Food Chemistry 128 (2011) 450–457

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Developmental changes in storage proteins and peptidyl prolyl cis–trans isomerase activity in grains of different wheat cultivars Tanima Dutta a,1, Harsimran Kaur a,1, Sandeep Singh b, Akanksha Mishra c, Jayant K. Tripathi c, Narpinder Singh b, Ashwani Pareek d, Prabhjeet Singh a,⇑ a

Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab 143005, India Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, Punjab 143005, India School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India d Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India b c

a r t i c l e

i n f o

Article history: Received 11 January 2011 Received in revised form 19 February 2011 Accepted 7 March 2011 Available online 12 March 2011 Keywords: Gliadins Glutenins Peptidyl-prolyl cis-trans isomerase Proteins Rheology Wheat

a b s t r a c t In the present study, storage proteins from five different wheat cultivars were extracted, fractionated and evaluated for their accumulation at different stages of development. SDS–PAGE analysis revealed that the accumulation of high molecular weight glutenin subunits was cultivar and stage dependent. However, low molecular weight glutenin subunits’ accumulation was not altered significantly after 16 days post anthesis in any of the cultivars. The rheological parameters (storage- and loss-modulus) of dough and gluten showed close association with either gliadins or glutenins. Peptidyl prolyl cis–trans isomerase (PPIase) activity, measured at different stages of grains development, showed variability with both the developmental stage and cultivar, and appeared to be primarily due to cyclophilins. Principal component analysis revealed the association of PPIase activity with either gliadin or total proteins, suggesting their significant role in the deposition of storage proteins in wheat. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Functionality of wheat flour for different applications is governed by the starch and proteins present in it. The quality and quantity of gluten proteins, which form a visco-elastic mass after the addition of water to the flour, determine the bread making quality of the wheat flour. The viscoelastic properties of wheat dough, which determine its application for different products, are largely determined by the structure and interaction of the storage proteins. Gluten consists of two types of proteins; gliadins and glutenins. The gliadins are a heterogeneous group of monomeric proteins, whereas glutenins are polymers consisting of high(HMW-GSs) and low-molecular weight glutenin subunits (LMWGSs). The HMW-GSs of glutenins and the ratio of gliadin/glutenin are recognised as the major determinants of dough and gluten properties (Popineau, Cornec, Lefebvre, & Marchylo, 1994). It has been demonstrated that glutenins contribute mainly to gluten’s elastic properties, while gliadins impart viscosity to the wheat dough (Ciaffi, Tozzi, Borghi, Corbellini, & Lafiandra, 1996).

⇑ Corresponding author. Tel.: +91 183 2258431; fax: +91 183 2258272. 1

E-mail address: [email protected] (P. Singh). These authors contributed equally.

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.052

The size and structure of glutenins is determined by biosynthetic mechanisms, which are under both genetic and environmental controls (Ciaffi et al., 1996). Our earlier studies revealed that the imposition of water stress at different stages of grain development in different Indian wheat cultivars resulted in a change in the pasting properties of starch, distribution of starch granules and profile of storage proteins (Singh, Singh, Singh, & Singh, 2008). The deposition of gliadins and glutenins in the mature grains was reported to be also affected by temperature stress (Randall & Moss, 1990) and varying concentration of nitrogen fertilisers (Paredez-Lopez, Corravioubas-Alvarez, & Barquin-Carmona, 1985). It is, thus, evident that the abiotic factors prevalent during the grain development period affect the storage protein composition of the mature grain and consequently the properties of the flour (Lukow & McVetty, 1991). Therefore, in order to develop strategies for manipulating glutenin structure, it is imperative that the regulation of gluten proteins should be studied at different stages of grain development in cultivars, which show variability with respect to dough characteristics. Furthermore, gluten proteins are rich in prolyl residues (10– 30%) (Van-Dijk et al., 1996) and about 6% of all Xaa-Pro (Xaa: other bulky amino groups preceding proline) peptide bonds show the cis conformation. The cis to trans conversion of the peptidyl prolyl bonds is a rate-limiting step in protein folding. Since peptidyl

T. Dutta et al. / Food Chemistry 128 (2011) 450–457

prolyl cis–trans isomerases (PPIases) are the only enzymes known, which catalyse the conversion of peptidyl prolyl bonds from cis to trans, the significance of these enzymes in the folding of gluten proteins by catalysing this conversion can not be ruled out. However, to our knowledge, studies on the relationship between PPIases activity and gluten proteins deposition have not yet been carried out. 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. The present study was therefore, initiated with an objective of analysing the changes in composition of grain storage proteins (albumins, gliadins and glutenins) and PPIase activity at different stages of development, and to evaluate the relationship between the protein fractions, PPIase activity and the dough rheological parameters in different wheat cultivars. 2. Materials and methods 2.1. Materials Seeds of five Indian wheat cultivars (GLUPRO, HPW-89, LOK-1, PBW-343 and RAJ-1482), varying in their dough characteristics, were procured from Punjab Agricultural University, Ludhiana (Punjab), India. Plants were raised in 5 l pots in the net house at Guru Nanak Dev University, Amritsar, as described earlier (Singh et al., 2008). Clay loam soil and farm manure (2:1 ratio) was mixed with 5 g per pot of N: P: K (5:2.5:1.2) and used for raising the plants. The ears were tagged on the day of anthesis and harvested at different stages of development as follows: 8, 12, 16, 20, and 25 days post anthesis (DPA) and finally at maturation. The grain samples were harvested in three replicates, with each replicate comprising of plants from three different pots. Harvested ears were stored in liquid nitrogen and used for the study. A portion of the mature grains of each cultivar was milled by using a Super Mill1500 (Newport Scientific, Warriewood, Australia) to obtain a whole-wheat meal. 2.2. Isolation and estimation of protein fractions Sequential extraction of gliadins, albumins, glutenins and other proteins was carried out as described in DuPont, Chan, Lopez, and Vensel (2005). Briefly, the powdered grains (1 g) were extracted thrice with 1-propanol-NaI solution and centrifuged. The residues were lyophilised for glutenin extraction, while the supernatants were pooled, precipitated with 0.1 M ammonium acetate-MeOH, and centrifuged to obtain a pellet of the gliadin fraction. The supernatant thus obtained was precipitated using chilled acetone and centrifuged to obtain the albumin/globulin fraction. The lyophilised residue was extracted twice with sodium dodecyl sulphate (SDS), dithiothrietol (DTT) and Tris (pH 8.0) solution, precipitated with ammonium acetate-MeOH and centrifuged to obtain the pellet of the glutenin fraction. The fractions were immediately lyophilised and stored at -80 °C for further analysis. For protein determination, lyophilised fractions were re-suspended in 2 mM DTT, 0.1% TritonX100 in 63 mM Tris (pH 6.8) and a Bradford assay was carried out in triplicates (Bradford, 1976). The protein content (%N  5.7) was also estimated by the micro-Kjeldahl method (AACC, 2000) using a nitrogen analyser (K-350, Buchi, Switzerland). 2.3. SDS–PAGE analysis The lyophilised samples were resuspended in the sample buffer [2% SDS, 5% ß-mercaptoethanol, 20% glycerol in 63 mM Tris (pH 6.8) and 0.1% bromophenol blue], incubated for 1 h at 22 °C with

451

shaking, centrifuged at 10,000g for 10 min and resolved by SDS– PAGE under reduced conditions, as described earlier (Laemmli, 1970). Gels were stained overnight with coomassie brilliant blueR250 and destained using 20% methanol. 2.4. Peptidyl prolyl cis–trans isomerase (PPIase) assay The total soluble proteins from the harvested tissues were extracted in ice-cold extraction buffer [5 mM Tris–Cl (pH-7.8), 0.015% Triton X-100] (5 ml/g F.W.) The extracts were centrifuged at 12,000g for 20 min at 4 °C. The supernatant, comprising of crude extract, was used for determining the PPIase activity. The total protein content of the samples was estimated by Bradford’s method using BSA as a standard (Bradford, 1976). PPIase activity was assayed at 15 °C for 360 s in a coupled reaction with chymotrypsin as described earlier (Fischer, Bang, & Mech, 1984). The one ml assay mixture contained 40 lM N-succinyl-ala-ala-pro-phep-nitroanilidine as test peptide, assay buffer [50 mM HEPES (pH 8.0), 150 mM NaCl, 0.05% Triton X-100] and 30–50 lg of total proteins. The reaction was initiated by the addition of chymotrypsin (300 lg/ml) and the change in absorbance at 390 nm was monitored using a spectrophotometer (Perkin-Elmer Lambda Bio20) equipped with a Peltier temperature control system. FK506binding proteins (FKBPs)- and Cyclophilins-associated PPIase activities were estimated by determining the inhibition of reaction in the presence of specific inhibitors, FK506 (30 lM) and cyclosporin A (CsA) (50 lM), respectively. The inhibitors were added to the assay mix 30 min before the start of the reaction and incubated at 4 °C. The PPIase activity was calculated as the product of the difference in the catalysed and uncatalysed first order rate constants (derived from the kinetics of the absorbance change at 390 nm) and the amount of substrate in each reaction (Breiman, Fawcett, Ghirardi, & Mattoo, 1992). 2.5. Dough preparation Doughs were prepared by mixing the whole-wheat meal and distilled water (60% w/w) to make stiff dough in a laboratory pin mixer (National Manufacturing Company, Lincoln, NE). 2.6. Gluten isolation Gluten was isolated by the Approved Method 38–10 (AACC, 2000). Meal was mixed with 60% (w/w) distilled water and kneaded to form a dough ball. Dough was soaked for 30 min and then gently washed with distilled water, until the washings became clear. The gluten was squeezed to remove excess water. 2.7. Dynamic rheology Oscillation measurements were performed using a RheoStress 6000 controlled stress rheometer (Haake, Karlsruhe, Germany) equipped with a Phoenix II P1-C25P refrigeration circulation bath. The plate and corresponding plate system were used with a diameter of 30 mm and a gap between the plates of 2 mm. The sample (dough or gluten) was placed between the plates of the rheometer and excess sample was carefully removed by using a sharp razor blade. A thin layer of silicon oil was applied to the exposed surface of the sample to prevent drying during testing. The sample was rested for 30 min to allow relaxation of stresses generated during sample loading before the measurement was taken. Linear viscoelastic region was determined by stress sweep tests (1 Hz at 25 °C) and stress value of 1 Pa was chosen for all the frequency tests. Frequency sweep tests (mechanical spectra) from 1 to 10 Hz were performed at 25 °C for samples of all the cultivars. The storage modulus (G0 ), loss modulus (G00 ) and phase angle (d)

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were derived at 25 °C for the dough samples. The moduli and phase angle were also determined for gluten samples during heating from 25 °C to 90 °C at the rate of 1 °C per minute followed by cooling to 25 °C at the same rate. Three replications were conducted for each dynamic test. 2.8. Statistical analysis The data reported are the average of triplicate biological repeats and were subjected to analysis of variance (ANOVA) by Duncan’s test (P < 0.05) using Minitab Statistical Software (State College, PA). Principal component analysis (PCA) was also carried out for determining the relationship between the different variables. The PCA results were graphically represented by the projection of the first two principal components. 3. Results and discussion 3.1. Isolation and SDS–PAGE analysis of different protein fractions Wheat flour contains different protein fractions such as gliadins, glutenins, globulins and albumins (Dupont et al., 2005). The isolation and separation of the different storage protein fractions becomes a challenge due to their cross contamination. Different methods for the fractionation of storage proteins were employed in this study and it was observed that amongst these, sequential extraction according to Dupont et al. (2005) resulted in the highest recovery of different protein fractions with minimal cross-contamination. The methods of Osborne (1924) or Fu and Sapirstein (1996), on the contrary, were observed to result in high levels of crosscontamination of gliadins and glutenins (data not shown). Depending upon the stage and cultivar, the residue protein fraction varied with the development stage and ranged from 2% to 20%, which is comparable with the previous findings (Triboi, Martre, & TriboiBlondel, 2003). In accordance with the earlier studies (Triboi et al., 2003), the residue fraction was pooled with the glutenin fraction. The separation of gliadins and glutenins from grains by sequential extraction was observed to be influenced by the developmental stage (Fig. 1). HMW-GSs of around 96,000 and 87,000 Da (shown by arrows) were observed in glutenin fractions from 12 DPA onwards in RAJ-1482. These proteins were also detected for up to 16 DPA in the gliadin fraction of this cultivar. LOK-1 showed HMW-GS of about 85,000 Da in glutenin fraction from 16 DPA until maturity. However, this subunit was observed until 25 DPA in the gliadin fraction in this cultivar, but was not detectable at maturity. PBW-343 showed the presence of HMW-GSs in gliadin fractions only at 16 DPA and 20 DPA. On the contrary, HMW-GSs were not detectable in the gliadin fractions at any of the developing stages in GLUPRO. These results clearly demonstrated that the extractability of polymeric proteins was affected by the developmental stage, which is in confirmation with the earlier findings (Gupta, Masci, Lafiandra, Bariana, & MacRitchie, 1996). Since solubility of individual subunits of glutenin and gliadin in aqueous alcohol is comparable (Veraverbeke, Larroque, Bekes, & Delcour, 2002), it is likely that HMW-GSs, which were fractionated along with gliadins at different stages of grain development in RAJ-1482, LOK-1 and PBW-343, may be the unpolymerised polypeptides which, however, requires further confirmation. Micro-Kjeldahl analysis carried out to determine the protein content in different fractions, revealed that the deposition of the different protein fractions at the different stages of grain development varied with the cultivar (Fig. 2). The total protein content in different cultivars ranged between 5–20% and an increase was observed in RAJ-1442 and PBW-343 with grain maturation. The gliadin accumulation reached a maximum at 25 DPA in LOK-1

and at 16 DPA in both RAJ-1482 and PBW-343, followed by a substantial decline until maturity. This was also accompanied by an increase in the accumulation of glutenins at these stages in these cultivars. GLUPRO, however, showed a biphasic accumulation pattern for both glutenins and gliadins. After decreasing between 8 and 12 DPA, the gliadin fraction in this cultivar increased till 25 DPA before declining again, while the glutenin accumulation depicted an inverse pattern. SDS–PAGE profiling of the different fractions showed the presence of several gliadin proteins from 8 DPA onwards in RAJ-1482, HPW-89, GLUPRO and LOK-1. However, in PBW-343 gliadins could not be detected by SDS–PAGE before 16 DPA, despite repeated attempts, which might also be due to degradative activity in this fraction at the earlier stages. The accumulation of several gliadin subunits was differentially affected by the development stage. For example, gliadin proteins of 65,000 and 62,000 Da in LOK-1 were barely detectable until 12 DPA, but showed enhanced accumulation from 16 DPA onwards (marked by arrows in Fig. 1). RAJ-1482 showed an increased accumulation of around 39,000 Da protein towards maturity, as compared to the earlier stages of the grain development (Fig. 1). Accumulation of HMW subunits of glutenins was also cultivar and stage dependent. This was evident since HMW-GSs were not observed up to 8 DPA and 12 DPA in RAJ-1482 and LOK-1, respectively. On the contrary, HMW-GSs showed accumulation from 8 DPA onwards in HPW89, PBW-343 and GLUPRO. The profile of LMW-GSs was not altered significantly after 16 DPA in any of the cultivars. Glutenin polymers can be dissociated into HMW-GSs and LMW-GSs after treatment with a disulphide reducing agent, dithiothrietol or ß-mercaptoethanol. SDS–PAGE analysis of the glutenin fractions showed the resolution of proteins with apparent molecular mass ranging from 80,000–120,000 Da and 30,000–50,000 Da (Fig. 1), which may correspond to HMW-GSs and LMW-GSs, respectively (Dupont et al., 2005). The molecular weight of glutenin subunits of different cultivars used in this study varied from 30,000–120,000 Da, which is in agreement with earlier reports (Veraverbeke et al., 2002). SDS–PAGE was unable to resolve several proteins in the range of 36,000–48,000 Da in the gliadin and glutenin fractions, which was also observed in earlier studies (Dupont et al., 2005). The presence of several bands with an apparent molecular mass between 50,000 and 65,000 Da in the gliadin fraction signified the presence of x-gliadins, whereas the proteins with molecular mass of 30,000–48,000 Da may correspond to a- and c- gliadins, as reported earlier (Dupont et al., 2005). The synthesis of HMW-GSs, LMW-GSs and gliadins in wheat was reported to occur concurrently, starting from as early as 7 DPA (Gupta et al., 1996), whereas other reports suggested that HMW-GS were synthesised earlier than gliadins (Skerritt, Lew, & Castle, 1988). Our observations however, indicate that the temporal synthesis of different gliadin and glutenin proteins was cultivar dependent. This is evident in PBW-343, wherein the accumulation of HMW-GSs was observed from 8 DPA, but the gliadins were not detectable before 16 DPA, thus supporting the observations of Skerritt et al. (1988). The synthesis of HMW-GSs, LMW-GSs and gliadins in HPW-89 on the contrary, was observed concurrently, which is in accordance with the earlier findings (Gupta et al., 1996). 3.2. Peptidyl prolyl cis–trans isomerase activity PPIase activity in plants is contributed mainly by cyclophilins and FKBPs (Romano, Gray, Horton, & Luan, 2005). Developmental regulation of PPIases in wheat grains has been reported only for a cyclophilin (Grimwade, Tatham, Freedman, Shewry, & Napier, 1996) and FKBP73 (Aviezer et al., 1998) genes, and that too only at a transcript level. However, expression analysis based on a few genes of the PPIase family may not reflect the real status of

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M

LMW-GS

66,000 43,000

66,000 43,000 29,000

18,400

18,400

97,400

HMW-GS

PBW-343

12 16 20 25 30 8 12 16 20 25 30

66,000

M 8

GLUPRO

12 16 20 25 30

97,400

8 12 16 20 25 30

43,000

29,000

29,000

18,400

18,400

LOK-1

12 16 20 25 30 8 12 16 20 25 30

HMW-GS

M 8

LMW-GS

LMW-GS

66,000

43,000

97,400

GLUTENINS

8 12 16 20 25 30

97,400

29,000

M 8

8 12 16 20 25 30

LMW-GS

97,400

HPW-89

HMW-GS

8 12 16 20 25 30 HMW-GS

M 8 12 16 20 25 30

GLIADINS

HMW-GS

RAJ-1482 GLIADINS GLUTENINS

43,000

LMW-GS

66,000

29,000

18,400

Fig. 1. SDS–PAGE analysis of gliadins and glutenins isolated from grains of different wheat cultivars at different stages of development (8, 12, 16, 20, 25 and 30 days post anthesis). M: Molecular weight markers.

biochemical activity since this family comprises of several genes that are regulated differentially (He, Li, & Luan, 2004). Furthermore, expression studies of the PPIases at the activity level are 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, it becomes imperative that the analysis of PPIase genes should be carried out at the activity level so as to understand their role in the accumulation of storage proteins in wheat. In the present study, PPIase activity in the crude protein extracts of developing grains was estimated by the coupling enzyme assay method using chymotrypsin for cleaving the test peptide (Fischer et al., 1984). Therefore, the PPIase activity analysed was primarily due to cyclophilins and FK506binding proteins (FKBPs), which were resistant to chymotrypsin. The present study revealed that the PPIase activity in the grains was cultivar dependent and regulated by the developmental stage (Fig. 2). PPIase activity was detectable at 8 DPA and onwards in all the cultivars. RAJ-1482, HPW-89 and LOK-1 grains showed the highest PPIase activity at 12 DPA, followed by a decline until 20 DPA. The PPIase activity in the grains of PBW-343 and GLUPRO decreased between 8 and 16 DPA, followed by an exponential increase. The variable changes in the PPIase activity during grain development may be due to differential temporal regulation of the different PPIase genes, which however, requires confirmation by analysing the expression of individual PPIases. PPIase activity of FKBPs and cyclophilins is inhibited by immunosuppressant drugs, FK506 and CsA, respectively (Harding, Galat, Uehlinh, & Schreiber, 1989). Since no cross inhibition by the two

drugs was reported (Harding et al., 1989), FK506 and CsA were, therefore, employed as specific inhibitors to determine the contribution of FKBPs and cyclophilins, respectively, to total PPIase activity in the crude extract of developing grains. PPIase activity at all stages of grain development in different cultivars, except at 25 DPA in LOK-1, was almost totally inhibited by CsA (Table 1). The combined inhibition by CsA (26%) and FK506 (25%) (data not shown) of grain PPIase activity at 25 DPA in LOK-1 was about 50%. It is likely that CsA- and FK506-insensitive PPIase activity at 25 DPA in LOK-1 may be due to the expression of another class of proteins, Parvulins (He et al., 2004). However, further studies are required to characterise the role of parvulins in the developing grains of wheat. These observations, thus, suggest that PPIase activity in the grains of different cultivars was primarily contributed by cyclophilins. These results are in agreement with earlier studies, which reported the abundance of a cyclophiln mRNA in the wheat grains at 5 DPA (Grimwade et al., 1996). The contribution of FKBPs to the total grain PPIase activity, however, can not be ruled out since the test peptide (N-succinyl-ala-ala-pro-phep-nitroanilidine) used in this study is cleaved less efficiently by FKBPs as compared to cyclophilins (Harrison & Stein, 1990), that might have resulted in an underestimation of the FKBP-associated PPIase activity. 3.3. Dough rheology The G0 of the dough from all the cultivars was greater than G00 , indicating predominance of an elastic character. Dough from

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RAJ-1482

50 40

1000

30 20

2000

60 1500 50 40 1000 30 20

500

500

10

10 0

0

0 0

5

10

15

20

25

30

0 0

35

5

10

20

25

30

35

Days Post Anthesis

Days Post Anthesis 80

80 2000

1500 50 40 1000 30 20

2000

60 1500

Percent (%)

60

GLUPRO

70

PPIase Activity (nmol/sec/g F.W.)

PBW-343

70

Percent (%)

15

50 40

1000

30 20

500

10

PPIase Activity (nmol/sec/g F.W.)

Percent (%)

1500

Percent (%)

60

HPW-89

70

PPIase Activity (nmol/sec/g F.W.)

70

2000

PPIase Activity (nmol/sec/g F.W.)

80

500

10

0

0 0

5

10

15

20

25

30

0

0 0

35

5

10

Days Post Anthesis

15

20

25

30

35

Days Post Anthesis

80

LOK-1

2000

60

Percent (%)

1500 50 40

1000

30 20

PPIase Activity (nmol/sec/g F.W.)

70

500

10 0

0 0

5

10

15

20

25

30

35

Days Post Anthesis Fig. 2. Accumulation of different protein fractions (%) and peptidyl prolyl cis–trans isomerase (PPIase) activity in the grains of wheat cultivars at different stages of development. (-- Total proteins, -j- Gliadins, -N- Glutenins, -d- Albumins and –  – PPIase activity).

Table 1 Percent inhibition of peptidyl prolyl cis–trans isomerase activity in the presence of cyclosporin A in different wheat cultivars at different stages of development. Cultivars

8 DPA

12 DPA

16 DPA

20 DPA

25 DPA

RAJ-1482 HPW-89 PBW-343 GLUPRO LOK-1

96.5a 100a 96a 100a 88a

100a 100a 94a 100a 100a

100a 97a 95a 100a 100a

100a 97a 94a 100a 100a

100a 96.5a 92a 100a 26b

Means with similar superscripts in a column do not differ significantly (p > 0.05).

RAJ-1482 showed the highest G0 and G00 , while PBW-343 showed the lowest values (Fig. 3). Phase angle (d) ranged between 19.5° and 22.5°, and was consistent with the values (18–24°) reported for European wheat cultivars (Bockstaele, Leyn, Eeckhout, & Dewettinck, 2008). An increase in phase angle value indicates a more viscous response as phase angle represents the degree by

which stress differs from strain. The majority of the cultivars did not show a large variation in the phase angle, probably due to the predominance of the elastic characteristics in the dough. G0 and G00 were positively correlated (r = 0.983, p 6 0.005) to each other while they showed negative correlations with the phase angle (r = 0.941, p 6 0.005). The G0 and G00 of dough showed a positive relationship with the glutenins (r = 0.815 for G0 and 0.828 for G00 , p 6 0.01), indicating a greater influence of the fraction on the viscoelastic properties of the dough. Variation in the viscoelasticity has been ascribed to the glutenin fraction (Schofield, 1994) and appears to be largely responsible for the differences in the dough properties of different wheat cultivars.

3.4. Gluten rheology The mechanical spectra showed typical solid-like character and frequency dependence of moduli, signifying relatively high overall

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60

40 RAJ-1482 GLUPRO HPW-89 LOK-1 PBW-343

20

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Time (min) Fig. 3. Rheological profiles (Storage modulus, G’) of doughs from different wheat cultivars.

chain mobility within the network. The G0 and G00 showed a frequency-dependence as both moduli increased with an increase in frequency. The typical viscoelastic behaviour of gluten observed during heating (25–90 °C) and cooling (90–25 °C) is illustrated in (Fig. 4). The average values of G0 and G00 of gluten and dough from the same cultivars showed a similar trend. During heating, the G0 progressively decreased to a minimum at a critical temperature i.e. between 60 °C and 70 °C. This indicates softening of the gluten. Beyond this critical temperature, further heating caused an increase in G0 in RAJ-1482 and GLUPRO cultivars. Similar effects of temperature on the G0 of gluten have been observed previously (Hayta & Schofield, 2005). However, both HPW-89 and PBW-343 did not show a significant increase in the G0 beyond the critical

8000

8000

G' , G" (Pa)

6000

G'

7000

G"

6000 5000

G' , G" (Pa)

RAJ-1482

7000 5000 4000 3000 2000 1000

GLUPRO

G"

3000 2000 1000 0

20

40

60

80

20

100

40

8000

8000

LOK-1

7000

G'

6000 5000

G"

4000 3000 2000 1000 0 20

60

80

100

Temperature (°C)

Temperature (°C)

G' , G" (Pa)

G'

4000

0

G' , G" (Pa)

G' in Pa (1x1000)

80

temperature. This behaviour may be attributed to the presence of higher proportions of gliadins (Khatkar, Fido, Tatham, & Schofield, 2002) compared to RAJ-1482 and GLUPRO (Fig. 2). It is thus evident from the observations of this study that the different cultivars showed a variability in their rheological behaviour, as well as storage protein deposition and PPIase activity, during grain development. In order to analyse the relationship amongst these, data were subjected to principal component analysis (PCA). PCA between different fractions of proteins and the rheological parameters of dough from matured grains revealed that G0 and G00 of dough were positively related to the glutenins, as indicated by their location on the same side of the loading plot (Fig. 5a). Both these moduli, however, were located on the opposite side to the gliadins, indicating a negative relationship with this protein fraction. PCA for the relationship between the PPIase activity and accumulation of different protein fractions at different stages of grain development was also performed (Fig. 5b).The loading plot clearly revealed that PPIase activity in HPW-89 and GLUPRO was related to the accumulation of gliadins. This relationship was stronger in HPW-89. The PPIase activity in RAJ1482 and PBW-343 was more closely associated with total proteins and glutenin fraction. PPIase activity of RAJ-1482 showed the strongest association with total proteins as compared to those of other cultivars. The presence of PPIase activity at different stages of grain development in all the cultivars, in addition to its close association with storage proteins, indicate that these enzyme(s) may be playing an important role in the deposition of storage proteins in wheat. However, cloning and biochemical characterisation of different PPIase genes, which is in progress, will further elucidate the contribution of these enzymes to storage protein deposition in the wheat grains.

PBW-343

7000 6000

G' G"

5000 4000 3000 2000 1000 0

40

60

80

20

100

40

60

80

100

Temperature (°C)

Temperature (°C) 8000

HPW-89

G' , G" (Pa)

7000

G'

6000

G"

5000 4000 3000 2000 1000 0

20

40

60

80

100

Temperature (°C) Fig. 4. Viscoelastic behaviour of glutens measured during heating (25–90 °C) and cooling (90–25 °C) from different wheat cultivars.

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(a)

Albumins

0.50

Second Component

G" dough G' dough

Gliadins

Total Proteins

0.25

0.00

-0.25

Phase angle Glutenins -0.50 -0.50

-0.25

0.00

0.25

0.50

First Component 1.0

1.0

HPW-89 Glutenins

0.5

0.0 PPIase Gliadins Albumins

-0.5

GLUPRO

Total Proteins

Second Component

Second Component

(b)

0.5 Gliadins

Albumins

0.0 PPIase

-0.5

Glutenins

Total Proteins

-1.0 -0.75

-1.0 -0.50

-0.25

0.00

0.25

0.50

-0.75

0.75

-0.50

-0.25

0.25

0.50

0.75

1.0

1.0

PBW-343

RAJ-1482

Total Proteins

Glutenins

0.5 Albumins

Total Proteins

0.0

PPIase

-0.5

Gliadins

-0.50

-0.25

0.00

0.25

PPIase

0.5 Glutenins

0.0 Gliadins Albumins

-0.5

-1.0

-1.0 -0.75

Second Component

Second Component

0.00

First Component

First Component

0.50

0.75

-0.75

-0.50

-0.25

First Component

0.00

0.25

0.50

0.75

First Component

Fig. 5. Principal component analysis showing (a) relationship between the rheological parameters of dough and different fractions of proteins separated from matured grains (30 DPA) and (b) relationships between the accumulation of total proteins, different protein fractions and peptidyl prolyl cis–trans isomerase activity at different stages of grain development (8–25 DPA).

4. Conclusions

Acknowledgements

The present study demonstrated that the developmental regulation of gliadins and glutenins accumulation varied with the cultivars. The dynamic rheological parameters of the dough and gluten were observed to be positively related to glutenin and negatively to gliadins. PPIase activity showed a close association with accumulation of either gliadins or storage proteins, thus signifying the importance of these enzymes in storage protein deposition. To the best of our knowledge, the present study is the first to report the association of PPIase activity with wheat storage proteins. This indicates that the identification and studies on regulation of individual PPIase genes may help in designing strategies for manipulating the wheat storage proteins and thus the dough and bread making quality.

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