Elsevier

Journal of Biotechnology

Volume 327, 10 February 2021, Pages 97-105
Journal of Biotechnology

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury

https://doi.org/10.1016/j.jbiotec.2021.01.003Get rights and content

Highlights

  • IRI3 transcript enhancement renders transgenic potato tolerant to freezing stress.

  • Reduced electrolyte leakage was found associated with IRI3 transcript abundance in transgenic potato lines.

  • Efficient recovery post freezing injury was observed in transgenic potato lines when compared with non-transgenic lines.

Abstract

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14–22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Introduction

Being sessile in nature, plants have to withstand all environmental stresses both biotic and abiotic. However, plants have evolved sophisticated protective mechanism and responses at cellular, molecular and physiological level that help them cope with all stress factors. Drastically changing temperature variations (heat, frost etc.) across the globe severely affect the production of crops including cultivated potato (Levy and Veilleux, 2007). Potato is the fourth major food crop worldwide and its production in Pakistan is being threatened by the recent severe and prolong frost spells. Potato is very sensitive species affected by freezing injury (Pino et al., 2008a; Duman and Wisniewski, 2014) causing up to 40 % yield losses while prolong exposure can lead to death of potato plant (Zhu, 2016). Frost not only affects the potato yield but also deteriorate its quality and this effect became more pronounced if the crop is at tuber formation stage (Iovene et al., 2004). A temperature below subzero reduces the plant’s efficiency in carrying out physiological processes causing reduced growth and leads to reduced agricultural productivity.

Plants at subzero temperatures, experience ice formation in intercellular spaces which causes freezing injury. This freezing injury causes cellular dehydration due to the fact that extremely low temperatures initiate the conversion of water molecules in ice crystals (Duman and Wisniewski, 2014) and cause structural damages of cells. The damaged cells then cannot perform normal physiological functions (i.e., respiration, transpiration and photosynthesis). Hence, reduced photosynthesis tend to reduce stored food in potato tuber (Bartels-Rausch et al., 2012) while evaporation tends to accelerate because of presence of damaged cells at leaf surfaces ((Kratsch and Wise, 2000; Li et al., 2015; Liu et al., 2019).

Although conventional breeding methods have contributed significantly towards trait improvement in potato but the efforts were limited towards development of tolerance against temperature stress. However; generation of transgenic plants through expression of abiotic stress tolerant transgenes proves to be an important approach. Several transcription factors have been utilized in order to create stress tolerance against freezing injury which includes transformation of potato with Arabidopsis CBF genes (AtCBF1−3) under the control of stress-inducible promoter for efficient transgene expression and resulted in significant tolerance of transgenic lines against freezing stress (Pino et al., 2008b); Invertase gene derived from yeast (Deryabin et al., 2003); Arabidopsis thaliana derived DREB1A transgene expression in potato under the control of rd29A promoter (Behnam et al., 2007); AtDREB1B transgene (Movahedi et al., 2012); and zinc finger transcription factor (SCOF-1) from soybean (Kim et al., 2016). Although, the expression of transcription factors render plants tolerant towards freezing stress but in this context, Ice Recrystallization inhibition (IRI) proteins also termed as antifreeze proteins have provided even better protection against freezing stress injury (Wallis et al., 1997; Griffith and Yaish, 2004; Walters et al., 2009).

Ice binding proteins (IBPs) adsorb to ice crystals and modify their growth that helps organisms avoid freeze injury. The plant IBPs are known as antifreeze proteins or IRI proteins (Bredow and Walker, 2017). Lolium perenne is a freeze-tolerant perennial ryegrass that can tolerate freezing temperatures upto −13 °C (Bredow et al., 2016). These proteins exhibit a specific property termed as ice recrystallization inhibition (IRI). More specifically, IRI proteins reduce the freezing point by hindering secondary nucleation event during Thermal hysteresis (TH) activity (Kristiansen and Zachariassen, 2005). These proteins have been identified in algae, fungi, arthropods, eubacteria and more than 30 plant species e.g. carrot (Wang et al., 2020), bitter sweet (Huang and Duman, 2002), winter rye (Yu and Griffith, 1999), and perennial ryegrass (Bredow et al., 2016) and wheat (Bredow and Walker, 2017).

The environmental stresses including the cold stress beyond the physiological threshold of a plant trigger increased accumulation of reactive oxygen species (ROS) in subjected cells (Weydert and Cullen, 2010). The lower levels of ROS are necessary for all oxygen metabolizing processes in a normal cell that include apoptosis, cell proliferation, and differentiation but enhanced levels prove cytotoxic (Lo et al., 1996). The over production of ROS leads to oxidative stress that disrupt the normal cellular metabolism in plant cells through oxidation of proteins, nucleic acids, membrane lipids (Weydert and Cullen, 2010). Several anti-oxidative defense mechanisms activate and coordinate in the plant to overcome the damages associated with the high concentration of ROS. These defense mechanisms may comprise of production of certain antioxidant enzymes like peroxidase dismutase,(POD), superoxide dismutase (SOD), catalase (CAT), Glutathione reductase (GR) and few plant metabolites that include proline (Soengas et al., 2018).

Here, we report improvement of local potato lines against freezing injury through expression of Lollium perenne derived IRI3 gene and evaluation of the transgenic potato lines for frost tolerance under cold stress treatment. Our findings suggested significantly enhanced frost tolerance in transgenic potato lines with desirable agronomic traits in potato.

Section snippets

Homology modeling and validation

The 3D structure of synthesized IRI3 gene originally derived from perennial ryegrass (Lolium perenne) was predicted through homology modeling (Fiser and Šali, 2003). BLASTp was used to select templates for modeling of the protein as described by Gish (1993). Seven variable templates; 3ULT, 4Z64, 6FG8, 4Z61, 4Z63, 6FG7, and 4Q3I with highest amino acid homology to query sequence were selected followed by an automated comparative protein modeling performed using Modeler9v13 (Fiser and Šali, 2003

Results

The primary aim of this study was to develop transgenic potato lines conferring frost tolerance in drastically changing climate. A synthetic ∼844 bp IRI3 gene was transformed in potato variety Diamant. Screening of transgene ‘IRI3′ for abiotic stress potential was evaluated through in-vitro cold stress treatment. Further the transgenic plant lines were evaluated in field with short spells of frost in January 2019 and were analyzed at biochemical and molecular level to reveal antioxidant

Discussion

With drastically changing climatic condition across the globe, temperature stress including heat, cold and freezing stress has emerged as potential threat to agriculture causing severe yield losses. The climatic models predict an increase in the frequency and severity of the late-frost periods in near future, which will further magnify the crop losses. The freezing temperatures cause intra- and extracellular ice formation provoking osmotic stress leading to cell death (Ambroise et al., 2019).

CRediT authorship contribution statement

Khadija Aaliya: Investigation. Idrees Ahmad Nasir: Supervision, Conceptulization. Anwar Khan: Writing - original draft. Nida Toufiq: Investigation. Iqra Yousaf: Validation. Olawale Samuel Adeyinka: Investigation. Sehrish Iftikhar: Software, Supervision. Abdul Munim Farooq: Investigation. Bushra Tabassum: Writing - review & editing, Data curation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by Higher Education Commission (HEC) under HEC Indigenous fellowship scheme.

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