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Plant Cell Reports

, Volume 38, Issue 12, pp 1551–1561 | Cite as

Characterization of watermelon anther and its programmed cell death-associated events during dehiscence under cold stress

  • Xiaolong Lyu
  • Shuna Chen
  • Nanqiao Liao
  • Jie Liu
  • Zhongyuan Hu
  • Jinghua Yang
  • Mingfang ZhangEmail author
Original Article

Abstract

Key message

The ‘neglected’ thermophile fruit crop of watermelon was first used as a model crop to study the PCD associated with anther dehiscence in cold-exposed condition during anther development.

Abstract

Anther dehiscence ensures normal pollen release and successful fertilization at fruit-setting stages in flowering plants. However, most researches pertinent to anther dehiscence are centered on model plant and/or major field crops under optimal growth condition. Due to anther indehiscence in cold condition, crop plants of thermophile tropical or subtropical fruit crops fail to accomplish timely pollination and fertilization, resulting in a great yield loss annually. Herein, we developed an ideal model crop for studying the programmed cell death (PCD) associated with anther dehiscence under low-temperature stress using the S-shaped spiral anther in watermelon as instead. Our results revealed that, including the tapetal cell layers, both cells of the interlocular septum and the stomium were blocked in PCD associated with anther dehiscence at 15 °C. Likewise, TUNEL assays visualized the evidence that low temperature at 15 °C interferes with not only the PCD of tapetal cells, but also the PCD of interlocular septum and stomium. Furthermore, the expressions of genes correlated with PCD of tapetum and stomium were significantly inhibited at 15 °C, suggesting that low temperature affects anther dehiscence by inhibiting PCD of sporophytic tissue-related gene expressions. The findings of the current research provide mechanistic insights into anther indehiscence leading to poor fruit-setting for thermophile fruit crop such as watermelon under adverse cold condition in flowering.

Keywords

Anther dehiscence Cold stress PCD Thermophile crop Watermelon 

Notes

Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant No. 31672175), the Key Science and Technology Program for Agricultural (Vegetable) New Variety Breeding of Zhejiang Province (2016C02051-4-1) and the Earmarked Fund for Modern Agro-Industry Technology Research System of China (CARS-26-17).

Author contribution statement

XL, ZH, and MZ conceived and designed the study. XL, SC and NL performed the experiments. XL, JY and JL analyzed the data, and XL and MZ wrote the paper. All authors reviewed the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

299_2019_2466_MOESM1_ESM.jpg (346 kb)
Supplementary Fig. S1. Mature anther size at 26 °C and 15 °C. (a-d) Schematic diagram of anther size measurement. Scale bars, 1 mm. (e) The size of mature anther. Mean ± standard deviation values were calculated from ten biological repetition. There’s no significant difference between the samples at 26 °C and 15 °C (P > 0.05; Welch’s t test). (JPEG 346 kb)
299_2019_2466_MOESM2_ESM.jpg (382 kb)
Supplementary Fig. S2. Anther endodermis secondary cell wall thickness at 26 °C and 15 °C. (a-b) Transverse section of anther endodermis cells at 26 °C (a) and 15 °C (b). Red arrow indicates the thickened secondary cell wall (scw). Scale bars, 2 μm. (c) Secondary cell wall thickness. One hundred cells from ten anthers were measured at each temperature condition. Mean ± standard deviation values were calculated from 100 repetition. There’s no significant difference between the samples at 26 °C and 15 °C (P > 0.05; Welch’s t-test). (JPEG 381 kb)
299_2019_2466_MOESM3_ESM.jpg (764 kb)
Supplementary Fig. S3. PCD in watermelon anther at microspore stages by TUNEL assays. Fluorescence microscope of cross-sections of watermelon anthers at 26 °C (a-f) and 15 °C (g-l). Green fluorescence indicates TUNEL-positive signals while red fluorescence indicates DAPI (4’,6-diamidino-2-phenylindole) staining. White arrows indicate tapetal cells tissues undergoing PCD. Except the autofluorescence from watermelon pollen wall, no green fluorescence signal was observable in the anther at young microspore stage at both 26 °C (a-c) and 15 °C (g-i). At later microspore stage, TUNELpositive signals were present in tapetum cells at 26 °C (d-f), suggesting that advanced PCD degeneration had occurred (white arrows), while no signal was present in anthers at 15 °C at later microspore stage (j-l). Scale bars, 50 μm. (JPEG 763 kb)
299_2019_2466_MOESM4_ESM.jpg (1.1 mb)
Supplementary Fig. S4. Pollen viability at 26 °C and 15 °C. (a-d) Viability of pollen grains as assayed with Alexander staining. Pollen grains collected from the mature anthers at both 26 °C (a) and 15 °C (c), as well as those collected from dehiscent anthers at 26 °C (b) were viable (purple-stained pollen grains), while that of the pollen grains collected from the indehiscent anthers after pollen mature stage at 15 °C (d) contains a lot of non-viable pollen grains (blue-stained pollen grains; red arrows). mp: mature pollen; pad: pollen after anther dehiscence; pia: pollen in the indehiscent anthers. Scale bars, 100 μm. (e) Reduced pollen viability in indehiscent anthers at 15 °C. Pollen grains were observed in three different fields of view with 10 biological repetitions. Results were plotted as mean ± standard deviation values calculated from 30 repetitions in percentage of pollen viability. The viability of pollen from indehiscent anther at 15 °C was significantly reduced compared to the pollen from dehiscent anthers at 26 °C. (***P < 0.001; Welch’s t-test). (JPEG 1174 kb)
299_2019_2466_MOESM5_ESM.xlsx (11 kb)
Supplementary Table S1. Primer sequences of detected genes for qRT-PCR analyses (XLSX 11 kb)
299_2019_2466_MOESM6_ESM.xlsx (12 kb)
Supplementary Table S2. PCD relevant genes in watermelon (XLSX 11 kb)

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Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of Germplasm Innovation and Molecular Breeding, College of Agriculture and BiotechnologyZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.Key Laboratory of Horticultural Plant Growth, Development & Quality ImprovementMinistry of AgricultureHangzhouPeople’s Republic of China

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