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Granule-bound starch synthase I (GBSSI) encoding gene Waxy (Wx), which largely regulates the amylose content of rice grains, is a master module determining rice eating and cooking quality (ECQ). Fine-tuning amylose level of grains is an ideal strategy to improve rice quality. Through fine editing of Wx a promoter and 5'UTR by CRISPR/Cas9 system, we created 14 types of novel Wx allelic variations, of which MT7 and MT13 were able to alter Wx expression and amylose content of grains. MT7 showed fragment deletion and base insertions in CAAT-boxes, hardly detectable expression levels of GBSSI mRNA and protein, and generated 5.87% amylose in grains. MT13 had fragment deletions in the A-box and the TATA-box, low expression levels of GBSSI mRNA and protein, and generated 9.61% amylose in grains. Besides of the amylose content, MT7 and MT13 significantly reduced protein content and increased lipid content of grains compared with Wx a . A comparison of MT7, MT13 and other allelic lines demonstrated the importance of base insertion around the second CAAT-box and 31bp-deletion following the second TATA-box in modulating Wx expression. Thus, our study generated two novel Wx allelic variations which significantly alter Wx expression and amylose content of rice grains, providing not only new germplasms for soft rice breeding, but also insights into candidate cis elements of Wx., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Tai Wang reports financial support was provided by Chinese Academy of Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The type of soft rice with low amylose content (AC) is more and more favored by consumers for its better eating and cooking quality, as people's quality of life continuously improves in China. The Wx gene regulates the AC of rice grains, thus affecting the degree of softness of the rice. Mei Meng B (MMB), Tian Kang B (TKB), and DR462 are three indica rice maintained lines with good morphological characters, but also with undesirably high AC. Therefore, CRISPR/Cas9 technology was used to edit the Wx gene of these lines to create a batch of soft rice breeding materials. New gene-edited lines MMB-10-2, TKB-21-12, and DR462-9-9, derived from the above parental lines, respectively, were selected in the T 2 generations, with an AC of 17.2%, 16.8%, and 17.8%, and gel consistency (GC) of 78.6 mm, 77.4 mm, and 79.6 mm, respectively. The rapid viscosity analysis (RVA) spectrum showed that the three edited lines had a better eating quality as compared to the corresponding wild type, and showing new characteristics, different from the high-quality soft rice popular in the market. There was no significant difference in the main agronomic traits in the three edited lines compared to the corresponding wild types. Moreover, the chalkiness of DR462-9-9 was reduced, resulting in an improved appearance of its polished rice. The present study created soft rice germplasms for breeding improved quality hybrid rice, without changing the excellent traits of their corresponding wild type varieties.

Background: The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5'splicing site of its 1st intron greatly affects this intron's splicing efficiency and defines two predominant Wx alleles, Wx a and Wx b . Wx a rice often harbours intermediate to high amylose contents, whereas Wx b rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content., Results: CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wx b ), X32 (Wx b ), X35 (Wx a ) and X55 (Wx lv ). Deletion of the 1st intron occurred in 8.6-11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wx b allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wx a and Wx lv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed., Conclusions: This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wx b allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality., (© 2022. The Author(s).)

The Waxy (Wx) gene that governs amylose synthesis is an old but widely used target in improving the quality of starchy crops. New genome-editing strategies are being deployed to create beneficial Wx alleles with finely tuned amylose content (AC). Precise targeting must be combined with traditional approaches to develop healthier and high-quality breeding lines., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: With the rapid development of modern agriculture, high-quality rice production and consumption has become the current urgent demand for the development of rice production. In this paper, the effects of late-stage nitrogen fertilizer application on rice quality were studied under the same genetic background. Wx near-isogenic lines were used as test materials to study the starch composition, amylopectin structure and cooking quality of rice., Results: Results showed that rice amylose content and gel consistency significantly differed when different Wx genes were tranformed into waxy rice. The law of apparent amylose content in rice is Wx a > Wx in > Wx b > wx at the same nitrogen level, while the trend of gel consistency was opposite to that of apparent amylose content, presenting obvious characteristics of Indica and Japonica varieties. As the amount of fertilizer application increased, apparent amylose content increased, gel consistency decreased, breakdown and peak viscosities dropped and setback viscosity and peak time increased. Moreover, the cooking quality of rice significantly decreased with the use of nitrogen fertilizer, especially under low-level nitrogen fertilizer application. Amylopectin structure varied significantly in different genotypes of the Wx gene, and the degree of branching was as follows: wx > Wx b > Wx in > Wx a . This result indicated that the closer to Indica rice, the fewer short chains of amylopectin. Starch crystallinity and swelling potential were negatively correlated with amylose content but significantly positively correlated with amylopectin branching degree, decreasing with the increase of late-stage nitrogen fertilization., Conclusion: Late-stage nitrogen fertilization reduced the cooking quality of rice by increasing amylose content and reducing amylopectin branching degree, which decreased starch crystallinity and aggravated pasting properties. Obviously, controlling late nitrogen application is essential to optimize rice quality. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)