Hai-Tao Huang，Ji-Zhong Yu，Xian-Bo Wang，Tie-Feng Zhou，Cun Ao，Wei Zhang

Tea Research Institute，Hangzhou Academy of Agricultural Sciences，Hangzhou，311115，China.

Tea plant belongs to the subtropical crops with weak cold-resistance，and often suffers cold and frost damage.After observation，Huang Haitao ＆ Li Xiangyun et al［1，2］found that tea plants were always subjected to the cold and frost damages in Hangzhou in recent years，and one of the damages caused 6667 hm2tea plant area frozen and 340，000，000 yuan lost on March 7 to 14，2010.Therefore，the research of the freeze injury defense on tea plant has been focus in tea industry in early spring，and would be of great significance to the harvest of tea farmers.

Identification and breeding of cold-resistant tea varieties are the most important measures for the prevention of freeze injury.There have been many literatures concerning the investigations of identification of tea varieties and resources with different approaches［3-5］(Fang Y.，Wang Y.G.＆ Hou Y.J.)，but much less on the characters and physiological and biochemical indices of the tea varieties with the different cold-resistance.The previous studies mostly used mature tea leaves as the research material，and no studies on the cold related enzyme activities and physiological and biochemical indices of the new tea shoots have been reported，which is very important to the freeze defense of tea plant in the early spring.Consequently，in order to provide the actual identification indices for breeding tea varieties with cold resistant new shoots，the physiological and biochemical characteristics of the new shoots such as protective enzyme activity and protein，soluble sugar and MDA，are observed among different tea varieties which identified as high，middle and low cold resistance based on the previous studies on the approaches of natural identification，anatomical structure of leaves and conductance.

1.Material and method

1.1.Plant material

Zhenong 113 and Cui Feng were selected as high cold resistant tea varieties，Wuniuzao and Yingshuang as middle cold resistant，Lechang Baimaocha and Xiangguliao baihao 16-14 as low cold resistant.New shoots with a bud and two leaves were sampled to determine the physiological and biochemical indices in October 2012，viz.enzyme activity and protein，polysaccharide and MDA level.

1.2.Enzyme activity

Enzyme preparation:1 g weighted fresh leaves and 0.6 g polyvinylpyrrolidone(PVPP)，added 20 ml precooling buffer，were homogenized at 14000 rpm at 4℃，and then centrifuged at 1776 rcf for 15 min.The supernatant was collected for the determination of PPO，POD，CAT and SOD activity.

PPO activity:catechol colorimetric method.One unit of PPO activity was defined as each 0.1 increment in absorbance per min and presented as Units mg-1protein min-1.

POD activity:the activity of POD was measured according to the method of guaiacol colorimetric.The 3 mL reaction mixture contained 20 mM guaiacol(1-hydroxy-2-methoxybenzene)，20 mM H2O2，50 mM phosphate buffer(pH 6.8)，and 100 L enzyme extract.The reaction was heated in boiling water bath for 3 min and started by adding 20 mM H2O2.One unit of POD activity was defined as each 0.1 increment in absorbance at 480 nm per min and presented as Units mg-1protein min-1.

CAT activity:UV spectrophotometry method.One unit of CAT activity was defined as each 0.01 decline in absorbance per min and presented as Units mg-1protein min-1.

SOD activity:the SOD activity was measured according to the method of nitro blue tetrazolium(NBT).100 microliters of enzyme extract was added to a 3 mL reaction solution containing 75 M NBT，2 M riboflavin，13 mM methionine，0.1 M ethylenediaminetetraacetic acid(EDTA)，and 50 mM phosphate buffer(pH 7.8).The solutions were placed under light at 3000 lx for 10 min.The absorbance of reacted solutions and non-reacted solutions at 560 nm was determined with a spectrophotometer.One unit of SOD activity was defined as the amount of SOD required to cause 50%inhibition of the rate of NBT reduction at 560 nm.

1.3.Physiological and biochemical indices

Protein content:Coomassie brilliant blue colorimetric method.

Soluble sugar and MDA content was measured using the method of thiobarbituric acid(TBA)method.A 2 mL MDA extract was added to 2 mL of 10%trichloroacetic acid containing 0.6%TBA.The solution was heated in boiling water bath for 30 min and then quickly cooled in ice bath.The solution was centrifuged at 40，000 r/min for 10 min.The absorbance of the supernatant was measured at 450，532 and 600 nm.The concentration was calculated according to A532 and an extinction coefficient of 155 for MDA.1 g chopped fresh leaves，add 2 ml 10%TCA and a small amount of quartz sand，grinding to homogenate，plus 8 ml TCA further grinding，4，000 g homogenate supernatant centrifugal 10 min，get 2 ml upper clear liquid(control add 2 ml distilled water)，adding 2 ml 0.6%TBA solution，mixed material in the boiling water bath on the reaction of 15 min，rapid cooling after centrifugation.The supernatant were measured by 532，600 and 450 nm wavelengths extinction ratio.Soluble sugar and MDA concentration was calculated according to the following formula，and convert the content per gram dry weight.

Soluble sugar content(mmol/L)=11.71 OD450

MDA level(μmol/L)=6.45(OD532-OD600)-0.56 OD450

1.4.Statistics and analysis

Experimental data were analyzed with the EXCEL statistical program.One-way ANOVA was used to test the significance of difference in measured variables between control and treatment.The Least-significant difference(LSD)was employed to test the significance of difference among treatments.Statistical significance of difference was based at the 0.05 or 0.01 level and p-values less than 0.05 were considered significant.

2.Results and analysis

2.1.Cold-resistance related enzyme activity in new shoots of different tea varieties

Different enzyme activities in different tea varieties were listed in detail in Table1.As seen from table 1，CAT activity of strong cold resistant tea varieties was slightly higher than the weak cold resistant，but there were no significance among them;There were no significant differences in the CAT activity between the six different cold resistant tea varieties，which showed that the activity of CAT enzyme was not closely associated with cold resistance of tea plants.

Table1.Enzyme activity of different cold resistant tea varieties.

POD enzyme activity of the strong cold resistant tea varieties was substantially higher than that of the weak cold resistant，and it reached the great significant level，while the POD activity of middle cold resistant tea varieties was between the two species，and the POD activity of the six different cold resistant tea varieties decreased in the order with cold resistance strength.It indicated that the cold resistance of tea plant was closely related to the activity of POD，therefore，the level of POD activity could be used as identification indices for cold resistance.

SOD enzyme activities were different among the different cold resistant tea varieties，but it has no regularity with the cold resistance.

PPO enzyme activity of the strong cold resistant tea varieties was significantly lower than that of the weak cold resistant，but the PPO activity of the middle cold resistant tea varieties was instable.Therefore，PPO activity is not closely associated with cold resistance of tea plants.

2.2.Physiological and biochemical characteristics of new shoots in different tea varieties

As seen from table 2，water soluble protein contents of strong and medium cold resistant tea varieties were substantially higher than the weak ones，and reached the significant level，but there were no significant difference between the strong and medium cold resistance tea varieties.It indicated that the water soluble protein content was certain associated with cold resistance，but the correlation is not strong.

Table2.Water soluble protein and sugar，and MDA level of different cold resistant tea varieties.

Soluble sugar content of strong cold resistant tea varieties was substantially higher than that of weak cold resistance，and reached the great significant level，then the soluble sugar content of medium cold resistant tea varieties was between the two species above.It suggested that soluble sugar and cold resistance of tea plants were closely related，therefore，the soluble sugar content could be used as identification indices for cold resistance of tea plant.

There were significant differences in MDA level among different cold resistant tea varieties，but there was no regularity between MDA level and cold-resistance of tea varieties.

3.Results and discussion

3.1.Enzyme activity and cold resistance of tea plant

According to the previous researchs，cold resistance of tea plants was closely associated with several enzyme activities，such as CAT，POD，SOD and PPO［12］(Wang Y.J.，2011).Huang H.T.，et al［13］(1986)found that CAT activity top level appeared early in strong cold resistant tea varieties.Huang J.A.，et al［7］(1990)observed several enzyme activities of mature leaves in 6 different cold resistant tea varieties，and found that strong cold resistance varieties could maintain a higher level of SOD and CAT enzyme activity.Luo J.W.［8］(2001)investigated CAT，POD and SOD activity of mature leaves in 6 different cold resistant tea varieties，and found CAT and SOD activity of tea had strong regularity with cold resistance，but there was no regularity between POD activity and cold resistance.

In the present paper，four enzyme activities of new shoots in autumn tea varieties were investigated，and results that the correlation of CAT，SOD and PPO activity with cold resistance was not strong，while POD was closely associated with cold tolerance.Consequently，POD activity level in fresh tea shoots could be used as identification indices for cold resistance.

3.2.Soluble protein，soluble sugar，MDA level and cold resistance of tea plant

Soluble protein，soluble sugar and MDA level were closely associated with cold resistance of tea plant.Watersoluble protein and soluble sugar content were positive related with cold resistance.Higher water-soluble protein or soluble sugar content，stronger cold-resistance of tea plant.MDA is the final decomposition products resulted from plant senescence or film lipid peroxidation after suffering stress，but was also related with cold-resistance［14］(Qiu L.L.，2012).Luo JW.，et al［8］(2001)found that through determining water-soluble protein and MDA level of mature leaves in six different cold resistant and found that strong cold resistant tea varieties had higher soluble protein content，and vice versa.MDA level was only closely related with the leaf age，and hence increased with the leaf senescence，however，there was no regularity with cold resistance among tea varieties.

In the present paper，results indicated tea varieties with strong cold resistance had higher water soluble protein content，with weak cold resistance had lower water soluble protein content，and with middle cold resistance also had higher water soluble protein content.Soluble sugar was positive related with cold resistance，and could be used as identification indices for cold resistance of tea plant;MDA level showed significant difference among different tea varieties，but no regularity with cold resistance，therefore，it couldn't be used as identification indices for cold resistance.

Early spring frost caused great damages to tea yield，quality and economic benefits，especially for Hangzhou with only one picking season in spring.Along with the exacerbation of frost damage in early spring，selecting tea varieties with cold-resistance，especially in early spring new shoot，was of great application value to resist"late spring clodness".

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