Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (16): 3369-3380.doi: 10.3864/j.issn.0578-1752.2021.16.001

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

Function of Sucrose Transporter OsSUT5 in Rice Pollen Development and Seed Setting

ZHANG YaWen1(),BAO ShuHui1,TANG ZhenJia1,WANG XiaoWen1,2,YANG Fang3,ZHANG DeChun4(),HU YiBing1()   

  1. 1College of Resources & Environmental Sciences, Nanjing Agricultural University, Nanjing 210095
    2College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    3State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072
    4Bio-technology Research Center, China Three Gorges University, Yichang 443002, Hubei
  • Received:2020-12-18 Accepted:2021-02-12 Online:2021-08-16 Published:2021-08-24
  • Contact: DeChun ZHANG,YiBing HU E-mail:zyw0615yaya@163.com;zhangdc227@163.com;huyb@njau.edu.cn

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Abstract:

【Objective】 Sucrose is the main form of photosynthates transported in the plant, and sucrose transporters (or sucrose carrier, SUT/SUC) play important roles in the transport of sucrose across the plasma membrane between cells and allocation of sucrose among different tissues and organs. The rice SUT family possesses 5 members. Knockout of the genes encoding OsSUT1, OsSUT2, OsSUT3 or OsSUT4 confers significant effects on rice indicating that the functions of these genes are indispensable. However, the physiological role of OsSUT5 has not been systematically characterized. This study aims at elucidating the function of OsSUT5 in rice growth and development and provides new evidence for a comprehensive understanding of SUT’s role in model plant rice. 【Method】In this study, the temporal and spatial expression pattern of OsSUT5 was analyzed via quantitative real-time PCR. The tissue localization of OsSUT5 in rice was tested via GUS represented expressions driven by the putative promoter of OsSUT5 and subcellular localization of transiently expressed OsSUT5-GFP fusion protein was observed in leaf cells of tobacco. In addition, CRISPR-Cas9 mediated gene editing was employed to create mutant lines of the gene for the characterization of OsSUT5. 【Result】Our results show that OsSUT5 was expressed in culm, leaf, inflorescence, and caryopsis of rice but it was predominantly expressed in inflorescence and developing caryopsis at the transcriptional level. At the protein level, it was prominently expressed in the vascular bundles of rice vegetative organs. In reproductive organs, the protein was mainly expressed in the anther and developing caryopsis, particularly in the scutellum and coleorhiza. Transient expression of OsSUT5-GFP fusion protein in epidermis cells of tobacco leaf indicated that it was localized on the plasma membrane. Compared with the wild-type control, three homozygous mutant lines of OsSUT5 created via CRISPR-Cas9 gene-editing system consistently showed reduced pollen viability, a lower percentage of germination rates. Accordingly, the percentage of unpollinated florets and seed-setting rate decreased significantly. Comparison of OsSUT5 mutant lines and the wild-type control showed that more chalk was observed in the mutant caryopses than that of the wild type. In the mutant lines, caryopsis length increased but 1000 grain weight didn’t show a significant difference between the mutants and the wild-type control based on statistics. 【Conclusion】These results indicate that OsSUT5 played an important role in pollen development and probably also in the fertilization process. Knockout of the gene affected the morphology and quality of rice caryopsis. Given the sucrose transport capacity of OsSUT5 and its plasma membrane localization, it can be deduced that function of OsSUT5 including its influence on rice pollen viability and endosperm development is related to its sucrose transport activity at the cellular level.

Key words: rice, OsSUT5, CRISPR-Cas9, pollen germination, seed setting rate, chalk

Fig. 1

Expression of OsSUT5 analyzed via qRT-PCR"

Fig. 2

Histochemical staining results of GUS expression in rice tissues driven by OsSUT5 prompter A: Root of 3 d seedling after germination; B: Cross-section of seedling stem; C: Leaf blade of mature plant; D: Floret about to blossom; E: Longitudinal section of 9 d caryopsis after pollination; F: The embryo of 9 d caryopsis after pollination. White arrow heads indicate vascular bundles; red arrow heads indicate the scutellum; yellow arrow heads indicate the coleorhiza. Bars=1 mm"

Fig. 3

Subcellular localization of OsSUT5-GFP fusion protein transiently expressed in tobacco leaf cells"

Fig. 4

Gene editing site certification and phenotype of the OsSUT5 mutant lines WT: Wild type. ossut5-1, ossut5-17, and ossut5-25 are three homozygous rice lines created via the gene editing system. A: OsSUT5 mutants and the wild-type rice comparison; B: Panicle comparison; C: Editing site identification; D: Caryopsis length comparison; E: A column diagram of grain composition; F: Caryopsis morphology comparison; G: A column diagram of caryopsis length; H: A column diagram of 1000 grain weight. Different lowercase letters in E, G, and H represent significantly different values (P<0.05). The same as below"

Fig. 5

Pollen viabilities tested by I2-KI staining and germination ratio in vitro of OsSUT5 mutants and wild-type rice A, B, C, D: Pollens of wild type rice and ossut5-1, ossut5-17, ossut5-25 mutants stained with KI-I2 solution observed under a microscope. E, F, G, H: Germination of wild type rice and ossut5-1, ossut5-17, ossut5-25 mutants in vitro observed under a microscope. I: Pollen viabilities. J: Pollen germination rates. Scale bars =200 μm"

Fig. 6

A phylogenetic tree (Neighbor-Joining) of SUT(C) proteins Red dots indicate rice SUTs; Blue dot indicates Arabidopsis SUT5. Accession numbers of 153 SUT(C) proteins in the figure are listed in the supplementary table 2"

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