Study On Carbon Assimilation Characteristics Of Pitaya And Its Response To Drought And Low Temperature Stress

Pitaya(Hylocereus)is a typical crassulacean acid metabolism(CAM)plant that is highly resistant to drought but sensitive to low temperature.Because of its high commodity value,drought resistance and inferiority tolerance,it has developed into an important pillar industry for farmers in Guizhou karst mountain areas to get rid of poverty.As a high quality and rare fruit tree,pitaya has been widely cultivated in China,but the related physiological basic research is very weak.Physiological studies on the interaction of pitaya in response to drought and low temperature stress are still lacking.In this study,aiming at the limiting factors of drought and winter low temperature in the karst landforms of Guizhou,the potted ‘Zihonglong' pitaya stems were used as experimental materials to study the growth condition,tissue structure,ultrastructure,antioxidant system,carbon assimilation and carbon metabolism changes under drought and low temperature stress.The physiological effects of drought stress on the response to low temperature of pitaya were explored,and the transcriptomic changes were also analyzed to find out the differential expression genes under drought and low temperature stress,verifying the association on the adaptation mechanisms of drought and low temperature in pitaya.At the same time,the effects of light conditions on the response of low temperature stress in the field pitaya were investigated.The main results are as follows:(1)Under severe soil drought(soil moisture content from 80% to 15%),the relative water content in the stem of pitaya has only slightly decreased(from 90% to 80%),indicating that under drought stress the pitaya stem has strong water retention capacity.This characteristic was related to the structural changes of pitaya under drought stress,including the increased of multiple epidermis thickness,vascular bundle,number of water storage cells and ratio of cortex to stem thickness.This structural alteration not only increased the transpiration resistance of pitaya,but also strengthened the storing and transporting abilityof water within plants.(2)Under drought stress,the pitaya's growth rapidly ceased crosswise(stopping watering for 4 weeks),and the elongation growth was only weakened under severe drought(12 weeks after stopping watering).The drought treatment significantly promoted the new root growth and elongation.After 8 weeks of stopping watering,the chlorophyll content in pitaya stem was significantly reduced.The photochemical quenching q P value was lower than the control at 4 to 16 weeks after stopping watering,and the Fv/Fm value and ?PSII were significantly decreased at 12 weeks after the watering was stopped.All results were showed that the chlorophyll content in the stem and the photochemical efficiency of photosystem II(PSII)decreased under drought,but PSII only suffered damage under severe drought.Drought treatment significantly reduced contents of starch and soluble sugar,indicating that drought caused a decrease in photosynthetic capacity,but did not trigger osmotic adjustment in the form of sugar accumulation.Drought treatment inhibited the activities of CAT,POD and SOD in the stem of pitaya,and decreased the content of GSH,but significantly increased the activity of APX and the content of ASA.We speculated that the pitaya scavenge reactive oxygen species mainly dependent on the As A-GSH circulatory system.(3)The stomata in pitaya of the well-watered and drought treatment both showed the circadian rhythm with closing during the day and opening at night,but the open frequency and stomatal apertura at each time in the control were higher than those in the drought treatment.Both soluble sugar and starch accumulated during the day and decreased at night,but the drought treatment was significantly lower.The chloroplast ultrastructure showed diurnal variation: the starch was numerous and large during the day,and decreased at the nighttime;liposomes increased at night and decreased at daytime,but the change of the granules was opposite to that of the liposome.The drought treatment lead to reduction in starch granules,increase in liposomes,and decrease in basal density.The diurnal variation rhythm of malic acid and citric acid in the stem of pitaya was exactly opposite to the circadian rhythm of starch and sugar.The organic acid content in the drought treatment was significantly reduced,and the fluctuation between day and night was also significantlyreduced.The PEPC enzyme activity in the stem was low at night and high during the day,while NADP-ME and RUBPC activities showed opposite results.Drought significantly reduced activities of PEPC and NADP-ME.These results indicated that under drought conditions,the carbon assimilation ability of pitaya was significantly decreased,but drought stress did not affect the circadian rhythm associated with carbon assimilation.Under the conditions of sufficient water and drought,CAM metabolism mode was dominant.Therefore,pitaya is a non-facultive CAM plant.(4)After the whole low temperature period and temperature recovery in spring,the chilling injury symptoms and mortality of the normal watering control plants were significantly higher than those under drought stress,and this difference was also reflected in the physiological index of low temperature injury(membrane leakage rate,REC,MDA and ROS content).The degree of low temperature damage was closely related to temperature,time and range of temperature rising up.The membrane permeability was significantly increased within 7 days below 0°C and within 14 days at 4°C,but not significantly increased within 14 days above 8°C.After adaptation at low temperature of5°C for 2 weeks,the higher the temperature rise up,the greater the damage degree was.When the recovery range was above 10°C,the damage was obvious: the membrane permeability and MDA content increased significantly.The above-mentioned low temperature and temperature change treatments were significantly less harmful to plants under drought stress than to the control plants.This effect may be significantly associated with that the drought treatment increased Pro content,APX enzyme activity and ASA content in the oxidative protection system,together with significantly reducing the GSH and superoxide anion content.Therefore,drought treatment can improve the cold tolerance of pitaya,which is related to the improvement of osmotic regulator(Pro)and the activity of As A-GSH circulating active oxygen scavenging system.(5)Under low temperature in winter,shading treatment significantly improved the cold tolerance of pitaya and reduced the symptoms of chilling damage.Compared with the control,shading treatment increased chlorophyll content and maximum photochemical efficiency(Fv/Fm),promoted the accumulation of Pro and ASA,increased APX activity,and decreased superoxide anion accumulation and membrane lipid peroxidation product,MDA,accumulation.(6)18 samples of Pitaya stem from six treatments(three replicates per treatment):No.1(room temperature and watering),No.2(room temperature and drought),No.3(low temperature and watering),No.4(low temperature and drought),No.5(watering after backing to room temperature from low temperature)and No.6(drought treatment after backing to room temperature from low temperature).After sequencing and assembling,a total of 187,530 transcripts and 88,568 unigenes were obtained.Aligning with 8 databases(Nr,GO,KEGG,COG,Swissprot,KOG,Pfam,egg NOG),35,490 unigene were annotated.Differential expression analysis indicated that there were 11,340 unigene showing differential expressed patterns,including between No.2(‘room temperature and drought')and No.4(‘low temperature and drought'),and between No.1(‘room temperature and watering')and No.3(‘low temperature and watering').A large number of unigenes were enriched in ‘response to stimulus',‘biological regulation' and ‘immune system process' of biological processes.In addition,as to regulatory gene family members in response to stress,such as ‘CBF'(8 unigenes),‘NAC'(35 unigenes),‘MYB'(66 unigenes),‘DREB'(7unigenes)and ‘AP2'(10 unigenes),expression pattern clustering analysis was performed,and q PCR analysis of selected key genes in response to drought and low temperature showed that LTI,KIN,DREB-1,ERD-1 and ERD-2 genes may play key roles in cold resistance of pitaya stem,while RD-2,APX-3 and APX-4 may play key roles in drought resistance.