Introduction

The large family of small heat shock proteins (sHSPs) are characterized by their strongly conserved α-crystallin domain (ACD), while their N- and C-terminal sequences are highly variable (Haslbeck et al. 2015; McLoughlin et al. 2016). The molecular weight of sHSP monomers lies in the range 12–42 kDa. Based on other domain sequences and their targeting signal, the family has been categorized into 21 subfamilies (Jaspard and Hunault 2016). Meanwhile, a categorization of plant sHSPs, based on their intracellular localization in the model plant Arabidopsis thaliana, recognizes 12 subfamilies: the members of CI-CVII are found in the cytoplasm/nucleus, while those of CVIII-CXII are found in various organelles (Wagner et al 2005; Jaspard and Hunault 2016).

As the immediate response to stress frequently involves the induction of sHSPs, these proteins have been considered to form part of the plant’s first line of defense against stress (Haslbeck et al. 2015). Various experiments have demonstrated that plant sHSPs are important not only for development but also for adaptation (Sun et al 2001; Mahesh et al. 2013; Kumar et al. 2016; Ali et al. 2019). In both A. thaliana and rice, a number of genes encoding a sHSP are ubiquitously transcribed and are inducible by biotic and abiotic stress (Sarkar et al. 2009; Waters 2013; Bernfur et al. 2017; Kuang et al. 2017). According to Zhang et al. (2013), the largest plant sHSP subfamily involved in the response to abiotic stress is CI. The contribution of the sHSPs to the plant response to soil salinity, a leading constraint over crop growth and productivity, has not been widely researched (Park et al. 2016; Jusovic et al. 2018). Several sHSP genes are known to be up-regulated by salt stress (Sun et al. 2001; Zhang et al. 2013; Li et al. 2016) and it has been shown for some of them that their constitutive expression can enhance salt tolerance (Sun et al. 2012; Kuang et al. 2017). Li et al. (2018) have reported that in plants subjected to high temperature stress, the product of the A. thaliana gene AtsHSP22 requires ABI1 protein phosphatase to support the auxin-regulated hypocotyl elongation. However, there is a lack of general understanding of either the mechanistic basis of sHSP-determined salinity tolerance, or whether these proteins only act in concert with the key abiotic stress ABA.

It has been estimated that some 340 Mha of arable land is affected by salinity, and a further 560 Mha is sodic (sodium-affected) (Kenneth 2002), underlining the importance to crop improvement of finding genetic solutions to combat salinity-induced crop productivity loss. The grain yield of most cultivars of the major staple crop bread wheat is reduced when the plants are grown on salinity-affected soil (Landi et al. 2017; Abhinandan et al. 2018). However, the current understanding of how the wheat plant deals with salt stress at the molecular level is still rudimentary (Abhinandan et al. 2018; Ahmad et al. 2018). Some genetic variation associated with the level of tolerance is known, and has been exploited by breeders to produce more tolerant wheats. An example is the cultivar SR3, bred by introgressing into the sensitive cultivar JN177 genetic material derived from a highly tolerant near relative (Xia et al. 2003). Here, an attempt was made to evaluate a candidate gene TaHSP17.6 responsible for the tolerance shown by SR3 identified by comparing the SR3 and JN177 transcriptomes.

Materials and Methods

Plant Materials and Stress Treatments

Grains of the salinity tolerant cultivar SR3 and of the sensitive cultivar JN177 were sterilized by immersion in 75% ethanol for 15 min, followed by three rinses in sterile water. Seedlings were raised for ten days in a greenhouse held at 25 °C and under a 16 h photoperiod, after which the remaining endosperm was removed and the plants transferred into half strength Hoagland's liquid medium containing either 0 or 200 mM NaCl for a period of 0, 0.5, 3, 6, 12 or 24 h (huor), or one containing 100 μM ABA for 0, 0.5, 3, 6, 12, 24 or 36 h. At each time point, the plants were harvested, the leaves were separated from the roots, the samples were snap-frozen in liquid nitrogen and then stored at -80 °C. Wild type (WT) ecotype Columbia-0(Col-0) A. thaliana and transgenic A. thaliana constitutively expressing TaHSP17.6 were subjected to stress by plating five-day-old seedlings on agar plates containing Murashige and Skoog (1962) medium with or without either 50 mM NaCl or 2 μM ABA. The plates were hold vertically, and the seedlings were allowed to grow for seven days under a 16 h photoperiod at 22/20 °C and 60% relative humidity.

cDNA Isolation and the Amplification of TaHSP17.6

RNA was isolated from samples of either leaf or root using the RNAiso Plus (Takara) reagent, following to the user manual. The first cDNA strand was synthesized using a primerscript RT reagent kit (Takara). TaHSP17.6 transcript was amplified using SR3 cDNA library using forward primer TaHSP17.6-1F and the reverse primer NT3 (primer sequences given in Table 1). The resulting amplicon was cloned using the T/A method (Invitrogen) and sequenced for validation. The TaHSP17.6 genomic sequence was amplified from SR3 genomic DNA using the primer pair TaHSP17.6-1F/R (Table 1). The amplification regime comprised a 94 °C/3 min pre-denaturation, followed by 35 cycles of 94 °C/1 min, 56 °C/1 min, 72 °C/1 min (72 °C/2 min for the genomic sequence), and completed with a final extension step of 72 °C/5 min.

Table 1 Primer sequences used in the experiments
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Transcriptional Profiling

The transcriptional profiling of TaHSP17.6 was achieved using a semi-quantitative RT-PCR (sRT-PCR) and a quantitative real time PCR (qRT-PCR) assay. The sRT-PCR assays used the TaActin (AB181991) as the reference while the gene-specific primer pair was TaHSP17.6-2F/R (Table 1); the amplification regime and volume referred to reported method (Qin et al. 2012). The 10 μL qRT-PCRs contained 1 μL cDNA, 5 μL 2 × SYBR Ex Taq mix (Takara) and 0.2 μM of both the forward and reverse primers (the same primer pair was used as for the sRT-PCRs). The amplification regime comprised a pre-denaturation step of 95 °C/30 s, followed by 45 cycles of 95 °C/15 s, 56 °C/15 s, 72 °C/20 s.

Creation of A. thaliana Plants Constitutively Expressing TaHSP17.6

To obtain the TaHSP17.6 open reading frame sequence, cDNA of SR3 was amplified using the TaHSP17.6-2F/-2R primer pair, and the amplicon digested with XbaI and KpnI. The resulting fragment was inserted into pCAMBIA super 1300 vector to place the transgene under the control of the CaMV 35S promoter. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 and from thence into A. thaliana using the floral dip method (Clough and Bent 1998). Genomic DNA was extracted from putative transgenic A. thaliana plants using a little modified CTAB method and used as a PCR template to validate the plants’ transgenic status. Each 10 μL PCR was pre-denatured (94 °C/3 min), then cycled 35 times through 94 °C/60 s, 56 °C/30 s, 72 °C/35 s, completing with a final elongation step (72 °C/5 min). The primer pair used was TaHSP17.6-2F/R (Table 1).

Quantification of Leaf Proline Content, Peroxidase Activity and Rate of Water Loss from Detached Leaves

The free proline content in the leaves of three week old A. thaliana plants was determined following the method given by Bates et al. (1973). Peroxidase (POD) activity was quantified by measuring the absorbance of reactions at 470 nm following Yang et al. (2015). The rate of water loss from the detached leaves of month old plants was estimated following Shin et al. (2019).

Results

Isolation and Sequence Analysis of a Wheat CI sHSP

Based on the identification of a gene encoding a heat shock protein which was differentially expressed between SR3 and JN177 (Liu et al. 2012), a PCR assay was conducted to recover the gene sequence from a cDNA library of SR3. Sequencing of the resulting amplicon revealed an amplicon of length 857 nt, predicted to encode a 158 residue protein of molecular mass 17.6 kDa. Aligning the cDNA and gDNA sequences showed that the gene is free of introns. The predicted peptide sequence included a canonical 92 residues ACD domain, sharing 97% identity with the Aeqilops tauschii CI sHSP XP020171799 and 83% with the maize CI sHSP NP001130454.1 (Fig. 1). Thus TaHSP17.6 was concluded to be a member of the wheat CI small heat shock protein subfamily. Given its molecular weight, the protein was designated TaHSP17.6 and its encoding gene TaHSP17.6.

Fig. 1
figure 1

Polypeptide sequences of related plant sHSPs. The residues highlighted by underlining form the ACD domain

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Transcriptional Profiling of TaHSP17.6

In the absence of salt stress, the abundance of TaHSP17.6 transcript in the roots was greater in SR3 plants than in JN177 ones (Fig. 2a). In response to the stress generated by the addition of 0.2 M NaCl to the medium, the gene was rapidly induced in the leaves, peaking at 0.5 h after the treatment was imposed (Fig. 2b); the abundance of transcript was also higher in SR3 leaves than in those of JN177. Notably, in roots, the two cultivars exhibited a contrasting response: in SR3, the gene was strongly transcribed for the first 3 h of the stress treatment, but in JN177, it was not significantly induced during this period (Fig. 2a). The two cultivars’ response to exogenously supplied ABA also differed. In JN177 roots, the gene was strongly up-regulated after 0.5 h ABA treatment (Fig. 2c), after which time the abundance of transcript declined gradually; in contrast, in SR3 roots, the peak level of transcription occurred 36 h after the imposition of stress (Fig. 2d). Similarly, the pattern of TaHSP17.6 transcription shown by the two cultivars leaves differed markedly: in JN177 the intensity of transcription declined over time, while in SR3 it rose (Fig. 2e, f).

Fig. 2
figure 2

Transcriptional profiling of TaHsp17.6 in wheat. The response of (a) the roots, (b) the leaves of JN177 and SR3 to the presence of 0.2 M NaCl. The response to the presence of 0.1 mM ABA of (c, d) the roots of (c) JN177, (d) SR3, and (e, f) the leaves of (e) JN177, (f) SR3. TaActin was used as the reference sequence. *Means differ significantly (p < 0.05) from the baseline value

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The Phenotypic Effect of Constitutively Expressing TaHSP17.6 in A. thaliana

Of the 20 independent A. thaliana lines constitutively expressing TaHSP17.6 obtained, three were retained to assess the phenotypic consequences of carrying the transgene. The presence of TaHSP17.6 transcript was confirmed in the selections (Fig. 3a). There was no observable phenotypic effect of the transgene when the plants were grown in the absence of stress (Fig. 3b), but when grown in the presence of 50 mM NaCl, all of the transgenic plants exhibited greater salinity tolerance than did WT plants (Col-0)—the former developing a greater number of lateral roots and forming larger leaves (Fig. 3c). A similar differential response was observed when the plants were challenged with 2 μM ABA (Fig. 3d). In the absence of stress, the transgenic plants accumulated more proline than did the WT ones (Fig. 3e) and exhibited a higher level of POD activity (Fig. 3f). Finally, the rate of water loss experienced by leaves detached from the transgenic plants was significantly lower than that measured from leaves detached from WT plants (Fig. 3g).

Fig. 3
figure 3

The phenotypic effect of constitutively expressing TaHSP17.6 in A. thaliana. a PCR validation of the presence of the transgene. The appearance of plants grown (b) in the absence of stress, (c) in the presence of 50 mM NaCl, (d) in the presence of 2 μM ABA. L2, L4, L5: three independent transgenic lines, W1, W2: weight of detached leaves (W1) prior to dehydration, (W2) during the dehydration process. CK control check

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Discussion

Plant CI sHSPs belong to a well studied family of proteins. Based on its polypeptide sequence (specifically the possession of the strongly conserved ACD domain and its distinctive N- and C-terminal residues, see Fig. 1), TaHSP17.6 clearly can be considered as a bona fide CI sHSP. Muthusamy et al. (2017) have cataloged the wheat complement of HSP20 proteins, identifying three of molecular weight 17.6 kDa, namely TaHSP17.6A (classified as a CII sHSP) and the two CI sHSPs TaHSP17.6B and TaHSP17.6C. The polypeptide sequence of the TaHSP17.6 reported here is identical with that of TaHSP17.6B and TaHSP17.6C.

A number of CI sHSPs are transcribed in an organ/tissue specific manner and/or are induced by environmental cues (Wagner et al. 2005; Kumar et al. 2016). Under non-stressful conditions, TaHSP17.6 was strongly transcribed in the leaves of JN177 (Fig. 2b), but hardly at all in its roots (Fig. 2a), in line with the behavior of TaHSP17.6B and TaHSP17.6C (Muthusamy et al. 2017). However, the gene behaved quite differently in SR3, where the transcript was abundant in the roots of non-stressed plants (Fig. 2a). Exposure to salt stress resulted in a marked and rapid induction of TaHSP17.6 in the leaves of both cultivars (Fig. 2b), and also—albeit somewhat later—in the roots of JN177 seedlings. The suggestion is therefore that the different expression profiles of TaHSP17.6 between two wheat cultivars make a contribution to the SR3 tolerance of salt stress. Plants can respond to salt stress in a manner which can be either ABA-dependent or -independent (Abhinandan et al. 2018). The phenotypic effect of constitutively expressing TaHSP17.6 in A. thaliana was to prompt plants supplied with exogenous ABA to form a higher number of lateral roots and to develop larger leaves (Fig. 3d), which implied that TaHSP17.6 operates within a pathway which is reliant on ABA signaling.

A number of examples have been presented in the literature to show that constitutively expressing a sHSP can enhance a plant’s tolerance of abiotic stress. Thus, the heterologous expression of the primula gene PfHSP17.1 in A. thaliana has a positive effect on tolerance to high temperature, salinity and drought (Zhang et al. 2013), while over-expressing HSP18.6 in rice increases the level of tolerance to both low and high temperature, salinity and drought (Wang et al. 2015). Finally, when the Populus trichocarpa gene PtHSP17.8 is constitutively expressed in A. thaliana, both high temperature and salinity tolerance are enhanced (Li et al. 2016). Similarly, as shown here, the constitutive expression in A. thaliana of TaHSP17.6 enhanced salinity tolerance, since it encouraged the development of both a stronger root system and larger leaves (Fig. 3b). As was also the case when HSP18.6 is over-expressed in rice (Wang et al. 2015), the TaHSP17.6 transgenic plants exhibited its higher level of salt stress tolerance without any obvious phenotypic defects, implying that TaHSP17.6 represents a potential candidate gene for engineering wheat to become more tolerant of saline soil.

Abiotic stress typically imposes a combination of osmotic and oxidative stress (Park et al. 2016; Abhinandan et al 2018), to which plants respond by both accumulating osmotica such as proline and soluble sugar and by promoting the enzymatic neutralization of oxidizing compounds. In rice plants over-expressing HSP18.6, the activity of the key enzymes catalase and superoxide dismutase is markedly heightened when the plants are subjected to either high temperature or drought (Wang et al. 2015). Similarly the heterologous expression of PtHSP17.8 in high temperature- and salinity-stressed A. thaliana has the effect of boosting the plants’ capacity to retain their hydration, as well as raising the activity of antioxidant enzymes and the tissue content of proline and soluble sugar (Li et al. 2016). Here, the TaHSP17.6 transgenics grown in the absence of stress were clearly more effective than WT plants both in terms of accumulating proline (Fig. 3e) and in the level of POD activity achieved (Fig. 3f): the result was that their leaves were better able to retain moisture when detached from the plant (Fig. 3g). Overall, constitutively expressing TaHSP17.6 appeared to enhance the plants’ tolerance of salt stress in an ABA-dependent manner.

Conclusion

Since soil salinity is a growing constraint over crop productivity, an important priority for the crop science community is to gain a holistic understanding of the molecular basis of tolerance. The sHSPs are considered to act as a first line of defense against stress, since they are produced rapidly by plants upon exposure to stress (Haslbeck et al. 2015). An sHSP gene which was differentially transcribed in a pair of closely related wheat cultivars, one of which was salinity tolerant and the other not, was used here to investigate the relevance of its product to the stress response. Constitutively expressing the gene in A. thaliana had the effect of boosting the plant’s tolerance of salinity: the transgenic plants were also more effective with respect to accumulating proline, neutralizing oxidizing compounds and retaining the hydration of detached leaves. The proposition is that TaHSP17.6 represents a promising candidate gene for breeding salinity tolerance in wheat.