Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (21): 4161-4174.doi: 10.3864/j.issn.0578-1752.2024.21.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Study on the Involvement of OsFWL3 in the Regulation of Metal Ion Transport and Accumulation in Rice

LIU YiHan1,2(), MU QingShan1,2(), HE Xiang1,2, CHEN Min2, HU Jin1,2, GUAN YaJing1,2()   

  1. 1 Hainan Institute, Zhejiang University, Sanya 572025, Hainan
    2 The Advanced Seed Institute, College of Agriculture and Biotechnology, Zhejiang University/Zhejiang Key Laboratory of Crop Germplasm Innovation and Utilization, Hangzhou 310058
  • Received:2024-04-30 Accepted:2024-06-28 Online:2024-11-01 Published:2024-11-10

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Abstract:

【Objective】FWL (Fruit Weight2.2-Like) gene is a negative regulator of cell proliferation, which not only regulates plant organogenesis and organ size, but also participates in the regulation of metal ion transport accumulation and signal transduction. Analyzing of the function of OsFWL3 gene is helpful to reveal the transport mechanism of trace metal elements in crops. It provides theoretical support for reducing heavy metal accumulation and improving crop quality. 【Method】The gene information, genome structure and phylogenetic tree of OsFWLs family were analyzed by bioinformatics method, and the expression profile of OsFWL3 gene was predicted. Two OsFWL3 knockout lines were obtained using CRISPR/Cas9 gene editing technology. Then wild type and Osfwl3 mutants were treated with ZnSO4 at seedling stage and filling stage, respectively. The phenotypes of plants and grains after treatment were analyzed, and the content variation of metal elements such as Zn was determined to explore the effects of OsFWL3 on the transport and accumulation of metal ions and seed quality. 【Result】The gene function of OsFWLs family is similar to some extent. OsFWL3 gene is highly expressed in anther and panicle, indicating that it is closely related to reproductive development of rice. The number of primary branches, grian length, grain thickness and 100-grain weight of Osfwl3 mutants are significantly larger than WT. OsFWL3 affects the content and distribution of Zn and other metal ions in rice seedlings and grains. The deletion of OsFWL3 gene affects the competitive transport of Zn, Cd and Mn from underground to above-ground, lower grain to central grain and husk to brown rice. 【Conclusion】OsFWL3 gene affects the distribution of Zn and other metal ions in rice grains and plants, and it plays an important role in regulating the growth and development of rice plants and grain size.

Key words: rice, OFWL3, ZnSO4 treatment, metal ion transport, metal ion accumulation, grain size

Table 1

Primers used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途 Purpose
OsFWL3-T-F
OsFWL3-T-R		 CGCCAGGGTTTTCCCAGTCACGAC
GTTGGAAACCACGTGTGATG		 Osfwl3突变体T-DNA鉴定
T-DNA identification of Osfwl3 mutants
OsFWL3-C-F
OsFWL3-C-R		 CCATGGTCCACCGACCTCTTT
CATCCCAGGGTACATCTCCGG		 Osfwl3突变体靶点鉴定
Target identification of Osfwl3 mutants
OsActin-qF
OsActin-qR		 TGGCATCTCTCAGCACATTCC
TGCACAATGGATGGGTCAGA		 实时荧光定量分析
RT-qPCR
OsFWL3-qF
OsFWL3-qR		 TTCGACATGAGCCTCGGATG
ATCCCAGGGTACATCTCCGG
OsFWL3-gRT1 TCCCGCACGACGACGACCCCgttttagagctagaaat CRISPR/Cas9载体构建
CRISPR/Cas9 vector construction
OsFWL3-U3T1 GTACGGCCTGCAGGAGACGCTTgccacggatcatctgc
OsFWL3-gRT2 GCGTCTCCTGCAGGCCGTACgttttagagctagaaat
OsFWL3-U6aT2 GGGGTCGTCGTCGTGCGGGACggcagccaagccagca

Fig. 1

Bioinformatics analysis of OsFWL3 and FWL gene family in rice A: Summary information of rice FWL gene family; B: Genome structure of rice FWL gene family; C: Phylogenetic tree of FWL gene family, Os: Oryza sativa L., Zm: Zea mays L., At: Arabidopsis thaliana (L.), Gm: Glycine max (Linn.), SL: Solanum lycopersicum L.; D: Prediction of OsFWL3 gene expression; E: The expression patterns of OsFWL3 in specific tissues determined by qRT-PCR analysis"

Fig. 2

OsFWL3 gene regulates plant growth and grain size in rice A: The target site of OsFWL3 gene; B: Two mutant types of Osfwl3; C: Plant phenotypes of Osfwl3 and WT, scale =20 cm; D: Spike phenotypes of Osfwl3 and WT, scale =2 cm; E: Plant height of Osfwl3 and WT; F: Number of primary branches per spike in Osfwl3 and WT; G: Grain phenotypes of Osfwl3 and WT, scale =1 cm; H: Grain length of Osfwl3 and WT; I: Grain width of Osfwl3 and WT; J: Grain thickness of Osfwl3 and WT; K: Hundred-grain weight of Osfwl3 and WT. Mean ±SD (n≥10) was used for statistical analysis by one-way ANOVA, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. The same as below"

Fig. 3

OsFWL3 affects the distribution of metal ions in rice grain A: Zn content in Osfwl3 and WT brown rice; B: Cd content in Osfwl3 and WT brown rice; C: Mg content in Osfwl3 and WT brown rice; D: Mn content in Osfwl3 and WT brown rice; E: Fe content in Osfwl3 and WT brown rice; F: Zn content in Osfwl3 and WT grain husk; G: Cd content in Osfwl3 and WT grain husk; H: Mg content in Osfwl3 and WT grain husk; I: Mn content in Osfwl3 and WT grain husk; J: Fe content in Osfwl3 and WT grain husk"

Fig. 4

OsFWL3 affects the accumulation and transport of Zn and other metal ions in rice seedling stage A: Seedling phenotypes of Osfwl3 and WT, scale=10 cm; B: Shoot height of Osfwl3 and WT; C: Root length of Osfwl3 and WT; D: Fresh weight of Osfwl3 and WT; E: Dry weight of Osfwl3 and WT; F: Zn content in the shoot and root parts of Osfwl3 and WT; G: Cd content in the shoot and root parts of Osfwl3 and WT; H: Mn content in the shoot and root parts of Osfwl3 and WT; I: Root to shoot ratio of Zn content in Osfwl3 and WT; J: Root to shoot ratio of Cd content in Osfwl3 and WT; K: Root to shoot ratio of Mn content in Osfwl3 and WT"

Fig. 5

OsFWL3 affected the accumulation and transport of Zn and other metal elements in rice filling stage A: Zn content in Osfwl3 and WT brown rice; B: Zn content in Osfwl3 and WT husk; C: Husk to brown rice ratio of Zn content in Osfwl3 and WT; D: Cd content in Osfwl3 and WT brown rice; E: Cd content in Osfwl3 and WT husk; F: Husk to brown rice ratio of Cd content in Osfwl3 and WT; G: Mn content in Osfwl3 and WT brown rice; H: Mn content in Osfwl3 and WT husk; I: Husk to brown rice ratio of Mn content in Osfwl3 and WT; J: Grain Zn content in the upper, middle and lower panicles of Osfwl3 and WT; K: Lower grain to central grain ratio of Zn content in Osfwl3 and WT; L: Grain phenotypes of upper, central and lower panicle in Osfwl3 and WT; M: Grain length of upper, central and lower panicle in Osfwl3 and WT; N: Grain width of upper, central and lower panicle in Osfwl3 and WT; O: Grain thickness of upper, central and lower panicle in Osfwl3 and WT; P: 100-grain weight of upper, central and lower panicle in Osfwl3 and WT"

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