EFFECT OF pH ON PROLINE CONTENTS AND PEROXIDASE ACTIVITY IN METABOLISM OF SUGARCANE CULTIVATED in vitro

 lsabela M. T. Piza*, Gluseppina P. Percira Lima**, Andréla Henrique* and Oswaldo G. Brasil**

* Profesores. Universidade Estadual Paulista. Instituto de Biociências.
Botucatu. SP. Brazil. ** CP: 545, CEP: 18618-000.

Summary

After autoclaving and during the culture and development period, changes in pH values in the culture medium occur, which can affect nutrient availability, altering protein and carbohydrate metabolism. In this work, the effect of different values of pH on in vitro development of sugarcane variety SP 801816 was evaluated. Plantiets were grown in MS liquid medium in different pH levels (4.5; 5.0 and 5.8). An evaluation was performed of the contents of soluble proteins, peroxidase activity, proline content, reducing sugars and total protein. Decrease in pH values were abserved in all treatments after autoclaving and at the end of the analyzed period, independently of the initially adjusted pH. These resuits suggest that pH changes promoted alterations in biochemical parameters, like nitrogen metabolism, peroxidase activity and reducing sugars. The different values of pH affected biochemical parameters tested, but visual development and multiplication of the plants was not affected.

Key Words: Biochemical parameters; tissue culture; Saccharum offícinarum

Resumen

Después del autoclavado y durante el período de cultivo, ocurren cambios en los valores de pH que pueden afectar la viabilidad de los nutrimentos, alterando su metabolismo. En este trabajo se evaluó el efecto de dife- rentes valores de pH en el crecimiento in vitro de la variedad de cafia de azúcar SP 801816. Las plántulas crecieron en medio MS líquido, en diferentes valores de pH (4,5; 5,0; 5,8). Se analizó el contenido de proteínas solubles, actividad peroxidasa, contenido de prolina, azúcares reductores y proteína total. Se observó alteraciones de pH, ajustados inicialmente debido a las distintas fases de preparación y el tiempo de crecimiento. Los resultados sugieren que las alteraciones de pH causaron modificaciones en los parámetros bioquímicos; plantas crecidas en diferentes valores de pH presentaron cambios en el metabolismo del nitrógeno, actividad peroxidasa y azúcares reductores. El decrecimiento del pH fue observado en todos los tratamientos durante el período de cultivo en relación al pH inicial. Los distintos pH afectaron los parámetros bioquímicos, más no el desarrollo y la multiplicación de las plantas.

Palabras Clave: Parámetros bioquímicos; cultivo de tejidos; Saccharum officinarum.

Recibido: julio 20, 2001.

Introducción

Some factors, such as mineral nutrient availability, pH, temperatura and luminous intensity affect growth and multiplication of plants in vitro Leifert et al., 1992).  

Adjustment of pH has bcen accepted as an importan step in the preparation of in vitro culture media. Usually the values of pH is adjusted prior to autoclaving and subsequeni changes in pH during preparation or during the culture period are ignored.  

Few works have been done to identify the effect of pH variations in the culture medium, when pH has been adjusted before the autoclaving to values between 5.0 and 5,8, either in solid or liquid medium. The most favorable pH for adequate growth and development of sugarcane is from 5.5 to 6.5. At low pH values symptoms of Ca deficiency in cultivated plants occur. Frequently, this condition leads to symptoms of phosphate or molybdenum deficiency and toxicity of Al or Mn (Epstein, 1997). lf one is to investigase effects of pH on plant growth and development in vitro, it is clearly important to know how pH changes over the culture period. The significance of such changes in pH has not been established. Differences in initial pH values have been found to affect the pattern of explants growth and the induction of roots on some species.  

Biochemical and physiological changes in tissues in response to severas kinds of stress can be verified through alterations in the arnine compounds such as proline, proteins and in the enzyme activity, such as peroxidase. 

The present work aimed at determining pH variations that occur during the in vitro development of sugarcane variety SP 801 816, and their effects on the metabolism through the analysis of proline, peroxidase activity, soluble total proteins, total protein, and reducing sugars.

 Material and methodos 

Plantiets of sugarcane variety SP 801816 were micropropagated in vitro in Murashige and Skoog (1962) ¡¡quid medium (MS), enriched with 20 g 1-1 sucrose, 0.1 g 1-1 myo-inositol, 0.2 mg 1-1 BAP, 0.1 mg 1-1 Kinetin and 1 mg 1-1 thiarnine. At the time of experiment settlement, plantiets had reached 75 days of cultivation, at which time the process of plant multiplication process began.

The medium was sterilized by autoclaving after pH adjustment. The levels of pH studied were 4.5; 5.0 and 5.8, with 4 repetitions and 4 collection timps, at 0, 15, 30 and 45 days after plating (150 plants were used in the experiment). At 30 days, the plants were siib cultured in the same medium. The experiment was carried out in growth chambers, with an average temperatura of 29 °C (28 ± 2 °C), photoperiod of 12 hours and density of 30 m mol m-2s-1.

Parameters used for analyzing the effect of the different levels of pH on plant development were proline (Torelo and Rice, 1986); peroxidase activity (Lima et al., 1999); soluble protein content in cytoplasm, pH near 6,7 (Bradford, 1976); total protein (% N x 6.25) A.O.A.C. (1995); reducing sugars (Nelson, 1944).

Data frorn the first experiment (levels ofph) were analyzed using Tukey's multiple comparison test. The others experiments were analyzed utilizing the highest degree polynomial required to adequately fit (P<0.005) changes over time for cach variable measured.

Result and discussion

Decrease in pH values (Table) were observed in al¡ the treatments after autoclaving and at the end of the analyzed period (45 days), independently on the initially adjusted pH. For all pH values there were significant differences between the initial pH and that of the culture period. After autoclaving, a medium decrease of 0.3 units was observed for all pH values. In the first 15 days of culture, pH levels were lower and significant differences were observed. Between 30 and 45 days, pH levels showed a small increase in relation to the previous values. These results can he related to the absorption of nutrients and to the process of water loss. Similar results were observed by Lal and Singh (1995) for sugarcane, where media pH decreased during autoclaving and this decline was greater at the lower end of the agar concentration used and appreciable changes were observed during the culture period. They concluded that this indicates that certain chemical changes brought about during the culture lead to pH varations.

Pasqual et al. (1992) observad pH reduction after the process of autoclaving medium either in the presence or absence of agar. Aceording to the authors, autoclaving caused a pH decrease of 1 unit in MS mediurn; at very low pH (less than 4) there were no alterations, and when pH was adjusted hefore the autoclaving, there was a decrease of 0,3 units during the first 24 hours generally and then remained constant for a storage period of 48 hours; after this time it was possible to find new changes or acidification. Such changes could be caused either by medium dehydration or mineral components precipitation or even by ion elimination by the plant in order to provide pH balance in the medium (Paiva et al, 1999).

In all the treatments plants developed normally; any damage caused by culture in acid pH was not noticed visually. This seems to be an indicado that plants of sugarcane resist micropropagation in pH values below 5.8, which is a common value. Plant development was considered satisfactory and multiplication rates were not affected.

Use of biochemical markers in the study of physiologic process as weil as in the study of many types of stress in plants are reported in the literatura. Biochemical indicators, such as changes in the enzyme activity and metabolic "pools", are particularly used as means of detection of plants primary alterations, where injuries can not be visually observed (Castillo, 1986).

In relation to total soluble protein of contents, all samples in the first day (time 0) showed the same levels soluble protein, close to 260 m g proteinlg fresh matter. Differences were observed in contents of soluble proteins. The level of pH 4.5 showed an increase of soluble proteins in the first 15 days with a later decrease at 30 and 45 days. At pH values of 5.0 and 5.8 similar trends were observed during the period of culture, with reductions in the first 15 days, later increases at 30 with no differences at 45 days. Vegetable proteins are continually degraded and synthesized, which is essential so that plants may reuse amino acids for maintenance of homeostasis and growth. Reductions in protein contents may reflect a delay in protein synthesis or an accelaration of its degradation, according to the increase in the amount of free amino acids or the inhibition of the incorporation of these amino acids in the proteins. Increased tritates observed in the pH values of 5.0 and 5.8 at 30 days could be related with a period of active multiplication of the plants, and consequently higher protein synthesis.

TABLE. Average pH values after autoclaving, and during
the culture in vitro period, of sugarcane variety SP 801816.

---

   Initial pH          pH after      pH after        pH after     pH 45 days
autoclaving    15 days       30 days

---

                                    4,5A                  4,40A            3,49C               3,76B           3,60B
                                             5,0A                  4,92A            3,48C               3,67B           3,64B
                                             5,8A                  5,73A            3,47C               3,60B           3,74B

---

                                       Letters in rows indicate calculated levels of significant differences Tukey 5%.

Similar results were reported by Martin and Rose (1976), when they cultivated cellular suspensions of Ipomea in pH 4.8, were they also identified a sligth increase in protein content.

pH 4,5 y- 0,0038X3- 0,4822x2 + 13,789x + 259 R 3=1
pH 5,0 y= -0,008SX3 + 0,5733x2 - 6,5444x + 259 R3=1
pH 5,8 y= -0,0074x3 + 0,4844X2 - 6,8778x + 259 R3=1                                

              FIGURE 1. Total soluble proteins contents (m g protein/g fresh matter)
                                           in sugarcane plants.

In relation to total proteins (Figure 2) increase in up to 30 days of culture in pH 4.5 was noticed, with a subsequent fali at 45 days. In pH 5.0 a sinall increase at 15 days with a posterior decrease at 30 and 45 days was noticed. In pH 5.8 a decrease in total protein tritates at 30 days with a increase at 45 days was noticed. This fact may suggest that pH influenced growth and development, mainly through its effect on nitrogen absorption and use, which can he confirmed by other biochemical analysis carried out.  

According to Martin and Rose (1976), in low pH values, nitrogen absorption by plants is higher during the first hours after inoculation of plant in the culture medium. Nitrate is not used in the first 40 hours after the absorption; therefore, after that period its utilization proceeds at a faster rate.  

In relation to proline levels (Figure 3) an increase in titrates occurred at 15 days of cultivation in pH 4.5, with a posterior fall at 30 and 45 days.

 pH 4,5 y =-6E-05x3 + 0,0023x2 + 0,0046x +3,1 R2=1
pH 5,0 y =-5E-05x3 + 0,0038x2 + 0,0589x +3,1 R2=1
               pH 4,5 y =-8E-05x3 + 0,004x2 + 0,0156x +3,1 R2=1               

FIGURE 2. Total protein contents in sugarcane plants.

 In pH 5.0 reductions oecurred at 15 days with a posterior increase at 30 and 45 days without significant differences between the last two collections. In pH 5.8 curves of the same same trend as pH 5.0, were observed.

The analysis of nitrogen compounds, such as soluble proteins and proline, revealed that the highest absorption and utilization were really in low pH values, in the first collections. Several species can present altemative strategies for adaptation to stress, among them the proline accumulation (Hare and Cress, 1997).

In relation to peroxidase activity (Figure 4) a reduction was observed in pH 4.5 at the first 15 days with increase to 30 days and a smalt decline at 45 days, probably caused by the stress that the plant suffered in the medium, which could have influenced mineral absorption. In the pH 5.0 enzyme concentration increased in all the collections up to 45 days. In pH 5.8 an inverse tend compared to pH 4.5 was observed, with an increase at 15 days, a fall at 30 days and a small elevation at 45 days, which may be explained probably by the fact that these plants, besides suffering new processes of adaptation to a medium that acidified with the time, they had also been in cultivation for a long time with a consequent deterioration of, their quality.

                               pH 4,5 y = 0,0003x3 - 0,0293x2 + 0,5927x + 6,36 R2=1
                               pH 5,0 y = -0,0004x3 + 0,0307x2 - 0,4951 x + 6,36 R2= 1
                               pH 5,8 y = -0,0003x3 + 0,0203x2 - 0,3518 x + 6,36 R2=1
           FIGURE 3. Proline contents (limois prolinelg fresh mater) in sugarcane plants.

lt become evident from severa¡ works that peroxidases are dependent on the ion calcium, which is related to different cellular processes, acting as a secondary carrier and on the regulation of many funetions (Penel, 1986). According to Epstein (1997) low pH can induce Ca deficiency; in this way, the enzyme may have presentes alterations in its activity because of the set pH/Ca relation, causing a stress in the plant.

The higher peroxidase activity observed in plants submitted to stress can indicate the ability of certain genotypes to degrade toxic substances, such as free radicals (peroxides) released under these conditions (Subhashini and Reddy, 1990).

                                  pH 4,5 y = - 0,0002x3 + 0,012x2 - 0,1697x + 1,71 R2 =1
                                  pH 5,0 y = - 2E-05x +3 0,0018x2 - 0,0232x + 1,71 R2 =1
                                  pH 5,8 y = 8E_OSX3 - 0,005 1 X2 + 0,0837x + 1,71 R2 =1
        FIGURE 4. Peroxidase activity (m mols H202 x 10-3/m g protein x min) in sugarcane plants.

Pieris et al. (1986) and Lima et al. (1999) suggest that there is a complex involvement of peroxidase in plants grown under stress. That enzyme could he used as a marker for incipient stress, or as an indicator in plants already injured. Aecording to Siegel (1993) peroxidase activity can present alterations in plants submitted to different kinds of stress, such as acid.

In relation to the reducing sugar contents (Figure 5), was noticed that in pH 4.5 there was a slight decreasing trend at 30 days with posterior increase at 45 days. In pH 5.0 a curve with a similar trend ocurred and in pH 5.8 a small decline at 15 days with a posterior elevation at 45 days was observed.

The content of reducing sugars in plants grown in pH 5.8 increased with culture time and no changes were observed in the last collection. In all treatments and in all collections, an increase ocurred in sugar levels at 45 days of culture, which might have been caused by the absorption and partition of sucrose in the medium. Leifert et al. (1992) reported that after autoclaving, sucrose suffers hydrolysis into fructose and glucose, in this way the plant could have absorbed higher contents of glucose andlor fructose. lt appears probable that the reducing sugars contents varied more due to medium sucrose availability than the tested pH levels.

                                   pH 4,5 y = 4E-05x3 - 0,0019x2+ 0,0167x + 0,58 R2=1
                                   pH 5,0 y = SE-05x3- 0,0025x2 + 0,0358x + 0,58 R2 =1
                                   pH 5,8 y = -6E-05x3+ 0,0044x2 - 0,0558x + 0,58 R2=1
        FIGURE 5. Reducing sugar contents (mg glucose/g fresh matter)in sugarcane plants.

Although the growth of plants cultivated in vitro, having as source reducing sugars carbohydrates (hexoses), is usually inferior to that verified with sucrose, there might be exceptions. Caldas et al (1998) conceded that if there were suerose hydrolysis, the growth would not be decreased.

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