Targeted mutagenesis of the vacuolar H⁺ translocating pyrophosphatase gene reduces grain chalkiness in rice

Abstract

SUMMARYGrain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V‐PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V‐PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V‐PPase. Here, we describe promoter targeting of the japonica V‐PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 suppressed V‐PPase activity, reduced grain chalkiness and impacted post‐germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V‐PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains.

A vacuolar proton pump controls post-germinative growth of rice (Oryza sativa ssp. japonica)

“…The resulting mutants showed 5 to 12-fold suppression of VPP5 in the caryopses, a relatively higher level of PPi, and enhanced expression of PPi-dependent enzyme genes, Fructose-6P 1phosphotransferase PPi (PFP), Pyruvate, orthophosphate dikinase (PPDK), and UGPase, indicating the effect of VPP5 on metabolic processes in the developing grains. Accordingly, vpp5 lines contained higher starch content and lower chalk in the mature grains, and most importantly, resisted chalk formation under high nighttime temperatures, a major inducer of grain chalkiness in rice (Gann et al, 2023). This study also found post-germinative growth defect in the vpp5 lines that was rescued by sucrose in the media.…”

“…Rice vpp5 mutants show downregulation of vacuolar H + translocating pyrophosphatase (V-PPase) gene in the developing caryopses, which correlates with the accumulation of PPi and an altered rate of starch metabolism during grain lling. These mutants showed a relatively slower post-germinative growth, which could be rescued by the exogenous supply of sucrose (Gann et al, 2023). By regulating cellular pH and PPi concentration, V-PPases regulate metabolism.…”

“…The process of starch degradation in the seed is initiated soon after the imbibition of water and continues until post-germinative growth (emergence and elongation of the seedling). Previously we reported that sucrose content in vpp5 germinating seeds was lower compared to WT that correlated with reduced rate of starch utilization (Gann et al, 2023). We measured the sucrose and starch contents in the germinated seeds at 3 and 6 DAG.…”

“…2b). Earlier we reported that the impacted postgerminative growth of vpp5 could be rescued by the addition of 1% sucrose in the media (Gann et al, 2023). Here, we analyzed the effect of 1% sucrose supplementation on the seedling length and fresh weight in 9-d-old seedlings grown in the media plates.…”

“…One of which is VPP5 that is expressed in the anther, ovary, embryo, and the endosperm during early grain lling stages. The physiological role of V-PPase in these tissues is unclear but its hyperactivity leads to chalky grains and its suppression ameliorates the trait including heat-induced grain chalkiness (Li et al, 2014;Gann et al, 2023).…”