Introduction

Plants being sessile organisms are highly influenced by different stresses exerted by the changing environment. Plant growth, development, survival and yield depends upon the mechanism with which they adapt to tolerate such unpropitious environmental conditions. In response to such conditions plants have evolved advanced and complex defence mechanisms. These responses may include the production of reactive oxygen species (ROS), change in redox potential or cellular level of Ca2+, disruption of ion homeostasis, and adjustment of membrane fluidity (Choudhury et al. 2017; Gilroy et al. 2016). Abiotic stresses are multigenic in nature and multiple stress responsive genes act in a synchronized manner to attain tolerance to abiotic stresses (Tuteja 2007). One such family of genes that provides tolerance to a plethora of abiotic stresses is the Universal Stress Proteins (USPs). These genes are known to be present in a profusion of organisms, ranging from bacteria to higher life forms like plants and animals (Vollmer et al. 2018).

The Universal Stress Protein (USP) was first discovered in Escherichia coli as a 13.5 kDa cytosolic protein which was induced under a broad range of nutrient starvation stresses (Nystrdm and Neidhardt 1992). Proteins of this class play a crucial role in cell survival during H2O2 stress, osmotic stress, heat shock, exposure to DNA-damaging agents and UV light treatment in Escherichia coli (Nystrom and Neidhardt 1994; Nachin et al. 2005). USP genes have been broadly classified into two categories based on the structural homology of the proteins encoded by these genes with MJ0577 protein of Methanocaldococcus jannaschii and USPA protein of Haemophilus influenza. USPs having structural homology with USPA protein of Haemophilus influenza lack the ATP-binding residues (Sousa and Mckay 2001). The USPs which have a structural homology with MJ0577 protein contain an ATP-binding motif G-2X-G-9X-G-(S/T) at their C-terminal region and a five β-strands—four α-helical α/β-core structure (Aravind et al. 2002; Siegele 2005). After the discovery of USP in E. coli, many proteins containing at least one USP domain, with a conserved set of 140–160 amino acid residues and other diverse functional motifs have been found from variety of other organisms including bacteria, archaea, plants, and metazoans (Forêt et al. 2011; Vollmer et al. 2018).

In plants, the USP family thrives abundantly and a single plant genome may harbour typically 20–50 members, that may go up to 142 as in Brassica napus. (Li et al. 2010; Chi et al. 2019). The Arabidopsis thaliana genome consists of 44 USP encoding genes (Kerk et al. 2003), which confer tolerance under different abiotic stress conditions. Hypoxia-Responsive Universal stress protein, HRU1 (At3g03270) is involved in ROS homeostasis under anoxia (Gonzali et al. 2015). Plants over-expressing At3g53990 gene show tolerance to heat shock and oxidative stress via redox-dependent chaperone activity (Jung et al. 2015). At3g53990, a cold stress responsive USP possesses strong nucleic acid melting activity and functions as a RNA chaperone to provide tolerance to cold stress (Melencion et al. 2017). Other AtUSPs are identified to be playing various roles in multiple abiotic stresses (Bhuria et al. 2019) but their detailed biochemical and function characterization is still limited in plants. Other than Arabidopsis thaliana, USPs have been studied in Solanum lycopersicum, SlRd2 a USP domain containing protein regulates Calcineurin B-like interacting protein kinase (SlCipk6)-mediated ROS generation. Solanum pennellii, SpUSP confers drought tolerance via regulation of stomatal closure, in seedling and adult stages in tomato.: (Loukehaich et al. 2012; Gutiérrez-Beltrán et al. 2017). Many of the UspA proteins have also been characterized in rice (Sauter et al. 2002), cotton (Maqbool et al. 2009) legumes (Sinha et al. 2016), and Salicornia (Udawat et al. 2016).

At the cellular level, USPs are essentially involved in protein scaffolding, preventing the denaturation of globular macromolecules, and aid in cellular protein transport (Vollmer et al. 2018). However, the precise molecular function and regulation of USPs under diverse abiotic stresses are still unknown in many plants. No literature till date has reported any characterization of USPs in Triticum aestivum. In this study we attempt to identify TaUSP genes within the genome of Triticum aestivum, and characterization of abiotic stress responsive TaUSP genes under different abiotic stress in yeast and A.thaliana heterologous systems.

Material and methods

Identification and phylogenetic analysis of USP genes

USP gene family members were identified by HMM search using wheat transcriptome data. We found 106 USP domain containing proteins encoded by 85 genes in the wheat genome. The putative USP genes were confirmed by using NCBI-CDD search (Marchler-Bauer et al. 2011). They were named as TaUSP_1A-1 to TaUSP_7D-2, according to their annotated gene IDs. For example, gene ID TraesCS1A02G106600.1 has been named as TaUSP_1A-1, in which 1A is chromosome number and 1 signifies the first transcript on it. Further, phylogenetic analysis was done, based on full-length protein sequences with single transcript per gene. Multiple sequence alignment was carried out using CLUSTALW program, and the phylogenetic tree was constructed using the MEGA7 software with Neighbor-Joining method.

Structural and domain analysis of abiotic stress responsive TaUSP gene members

The HMM profiles for USP (PF00582) were retrieved from Pfam database74 https://pfam.xfam.org. Each protein sequence was checked on wheat expression database (http://www.wheat-expression.com). Six TaUSP genes were selected based on their expression levels under different abiotic stresses. Search was performed for these six genes against Ensembl Genomes 49 (http://plants.ensembl.org/index.html). Their chromosomal location, structural organisation (introns, exons, and transcript length) and isoelectric points of the proteins encoded by the selected TaUSP gene family members were analysed. The conserved motifs in the selected TaUSP gene family members were identified using a motif-based sequence analysis tool, MEME suite 5.3.0 (http://meme-suite.org). The sequences were searched for a total of 5 motifs along with the default parameters.

Plant growth conditions and stress treatment

Bread wheat (T. aestivum) variety PBW343 was used in this study. Seeds were surface-sterilized with 4% sodium hypochlorite for 20 min, followed by five washes with autoclaved RO water. Seeds were then grown on a cotton bed in a growth chamber (Conviron, Canada) maintained at 22 ± 1 °C with a 16-h photoperiod. After 10 days the seedlings were subjected to different abiotic stresses such as cold (4 °C for 24 h), salt (200 mM NaCl for 24 h), heat (42 °C for 2 h) and drought (200 mM mannitol for 24 h)(Amoah et al. 2019; Chauhan et al. 2011; Khurana et al. 2013; Hamdi et al. 2020). After the stress treatment, seedlings of control and treated plants were frozen in liquid nitrogen and stored at − 80 °C until RNA isolation. The seeds of the transgenic A. thaliana were germinated on half strength MS media and allowed to grow for 7 days in a growth chamber maintained at 22 °C with a photosynthetic flux density of 300 μ mol m−2 s−1, 60% humidity, and a photoperiod duration of 16/10 h light/dark phase. For drought stress, the plants were transferred to a 200 mM mannitol, supplemented MS media plates after 7 days. Phenotype was observed after 7 days. For expression level of TaUSP_5D-1 in reproductive tissues, T. aestivum plants were grown in open fields during growth seasons and tissue samples were collected for RNA isolation.

RNA isolation and expression analysis

RNA isolation was done by using RNeasy plant mini kit (Qiagen, Germany) as per the instructions. On-column DNaseI treatment was given to the RNA samples to remove any genomic DNA contamination. Thereafter 2 μg RNA was used for the synthesis of cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, United States). For RT-PCR analysis the cDNA was mixed with 200 nM of each primer and SYBR Green PCR Master Mix (Applied Biosystems). ABI Prism 7000 sequence detection system and software (PE Applied Biosystems) was used for the analysis as per the manufacturers’ instructions. Relative fold-change was calculated using expression of TaGAPDH, a housekeeping gene as the reference point. Three biological replicates and three technical replicates were used for plotting the graphs (Meena et al. 2022).

Cloning of abiotic stress responsive TaUSP gene family members

For cloning purpose full length CDSs of all the six abiotic stress responsive TaUSP gene family members (500–550 bp) were amplified with their respective gene specific primers. Control cDNA was isolated from 10-day-old seedlings of T. aestivum (cv. PBW343) and used as the template for gene amplification. The amplified products were cloned into the Gateway entry vector (pENTR™/D-TOPO) and then into the destination vectors pGBKT7 and pGADT7 under T7 promoter for yeast-2-hybrid assay in S. cerevisiae strain AH109. All the six genes were also cloned into destination vector pSITE-3CA under CaMV35S promoter for subcellular localization. The cloning was done following the Gateway™ cloning strategy (Directional TOPO cloning kit and LR clonase enzyme mix II kit, Invitrogen Inc., United States). Abiotic stress responsive TaUSP genes were further cloned into p426GPD, between EcoRI and HindIII (for TaUSP_1A-3, TaUSP_3B-1 and TaUSP_6D-5) and between BamHI and HindIII (for TaUSP_1B-1, TaUSP_1D-1 and TaUSP_5D-1) under GPD promoter for stress assays. Stress assays were done in S. cerevisiae strain BY4741. For generating overexpression lines full length CDS of TaUSP_5D-1 was cloned into gateway destination vector pMDC32, under CaMV35S promoter.

Subcellular localization of abiotic stress responsive TaUSP genes

For subcellular localization pSITE-3CA-TaUSP constructs with the CDS fused in frame with the N-terminal of YFP were used. Each construct was coated on gold particles and bombarded at a pressure of 1100 psi on the onion epidermal peels, plated on 1/2MS media (Lee et al. 2008; Nebenführ 2014). The PDS-1000 bombardment system (Bio-Rad, Canada) was used for the purpose. After bombardment the onion peels were incubated for 12 h at 27 °C in dark. After the incubation period, fluorescence was observed in confocal microscope (Leica, Germany). As the fluorescence seemed to appear in some organelles, expression of each gene was further analysed with the help of ER and Golgi organelle markers.

Yeast-two-hybrid interaction of proteins encoded by abiotic stress responsive TaUSP genes

Proteins encoded by each of the six TaUSP gene family members were checked for the interaction with every other selected member of the family. Each gene was cloned into gateway destination vectors pGBKT7(DBD) and pGADT7(AD). To check for the interaction between two proteins, constructs were used for co-transformation into S. cerevisiae strain AH109, harboring the HIS3 reporter gene. Yeast supplement dropout media, lacking leucine and tryptophan (-LW) was used to select the transformants. Further, the interactions were checked with dilutions on selective media, lacking histidine, leucine and tryptophan (-HLW), other medium lacking adenine, histidine, leucine and tryptophan (-AHLW) and one medium lacking histidine, leucine and tryptophan (-HLW) supplemented with of 1 mM 3AT (3-Amino-1,2,4-triazole) (Meena et al. 2020).

In-silico promoter analysis for abiotic stress responsive TaUSP genes

Genomic sequences of all the six members were fetched from the EnsemblePlant database (http://plants.ensembl.org/index.html). A 1.5 kb region, upstream to the transcription start site was taken. The upstream sequence for each of the six members was then searched for various abiotic stress responsive elements using the Plant Care and PLACE databases (Suppl. Table 1) (Lescot et al. 2002).

Autoactivation assay of TaUSP_5D-1 and BiFC assay for TaUSP_5D-1 interactions

USP genes are known to have DNA binding activity (Melencion et al. 2017). So the DNA binding activity of the selected TaUSP genes was checked via yeast-2-hybrid. TaUSP genes (CDS) were cloned into pDEST-pGBKT7. Yeast cells (strain AH109) were co-transformed with these constructs and with EV pDEST-pGADT7. The DNA binding activity was analysed by drop-out assays on yeast supplement dropout media, lacking histidine, leucine and tryptophan (-HLW) and -HLW with X-α-gal. The activity was also checked by transforming yeast cells with pGBKT7- TaUSP_5D-1 construct and then doing a drop out assay on SD/-HW medium (Meena et al. 2022). Because of DNA binding activity of TaUSP_5D-1, its interactions with protein encoded by other TaUSP genes were further confirmed via BiFC assay. For BiFC assay TaUSP_5D-1 was cloned in pSITE-nEYFP-N1 and genes encoding interacting partners were cloned in pSITE-cEYFP-N1. Both the constructs were coated on gold particles and bombarded with a pressure of 1100 psi on the onion epidermal peels, plated on 1/2MS medium (Lee et al. 2008). All other steps were same as in the localization procedure.

Stress tolerance assays in yeast

For stress tolerance assays, yeast strain BY4741 harboring p426GPD EV and p426GPD-TaUSP constructs were grown in liquid synthetic defined medium lacking uracil containing 2% Glc (w/v) for 24 h at 30 °C. Subsequently, they were diluted to the different concentrations (10−1, 10−2, 10−3, and 10−4) with uniform initial OD600 of 0.4 and 10 μL of each dilution was spotted onto solid YPD medium, supplemented with the different stress agents. Yeast dropouts were allowed to grow for 3 days at 30 °C on SD/-Ura medium supplemented with DTT, LiCL2 H2O2 (Gutiérrez-Beltrán et al. 2017). For temperature stress, yeast cultures overexpressing TaUSP genes were grown overnight in SD/-Ura medium and O.D600 was adjusted to 0.4. These cultures were then grown for 1 h at 42 °C and 46 °C for heat stress. For cold stress cultures were grown at 0 °C and -20 °C for 1 h (Wang et al. 2017; Jiang et al. 2009; Qin et al. 2015).

Overexpression of TaUSP 5D-1 in Arabidopsis thaliana

The floral dip method was used to transform Arabidopsis plants with the GV3101 strain of Agrobacterium tumefaciens carrying the pMDC32- TaUSP 5D-1 (Zhang et al. 2006). After successive generations of selection on MS-agar medium, supplemented with 15 mg/l hygromycin, positive transgenic plants were identified. PCR using gene-specific primers was used to confirm the plants. Members of the T3 generation with 100% hygromycin resistance were considered homozygous. The level of ectopic expression in homozygous plants was checked by RT-PCR analysis.

Histochemical ROS detection

Nitro blue tetrazolium (NBT) and 3,3′-Diaminobenzidine (DAB) staining was done to measure the amount of reactive oxygen species (ROS), produced in response to DS in transgenic Arabidopsis and WT plants (Agarwal and Khurana 2018). For this, 3-week-old Arabidopsis WT and overexpression transgenic seedlings were exposed to DS (200 mM mannitol for 3 days), and then the plants were overnight stained by incubating them in NBT and DAB stains separately. The following day, the seedlings were washed with water and then dipped in a bleaching solution to remove the chlorophyll (ethanol, acetic acid, and glycerol in a ratio of 3:1:1). Following the wash, plants were observed under a bright field light microscope (Leica) and images were acquired for the comparison of ROS in transgenics and the WT Arabidopsis plants.

Results

Identification, phylogenetic and structural analysis of USP genes in Triticum aestivum

A total of 108 USP domain containing proteins encoded by 85 genes were found to be present in the wheat genome, using HMM search and Triticum aestivum ensemble genome_52. The size of TaUSP gene ranged from 423 bp (TaUSP_2D-6) to 1989 bp (TaUSP_6D-5) and the molecular weights of proteins varied from 15.23 kDa (TaUSP_2D-6) to 71.93 kDa (TaUSP_4A-1). The predicted theoretical isoelectric points, ranged from 4.67 (TaUSP_2A-5) to 11.83 (TaUSP_3B-2). Structural and molecular parameters for selected TaUSP encoding proteins are summarised in Table 1. Phylogenetic analysis revealed that TaUSP genes can be grouped into four groups based on their sequence similarity (Fig. 1). Gene structure analysis found that most of these USPs had four exons except TaUSP_1B-1.1, with three exons (Fig. 2a). These genes are found to be diversified in their functions with the presence of different domains, like Ubox, protein kinase catalytic domain, N-terminal serine threonine kinase, adenine nucleotide alpha hydrolases (Suppl. File 4), along with the USP domain. Proteins encoded by selected abiotic stress responsive TaUSPs were analysed for such domains and two prominent domains, USP domain and AANH_like superfamily domain were found in these proteins (Fig. 2b). Sequence analysis of the encoded proteins revealed that except for TaUSP_1B-1, all the abiotic stress response TaUSP genes code for proteins which contain a ATP binding motif similar to MJ0577 Methanocaldococcus jannaschii USP protein (Fig. 3a) (Chi et al. 2019). Phylogenetic analysis of abiotic stress responsive TaUSP genes revealed that they are more closely related to gene encoding MJ0577 protein than Haemophilus influenza_USPA homolog (Fig. 3b).

Table 1 Description of abiotic stress responsive TaUSPs in Triticum aestivum
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Fig. 1
figure 1

Phylogenetic analysis of USP gene family in Triticum aestivum. Sequence alignment for total TaUSP protein sequences was done by CLUSTALW program and phylogenetic tree was constructed based on Neighbor-Joining method using MEGA7. Selected Abiotic stress responsive TaUSPs are highlighted in boxes

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Fig. 2
figure 2

Structural and domain analysis of selected TaUSP members. a Gene structure analysis of selected TaUSP members. Exons are depicted using solid boxes and introns are shown using lines. b Protein domain structure was constructed using MEME Suite software

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Fig. 3
figure 3

Protein sequence comparison analysis of selected TaUSPs with MJ0577 protein and H. influenzae USP. a Nucleotide sequence analysis for ATP binding motif in TaUSPs, MJ0577 USP and H. influenzae USP. b Phylogenetic analysis based on protein sequence homology depicting evolutionary relation between MJ0577, TaUSPs and H. influenzae USP

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Expression profile of abiotic stress responsive TaUSP genes under different abiotic stresses

Six genes, based on the transcript levels under abiotic stress on online wheat expression database were selected for further analysis. Their roles in different abiotic stresses were validated via quantitative real-time PCR (qPCR). Ten-day-old Triticum aestivum var. PBW343 seedlings were subjected to the above mentioned abiotic stresses. These members expressed differentially under different abiotic stresses. TaUSP_1A-3 and TaUSP_1D-1 were upregulated in cold stress and downregulated in heat stress. TaUSP_3B-1 was upregulated in salt and drought stress condition, while TaUSP_6D-5 was upregulated solely under heat stress and downregulated in all other stress treatments (Fig. 4). Transcript levels of TaUSP_5D-1 were also checked in reproductive tissues of Triticum aestivum var. PBW343 (Suppl. Figure 8).

Fig. 4
figure 4

Relative expression of different abiotic stress responsive TaUSPs in 10-day old Triticum aestivum seedlings under heat stress (HS), cold stress (CS), salt stress (SS) and drought stress (DS). Error bars are plotted using the standard error of three biological replicates. Three technical replicates were run for each biological replicate. The asterisk indicates the fold-changes calculated as significant (Students’ T test; p value \(\le\) 0.05)

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Subcellular localization of TaUSP proteins

Subcellular localization of TaUSP proteins was done to narrow down the possible physiological functions of abiotic stress responsive TaUSP genes. All the gene constructs encoding TaUSP proteins fused to YFP in frame were bombarded on onion epidermal peels. YFP signal for the TaUSP-YFP proteins was observed in cytoplasmic structures resembling ER and Golgi bodies (Fig. 5). To confirm this result further, co-localization with ER and Golgi body specific organelle markers was done. Proteins encoded by abiotic stress responsive TaUSP genes were predominantly localized in ER with some small traces in the Golgi bodies. These results were relatable to the cellular functions of USP being associated with protein scaffolding, holding and preventing the denaturation of globular macromolecules, and cellular protein transport (Vollmer et al. 2018).

Fig. 5
figure 5

Subcellular localisation of abiotic stress responsive TaUSP proteins along with different organelle markers. CDS of TaUSPs were cloned in frame with YFP protein and the expression was and observed in onion epidermal cells under confocal microscope. ER and Golgi body markers were used to locate the protein in onion epidermis cells. YFP yellow fluorescent protein

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Yeast-two-hybrid and BiFC assays for TaUSP_5D-1

USP proteins are known to form homo-dimers and hetero-dimers amongst themselves (Nachin et al. 2008). Therefore, proteins encoded by abiotic stress responsive TaUSP genes were checked for homo- and hetero-dimerization in S. cerevisiae strain AH109. In total 7 interactions were found, 3 being homo and 4 heterologous interactions (Fig. 6). The strength of interactions was checked by dropout assays up to a dilution of 10–4 on dropout yeast media, -LW, -HLW, -AHLW and 3AT (1 mM). Proteins encoded by TaUSP_1A-3, TaUSP_1D-1 and TaUSP_5D-1 were found to form homodimers. TaUSP_6D-5 and TaUSP_5D-1 had the maximum number of interactions amongst the candidate proteins. TaUSP_5D-1 and TaUSP_6D-5 were found to be forming heterodimers with three other abiotic stress responsive TaUSP proteins, respectively. Certain USP proteins are known to bind DNA (Melencion et al. 2017), so construct having all abiotic stress responsive pDEST-pGKT7:TaUSP genes were used with empty vector pDEST-pGADT7. Only TaUSP_5D-1 was found to have the property of autoactivation when co-transformed with empty vector pDEST-pGAD (Fig. 7a). To confirm the growth of yeast cells transformed with pGBKT7:TaUSP_5D-1, was checked on SD/-HW media. Further, all the interactions for TaUSP_5D-1 were confirmed with BiFC assay in onion epidermal peels and were found to be positive (Fig. 7b).

Fig. 6
figure 6

Yeast-2-hybrid assay for homodimerization and heterodimerization study of TaUSPs. Yeast cells were co-transformed with pGBKT and pGAD TaUSP constructs and the growth was analyzed on SD/-Leu/-Trp (-LW), SD/-Leu/-Trp/-His(-HLW), SD/-Leu/-Trp/-His/-Ade (-AHLW) and SD/-Leu/-Trp/-His(-HLW) + 1 mM 3-aminotriazole (3-AT) media. Interaction between pGAD-T-Antigen and pGBKT-p53 was used as positive control, and interaction between pGAD-T-Antigen and pGBKT-Lam was used as a negative control

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Fig. 7
figure 7

DNA binding activity check in yeast. a. Growth of yeast cells harbouring pDEST-pGBKT7::TaUSP_5D-1 and pDEST-pGADT7 EV was analysed on SD/-Leu/-Trp, SD/-His/-Leu/-Trp and SD/-His/-Leu/-Trp supplemented with X-⍺-GAL. pGADT7-T/pGBKT-53 was taken as positive interaction control and pGADT7-T/pGBKT-Lam was taken as negative interaction control. Growth of yeast cells transformed with fusion constructs pDEST- GBKT7:: TaUSP_5D-1, harbouring histidine reporter gene was analysed on SD/-Trp (-W) medium and on SD/-Trp/-His (-HW) medium. Yeast cells transformed with the empty vector pGBKT7 (EV) alone were used as a negative control and pDEST -pGBKT::TaHsfA6b were taken as positive control respectively. b. BiFC assay in onion epidermal cells to confirm TaUSP_5D-1 interactions

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Stress assays in TaUSP overexpressing yeast

To functionally validate the abiotic stress responsive TaUSP genes in mediating different stress responses, yeast model system was used. Overexpression of abiotic stress responsive TaUSP in BY4741 (Matα met15Ɗ0 his3Ɗ1 ura3Ɗ1 leu2Ɗ0) imparted tolerance to the yeast for a variety of stresses. The comparison was done with the yeast transformed with empty vector p426GPD. All abiotic stress responsive TaUSP: p426GPD constructs were transformed into S. cerevisiae strain BY4741 and treated with different stress conditions. TaUSP_1A-3 overexpressing yeast was found to be more tolerant to cold stress (0 °C), even at freezing stress of (− 20 °C), as compared to yeast transformed with empty vector. TaUSP_5D-1 and TaUSP_6D-5 transformed yeast cells were found to be better in growth in comparison to empty vector transformed yeast cells even at a sub-lethal temperature of 46 °C (Fig. 8a). Because abiotic stress responsive TaUSP genes were majorly localized in ER, so DTT stress was given to the yeast to check for their probable role in UPR, and it was found that TaUSP_3B-1 and TaUSP_6D-5 imparted tolerance to DTT stress in yeast (Fig. 8c). TaUSP_3B-1 was found to be providing tolerance to yeast for H2O2 and LiCl2 stress (Fig. 8d and b). TaUSP_6D-5 and TaUSP_1D-1 overexpressing yeast cells were growing better in oxidative stress (H2O2) than the empty vector transformed yeast cells (Fig. 8b).

Fig. 8
figure 8

Overexpression of TaUSP genes impart tolerance to S.cerevisiae under different stress conditions. The strain BY4741 was transformed with p426GPD (EV), p426GPD:TaUSP_1A-3, p426GPD:TaUSP_5D-1, p426GPD:TaUSP_6D-5, p426GPD:TaUSP3B-1. All mentioned TaUSP gene constructs and EV were grown in liquid culture for, a heat stress (42 °C and 46 °C for 1 h) and cold stress (0 °C and − 20 °C for 1 h), serially diluted samples were spotted on plates of SD/-Ura media. Yeast dropouts were allowed to grow for 3 days at 30 °C on SD/-Ura media for b oxidative stress (3 mM H2O2), c ER stress (30 mM DTT) and d Ionic homeostatis disruption (300 mM LiCl2)

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Overexpression of TaUSP_5D-1 enhanced drought tolerance in A. thaliana

To elucidate the functional role of TaUSP gene in plants, overexpression transgenic lines of Arabidopsis were generated for TaUSP_5D-1. These lines were confirmed using gene-specific semi quantitative PCR and real-time PCR in transgenic line and WT (Suppl. Figure 2). Overexpression lines showed drought tolerance and better lateral root growth under drought stress (200 mM mannitol) at seedling stage. Since abiotic stress accelerates the ROS accumulation in plants, leading to oxidative damage, ROS levels were checked via DAB and NBT staining in WT and transgenic lines. Transgenic lines showed lesser ROS levels as compared to the WT plants. Further transcription profiling for drought stress responsive genes was done in WT and transgenic lines (Agarwal and Khurana 2020). At.RD22, At.DREB2A and At.RD29B were found to be upregulated in transgenic lines as compared to the WT plants under control conditions (Fig. 9).

Fig. 9
figure 9

Phenotypical and physiological analysis of TaUSP_5D-1 overexpression Arabidopsis transgenics. a Transgenic plants overexpressing TaUSP_5D-1 under drought conditions. Plants were grown under control conditions for 7 days, then transferred to mannitol (200 mM)-supplemented MS media. Phenotype with increased number of lateral roots in transgenics was observed after seven-days. b Two-week-old seedling of WT and transgenics were given drought stress for three days on 200 mM mannitol-supplemented MS medium and stained with DAB and NBT to analyse the H2O2 and superoxide anion accumulation, respectively, after the stress treatment c Expression analysis of drought stress marker genes. Transcript analysis of drought stress marker genes in WT and transgenics stress conditions. Transcript levels were normalized to WT, and AtActin was used as a housekeeping gene. Values represent data from three biological replicates and three technical replicates. Error bars indicate values ± SD. Asterisks on top of the error bars represent the significance levels (Student’s t-test; p-value ≤ 0.05)

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Discussion

The identification of total TaUSP genes and characterization of abiotic stress responsive TaUSP genes is an important step for understanding their downstream signalling and to identify the pathways they regulate. No assigned function of these proteins in wheat was priorly available. The large number of TaUSP genes made us to prioritize the characterization of abiotic stress responsive TaUSP genes.

In E. coli, the UspA protein accumulates in response to a wide range of stresses in organisms, providing survival advantage under adverse growth conditions (Freestone et al. 1997; Jung et al. 2015). Members of the UspA family have been found to regulate plant response to various abiotic stress conditions (Loukehaich et al. 2012: Gonzali et al. 2015: Jung et al. 2015: Gutiérrez-Beltrán et al. 2017: Melencion et al. 2017 and Chi et al. 2019). In wheat TaUSP genes appear to actively impart tolerance to the abiotic stress treatments. The expression pattern of these genes was interesting to study because of varied response of different TaUSPs in the same stress (Fig. 4). TaUSP_1A-3 and TaUSP_1D-1 were upregulated in cold stress but downregulated in heat stress condition. However, TaUSP_6D-5 showed the exact opposite pattern of transcript levels in the same afore mentioned stresses. Same was the case with TaUSP_3B-1 and TaUSP_6D-5 in drought and salt stress. These observations point towards the fact that different abiotic stress responsive TaUSP genes may respond to same stress through a different downstream pathway.

Although it has been reported that UspA proteins contain a dimerization domain in their sequence, little is known about its biological relevance. E.coli Usp proteins have been shown to form homodimers and/or heterodimers in vivo, resulting in adaptation to different stresses (Nachin et al. 2008; Heermann et al. 2009). Abiotic stress responsive TaUSP genes were also found to be forming hetero/ homo dimers (Fig. 6), suggesting that these genes crosstalk among themselves to respond to various stress conditions. Homo-dimerization is an essential need for these genes, in certain plants, to function for better adaptability towards external stresses (Gutiérrez-Beltrán et al. 2017). However the Y2H assay data for wheat USPs showed that not all TaUSPs essentially formed homodimers, some of them rather only formed heterodimers but these heterodimer formations were preferentially within the same clade of genes (Fig. 1 and Fig. 6).

All abiotic stress responsive TaUSP genes, except TaUSP_1B-1, were found to be closely related to gene encoding MJ0577, and code for proteins with ATP binding motif at their C-terminal as is present in MJ0577 (Fig. 3). The ATP binding functionality in a few members of the UspA family has brought about the hypothesis that nucleotide binding USP proteins ought to feature as molecular switches, through sensing ATP levels for the duration of stress to detect cell metabolic status (O’Toole and Williams 2003; Persson et al. 2007; Drumm et al. 2009). Autoadenylation is found in bacterial USPs in stationary phase (Weber and Jung 2006) and it has been proven to be a key aspect for microbial survival in O2 depletion during the course of growth arrest and virulence. In fact, it has been demonstrated that the capacity of UspA protein Rv2623 from M. tuberculosis to regulate its growth and latency in the host, depends on its ATP-binding activity (Drumm et al. 2009). Presence of the ATP binding motifs in abiotic stress responsive TaUSPs might be the stress sensing mechanism used by these USP proteins which is majorly conserved in euryarchaeota and plant kingdom. TaUSP_5D-1 also shows some DNA binding activity which implies its role in DNA damage repair under stress conditions (Fig. 7a), however, this needs to be further studied for a better understanding.

TaUSPs were majorly localized in endoplasmic reticulum and some residual amount in golgi bodies in the cell. ER is known to be the major site for synthesis and protein folding in cells (Gidalevitz et al. 2013). During different abiotic stress conditions, demand for protein folding exceeds the production capacity, which leads to accumulation of unfolded or incorrectly folded proteins resulting in ER stress (Fernández-Bautista et al. 2017).Various stresses like heat, drought and salt lead to ER stress. Localization of TaUSPs in ER (Fig. 5), led to speculation of their involvement in UPR stress. To check for the same, DTT (ER stressor) treatment was given to TaUSP OE yeast. Transformation of yeast with TaUSP_3B-1 and TaUSP_6D-5 imparted better growth under DTT stress, pointing towards the possibility of their roles in UPR under drought and heat stress, respectively, in plants. Transformation with TaUSP_3B-1 also imparted better growth to the yeast under LiCl2, depicting its role in maintaining ion homeostasis in cells under salt stress (Gutiérrez-Beltrán et al. 2017). In the yeast system TaUSP_6D-5 and TaUSP_5D-1 imparted tolerance to heat stress even at a sub-lethal temperature of 46 °C, and TaUSP_1A-3 imparted tolerance even at a temperature of 0 °C and − 20 °C (Fig. 8). To elucidate the function of USP genes in planta, gain of function approach in A.thaliana was used. Overexpression lines for TaUSP_5D-1 were generated and analyzed for their phenotype and tolerance in drought stress conditions. It was found that plants overexpressing the selected TaUSP better performed under drought condition and had a dense rooting system due to a greater number of lateral roots as compared to the WT plants. Results reveal that TaUSP_5D-1 provides drought tolerance to plants via improving lateral root system of the plant. At molecular level transgenic lines even showed less ROS accumulation under stress condition as compared to the WT (Fig. 9).

In conclusion, wheat abiotic stress responsive USP genes are good candidates for imparting abiotic stress tolerance to crops through genetic engineering. TaUSP_6D-5 and TaUSP_3B-1 connect heat stress and drought stress to UPR, respectively in the yeast system. The TaUSP_6D-5 also stands as a good candidate gene for heat stress tolerance and should be further studied for the downstream singling pathway for its functioning. Similarly, TaUSP_3B-1 providing tolerance to LiCl2 in yeast and its upregulation in drought and salt stress suggests the possibility of its function in maintaining cellular homeostasis for imparting drought and salt stress in plants. Further overexpression of TaUSP_5D-1 in plant imparts drought stress tolerance via alteration in root morphology. The other abiotic stress responsive TaUSP genes can also be used to engineer crops to impart tolerance to cold stress (TaUSP_1A-3 and TaUSP_1D-1), drought and salt stress.