Xiong JING,Yongxia BAI,Man SHEN*,Pingsheng LENG 1.Plant Science and Technology College,Beijing University of Agriculture,Beijing 102206,China;2.Key Laboratory of New Technology in Agricultural Application of Beijing,Beijing University of Agriculture,Beijing 102206,China;.Landscape Architecture College,Beijing University of Agriculture,Beijing 102206,China

Chrysanthemum is a perennial flower ofDendranthema,Asteraceae,and ground-cover chrysanthemum (Dendranthema×grandiflorumKitamura)is a new variety group in chrysanthemum family,which was bred by Professor Chen Junyu from Beijing Forestry University in the 1980s[1].It has dwarf plant,long florescence,close flower shape and strong stress resistance,and is able to bear extensive cultivation,So this mum variety group is used well as groundcover plant.In recent years,the researches about ground-cover chrysanthemum mainly focus on introduction,breeding,cultivation[2-4],drought resistance[5-6]and saline alkali resistance[7],and there are few reports about cold resistance.The ground-cover chrysanthemum ‘Zixunzhang’was used as the experimental material in this study.By determining physiological and biochemical indexes of leaves,emergent rhizomesand rootsfrom ‘Zixunzhang’which grew in the open field during overwintering period,we tried to analyze correlation between cold tolerance of ground-cover chrysanthemum and these physiological and bio-chemical indexes,which could provide test proof for correctly identifying cold tolerance of different varieties.

Materials and Methods

Test materials

The ground-cover chrysanthemum ‘Zixunzhang’ was used as the experimental material in this study,which was planted in open field of nursery of Science and Technology Park,Beijing University of Agriculture in spring of 2011.From September of 2011 to May of 2012,leaves and emergent rhizomes of ground-cover chrysanthemum were sampled every 15 days,while roots were sampled every 30 days.Relative water content,water content,root activity,soluble protein content and activities of three protection enzymes(POD,SOD and CAT)from the fresh materials were measured,and samples used for measuring other related indexes were conserved at-80℃.

Test and data analysis methods

Referring to the methods of Li Hesheng et al.[8-9],the below indexes were determined.Relative water content and water content were determined by drying method;SOD enzyme activity measurement used NBT photo reduction method;POD enzyme activity measurementused guaiacol method;CAT enzyme activity measurement used ultraviolet absorption method;soluble sugar content was determined by anthrone method;soluble protein content was determined by Kaumas blue staining method;proline content measurement used acid three indene keton colorimetric method;root activity was determined by TTC method.Variance analysis and correlation analysis of test data were conducted by Microsoft Excel and SPSS 17.0 software.

Results and Analysis

Changes of atmosphere temperature and surface temperature in open cultivation field during overwintering period

According to atmosphere temperature every day from September of 2011 to May of 2012 recorded by weather forecast,monthly average maximum,minimum and average atmosphere temperature were obtained(Fig.1).Right angle thermometer inserting into underground 20 cm was used to record surface temperature,and monthly average minimum,maximum and average temperature at underground 20 cm were obtained(Fig.2).Seen from Fig.1,atmosphere temperature continuously declined in winter.Average minimum atmosphere temperature in December was-5.4℃,and daily minimum atmosphere temperature reached-10℃.To January,atmosphere temperature reached the minimum.Monthly average minimum atmosphere temperature in January reached-8.3℃,and daily minimum atmosphere temperature was-13℃,which then gradually rose.Seen from Fig.2, surface temperature also reached the minimum in January,and monthlyaverageminimum surface temperature in January was-3.4℃,which then gradually rose.

Morphological changes of leaves,emergent rhizomes and roots from‘Zixunzhang’growing in open field during overwintering period

During overwintering period,morphological changes of leaves,emergent rhizomes and roots from‘Zixunzhang’were shown as Fig.3.Leaves of‘Zixunzhang’ still maintained green in the end of October,but basically withered after early frost in middle dekad of November.Emergent rhizomes sprouting tillers still grew in middle dekad of November,and some emergentrhizomesstillmaintained green after snowfall in December.But as low temperature below zero celsius continued to middle dekad of December,emergent rhizomes at rhizosphere basically died,which germinated again until next April.Roots always maintained activity during whole overwintering period.Although lateral roots had black and dead phenomenon during January-February,most of principal roots still maintained white or light brown.Seen from morphological observation results,it seems that cold tolerance of ground-cover chrysanthemum mainly depended on the vitality of root system.

Changes ofroots activity from‘Zixunzhang’mum which grew in open field during the overwintering

Perennial flower mainly depends on roots activity to overwinter in open field.Thence,vitality state of plant roots can reflect cold tolerance of plant in certain degree[10].Seen from Fig.4,as temperature slowly declined,vitality value of roots slowly declined before December 23.But vitality value of roots fell after significantly rose in January when temperature was the minimum and on March 18 when temperature abruptly declined,and increase magnitudes were 19.21% and 81.18% respectively.It was suggested that maybe prior slow temperature reduction made roots obtain certain lowtemperature domestication,thereby causing that low temperature in short time stimulated root vitality rising.

Changes of water content of‘Zixunzhang’mum which grew in open field during overwintering process

Water content of plant tissue declines in late autumn and early winter,which is conducive to accumulation of dry matter and improvement of cold resistance[11].Seen from Table 1,as winter temperature declined,relative water contents of aboveground part and water content of underground part from ‘Zixunzhang’ all presented declining tendency,and gradually rose again after next spring.Relative water contents of leaves and emergent rhizomes reached the minimum before withered.Water content of root started to slowly rise after reaching the minimum on January 15,in which water content of root abruptly rose on December 23.It was speculated that maybe snowfall on December 2 increased soil water content,causing water content of root rose in short time.

Table 1 Changes in water contents of leaves,emergent rhizomes and roots of‘Zixunzhang’mum during the overwintering %

Table 2 Changes in SOD activities of leaves,emergent rhizomes and roots of‘Zixunzhang’mum during the overwintering U/g

Changes of protection enzyme activity of ‘Zixunzhang’mum during the overwintering

As temperature declined,SOD and CAT enzyme activities of leaves presented rise-decline tendency(Table 2,Table 3),while POD enzyme presented decline-rise tendency(Table 4).SOD enzyme activity significantly rose on October 15,and increase magnitude reached 309.81%,while POD enzyme activity significantly declined at this time,and decline magnitude was 32.28%.Both SOD and CAT enzyme activities declined after rising to the maximum on October 30,while POD rose after declining to the minimum on October 15.As temperature declined,enzyme activity of leaves had significant difference.

Both SOD and CAT enzyme activities of emergent rhizomes overall presented rise-decline-rise tendency,while POD presented decline-rise tendency.SOD enzyme activity significantly declined on December 2,and decline magnitude reached 39.90%.CAT enzyme activity significantly rose on October 30,with increase magnitude of 46.77%,then gradually declined,and reached the minimum on December 2.POD significantly declined on October 30,with decline magnitude of 58.92%,then gradually rose,and reached the maximum on December 2.After next spring,each enzyme activity gradually rose and recovered normal.Seen from the above analysis,abrupt temperature reduction on December 2 had larger impact on enzyme activity change of emergent rhizomes.

SOD and CAT enzyme activities of leaves and emergent rhizomes had consistent change rule,while POD had contrary rule.It was speculated that maybe the three protective enzymes alternately played role under low temperature,to commonly resist the impact of low temperature on plant metabolism.

SOD enzyme activity of root significantly declined on December 2,and decline magnitude reached 71.39%.CAT enzyme activity significantly rose on January 3,with increase magnitude of 454%,and significantly declined on March 30.POD activity reached the maximum on March 30,and increase magnitude was 150.94%.It was clear thatenzyme activity ofrootonly changed greatly in January and when temperature abruptly declined,and total change tendency during whole overwintering period was not very significant.

Changes of osmotic adjustment substance of ‘Zixunzhang’ mum during the overwintering

Seen from Table 5 to Table 7,as variation of winter outdoor temperature,soluble sugar of leaves accumulated,while soluble protein and proline content presented declining tendency.Before leaves withered,its soluble protein and soluble sugar reached the minimum and maximum respectively.Proline content significantly declined on October 15,with decline magnitude of 77.55%.

Soluble protein and proline content of emergent rhizomes overall presented decline-rise-decline-rise tendency,and soluble protein reached the maximum on November 29.Proline content significantly declined on October 30,with decline magnitude of 66.53%,and reached the maximum on December 2.Soluble sugar content presented rise-decline-rise-decline tendency,and significantly rose on October 30,with increase magnitude of 280.96%,and reached the maximum on December 2.After next spring,content of each osmotic adjustment substance of emergent rhizomes gradually recovered to the October level.

Seen from the above analysis,soluble protein and proline of leaves and emergent rhizomes had consistent variation,and they had contrary change tendency with that of soluble sugar content.The common point was that osmotic adjustment substances reached the extreme value before plant withered and temperature was the lowest.

Soluble protein content of root changed stably,and overall presented rise-decline tendency.Soluble protein content of root reached the maximum(31.61 mg/g)on December 2,and had transient increase on March 30.It was speculated that it was caused by abrupt temperature reduction on March 18.Soluble sugarcontent reached the maximum (71.79 mg/g)on January 3,and then gradually declined,in which it significantly rose on November 20,with increase magnitude of 389.51%.Change of proline was not stable,and it significantly rose on November 20,December 23 and February 22.As temperature declined,osmotic adjustment substances from root all increased somewhat.

Correlation analysis among the measured indexes of‘Zixunzhang’mum

Table 8 showed correlation coefficients among the measured indexes in natural temperature reduction process.Seen from Table 8,water content and relative water content presented significantly positive correlation,while POD and SOD presented extremely significant positive correlation.Soluble protein presented extremely significant positive correlation with water content,and extremely significant negative correlation with SOD and POD.Soluble sugar presented extremely significant negative correlation with relative water content and water content,and extremely significant positive correlation with SOD.Proline presented significantly negative correlation with relative water content,and extremely significant positive correlation with SOD.Root activity presented extremely significant positive correlation with relative water content and CAT,and significantly negative correlation with soluble sugar and soluble protein.Seen from the above analysis,correlation among relative water content,SOD,soluble protein,soluble sugar and root activity was higher,followed by that among water content,proline,POD and CAT.

Discussion

Change of the plant root function is bound to affect physiological characteristics of aboveground leaves[12].Root activity can change under temperature.In the research,it was found that root activity slowly declined inslow temperature reduction process during September-October.But in January when the temperature was the lowest and latter period of winter when temperature abruptly declined after rise,root activity had falling phenomenon after transient rise.It illustrated that certain low temperature could cause decline of root activity,but slow temperature reduction might make roots obtain low temperature domestication at certain degree.When encountering low temperature with larger reduction magnitude,protective enzyme activity in plant roots was activated because of low temperature domestication within certain low temperature range,thereby causing that root activity rose.

Table 3 Changes in CAT activities of leaves,emergent rhizomes and roots of‘Zixunzhang’ mum during the overwintering U/(g﹒min)

Table 4 Changes in POD activities of leaves,emergent rhizomes and roots of‘Zixunzhang’ mum during the overwintering U/(g﹒min)

Table 5 The changes in soluble protein contents of leaves,emergent rhizomes and roots of‘Zixunzhang’ mum during the overwintering mg/g

Table 6 The changes in soluble sugar contents of leaves,emergent rhizomes and roots of‘Zixunzhang’mum during the overwintering mg/g

When water content of plant declines,the amount of intracellular osmotic matter relatively increases,which can increase cell concentration,decline osmotic potential,make freezing possibility decline,and enhance the ability of resisting coldness and preventing freeze[13].In the research,it was found that relative water content of aboveground part and water content of underground part overall presented declining tendency in overwintering process,which may be conducive to plant resisting the injury of low temperature.The conclusion was consistent with research results of Bao Siweiet al[14-15].

When plant suffers cold injury,some enzymes andnonenzymes cleaning free radicals and reactive oxygen in plant can regulate membrane permeability and increase structure and function stability of membrane,to protect plant cell from injury[16].Under low temperature,SOD,CAT and POD enzyme activities in plant are directly related to plant’s ability resisting low temperature injury[17].In the research,it was found that SOD and CAT enzyme activities of leaves and emergent rhizomes overall presented the tendency of first rising and then declining,while POD presented the tendency of first declining and then rising.It was suggestedthatthese three enzymes maybe alternately play role.Enzyme activity of root had small difference during whole overwintering period except at several points with larger temperature reduction magnitude.It was speculated that low temperature had smaller impact on these protective enzymes in the root.

In plant metabolism process,plant can ease the injury of low temperature and regulate cell osmotic equilibrium by accumulating or decomposing osmotic adjustment substances[16].The test result found that leaves could accumulate a lot of soluble sugar,while soluble protein and proline content overall presented declining tendency.It was speculated that when leaves entered into the aging stage,decomposing velocity was larger than synthesis velocity.Maybe it was caused by these matters transferring from aboveground part to underground part.Total tendency displayed that contents of osmotic adjustment substances of emergent rhizomes and roots overall presented rise-decline tendency,showing that winter low temperature induced emergent rhizomes and roots synthesizing a lot of osmotic adjustment substances to improve their own cold resistance.

For ground-cover chrysanthemum,its physiological and biochemical indexes changed at the amountduring overwintering period,which was related to low temperature.According to the result of correlation analysis method,it was initially judged that relative water content,SOD,soluble protein,soluble sugar,root system activity of ground-cover chrysanthemum were closely related to its cold resistance,followed by water content,proline,POD and CAT.

Table 7 The changes in proline contents of leaves,emergent rhizomes and roots of‘Zixunzhang’mum during the overwintering mg/g

Cold tolerance of ground-cover chrysanthemum is affected by integrated effect of its physiological and biochemicalcharacteristics,and its cold resistant mechanism is complicated[18].It is not accurate to judge cold resistance of ground-cover chrysanthemum only by single cold resistance index.Therefore,it has important actual significance for identifying cold resistance of ground-cover chrysanthemum by multiple physiological and biochemical indexes.

Table 8 Relative coefficient between each pair of indexes of‘Zixunzhang’ mum during the overwintering

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