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A comparative proteomics analysis in roots of soybean to compatible symbiotic bacteria under flooding stress

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Abstract

A proteomics approach was used to evaluate the effects of flooding stress on early symbiotic interaction between soybean roots and soil bacteria, Bradyrhizobium japonicum. Three-day-old soybean was inoculated with B. japonicum followed by flooding. The number of root hairs in seedlings, without or with flooding stress, was increased after 3 days of inoculation. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 219 protein spots, 14 and 8 proteins were increased and decreased, respectively, by inoculation under flooding compared with without flooding. These proteins were compared in untreated and flooded seedlings. Increased level of 6 proteins in flooded seedlings compared with untreated seedlings was suppressed by inoculation in seedlings under flooding. They were related to disease/defense, protein synthesis, energy, and metabolism. Differential abundance of glucan endo-1,3-beta-glucosidase, phosphoglycerate kinase, and triosephosphate isomerase, based on their localization in middle and tip of root, indicated that they might be related to increase in number of root hairs. These results suggest that disease/defense, energy, and metabolism-related proteins may be particularly subjected to regulation in flooded soybean seedlings, when inoculated with B. japonicum and that this regulation may lead to increase in number of root hair during early symbiotic differentiation.

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Abbreviations

2-DE:

Two-dimensional polyacrylamide gel electrophoresis

CBB:

Coomassie brilliant blue

MS:

Mass spectrometry

LC:

Liquid chromatography

pI :

Isoelectric point

IEF:

Isoelectric focusing

MALDI-TOF:

Matrix-assisted laser desorption ionization time-of-flight

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Acknowledgments

The authors thank the students exchange programme between Kohat University of Science and Technology, Pakistan, and University of Tsukuba, Japan, for providing scholarship. They are grateful to the National Institute of Crop Science of Japan for all experimental support during this project. The authors thank Dr. Yohei Nanjo and Dr. Keito Nishazawa for their valuable discussion. They are also thankful to Dr. Kentaro Kawaguchi and Dr. Md. Emdadul Haque for support during microscopic analysis. This work was supported by the grants from National Agriculture and Food Research Organization, Japan. Bacterial strain, B. japonicum MAFF 211342 was obtained from the Genebank at National Institute of Agrobiological Sciences (Tsukuba, Japan).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. National Institute of Crop Science, National Agriculture and Food Research Organization, 2-1-18 Kannondai, Tsukuba, 305-8518, Japan

    Amana Khatoon, Afshin Salavati & Setsuko Komatsu

  2. Department of Plant Sciences, Kohat University of Science and Technology, Kohat, 26000, Pakistan

    Amana Khatoon & Shafiq Rehman

  3. Department of Plant Breeding and Biotechnology, Ramin University of Agriculture and Natural Resources (Khuzestan), Ahwaz, Iran

    Afshin Salavati

Authors
  1. Amana Khatoon
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  2. Shafiq Rehman
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  3. Afshin Salavati
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  4. Setsuko Komatsu
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Corresponding author

Correspondence to Setsuko Komatsu.

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Supplementary Table 1 (DOCX 119 kb)

726_2012_1333_MOESM2_ESM.pptx

Supplementary Fig 1. Experimental design of effects of flooding stress on early symbiotic interaction in soybean roots inoculated with compatible bacteria. Experimental design representing physiological (A) and proteomics (B) work flow. Three-day-old soybean was inoculated with B. japonicum without or with flooding stress. Untreated soybean served as control. a, b, c and d represent, no treatment, inoculation, inoculation under flooding and flooding treatment, respectively. Time of sowing, inoculation, initiation of flooding, and sampling are specified by downward triangles, inverted arrow, upward triangles, and circles, respectively. Statistical analyses were performed between the treatments as shown by the lines connecting the treatments vertically, using Student’s t test and one-way ANOVA Duncan’s multiple comparisons test when 2 or more than 2 treatments were compared, respectively. (PPTX 53 kb)

726_2012_1333_MOESM3_ESM.pptx

Supplementary Fig. 2. Comparision of shoot length and fresh weight of soybean seedlings between untreated seedlings, inoculated seedlings, seedlings inoculated under flooding, and flooded seedlings. Three-day-old soybean was inoculated with B. japonicum followed by flooding. Untreated soybean served as control. Lengths and fresh weights of epicotyls (E) and hypocotyl (H) were measured 0, 3 and 6 days after treatment. Each bar represents the average ± SE of 18 seedlings. White, black, squared, and dotted bars represent untreated seedlings, inoculated seedlings, inoculated seedlings under flooding, and flooded seedlings, respectively. Statistical analysis was performed by one-way ANOVA Duncan’s multiple comparisons test. Different letters above the bars indicate a statistically significant difference (P < 0.05). (PPTX 46 kb)

726_2012_1333_MOESM4_ESM.pptx

Supplementary Fig. 3. Morphological comparison of root hair growth response between untreated seedlings, inoculated seedlings, seedlings inoculated under flooding, and flooded seedlings. Three-day-old soybean was inoculated with B. japonicum and flooded. Soybean without any treatment served as control. Root samples were collected 3 and 6 days after treatment and stained with methylene blue. Number of root hairs per 200 μm2 of root was determined under light microscope (4X, 10X, 20X). (PPTX 1023 kb)

726_2012_1333_MOESM5_ESM.pptx

Supplementary Fig. 4. Comparison of 2-DE pattern of protein abundance in soybean roots between untreated seedlings, inoculated seedlings, seedlings inoculated under flooding, and flooded seedlings. Three-day-old soybean was inoculated with B. japonicum and flooded. Untreated soybean served as control. Proteins were extracted from roots 3 days after treatment, separated by 2-DE and stained by CBB. The abundance pattern of 22 protein spots, which differentially changed in soybean roots inoculated with B. japonicum under flooding, was compared with untreated and flooded seedlings. Open circles show protein spots with altered abundance. Upward and downward arrows indicate increased and decreased abundance, respectively. Spot numbers are same as in Fig. 2. (PPTX 693 kb)

726_2012_1333_MOESM6_ESM.pptx

Supplementary Fig. 5. Comparison of protein abundance in soybean roots between untreated seedlings, inoculated seedlings, seedlings inoculated under flooding, and flooded seedlings. Three-day-old soybean was inoculated with B. japonicum and flooded. Untreated soybean served as control. Three days after treatments, proteins were extracted from roots of untreated seedlings (white bars), inoculated seedlings (black bars), inoculated seedlings under flooding (squared bars), and flooded soybean seedlings (dotted bars). The differentially changed proteins were quantified using PDQuest software and plotted as the relative intensity. Results are presented as mean ± SE of relative protein intensity for gels from three biological replicates. The results were compared using Student’s t test between the treatments as shown by line with asterisks above the bars, indicating significant differences between treatments (*P < 0.05, **P < 0.01). (PPTX 73 kb)

726_2012_1333_MOESM7_ESM.pptx

Supplementary Fig. 6. Photograph of soybean seedling showing different portions of root. Three-day-old soybean was inoculated with B. japonicum followed by flooding. Middle and tip of roots were taken for differential abundance analysis of proteins related to root hair number and morphology in respective portions of roots. (PPTX 150 kb)

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Khatoon, A., Rehman, S., Salavati, A. et al. A comparative proteomics analysis in roots of soybean to compatible symbiotic bacteria under flooding stress. Amino Acids 43, 2513–2525 (2012). https://doi.org/10.1007/s00726-012-1333-8

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