Molecular evolution and structural variations in nuclear encoded chloroplast localized heat shock protein 26 (sHSP26) from genetically diverse wheat species

Highlights

• Identification and in silico characterization of choloroplast specific HSP26 allelic variants in wild relatives of wheat.

• A novel HSP26 gene discovered in DD-genome assembly of diploid progenitor wheat, the orthologue of which -is not present in hexaploid wheat genome.

• First report -on the isolation of a novel chloroplast specific sHSP26 protein from Ae. tauschiiaccession pau14376 (GenBank:MN119514).

• A 9-bp indel in TmHSP26-1(GA) translated into a deletion of SPM amino acid segment in chloroplast specific conserved consensus region III.

• High degree of divergence in nucleotide sequence between cultivated and wild species appeared in the form of higher ω values (Ka/Ks >1).

Abstract

Heat shock proteins are an important class of molecular chaperones known to impart tolerance under high temperature stress. sHSP26, a member of small heat shock protein subfamily is specifically involved in protecting plant’s photosynthetic machinery. The present study aimed at identifying and characterizing sequence and structural variations in sHSP26 from genetically diverse progenitor and non-progenitor species of wheat. In silico analysis identified three paralogous copies of TaHSP26 to reside on short arm of chromosome 4A while one homeologue each was localized on long arm of chromosome 4B and 4D of cultivated bread wheat. Wild DD-genome donor Aegilops tauschii carried an additional sHSP26 gene (AET4Gv20569400) which was absent in the cultivated DD genome of bread wheat. In vitro amplification of this novel gene in wild accessions of Ae. tauschii and synthetic hexaploid wheat but not in cultivated bread wheat validated this finding. Further, significant length polymorphism could be identified in exon1 from diverse sHSP26 sequences. Multiple sequence alignment of procured sequences revealed numerous sSNPs and nsSNPs. D3A, P125 L, Q242 K were designated as homeolog specific- while A49 G as non-progenitor specific amino acid replacements. A 9-bp indel in TmHSP26-1(GA) translated into a deletion of SPM amino acid segment in chloroplast specific conserved consensus region III. High degree of divergence in nucleotide sequence between cultivated and wild species appeared in the form of higher ω values (Ka/Ks >1) indicating positive selection during the course of evolution. Phylogenetic analysis elucidated ancestral relationships between wheat sHSP26 proteins and orthologous proteins across plant kingdom. Overall, data mining approach may be employed as an effective pre-breeding strategy to identify and mobilize novel stress responsive genes and distinct allelic variants from wider germplasm collections of wheat to enhance climate resilience of present day elite wheat cultivars.

Introduction

Wheat, a cool season crop, has been found to suffer enormously due to any change in ambient temperature in its growth environment. Global warming has resulted in a slow but gradual increase in daily atmospheric temperature. Lobell and Field (2007) reported that wheat yield decreased globally by ∼5.4% per 1⁰C rise in mean minimum or maximum temperature from 1991 to 2002. Further, climate change is predicted to have a significant impact on temperature (estimated to increase by ±2⁰C by 2050) and precipitation profiles in wheat growing regions around the world. Accordingly there is an urgent need to identify new sources of genetic variation and develop wheat cultivars that are resilient to climatic uncertainties.

Wheat experiences heat stress to varying degrees during different phenological stages of its development. Photosynthesis is one of the most sensitive physiological processes to elevated temperatures as any sudden spike in day temperature alters structural integrity and normal functioning of the chloroplast (Hugly et al., 1989.; Olmos et al., 2007). Oxidative burst, as one of the by-product of heat stress induced redox perturbation leads to lipid peroxidation in thylakoids membranes resulting in increased membrane fluidity and dislodging of light harvesting complex, especially photosystem II (Al‐Khatib and Paulsen, 1984; Prasad et al., 2008). This impaired steady state physiology is accompanied by protein degradation and inactivation of the enzymes involved in chlorophyll biosynthesis, tetrapyrrole metabolism (Tewari and Tripathi, 1998) and carbon assimilation (Xue et al., 2008). Accelerated leaf senescence reduces net supply of photo assimilates to the developing kernels affecting starch deposition. Accordingly, wheat grown in tropical climates of India is particularly prone to yield penalty on account of rising diurnal temperature and concomitant drought stress developed due to increased transpiration and surface evaporation especially during the grain filling period.

Of the diverse endogenous tolerance mechanisms developed by the plants during the course of evolution, production of heat shock proteins under high temperature stress is well known. Heat shock proteins are an evolutionary conserved class of proteins, divided into sub-families based on their apparent molecular weight i.e. HSP100, HSP90, HSP70, HSP60 and small HSPs (Boston et al., 1996). They have been found to play a very significant role in preventing misfolding of native proteins and aggregation of denatured proteins under stress (Nakamoto and Vigh, 2007). Of these, small heat shock proteins (size ranging from 15 kDa to 40 kDa) have been recognized as the first line of defense in the cell when proteins begin to misfold (Hilton et al., 2012). Sixteen sub-families of sHSPs have been identified in higher plants, with 11 cytoplasmic or nuclear localized (CI –CXI) and five organelle-localized, each of which is known to encode a protein destined for a specific cellular compartment (Waters et al., 1996). sHSP26 is a nuclear encoded protein that consists of a variable amino terminal region, a more conserved carboxyl terminal region (that harbors heat shock domain, consensus region I, consensus region II) and a consensus region III, also referred to as methionine bristles. The N-terminal region bearing the leader sequence (specific for each sub-cellular organelle) is highly diverse, with functional role implicated in substrate binding (Giese et al., 2005). The 90–100 amino acid long HSP20 domain (α-crystalline domain) is essentially conserved among different subfamilies of sHSP and is suggested to be important for oligomerization (Sun and MacRae, 2005; Basha et al., 2012). However, methionine rich amphipathic α-helix has been found to be exclusively present in sHSP polypeptides functional in chloroplast (Chen and Vierling, 1991; Waters, 1995). The ability of these methionine bristles to undergo oxidation dependent conformational changes may protect chloroplast from oxidative stress (Harndahl et al., 1999). sHSP26 is synthesized as a precursor polypeptide, which when transported to chloroplast gets associated with either soluble stroma portion or grana region of the thylakoid membrane and plays its part in enhancing the photochemical efficiency of PSII against heat and accompanied oxidative stress (Lee et al., 2000; Chauhan et al., 2012; Kim et al., 2012). The first complete sequence of this low molecular weight (LMW) chloroplast HSP from wheat was reported in 1991 (Weng et al., 1991), followed by the identification of its novel isoform in 1993 (Nguyen et al., 1993).

Up-regulation in the expression of heat stress responsive transcription factors (HSFs) and induction of heat shock proteins (HSPs) upon exposure to elevated temperature is well established (Chauhan et al., 2011; Waters, 2013; Abu-Romman, 2015). In order to understand the importance of HSP26 in acquired thermotolerance, a few studies concerned with development of transgenic (Chauhan et al., 2012), promoter analysis (Khurana et al., 2013), variability in the expression of heat shock proteins in durum (Yildiz and Terzi, 2008; Rampino et al., 2009) and wild species (Rampino et al., 2018) have been carried out in wheat. Hu et al., 2015a demonstrated ZmHSP26 to strongly interact with four chloroplastic proteins, namely ATP synthase subunit β, chlorophyll a–b binding protein, oxygen evolving enhanced protein I and PSI reaction center subunit IV to protect chloroplast under high temperature stress. Comastri et al., 2018 has recently isolated and molecularly characterized homologs of sHSP26 from T. durum cultivars and identified SNPs using TILLING online resource and KASP markers.

The structural-, functional- and evolutionary relationships of sHSPs across the plant kingdom have been extensively studied past two decades and are well documented (Waters 1195, 1996, Waters et al., 2008a; Safdar et al., 2012; Bondino et al., 2012). In recent years, several studies have reported genome wide identification of HSPs in general and sHSP gene family in particular in different plants like Arabidopsis (Scharf et al., 2001; Siddique et al., 2008), sugarcane (Borges et al., 2007), populus (Waters et al., 2008a), rice (Sarkar et al., 2009), CAM plants (Shakeel et al., 2012), soybean (Lopes-Caitar et al., 2013), pepper (Guo et al., 2015), barley (Pandey et al., 2015), setaria (Singh et al., 2016), wheat (Pandey et al., 2015; Muthusamy et al., 2017; Wang et al., 2017). Genome wide analysis of HSFs in T.aestivum (Xue et al., 2014), T.urartu and Ae. tauschii with spatial variation in expression has also been conducted (Yang et al., 2014). Though Muthusamy et al., 2017 carried out a comprehensive analysis of sHSP gene family in bread wheat genome and Wang et al., 2017 gave an evolutionary angle to the theme by including sHSPs sequences from related wild wheat species, but exploring the degree of sequence divergence and extent of structural variations in chloroplast specific sHSP26 as the molecular basis of the inherent thermo tolerant nature of wild wheat relatives did not emerge as the primary focus of these studies.

Wheat is an allohexaploid, combining genome of three diploid grass species, A-genome of T. urartu, B-genome from a species within the sitopsis section and related to Ae. speltoides and D-genome of Ae. tauschii (Dvorak and Zhang, 1992; Feldman et al., 1995). Wild wheat species have been found to be more tolerant to abiotic stress constraints (like high temperature, drought, and salinity stress) than cultivated wheat (Vierling and Nguyen, 1990, 1992, Ehdaie and Waines, 1992; Zaharieva et al., 2001; Dulai et al., 2006; Gupta et al., 2010; Pradhan et al., 2012; Awlachew et al., 2016; Suneja et al., 2015, 2017; Suneja et al., 2019). Wheat breeding efforts have generally been directed towards assessing acquired thermotolerance potential of genetically diverse accessions or genotypes in a segregating population using different empirical traits like cell membrane stability (Ibrahim and Quick, 2001), TTC based cell viability (Fokar et al., 1998), chlorophyll fluorescence, stay green habit, canopy temperature depression (Reynolds et al., 1998). Research into physiological and biochemical basis of crop adaptation to stress is well established (Fischer and Turner, 1978; Richards et al., 2002) with molecular technologies adding a new dimension to it (Chaves et al., 2003). Since the rice genome was completely sequenced more than ten years ago, the reference genome sequence of several other major crops- including barley, millet, maize, sorghum, pearl millet, brachypodium, secale cereale, soybean, chickpea, potato, tomato, brassica etc. have been reported (Bolger et al., 2014). International wheat genome sequencing consortium has already sequenced 16 Gb long genome of hexaploid wheat (IWGSC, 2014; Marcussen et al., 2014). Genome sequence of Ae. tauschii (Luo et al., 2012; Jia et al., 2013) and T. urartu (Ling et al., 2013) has also been published.

Successful implementation of international efforts like 1000 Genome project, HapMap Project (in humans) and 1001 Genomes for Arabidopsis (Weigel and Mott, 2009) have demonstrated the -significance of single nucleotide polymorphism (SNPs) as the basis of genetic variation within a species or a population and subsequently brought re-sequencing efforts to the forefront. With sequencing becoming cost effective (and molecular applications like Genotyping by Sequencing (GBS) proving their mettle in revealing intraspecific variation (Vikram et al., 2016; Singh et al., 2018), followed by parallel advancement in genome analysis tools, rapid inspection of sequence variation is now within the reach of crop scientists. Data mining approach may thus be used as a surrogate to identify new sources of genetic variability to be utilized in pre-breeding programs to improve plant performance for agronomic traits of economic importance. With the annotated genome sequence of wheat and its wild relatives becoming publically available, gene information can now be effectively utilized for dissecting specific traits of interest. Allelic richness of wild species and old landraces can accordingly be examined for the key genetic loci to identify a potentially adaptive allele, which if introduced into a useful genetic background, may add to the efficiency of persisting stress tolerance mechanisms and enhance overall performance of the elite cultivar (Bansal et al., 2014; Brozynka et al., 2016). Wide hybridization, alien introgression and cisgenics offer good prospects for using retrieved genomic information to breed heat-tolerant crops, particularly wheat.

Therefore, the present study was planned to identify genomic homeologs and orthologs from genetically diverse wild and cultivated relatives of wheat and explore the extent of sequence level variations in sHSP26 gene accumulated during the course of wheat polyploidization. Sequences from whole genome assemblies of released cultivars- Cadenza, Claire, Robigus, Paragon (hexaploid bread wheat) and Kronos, Svevo (tetraploid durum wheat) were also included (keeping in mind the importance of resequencing) to further confirm species specific SNPs identified from Chinese Spring database (and identify cultivar-specific differences, if any) . Allelic diversity in the coding region of sHSP26 sequences provided novel insights into molecular evolution of sHSP26 gene within the triticeae tribe. Amplification of in silico identified sub-genome specific copies of sHSP26 gene and its homologs across 18 wild and cultivated wheat accessions allowed validation of key results obtained from data mining approach. It also shed light on how diversity in the nucleotide sequence translated into differences in amino acid composition of conserved domains and motifs that might influence functionality of this heat shock protein under stress. Estimation of Ka/Ks between all gene pairs and phylogenetic analysis illustrated sub-genome specific clustering of sHSP26 proteins and their evolutionary diversification.