Heat treatment at 65°C induces the formation of recombinant <i>Tc</i>CYS4 dimers.

<p><b>A</b>, native gel of <i>Tc</i>CYS4 incubated at temperatures ranging between 45 and 80°C for 10 minutes. <b>B</b>, percentages of dimers and monomers of <i>Tc</i>CYS4 obtained by densitometry of the gels of A in triplicate using the software Image2D Platinum 7.0. <b>C</b>, size exclusion chromatography of <i>Tc</i>CYS4 after heat treatment. The retention volumes for the standards are indicated as Al, BSA with 66.5 kDa; Ov, ovalbumin with 45 kDa; Ch, Chymotrypsinogen A with 25 kDa; AR, ribonuclease A with 13.8 kDa. The size expected for monomers, dimers ande trimers for His-tagged protein is approximately 24, 48 and 72 kDa, respectively. <b>D</b>, Spectral profiles by circular dichroism at the wavelengths from 190 to 250 nm of the <i>Tc</i>CYS4 treated at 20°C (solid line), 65°C (dashed line), and 90°C (dotted line) showing that the protein undergoes minor alterations at 65°C, and, when analyzed at 90°C, it undergoes loss of structure when compared with the protein at 20°C.</p

The Activity of <i>Tc</i>CYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa, Caused by the Fungus <i>Moniliophthora perniciosa</i>

<div><p>The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from <i>Theobroma cacao</i> L. previously identified in ESTs libraries of the interaction with the fungus <i>Moniliophthora perniciosa</i> and named <i>Tc</i>CYS1, <i>Tc</i>CYS2, <i>Tc</i>CYS3 and <i>Tc</i>CYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. <i>Tc</i>CYS1 was sensitive to temperatures above 50°C, while <i>Tc</i>CYS2, <i>Tc</i>CYS3, and <i>Tc</i>CYS4 were thermostable. <i>Tc</i>CYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that <i>Tc</i>CYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. <i>Tc</i>CYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. <i>Tc</i>CYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of <i>Tc</i>CYS4 oligomers. A variation in the titratable acidity was observed in tissues of <i>T</i>. <i>cacao</i> during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of <i>Tc</i>CYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.</p></div

pH 5 induces the formation of <i>Tc</i>CYS4 dimers.

<p><b>A</b>, Increases in absorbance of the reactions with <i>Tc</i>CYS4 (+<i>Tc</i>CYS4) and without <i>Tc</i>CYS4 (-<i>Tc</i>CYS4) at different pHs, showing that the inhibitory potential of <i>Tc</i>CYS4 was higher between pH 4 and 9. <b>B</b>, papain inhibition percentage of <i>Tc</i>CYS4 generated from A. The bars correspond to the standard error of the mean (n = 3). <b>C</b>, residual activity of papain in reactions containing <i>Tc</i>CYS4 under influence by the pHs 4, 5, 6, 7, and 8 associated with the heat treatment from 45 to 80°C. The bars correspond to the standard deviation of the mean (n = 3). Means followed by the same capital letters, for the same treatment of temperature, do not differ by the Scott-Knott test at 5% probability. Means followed by the same lowercase letters, for the same pH treatment, do not differ by the Scott-Knott test at 5% probability. <b>D</b>, profile of ellipticity by circular dichroism of <i>Tc</i>CYS4 at 26°C and pH 6 (solid line), 65°C and pH 6 (dashed line), 26°C and pH 5 (dotted line), and 65°C and pH 5 (dash and dot), showing that the treatments present similar profiles but the proteins at pH 5 present lower signals.</p

Inhibitory profile of the four <i>T</i>. <i>cacao</i> cystatins after heat treatment.

<p><i>Tc</i>CYS1 protein(■) is heat unstable, and the <i>Tc</i>CYS3 protein(▲) is heat stable. <i>Tc</i>CYS2 proteins(●) and <i>Tc</i>CYS4 (♦) also presented heat-stable behavior; however, they present reduction of inhibitory potential after treatments in the temperature range between 60 and 70°C. The vertical bars correspond to the standard deviations of the mean (n = 3).</p

The<i>Tc</i>CYS4 dimers are stable after storage at 8°C.

<p>A, residual activity of papain in reactions containing aliquots of the protein treated at 0, 0.4, 0.5, 1, 2, 2, 4, 5, and 10 minutes at 65°C. The bars correspond to the standard deviation of the mean (n = 4). <b>B</b>, Percentage of residual activity of papain in reactions with <i>Tc</i>CYS4 treated at 65°C and stored at 8°C (Control) over 20 days. The bars correspond to the standard deviations of the mean (n = 3). <b>C</b>, percentage of residual activity of papain in reactions with <i>Tc</i>CYS4 treated at 65°C, 80°C, and 65°C for 10 minutes and then treated at 80°C for 10 minutes, showing that the <i>Tc</i>CYS4 dimers were reconverted to monomers, when treated at 80°C. The activity of <i>Tc</i>CYS4-His-tag (histidine tailed) was similar to that of the protein <i>Tc</i>CYS4 (without histidine tail), demonstrating that the histidine tag does not interfere with the protein’s activity. The bars correspond to the standard deviation of the mean (n = 4). Means followed by the same capital letters, for the same inhibitor,do not differ among themselves by the Scott-Knott test at 5% probability. Means followed by the same lowercase letter, for the same treatment, do not differ among themselves by t test at 5% probability.</p

Unfolding and refolding of TcCYS4 expressed in percentage of structue.

<p>Unfolding of <i>Tc</i>CYS4 by heating from 26 to 92°C previously incubated at 8°C (empty circle) and 65°C (full circle), and refolding of this protein from 92 to 26°C previously incubated at 8°C (empty square) and 65°C (full square), showing that the protein pretreated at 65°C re-coils, similarly to protein pretreated at 8°C. Three spectra were performed to each treatment.”</p

Variation of acidity in infected cacao leaves.

<p><b>A</b>, image of the cocoa leaves with the same level of expansion and at each stage of development of the disease.<b>B</b>, Level of titratable acidity (TA) in healthy leaves and three developmental stages of witches’ broom disease. The bars correspond to the standard deviation of the mean (n = 5). Means followed by the same letters do not differ by the Scott-Knott test at 5% probability.</p