Next Article in Journal
Nutritional Facts and Health/Nutrition Claims of Commercial Plant-Based Infant Foods: Where Do We Stand?
Next Article in Special Issue
Transcriptome and Metabolome Analyses Reveal New Insights into the Regulatory Mechanism of Head Milled Rice Rate
Previous Article in Journal
Edible Flower Species as a Promising Source of Specialized Metabolites
Previous Article in Special Issue
Combined Effects of Different Alleles of FLO2, Wx and SSIIa on the Cooking and Eating Quality of Rice
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 11
Issue 19
10.3390/plants11192530
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Control of Grain Shape and Size in Rice by Two Functional Alleles of OsPUB3 in Varied Genetic Background

by
Zhu-Hao Li
1,†,
Shi-Lin Wang
1,†,
Yu-Jun Zhu
1,
Ye-Yang Fan
1,
De-Run Huang
1,
Ai-Ke Zhu
2,
Jie-Yun Zhuang
1,
Yan Liang
1,* and
Zhen-Hua Zhang
1,*
1
State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China
2
Nanchong Academy of Agricultural Sciences, Nanchong 637000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2022, 11(19), 2530; https://doi.org/10.3390/plants11192530
Submission received: 29 August 2022 / Revised: 20 September 2022 / Accepted: 23 September 2022 / Published: 27 September 2022
(This article belongs to the Special Issue Germplasm Enhancement and Breeding for Rice Quality Improvement)
Browse Figures
Versions Notes

Abstract

:
Grain shape and size are key determinants of grain appearance quality and yield in rice. In our previous study, a grain shape QTL, qGS1-35.2, was fine-mapped using near-isogenic lines (NILs) derived from a cross between Zhenshan 97 (ZS97) and Milyang 46 (MY46). One annotated gene, OsPUB3, was found to be the most likely candidate gene. Here, knockout and overexpression experiments were performed to investigate the effects of OsPUB3 on grain shape and size. Four traits were tested, including grain length, grain width, grain weight, and the ratio of grain length to width. Knockout of OsPUB3 in NILZS97, NILMY46, and another rice cultivar carrying the OsPUB3MY46 allele all caused decreases in grain width and weight and increases in the ratio of grain length to width. Results also showed that the magnitude of the mutational effects varied depending on the target allele and the genetic background. Moreover, it was found that NILZS97 and NILMY46 carried different functional alleles of OsPUB3, causing differences in grain shape rather than grain weight. In the overexpression experiment, significant differences between transgenic-positive and transgenic-negative plants were detected in all four traits. These results indicate that OsPUB3 regulates grain shape and size through a complex mechanism and is a good target for deciphering the regulatory network of grain shape. This gene could be used to improve grain appearance quality through molecular breeding as well.

1. Introduction

Rice (Oryza sativa L.) provides a staple food source for more than half the world’s population. Grain shape and size are important appearance quality of rice, which are one of the most direct characteristics for consumers and influence the market value of grain products. In general, long and slender grains have higher competitive market value, which is preferred by consumers in most regions of the world [1,2,3,4]. Grain size is also a major determinant of grain weight, one of the three yield components (number of panicles per plant, number of grains per panicle, and grain weight). Therefore, understanding the genetic basis of grain shape and size is vital for improving grain quality and yield of rice.
Grain shape and size are largely determined by grain length and width, which are controlled by a large number of quantitative trait loci (QTL). At least 25 QTLs for grain shape and size in rice have been cloned. Fifteen of them mainly regulated grain length, including qTGW1.2b [5], GS2/GL2 [6,7], OsLG3 [8], OsLG3b/qLGY3 [9,10], GS3.1 [11], GS3 [12], SG3 [13], GL3.1/qGL3 [14,15], qTGW3 [16], qGL5 [17], TGW6 [18], GW6a [19], GL6 [20], GLW7 [21] and GL10/OsMADS56 [22,23]. Seven other QTLs mainly regulated grain width, including GW2 [24], TGW2 [25], GS5 [26], GSE5 [27], GW6 [28], GW8 [29] and GW10 [30]. The remaining three genes, including GSA1 [31], GL7/GW7 [32,33], and GS9 [34], exhibited similar effects on grain length and width. All of these QTLs conferred significant influence on grain weight, except GL7/GW7 and GS9. Characterization of these QTLs has greatly enriched our knowledge of the genetic control of grain size in rice. Some elite alleles of these QTLs have been tried for targeted improvement of rice by genome editing system [3,35,36,37]. However, much more effort is needed to fully understand the regulatory mechanisms of grain shape and size [4,38,39]. Moreover, the identification of more genes could provide the flexibility needed to design various rice grain shapes [4].
Ubiquitination is a post-translational modification that regulates protein stability. Ubiquitination requires a series of enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3) [40,41,42]. E1 activates ubiquitin and transfers it to E2. E3 promotes the transfer of the ubiquitin conjugated by E2 to the substrate. In the ubiquitination process, the substrate is mainly recognized by E3 which is usually classified into three groups: the RING and U-box type, the HECT type, and the RBR type [43]. In addition, ubiquitination is dynamic and can be removed by deubiquitinating enzymes [43]. The ubiquitin-proteasome pathway is known to play an important role in regulating grain shape and size. GW2 was the first cloned QTL for grain width and weight, which encodes a RING-type E3 ubiquitin ligase [24]. A recent study revealed that GW2 could ubiquitinate a glutaredoxin protein WG1 and direct WG1 to the proteasome for degradation. The degradation eliminated the repression of the transcriptional activity of OsbZIP47 by WG1 and promoted the transcription of downstream genes, which consequently regulated grain shape and weight [44]. Another gene, WTG1/qNPT1 identified as a major QTL determining the “new plant type” architecture [45], encoded a deubiquitinating enzyme with homology to human OTUB1 and negatively regulated grain width and weight [46]. WTG1/qNPT1 could physically interact with the E2 ubiquitin-conjugating protein OsUBC13 and transcription factor OsSPL14. This interaction affected ubiquitination and proteasomal degradation of OsSPL14 [45].
A minor QTL for grain shape, qGS1-35.2, was previously fine-mapped into a 57.7-kb region on chromosome 1 using near-isogenic lines (NILs) derived from a cross between indica rice cultivars Zhenshan 97 (ZS97) and Milyang 46 (MY46). One annotated gene, Os01g0823900 encoding a U-box type E3 ubiquitin ligase OsPUB3, was found to be the most likely candidate gene [47]. In order to investigate the effect of OsPUB3 on grain shape and size, we performed knockout and overexpression experiments in the present study. Knockout of OsPUB3 in three rice cultivars carrying the ZS97 or MY46 allele all caused changes in grain shape and size, and the magnitude of a mutational effect varied depending on the target allele and genetic background. Overexpression of OsPUB3 also brought about changes in grain shape and size, whereas the effect of overexpression was not exactly opposite to the effect of knockout experiments. These results suggested that OsPUB3 regulates grain development through a complex mechanism and is a good target gene for deciphering the regulatory network of grain shape and size in rice. In addition, OsPUB3 may coordinate the trade-off between grain weight and other yield traits. Knockout reduced grain weight but increased panicle or grain number, resulting in stable grain yield. Therefore, OsPUB3 could be used to improve grain appearance quality without yield penalty. Overall, our study provides a new genetic resource to improve grain appearance quality and explore the regulatory framework for grain shape and size.

2. Results

2.1. Knockout Mutants of OsPUB3 Produced from Three Rice Cultivars

The CRISPR/Cas9 system was used to produce mutants for validating the effects of OsPUB3. OsPUB3 protein contained a U-box motif and Armadillo (ARM) repeats in the central region. A site located in the distal N-terminal region was selected as the target for CRISPR/Cas9 gene editing (Figure 1A). To compare the effects of the two parental alleles, OsPUB3ZS97 and OsPUB3MY46, and to test the effects of OsPUB3 in different genetic backgrounds, three recipients were used. They are NILZS97 and NILMY46 previously used for fine-mapping qGS1-35.2, and another indica rice cultivar carrying the OsPUB3MY46 allele, Zhonghui 161 (ZH161). NILZS97 and NILMY46 have a small difference in grain shape and size, whereas the grain of ZH161 is much thinner and smaller (Figure 2).
A total of twelve independent T0 mutants were obtained, including three homozygous and nine biallelic mutants (Figure 1B). For NILZS97, two homozygous mutants and one transgenic-negative control were identified. KO-ZS-1 had a 1 bp deletion and KO-ZS-2 had a 1 bp insertion. For NILMY46, one homozygous and six biallelic mutants were identified. No negative transformant was found. The homozygous mutant had a 1 bp insertion, and each of the biallelic mutants contained a 1 bp insertion and a 1 bp deletion. For ZH161, three biallelic mutants and one negative transformant were identified. KO-ZH-1 contained a 1 bp insertion and a 1 bp deletion, KO-ZH-2 contained a 1 bp insertion and a 22 bp deletion, and KO-ZH-3 had 22 bp and 35 bp deletions. The mutations all led to a frameshift and produced premature stops before the U-box motif and ARM repeats (Figure S1).

2.2. Phenotypic Change Due to OsPUB3 Knockout

Phenotypic changes resulting from the knockout of OsPUB3 were tested in 2021. Three sets of rice materials were used: (a) recipient NILZS97, T1 lines of the two homozygous mutants, and a transgenic-negative control (CKZS97); (b) recipient NILMY46, a T1 line of the homozygous mutant, and six T1 populations segregating the biallelic mutations; (c) recipient ZH161, six T2 homozygous mutants derived from the three biallelic mutants, and a transgenic-negative control (CKZH161). All these populations were measured for four traits of grain shape and size, including grain length (GL), grain width (GW), 1000 grain weight (TGW), and the ratio of grain length to width (RLW). The ZH161-type lines were additionally measured for other five traits, including the number of panicles per plant (NP), number of spikelets per panicle (NSP), number of grains per panicle (NGP), grain yield per plant (GY), and heading date (HD). Student’s t-test was performed to analyze phenotypic differences between mutants and controls.
The two homozygous mutants of NILZS97 both showed significant decreases in GL, GW, and TGW compared with the recipient and CK (Table 1). Decreases in KO-ZS-1 and KO-ZS-2 over CKZS97 were 0.102 and 0.204 mm for GL, 0.080 and 0.122 mm for GW, and 2.14 and 2.67 g for TGW, respectively. The CK itself decreased over the recipient NILZS97 on these traits. Thus, decreases in the two mutants over NILZS97 were larger, becoming 0.266 and 0.369 mm for GL, 0.091 and 0.133 mm for GW, and 3.02 and 3.55 g for TGW. Because changes in GL and GW had the same direction, their influences on the GL/GW ratio were mitigated. The two mutants showed non-significant differences with NILZS97 and significant increases over CKZS97.
The NILMY46 mutants were only tested against the recipient due to unavailable transgenic CK. In each of the six segregating populations, non-significant phenotypic variation was found among the three genotypic groups (data not shown); thus, data of the three genotypes were merged. Together with the homozygous mutant KO-MY-1, a total of seven mutants were analyzed (Table 1). Compared with NILMY46, the mutants all showed decreases in GL, GW, and TGW, which were significant except for GL in KO-MY-1 and KO-MY-3. The decreases in GL, GW, and TGW ranged from 0.010–0.270 mm, 0.065–0.218 mm, and 1.72–4.23 g, and averaged 0.124 mm, 0.143 mm, and 3.02 g, respectively. Compared with the differences between NILZS97 and its mutants, these effects had the same direction, and the decreases were smaller on GL, larger on GW, and similar on TGW. Accordingly, increases in the GL/GW ratio over the control were much larger in the mutants of NILMY46 than that of NILZS97.
Among the six homozygous mutants of ZH161, all and five showed significant decreases in GW and TGW over the two controls. Compared with ZH161 and CKZH161, the decreases were averaged as 0.093 and 0.068 mm for GW and 0.052 and 0.072 g for TGW, respectively (Table 1). For GL and RLW, four and six mutants showed significant increases over the two controls. Compared with ZH161 and CKZH161, the increases were averaged as 0.215 and 0.114 mm for GL and 0.184 and 0.111 for RLW, respectively. The mutational directions, compared with that detected in the NILZS97 and NILMY46 populations, were the opposite for GL but the same for the other three traits. Variations among the three experiments were also found in the magnitude of mutational effects on GW, TGW, and RLW. In accordance with the thinner and smaller grains of ZH161 than NILZS97 and NILMY46, the knockout caused fewer decreases in GW and TGW when using ZH161 as the recipient, especially in TGW. For RLW, knockout on the OsPUB3MY46 allele carried by ZH161 and NILMY46 caused much larger increases than on the ZS97 allele carried by NILZS97. As for the five traits that were only tested in the ZH161 population, the most consistent results were found in the differences in NP, NSP, and NGP between mutants and CKZH161. General decreases in NP and increases in NSP and NGP were observed (Table S1).
In summary, the knockout of OsPUB3 in the three recipients all resulted in increased RLW and decreased GW and TGW, whereas the effects on GL were less consistent. Meanwhile, the magnitude of the mutational effects on GW, TGW, and RLW varied depending on the target allele as well as the genetic background. NILMY46 is the only recipient on which OsPUB3 knockout caused large changes for all the RLW, GW, and TGW.

2.3. Effects of Expressing OsPUB3 with Rice Actin 1 Promoter

Overexpression transgenic plants were generated to further validate the effects of OsPUB3. Since the knockout of OsPUB3 in NILMY46 caused the largest effect, we amplified the coding sequence of OsPUB3 from MY46 and introduced it into NILMY46 driven by a rice Actin 1 promoter. Four independent T1 populations, namely OE-1 to OE-4, were selected to measure the four grain-shape traits. The Student’s t-test was performed to determine phenotypic differences between negative and positive transgenic plants in each T1 population.
Significant differences were detected for all four traits in at least two populations (Table 2). For GL, a significant increase in positive plants over negative plants was observed in each population. The increases ranged from 0.049 to 0.104 mm. For GW, a decrease in positive plants over negative plants was shown in each population but only significant in two populations, OE-1 and OE-4, reduced by 0.039 and 0.042 mm, respectively. For TGW, a significant increase in positive plants over negative plants was detected in two populations, OE-2 and OE-4, rising by 0.84 and 0.46 g, respectively. The other two populations showed a non-significant increase or decrease. For RLW, an increase in positive plants over negative plants was observed in each population, but only significant in three populations, OE-1, OE-3, and OE-4. The significant increases ranged from 0.051 to 0.074. These results showed that overexpression of the OsPUB3MY46 allele in NILMY46 increased GL but decreased GW. Due to the effect being larger on GL than on GW, increases were observed for RLW and TGW.

3. Discussion

Most important agronomic traits are controlled by a few major QTLs and many minor QTLs. Only a small number of genes underlying minor QTLs have been isolated in rice [5,13,23,48,49]. In our previous study, minor QTL qGS1-35.2 was fine-mapped using NIL populations derived from a cross between ZS97 and MY46. One annotated gene, OsPUB3, was found to be the most likely candidate gene. In these NIL populations, the ZS97 allele had a slight effect causing larger GL and smaller GW compared to the MY46 allele, thereby qGS1-35.2 exhibited a significant effect on RLW [47]. In the present study, the effect of OsPUB3 was investigated through knockout and overexpression experiments. Knockout of OsPUB3 in NILZS97, NILMY46, and ZH161, another cultivar carrying the OsPUB3MY46 allele, caused consistent effects on RLW, GW, and TGW, as well as significant but less consistent effects on GL. These effects, reflecting differences between function and loss-of-function alleles, are much larger than those detected in the fine-mapping of qGS1-35.2 that reflected the differences between the functional OsPUB3ZS97 and OsPUB3MY46 alleles. Comparing the mutational effects on the four traits between NILZS97 and NILMY46, changes of 3.28 and 3.02 g in TGW are the closest, and that of 0.000 and 0.098 in RLW are the furthest. This is in agreement with the previous result that the effect of qGS1-35.2 was significant on grain shape and non-significant on grain weight. These results also confirm our previous hypothesis that the small effect of a minor QTL is a result of the small contrast between partially function alleles carried by the parental lines [5]. There is abundant genetic variation in natural populations, and different alleles of a gene may have different functions [50,51,52]. The identification and screening of different alleles are helpful to rice improvement.
Overexpression and knockout/knockdown of a same gene usually causes opposite effects [10,11,13,15,17,20,23,25,27,28,31,34]. However, we surprisingly discovered that overexpressing and knockout OsPUB3MY46 both decreased GW. This implied that OsPUB3 regulated GW by a rare molecular mechanism. OsPUB3 encodes a U-box type E3 ubiquitin ligase, which has been verified to have ubiquitination activities [53]. There are very few examples of overexpression and knockout/knockdown of the same gene exhibiting a similar phenotype [54,55,56,57,58,59,60,61]. Interestingly, three of them were found to be involved in the ubiquitin-proteasome pathway. SPL35 regulated rice defense response by interacting with a ubiquitin-conjugating E2 enzyme. Both overexpression and knockdown of SPL35 caused the lesion to mimic the phenotype [61]. UNC-45 functioned the organization of myosin. Loss of function of UNC-45 resulted in paralyzed animals. The overexpressing UNC-45 promoted the nonnative myosin conformation that was degraded by ubiquitination complexes, displaying a paralysis phenotype as well [55,57,59]. Usp28 encoded a deubiquitinase that stabilized the subunit of SCF ubiquitin ligase, Fbw7. At the highest level, Usp28 could also stabilize substrates of Fbw7, proto-oncogenes. Knockout of Usp28 triggered Fbw7 degradation and accumulation of Fbw7 substrates, resulting in oncogenic transformation. Overexpressing Usp28 stabilized both Fbw7 and its substrates, also causing oncogenic transformation [58]. These examples illustrated that ubiquitination exquisitely regulates protein turnover and homeostasis through a complex interaction system, though the regulatory mechanisms of these genes were different. Whether OsPUB3 regulated GW through one of the similar molecular mechanisms described above or through other mechanisms remains to be explored.
Our results also showed that the effects of OsPUB3 varied depending on genetic background. Knockout of OsPUB3MY46 decreased GL in the NIL background but increased the trait in the ZH161 background. As for GW, the magnitude of the mutational effect was much larger in the NIL background than that in the ZH161 background, though knockout OsPUB3MY46 always exhibited decreasing effects. Consequently, the decrease in TGW was approximately five-fold greater in the NIL background than that in the ZH161 background, while the increase in RLW in the NIL background was only half of that in the ZH161 background. The grains of ZH161 were much thinner and smaller compared to those of NIL. The effect variation of OsPUB3 in the two backgrounds may be related to the QTLs responsible for the difference in grain shape between the two varieties. The genetic interaction between OsPUB3 and these QTLs would be a good starting point to investigate the molecular mechanisms underlying the role of OsPUB3 in regulating grain shape and size. Overall, OsPUB3 regulates grain development through a complex mechanism and is a good target gene for deciphering the regulatory network of grain shape and size in rice.
Grain shape is an important appearance quality, which greatly influences the market value of rice products. Large RLW generally confers better appearance quality and competitive market value. Some breeding programs recently even attempted to convert the traditional short and bold grains of japonica rice into long and slender grains because of their excellent appearance quality [55,56]. Due to a more obvious reduction in grain width or increase in grain length, overexpressing OsPUB3 and knockout in different genetic backgrounds always increased the RLW. Moreover, OsPUB3 seems to coordinate the trade-off between grain weight and other yield traits. The knockout of OsPUB3 reduced grain weight but increased the number of panicles or grains, resulting in a stable grain yield. Therefore, OsPUB3 could be used to improve grain appearance quality without yield penalty through genetically modified breeding.

4. Materials and Methods

4.1. Knockout OsPUB3 in Three Rice Cultivars

The CRISPR/Cas9 system was used to knockout OsPUB3. One target, located at +74 to +93 in the coding region (Figure 1A), was selected using the web-based tool CRISPR-GE (http://skl.scau.edu.cn, accessed on 6 March 2018). The oligonucleotides 3900-cri (Table S2) were designed and ligated into the BGK03 vector (BIOGLE Co., Ltd., Hangzhou, China) according to the manufacturer’s instructions. The original BGK03 vector contains a rice U6 promoter for activating the target sequence, a Cas9 gene driven by the maize ubiquitin promoter, and a hygromycin marker gene driven by the Cauliflower mosaic virus 35S promoter.
Three transgenic recipients were used, including NILZS97, NILMY46, and ZH161. The NILZS97 and NILMY46 were derived from the cross between ZS97 and MY46, which were previously used for fine-mapping qGS1-35.2. ZH161 is an indica rice cultivar carrying the OsPUB3MY46 allele. The ORF sequence of OsPUB3 in ZH161 was analyzed according to the following procedure. Genomic DNA was extracted from a 2 cm long leaf sample of ZH161 using DNeasy Plant Mini Kit (QIAGEN, Hilden, German). Full-length ORF of OsPUB3 was amplified using the primer pairs 3900-seq (Table S2) and the product was sequenced by the Sanger method.
The CRISPR/Cas9 constructs were separately introduced into three recipients using Agrobacterium tumefaciens-mediated transformation. Genomic DNA of T0 plants was extracted from a 2 cm long leaf sample using the DNeasy Plant Mini Kit. They were assayed with the hygromycin gene marker Hyg (Table S2). The OsPUB3 gene fragment was amplified from each Hyg-positive plant using the primer pairs 3900-cri-seq (Table S2). The product was directly sequenced by the Sanger method and decoded using the web-based tool DSDecodeM (http://skl.scau.edu.cn/dsdecode, accessed on 10 May 2019 and 27 July 2020).

4.2. Expressing OsPUB3 with Rice Actin 1 Promoter

Total RNA was extracted from leaves of MY46 using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). The 1st strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). The OsPUB3 cDNA fragment that contained the full coding sequence was amplified using the primer pairs 3900-oe (Table S2). The product was recombined in the pCAMBIA2300 vector using an In-Fusion Advantage Cloning kit (Clontech, Osaka, Japan). The original pCAMBIA2300 vector comprised a rice Actin1 promoter for activating the target sequence and a neomycin marker gene driven by CaMV 35S promoter. The overexpression construct was introduced into NILMY46 using Agrobacterium tumefaciens-mediated transformation. Total DNA was extracted from a 2 cm long leaf sample of transgenic plants using the method of Zheng et al. [62]. Genotypes of transgenic plants were assayed with the neomycin gene marker Neo marker (Table S2).

4.3. Field Experiments and Phenotyping

All the rice materials were tested in 2021 at paddy field in the China National Rice Research Institute in Hangzhou, Zhejiang Province, China. Seeds were sown in paddy fields in May and raised in wet bed condition for 26 days. Then, the plants were transplanted with spacing of 16.7 cm between plants and 26.7 cm between rows. In each population, the plants were randomly planted. Normal agricultural practice was employed in field management. From transplanting to harvesting, irrigation was applied to maintain a well-watered condition except that the water was drained for five days at the maximum tillering stage and before harvesting.
For knockout experiment in NILZS97, each of the four T1 homozygous lines contained 10 plants (Table 1). For knockout experiment in NILMY46, 12 plants were planted for the control NILMY46 and 36 T1 plants were grown for each of the six segregating populations and one homozygous line. Genotyping was performed using the marker 3900-cri-seq, and those showing an ambiguous genotype were removed. For knockout experiment in ZH161, each of the eight T2 homozygous lines contained 30 plants. For overexpression experiment, 30 T1 plants were grown for each population. Genotyping was performed using the Neo marker.
In October, plants in each population were individually harvested at maturity for phenotyping. Four traits of grain shape and size, including GL, GW, TGW, and RLW, were measured in all experiments. Approximately 6 g of fully filled grains were divided into two halves and measured for the four traits using an automatic seed counting and analyzing instrument (Model SC-G, Wanshen Ltd., Hangzhou, China). Other five traits, including NP, NSP, NGP, GY, and HD, were additionally measured in the ZH161 knockout experiment. The Student’s t-test was performed to determine phenotypic differences between mutants and controls in the knockout experiments and between negative and positive transgenic plants in the overexpression experiment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11192530/s1, Figure S1: Amino acid sequences of OsPUB3 in the three recipients (NILZS97, NILMY46, and ZH161) and their homozygous mutants; Table S1: Four yield traits and heading date in ZH161, the transgenic-negative control, and knockout mutants of OsPUB3; Table S2: Primers used in this study.

Author Contributions

J.-Y.Z., Z.-H.Z. and Y.L. conceived and designed the research; Z.-H.Z. and S.-L.W. constructed the populations; S.-L.W., Z.-H.L. and Y.-Y.F. performed genotyping; S.-L.W., Z.-H.L., Y.-J.Z., D.-R.H. and A.-K.Z. conducted phenotyping; J.-Y.Z., Z.-H.Z. and S.-L.W. analyzed the data; J.-Y.Z., Z.-H.Z., Y.L. and Z.-H.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Sichuan Province Regional Innovation Cooperation Project (2022YFQ0111), the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2021-CNRRI), the Central Public-interest Scientific Institution Basal Research Fund (CPSIBRF-CNRRI-202112), the Science Technology Department of Zhejiang Province to the Zhejiang Agricultural Key Breeding Project (2021C02063-4), and the China National Rice Research Institute Key Research and Development Project (CNRRI-2020-01).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Calingacion, M.; Laborte, A.; Nelson, A.; Resurreccion, A.; Concepcin, J.C.; Daygon, V.D.; Mumm, R.; Reinke, R.; Dipti, S.; Bassinello, P.Z.; et al. Diversity of global rice markets and the science required for consumer-targeted rice breeding. PLoS ONE 2014, 9, e85106. [Google Scholar] [CrossRef]
  2. Huang, H.X.; Qian, Q. Progress in genetic research of rice grain shape and breeding achievements of long-grain shape and good quality japonica rice. Chin. J. Rice Sci. 2017, 31, 665–672. [Google Scholar]
  3. Mao, T.; Zhu, M.D.; Sheng, Z.H.; Shao, G.N.; Jiao, G.A.; Mawia, A.M.; Ahmad, S.; Xie, L.H.; Tang, S.Q.; Wei, X.J.; et al. Effects of grain shape genes editing on appearance quality of erect-panicle geng/japonica rice. Rice 2021, 14, 74. [Google Scholar] [CrossRef]
  4. Zhao, D.; Zhang, C.; Li, Q.; Liu, Q. Genetic control of grain appearance quality in rice. Biotechnol. Adv. 2022, 60, 108014. [Google Scholar] [CrossRef]
  5. Chan, A.N.; Wang, L.L.; Zhu, Y.J.; Fan, Y.Y.; Zhuang, J.Y.; Zhang, Z.H. Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice. Theor. Appl. Genet. 2021, 134, 327–337. [Google Scholar] [CrossRef]
  6. Che, R.; Tong, H.; Shi, B.; Liu, Y.; Fang, S.; Liu, D.; Xiao, Y.; Hu, B.; Liu, L.; Wang, H.; et al. Control of grain size and rice yield by GL2-mediated brassinosteroid responses. Nat. Plants 2015, 2, 15195. [Google Scholar] [CrossRef]
  7. Hu, J.; Wang, Y.; Fang, Y.; Zeng, L.; Xu, J.; Yu, H.; Shi, Z.; Pan, J.; Zhang, D.; Kang, S.; et al. A rare allele of GS2 enhances grain size and grain yield in rice. Mol. Plant 2015, 8, 1455–1465. [Google Scholar] [CrossRef]
  8. Yu, J.; Xiong, H.; Zhu, X.; Zhang, H.; Li, H.; Miao, J.; Wang, W.; Tang, Z.; Zhang, Z.; Yao, G.; et al. OsLG3 contributing to rice grain length and yield was mined by Ho-LAMap. BMC Biol. 2017, 15, 28. [Google Scholar] [CrossRef]
  9. Liu, Q.; Han, R.; Wu, K.; Zhang, J.; Ye, Y.; Wang, S.; Chen, J.; Pan, Y.; Li, Q.; Xu, X.; et al. G-protein βγ subunits determine grain size through interaction with MADS-domain transcription factors in rice. Nat. Commun. 2018, 9, 852. [Google Scholar] [CrossRef]
  10. Yu, J.; Miao, J.; Zhang, Z.; Xiong, H.; Zhu, X.; Sun, X.; Pan, Y.; Liang, Y.; Zhang, Q.; Abdul Rehman, R.M.; et al. Alternative splicing of OsLG3b controls grain length and yield in japonica rice. Plant Biotechnol. J. 2018, 16, 1667–1678. [Google Scholar] [CrossRef]
  11. Zhang, Y.M.; Yu, H.X.; Ye, W.W.; Shan, J.X.; Dong, N.Q.; Guo, T.; Kan, Y.; Xiang, Y.H.; Zhang, H.; Yang, Y.B.; et al. A rice QTL GS3.1 regulates grain size through metabolic-flux distribution between flavonoid and lignin metabolons without affecting stress tolerance. Commun. Biol. 2021, 4, 1171–1185. [Google Scholar] [CrossRef]
  12. Fan, C.; Xing, Y.; Mao, H.; Lu, T.; Han, B.; Xu, C.; Li, X.; Zhang, Q. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor. Appl. Genet. 2006, 112, 1164–1171. [Google Scholar] [CrossRef]
  13. Li, Q.; Lu, L.; Liu, H.; Bai, X.; Zhou, X.; Wu, B.; Yuan, M.; Yang, L.; Xing, Y. A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice. Theor. Appl. Genet. 2020, 133, 2387–2399. [Google Scholar] [CrossRef]
  14. Qi, P.; Lin, Y.S.; Song, X.J.; Shen, J.B.; Huang, W.; Shan, J.X.; Zhu, M.Z.; Jiang, L.; Gao, J.P.; Lin, H.X. The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3. Cell Res. 2012, 22, 1666–1680. [Google Scholar] [CrossRef]
  15. Zhang, X.; Wang, J.; Huang, J.; Lan, H.; Wang, C.; Yin, C.; Wu, Y.; Tang, H.; Qian, Q.; Li, J.; et al. Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice. Proc. Natl. Acad. Sci. USA 2012, 109, 21534–21539. [Google Scholar] [CrossRef]
  16. Hu, Z.; Lu, S.J.; Wang, M.J.; He, H.; Sun, L.; Wang, H.; Liu, X.H.; Jiang, L.; Sun, J.L.; Xin, X.; et al. A novel QTL qTGW3 encodes the GSK3/SHAGGY-like kinase OsGSK5/OsSK41 that interacts with OsARF4 to negatively regulate grain size and weight in rice. Mol. Plant 2018, 11, 736–749. [Google Scholar] [CrossRef]
  17. Qiao, J.; Jiang, H.; Lin, Y.; Shang, L.; Wang, M.; Li, D.; Fu, X.; Geisler, M.; Qi, Y.; Gao, Z.; et al. A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice. Mol. Plant 2021, 14, 1683–1698. [Google Scholar] [CrossRef]
  18. Ishimaru, K.; Hirotsu, N.; Madoka, Y.; Murakami, N.; Hara, N.; Onodera, H.; Kashiwagi, T.; Ujiie, K.; Shimizu, B.; Onishi, A.; et al. Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield. Nat. Genet. 2013, 45, 707–711. [Google Scholar] [CrossRef]
  19. Song, X.J.; Kuroha, T.; Ayano, M.; Furuta, T.; Nagai, K.; Komeda, N.; Segami, S.; Miura, K.; Ogawa, D.; Kamura, T.; et al. Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice. Proc. Natl. Acad. Sci. USA 2015, 112, 76–81. [Google Scholar] [CrossRef]
  20. Wang, A.; Hou, Q.; Si, L.; Huang, X.; Luo, J.; Lu, D.; Zhu, J.; Shangguan, Y.; Miao, J.; Xie, Y.; et al. The PLATZ transcription factor GL6 affects grain length and number in rice. Plant Physiol. 2019, 180, 2077–2090. [Google Scholar] [CrossRef]
  21. Si, L.; Chen, J.; Huang, X.; Gong, H.; Luo, J.; Hou, Q.; Zhou, T.; Lu, T.; Zhu, J.; Shangguan, Y.; et al. OsSPL13 controls grain size in cultivated rice. Nat. Genet. 2016, 48, 447–456. [Google Scholar] [CrossRef] [PubMed]
  22. Zhan, P.L.; Ma, S.P.; Xiao, Z.L.; Li, F.P.; Wei, X.; Lin, S.J.; Wang, X.L.; Ji, Z.; Fu, Y.; Pan, J.H.; et al. Natural variations in grain length 10 (GL10) regulate rice grain size. J. Genet. Genomics 2022, 49, 405–413. [Google Scholar] [CrossRef]
  23. Zuo, Z.W.; Zhang, Z.H.; Huang, D.R.; Fan, Y.Y.; Yu, S.B.; Zhuang, J.Y.; Zhu, Y.J. Control of thousand-grain weight by OsMADS56 in rice. Int. J. Mol. Sci. 2021, 23, 125. [Google Scholar] [CrossRef]
  24. Song, X.J.; Huang, W.; Shi, M.; Zhu, M.Z.; Lin, H.X. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat. Genet. 2007, 39, 623–630. [Google Scholar] [CrossRef] [PubMed]
  25. Ruan, B.; Shang, L.; Zhang, B.; Hu, J.; Wang, Y.; Lin, H.; Zhang, A.; Liu, C.; Peng, Y.; Zhu, L.; et al. Natural variation in the promoter of TGW2 determines grain width and weight in rice. New Phytol. 2020, 227, 629–640. [Google Scholar] [CrossRef] [PubMed]
  26. Li, Y.; Fan, C.; Xing, Y.; Jiang, Y.; Luo, L.; Sun, L.; Shao, D.; Xu, C.; Li, X.; Xiao, J.; et al. Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nat. Genet. 2011, 43, 1266–1269. [Google Scholar] [CrossRef] [PubMed]
  27. Duan, P.; Xu, J.; Zeng, D.; Zhang, B.; Geng, M.; Zhang, G.; Huang, K.; Huang, L.; Xu, R.; Ge, S.; et al. Natural variation in the promoter of GSE5 contributes to grain size diversity in rice. Mol. Plant 2017, 10, 685–694. [Google Scholar] [CrossRef]
  28. Shi, C.L.; Dong, N.Q.; Guo, T.; Ye, W.W.; Shan, J.X.; Lin, H.X. A quantitative trait locus GW6 controls rice grain size and yield through the gibberellin pathway. Plant J. 2020, 103, 1174–1188. [Google Scholar] [CrossRef]
  29. Wang, S.; Wu, K.; Yuan, Q.; Liu, X.; Liu, Z.; Lin, X.; Zeng, R.; Zhu, H.; Dong, G.; Qian, Q.; et al. Control of grain size, shape and quality by OsSPL16 in rice. Nat. Genet. 2012, 44, 950–954. [Google Scholar] [CrossRef]
  30. Zhan, P.L.; Wei, X.; Xiao, Z.L.; Wang, X.L.; Ma, S.P.; Lin, S.J.; Li, F.P.; Bu, S.H.; Liu, Z.P.; Zhu, H.T.; et al. GW10, a member of P450 subfamily regulates grain size and grain number in rice. Theor. Appl. Genet. 2021, 134, 3941–3950. [Google Scholar] [CrossRef]
  31. Dong, N.Q.; Sun, Y.W.; Guo, T.; Shi, C.L.; Zhang, Y.M.; Kan, Y.; Xiang, Y.H.; Zhang, H.; Yang, Y.B.; Li, Y.C.; et al. UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice. Nat. Commun. 2020, 11, 2629–2645. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, S.; Li, S.; Liu, Q.; Wu, K.; Zhang, J.; Wang, S.; Wang, Y.; Chen, X.; Zhang, Y.; Gao, C.; et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nat. Genet. 2015, 47, 949–954. [Google Scholar] [CrossRef] [PubMed]
  33. Wang, Y.; Xiong, G.; Hu, J.; Jiang, L.; Yu, H.; Xu, J.; Fang, Y.; Zeng, L.; Xu, E.; Xu, J.; et al. Copy number variation at the GL7 locus contributes to grain size diversity in rice. Nat. Genet. 2015, 47, 944–948. [Google Scholar] [CrossRef] [PubMed]
  34. Zhao, D.S.; Li, Q.F.; Zhang, C.Q.; Zhang, C.; Yang, Q.Q.; Pan, L.X.; Ren, X.Y.; Lu, J.; Gu, M.H.; Liu, Q.Q. GS9 acts as a transcriptional activator to regulate rice grain shape and appearance quality. Nat. Commun. 2018, 9, 1240. [Google Scholar] [CrossRef] [PubMed]
  35. Zhou, J.; Xin, X.; He, Y.; Chen, H.; Li, Q.; Tang, X.; Zhong, Z.; Deng, K.; Zheng, X.; Akher, S.A.; et al. Multiplex QTL editing of grain-related genes improves yield in elite rice varieties. Plant Cell Rep. 2019, 38, 475–485. [Google Scholar] [CrossRef]
  36. Zhao, D.; Liu, J.; Ding, A.; Zhang, T.; Ren, X.; Zhang, L.; Li, Q.; Fan, X.; Zhang, C.; Liu, Q. Improving grain appearance of erect-panicle japonica rice cultivars by introgression of the null gs9 allele. J. Integr. Agric. 2021, 20, 2032–2042. [Google Scholar] [CrossRef]
  37. Huang, H.; Ye, Y.; Song, W.; Li, Q.; Han, R.; Wu, C.; Wang, S.; Yu, J.; Liu, X.; Fu, X.; et al. Modulating the C-terminus of DEP1 synergistically enhances grain quality and yield in rice. J. Genet. Genomics 2022, 49, 506–509. [Google Scholar] [CrossRef]
  38. Li, N.; Xu, R.; Li, Y. Molecular networks of seed size control in plants. Annu. Rev. Plant Biol. 2019, 70, 435–463. [Google Scholar] [CrossRef]
  39. Jiang, H.; Zhang, A.; Liu, X.; Chen, J. Grain size associated genes and the molecular regulatory mechanism in rice. Int. J. Mol. Sci. 2022, 23, 3169. [Google Scholar] [CrossRef]
  40. Deshaies, R.J.; Joazeiro, C.A.P. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 2009, 78, 399–434. [Google Scholar] [CrossRef]
  41. Schulman, B.A.; Harper, J.W. Ubiquitin-like protein activation by E1 enzymes: The apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 2009, 10, 319–331. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Ye, Y.H.; Rape, M. Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 2009, 10, 755–764. [Google Scholar] [CrossRef] [PubMed]
  43. Komander, D.; Rape, M. The ubiquitin code. Annu. Rev. Biochem. 2012, 81, 203–229. [Google Scholar] [CrossRef] [PubMed]
  44. Hao, J.Q.; Wang, D.K.; Wu, Y.B.; Huang, K.; Duan, P.G.; Li, N.; Xu, R.; Zeng, D.L.; Dong, G.J.; Zhang, B.L.; et al. The GW2-WG1-OsbZIP47 pathway controls grain size and weight in rice. Mol. Plant 2021, 14, 1–15. [Google Scholar] [CrossRef] [PubMed]
  45. Wang, S.S.; Wu, K.; Qian, Q.; Liu, Q.; Li, Q.; Pan, Y.J.; Ye, Y.F.; Liu, X.R.; Wang, J.; Zhang, J.Q.; et al. Non-canonical regulation of SPL transcription factors by a human OTUB1-like deubiquitinase defines a new plant type rice associated with higher grain yield. Cell Res. 2017, 27, 1142–1156. [Google Scholar] [CrossRef]
  46. Huang, K.; Wang, D.K.; Duan, P.G.; Zhang, B.L.; Xu, R.; Li, N.; Li, Y.H. WIDE AND THICK GRAIN 1, which encodes an otubain-like protease with deubiquitination activity, influences grain size and shape in rice. Plant J. 2017, 91, 849–860. [Google Scholar] [CrossRef]
  47. Dong, Q.; Zhang, Z.H.; Wang, L.L.; Zhu, Y.J.; Fan, Y.Y.; Mou, T.M.; Ma, L.Y.; Zhuang, J.Y. Dissection and fine-mapping of two QTL for grain size linked in a 460-kb region on chromosome 1 of rice. Rice 2018, 11, 44. [Google Scholar] [CrossRef]
  48. Zheng, K.L.; Huang, N.; Bennett, J.; Khush, G.S. PCR-Based Marker Assisted Selection in Rice Breeding: IRRI Discussion Paper Series No. 12; International Rice Research Institute: Los Banos, CA, USA, 1995. [Google Scholar]
  49. Hori, K.; Matsubara, K.; Yano, M. Genetic control of flowering time in rice: Integration of Mendelian genetics and genomics. Theor. Appl. Genet. 2016, 129, 2241–2252. [Google Scholar] [CrossRef]
  50. Chen, J.Y.; Zhang, H.W.; Zhang, H.L.; Ying, J.Z.; Ma, L.Y.; Zhuang, J.Y. Natural variation at qHD1 affects heading date acceleration at high temperatures with pleiotropism for yield traits in rice. BMC Plant Biol. 2018, 18, 112. [Google Scholar] [CrossRef]
  51. Zhang, J.; Zhou, X.; Yan, W.; Zhang, Z.; Lu, L.; Han, Z.; Zhao, H.; Liu, H.Y.; Song, P.; Hu, Y.; et al. Combinations of the Ghd7, Ghd8 and Hd1 genes largely define the ecogeographical adaptation and yield potential of cultivated rice. New Phytol. 2015, 208, 1056–1066. [Google Scholar] [CrossRef]
  52. Lin, S.J.; Liu, Z.P.; Zhang, K.; Yang, W.F.; Zhan, P.L.; Tan, Q.Y.; Gou, Y.J.; Ma, S.P.; Luan, X.; Huang, C.B.; et al. GL9 from Oryza glumaepatula controls grain size and chalkiness in rice. Crop J. 2022. [Google Scholar] [CrossRef]
  53. Pan, L.X.; Sun, Z.Z.; Zhang, C.Q.; Li, B.; Yang, Q.Q.; Chen, F.; Fan, X.L.; Zhao, D.S.; Lv, Q.M.; Yuan, D.Y.; et al. Allelic diversification of the Wx and ALK loci in indica restorer lines and their utilization in hybrid rice breeding in China over the last 50 years. Int. J. Mol. Sci. 2022, 23, 5941. [Google Scholar] [CrossRef] [PubMed]
  54. Byun, M.Y.; Cui, L.H.; Oh, T.K.; Jung, Y.J.; Lee1, A.; Park, K.Y.; Kang, B.G.; Kim, W.T. Homologous U-box E3 ubiquitin ligases OsPUB2 and OsPUB3 are involved in the positive regulation of low temperature stress response in rice (Oryza sativa L.). Plant Sci. 2017, 8, 16. [Google Scholar] [CrossRef] [PubMed]
  55. Samuel, M.A.; Ellis, B.E. Double jeopardy: Both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive. Plant Cell 2022, 14, 2059–2069. [Google Scholar] [CrossRef]
  56. Hoppe, T.; Cassata, G.; Barral, J.M.; Springer, W.; Hutagalung, A.H.; Epstein, H.F.; Baumeister, R. Regulation of the myosin-directed chaperone UNC-45 by a novel E3/E4-multiubiquitylation complex in C. elegans. Cell 2004, 118, 337–349. [Google Scholar] [CrossRef] [PubMed]
  57. Kelley, D.R.; Skinner, D.J.; Gasser, C.S. Roles of polarity determinants in ovule development. Plant J. 2009, 57, 1054–1064. [Google Scholar] [CrossRef] [PubMed]
  58. Bernick, E.P.; Zhang, P.J.; Du, S.J. Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos. BMC Cell Biol. 2010, 11, 70. [Google Scholar] [CrossRef] [PubMed]
  59. Schülein-Völk, C.; Wolf, E.; Zhu, J.; Xu, W.S.; Taranets, L.; Hellmann, A.; Jänicke, L.A.; Diefenbacher, M.E.; Behrens, A.; Eilers, M.; et al. Dual regulation of Fbw7 function and oncogenic transformation by Usp28. Cell Rep. 2014, 9, 1099–1109. [Google Scholar] [CrossRef]
  60. Landsverk, M.L.; Li, S.M.; Hutagalung, A.H.; Najafov, A.; Hoppe, T.; Barral, J.M.; Epstein, H.F. The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans. J. Cell Biol. 2007, 177, 205–210. [Google Scholar] [CrossRef]
  61. Wang, P.P.; Zhou, Z.H.; Hu, A.C.; Albuquerque, C.P.D.; Zhou, Y.; Hong, L.X.; Sierecki, E.; Ajiro, M.; Kruhlak, M.; Harris, C.; et al. Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt. Mol. Cell 2014, 54, 378–391. [Google Scholar] [CrossRef]
  62. Ma, J.; Wang, Y.F.; Ma, X.D.; Meng, L.Z.; Jing, R.N.; Wang, F.; Wang, S.; Cheng, Z.J.; Zhang, X.; Jiang, L.; et al. Disruption of gene SPL35, encoding a novel CUE domain-containing protein, leads to cell death and enhanced disease response in rice. Plant Biotechnol. J. 2019, 17, 1679–1693. [Google Scholar] [CrossRef] [Green Version]
Plants 11 02530 g001 550
Figure 1. Knockout of OsPUB3 in three rice cultivars. (A) The schematic of OsPUB3 gene structure and the CRISPR/Cas9 target site. OsPUB3 has only one exon indicated by black rectangles. The translation initiation codon (ATG) and termination codon (TGA) are shown. OsPUB3 protein contains a U-box motif and an Armadillo repeat. (B) Sequence mutations in the target region. Mutations are indicated by blue letters. KO-ZS-1 and KO-ZS-2 were two homozygous mutants in the NILZS97 background. KO-MY-1 was a homozygous mutant in the NILMY46 background; KO-MY-2 to KO-MY-7 were six biallelic mutants in the NILMY46 background. KO-ZH-1, KO-ZH-2 and KO-ZH-3 were three biallelic mutants in the ZH161 background.
Figure 1. Knockout of OsPUB3 in three rice cultivars. (A) The schematic of OsPUB3 gene structure and the CRISPR/Cas9 target site. OsPUB3 has only one exon indicated by black rectangles. The translation initiation codon (ATG) and termination codon (TGA) are shown. OsPUB3 protein contains a U-box motif and an Armadillo repeat. (B) Sequence mutations in the target region. Mutations are indicated by blue letters. KO-ZS-1 and KO-ZS-2 were two homozygous mutants in the NILZS97 background. KO-MY-1 was a homozygous mutant in the NILMY46 background; KO-MY-2 to KO-MY-7 were six biallelic mutants in the NILMY46 background. KO-ZH-1, KO-ZH-2 and KO-ZH-3 were three biallelic mutants in the ZH161 background.
Plants 11 02530 g001
Plants 11 02530 g002 550
Figure 2. Grains of the recipients and knockout mutants. Bar = 10 mm.
Figure 2. Grains of the recipients and knockout mutants. Bar = 10 mm.
Plants 11 02530 g002
Table
Table 1. Phenotypic change due to OsPUB3 knockout.
Table 1. Phenotypic change due to OsPUB3 knockout.
PopulationNo. ofGrain Length (mm)Grain Width (mm)1000-Grain Weight (g)Ratio of Grain Length to Width
PlantsMean ± SDD1 aD2 bMean ± SDD1D2Mean ± SDD1D2Mean ± SDD1D2
NILZS97108.387 ± 0.074 3.074 ± 0.036 27.33 ± 0.82 2.738 ± 0.022
CKZS97108.222 ± 0.068 3.063 ± 0.040 26.45 ± 0.37 2.696 ± 0.040
KO-ZS-1108.121 ± 0.060−0.266 **** c−0.102 **2.983 ± 0.068−0.091 ***−0.080 **24.31 ± 1.18−3.02 ****−2.14 ****2.737 ± 0.053−0.0010.040 *
KO-ZS-2108.018 ± 0.067−0.369 ****−0.204 ****2.941 ± 0.032−0.133 ****−0.122 ****23.78 ± 0.34−3.55 ****−2.67 ****2.738 ± 0.0200.0000.042 ****
NILMY46128.417 ± 0.018 3.109 ± 0.008 27.97 ± 0.16 2.715 ± 0.007
KO-MY-1368.407 ± 0.012−0.010 2.934 ± 0.007−0.175 **** 24.57 ± 0.16−3.40 **** 2.884 ± 0.0090.169 ****
KO-MY-2348.367 ± 0.015−0.050 * 3.014 ± 0.007−0.095 **** 26.25 ± 0.09−1.72 **** 2.790 ± 0.0080.075 ****
KO-MY-3358.389 ± 0.015−0.028 2.891 ± 0.015−0.218 **** 24.03 ± 0.24−3.94 **** 2.916 ± 0.0130.201 ****
KO-MY-4368.314 ± 0.015−0.103 **** 2.891 ± 0.015−0.218 **** 23.74 ± 0.22−4.23 **** 2.895 ± 0.0130.180 ****
KO-MY-5348.166 ± 0.017−0.251 **** 3.044 ± 0.007−0.065 **** 25.77 ± 0.08−2.20 **** 2.695 ± 0.008−0.020
KO-MY-6298.260 ± 0.018−0.157 **** 3.004 ± 0.009−0.105 **** 25.69 ± 0.14−2.28 **** 2.763 ± 0.0090.048 ****
KO-MY-7358.147 ± 0.014−0.270 **** 2.984 ± 0.006−0.125 **** 24.59 ± 0.10−3.38 **** 2.746 ± 0.0060.031 **
ZH161307.927 ± 0.091 2.578 ± 0.041 20.24 ± 0.53 3.092 ± 0.040
CKZH161308.029 ± 0.097 2.552 ± 0.042 20.44 ± 0.35 3.164 ± 0.072
KO-ZH-1a308.116 ± 0.1290.189 ****0.087 **2.498 ± 0.034−0.080 ****−0.054 ****19.94 ± 0.54−0.30 *−0.50 ****3.271 ± 0.0850.179 ****0.106 ****
KO-ZH-1b308.209 ± 0.1030.282 ****0.180 ****2.510 ± 0.041−0.068 ****−0.043 ****20.53 ± 0.610.29 *0.093.292 ± 0.0560.201 ****0.128 ****
KO-ZH-2a308.049 ± 0.0990.122 **0.0202.495 ± 0.031−0.082 ****−0.057 ****19.90 ± 0.48−0.34 **−0.53 ****3.246 ± 0.0550.154 ****0.081 ****
KO-ZH-2b307.904 ± 0.103−0.023−0.125 ****2.475 ± 0.042−0.103 ****−0.077 ****19.32 ± 0.42−0.92 ****−1.11 ****3.213 ± 0.0710.122 ****0.049 **
KO-ZH-3a308.089 ± 0.0810.162 ****0.060 **2.457 ± 0.051−0.120 ****−0.095 ****19.58 ± 0.46−0.66 ****−0.86 ****3.314 ± 0.0650.222 ****0.149 ****
KO-ZH-3b308.156 ± 0.0850.229 ****0.127 ****2.472 ± 0.037−0.106 ****−0.080 ****19.85 ± 0.36−0.39 ***−0.58 ****3.320 ± 0.0490.229 ****0.156 ****
a D1, increase or decrease over the recipient. b D2, increase or decrease over the transgenic-negative control (CK). c * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Table
Table 2. Phenotypic performance of OsPUB3 driven by an Actin 1 promoter.
Table 2. Phenotypic performance of OsPUB3 driven by an Actin 1 promoter.
PopulationGenotype aNo. ofGrain Length (mm)Grain Width (mm)1000-Grain Weight (g)Ratio of Grain Length to Width
PlantsMean ± SDD bMean ± SDDMean ± SDDMean ± SDD
OE-1−118.355 ± 0.060 3.124 ± 0.027 27.71 ± 0.49 2.683 ± 0.018
+198.404 ± 0.0750.049 *c3.085 ± 0.046−0.039 **27.65 ± 0.59−0.072.733 ± 0.0390.051 ***
OE-2−78.271 ± 0.106 3.120 ± 0.045 26.25 ± 0.77 2.661 ± 0.027
+238.344 ± 0.0760.073 *3.108 ± 0.058−0.01227.09 ± 0.630.84 **2.695 ± 0.0540.033
OE-3−58.352 ± 0.058 3.081 ± 0.066 26.85 ± 0.91 2.720 ± 0.041
+258.435 ± 0.0690.083 **3.049 ± 0.057−0.03226.99 ± 0.710.152.777 ± 0.0410.057 **
OE-4−78.305 ± 0.055 3.089 ± 0.030 27.03 ± 0.40 2.696 ± 0.037
+228.409 ± 0.0810.104 **3.047 ± 0.024−0.042 ***27.49 ± 0.420.46 **2.770 ± 0.0390.074 ****
a −, negative transgenic plants; +, positive transgenic plants. b D, increase or decrease over the negative transgenic plants. c * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Li, Z.-H.; Wang, S.-L.; Zhu, Y.-J.; Fan, Y.-Y.; Huang, D.-R.; Zhu, A.-K.; Zhuang, J.-Y.; Liang, Y.; Zhang, Z.-H. Control of Grain Shape and Size in Rice by Two Functional Alleles of OsPUB3 in Varied Genetic Background. Plants 2022, 11, 2530. https://doi.org/10.3390/plants11192530

AMA Style

Li Z-H, Wang S-L, Zhu Y-J, Fan Y-Y, Huang D-R, Zhu A-K, Zhuang J-Y, Liang Y, Zhang Z-H. Control of Grain Shape and Size in Rice by Two Functional Alleles of OsPUB3 in Varied Genetic Background. Plants. 2022; 11(19):2530. https://doi.org/10.3390/plants11192530

Chicago/Turabian Style

Li, Zhu-Hao, Shi-Lin Wang, Yu-Jun Zhu, Ye-Yang Fan, De-Run Huang, Ai-Ke Zhu, Jie-Yun Zhuang, Yan Liang, and Zhen-Hua Zhang. 2022. "Control of Grain Shape and Size in Rice by Two Functional Alleles of OsPUB3 in Varied Genetic Background" Plants 11, no. 19: 2530. https://doi.org/10.3390/plants11192530

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop