Molecular Mechanism Underlying Plant Response to Cold Stress

Low temperature stress is one of the most important factors limiting plant growth and geographical distribution.In order to adapt to low temperature,plants have evolved strategies to acquire cold tolerance,known as,cold acclimation.Current molecular and genomic studies have reported that annual herbaceous and perennial woody plants share similar cold acclimation mechanisms.However,woody perennials also require extra resilience to survive cold winters.Thus,trees have acquired complex dynamic processes to control the development of dormancy and cold resistance,ensuring successful tolerance during the coldest winter season.In this review,we systemically described how woody plants perceive and transduce cold stress signals through a series of physiological changes such as calcium signaling,membrane lipid,and antioxidant changes altering downstream gene expression and epigenetic modification,ultimately bud dormancy.We extended the discussion and reviewed the processes endogenous phytohormones play in regulating the cold stress.We believe that this review will aid in the comprehension of underlying mechanisms in plant acclimation to cold stress.

Functional characterization of MdMYB73 reveals its involvement in cold stress response in apple calli and Arabidopsis

Recent studies have shown that the R2R3-MYB transcription factor MdMYB73 is involved in salt stress response in apple. However, no research was done whether MdMYB73 mediated cold tolerance in apple or not. In this study, we found that the expression of MdMYB73 was obviously induced by cold stress. Functional analysis showed that MdMYB73 significantly increased cold tolerance in transgenic apple calli and Arabidopsis. Quantitative real-time PCR （qRT-PCR） assay indicated that the expression levels of cold-responsive genes including MdCBF2, MdCBF3, MdCBF4, and MdCBF5 were obviously enhanced in MdMYB73 transgenic calli, suggesting that MdMYB73 increased cold tolerance via C-repeat binding factor （CBF） cold response pathway. Finally, we found that soluble sugar, which provides an osmoticum for cells, was increased in MdMYB73 transgenic calli compared to that in the wild type control. These findings provide a new insights into how MdMYB73 is involved in cold stress response.

Effects of Acute and Chronic Cold Stress on Antioxidant Function in Intestinal Tracts of Chickens

This study was to investigate the effects of cold stress on the contents of total antioxidant capacity （T-AOC） malondialdehyde （MDA） and superoxide dismutase （SOD） in duodenum, jejunum and ileum of chickens. A total of 80 15-dayold male chickens were treated by cold stress with the duration of the acute cold stress being 1, 3, 6, 12, and 24 h, and the chronic cold stress was 5, 10, and 20 days, respectively. Cold stress temperature was （12±1）℃. The chemical colorimetric method was used to detect the changes of the T-AOC, SOD activities and MDA contents. The results showed that compared with the corresponding control group, effects of acute cold stress on the T-AOC in duodenum, jejunum and ileum of chickens significantly （P〈0.05） increased firstly and then decreased. Under chronic cold stress, the T-AOC significantly （P〈0.05） decreased. Under acute cold stress and chronic cold stress, the MDA contents significantly （P〈0.05） increased in duodenum, jejunum and ileum of chickens. The effects of acute cold stress on the SOD activities in duodenum, jejunum and ileum of chickens significantly （P〈0.05） increased firstly and then decreased Under chronic cold stress the SOD activities significantly （P〈0.05） decreased in jejunum and ileum, but significantly （P〈0.05） decreased firstly and then increased in duodenum.

p38 MAPK is a Component of the Signal Transduction Pathway Triggering Cold Stress Response in the MED Cryptic Species of Bemisia tabaci

Cold stress responses help insects to survive under low temperatures that would be lethal otherwise.This phenomenon might contribute to the invasion of some Bemisia tabaci cryptic species from subtropical areas to temperate regions.However,the molecular mechanisms regulating cold stress responses in whitefly are yet unclear.Mitogen-activated protein kinases（MAPKs）which including p38,ERK,and JNK,are well known for their roles in regulating metabolic responses to cold stress in many insects.In this study,we explored the possible roles of the MAPKs in response to low temperature stresses in the Mediterranean cryptic species（the Q-biotype）of the B.tabaci species complex.First,we cloned the p38 and ERK genes from the whitefly cDNA library.Next,we analyzed the activation of MAPKs during cold stress in the Mediterranean cryptic species by immuno-blotting.After cold stress,the level of phospho-p38 increased but no significant change was observed in the phosphorylation of ERK and JNK,thus suggesting that the p38 might be responsible for the defense response to low temperature stress.Furthermore,we demonstrated that：i）3 min chilling at 0°C was sufficient for the activation of p38 MAPK pathway in this whitefly;and ii）the amount of phosphorylated p38 increased significantly in the first 20 min of chilling,reversed by 60 min,and then returned to the original level by 120 min.Taken together,our results suggest that the p38 pathway is important during response to low temperature stress in the Mediterranean cryptic species of the B.tabaci species complex.

Genetic Control of Germination Ability under Cold Stress in Rice

An F9 recombinant inbred lines （RIL） population, derived from a cross between IR28 （Oryza sativa L. spp. indica） and Daguandao （O. sativa L. spp. japonica）, was used to construct a molecular linkage map and to identify germination ability including the traits of imbibition rate, germination rate, germination index, root length, shoot length and seed vigor at 14℃ for 23 d. A composite interval mapping approach was applied to conduct genetic analysis for germination ability. The frequency distributions of the germination ability traits under the cold stress in the RIL population showed continuous segregation, suggesting they were quantitative traits controlled by several genes. A total of seven QTLs were identified on chromosomes 4 6 and 9, including two for imbibition rate （qIR-6, qIR-9）, one for germination rate （qGR-4）, two for germination index （qGI-4-1, qGI-4-2） and two for root length （qRL-4-1, qRL-4-2）. There were no detected QTLs controlling shoot length and seed vigor. The phenotypic variance explained by a single QTL ranged from 9.1% to 37.0%, and two major QTLs, qlR-6 and qGI-4-2, accounted for over 30% of the phenotypic variance. The expressions of QTLs were developmentally regulated and growth stage-specific. Most of the QTLs observed here were located in the regions similar to the QTLs for rice cold tolerance reported previously, indicating that these QTLs were reliable. However, qRL-4-2 is not reported before.

Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis

Eugenol is a natural phenolic compound known for its health-promoting properties and its ability to add a floral scent to tea plants.Plant eugenol glycosides have been identified and shown to make important contributions to fruit floral quality.However,the details of their biosynthesis and metabolism in tea plants are still unknown.Here,eugenol glucoside was unambiguously identified as a native metabolite in the tea plant,and its biosynthesis was shown to be induced by low temperature treatment.Through the analysis of UGTs induced by low temperature,the glycosyltransferase CsUGT78A15 was identified in tea,and its encoded protein was shown to catalyze the glucosylation of eugenol.Vmax/Km ratios showed that eugenol was the most suitable substrate for CsUGT78A15.Sugar donor preference analysis showed that CsUGT78A15 had a higher selectivity for glucose,followed by galactose and glucuronic acid.The expression of CsUGT78A15was correlatedwith the accumulation of eugenol glucoside in different tissues and genotypes of tea.Down-regulation of CsUGT78A15 led to a decreased eugenol glucoside content under cold stress,indicating that CsUGT78A15 plays an important role in the biosynthesis of eugenol glucoside under cold stress.The identification of eugenol glucoside in the tea plant and the discovery of a cold stress-induced eugenol glucosyltransferase in tea provide the foundation for the improvement of tea flavor under cold stress and the biotechnological production of eugenol glucoside.

Genome-wide identification and cold stress-induced expression analysis of the CBF gene family in Liriodendron chinense

Cold-resistance pathways that operate in model plants such as Arabidopsis thaliana and Oryza sativa have been studied extensively.It has been found that CBF genes play an important role in plant cold resistance.Liriodendron chinense,a tree known for its graceful tree shape and widely spread in south China,has weak cold tolerance.However,little is known about its response to cold.To further study the function of L.chinense CBF gene family,we started by characterizing all members of this gene family in the L.chinense genome and their expression profiling.Phylogenetic analysis found that 14 CBF genes in L.chinense are more closely related to their homologues in woody plants and A.thaliana than those in O.sativa.Cis-acting elements and GO analysis showed that some LcCBF genes participated in the biological process of cold stress response.The transcriptomic and RT-qPCR data showed that most of LcCBF genes displayed an initially increasing and subsequently decreasing trend during cold stress course and the expression profile of each member was different.Some LcCBF genes exhibited a different abundance in callus,root,stem and leaf tissues.The structure and expression characteristics of LcCBF genes imply that they may have similar and different functions in response to cold stress conditions.The identification and analysis of LcCBF gene family have laid the foundation for future studies into L.chinense cold stress mechanisms and for the cultivation of cold-resistance cultivars.

Heterologous Expression of Camellia sinensis Late Embryogenesis Abundant Protein Gene 1(CsLEA1)Confers Cold Stress Tolerance in Escherichia coli and Yeast

Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.

Differences in biochemical responses to cold stress in two contrasting varieties of rape seed (Brassica napus L.)

Changes in isozymes of peroxidase, catalase, superoxide dismutase, polyphenol oxidase, and esterase and activities of per- oxidase and polyphenol oxidase in two annual Brassica varieties （AS-3 and CON, cold-tolerant and cold-sensitive, respectively） after 4℃ treatment were assayed. After the low temperature treatment, PAGE patterns of isozymes of peroxidase, catalase, superoxide dismutase, polyphenol oxidase, and esterase showed that the expressions and activities of these enzymes in the AS-3 variety were higher than the CON variety. Under normal and cold conditions, peroxidase and esterase activities of AS-3 were higher than those of CON. The relative freezing tolerance of these two cultivars was discussed.

Effects of cold stress on hypothalamic corticotrophin-releasing and thyrotropin-releasing hormone messenger RNA levels in chickens

There is increasing evidence that stress can activate the hypothalamic-pituitary-adrenal-axis and hypothalamic-pituitary- thyroid-axis, and further affect the synthesis and secretion of corticotrophin-releasing hormone （CRH） and thyrotropin-releasing hormone （TRH）. To evaluate the effect of cold stress on the hypothalamic CRH and TRH messenger RNA （mRNA） levels in Yisha chickens, male Yisha chickens were subjected to acute （1, 6, 12 h） and chronic （5, 10, 20 d） cold stress （12±1）℃. Hypothalami were collected for assessment of mRNA levels by semi-quantitative RT-PCR. Acute stress resulted in a significant decrease of CRH mRNA levels at 6 and 12 h, and a significant increase of TRH mRNA levels at every stress time point. Chronic cold stress resulted in a significant increase of CRH mRNA levels and a significant decrease of TRH mRNA levels compared with the control group at every stress time point. The results suggest that the two genes differently respond to cold stress at the mRNA levels. And the different degrees of cold stress will produce different effects on the identical gene.

Overexpression of Arabidopsis thaliana malonyl-CoA synthetase gene enhances cold stress tolerance by activating mitogen-activated protein kinases in plant cells

Malonyl-CoA synthetases may modulate cell responses to abiotic stress by regulating stress-related signaling transduction pathways or activating expression of transcription factors.However,the molecular mechanism of cold stress tolerance enhanced by malonyl-CoA synthetase is not fully understood.Here,we report that overexpression of the Arabidopsis thaliana malonyl-CoA synthetase gene AAE13.1 resulted in increased cell viability and growth rate and decreased thiobarbituric acid reactive substances under cold stress in rice(Oryza sativa L.),tobacco(Nicotiana tabacum),and slash pine(Pinus elliottii Engelm.).AAE13.1 was associated with cold stress tolerance by increasing the activity of ascorbate peroxidase,catalase,polyphenol oxidase,and peroxidase and the accumulation of acid phosphatase and alkaline phosphatase.Among six rice mitogenactivated protein kinase(MAPK)genes examined,AAE13.1 overexpression increased the expression of OsMAPK genes during cold stress.AAE13.1 activated expression of stressresponse genes OsMAPK1,OsMAPK2,and OsMAPK3,indicating that AAE13.1 enhances cold stress tolerance by regulating expression of MAPK genes in plant cells.These results increase our understanding of cold stress tolerance in species of monocotyledons,dicotyledons,and gymnosperms.

Physiological Responses of Dendrobium officinale under Exposure to Cold Stress with Two Cultivars

This study aimed to explore the cold tolerance of two cultivars of Dendrobium officinale(MG1,MG2)grown in different regions of China.Under-2℃ incubation,cultivar MG1 remained active after 3 d,and continued to grow after returning to room temperature.However,MG2 could only maintain its activity after 2 d treatment at−2℃,and the seedlings died with the low temperature treatment time.Investigation of the characteristics of the plants grown in the south(Hangzhou)or north(Zhengzhou)of China indicated that the leaves of MG1 also had reduced stomatal density,the highest thickness,and a compact microstructure.The contents of proline and soluble sugars were higher in MG1 than those in MG2.The cultivar MG1 had higher SOD enzyme activity than MG2,while CAT and POD activities in samples from Zhengzhou were higher than those from Hangzhou.The contents of polysaccharides and alkaloids in stems of in MG1 were higher than those in MG2,while the content of flavonoids in the Zhengzhou samples was higher than that in the Hangzhou samples.In addition,plant heights,stem diameters,and chlorophyll content were higher in MG1.Overall,MG1 had better cold resistance than MG2.MG1 is a cold tolerant cultivar with thick leaves and reduced stomatal density,higher contents of soluble sugars,proline,CAT,POD,polysaccharides,flavonoids and alkaloids,which together make it more adaptable to low temperatures.Thus,the cultivar MG1,with its demonstrated cold tolerance,can accordingly be grown on a large scale in cold regions,thereby expanding the available planting area for this important traditional medicinal plant to meet the increasing commercial demand for it.

Therapeutic potential of gasotransmitters for cold stress-related cardiovascular disease

Growing evidence has shown that exposure to low ambient temperature poses a huge challenge to human health globally.Actually,cold stress is closely associated with a higher incidence of cardiovascular morbidity and mortality in winter or in cold regions.Cellular and molecular mechanisms underlying cardiovascular complications in response to cold exposure have yet to be fully clarified.Considering that cold exposure is an important risk of cardiovascular complications,it is necessary to clarify the molecular mechanism of cold stress-induced cardiovascular diseases and to develop effective intervention strategies.Hydrogen sulfide(H2S),nitric oxide(NO),and carbon monoxide(CO)are well-known gasotransmitters that are endogenously produced in many biological systems.Accumulating studies have demonstrated that these gasotransmitters play a critical role in a wide spectrum of physiological and/or pathophysiological processes by regulating numerous signaling pathways.These gas signal molecules are emerging as important players in cardiovascular homeostasis,and disruption of these gasotransmitters is critically implicated in cardiovascular anomalies,such as hypertension,atherosclerosis,myocardial ischemia,heart failure,and stroke.Also,evidence is emerging that H2S,NO,and CO may be involved in the pathologies of cold stress-induced cardiovascular ailments.In this review,we aim to highlight and discuss the recent advances towards the development of gasotransmitters-based therapeutics for cold stress-related cardiovascular pathogenesis.We believe that the effects of H2S,NO,and CO on cardiovascular regulation under cold environment will attract tremendous interest in the near future as they serve as novel regulators of cardiovascular biology in cold environment.

Therapeutic effect of bone marrow mesenchymal stem cells on cold stress induced changes in the hippocampus of rats

The present study aims to evaluate the effect of bone marrow mesenchymal stem cells on cold stress induced neuronal changes in hippocampal CA1 region of Wistar rats. Bone marrow mes- enchymal stem cells were isolated from a 6-week-old Wistar rat. Bone marrow from adult femora and tibia was collected and mesenchymal stem cells were cultured in minimal essential medium containing 10% heat-inactivated fetal bovine serum and were sub-cultured. Passage 3 cells were analyzed by flow cytometry for positive expression of CD44 and CD90 and negative expression of CD45. Once CD44 and CD90 positive expression was achieved, the cells were cultured again to 90% confluence for later experiments. Twenty-four rats aged 8 weeks old were randomly and evenly divided into normal control, cold water swim stress （cold stress）, cold stress ＋ PBS （intra- venous infusion）, and cold stress ＋ bone marrow mesenchymal stem cells （1 x 106; intravenous infusion） groups. The total period of study was 60 days which included 1 month stress period followed by 1 month treatment. Behavioral functional test was performed during the entire study period. After treatment, rats were sacrificed for histological studies. Treatment with bone marrow mesenchymal stem cells significantly increased the number of neuronal cells in hippocampal CA 1 region. Adult bone marrow mesenchymal stem cells injected by intravenous administration show potential therapeutic effects in cognitive decline associated with stress-related lesions.

Effects of Heat Shock Factor AtHsfA1a on Programmed Cell Death in Arabidopsis thaliana under Cold Stress

[Objective] This study aimed to investigate the effects of heat shock factor AtHsfAla on programmed cell death in Arabidopsis thaliana under cold stress. [ Method] AtHsfAla-silenced transgenic （NT） and wild-type （WT） A. thaliana seedlings were used as experimental materials to induce the formation of callus; the callus were cultured to single cells by suspension culture, subjected to cold stress, stained with DAPI, prepared into cell smears and observed under a fluorescence microscope. [ Result] Under cold stress, cell nucleus of wild-type A. thaliana displayed morphological changes, but no apoptotic bodies were found; apoptotic bodies were observed in AtHsfAla-silenced transgenic A. thaliana cells, and the cytoplasm was remarkably concentrated. [ Conclusion] Under cold stress, heat shock factor AtHsfAla exerted inhibitory effects on programmed cell death in A. thaliana, which was of great significance for clarifying the mechanism of stress responses in plants.

Antioxidant metabolic system and comparative proteomics analysis in winter turnip rape(Brassica rapa L.)under cold stress

Winter turnip rape(Brassica rapa L.)is widely cultivated in winter in Northwest China,however,its cold-tolerant mechanism remains insufficiently understood.In this study,winter turnip rape cultivar Longyou 7,a cold-tolerant variety,was used as material,whose accumulation of H_(2)O_(2) and O_(2)^(·-),antioxidant enzyme activity as well as differences in protein expression based on two-dimensional gel electrophoresis(2-DE)technique under -4℃ stress were analyzed.Results showed that,production of H_(2)O_(2) and O_(2)^(·-) were increased in Longyou 7 leaves,simultaneously,SOD and POD activities were also obviously rosed up,but the activities of CAT and APX were gradually reduced with the temperature.Thirty-six differential protein spots were successfully identified between control and treatments group by using mass spectrometry analysis.Among them,4 differential protein spots were induced under cold stress,and 2 were inhibited at-4℃.Functional analysis found that these identified proteins mainly participated in photosynthesis,carbohydrate metabolism,defense,protein synthesis,enzyme activity,redox and membrane metabolism,respectively.Additionally,13 proteins'function were still unknown.In conclusion,strong antioxidant capacity and cell defense ability might play important roles in Longyou 7 response to cold stress.

Effect of aging on cardiovascular responses to cold stress in humans

Cold exposure increases the risk of adverse events related to cardiovascular causes,especially in the elderly.In this review,we focus on recent findings concerning the impact of aging on the regulatory mechanisms of cold-induced cardiovascular responses.In response to cold exposure,the initial physiological thermoregulation in healthy young persons,such as cutaneous vasoconstriction to reduce heat loss,is attenuated in older individuals,resulting in a reduced ability of the older persons to maintain body temperature in cold environment.Impaired sympathetic skin response,reduced noradrenergic neurotransmitter synthesis,insufficient noradrenergic transmitters,and altered downstream signaling pathways inside the vascular smooth muscle may be among the underlying mechanisms for the maladaptive vasoconstrictive response to cold stress in the elderly.The increase in blood pressure during cold exposure in young persons may be further augmented in aging adults,due to greater central arterial stiffness or diminished baroreflex sensitivity with aging.Cold stress raises myocardial oxygen demand caused by increased afterload in both young and old adults.The elderly cannot adjust to meet the increased oxygen demand due to reduced left ventricular compliance and coronary blood flow with advancing age,rendering the elderly more susceptible to hypothermia-induced cardiovascular complications from cold-related diseases.These age-associated thermoregulatory impairments may further worsen patients'health risk with existing cardiovascular diseases such as hypertension,coronary artery disease,and heart failure.We searched PubMed for papers related to cold stress and its relationship with aging,and selected the most relevant publications for discussion.

BH3-only protein Bim is involved in myocardial injury induced by co-stress of ischemia and cold stress in rats

Objectives To investigate the effect of co-exposure of myocardial ischemia and cold stress on myocardial injury in rats and the relative mechanism.Methods Myocardial ischemia model was established by ligation of left coronary artery.SD rats were randomly allocated to 4 groups; sham+normal temperature(S group),sham+cold stress(SC group),myocardial ischemia+ normal temperature(Ⅰgroup), myocardial ischemia+cold stress(IC group).On the condition of 26℃,SC and IC groups were keeped in a 4℃artificial chamber for 8h(8;00-16:00) for 4 consecu- tive days.Car diac function was assessed by echocardiography;pathological change was analyzed by HE staining;myocardial infarct size was determined by TTC staining;Bim,Caspase-3 expression in myocardium was determined by western blotting.Results It was demonstrated that co-exposure of myocardial ischemia and cold stress could significantly make the cardiac muscle in abnormal shape,increase the infarct size and the expression of Bim and Caspase-3.Conclusions Co-exposure of myocardial ischemia and cold stress may aggravate the cardiac injury,pro- apoptosis protein Bim is involved.

Study on the physiological indices of Pinus sibirica and Pinus koraiensis seedlings under cold stress

Although Pinus sibirica and Pinus koraiensis are resistant to cold or low temperatures in the cold temperate regions of the northern hemisphere,the former has a stronger cold resistance.Research has been limited to the comparison of physiological responses of the two species to cold stress.In this study,5-year-old seedlings of P.sibirica and P.koraiensis were subjected to six temperature treatments,[20℃(control),0℃,-20℃,-40℃,-60℃,and-80℃],under different stress periods(6,12,24,and 48 h).The results showed that differences in each physiological index were significant between P.sibirica and P.koraiensis,except for the permeability of cell membranes,reactive oxygen species,proline and soluble proteins.An ANOVA test indicated that there were extreme differences among the temperatures for each index,stress time and temperature 9 time for most indices.All indices showed a similar trend for P.sibirica and P.koraiensis with decreasing temperature or the extension of stress time.Soluble sugars and proline increased at 0 to-20℃and then remained unchanged with temperature decline.Other indices showed an increase from 20 to-20℃,stable from-20 to-40℃and a decrease from-40 to-80℃.All the indices increased and then declined along with the prolonged cold stress time,except for the control.From 0 to-40℃,the permeability of cell membranes,relative conductivity,reactive oxygen species and malonaldehyde of P.koraiensis seedlings were higher than in P.sibirica,but superoxide dismutase,peroxidase,catalase activity and soluble sugars,proline,and soluble proteins content emerged as opposite.This study compared the physiological mechanism responses to cold stress between P.sibirica and P.koraiensis to provide the basis for the introduction,distribution,and genetic improvement of these coniferous species.

Effects of acupuncturing Tsusanli (S_T36) on expression of nitric oxide synthase in hypothalamus and adrenal gland in rats with cold stress ulcer

AIM: To study the protective effect of acupuncturing Tsusanli (ST36) on cold stress ulcer, and the expression of nitric oxide synthase (NOS) in hypothalamus and adrenal gland.METHODS: Ulcer index in rats and RT-PCR were used to study the protective effect of acupuncture on cold stress ulcer, and the expression of NOS in hypothalamus and adrenal gland. Images were analyzed with semi-quantitative method.RESULTS: The ulcer index significantly decreased in rats with stress ulcer. Plasma cortisol concentration was up regulated during cold stress, which could be depressed by pre-acupuncture. The expression of NOS1 in hypothalamus increased after acupuncture. The increased expression of NO$2 was related with stress ulcer, which could be decreased by acupuncture. The expression of NOS3 in hypothalamus was similar to NOS2, but the effect of acupuncture was limited. The expression of NOS2 and NOS3 in adrenal gland increased after cold stress, only the expression of NOS1 could be repressed with acupuncture. There was no NOS2 expression in adrenal gland in rats with stress ulcer.CONCLUSION: The protective effect of acupuncturing Tsusanli (Sr36) on the expression of NOS in hypothalamus and adrenal gland can be achieved.