CRISPR/Cas9 based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature

CRISPR/Cas9 based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CRISPR/Cas9 based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature Flávia Barbosa Silva Botelho, Soumen Nandy, Vibha Srivastava This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6229149/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 May, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Warming trend of the climate has been linked with yield losses in many crops. Like other cereals, rice is highly sensitive to above-typical temperatures during reproductive and grain filling stages. In fact, nighttime temperatures have risen faster than daytime temperatures in many parts of the world. For this reason, rice studies in recent years have focused on the effect of high nighttime temperature (HNT) on grain yield. Several studies have shown that HNT disturbs key processes in reproductive development and grain filling that lead to reduced spikelet fertility (SF) and enhanced grain chalkiness (Srivastava et al., 2024). Chalkiness is the opaque area on the grain, and it is not just an appearance issue, it also impacts milling quality. Above the generally acceptable chalk values (6-10%), every 1% increase in chalkiness leads to 1% decline in head rice yield (HRY) (Zhao and Fitzgerald 2013). Therefore, breeding HNT tolerance is vital for safeguarding grain yields from heat waves in the future. However, breeding efforts have been impeded by the lack of reliable tolerance alleles in modern cultivars. Breeding is also complicated by the complex nature of HNT tolerance as not only SF and grain quality traits, but other yield components such as panicle length, grain width, grain size, and grain weight are also affected by HNT in a genotype-dependent manner. Not surprising, hundreds of QTLs have been identified in the genomics studies. Of which, Chalk5 (Os05g0156900) is most notable as it stands as one of the few functionally validated QTL (Fan et al., 2024; Gann et al., 2023; Li et al., 2014). Figures Figure 1 Key Message Transcriptional modulation of the vacuolar H + translocating pyrophosphatase expressed specifically in the endosperm and reproductive tissue of rice improves its spikelet fertility and reduces grain chalkiness under high nighttime temperature. Full Text Chalk5 was identified in chalky indica rice. It encodes a vacuolar H + translocating pyrophosphatase (V-PPase) that is most strongly expressed in the reproductive tissue and developing caryopses (Li et al., 2014). V-PPase regulates metabolic activities by maintaining cellular pH and preventing the buildup of inorganic pyrophosphate (PPi). Li et al. (2014) found that hyperactivity of Chalk5 in young caryopses contributes to chalkiness in mature grains, and this hyperactivity is based on promoter elements found in indica rice that develop chalky kernels. These promoter elements are absent in the V-PPase allele of non-chalky rice, referred to as VPP5 to distinguish it from Chalk5 associated with chalky trait. However, transcriptional modulation of VPP5 in non-chalky rice, Nipponbare, by CRISPR/Cas9 based mutagenesis of its promoter led to substantial reduction in grain chalkiness under HNT (Gann et al., 2023). This was very encouraging because most modern cultivars succumb to undesirable levels of chalkiness under HNT that is a major threat to rice production (Srivastava et al., 2024). Here, we determined whether the CRISPR based vpp5 allele is a reliable source of HNT tolerance by (a) analyzing yield traits of the Nipponbare line harboring vpp5 allele (Nip_vpp5), (b) validating the effect of VPP5 promoter mutagenesis in Kitaake rice through CRISPR/Cas9, and (c) exploring the underlying mechanism by RNA-seq. For phenotypic analysis, plants were maintained in the greenhouse (average seasonal temperature: 27.8 o C daytime/25.8 o C nighttime) until booting stage, and then half of them were transferred into HNT growth chamber (30 o C daytime/28 o C nighttime, 14 h photoperiod) until harvest. Nip_vpp5 plants generally took 4 – 5 days longer to flower in both greenhouse (GH) and HNT conditions compared to the wildtype (Nip_WT), and average height of Nip_vpp5 plants at the seed filling stage was significantly lower than that of Nip_WT ( Fig. S1a-c ), but no significant effect of vpp5 mutation on panicle number and panicle length was observed ( Fig. S1d-e ). However, SF was significantly improved. Although detrimental effect of HNT on SF was observed in both genotypes, Nip_vpp5 showed higher SF compared to Nip_WT ( Fig. 1a; Fig. S2a ). As observed in our previous study (Gann et al., 2023), Nip_vpp5 showed significantly lower (~3-fold lower) grain chalkiness compared to Nip_WT ( Fig. 1b; Fig. S2b ). Next, a negative effect of HNT on grain weight was observed in both genotypes, but significant differences between genotypes within a growing condition were not found ( Fig. S3a ). Finally, milled rice yield (MRY) and head rice yield (HRY) were significantly higher for Nip_vpp5 under HNT, but no significant difference was observed for grains ripened in the greenhouse ( Fig. 1c-d ). Interestingly, a small but significant increase in grain length was observed in Nip_vpp5 milled grains compared to Nip_WT ( Fig. S3b ). Overall, this analysis illuminated a positive effect of vpp5 mutation on major yield components: SF, MRY, and HRY. Next, we determined the effect of vpp5 mutation in rice cv. Kitaake. Using the CRISPR/Cas9 vector and transformation methods described earlier (Gann et al., 2023), we developed two vpp5 mutants in Kitaake (Kit_vpp5-6 and Kit_vpp5-7). Kit_vpp5-6 contained a biallelic homozygous insertion-deletion consisting of 256 bp deletion and 13 bp insertion, and Kit_ vpp5-7 contained biallelic heterozygous 240 bp deletion with or without 2 bp insertion between the targeted sites ( Fig. S4a-b ). Gene expression analysis by qPCR showed that VPP5 was downregulated in the caryopses of both lines; however, a significant difference ( p <0.01) was observed only in Kit_vpp5-6 ( Fig. S4c ). Based on this, Kit_vpp5-6 was selected for the yield analysis. First generation seeds (T1) of Kit_vpp5-6, referred to as Kit_vpp5, hereafter, were sown in the greenhouse and the plants at booting stage were treated with HNT as described above. Notably, Kit_vpp5 showed improved SF in the greenhouse and reduced chalkiness under HNT compared to the wildtype (Kit_WT) ( Fig. 1e-f ), indicating a positive effect of vpp5 mutation on these traits. This is highly significant as it validates the effect of vpp5 mutation on two major yield traits. Finally, MRY and HRY in Kit_vpp5 was variable but significantly higher than that of Kit_WT in at least one environment ( Fig. 1g-h ). Overall, the positive effect of vpp5 on grain quality and yield was observed in rice varieties, which are generally non-chalky, but succumb to chalkiness under HNT. Previous studies suggested that V-PPase affects starch related processes (Li et al., 2014; Gann et al., 2023). Corroborating with that, Nip_vpp5 showed a slower rate of post-germinative growth that correlated with delayed starch hydrolysis (Gann et al., 2025). Here, we carried out RNA-seq analysis in developing caryopses to understand transcriptional changes associated with improved grain quality in Nip_vpp5. We selected caryopses at 10 days after flowering (10DAF) as VPP5 is highly expressed at this stage (Gann et al., 2023). Principal component analysis showed >50% variance in the transcriptional profile of Nip_vpp5 and Nip_WT, and differential expression analysis found 615 differentially-expressed genes (FDR1) ( Fig. S5a-b ). As expected, the key starch pathway genes were differentially expressed ( Fig. S5c ); however, gene enrichment analysis showed that heat response processes were downregulated. Specifically, gene ontology (GO) terms, ‘response to temperature stimulus’, ‘response to heat’, and ‘protein folding’ were highly significant (FDR<0.01) ( Fig. S5d ). These pathways include heat shock protein (HSP) and heat shock transcription factor (HSF), 16 of which were downregulated (FDR1) in Nip_vpp5 ( Fig. 1i ). We validated this data by qPCR on a subset of these genes ( Fig. 1j ). HSPs and HSF play a major role in heat stress response, but they also exert developmental controls in non-stress condition, e.g. , small HSP (sHSP) accumulate in embryo and endosperm during seed development (Waters and Vierling, 2020). Further, since heat induces HSP expression in rapidly growing cells and heat is also a major inducer of grain chalkiness in most cultivars, a correlation of HSP expression and chalkiness is logical. This hypothesis is supported by studies that showed upregulation of HSP and HSF in rice caryopses under elevated temperature and accumulation of small HSP in chalky kernels (Yamakawa et al., 2007; Yamakawa and Hakata, 2010). Thus, downregulation of sHSP genes in Nip_vpp5 arguably contributes to reduced chalkiness. Next, we found that a set of 13 kDa prolamin genes was upregulated in Nip_vpp5, while 11S globulin was downregulated ( Fig. 1i, k ). Lower abundance of 13 kDa prolamins and higher abundance of 11S globulin have been linked with chalky appearance of the grains (Lin et al., 2017; Yamakawa et al., 2007; Yamakawa and Hakata, 2010). Prolamin is the major component of protein bodies in rice endosperm, and its deficiency could impact their morphology. Disturbance in protein body number and morphology was pointed out by Li et al. as the basis of chalky endosperm. In conclusion, this study showed that CRISPR based modulation of VPP5 generated a reliable allele for improved SF and reduced chalkiness under HNT. The underlying mechanism involves a range of metabolic changes that are not limited to starch biosynthesis and include differential accumulation of prolamin and sHSP. Declarations Acknowledgements: This work was supported by grants from National Science Foundation (NSF EPSCoR RII Grant No.1826836) and National Institute of Food and Agriculture (USDA-NIFA Grant No. 2023-11092). Data availability: Please contact authors for the availability of additional data on differential gene expression. Contributions: FB performed phenotypic analysis, SN carried out gene expression analysis, and VS analyzed all data and wrote the paper. References Fan G, Jiang J, Long Y, Wang R, Liang F, Liu H, Xu J, Qiu X, Li Z (2024) Generation of Two-Line Restorer Line with Low Chalkiness Using Knockout of Chalk5 through CRISPR/Cas9 Editing. Biology (Basel)13:617. https://doi.org/10.3390/biology13080617 Gann PJI, Dharwadker D, Cherati SR, Vinzant K, Khodakovskaya M, Srivastava V (2023) Targeted mutagenesis of the vacuolar H + translocating pyrophosphatase gene reduces grain chalkiness in rice. Plant J. 115:1261-1276. https://doi.org/10.1111/tpj.16317. Gann PJI, Nandy S, Botelho FBS, Vinzant K, Khodakovskaya M, Srivastava V (2025) A vacuolar proton pump controls the post-germinative growth of rice ( Oryza sativa ssp. japonica ). Plant Growth Regul. https://doi.org/10.1007/s10725-025-01290-x Li Y, Fan C, Xing Y, Yun P, Luo L, Yan B, Peng B, Xie W, Wang G, Li X, Xiao J, Xu C, and He Y (2014) Chalk5 encodes a vacuolar H + -translocating pyrophosphatase influencing grain chalkiness in rice. Nat Genet 46:398–404. https://doi.org/10.1038/ng.2923 Lin Z, Wang Z, Zhang X, Liu Z, Li G, Wang S, Ding Y (2017) Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation. Plant Cell Physiol. 58:560-573. https://doi.org/10.1093/pcp/pcx001 Srivastava V, De Guzman C, Fernandes SB (2024) Beat the heat: Breeding, genomics, and gene editing for high nighttime temperature tolerance in rice. Curr. Opin. Plant Biol. 82:102659. https://doi.org/10.1016/j.pbi.2024.102659 Waters ER and Vierling E (2020) Plant small heat shock proteins – evolutionary and functional diversity. New Phytol. 227: 24-37. https://doi.org/10.1111/nph.16536. Yamakawa H, Hirose T, Kuroda M, Yamaguchi T (2007) Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiol. 144:258-77. https://doi.org/10.1104/pp.107.098665. Yamakawa H and Hakata M (2010) Atlas of rice grain filling-related metabolism under high temperature: joint analysis of metabolome and transcriptome demonstrated inhibition of starch accumulation and induction of amino acid accumulation. Plant Cell Physiol. 51:795-809. https://doi.org/10.1093/pcp/pcq122 Zhao X and Fitzgerald M (2013) Climate Change: Implications for the Yield of Edible Rice. PLoS ONE 8(6): e66218. https://doi.org/10.1371/journal.pone.0066218 Supplementary Files SupplementaryFig.S1S5.pptx Cite Share Download PDF Status: Published Journal Publication published 10 May, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 13 Apr, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers invited by journal 17 Mar, 2025 Editor assigned by journal 17 Mar, 2025 First submitted to journal 14 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6229149","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430083001,"identity":"fa6263da-ad3d-4ab6-b59d-196f247080bf","order_by":0,"name":"Flávia Barbosa Silva Botelho","email":"","orcid":"","institution":"University of Arkansas Division of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Flávia","middleName":"Barbosa Silva","lastName":"Botelho","suffix":""},{"id":430083002,"identity":"67f74722-b2de-4898-87b3-2316b046338b","order_by":1,"name":"Soumen Nandy","email":"","orcid":"","institution":"University of Arkansas Division of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Soumen","middleName":"","lastName":"Nandy","suffix":""},{"id":430083003,"identity":"88a0f8dc-4a6c-4a44-b6c5-5e66ae717274","order_by":2,"name":"Vibha Srivastava","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACZh42IGkDxGxQTKSWNFK0MIC1HCZBi8Fx3mMPfvw5n9g/+1gCw4eyw0RoOcyXbtjbdjtxxrm0A4wzzhGhRbKZx0yCt+F2YsMZ9gZm3jYitUj++XMucT5Iy19itPAz85hJ87AdSNxwhu0AMyNxWvjSpGXbko03nmFLONhzLp2wFjb+s8ck3/yxk513hs3wwY8ya8JaUMABEtWPglEwCkbBKMAFAKZGN009OANgAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3031-6984","institution":"University of Arkansas Fayetteville: University of Arkansas","correspondingAuthor":true,"prefix":"","firstName":"Vibha","middleName":"","lastName":"Srivastava","suffix":""}],"badges":[],"createdAt":"2025-03-14 20:59:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6229149/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6229149/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03504-y","type":"published","date":"2025-05-10T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79254787,"identity":"777e5756-7d10-4f68-9c2e-c7ec48efe590","added_by":"auto","created_at":"2025-03-26 08:51:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":188388,"visible":true,"origin":"","legend":"\u003cp\u003eGrain quality and yield analysis of Nipponbare(Nip) or Kitaake(Kit) rice lines harboring \u003cem\u003evpp5\u003c/em\u003e mutation: Nip_vpp5 (\u003cstrong\u003ea-d\u003c/strong\u003e) and Kit_vpp5 (\u003cstrong\u003ee-f\u003c/strong\u003e). The experiment was conducted in two environments: greenhouse (GH) and growth chamber set at high nighttime temperature (HNT). (\u003cstrong\u003ea, e\u003c/strong\u003e) Spikelet fertility, (\u003cstrong\u003eb, f\u003c/strong\u003e) percent chalk per grain, (\u003cstrong\u003ec, g\u003c/strong\u003e) milled rice yield (MRY), and (\u003cstrong\u003ed, h\u003c/strong\u003e) head rice yield (HRY: number of unbroken kernels), (\u003cstrong\u003ei\u003c/strong\u003e) expression pattern of heat-shock protein (HSP) and heat stress transcription factor (HSF) genes in Nip_vpp5 and Nip_WT. Expression values were obtained from RNA-seq analysis using two biological replicates of each genotype, (\u003cstrong\u003ej-k\u003c/strong\u003e) gene expression analysis by real time qPCR on heat shock protein (HSP), 11S globulin (Glob.), and 13 KDaprolamin (Prol.) genes in the developing caryopses. Relative expression was calculated against rice ubiquitin 2 gene. Primers are shown in Fig. S5e. Error bars in (a) and (e) show standard deviation of data from 15 plants in each genotype/environment. Percent chalk and milling yield were determined on 3 replicates of 100 grains each on WinSEEDLETM 2024 and ZavvariaPAZ-1DTA mill, respectively. Significance in Tukey’s multiple comparison at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 is shown by capital letters. Error bars in (j-k) show standard derivation based on 2 – 4 replicates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6229149/v1/a5ca98d02b33272f7b0c727d.png"},{"id":82537516,"identity":"0de1bca2-ba64-410b-a82e-214018667398","added_by":"auto","created_at":"2025-05-12 16:07:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":523155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6229149/v1/f9df7702-64ef-4e12-88a6-85e1a26c373c.pdf"},{"id":79254795,"identity":"ad20e53e-2d20-426d-9f3d-5296d7bba633","added_by":"auto","created_at":"2025-03-26 08:51:41","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5858528,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S1S5.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6229149/v1/dbe5d2a9814a587951386e54.pptx"}],"financialInterests":"","formattedTitle":"CRISPR/Cas9 based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature","fulltext":[{"header":"Key Message","content":"\u003cp\u003eTranscriptional modulation of the vacuolar H\u003csup\u003e+\u003c/sup\u003e translocating pyrophosphatase expressed specifically in the endosperm and reproductive tissue of rice improves its spikelet fertility and reduces grain chalkiness under high nighttime temperature.\u003c/p\u003e"},{"header":"Full Text","content":"\u003cp\u003e\u003cem\u003eChalk5\u0026nbsp;\u003c/em\u003ewas identified in chalky \u003cem\u003eindica\u003c/em\u003e rice. It encodes a vacuolar H\u003csup\u003e+\u003c/sup\u003e translocating pyrophosphatase (V-PPase) that is most strongly expressed in the reproductive tissue and developing caryopses (Li et al., 2014). V-PPase regulates metabolic activities by maintaining cellular pH and preventing the buildup of inorganic pyrophosphate (PPi). Li et al. (2014) found that hyperactivity of \u003cem\u003eChalk5\u003c/em\u003e in young caryopses contributes to chalkiness in mature grains, and this hyperactivity is based on promoter elements found in \u003cem\u003eindica\u0026nbsp;\u003c/em\u003erice that develop chalky kernels. These promoter elements are absent in the \u003cem\u003eV-PPase\u003c/em\u003e allele of non-chalky rice, referred to as \u003cem\u003eVPP5\u0026nbsp;\u003c/em\u003eto distinguish it from \u003cem\u003eChalk5\u003c/em\u003e associated with chalky trait. However, transcriptional modulation of \u003cem\u003eVPP5\u003c/em\u003e in non-chalky rice, Nipponbare, by CRISPR/Cas9 based mutagenesis of its promoter led to substantial reduction in grain chalkiness under HNT (Gann et al., 2023). This was very encouraging because most modern cultivars succumb to undesirable levels of chalkiness under HNT that is a major threat to rice production (Srivastava et al., 2024). \u0026nbsp;Here, we determined whether the CRISPR based \u003cem\u003evpp5\u0026nbsp;\u003c/em\u003eallele is a reliable source of HNT tolerance by (a) analyzing yield traits of the Nipponbare line harboring \u003cem\u003evpp5\u003c/em\u003e allele (Nip_vpp5), (b) validating the effect of \u003cem\u003eVPP5\u0026nbsp;\u003c/em\u003epromoter mutagenesis in Kitaake rice through CRISPR/Cas9, and (c) exploring the underlying mechanism by RNA-seq.\u003c/p\u003e\n\u003cp\u003eFor phenotypic analysis, plants were maintained in the greenhouse (average seasonal temperature: 27.8\u003csup\u003eo\u003c/sup\u003eC daytime/25.8\u003csup\u003eo\u003c/sup\u003eC nighttime) until booting stage, and then half of them were transferred into HNT growth chamber (30\u003csup\u003eo\u003c/sup\u003eC daytime/28\u003csup\u003eo\u003c/sup\u003eC nighttime, 14 h photoperiod) until harvest. Nip_vpp5 plants generally took 4 \u0026ndash; 5 days longer to flower in both greenhouse (GH) and HNT conditions compared to the wildtype (Nip_WT), and average height of Nip_vpp5 plants at the seed filling stage was significantly lower than that of Nip_WT (\u003cstrong\u003eFig. S1a-c\u003c/strong\u003e), but no significant effect of\u003cem\u003e\u0026nbsp;vpp5\u003c/em\u003e mutation on panicle number and panicle length was observed (\u003cstrong\u003eFig. S1d-e\u003c/strong\u003e). However, SF was significantly improved. Although detrimental effect of HNT on SF was observed in both genotypes, Nip_vpp5 showed higher SF compared to Nip_WT (\u003cstrong\u003eFig. 1a; Fig. S2a\u003c/strong\u003e). As observed in our previous study (Gann et al., 2023), Nip_vpp5 showed significantly lower (~3-fold lower) grain chalkiness compared to Nip_WT (\u003cstrong\u003eFig. 1b; Fig. S2b\u003c/strong\u003e). Next, a negative effect of HNT on grain weight was observed in both genotypes, but significant differences between genotypes within a growing condition were not found (\u003cstrong\u003eFig. S3a\u003c/strong\u003e). Finally, milled rice yield (MRY) and head rice yield (HRY) were significantly higher for Nip_vpp5 under HNT, but no significant difference was observed for grains ripened in the greenhouse (\u003cstrong\u003eFig. 1c-d\u003c/strong\u003e). Interestingly, a small but significant increase in grain length was observed in Nip_vpp5 milled grains compared to Nip_WT (\u003cstrong\u003eFig. S3b\u003c/strong\u003e). Overall, this analysis illuminated a positive effect of \u003cem\u003evpp5\u0026nbsp;\u003c/em\u003emutation on major yield components: SF, MRY, and HRY.\u003c/p\u003e\n\u003cp\u003eNext, we determined the effect of \u003cem\u003evpp5\u003c/em\u003e mutation in rice cv. Kitaake. Using the CRISPR/Cas9 vector and transformation methods described earlier (Gann et al., 2023), we developed two \u003cem\u003evpp5\u0026nbsp;\u003c/em\u003emutants in Kitaake (Kit_vpp5-6 and Kit_vpp5-7). Kit_vpp5-6 contained a biallelic homozygous insertion-deletion consisting of 256 bp deletion and 13 bp insertion, and Kit_ vpp5-7 contained biallelic heterozygous 240 bp deletion with or without 2 bp insertion between the targeted sites (\u003cstrong\u003eFig. S4a-b\u003c/strong\u003e). Gene expression analysis by qPCR showed that \u003cem\u003eVPP5\u003c/em\u003e was downregulated in the caryopses of both lines; however, a significant difference (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;0.01) was observed only in Kit_vpp5-6 (\u003cstrong\u003eFig. S4c\u003c/strong\u003e). Based on this, Kit_vpp5-6 was selected for the yield analysis. First generation seeds (T1) of Kit_vpp5-6, referred to as Kit_vpp5, hereafter, were sown in the greenhouse and the plants at booting stage were treated with HNT as described above. Notably, Kit_vpp5 showed improved SF in the greenhouse and reduced chalkiness under HNT compared to the wildtype (Kit_WT) (\u003cstrong\u003eFig. 1e-f\u003c/strong\u003e), indicating a positive effect of \u003cem\u003evpp5\u003c/em\u003e mutation on these traits. \u0026nbsp;This is highly significant as it validates the effect of\u003cem\u003e\u0026nbsp;vpp5\u003c/em\u003e mutation on two major yield traits. Finally, MRY and HRY in Kit_vpp5 was variable but significantly higher than that of Kit_WT in at least one environment (\u003cstrong\u003eFig. 1g-h\u003c/strong\u003e). Overall, the positive effect of \u003cem\u003evpp5\u003c/em\u003e on grain quality and yield was observed in rice varieties, which are generally non-chalky, but succumb to chalkiness under HNT.\u003c/p\u003e\n\u003cp\u003ePrevious studies suggested that V-PPase affects starch related processes (Li et al., 2014; Gann et al., 2023). Corroborating with that, Nip_vpp5 showed a slower rate of post-germinative growth that correlated with delayed starch hydrolysis (Gann et al., 2025). Here, we carried out RNA-seq analysis in developing caryopses to understand transcriptional changes associated with improved grain quality in Nip_vpp5. We selected caryopses at 10 days after flowering (10DAF) as \u003cem\u003eVPP5\u003c/em\u003e is highly expressed at this stage (Gann et al., 2023). Principal component analysis showed \u0026gt;50% variance in the transcriptional profile of Nip_vpp5 and Nip_WT, and differential expression analysis found 615 differentially-expressed genes (FDR\u0026lt;0.05, |log2FC|\u0026gt;1) (\u003cstrong\u003eFig. S5a-b\u003c/strong\u003e). As expected, the key starch pathway genes were differentially expressed (\u003cstrong\u003eFig. S5c\u003c/strong\u003e); however, gene enrichment analysis showed that heat response processes were downregulated. Specifically, gene ontology (GO) terms, \u0026lsquo;response to temperature stimulus\u0026rsquo;, \u0026lsquo;response to heat\u0026rsquo;, and \u0026lsquo;protein folding\u0026rsquo; were highly significant (FDR\u0026lt;0.01) (\u003cstrong\u003eFig. S5d\u003c/strong\u003e). These pathways include heat shock protein (HSP) and heat shock transcription factor (HSF), 16 of which were downregulated (FDR\u0026lt;0.05, |log2FC|\u0026gt;1) in Nip_vpp5 (\u003cstrong\u003eFig. 1i\u003c/strong\u003e). We validated this data by qPCR on a subset of these genes (\u003cstrong\u003eFig. 1j\u003c/strong\u003e). HSPs and HSF play a major role in heat stress response, but they also exert developmental controls in non-stress condition, \u003cem\u003ee.g.\u003c/em\u003e, small HSP (sHSP) accumulate in embryo and endosperm during seed development (Waters and Vierling, 2020). Further, since heat induces \u003cem\u003eHSP\u003c/em\u003e expression in rapidly growing cells and heat is also a major inducer of grain chalkiness in most cultivars, a correlation of HSP expression and chalkiness is logical. This hypothesis is supported by studies that showed upregulation of HSP and HSF in rice caryopses under elevated temperature and accumulation of small HSP in chalky kernels (Yamakawa et al., 2007; Yamakawa and Hakata, 2010). Thus, downregulation of sHSP genes\u003cem\u003e\u0026nbsp;\u003c/em\u003ein Nip_vpp5 arguably contributes to reduced chalkiness. Next, we found that a set of 13 kDa prolamin genes was upregulated in Nip_vpp5, while 11S globulin was downregulated (\u003cstrong\u003eFig. 1i, k\u003c/strong\u003e). Lower abundance of 13 kDa prolamins and higher abundance of 11S globulin have been linked with chalky appearance of the grains (Lin et al., 2017; Yamakawa et al., 2007; Yamakawa and Hakata, 2010). Prolamin is the major component of protein bodies in rice endosperm, and its deficiency could impact their morphology. Disturbance in protein body number and morphology was pointed out by Li et al. as the basis of chalky endosperm. In conclusion, this study showed that CRISPR based modulation of \u003cem\u003eVPP5\u0026nbsp;\u003c/em\u003egenerated a reliable allele for improved SF and reduced chalkiness under HNT. The underlying mechanism involves a range of metabolic changes that are not limited to starch biosynthesis and include differential accumulation of prolamin and sHSP.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from National Science Foundation (NSF EPSCoR RII Grant No.1826836) and National Institute of Food and Agriculture (USDA-NIFA Grant No. 2023-11092).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e Please contact authors for the availability of additional data on differential gene expression.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions:\u0026nbsp;\u003c/strong\u003eFB performed phenotypic analysis, SN carried out gene expression analysis, and VS analyzed all data and wrote the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFan G, Jiang J, Long Y, Wang R, Liang F, Liu H, Xu J, Qiu X, Li Z (2024) Generation of Two-Line Restorer Line with Low Chalkiness Using Knockout of \u003cem\u003eChalk5\u003c/em\u003e through CRISPR/Cas9 Editing. Biology (Basel)13:617. https://doi.org/10.3390/biology13080617\u003c/li\u003e\n\u003cli\u003eGann PJI, Dharwadker D, Cherati SR, Vinzant K, Khodakovskaya M, Srivastava V (2023) Targeted mutagenesis of the vacuolar H\u003csup\u003e+\u003c/sup\u003e translocating pyrophosphatase gene reduces grain chalkiness in rice. Plant J. 115:1261-1276. https://doi.org/10.1111/tpj.16317.\u003c/li\u003e\n\u003cli\u003eGann PJI, Nandy S, Botelho FBS, Vinzant K, Khodakovskaya M, Srivastava V (2025) A vacuolar proton pump controls the post-germinative growth of rice (\u003cem\u003eOryza sativa\u003c/em\u003e ssp. \u003cem\u003ejaponica\u003c/em\u003e). Plant Growth Regul. https://doi.org/10.1007/s10725-025-01290-x\u003c/li\u003e\n\u003cli\u003eLi Y, Fan C, Xing Y, Yun P, Luo L, Yan B, Peng B, Xie W, Wang G, Li X, Xiao J, Xu C, and He Y (2014) \u003cem\u003eChalk5\u003c/em\u003e encodes a vacuolar H\u003csup\u003e+\u003c/sup\u003e-translocating pyrophosphatase influencing grain chalkiness in rice. Nat Genet 46:398\u0026ndash;404. https://doi.org/10.1038/ng.2923\u003c/li\u003e\n\u003cli\u003eLin Z, Wang Z, Zhang X, Liu Z, Li G, Wang S, Ding Y (2017) Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation. Plant Cell Physiol. 58:560-573. https://doi.org/10.1093/pcp/pcx001\u003c/li\u003e\n\u003cli\u003eSrivastava V, De Guzman C, Fernandes SB (2024) Beat the heat: Breeding, genomics, and gene editing for high nighttime temperature tolerance in rice. Curr. Opin. Plant Biol. 82:102659. https://doi.org/10.1016/j.pbi.2024.102659\u003c/li\u003e\n\u003cli\u003eWaters ER and Vierling E (2020) Plant small heat shock proteins \u0026ndash; evolutionary and functional diversity. New Phytol. 227: 24-37. https://doi.org/10.1111/nph.16536.\u003c/li\u003e\n\u003cli\u003eYamakawa H, Hirose T, Kuroda M, Yamaguchi T (2007) Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiol. 144:258-77. https://doi.org/10.1104/pp.107.098665.\u003c/li\u003e\n\u003cli\u003eYamakawa H and Hakata M (2010) Atlas of rice grain filling-related metabolism under high temperature: joint analysis of metabolome and transcriptome demonstrated inhibition of starch accumulation and induction of amino acid accumulation. Plant Cell Physiol. 51:795-809. https://doi.org/10.1093/pcp/pcq122\u003c/li\u003e\n\u003cli\u003eZhao X and Fitzgerald M (2013) Climate Change: Implications for the Yield of Edible Rice. PLoS ONE 8(6): e66218. https://doi.org/10.1371/journal.pone.0066218\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6229149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6229149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWarming trend of the climate has been linked with yield losses in many crops. Like other cereals, rice is highly sensitive to above-typical temperatures during reproductive and grain filling stages. In fact, nighttime temperatures have risen faster than daytime temperatures in many parts of the world. For this reason, rice studies in recent years have focused on the effect of high nighttime temperature (HNT) on grain yield. Several studies have shown that HNT disturbs key processes in reproductive development and grain filling that lead to reduced spikelet fertility (SF) and enhanced grain chalkiness (Srivastava et al., 2024). Chalkiness is the opaque area on the grain, and it is not just an appearance issue, it also impacts milling quality. Above the generally acceptable chalk values (6-10%), every 1% increase in chalkiness leads to 1% decline in head rice yield (HRY) (Zhao and Fitzgerald 2013). Therefore, breeding HNT tolerance is vital for safeguarding grain yields from heat waves in the future. However, breeding efforts have been impeded by the lack of reliable tolerance alleles in modern cultivars. Breeding is also complicated by the complex nature of HNT tolerance as not only SF and grain quality traits, but other yield components such as panicle length, grain width, grain size, and grain weight are also affected by HNT in a genotype-dependent manner. Not surprising, hundreds of QTLs have been identified in the genomics studies. Of which, \u003cem\u003eChalk5\u003c/em\u003e (Os05g0156900) is most notable as it stands as one of the few functionally validated QTL (Fan et al., 2024; Gann et al., 2023; Li et al., 2014).\u003c/p\u003e","manuscriptTitle":"CRISPR/Cas9 based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 08:51:36","doi":"10.21203/rs.3.rs-6229149/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-04-13T04:17:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-20T10:20:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-17T18:10:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-17T10:05:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-03-14T16:59:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a3bb2705-2063-4337-a112-4a1fec6acb59","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:01:55+00:00","versionOfRecord":{"articleIdentity":"rs-6229149","link":"https://doi.org/10.1007/s00299-025-03504-y","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-05-10 15:57:34","publishedOnDateReadable":"May 10th, 2025"},"versionCreatedAt":"2025-03-26 08:51:36","video":"","vorDoi":"10.1007/s00299-025-03504-y","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03504-y","workflowStages":[]},"version":"v1","identity":"rs-6229149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6229149","identity":"rs-6229149","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}