Skip to main content

Metabolite signatures of grasspea suspension-cultured cells illustrate the complexity of dehydration response

Planta volume 250, pages 857–871 (2019)Cite this article

Abstract

Main conclusion

This represents the first report deciphering the dehydration response of suspension-cultured cells of a crop species, highlighting unique and shared pathways, and adaptive mechanisms via profiling of 330 metabolites.

Abstract

Grasspea, being a hardy legume, is an ideal model system to study stress tolerance mechanisms in plants. In this study, we investigated the dehydration-responsive metabolome in grasspea suspension-cultured cells (SCCs) to identify the unique and shared metabolites crucial in imparting dehydration tolerance. To reveal the dehydration-induced metabolite signatures, SCCs of grasspea were exposed to 10% PEG, followed by metabolomic profiling. Chromatographic separation by HPLC coupled with MRM-MS led to the identification of 330 metabolites, designated dehydration-responsive metabolites (DRMs), which belonged to 28 varied functional classes. The metabolome was found to be constituted by carboxylic acids (17%), amino acids (13.5%), flavonoids (10.9%) and plant growth regulators (10%), among others. Pathway enrichment analysis revealed predominance of metabolites involved in phytohormone biosynthesis, secondary metabolism and osmotic adjustment. Exogenous application of DRMs, arbutin and acetylcholine, displayed improved physiological status in stress-resilient grasspea as well as hypersensitive pea, while administration of lauric acid imparted detrimental effects. This represents the first report on stress-induced metabolomic landscape of a crop species via a suspension culture system, which would provide new insights into the molecular mechanism of stress responses and adaptation in crop species.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

ABA:

Abscisic acid

DRM:

Dehydration-responsive metabolite

DW:

Dry weight

FDA:

Fluorescein diacetate

FW:

Fresh weight

GA:

Gibberellic acid

JA:

Jasmonic acid

MDA:

Malondialdehyde

PC:

Principal component

PEG:

Polyethylene glycol

SA:

Salicylic acid

SCC:

Suspension-cultured cell

TFC:

Total flavonoid content

TPC:

Total phenolic content

References

  • Alexieva V, Sergiev I, Mapelli S, Karanov E (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ 24:1337–1344

    Article  CAS  Google Scholar 

  • Barpete S (2015) Genetic associations, variability and diversity in biochemical and morphological seed characters in indian grass pea (Lathyrus sativus L.) accessions. Fresen Environ Bull 24:492–497

    CAS  Google Scholar 

  • Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207

    Article  CAS  Google Scholar 

  • Begum AA, Leibovitch S, Migner P, Zhang F (2001) Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments. J Exp Bot 52:1537–1543

    Article  CAS  PubMed  Google Scholar 

  • Bowne JB, Erwin TA, Juttner J, Schnurbusch T, Langridge P, Bacic A, Roessner U (2012) Drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the metabolite level. Mol Plant 5:418–429

    Article  CAS  PubMed  Google Scholar 

  • Brenner ED, Stahlberg R, Mancuso S, Vivanco JM, Baluska F, van Volkenburgh E (2006) Plant neurobiology: an integrated view of plant signaling. Trends Plant Sci 11:413–419

    Article  CAS  PubMed  Google Scholar 

  • Carpita N, Sabularse D, Montezinos D, Delmer DP (1979) Determination of the pore size of cell walls of living plant cells. Science 205:1144–1147

    Article  CAS  PubMed  Google Scholar 

  • Chattopadhyay A, Subba P, Pandey A et al (2011) Analysis of the grasspea proteome and identification of stress-responsive proteins upon exposure to high salinity, low temperature, and abscisic acid treatment. Phytochemistry 72:1293–1307

    Article  CAS  PubMed  Google Scholar 

  • Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Res 17:121

    Article  CAS  PubMed  Google Scholar 

  • Das A, Rushton PJ, Rohila JS (2017) Metabolomic profiling of soybeans (Glycine max L.) reveals the importance of sugar and nitrogen metabolism under drought and heat stress. Plants 6:21

    Article  CAS  PubMed Central  Google Scholar 

  • Degenkolbe T, Do PT, Kopka J, Zuther E, Hincha DK, Kohl KI (2013) Identification of drought tolerance markers in a diverse population of rice cultivars by expression and metabolite profiling. PLoS One 8:e63637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dias C, Estruch SB, Graham SA et al (2016) BCL11A haploinsufficiency causes an intellectual disability syndrome and dysregulates transcription. Am J Hum Genet 99:253–274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Farag MA, Huhman DV, Dixon RA, Sumner LW (2008) Metabolomics reveals novel pathways and differential mechanistic and elicitor-specific responses in phenylpropanoid and isoflavonoid biosynthesis in Medicago truncatula cell cultures. Plant Physiol 146:387–402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferri M, Righetti L, Tassoni A (2011) Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell cultures. J Plant Physiol 168:189–195

    Article  CAS  PubMed  Google Scholar 

  • Geng S, Misra BB, Armas E, Huhman DV, Alborn HT, Sumner LW, Chen S (2016) Jasmonate-mediated stomatal closure under elevated CO2 revealed by time-resolved metabolomics. Plant J 88:947–962

    Article  CAS  PubMed  Google Scholar 

  • Geng S, Yu B, Zhu N, Dufresne C, Chen S (2017) Metabolomics and proteomics of Brassica napus guard cells in response to low CO2. Front Mol Biol-Metabolomics 4:51

    Article  CAS  Google Scholar 

  • Ghatak A, Chaturvedi P, Weckwerth W (2018) Metabolomics in plant stress physiology. In: Varshney R, Pandey M, Chitikineni A (eds) Plant genetics and molecular biology. Advances in biochemical engineering/biotechnology. Springer, Cham, pp 187–236

  • Golldack D, Li C, Mohan H, Probst N (2014) Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci 5:151

    Article  PubMed  PubMed Central  Google Scholar 

  • Gong XO, Bisson MA (2002) Acetylcholine-activated Cl-channel in the Chara tonoplast. J Membrane Biol 188:107–113

    Article  CAS  Google Scholar 

  • Grima G, Benz B, Parpura V, Cuenod M, Do KQ (2003) Dopamine-induced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophrenia Res 62:213–224

    Article  Google Scholar 

  • Grzesiak S, Iijima M, Kono Y, Yamauchi A (1997) Differences in drought tolerance between cultivars of field bean and field pea. A comparison of drought-resistant and drought-sensitive cultivars. Acta Physiol Plant 19:349–357

    Article  Google Scholar 

  • Gupta S, Wardhan V, Kumar A, Rathi D, Pandey A, Chakraborty S, Chakraborty N (2015) Secretome analysis of chickpea reveals dynamic extracellular remodeling and identifies a Bet v1-like protein, CaRRP1 that participates in stress response. Sci Rep 5:18427

  • Gurung AM, Pang EC, Taylor PW (2002) Examination of Pisum and Lathyrus species as sources of ascochyta blight resistance for field pea (Pisum sativum). Aust J Plant Pathol 31:41–45

    Article  Google Scholar 

  • Hernandez G, Ramírez M, Valdes-Lopez O et al (2007) Phosphorus stress in common bean: root transcript and metabolic responses. Plant Physiol 144:752–767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hillocks RJ, Maruthi MN (2012) Grass pea (Lathyrus sativus): is there a case for further crop improvement? Euphytica 186:647–654

    Article  Google Scholar 

  • Hincha DK, Oliver AE, Crowe JH (1999) Lipid composition determines the effects of arbutin on the stability of membranes. Biophy J 77:2024–2034

    Article  CAS  Google Scholar 

  • Hochberg U, Degu A, Toubiana D, Gendler T, Nikoloski Z, Rachmilevitch S, Fait A (2013) Metabolite profiling and network analysis reveal coordinated changes in grapevine water stress response. BMC Plant Biol 13:184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jia W, Zhang J (2008) Stomatal movements and long-distance signaling in plants. Plant Signal Behav 3:772–777

    Article  PubMed  PubMed Central  Google Scholar 

  • Johnson WE, Li C, Rabinovic A (2007) Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8:118–127

    Article  PubMed  Google Scholar 

  • Khashimova ZS (2003) Hydroxyproline-containing plant proteins. Chem Nat Compd 39:229–236

  • Kisnieriene V, Ditchenko T, Kudryashov A, Sakalauskas V, Yurin V, Ruksenas O (2012) The effect of acetylcholine on Characeae K+ channels at rest and during action potential generation. Open Life Sci 7:1066–1075

    CAS  Google Scholar 

  • Kumar S, Bejiga G, Ahmed S, Nakkoul H, Sarker A (2011) Genetic improvement of grass pea for low neurotoxin (β-ODAP) content. Food Chem Toxicol 49:589–600

    Article  CAS  PubMed  Google Scholar 

  • Lefevre I, Gratia E, Lutts S (2001) Discrimination between the ionic and osmotic components of salt stress in relation to free polyamine level in rice (Oryza sativa). Plant Sci 161:943–952

    Article  CAS  Google Scholar 

  • Maruyama K, Urano K, Yoshiwara K et al (2014) Integrated analysis of the effects of cold and dehydration on rice metabolites, phytohormones and gene transcripts. Plant Physiol 164:1759–1771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Misra BB, Assmann SM, Chen S (2014) Plant single-cell and single-cell-type metabolomics. Trends Plant Sci 19:637–646

    Article  CAS  PubMed  Google Scholar 

  • Moschen S, Bengoa Luoni S, Di Rienzo JA et al (2016) Integrating transcriptomic and metabolomic analysis to understand natural leaf senescence in sunflower. Plant Biotechnol J 14:719–734

    Article  CAS  PubMed  Google Scholar 

  • Moyankova D, Mladenov P, Berkov S, Peshev D, Georgieva D, Djilianov D (2014) Metabolic profiling of the resurrection plant Haberlea rhodopensis during desiccation and recovery. Physiol Plant 152:675–687

    Article  CAS  PubMed  Google Scholar 

  • Nam KH, Shin HJ, Pack IS, Park JH, Kim HB, Kim CG (2015) Metabolomic changes in grains of well-watered and drought-stressed transgenic rice. J Food Sci Agric 96:807–814

    Article  CAS  Google Scholar 

  • Nützmann HW, Huang A, Osbourn A (2016) Plant metabolic clusters–from genetics to genomics. New Phytol 211:771–789

    Article  PubMed  PubMed Central  Google Scholar 

  • Ogawa-Ohnishi M, Matsushita W, Matsubayashi Y (2013) Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana. Nat Chem Biol 9:726

  • Oliver SG, Winson MK, Kell DB, Baganz F (1998) Systematic functional analysis of the yeast genome. Trends Biotechnol 16:373–378

    Article  CAS  PubMed  Google Scholar 

  • Oliver AE, Hincha DK, Tsvetkova NM, Vigh L, Crowe JH (2001) The effect of arbutin on membrane integrity during drying is mediated by stabilization of the lamellar phase in the presence of nonbilayer-forming lipids. Chem Phys Lipids 111:37–57

    Article  CAS  PubMed  Google Scholar 

  • Ollas C, Arbona V, Gomez-Cadenas A (2015) Jasmonoyl isoleucine accumulation is needed for abscisic acid build-up in roots of Arabidopsis under water stress conditions. Plant Cell Environ 38:2157–2170

    Article  CAS  PubMed  Google Scholar 

  • Owen C, Patron NJ, Huang A, Osbourn A (2017) Harnessing plant metabolic diversity. Curr Opin Chem Biol 40:24–30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Padulosi S, Thompson J, Rudebjer P (2013) Fighting poverty, hunger and malnutrition with neglected and underutilized species: Needs, challenges and the way forward. Bioversity International, Rome

    Google Scholar 

  • Pari L, Shagirtha K (2012) Hesperetin protects against oxidative stress related hepatic dysfunction by cadmium in rats. Exp Toxicol Pathol 64:513–520

    Article  CAS  PubMed  Google Scholar 

  • Paul MJ, Cockburn W (1989) Pinitol, a compatible solute in Mesembryanthemum crystallinum L.? J Exp Bot 40:1093–1098

  • Phang JM, Liu W, Zabirnyk O (2010) Proline metabolism and microenvironmental stress. Annu Rev Nutr 30:441–463

  • Plaza-Wuthrich S, Blosch R, Rindisbacher A, Cannarozzi G, Tadele Z (2016) Gibberellin deficiency confers both lodging and drought tolerance in small cereals. Front Plant Sci 7:643

  • Pluskal T, Uehara T, Yanagida M (2012) Highly accurate chemical formula prediction tool utilizing high-resolution mass spectra, MS/MS fragmentation, heuristic rules, and isotope pattern matching. Anal Chem 84:4396–4403

    Article  CAS  PubMed  Google Scholar 

  • R Development Core Team (2011) R: A language and environment for statistical computing. R foundation for statistical computing. Vienna, Austria. http://www.R-project.org

  • Rabara RC, Tripathi P, Rushton PJ (2017) Comparative metabolome profile between tobacco and soybean grown under water-stressed conditions. BioMed Res Int 2017:3065251

  • Rathi D, Chakraborty S, Chakraborty N (2015) Proteomics of an orphan legume, grasspea: current status and future strategy. Plant Tissue Cult Biotechnol 25:117–141

    Article  Google Scholar 

  • Rathi D, Pareek A, Gayali S, Chakraborty S, Chakraborty N (2018) Variety-specific nutrient acquisition and dehydration-induced proteomic landscape of grasspea (Lathyrus sativus L.). J Proteomics 183:45–57

    Article  CAS  PubMed  Google Scholar 

  • Rathi D, Gayali S, Pareek A, Chakraborty S, Chakraborty N (2019) Transcriptome profiling illustrates expression signatures of dehydration tolerance in developing grasspea seedlings. Planta. https://doi.org/10.1007/s00425-018-03082-2

  • Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R (2004) When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol 134:1683–1696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sagane Y, Nakagawa T, Yamamoto K, Michikawa S, Oguri S, Momonoki YS (2005) Molecular characterization of maize acetylcholinesterase. A novel enzyme family in the plant kingdom. Plant Physiol 138:1359–1371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sanchez DH, Schwabe F, Erban A, Udvardi MK, Kopka J (2012) Comparative metabolomics of drought acclimation in model and forage legumes. Plant Cell Environ 35:136–149

    Article  PubMed  Google Scholar 

  • Sgherri C, Stevanovic B, Navari-Izzo F (2004) Role of phenolics in the antioxidative status of the resurrection plant Ramonda serbica during dehydration and rehydration. Physiol Plant 122:478–485

    Article  CAS  Google Scholar 

  • Sharma S, Verslues PE (2010) Mechanisms independent of abscisic acid (ABA) or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery. Plant Cell Environ 33:1838–1851

  • Silvestre S, de Sousa Araújo S, Vaz Patto MC, Marques da Silva J (2014) Performance index: an expeditious tool to screen for improved drought resistance in the Lathyrus genus. J Integr Plant Biol 56:610–621

    Article  PubMed  Google Scholar 

  • Stoddard FL, Balko C, Erskine W, Khan HR, Link W, Sarker A (2006) Screening techniques and sources of resistance to abiotic stresses in cool-season food legumes. Euphytica 147:167–186

    Article  Google Scholar 

  • Suau R, Cuevas A, Valpuesta V, Reid MS (1991) Arbutin and sucrose in the leaves of the resurrection plant Myrothamnus flabellifolia. Phytochemistry 30:2555–2556

    Article  CAS  Google Scholar 

  • Sugiyama KI, Tezuka T (2011) Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus. Plant Signal Behav 6:1545–1553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suguiyama VF, Silva EA, Meirelles ST, Centeno DC, Braga MR (2014) Leaf metabolite profile of the Brazilian resurrection plant Barbacenia purpurea Hook. (Velloziaceae) shows two time-dependent responses during desiccation and recovering. Front Plant Sci 5:96

  • Sun C, Gao X, Fu J, Zhou J, Wu X (2015) Metabolic response of maize (Zea mays L.) plants to combined drought and salt stress. Plant Soil 388:99–117

    Article  CAS  Google Scholar 

  • Szabo E, Thelen A, Petersen M (1999) Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Rep 18:485–489

    Article  CAS  Google Scholar 

  • Talukdar D (2011) Morpho-physiological responses of grass pea (Lathyrus sativus L.) genotypes to salt stress at germination and seedling stages. Legume Res 34:232–241

    Google Scholar 

  • Talukdar D (2012) Total flavonoids, phenolics, tannins and antioxidant activity in seeds of lentil and grass pea. Int J Phytomed 4:537

    CAS  Google Scholar 

  • Watson BS, Bedair MF, Urbanczyk-Wochniak E et al (2015) Integrated metabolomics and transcriptomics reveal enhanced specialized metabolism in root border cells. Plant Physiol 167:1699–1716

  • Wisniewska J, Tretyn A (2003) Acetylcholinesterase activity in Lycopersicon esculentum and its phytochrome mutants. Plant Physiol Biochem 41:711–717

    Article  CAS  Google Scholar 

  • Yamamoto K, Sakamoto H, Momonoki YS (2011) Maize acetylcholinesterase is a positive regulator of heat tolerance in plants. J Plant Physiol 168:1987–1992

    Article  CAS  PubMed  Google Scholar 

  • Yobi A, Wone BW, Xu W, Alexander DC, Guo L, Ryals JA, Oliver MJ, Cushman JC (2013) Metabolomic profiling in Selaginella lepidophylla at various hydration states provides new insights into the mechanistic basis of desiccation tolerance. Mol Plant 6:369–385

    Article  CAS  PubMed  Google Scholar 

  • Yuan L, Wang L, Han Z, Jiang Y, Zhao L, Liu H, Yang L, Luo K (2012) Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants. J Exp Bot 63:2513–2524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Council of Scientific and Industrial Research (CSIR), Govt. of India, for providing pre-doctoral fellowship to D.R. and the University Grant Commission (UGC), Govt. of India, for providing pre-doctoral fellowship to A.P. This work was supported by Grants (38/1385/14/EMR-II) from the Council of Scientific and Industrial Research (CSIR), Govt. of India.

Author information

Authors and Affiliations

  1. National Institute of Plant Genome Research, Jawaharlal Nehru University Campus, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Divya Rathi, Akanksha Pareek, Subhra Chakraborty & Niranjan Chakraborty

  2. Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610, USA

    Tong Zhang, Qiuying Pang & Sixue Chen

  3. Proteomics and Mass Spectrometry Facility, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32610, USA

    Sixue Chen

Authors
  1. Divya Rathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akanksha Pareek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiuying Pang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sixue Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Subhra Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Niranjan Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Niranjan Chakraborty.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rathi, D., Pareek, A., Zhang, T. et al. Metabolite signatures of grasspea suspension-cultured cells illustrate the complexity of dehydration response. Planta 250, 857–871 (2019). https://doi.org/10.1007/s00425-019-03211-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-019-03211-5

Keywords

  • Dehydration
  • Hardy legume
  • Metabolite signature
  • Non-targeted metabolomics
  • Suspension culture
  • Underutilized species