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Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface

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Abstract

Main conclusion

Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves.

Abstract

Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17 °C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

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Data availability statement

All data generated or analysed in this study is included in this manuscript and its supplementary information files. Raw data, including biological replicates, are available from the corresponding author on reasonable request.

Abbreviations

AFP:

Antifreeze protein

BJ:

Brassica juncea

FTIR:

Fourier transform infrared spectroscopy

ICM:

Ice crystal morphology

IRI:

Ice recrystallization inhibition

PR:

Pathogenesis-related

TH:

Thermal hysteresis

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Acknowledgements

This study was partially supported by Institute of Eminence (IoE), University of Delhi, FRP grant (IoE/FRP/LS/2020/27 & IoE/2021/12/FRP) and SAP grant F.3-5/2018/DRS-II (SAP II). SP availed senior research fellowship from Council of Scientific and Industrial Research (CSIR), India, MA received scholarship from University Grant Commission (UGC), India and Abhishek availed junior research fellowship from Department of Biotechnology (DBT), India.

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  1. Kailash Yadav and Meenakshi Arya have contributed equally.

Authors and Affiliations

  1. Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi, 110007, India

    Kailash Yadav, Preet Manchanda, Abhishek Kumar & Renu Deswal

  2. Department of Botany, Dyal Singh College, University of Delhi, Delhi, 110003, India

    Meenakshi Arya

  3. Department of Botany, Acharya Narendra Dev College, University of Delhi, Delhi, 110019, India

    Satya Prakash

  4. Department of Botany, Gargi College, University of Delhi, Delhi, 110049, India

    Bhavana Sharma Jha

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  1. Kailash Yadav
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425_2023_4203_MOESM3_ESM.pdf

Supplementary file3 Fig. S1 STRING analysis of three putative abundant AFPs. a Thaumatin like protein. b Chitinase. c Osmotin like protein. Fig. S2 a Ice crystal morphologies in B. juncea, B. nigra, B. rapa and R. sativus cuticle samples at different dilutions (0.1 mg/mL to 0.0125 mg/mL), after heat treatment and after proteinase K treatment. b Bar graphs showing TH values in B. juncea, B. nigra, B. rapa, and R. sativus cuticle samples at a concentration of 0.1 mg/mL All the samples are mean of three technical replicates (n=3, ±SD). Fig. S3 RuBisCO (chloroplastic marker) immunoblotting and G6PDH assay (cytosolic marker) to assess purity of isolated CPE fraction. a Cuticle proteins and crude leaf proteins were transferred to NC membrane. Blot was developed using anti-RuBisCO antibodies earlier developed in the lab. b G6PDH activity assay of CPE fraction. Activity of G6PDH activity in the CPE fraction is expressed as a % of activity in crude fraction. Fig. S4 Thin layer chromatography showing the presence of polysaccharides, lipids and amino acids in BJ ABC supernatant. Mannose and xylose sugars have been used as positive control for polysaccharides and negative control for lipids and proteins. Fig. S5 FTIR spectra of ABC pellet (a), pronase treated ABC supernatant (b), ZipTip processed ABC supernatant (c). FTIR spectra of pronase treated ABC supernatant and ZipTip processed ABC supernatant are compared with xylose FTIR spectra. In all, 32 scans per sample were done to reach desirable spectra. Air was included as a blank. Graph shows wave number on x-axis and transmittance on y-axis. Origin software, version 8.6 was used for plotting the graph (PDF 377 KB)

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Yadav, K., Arya, M., Prakash, S. et al. Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface. Planta 258, 44 (2023). https://doi.org/10.1007/s00425-023-04203-2

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