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Gene expression and localization of arabinogalactan proteins during the development of anther, ovule, and embryo in rice

  • Tengfei Ma
  • Fang Dong
  • Die Luan
  • Hengjin Hu
  • Jie ZhaoEmail author
Original Article
  • 71 Downloads

Abstract

Arabinogalactan proteins (AGPs) are hyperglycosylated members of the hydroxyproline-rich glycoprotein (HRGP) superfamily and are widely distributed throughout the plant kingdom. In Oryza sativa (rice), the gene expression and biological function of AGPs only have received minimal research attention. Here, we used qRT-PCR to detect the expression patterns of OsAGP genes in various organs, and found that six genes were preferentially expressed in panicles, three genes were specifically expressed in anthers, and one gene in the stigma. Furthermore, using four specific monoclonal antibodies (JIM8, JIM13, LM2, MAC207), we observed the distribution of AGPs in rice anthers, ovules, and embryos. In anthers, the strong fluorescence signals of AGPs were present in tapetum cells, pollen mother cells, and mature pollens, suggesting that AGPs might be related to the development of anther and pollen. In ovules, signals of AGPs were specifically distributed in the three micropylar megaspores of the tetrad, and with intense signals in the egg cell and synergid cells in the mature embryo sac. This suggests that AGPs may be involved in megaspore determination and double fertilization. In embryos, the immunological signals of AGPs appeared in peripheral and inner cells at the early stage, and in the scutellum, plumule, and radicle at the late stage, indicating that AGPs may be associated with organ differentiation and maturation of embryos. In this study, we revealed that AGPs were widely distributed in rice anthers, ovules, and embryos, which lays a foundation for the functional investigation of AGPs in various processes of sexual reproduction.

Keywords

Rice Arabinogalactan protein Gene expression Localization 

Abbreviations

ACs

archesporial cells

AGPs

Arabinogalactan proteins

DAP

Day after pollination

En

Endothecium

GPI

Glycosylphosphatidylinositol

GO

Gene ontology

ML

Middle layer

MMCs

Megaspore mother cells

MP

Mature pollen

NE

Nucellus epiderm

PCD

Programmed cell death

PMC

Pollen/microspore mother cell

PPCs

Primary parietal cells

PSCs

Primary sporogenous cells

qRT-PCR

Quantitative real-time PCR

Ta

Tapetum

Notes

Funding information

This research was supported by the National Natural Science Foundation of China (2016M590711, 31670312) and the Special Doctorial Program Funds of the Ministry of Education of China (20130141130008).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2019_1349_Fig7_ESM.png (716 kb)
Fig. S1

The expression patterns of reproductive organs-preferential/specific genes. The genes are divided into eight clusters according to their expression patterns: (A) preferentially expressed in Pr-In, Pistil and seeds; (B) preferentially expressed in Pr-In; (C) preferentially expressed in Pr-In, Pistil, Po-In and S1; (D) weakly expressed in reproductive tissues; (E) not expressed in all tissues; (F) preferentially but weakly expressed in Pr-In and seeds; (G) preferentially expressed in Anther and Po-In; (H) preferentially expressed in Pr-In, Po-In and S1, or preferentially expressed in S2, Em and En; (I) preferentially expressed in Pr-In, Po-In, Pistil and S1. The color scale was shown at the bottom. Pr-In, Pre-emergence inflorescence, panicle before emerging from the sheath of the flag leaf; Po-In, Post-emergence inflorescence, panicle after emerging from the sheath of the flag leaf; Anther, anther before pollination; Pistil, pistil before pollination; S1, seed at 5 days after pollination; S2, seed at 10 days after pollination; Em, embryo of seeds at 25 days after pollination; En, endosperm of seeds at 25 days after pollination. (PNG 716 kb)

709_2019_1349_MOESM1_ESM.tif (11.6 mb)
High Resolution Image (TIF 11849 kb)
709_2019_1349_Fig8_ESM.png (1.6 mb)
Fig. S2

Real-time PCR analysis of reproductive organs-preferentially OsAGP genes. R, 60-day-old roots; L, 60-day-old leaves; S, 60-day-old stems; An, anthers from 28-cm panicles; St, stigmas from 28-cm panicles; Ov, ovaries from 28-cm panicles; P1,0–3 cm panicles; P3, 5-10 cm panicles; P6, 22-30 cm panicles; S1, seeds at 1 day after pollination; S2, seeds at 10 days after pollination; S3, seeds at 30 days after pollination. Error bars indicate standard deviations of independent biological replicates (n = 2 or more). (PNG 1639 kb)

709_2019_1349_MOESM2_ESM.tif (38.3 mb)
High Resolution Image (TIF 39183 kb)
709_2019_1349_Fig9_ESM.png (8.7 mb)
Fig. S3

Fluorescence microscopy of rice ovaries at different stages labelled with mAbs LM2 and MAC207. (A, B) Rice ovaries at the pre-meiotic stage of macrosporogenesis labelled with LM2 (A) and MAC207 (B), respectively. (C, D) Rice ovaries at the tetrads stage of macrosporogenesis labelled with LM2 (C) and MAC207 (D), respectively. (E, F) Rice ovaries at the binucleate embryo sac stage of macrosporogenesis labelled with LM2 (E) and MAC207 (F), respectively. (G, H) Rice ovaries at the mature embryo sac stage of macrosporogenesis labelled with LM2 (G) and MAC207 (H), respectively. Ap, antipodal cells; E, egg cell; ES, embryo sac; II, inner integument; MMC, macrospore mother cell; MP, micropyle; MT, megaspore tetrad; OI, outer integument; S, synergids. Scale bars = 25 μm in A-F; Scale bars = 50 μm in G and H. (PNG 8900 kb)

709_2019_1349_MOESM3_ESM.tif (46.9 mb)
High Resolution Image (TIF 48069 kb)
709_2019_1349_Fig10_ESM.png (22.2 mb)
Fig. S4

The reaction of β-GlcY and rice pistils/embryos. (A) 1DAP ovary, there was distribution of AGPs on the stigmas. (B) 3DAP ovary, there was distribution of AGPs on the wilting stigmas. (C) The ventral side of 5 DAP embryo, AGPs were concentrated in the radicle sheath, the embryo sheath, and the top of the embryo. (D) The reverse side of 5 DAP embryo, there was also the distribution of AGPs. (E) The ventral side of 8 DAP embryo, AGPs were distributed in the coleorhiza, the coleoptile, and the epithelial cells of the scutellum. (F) The reverse side of 8 DAP embryo, there was also the distribution of AGPs. (G) The ventral side of 10 DAP embryo, AGPs were weakly distributed in the coleorhiza, the coleoptile, and the epithelial cells of the scutellum. (H) The reverse side of 10 DAP embryo, AGPs can be detected. (I) The ventral side of 15 DAP embryo, the distribution of AGPs was enhanced again, and the distribution was concentrated in the coleorhiza, the coleoptile, and the epithelial cells of the scutellum. (J) The reverse side of 15 DAP embryo, there was also the enhanced distribution of AGPs. (a-j) The controls with β-GlcY-untreated. Scale bars = 1 mm in A, B, a, b, G-J, g-j; Scale bars = 0.25 mm in C, D, c, d; Scale bars = 0.5 mm in E, F, e, f. (PNG 22758 kb)

709_2019_1349_MOESM4_ESM.tif (59.6 mb)
High Resolution Image (TIF 60996 kb)
709_2019_1349_Fig11_ESM.png (684 kb)
Figure S5

Gene ontology (GO) annotations of all detected genes. The histogram shows the result of AGP genes to the secondary classification of GO terms. The left y-axis indicates the percent of genes in a functional term. The right y-axis indicates the number of genes in a functional term. (PNG 684 kb)

709_2019_1349_MOESM5_ESM.tif (56.9 mb)
High Resolution Image (TIF 58232 kb)
709_2019_1349_MOESM6_ESM.xlsx (64 kb)
Table S1 Primers used in real-time PCR. (XLSX 63 kb)
709_2019_1349_MOESM7_ESM.xlsx (60 kb)
Table S2 Microarray analysis of AGP genes in rice. (XLSX 60 kb)
709_2019_1349_MOESM8_ESM.xlsx (15 kb)
Table S3 RNAseq analysis of AGP genes in rice. (XLSX 15 kb)
709_2019_1349_MOESM9_ESM.docx (26 kb)
Table S4 Informations of 24 sexual organs-preferential/specific AGP genes. (DOCX 26 kb)
709_2019_1349_MOESM10_ESM.doc (62 kb)
Table S5 Protein backbones of 24 sexual organs-preferential/specific OsAGPs. (DOC 62.5 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Tengfei Ma
    • 1
  • Fang Dong
    • 1
  • Die Luan
    • 1
  • Hengjin Hu
    • 1
  • Jie Zhao
    • 1
    Email author
  1. 1.State Key Laboratory of Hybrid Rice, College of Life SciencesWuhan UniversityWuhanChina

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