A  Novel Biochemical Method for Screening of Cold Tolerance in Rice a

Shyam K. Agrawal¹, Ganesh K. Agrawal¹ and Vishwanath P. Agrawal^1,2

¹ Research Laboraory for Agricultural Biotechnology and Biochemistry (RLABB), P.O. Box 2128, Kathmandu, Nepal; ² Royal Nepal Academy of Science and Technology (RONAST). P. O. Box 3323, Khumaltar, Lalitpur, Nepal

ABSTRACT

The present biochemical method involves quantification of Ribulose- 1,5-biphosphate carboxylase/oxygenase  (RuBisCo). It is  a simple, sensitive, rapid and a non-destructive  method for screening of cold tolerance in rice (Oryza sativa L.). Using this method, rice lines can be categorized into cold tolerant (CT), moderately cold tolerant (MCT), and cold sensitive (CS). In this method, 12 day old rice seedlings grown at 26ºC are treated at 12ºC for 5 days under a 12/12 h light/dark regime, and the level of total RuBisCo is measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of leaf protein followed by coomassie staining with subsequent determination of absorbance at 595 nm of the dye eluted from the bands of large and small subunits (LSU and SSU) of RuBisCo. Verification of this method has been done with 60 rice cultivars of different genotypes including CT, MCT, and CS cultivars. This RuBisCo method provides a convenient, inexpensive, and reliable biochemical procedure for distinguishing rice varieties on basis of cold tolerance and/or sensitivity. This is the also the first and only known method till date for screening of cold tolerance in rice.

INTRODUCTION

The enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (usually called RuBisCo, EC: 4.1.1.39), one of the most abundant proteins in the biosphere plays a pivotal role in photosynthesis, essential for almost all life on earth, as the carbohydrates produced during photosynthesis are the ultimate source of energy for virtually all non-photosynthetic organisms. It forms the bridge between life and the lifeless, creating organic carbon from the inorganic carbon dioxide in the air. The dark phase of photosynthesis, called the Calvin cycle, starts with the reaction of carbon dioxide and ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate, a highly exergonic reaction (DGº` = -12.4 kcal/mol) catalyzed by RuBisCo . This enzyme is located on the stromal surface of thylakoid membranes (Stryer 1995).  In spite of its central role, RuBisCo is remarkably inefficient. Typical enzymes can process thousand molecules per second, but RuBisCo fixes only about three carbon dioxide molecules per second. Plant cells compensate for this slow rate by building lots of the enzyme. Chloroplasts are filled with RuBisCo, which comprises half of the protein. This makes RuBisCo the most plentiful single enzyme on the Earth. RuBisCo in chloroplasts consists of eight large subunits (L, 55 kDa) and eight small ones (S, 13 kDa). Each L chain contains a catalytic site and a regulatory site. The S chains enhance the catalytic activity of the L chains.

Rice, one of the most important cereal crops, is the basic food of more than 3 billion people and it accounts for 50-80% of their daily calories intake. Besides its immense economical importance, rice has become a model system for genomics because of its relatively small genome size of 440 mega base pairs in the graminaceous family, and because of its closeness to other major cereal crops (Gale and Devos, 1998).

Cold temperature, a common environmental stress in the temperate regions, affects several biochemical, physiological and metabolic functions in plants (Murata, 1969; Levitt, 1980; Wang, 1982; Murata and Yamada, 1984; Roughan, 1985; Guy, 1990). Many plants of tropical origin cannot tolerate cold and can be damaged even at moderate temperatures that are below 20ºC (Graham and Patterson, 1982). Photosynthesis is one of the first processes to be affected when cold sensitive plants are exposed to low temperature (Levitt, 1980; Wang, 1982).

It has been reported that in rice plants growth rate and metabolism are markedly inhibited even at temperatures above the chilling temperature in the range of 15-20ºC (Kabaki et. al., 1982; Takanashi et. al., 1987) but the mechanism for the effects of low  temperature stress on growth and the accompanying metabolic changes remains unclear. It is generally recognized that the patterns of protein synthesis and mRNA level change when plants are exposed to cold temperatures (Thomashow, 1990; Koga-ban et. al., 1991). It has been shown earlier that cold treatment at seedling stage suppresses mRNA and protein levels of both large and small  subunits of RuBisCo in rice and this suppression of protein level is more pronounced in the cold sensitive  rice cultivars than in cold tolerant rice cultivars (Hahn and Walbot, 1989). Komatsu et al (1999) have reported that under cold stress RuBisCo is strongly phophorylated in cold tolerant rice compared to cold sensitive rice.

To improve rice cultivars against cold, traditional breeding can take several years to bring about any fruitful results. Therefore, tissue culture based techniques will be needed to reduce the time period required to release an improved rice variety/ cultivar. However, one of the serious problems of  tissue culture based techniques is the “somaclonal variation”. Somaclonal variation is defined as  genetic and phenotypic variation among clonally propagated plants of a single donor clone (Veilleux and Johnson, 1998; Olhoft and Phillips, 1999; Matzke and Matzke, 2000). Keeping the above problem(s) in mind, suitable biochemical and molecular marker(s) are needed for efficient screening of regenerated plants from tissues culture. To the best of our knowledge, there is no method available so far for screening of cold tolerance in rice, and in plants, in general.

 The main aim  of the resent research is to study  the response of RuBisCo to cold stress and thus to develop an appropriate biochemical marker for screening of cold tolerance in rice seedlings.

 

MATERIALS AND METHODS

SEED COLLECTION
Rice seeds were obtained from National Agriculture Research Council (NARC), Kathmandu, Nepal and International Rice Research Institute (IRRI), Philippines. Classification  of rice cultivars in CT, MCT, and CS categories were done on the basis of agronomic history and supplier’s recommendations/suggestions. Rice seeds were grown under controlled conditions (12h/12h Light/Dark regime) for 12 days in jars on moist filter papers at 26ºC.

COLD TREATMENT
Cleaned 12 day old seedlings (two leaf stage) were introduced into test tubes (25 x 150 mm) containing 10 ml Yoshida's nutrient solution (Yoshida et al, 1976) containing 114 ppm ammonium nitrate, 50 ppm sodium dihydrogen phosphate dihydrate, 89 ppm potassium sulfate, 111 ppm calcium chloride, 405 ppm magnesium sulfate 7 H₂O) and micro nutrients and subjected to cold by shifting to a constant temperature of 12ºC (12h/12h Light/Dark regime) for 5 days. For control, seedlings were grown for the same interval at 26ºC Yoshida` solution was changed every alternate day.

SAMPLE PREPARATION
Leaves of rice seedlings (20 mg) were ground at room temperature in 1 ml of grinding buffer [100 mM sodium phosphate buffer (pH 7.0) containing 0.7% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF)]. After centrifugation, 78 μl supernatant was mixed with 22 μl blue juice [9% (v/v) 2-mercaptoethanol, 22.7% (w/v) sucrose, 4.5% (w/v) SDS and 0.01% (w/v) bromophenol blue], and samples were heated in boiling water bath for 30 seconds. Protein determination with rest of the untreated supernatant was done according to Bradford method (1976) using BSA as a standard. To avoid variation, if any, among the seedlings, leaves of five individual seedlings were ground together.

SDS-PAGE
Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (Blackshear, 1984). The gel system  consisted of a 15% (w/v) resolving polyacrylamide gel (pH 8.9) and 3.04% (w/v) stacking polyacrylamide gel (pH 6.9), respectively.  Lengths of resolving and stacking gels were 10 and 2 cm, respectively. Fifteen microliter of prepared sample was loaded per well and the electrophoresis was carried out at room temperature at a constant voltage of 100 and 125 V for stacking and resolving gels, respectively. The electrophoresis was carried out until the dye (bromophenol blue) migrated 9.5 cm into the resolving gel.

DETERMINATION OF AMOUNTS OF LSU AND SSU IN PAGE BANDS

In order to stain LSU and SSU of RuBisCo, a direct staining method reported by Zehr et al. (1989) was used with some modification. For quantification of dye content of stained LSU and SSU bands, the method of Ball (1986) was used.

·        Soak the gels after PAGE in 20% TCA for 1 hr.

·        Wash the gels with H₂O three times for  5 minutes each.

·        Lightly agitate the gels for 24 hrs. in a staining solution containing 0.002% coomasie brilliant blue R-250, 10% ethanol and 5% acetic acid.

·        Carefully cut out the stained bands corresponding to large and small subunits of RuBisCo from the gel with razor blade and introduce in a  screw cap glass test tubes (7 x 100 mm) having 3 ml of eluting dye solution (3% SDS in 50% isopropanol).

·        Tightly close the  screw  cap of the tube and incubate it at 37⁰C in water bath for 24h without agitation.

·        Measure the absorbance of the resulting solution at 595 nm. And calculate the amount of protein from a calibration curve.

• Use the following formula  to calculate the amount of RuBisCo and its subunits as percentage of respective controls:

LSU(%) or SSU(%)  =  100 x A595 (cold treated sample)

       A595(control sample)

         Total RuBisCo (%) = [LSU (%) + SSU (%)] / 2

RESULTS AND DISCUSSION

 The main purpose of this study was to directly compare the effect of cold on levels of LSU and SSU of RuBisCo in CT, MCT, and CS rice cultivars in order to develop a biochemical marker for cold tolerance. For this purpose, 12 day old rice seedlings grown at 26ºC were treated at 12ºC (12h/12h Light/Dark regime) for 5 days and the levels of LSU and SSU of RuBisCo were measured by SDS-PAGE (Figure 1) with subsequent quantitative estimation of protein level by eluting dye from the LSU and SSU bands of RuBisCo (Table I).

Table I : Quantitative analysis of effect of cold on RuBisCo LSU and SSU protein bands of Rice cultivars

Values given  in parenthesis are based on the agronomic characteristics.
^a  Classification on the basis of visual estimation of the LSU and SSU bands separated by SDS – PAGE - coomasie staining; ^b   Classification on the basis of  dye estimation of LSU and SSU bands separated by SDS – PAGE - coomasie staining.

It was observed that due to cold stress levels of both subunits drastically decreased in CS, remained almost constant or slightly suppressed in MCT and increased in CT lines (Figure 1 & Table I ). It is the first report to the best of our knowledge on the cold induced increase in RuBisCo LSU and SSU in rice and in plants in general.

Cold induced decrease in levels of RuBisCo subunits can be explained in terms of (a) decreased mRNA and protein synthesis (Hahn and Walbot, 1989), (b) degradation by proteolytic enzymes known to be present in chloroplats (Yoshida and Minamikawa,1996, Musgrove et. al., 1989; Bushnell et. al., 1993) and (c) damage under  oxidative stress (Desimone et. al., 1996; Ishida et.al., 1997, Stadtman, 1990).

In order to examine the possibility of proteolytic degradation of RuBisCo during post cold treatment period, a fixed quantity of bovine serum albumin (BSA) was added to the rice leaves homogenizing buffer; proteins were then resolved by SDS-PAGE and detected using direct staining (Figure 2). Results  showed  that LSU and SSU were not degraded after cold stress as no bands, other than the LSU and SSU were stained by this method and  the amount of BSA remained almost constant in all samples analyzed (Figure 2).

Table II:  Classification of Rice cultivars in cold tolerant (CT), moderately cold tolerant (MCT) and cold sensitive (CS) groups using RuBisCo method

¹  On the basis of agronomic history, suppliers’ recommendations and l and suggestions of local people.
²  On the basis of RuBisCo method. S.No. 1-33: obtained from NARC, S.No. 34-43: received from IRRI., S.No. 44-57: collected by RLABB.

Using this method (SDS-PAGE followed by dye estimation) 60  rice cultivars were screened for their cold tolerance and grouped into CT (LSU + SSU > 100% of the control), MCT (LSU + SSU = 50 - 100% of the control), and CS (LSU + SSU < 50% of the control). Results obtained  revealed that RuBisCo method (visual  estimation of the bands separated by SDS-PAGE or quantitative estimation of dye eluted from LSU and SSU bands) compares satisfactorily with the suppliers recommendation/agronomic history (Table I1 ).

Finally, the results indicate that the effect of cold can be used to identify CS and CT rice. Moreover, the RuBisCo method can be used for relatively rapid screening of rice genotypes. The added advantage is that this method is non-destructive, that is, after screening seedlings for cold tolerance / sensitivity, the seedlings can be further grown in pots or fields for seed multiplication or breeding purposes.

To the best of our knowledge, this is the first report of any biochemical method capable of classifying unequivocally rice genotypes in CT, MCT, and CS. Moreover, this is also the first time that any one has reported cold induction of RuBisCo in rice.

CONCLUSIONS

 The results presented in this study show, firstly a novel RuBisCo method for screening cold tolerance/sensitivity in rice seedlings, and secondly present a reliable, cheap and efficient biochemical method for determining cold  tolerance or sensitivity in plants.

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