Wheat is one of the most important cereal crops and its end-products like breads, noodles, pasta and other baked products are consumed globally and have become staple diet. The viscoelastic properties of wheat dough are primarily dependent upon the interaction of gluten proteins. Gluten proteins consist of gliadins, which provide viscous property to wheat dough, and glutenins, which contribute towards elasticity of the dough (Ciaffi et al., 1996). Storage protein deposition is affected by environmental conditions during the grain development period (Randall & Moss, 1990; Lukow & McVetty, 1991). For controlling the variation in wheat flour, it is imperative that the regulatory factors responsible for formation, folding and polymerization of gluten proteins should be studied. In nature, folding of proteins is mediated by an array of proteins that act as molecular chaperones or foldases (Fischer and Schmid, 1999).
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The wheat gluten proteins are proline rich (10-30%) (Van-Dijk et al., 1997) and about 6% of all Xaa-Pro (Xaa: other bulky amino groups preceding proline) peptide bonds show the cis conformation. Peptidyl prolyl cis-trans isomerases (PPIases) are the only enzymes known to catalyse cis-trans isomerisation of peptidyl prolyl bonds which is a rate-limiting step in protein folding (Fischer et al., 1989). Understanding the role of PPIases in gluten protein deposition in wheat could help in developing strategies for manipulating the storage proteins desired for different food products by breeding and/or genetic engineering strategies.
Peptidyl-prolyl cis-trans isomerases comprise of three distinct classes of proteins- cyclophilins, which bind to the immunosuppressive drug cyclosporin A (CsA) (Handshumacher et al., 1984); FK506-binding proteins (FKBPs), which bind the macrolide drugs FK506 and rapamycin (Harding et al., 1989); and the parvulin family (Dolonski and Heitman, 1997). Due to their drug binding activities, cyclophilins and FKBPs are also known as immunophilins. The FKBPs are conserved in all organisms from prokaryotes to higher plants and mammals (Gasser et al., 1990). Rice genome is reported to contain largest number of FKBP members (Ahn et al., 2010). FKBPs, beside folding of proteins, are also involved in many other cellular processes such as cell signalling (Luan et al., 1998), protein complex formation (Pratt and Toft, 1997; Reynold et al., 1999), regulation of plant growth and development (Geisler et al., 2004), stress response (Kurek et al., 1999; Yu et al., 2012) and in redox control of photosynthesis (Gupta et al., 2002; Gopalan et al., 2004). Two multidomain FKBPs, FKBP73 and FKBP77, were cloned earlier from wheat (Avezier et al., 1998). These proteins were also demonstrated to play role in signal transduction through their interaction with mammalian p23 and plant HSP90 (Owens-Grillo et al., 1996; Reddy et al., 1998). Recently, genes encoding three single-domain wheat FKBPs, TaFKBP13, TaFKBP16-1 and TaFKBP16-3 were cloned and characterized by Gollan et al. (2011). TaFKBP13 was the first active lumenal FKBP reported in cereals, whereas, TaFKBP16-1 and TaFKBP16-3 did not show any PPIase activity (Gollan et al., 2011). These FKBPs were also implicated in assembly of photosytem complexes and thylakoid membrane complexes (Gollan et al., 2011). It is evident that information on FKBPs which have been cloned and characterized from wheat is limited (Aviezer et al., 1998; Bhave et al., 2011). Further, their role in gluten protein deposition has also not been explored as yet. Therefore, the present study was carried out with the following objectives.
- To analyze differences in deposition of gluten storage protein in grains at different stages of development in Indian wheat cultivars having varied protein content.
- Developmental changes in total PPIase activity and its correlation with storage protein deposition.
- To study the contribution of cyclophilins and FKBPs towards total PPIase activity in developing grains by inhibition assays employing cyclosporin A and FK506 as specific inhibitors, respectively.
- Cloning and characterization of FKBP genes and their expression analysis.
Salient findings of the study
Different hexaploid wheat (Triticum aestivum) cultivars (GLUPRO, LOKI, HPW89), which varied in their protein content, were selected for this study. The grains were harvested at different stages of development viz. 8, 12, 16, 20, 25 days post anthesis (DPA) and maturation. The isolation and separation of different storage protein fractions from the wheat grains pose a challenge due to their cross contamination. Therefore, different methods, which were reported earlier by Osborne (1924) and Fu and Saperstein (1996) were tried. These methods did not result in isolation of pure fractions of gliadins and glutenins from the grains of cultivars used in this study. However, the method reported by DuPont (2005) resulted in highest recovery of different protein fractions with minimal cross-contamination. The reducing SDS-PAGE analysis demonstrated that the accumulation of gliadins in the cultivars of wheat included in this study was affected by the developmental stage of the grain. Present study also demonstrated that accumulation of high molecular weight subunits of glutenins (HMW-GSs) was also cultivar- and stage dependent. The profile of high molecular weight subunits of glutenins (LMW-GSs) was not altered significantly after 16 DPA in any of the three cultivars. Contrary to gliadins and glutenins, the albumins in the present study did not show any significant inter-cultivar variability. Further, the accumulation of albumins in all the three cultivars started after 12 DPA and increased up to maturation. The different albumins may consist of proteins involved in important cellular functions like protein folding, plant defence mechanism, stress response, etc. (Merlino et al., 2009) and, therefore, must be conserved in nature, which explains the lack of intercultivar variation in the three cultivars analysed in this study.
Developmental regulation of PPIases in wheat grains has been reported for cyclophilin (Grimwade et al., 1996) and FKBP73 (Aviezer et al., 1998) at transcript and protein level, respectively. Expression studies of PPIases at activity level are however lacking also important because the transcript levels may not always culminate in higher levels of protein or activity due to post-transcriptional regulation (Arnholdt-Schmitt, 2004). Therefore, to elucidate the role of PPIase genes in accumulation of storage proteins in wheat grain, PPIase assays were performed by using crude protein extract of developing grains, and activity was estimated by a coupled enzyme assay method using chymotrypsin for cleaving the test peptide N-succinyl-ala-ala-pro-phe-p-nitroanilidine (Fischer et al., 1984). Principal Component Analysis (PCA) revealed that PPIase activity in cvs. HPW 89 and GLUPRO was related to the accumulation of gliadins. The presence of PPIase activity at different stages of grain development in all the cultivars and its close association with storage proteins indicated that these enzyme(s) may be playing an important role in deposition of storage proteins in wheat.
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PPIase activity of FKBPs and cyclophilins is inhibited by immunosuppressant drugs FK506 and CsA, respectively (Harding et al., 1989). Since no cross inhibition by the two drugs is reported (Harding et al., 1989), we, therefore, employed CsA and FK506 as specific inhibitors to determine the contribution of these two classes of proteins to total grain PPIase activity. Except at 25 DPA in LOK I, the PPIase activity at all stages of grain development in the three cultivars was almost totally inhibited by CsA. These observations, thus, suggest that PPIase activity in the grains, except at 25 DPA in LOK-1, was primarily due to cyclophilins. Since FK506-inhibitable activity in the crude protein extracts of the three cultivars was negligible, therefore, to further investigate the reason for this observation, cloning of FKBP genes, which are expressed in the developing grains, was attempted. Sequence of an active FKBP type-1 domain of wFKBP73 (accession number X86903.1) comprising of 95 (50-145) amino acid (a.a.) residues (Blecher et al., 1996) was used as a query, which resulted in identification of hundreds of different putative FKBP sequences in T. aestivum. These sequences were retrieved from NCBI and subjected to TBLASTn using TIGR Plant Transcript Assemblies database (TADB; http://plantta.jcvi.org/) for wheat. Of the several retrieved sequences from TIGR, three different cDNAs, TaFKBP15-1, TaFKBP16-1 and TaFKBP20-1, which showed longest open reading frame (ORFs), were selected for cloning using the RNA isolated from the developing grains harvested at 16 DPA. The study successfully resulted in cloning of three FKBP genes from Indian wheat. Bioinformatics analysis of the cloned cDNAs revealed that TaFKBP16-1 consists of an ORF of 408 bp encoding a protein of 135 a.a. residues with molecular weight (M.W.) and pI of 15.26 kDa and 5.75, respectively. The 561 bp and 477 bp ORFs of TaFKBP20-1 and TaFKBP15-1, respectively, were predicted to encode proteins of 186 and 157 a.a. residues, respectively, with M.W. and pI of 19.95 kDa and 6.77, and 16.61 kDa and 8.96, respectively. In silico analysis of a.a. sequences of the cloned TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 revealed that the FKBP domains architecture, though conserved in these proteins, also show variability observed in their secondary structures. Further, analysis of signal peptide using different online tools predicted localization of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 to nucleus, possibly cytosol and ER, respectively.
Compared to human homologue, hFKBP12, both TaFKBP15-1 and TaFKBP20-1 showed presence of all the essential residues (Y26, F36, F46, W59, Y82 and F99) required for PPIase activity, as compared to only three (Y26, Y82 and F99) in TaFKBP16-1. TaFKBP15-1 is 40% and 38% similar to TaFKBP16-1 and TaFKBP20-1, respectively, whereas, TaFKBP20-1 is 30% similar to TaFKBP16-1. The variability observed in these FKBPs in wheat suggests that these proteins may be playing specific roles in the cells, which need to be investigated further. The recombinant TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 proteins were expressed in E. coli BL21-CodonPlus(DE3)pLysS and purified by Ni-NTA affinity chromatography. The recombinant nature of the purified proteins was validated by immunoblotting studies using anti-His antibody, which resulted in detection of the specific bands corresponding to the respective purified FKBP proteins. For biochemical characterization of three proteins, PPIase assays were performed. None of the three purified FKBP proteins showed any detectable PPIase activity since the first order rate constant (0.013 s-1) in presence of up to 2 µg of each of the three purified proteins was similar to the first order rate constant (0.0135 s-1) observed for the uncatalysed control (in the absence of protein). To determine the reasons for lack of PPIase activity, despite the presence of conserved a.a. residues, TaFKBP20-1 and TaFKBP16-1 were subjected to chymotrypsin susceptibility assay, which is used for determining the PPIase activity. The assays revealed that both the proteins were cleaved by chymotrypsin which could be one of the reasons for the absence of PPIase activity in the two proteins. Lack of detectable activity inTaFKBP15-1, however, could be because of improper refolding due to the use of urea which was employed for solubilization of this protein during purification.
FKBPs in plants have been implicated in various stress responses (Kurek et al., 1999; Sharma and Singh, 2003; Magiri et al., 2006). Ca2+ is one of the most important secondary messengers in eukaryotes, which plays an important role in different signal transduction pathways under stress conditions (Reddy, 2001). A number of multi-domain FKBPs viz., MzFKBP66, AtFKBP62, AtFKBP65, wFKBP73 and wFKBP77 have been reported to interact with CaM in plants (Vucich and Gasser 1996; Hueros et al., 1998; Kurek et al., 2002; Aviezer-Hagai et al. 2007). We, therefore, also analysed the CaM-binding property of the cloned FKBPs. CaM gel-overlay assay demonstrated that of the three proteins, only the purified TaFKBP15-1 interacted with CaM, which was dependent on the presence of Ca2+. Real-time PCR analysis of TaFKBP20-1 and TaFKBP15-1 in developing grains of wheat revealed that expression of these genes is regulated developmentally- and is cultivar-dependent.
The lack of PPIase activity observed for TaFKBP16-1, TaFKBP20-1 and TaFKBP15-1 indicates that ability to catalyse cis-trans isomerisation of peptidyl prolyl bond may not be a conserved feature of plant FKBPs, since other plant orthologues viz., TaFKBP16-1, TaFKBP16-3, AtFKBP20-2, AtFKBP42were also found to be inactive (Gollan et al., 2011; Lima et al., 2006, Edvardsson et al., 2007; Kamphausen et al., 2002). However, despite lack of PPIase activity, these FKBPs may be involved in other cellular functions such as cell signalling, stress response, photosynthesis and plant development, as reported for other orthologues (Sigal and Dumnot, 1992; Geisler et al., 2003; Gollan et al., 2011; Lima et al., 2006).
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