Hydrogen sulfide is involved in the chilling stress response in Vitis vinifera L .

Hydrogen sulfide (H2S) is a novel gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO) [1]. Many data indicate that most of the endogenously synthesized H2S occurred via L-cysteine desulfhydrase (LCD, EC4.4.1.1) and D-cysteine desulfhydrase (DCD, EC4.4.1.15) in high plants [2–5]. Recent studies show that H2S is involved not only in plant responses to drought and copper stresses but also in tolerance to salinity, heat, cadmium, boron and chromium stresses [6–10]. It is reported that exogenous H2S can enhance the resistance of plants to drought stress [11–14] and copper stress [15,16] by improving the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). However, whether endogenous H2S is involved in plant cold stress response is poorly known. Signal molecules are involved in perception and transduction of low temperature signal and mediate chilling adaptive responses through physiological processes and transcription factors [17–20]. Recent research indicated that multiple transcription factors and inducers, such as C-repeat-binding factor (CBF), inducers of CBF expression (ICE) and cold-regulated (COR) genes, played pivotal roles in the resistance to low temperature stress [21–23]. Many signal molecules can improve plant tolerance to low temperature stress by altering the expression levels of COR, CBFs and ICE1 genes. For example, ethylene molecules negatively regulate cold tolerance by repressing expression of CBFs [25]. Inositol 1, 4, 5-trisphosphate (IP3) and Ca2+ induce CBFs and COR expression in plant [25]. NO positively regulate the expression of cold related genes COR15a, LT130 and LTI78 in Arabidopsis under low temperature conditions [17]. However, whether H2S regulates expression of these cold related genes in the response of chilling adaptive is still poorly understood. Different species of grape exhibit varying level of resistance to cold hardiness, ranging from the cold susceptive grape (‘Maoputao’, Vitis quinqanguoari Rehd. cv. Maoputao) to the high resistant grape ‘Zuoshan1’ (Vitis amurensis Rupr. cv. Zuoshan1) [26]. Even though the many researches about low temperature have been conducted in grape [26], the mechanism of low temperature resistance in grape at molecular level as well as the signal transduction pathways reminds unclear. In this paper, exogenous H2S was studied in cold-tolerant grapevine ‘F-242’ in response of cold stress. Our results showed that H2S was involved in cold stress response in grape by regulating superoxide anion radical content, MDA content, the relative permeability of cell membrane and SOD activity, as well as expression of VvICE1 and VvCBF3.

Signal molecules are involved in perception and transduction of low temperature signal and mediate chilling adaptive responses through physiological processes and transcription factors [17][18][19][20].Recent research indicated that multiple transcription factors and inducers, such as C-repeat-binding factor (CBF), inducers of CBF expression (ICE) and cold-regulated (COR) genes, played pivotal roles in the resistance to low temperature stress [21][22][23].Many signal molecules can improve plant tolerance to low temperature stress by altering the expression levels of COR, CBFs and ICE1 genes.For example, ethylene molecules negatively regulate cold tolerance by repressing expression of CBFs [25].Inositol 1, 4, 5-trisphosphate (IP3) and Ca 2+ induce CBFs and COR expression in plant [25].NO positively regulate the expression of cold related genes COR15a, LT130 and LTI78 in Arabidopsis under low temperature conditions [17].However, whether H 2 S regulates expression of these cold related genes in the response of chilling adaptive is still poorly understood.
Different species of grape exhibit varying level of resistance to cold hardiness, ranging from the cold susceptive grape ('Maoputao' , Vitis quinqanguoari Rehd.cv.Maoputao) to the high resistant grape 'Zuoshan1' (Vitis amurensis Rupr.cv.Zuoshan1) [26].Even though the many researches about low temperature have been conducted in grape [26], the mechanism of low temperature resistance in grape at molecular level as well as the signal transduction pathways reminds unclear.In this paper, exogenous H 2 S was studied in cold-tolerant grapevine Fu et al. / Hydrogen sulfide response to chilling in 1/2 MS medium, under a 16 h light/8 h dark photoperiod at 25°C.Four-weeks old seedlings were exposed to 4°C and 25°C for 0, 1, 3, 5, 7, 9, 11 and 22 h, respectively.Fully expanded leaves were harvested to determine cell membrane permeability, content of H 2 S, MDA and superoxide anion radical, activities of L-/D-cysteine desulfhydrase and SOD, and the expression levels of VvLCD and VvDCD genes.
Meanwhile, the plantlets were treated with 0.1 mM HT and NaHS (distilled water as a control) for 1 h, and then measured the cell membrane permeability, MDA content, superoxide anion radical content and SOD activities at their burst time under 4°C and 25°C.In addition, the seedlings treated (spraying) with the same concentration of HT and NaHS were exposed to 4°C for 7 h and 3 d.After that, leaves were harvested and immediately frozen in liquid nitrogen for RNA extraction.The expression levels of VvICE1 and VvCBF3 genes were determined by quantitative RT-PCR.

Measurement of H 2 S content
Measurement of H 2 S content was performed as described by by Liu et al. [27].

Superoxide anion radical content measurement
Measurement of superoxide anion radical content was conducted referring to Zhao and Zhou [28] with some modifications.0.5 g leaves was homogenized with 5 ml 50 mM potassium phosphate buffer at 4°C and centrifuged at 10000 rpm for 10 min.Mixed 0.5 ml 50 mM potassium phosphate buffer 1 ml 1 mM hydroxylamine hydrochloride (Sigma, USA) and 0.5 ml crude extract in reaction tubes, incubated at 25°C for 1 h, then added 1 ml of 17 mmol l −1 sulfanilic acid and 1 ml of 7 mmol l −1 α-naphthylamine (Sigma, USA) in reaction tubes, incubated at 25°C for 20 min, and the absorbance was read at 530 nm.Converted the photometric value to nitrite content via standard curve of nitrite and hydroxylamine reaction, and the content equaled to half of nitrite was measured.

MDA content measurement
Lipid peroxidation was estimated by concentration of thiobarbituric acid reactive substances (TBARS) [29].First, 0.1 g leaves was homogenized with 1 ml 10% (w/v) trichloroacetic acid (TCA; Sigma, USA) and the homogenate was centrifuged at 12000 rpm for 10 min.Then 500 μl of the supernatant was mixed with 500 μl 10% (w/v) TCA containing 0.6% (w/v) thiobarbituric acid (TBA; Sigma, USA).The mixture was incubated in boiling water for 15 min, cooled to room temperature, and centrifuged at 12000 rpm for 10 min.Absorbance of the supernatant was measured at 532 nm, and the non-specific absorbance was measured at 600 nm.The MDA content was determined using a molar extinction coefficient of 155 mM −1 cm −1 [30].

Membrane permeability measurement
Harvested leaves were washed with deionized water and slightly dried with filter paper.Leaf-disc was taken by punching bear, and then incubated in deionized water at 25°C for 1 h.Electrical conductivity (EC1) of the extravasation solution was measured using a conductivity meter (YSI model 55).Total ionic strength was determined after heating the solution in a 100°C water bath for 10 min, and the electrical conductivity (EC2) was measured after cooling the solution to 25°C as described by Welti et al. [31].Membrane relative permeability was calculated by the formula EC1/EC2 × 100%.

SOD activity measurement
Measurement of SOD activity was performed as described by Donahue et al. [32].First, 0.5 g leaves was homogenized with 5 ml 50 mM potassium phosphate buffer at 4°C and centrifuged at 10000 rpm for 10 min.200 μl crude extract was mixed with 3 ml 50 mM potassium phosphate buffer in reaction tubes and then illuminated (fluorescent light, 40 W) for 20 min.The absorbance was measured at 560 nm, taken the reaction solution without illumination as a control.A calibration curve with commercial SOD (Sigma, USA) was utilized to calculate SOD activity.One unit of SOD was defined as the amount of enzyme required to cause a 50% reduction of nitrotetrazolium blue chloride.

Data processing and statistical analysis
The DPS data processing system was used to carry out the significance analysis of the data.P value <0.05 was considered statistically significant.

Chilling treatment increased endogenous H 2 S level and activity of L-/D-cysteine desulfhydrase in grape leaves
Endogenous H 2 S content and the activity of VvLCD and VvDCD were detected in grape leaves in order to investigate H 2 S function in response to chilling temperature.When the grape seedlings were exposed to 4°C, H 2 S content did not change within the first three hours of cold treatment, but increased and reached the peak 5 h after the start of cold treatment, then deceased sharply to the normal level (P < 0.05; Fig. 1a).Additionally, the activity of VvL/DCD was measured at the same treatment condition to determine if the enzyme was involved in H 2 S accumulation.Similarly to the accumulation pattern of H 2 S, the activity of VvLCD and VvDCD were enhanced and
Furthermore, expression level of VvLCD and VvDCD were analyzed by quantitative RT-PCR.Expression pattern were similar between VvLCD and VvDCD, which reached their peak at 3 h and 5 h, respectively, then gradually declined to almost normal expression levels at 11 h cold treatment (Fig. 4).These results were in good agreement the profiles of H 2 S accumulation and corresponding H 2 S synthetase activity (Fig. 1).In conclusion, there is a hypothesis that H 2 S is involved in the chilling signaling pathway of grapevine 'F-242' .

Effects of NaHS and HT on the levels of superoxide anion radical, MDA, the relative permeability of cell membrane and SOD activity in grape leaves
Superoxide anion radical content, MDA levels, the relative permeability of cell membrane and SOD activity were identified in grape seedlings treated with NaHS and HT.The data revealed that the superoxide anion radical content (Fig. 5a), MDA content (Fig. 5b), the relative permeability of cell membrane (Fig. 5c) and SOD activity (Fig. 5d) were all significantly increased at 3 h, 5 h, 11 h and 3 h, respectively, in the grape leaves after 4°C treatment.While a decline occurred for superoxide anion radical, MDA and the relative permeability of the plasma membrane in the seedlings pretreated with NaHS (Fig. 5e-g).As was expected, the seedlings pretreated with HT presented the contrary results (Fig. 5e-g).However, SOD activity showed opposite changes, with the increase of SOD by NaHS addition and decreased by HT pretreatment (Fig. 5h).
Thus, it was suggested that H 2 S could reduce the level of superoxide anion radical and MDA, and improve SOD activity and the plasma membrane stability of grape leaves under low temperature condition.

Effects of NaHS and HT on transcription levels of VvICE1 and VvCBF3
To explore the possible targets of H 2 S during chilling acclimation, transcription levels of cold-responsive genes VvICE1 and VvCBF3 were analyzed in 4°C acclimated 'F-242' seedlings that were pretreated with NaHS and HT.At normal temperature   (25°C), expression of VvICE1 and VvCBF3 in 'F-242' were at low levels whether NaHS and HT were applied or not (Fig. 6).However, cold treatment significantly enhanced expression levels of VvICE1 and VvCBF3, which were further induced by the application of NaHS, but suppressed by HT (Fig. 6).Thus, there is a hypothesis that H 2 S play an important role in grape responses to cold stress by modulating VvICE1 and VvCBF3 expression.

Discussion
Recent studies in plants revealed that H 2 S plays multiple roles in the modulation of various physiological processes [3], such as copper, cadmium, drought, salinity and heat et al. [6][7][8][9][10].In addition, our results indicate that the content of H 2 S was rapidly increased by low temperature.All the results reveal that H 2 S may be an important signaling molecule in plant responses to abiotic stresses.
In mammals, H 2 S is produced from L-cysteine by at least four separate pathways, such as cystathionine β synthetase (CBS, EC 4.2.1.22),cystathionine γ lyase (CSE, EC 4.4.1.1),cysteine aminotransferase (CAT, EC 2.6.1.3)and cysteine lyase (CL, EC 4.4.1.10)[34], and CBS and CSE consistently demonstrated to produce H 2 S in mammalian tissues [1,35].Similar to animals, the homologs of CBS (LCD) and CSE (DCD) also are found in plants, and they are reported mainly responsible for generating H 2 S [4,36].LCD (At3g62130) and DCD (At1g48420) genes have been isolated from Arabidopsis [2,37].Meanwhile, the homolog genes of CDes also were isolated from B. napus and O. sativa [38].In our study, we isolated LCD and DCD genes from 'F-242' .Moreover, we also find the transcription levels and enzyme activities of LCD and DCD were induced by 4°C acclimation.However, inhibitors of LCD and DCD pathways could inhibit the accumulation of H 2 S. Therefore, we conclude that VvLCD and VvDCD genes contribute to the major production of H 2 S in grape at low temperature.
To confirm the functions of H 2 S in grape response to chilling stress, we analyzed superoxide anion radical content, MDA  content, the relative permeability of the plasma membranes and SOD activity in NaHS-and HT-pretreated grape seedlings under low temperature.The results demonstrated that H 2 S participated in the response of grape to chilling by protecting membrane integrity and enhancing SOD activities related to cold resistance.Meanwhile, the study of Li et al. shows that spraying NaHS improves heat tolerance in maize by alleviating the increase of electrolyte leakage and MDA [39].In addition, Zhang and his colleagues indicate that spraying NaHS delays excessive accumulation of MDA and reactive oxygen species, but enhance SOD activity against cooper stress and droughtinduced oxidative stress in wheat seeds and soybean seedlings [11,15].All these findings suggest that H 2 S possibly use the similar mechanism to resist various stresses in different plants.
Transcription factors play important roles in chilling stress response in plants.For instance, the expression levels of ICE1 and CBF3 can be enhanced in different freezing temperatures [40].Ectopically expression of AtCBFs and AtICE1 in different plant species can enhance chilling tolerance [41,42], and ectopically expression of CBFs from other plants is able to enhance the freezing tolerance of transgenic Arabidopsis [20,43].In the present work, it is shown that the transcription levels of VvICE1 and VvCBF3 were induced greatly and reached their peak at 7 h and 3 d, respectively, under low temperature in 'F-242' (data not shown).In addition, NaHS significantly induced VvICE1 and VvCBF3 at low temperature.It indicates that H 2 S is involved in grapevine chilling stress response by modulating VvICE1 and VvCBF3 transcription.Recently, it is shown that DREB1/CBF3 and RD29A (also known as COR78 or LTI78, a target gene of CBF3) can be induced by both cold and drought in plants [44][45][46].In addition, H 2 S up-regulates the expression of RD29A to improve drought resistance in Arabidopsis [13].We guess from these results that, in plants, the genes regulated by H 2 S could participate not only in drought stress but also in cold stress.
To further investigate the physiological function and regulatory mechanism of H 2 S in response to low temperature, some work could be conducted, such as grape genetic transformation, RNAi technology to generate the VvL-CD and VvD-CD mutants and transgenic grape plants for the genetic controls of H 2 S responses to low temperature in the future studies.

Fig. 1
Fig. 1 Detection of endogenous H 2 S contentand L/DCD activity in 'F-242' leaves under chilling stress.The values represent the average of three independent samples.a Effects of chilling on H 2 S content.b Effects of chilling on LCD activity.c Effects of chilling on DCD activity.d Effects of H 2 S synthesis inhibitors (AOA, NH 2 OH and C 3 H 3 KO 3 +NH 3 ) on chilling-induced H 2 S content.Error bars indicate ±SE.* indicates significant differences at P < 0.05 (Student's t-test).

Fig. 4
Fig. 4 Expression patterns of VvLCD and VvDCD in 'F-242' under chilling stress.The relative expression of VvLCD and VvDCD was quantified in comparison with the VvACTIN using quantitative RT-PCR with gene-specific primers (see "Material and methods").The values represent the average of three independent samples.Error bars indicate ±SE.* indicates significant differences at P < 0.05 (Student's t-test).

Fig. 5
Fig. 5 Effects of low temperature and H 2 S on several parameters involved in cold tolerance in V. vinifera L. 'F-242' .The values represent the average of three independent samples.a Effect of low temperature on superoxide anion radical content.b Effect of low temperature on MDA content.c Effect of temperature on the relative permeability of cell membrane.d Effect of low temperature on SOD activity.e Effect of NaHS and HT on superoxide anion radical content.f Effect of NaHS and HT on MDA content.g Effect of NaHS and HT on the relative permeability of cell membrane.h Effect of NaHS and HT on SOD activity.Error bars indicate ±SE.* indicates significant differences at P < 0.05 (Student's t-test).

Fig. 6
Fig. 6 Effects of HT and NaHS on VvICE1 (a) and VvCBF3 (b) expression patterns under chilling stress in V. vinifera L. 'F-242' .The relative expression of VvICE1 and VvCBF3 was quantified in comparison with the VvACTIN using quantitative RT-PCR with gene-specific primers (see "Material and methods").The values represent the average of three independent samples.Error bars indicate ±SE.* indicates significant differences at P < 0.05 (Student's t-test).