-core-endosperm seeds were nearly to 3:1 in both crosses, indicating that white-core endosperm phenotype is controlled by a nuclear recessive gene (Table S1). Furthermore, seeds from each F2line of/TN67 were self-pollinated for five generationsvia the single seed descent method to construct a RIL-F7population (Fig. 1-A). We selected 26 translucent endosperm (TE) lines and 26 white-core endosperm (WE) lines, of which the endosperm phenotype was stably inherited in three generations (F5, F6and F7). Genomic DNA from each line was equally mixed to construct a bulked TE DNA pool and a bulked WE DNA pool. The two bulked DNA pools,and TN67 were then subjected for next generation sequencing (Novegene, Tianjin, China). A total of 3 032 837 SNPs and 470 038 insertion/ deletions (InDels) were identified between the two parents. Absolute ΔSNP index analysis indicated that a significant peak nearly to 1.0 located on chromosome 4 (Fig. 1-B). In this region, 27 SNPs and 6 InDels were predicted to be candidate sites with 99% possibly responsible for the chalky endosperm. Among them, four SNPs in three genes (,and) contributed to nonsynonymous amino acid changes, and a thymine deletion incaused frame shift mutation (Table S2). To verify whether these SNPs were natural variations, we amplified and sequenced these five sites in GLA4,and TN67. The results showed that GLA4 andhad the same SNPs in,and. However, the thymine deletion inwas only observed in, while GLA4 and TN67 showed the same sequence with the reference sequence (9311) (Fig. 1-C). The 1-bp deletion generated a frame-shift mutation and resulted in another 11 aa before a stop codon occurred in(Fig. 1-D). Furthermore, we developed a derived cleaved amplified polymorphism sequence (dCAPS)marker to recognize the 1-bp deletion in(Table S3), and the amplified PCR products ofwere effectively cut by the restriction enzymeI, while those of GLA4 and TN67 were not cut by?. Then, we checked the genotype of the RIL population using the dCAPS marker, finding that the PCR products from lines with all the white-core endosperm were cut byI, while those from translucent- endosperm lines were not digested byI (Fig. 1-E). Thus, we speculated thatwasthe causal gene for the white-core endosperm in. Functional annotation revealed thatwas the previously reported gene, which encodes a tetratricopeptide repeat (TPR) domain containing protein (She et al, 2010). In the present study, the second and the third TPR domains (975–1 008 aa and 1 017– 1 050 aa) of the full-length protein of 1 720 aa were lost in(Fig. 1-D).

Fig. 1. One base pair deletioninresults in white-core endosperm mutant.

A, Schematic of bulked-segregant analysis (BSA) for detection of candidate genes for white-core endosperm (WE) in. Twenty-six translucent endosperm (TE) lines and 26 WE lines selected from a recombinant inbred line (RIL)-F7population were used for germination. Ten-day-old seedlings were used to extract genomic DNA individually, which was equally mixed to make the bulked TE DNA pool and WE DNA pool. Genomic DNA of the two bulked DNA pools,and Tainong 67 (TN67), were subjected for next generation sequencing. B, Absolute ΔSNP index plots for white-core endosperm. Absolute ΔSNP index = |SNP indexWE pool– SNP indexTE pool|. The significant peak (absolute ΔSNP index was around 1.0) was marked in the red cycle. C, Sanger sequencing and comparison of five candidate sites in four genes in Guanglu’ai 4 (GLA4),and TN67. D, Gene structure and mutation site of. A thymine nucleotide deletion incauses premature termination of translation after 983 aa and has an additional 11 aa resulting from the frame shift mutation. The coding sequence for tetratricopeptide repeat domain is located in the red box. E, One base pair deletion inis associated with white-core endosperm in the RIL population. For the different lines, PCR products from WE-RIL are recognized and cut by the restriction enzyme? as, while PCR products from TE-RIL cannot be cut by?as TN67.

Fig. 2. Complementary tests confirm thatlargely rescues white-core endosperm to translucent in.

A, FLO2-3×HA-TurboID fusion protein express intransgenic plants (CL#1 to CL#5). Total protein was extracted from young grains at 10 d after flowering and analyzed the expression of the fusion protein by immunoblot with anti-HA antibody.B, Genetic complementation ofinrescues grain appearance. CL#1 and CL#2 mean complementary lines 1 and 2, respectively.

To confirm whetherwas the causal gene forphenotypes,full-length coding sequence without a stop codon (5 160 bp) ofwas amplified from the total cDNA of GLA4 and cloned into a binary vector pCAMBIA1390. A 3×HA tag and a TurboID tag were fused to the C-terminal ofin frame. The Ubi-FLO2-3×HA-TurboID vector was transformed intovia- mediated transformation. We extracted the total proteins from the seeds of 10 T0transgenic plants at 10 d after flowering (DAF)and checked the expression of FLO2 by immunoblot using the anti-HA antibody. Positive signals were detected in five plants (Fig. 2-A). Some of T1seedsfrom these T0transgenic lines displayed translucent seeds (Fig. 2-B). The separation ratios of translucent seeds to white-core seeds were nearly to 3:1 (data not shown) in some lines, indicating that a single copy ofwas transformed into these transgenic lines.We further checked the expression of the FLO2-3×HA-TurboID fusion protein from the seeds of T1stable transgenic plants at 10 DAF using anti-HA antibody, and found that all of them were transgenic plants and the resulting seeds displayed translucent endosperm (data not shown). Thus, complementary assays confirmed thatis the causal gene for the white-core endosperm in.

Previously, we checked the expression of SSRGs in, and the expression levels of,,,,,,,andwere significantly down-regulated during seed development (Chen et al, 2020), which is in agreement with the results of She et al (2010). The disorder of SSRGs regulated byinresulted in the alternations of amylopectin structure and physiochemical properties in. The TPR domain of FLO2 mediates protein-protein interactions with bHLH and LEA transcription factors, which play a crucial role in regulating the expression of SSRGs (She et al, 2010). Suzuki et al (2020) identified that FLOC1 interacts with FLO2 via the TPR motif, and knock-down expression of FLOC1 shows significantly reduced fertility and generation of seeds with abnormal features. However, as a regulatory factor protein, more regulatory factors interacting with FLO2 remain unknown. In recent years, attention has been paid to identify the phosphorylated protein involved in the regulation of protein-protein interactions in starch biosynthesis (Crofts et al, 2017). Pang et al (2021) found that there are 24 residues phosphorylated atinrice GLA4 and 9311. Nine sites show significant down-regulation in 9311, while one site shows up-regulation in GLA4 in response to high temperature stress. She et al (2010) reported thatis also involved in heat tolerance during seed development. It offers a clue that phosphorylation of FLO2 might be responsible for the heat tolerance in rice. Furthermore, we are interested in identifying new protein complexes involving FLO2 to investigate phosphorylation of FLO2 in regulation of protein-protein interaction. In this study, we fused a TurboID tag at the C-terminal of FLO2 to develop a FLO2-3×HA-TurboID fusion protein. TurboID is a newly developed proximity catalytic enzyme. Supplementation of biotin will activate TurboID to proximity label the nearby proteins in living cells (Yang et al, 2020). In combination with mass spectrometry, many potential FLO2 interacting proteins will be identified, facilitating the identification of FLO2 related protein complex involved in rice endosperm starch biosynthesis. Moreover, we have generated the mutants of three branching enzyme genes by genome editing technology (Tappiban et al, 2022),and the combination ofand these mutants may generate new rice lines that have high resistant starch content and better eating quality.

Acknowledgements

This study was financial supported by the Zhejiang Provincial Natural Science Foundation (Grant No. LZ21C130003) and National Natural Science Foundation of China (Grant No. 31961143016).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://ricescience.org.

File S1. Methods.

Table S1. Segregation ratios of translucent and white-core endosperm plants in two F2populations.

Table S2. Genotypic variations of four single nucleotide polymorphisms in three genes and one base pair deletion in.

Table S3. Primers used in this study.

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s: Xu Feifei (xuxufei@zju.edu.cn); Cao Liyong (caoliyong@caas.cn)

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24 April 2022