Abstract
Drought is one of the main environmental constraints that can reduce plant yield. Nitric oxide (NO) is a signal molecule involved in plant responses to several environmental stresses. The objective of this study was to investigate the cytoprotective effect of a single foliar application of 0, 1, 10 or 100 µM of the NO donor sodium nitroprusside (SNP) in sunflower plants under water stress. Water stressed plants treated with 1μM SNP showed an increase in the relative water content compared with 0 μM SNP. Drought reduced the shoot dry weight but SNP applications did not result in alleviation of drought effects. Neither drought nor water stress plus SNP applications altered the content of photosynthetic pigments. Stomatal conductance was reduced by drought and this reduction was accompanied by a significant reduction in intercellular CO2 concentration and photosynthesis. Treatment with SNP did not reverse the effect of drought on the gas exchange characteristics. Drought increased the level of malondialdehyde (MDA) and proline and reduced pirogalol peroxidase (PG-POD) activity, but did not affect the activity of superoxide dismutase (SOD). When the water stressed plants were treated with 10 μM SNP, the activity of PG-POD and the content of proline were increased and the level of MDA was decreased. The results show that the adverse effects of water stress on sunflower plants are dependent on the external NO concentration. The action of NO may be explained by its ability to increase the levels of antioxidant compounds and the activity of ROS-scavenging enzymes.
photosynthesis; reactive oxygen species; antioxidant enzymes; proline; malondialdehyde
1 INTRODUCTION
Lower rainfall is observed in many regions worldwide, causing many negative plant
responses such as lower dry weight accumulation and lower rates of carbon
assimilation (Álvarez et al., 2011Álvarez, S., Navarro, A., Nicolas, E., & Sanchez-Blanco, M. J.
(2011). Transpiration, photosynthetic responses, tissue water relations and dry
mass partitioning in plants during drought conditions.
CallistemonScientia Horticulturae, 129, 306-312.
http://dx.doi.org/10.1016/j.scienta.2011.03.031.
http://dx.doi.org/10.1016/j.scienta.2011...
). Water
stress also causes an overproduction of a variety of reactive oxygen species (ROS)
such as hydrogen peroxide and singlet oxygen which are potentially harmful to all
cellular components. Plant cell evolved an antioxidant system composed of
nonenzymatic and enzymatic components that play a critical role in neutralizing the
free radicals which can affect the cellular stability. The ability to maintain a
high antioxidant system activity under water stress depends on plant species, stress
intensity and duration (DaCosta & Huang,
2007DaCosta, M., & Huang, B. (2007). Changes in antioxidant enzyme
activities and lipid peroxidation for bentgrass species in response to drought
stress. Journal of the American Society for Horticultural Science, 132,
319-326.), with the high antioxidant enzyme activity levels been positively
related to drought resistance (Ghahfarokhi et al.,
2015Ghahfarokhi, M. G., Mansurifar, S., Taghizadeh-Mehrjardi, R.,
Saeidi, M.; Jamshidi, A. M., & Ghasemi, E. (2015). Effects of drought stress
and rewatering on antioxidant systems and relative water content in different
growth stages of maize ( L.) hybrids. Zea maysArchives of
Agronomy and Soil Science, 61, 493-506.
http://dx.doi.org/10.1080/03650340.2014.943198
http://dx.doi.org/10.1080/03650340.2014....
; Sairam & Srivastava,
2001Sairam, R. K., & Srivastava, G. C. (2001). Water stress
tolerance of wheat ( L.): variations in hydrogen peroxide accumulation and
antioxidant activity in tolerant and susceptible genotypes. Triticum
aestivumJournal Agronomy & Crop Science, 186, 63-70.
http://dx.doi.org/10.1046/j.1439-037x.2001.00461.x.
http://dx.doi.org/10.1046/j.1439-037x.20...
).
Under a variety of stresses, active solute accumulation of compatible solutes such as
proline is claimed to be an effective stress tolerance mechanism. Proline works as
both an osmoprotectant and as a redox-buffering agent possessing antioxidant
property under conditions of stress (KaviKishor
& Sreenivasulu, 2014KaviKishor, P. B., & Sreenivasulu, N. (2014). Is proline
accumulation per se correlated with stress tolerance or is proline homeostasis a
more critical issue? Plant, Cell & Environment, 37, 300-311.
http://dx.doi.org/10.1111/pce.12157. PMid:23790054
http://dx.doi.org/10.1111/pce.12157...
). Accumulation of proline under drought stress
was found in several plants, particularly in young leaves (Cechin et al., 2006Cechin, I., Rossi, S. C., Oliveira, V. C., & Fumis, T. F.
(2006). Photosynthetic responses and proline content of mature and young leaves
of sunflower plants under water deficit. Photsynthetica, 44, 143-146.
http://dx.doi.org/10.1007/s11099-005-0171-2.
http://dx.doi.org/10.1007/s11099-005-017...
). Furthermore, foliar applied proline
ameliorated the adverse effects of water stress on growth and photosynthetic
capacity of two maize cultivars (Ali et al.,
2007Ali, Q., Ashraf, M., & Athar, H. U. R. (2007). Exogenously
applied proline at different growth stages enhances growth of two maize
cultivars grown under water deficit conditions. Pakistan Journal of Botany, 39,
1133-1144.). The osmolyte accumulations in plant cells may therefore be
important in maintaining various physiological processes in operation even under
stressed conditions.
Nitric oxide (NO), a molecule that is highly diffusible through cellular membrane due
to its lipophilic nature, is involved in several physiological, biochemical and
developmental processes in plants (Krasylenko et
al., 2010Krasylenko, Y. A., Yemets, A. I., & Blume, Y. B. (2010).
Fuctional role of nitric oxide in plants. Russian Journal of Plant Physiology,
57, 451-461. http://dx.doi.org/10.1134/S1021443710040011.
http://dx.doi.org/10.1134/S1021443710040...
and references therein). NO is itself a reactive nitrogen
species (RNS) produced in a variety of cells and its effects on different types of
cells have proved to be either protective or toxic, depending on its concentration
(Beligni & Lamattina, 1999Beligni, M., & Lamattina, L. (1999). Is nitric oxide toxic or
protective? Trends in Plant Science, 4, 299-300.
http://dx.doi.org/10.1016/S1360-1385(99)01451-X. PMid:10431217
http://dx.doi.org/10.1016/S1360-1385(99)...
). Besides
proline (Kahlaoui et al., 2014Kahlaoui, B., Hachicha, M., Rejeb, S., Rejeb, M. N., Hanchi, B.,
& Misle, E. (2014). Response of two tomato cultivars to field-applied
proline under irrigation with saline water: Growth, chlorophyll fluorescence and
nutritional aspects. Photosynthetica, 52, 421-429.
http://dx.doi.org/10.1007/s11099-014-0053-6.
http://dx.doi.org/10.1007/s11099-014-005...
), other
chemicals such as sodium nitroprusside (NO donor) are currently being applied to
plants exposed to stressful conditions in order to improve growth and yield (Farooq et al., 2009Farooq, M., Basra, S. M. A., Wahid, A., & Rehman, H. (2009).
Exogenously nitric oxide enhances the drought tolerance in fine grain aromatic
rice ( L.). Oryza sativaJournal Agronomy & Crop Science,
195, 254-261.
http://dx.doi.org/10.1111/j.1439-037X.2009.00367.x.
http://dx.doi.org/10.1111/j.1439-037X.20...
). In recent years,
evidences have accumulated showing that exogenous NO can alleviate the harmful
effects of environmental stresses in plants such as water stress (Boogar et al., 2014Boogar, A. R., Salehi, H., & Jowkar, A. (2014). Exogenous nitric
oxide alleviates oxidative damage in turfgrasses under drought stress. South
African Journal of Botany, 92, 78-82.
http://dx.doi.org/10.1016/j.sajb.2014.02.005.
http://dx.doi.org/10.1016/j.sajb.2014.02...
; Farooq et al., 2009Farooq, M., Basra, S. M. A., Wahid, A., & Rehman, H. (2009).
Exogenously nitric oxide enhances the drought tolerance in fine grain aromatic
rice ( L.). Oryza sativaJournal Agronomy & Crop Science,
195, 254-261.
http://dx.doi.org/10.1111/j.1439-037X.2009.00367.x.
http://dx.doi.org/10.1111/j.1439-037X.20...
; Liao et
al., 2012Liao, W.-B., Huang, G.-B., Yu, J.-H., & Zhang, M.-L. (2012).
Nitric oxide and hydrogen peroxide alleviate drought stress in marigold explants
and promote its adventitious root development. Plant Physiology and
Biochemistry, 58, 6-15. http://dx.doi.org/10.1016/j.plaphy.2012.06.012.
PMid:22771430
http://dx.doi.org/10.1016/j.plaphy.2012....
). Therefore, the objective of the present study was to
investigate whether sodium nitroprusside (SNP), a NO donor, plays an important role
in protecting sunflower plants against water stress, as assessed by dry weight
accumulation, gas exchange characteristics and antioxidant enzyme activities.
2 MATERIALS AND METHODS
Plant material and growth conditions
Seeds of sunflower (Helianthus annuus L. variety IAC-Iarama) were sown in 4 dm3 pots filled with a 47:13:40% mixture of pinus bark: vermiculite: peat enriched with minerals. Seedlings were thinned to one per pot after emergence and were grown in a greenhouse under natural photoperiod. Maximum day and night temperatures were close to 32 and 18 °C, respectively.
Plants were supplied with tap water according to the requirements and supplemented with 250 mL of 70% full strength Long Ashton nutrient solution (Hewitt, 1966Hewitt, E. J. (1966). Sand and water culture methods used in the study of plant nutrition. England: Commonwealth Agricultural Bureaux, Farnham Royal.) 16 and 23 days after planting. After 27 days of sowing, the plants were separated into five distinct groups: (1) plants well hydrated and without SNP (control), (2) plants under water stress and without SNP, (3) plants under water stress with 1μM of SNP, (4) plants under water stress with 10 μM of SNP and (5) plants under water stress with 100 μM of SNP. The well watered plants were watered daily according to the previous conditions, whereas the stressed plants had the water supply discontinued. Applications of SNP coincided with the onset of drought stress. The plants received a single dose of SNP, at 1, 10 or 100 μM concentrations. The well watered plants were sprayed with distilled water.
Gas exchange measurements
On the 3rd day after the induction of stress, a portable infra-red gas analyzer (LCpro, ADC, Hoddesdon, UK) was used for measurements of photosynthesis (A), stomatal conductance to water vapor (gs ), transpiration (E) and intercellular CO2 concentration (Ci ) on the youngest fully expanded leaf. Measurements were made inside the greenhouse and a photosynthetic active radiation (PAR) of 1000 μmol m–2 s–1 was supplied by a light unit mounted on the top of leaf chamber. The leaf was kept under this PAR until a steady-state rate was achieved.
Chemical analysis
The photosynthetic pigments were measured on leaf discs of known area from a leaf
next to the leaf used for gas exchange. Pigments were extracted in 80% aqueous
acetone and the content was calculated according to the equations proposed by
Lichtenthaler (1987)Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: pigment
photosynthetic biomembranes. Methods in Enzymology, 148,
362-385.. The proline
content was determined according to the method described by Bates et al. (1973)Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid
determination of free proline for water-stress studies. Plant and Soil, 39,
205-207. http://dx.doi.org/10.1007/BF00018060.
http://dx.doi.org/10.1007/BF00018060...
and modified by Torello & Rice (1986)Torello, W. A., & Rice, L. A. (1986). Effects of NaCl stress on
proline and cation accumulation in salt sensitive tolerance turfgrass. Plant and
Soil, 93, 241-247. http://dx.doi.org/10.1007/BF02374226.
http://dx.doi.org/10.1007/BF02374226...
from oven dried
and fine powder leaves and the concentration expressed on a leaf dry weight
basis by using proline as standard.
The activity of pirogalol peroxidase (PG-POD; EC 1.11.1.7), superoxide dismutase
(SOD; EC 1.15.1.1) and lipid peroxidation were determined on leaves next to
those used for the above analysis. The extraction of PG-POD and SOD were
determined according to Ekler et al.
(1993)Ekler, Z., Dutka, F., & Stephenson, G. R. (1993). Safener
effects on acetochlor toxicity, uptake, metabolism and glutathione S-transferase
activity in maize. Weed Research, 33, 311-318.
http://dx.doi.org/10.1111/j.1365-3180.1993.tb01946.x.
http://dx.doi.org/10.1111/j.1365-3180.19...
. The activity of the enzymes was determined according to Teisseire & Guy (2000)Teisseire, H., & Guy, V. (2000). Cooper-induced changes in
antioxidant enzymes activities in frond of duckweed (). Lemma
minorPlant Science, 153, 65-72.
http://dx.doi.org/10.1016/S0168-9452(99)00257-5.
http://dx.doi.org/10.1016/S0168-9452(99)...
and Beauchamp & Fridovich (1971)Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase:
improved assays and an assay applicable to acrylamide gels. Analytical
Biochemistry, 44, 276-287. http://dx.doi.org/10.1016/0003-2697(71)90370-8.
PMid:4943714
http://dx.doi.org/10.1016/0003-2697(71)9...
cited by
Bor et al. (2003)Bor, M., Özdemir, F., & Türkan, I. (2003). The effects of the
salt stress on lipid peroxidation and antioxidants in leaves of sugar beet L.
Beta vulgaris L. and wild beet Beta
maritimePlant Science, 164, 77-84.
http://dx.doi.org/10.1016/S0168-9452(02)00338-2.
http://dx.doi.org/10.1016/S0168-9452(02)...
, respectively. The
activity of PG-POD was expressed as μM min–1 mg–1 protein.
SOD activity was assayed by measuring its ability to inhibit the photochemical
reduction of nitro blue tetrazolium (NBT). Protein content was measured using
casein as standard according to the method of Bradford (1976)Bradford, M. M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Analytical Biochemistry, 72, 248-254.
http://dx.doi.org/10.1016/0003-2697(76)90527-3. PMid:942051
http://dx.doi.org/10.1016/0003-2697(76)9...
. The level of lipid peroxidation was measured in
terms of malondialdehyde (MDA) content, a product of lipid peroxidation,
according to the method described by Heath
& Packer (1968)Heath, R. L., & Packer, L. (1968). Photoperoxidation in isolated
chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives
of Biochemistry and Biophysics, 125, 189-198.
http://dx.doi.org/10.1016/0003-9861(68)90654-1. PMid:5655425
http://dx.doi.org/10.1016/0003-9861(68)9...
. The MDA content was calculated by its extinction
coefficient of 155 mmol L–1 cm–1 and expressed as nmol MDA
per g fresh weight.
Relative water content and dry mass determination
At the end of the experiment (4 days after stress induction), the leaf relative water content (RWC) was determined as:
where FW is the fresh weight obtained immediately after the removal of leaf discs; TW is the turgid weight determined after rehydration of the discs for 3 h and DW is the dry weight obtained after drying the discs in an oven at 60 °C for 48 hours. Plants of each treatment were selected randomly for shoot dry weight determinations. The sunflower plants were divided into stem and leaves before been oven dried at 60 °C for 48 hours.
Statistical analysis
The statistical tests were conducted using the Software Statistical Package for the Social (SPSS/PC) version 9.0 at 5% significance level. Quantitative changes of different parameters were analyzed through analysis of variance (ANOVA), with Tukey’s honestly significant difference multiple comparison test being used to determine significant differences among treatments.
3 RESULTS AND DISCUSSION
Similar to other environmental stresses, drought affects many physiological and
metabolic processes within plants thus resulting in lower dry weight accumulation.
Plant response to stress is first characterized by a rapid inhibition, followed by
adaptation to the new condition (Skirycz &
Inzé, 2010Skirycz, A., & Inzé, D. (2010). More from less: plant growth
under limited water. Current Opinion in Biotechnology, 21, 197-203.
http://dx.doi.org/10.1016/j.copbio.2010.03.002. PMid:20363612
http://dx.doi.org/10.1016/j.copbio.2010....
). After four days of stress imposition, leaf and shoot dry
weight and RWC were reduced about 23%, 22% and 13%, respectively but stem dry weight
accumulation was not affected (Table 1). The
observed reduction in leaves dry weight is a result of high sensitivity of young
leaves to changes in water supply (Cechin et al.,
2006Cechin, I., Rossi, S. C., Oliveira, V. C., & Fumis, T. F.
(2006). Photosynthetic responses and proline content of mature and young leaves
of sunflower plants under water deficit. Photsynthetica, 44, 143-146.
http://dx.doi.org/10.1007/s11099-005-0171-2.
http://dx.doi.org/10.1007/s11099-005-017...
) since cell division and elongation are influenced by leaf water
status or cell turgor (Heckenberger et al.,
1998Heckenberger, U., Roggatz, U., & Schurr, U. (1998). Effect of
drought stress on the cytological status in Ricinus
communis.Journal of Experimental Botany, 49, 181-189.
http://dx.doi.org/10.1093/jxb/49.319.181.
http://dx.doi.org/10.1093/jxb/49.319.181...
). Applications of SNP in wheat seedlings has been found to confer
water deficit tolerance by maintaining more water than the well watered plants
(García-Mata & Lamattina, 2001García-Mata, C., & Lamattina, L. (2001). Nitric oxide induces
stomatal closure and enhances the adaptive plant responses against drought
stress. Plant Physiology, 126, 1196-1204.
http://dx.doi.org/10.1104/pp.126.3.1196. PMid:11457969
http://dx.doi.org/10.1104/pp.126.3.1196...
). In
this study, althought short-term SNP applications of 1μM under water stress resulted
in an increase of 7% in RWC compared with 0 μM SNP this did not result in
improvement of plant growth. Applications of NO have been used to improve crop
growth under various abiotic stresses, with contradictory results. Foliar-applied NO
has been reported to enhance growth only under non-stressed conditions (Kausar et al., 2013Kausar, F., Shahbaz, M., & Ashraf, M. (2013). Protective role of
foliar-applied nitric oxide in under saline stress. Triticum
aestivumTurkish Journal of Botany, 37, 1155-1165.
http://dx.doi.org/10.3906/bot-1301-17.
http://dx.doi.org/10.3906/bot-1301-17...
) or under both
non-stressed and salt stressed conditions (Uchida
et al., 2002Uchida, A., Jagendorf, A. T., Hibino, T., Takabe, T., & Takabe,
T. (2002). Effects of hydrogen peroxide and nitric oxide on both salt and heat
stress toerance in rice. Plant Science, 163, 515-523.
http://dx.doi.org/10.1016/S0168-9452(02)00159-0.
http://dx.doi.org/10.1016/S0168-9452(02)...
). This conflicting results might be related to many factors
such as species, SNP concentration and duration of applications. In this study,
water stress did not occur abruptly, but developed slowly and increased with time in
intensity. The lack of 10 µM SNP beneficial effect on sunflower plant dry weight
accumulation under water stress as seen for proline, PG-POD and MDA (Figure 1) might be ascribed to the fact that the
dry weight was measured after 4 days of the beginning of water stress. Under short
period of water stress the physiological responses are easily seen whereas
morphological changes are not.
Relative water content (RWC; %), dry weight (g plant–1) of leaves, stem and shoot, and total chlorophyll (Chl) and carotenoids (Car) concentrations (g m–2) of well watered (WW) or water-stresed plus SNP (WS+SNP) sunflower plants. SNP, as NO donor, was added on the surface of the leaves. Values are means±SE of 5-6 plants. Values sharing the same letters within the same row are not significantly different at 5% significance level
Proline content (a), pirogalol peroxidase (PG-POD) activity (b), superoxide dismutase (SOD) activity (c) and malondialdehyde (MDA) content (d) of well watered (WW) or water-stresed plus SNP (WS+SNP) sunflower plants. SNP, as NO donor, was added on the surface of the leaves. Values are means±SE of 4 plants. Values sharing the same letters are not significantly different at 5% significance level.
David et al. (1998)David, M. M., Coelho, D., Barrote, I., & Correia, M. J. (1998).
Leaf age effects on photosynthetic activity and sugar accumulation in droughted
and rewatered plants. Lupinus albusAustralian Journal of Plant
Physiology, 25, 299-306. http://dx.doi.org/10.1071/PP97142.
http://dx.doi.org/10.1071/PP97142...
found that dehydration
increased the chlorophyll content in young leaves and that it was slightly decreased
in the older ones, suggesting that the contribution of mesophyll limitations in the
inhibition of photosynthesis increases with leaf age. The carotenoids that are
intrinsic components of chloroplastic membranes have been reported to decrease
(Yildiz-Aktas et al., 2009Yildiz-Aktas, L., Dagnon, S., Gurel, A., Gesheva, E., & Edreva,
A. (2009). Drought tolerance in cotton: involvement of non-enzymatic
ROS-scavenging compounds. Journal Agronomy & Crop Science, 195, 247-253.
http://dx.doi.org/10.1111/j.1439-037X.2009.00366.x.
http://dx.doi.org/10.1111/j.1439-037X.20...
) or to
increase significantly (Guha et al., 2012Guha, A., Sengupta, D., Rasineni, G. K., & Reddy, A. R. (2012).
Non-enzymatic antioxidant defence in drought-stressed mulberry ( L.) genotypes.
Morus indicaTrees, 26, 903-918.
http://dx.doi.org/10.1007/s00468-011-0665-4.
http://dx.doi.org/10.1007/s00468-011-066...
)
under drought. No significant changes in the concentration of total chlorophyll or
carotenoids were observed in sunflower plants under water stress without SNP when
compared with non-stressed plants without SNP (Table 1). It has been found that NO applications increased leaf
chlorophyll of plants under water stress, suggesting that NO treatment protected the
photosynthetic apparatus (Fan & Liu,
2012Fan, Q. J., & Liu, J. H. (2012). Nitric oxide is involved in
dehydration/drought tolerance in seedlings through regulation of antioxidant
systems and stomatal response. Poncirus trifoliataPlant Cell
Reports, 31, 145-154. http://dx.doi.org/10.1007/s00299-011-1148-1.
PMid:21938448
http://dx.doi.org/10.1007/s00299-011-114...
). This phenomenon was not observed in the present study. The
inconsistent findings observed in plant responses to water stress and to application
of NO may be related to the different approaches used by the authors or to the
variation in sensitivity among species.
The RWC of leaves and photosynthesis are under control of stomata and stomata closure
is one of the first responses to drought. A small decline in stomatal conductance
under mild water stress may have protective effects against stress by saving water
and improving water-use efficiency by the plant. Sunflower plants showed a
remarkable reduction in stomata conductance without significant reduction in
transpiration rate after three days of water stress which were not altered by SNP
concentration (Figures 2a,b). This results
contrast to those reported by García-Mata &
Lamattina (2001)García-Mata, C., & Lamattina, L. (2001). Nitric oxide induces
stomatal closure and enhances the adaptive plant responses against drought
stress. Plant Physiology, 126, 1196-1204.
http://dx.doi.org/10.1104/pp.126.3.1196. PMid:11457969
http://dx.doi.org/10.1104/pp.126.3.1196...
who found that exogenous NO was able to induce stomatal
closure under water stress. In addition, the authors showed that the stomata closure
was correlated with a 10% increase in RWC. However, through elegant experiments,
Ribeiro et al. (2009)Ribeiro, D. M., Desikan, R., Bright, J., Confraria, A., Harrison,
J., Hancock, J. T., Barros, R. S., Neill, S. J., & Wilson, I. D. (2009).
Differential requirement for NO during ABA-induced stomatal closure in turgid
and wilted leaves. Plant, Cell & Environment, 32, 46-57.
http://dx.doi.org/10.1111/j.1365-3040.2008.01906.x.
PMid:19021879
http://dx.doi.org/10.1111/j.1365-3040.20...
reported that the
effect of NO depends on the hydration conditions of the tissues. The authors
demonstrated that the NO is not involved in the stomata closure under water stress
conditions in Arabidopsis thaliana but in well-hydrated tissues.
Althought in the present study the effect of NO in well-hydrated plants was not
evaluated it seems that the observed effect of NO in water stressed plants is
similar to that found by Ribeiro et al.
(2009)Ribeiro, D. M., Desikan, R., Bright, J., Confraria, A., Harrison,
J., Hancock, J. T., Barros, R. S., Neill, S. J., & Wilson, I. D. (2009).
Differential requirement for NO during ABA-induced stomatal closure in turgid
and wilted leaves. Plant, Cell & Environment, 32, 46-57.
http://dx.doi.org/10.1111/j.1365-3040.2008.01906.x.
PMid:19021879
http://dx.doi.org/10.1111/j.1365-3040.20...
.
Stomatal conductance (gs , a), transpiration (E, b), photosynthesis (A, c), and intercellular CO2 concentration (Ci , d) of well watered (WW) or water-stresed plus SNP (WS+SNP) sunflower plants. SNP, as NO donor, was added on the surface of the leaves. Values are means±SE of 6 plants. Values sharing the same letters are not significantly different at 5% significance level.
Stomatal closure without a change in mesophyll capacity results in lower
concentration of intercellular CO2. In this study, the reduction in
stomata conductance under water stress without or with application of SNP was
accompanied by a significant reduction in the concentration of intercellular
CO2 and photosynthesis (Figures
2d,c). By removing the lower epidermis of sunflower leaves, Tang et al. (2002)Tang, A. C., Kawamitsu, Y., Kanechi, M., & Boyer, J. S. (2002).
Photosynthetic oxygen evolution at low water potential in leaf discs lacking an
epidermis. Annals of Botany, 89, 861-870. http://dx.doi.org/10.1093/aob/mcf081.
PMid:12102512
http://dx.doi.org/10.1093/aob/mcf081...
were able to demonstrate
that CO2 depletion was responsible for reduction in photosynthesis only
in the early phases of water stress. However, Tezara et al. (1999)Tezara, W., Mitchell, V. J., Driscoll, S. D., & Lawlor, D. W.
(1999). Water stress inhibits plant photosynthesis by decreasing coupling factor
and ATP. Nature, 401, 914-917. http://dx.doi.org/10.1038/44842.
http://dx.doi.org/10.1038/44842...
stated that metabolic inhibition of photosynthesis
also takes place at mild water stress and it becomes more important as the water
stress intensifies. Although the decline in photosynthesis and stomata conductance
are often taken to indicate that photosynthesis is affected via stomatal limitation
it is interesting to note that in this study the reduction in stomata conductance
was higher than in the concentration of intercellular CO2. Substantial
increase in intercellular CO2 concentration was observed with prolonged
water stress in sunflower plants, suggesting that the photosynthesis came gradually
under control of mesophyll metabolism (Cechin et
al., 2008Cechin, I., Corniani, N., Fumis, T. F., & Cataneo, A. C. (2008).
Ultraviolet-B and water stress effects on growth, gas exchange and oxidative
stress in sunflower plants. Radiation and Environmental Biophysics, 47, 405-413.
http://dx.doi.org/10.1007/s00411-008-0167-y. PMid:18404272
http://dx.doi.org/10.1007/s00411-008-016...
). In present study, the decrease in photosynthesis observed in
sunflower plants under three days of water stress with or whitought SNP was likely
due to stomatal closure rather than reflecting a reduction in chloroplast activity,
since intercellular CO2 concentration decreased in response to water
restriction.
NO is an important signaling molecule with diverse functions in plants and its
production can be triggered by several abiotic stresses (Fan & Liu, 2012Fan, Q. J., & Liu, J. H. (2012). Nitric oxide is involved in
dehydration/drought tolerance in seedlings through regulation of antioxidant
systems and stomatal response. Poncirus trifoliataPlant Cell
Reports, 31, 145-154. http://dx.doi.org/10.1007/s00299-011-1148-1.
PMid:21938448
http://dx.doi.org/10.1007/s00299-011-114...
; Yang et
al., 2011Yang, W., Sun, Y., Chen, S., Chen, F., Fang, W., & Liu, Z.
(2011). The effect of exogenously apllied nitric oxide on photosynthesis and
antioxidant activity in heat stressed chrysanthemum. Biologia Plantarum, 55,
737-740. http://dx.doi.org/10.1007/s10535-011-0178-4.
http://dx.doi.org/10.1007/s10535-011-017...
) or its endogenous level be increased by applications of NO
donors under both normal and stress conditions (Boyarshinov & Asafova, 2011Boyarshinov, A. V., & Asafova, E. V. (2011). Stress responses of
wheat leaves to dehydration: participation of endogenous NO and effect of sodium
nitroprusside. Russian Journal of Plant Physiology: a Comprehensive Russian
Journal on Modern Phytophysiology, 58, 1034-1039.
http://dx.doi.org/10.1134/S1021443711060033.
http://dx.doi.org/10.1134/S1021443711060...
; Fan
& Liu, 2012Fan, Q. J., & Liu, J. H. (2012). Nitric oxide is involved in
dehydration/drought tolerance in seedlings through regulation of antioxidant
systems and stomatal response. Poncirus trifoliataPlant Cell
Reports, 31, 145-154. http://dx.doi.org/10.1007/s00299-011-1148-1.
PMid:21938448
http://dx.doi.org/10.1007/s00299-011-114...
). Increase in endogenous NO via application of NO donors
has been shown to improve the photosynthetic performance in water stressed plants
(Fan & Liu, 2012Fan, Q. J., & Liu, J. H. (2012). Nitric oxide is involved in
dehydration/drought tolerance in seedlings through regulation of antioxidant
systems and stomatal response. Poncirus trifoliataPlant Cell
Reports, 31, 145-154. http://dx.doi.org/10.1007/s00299-011-1148-1.
PMid:21938448
http://dx.doi.org/10.1007/s00299-011-114...
), the improvement
was associated with an increase in photosynthetic pigments. In this study, foliar
spray of SNP failed to induce increase in photosynthetic pigments and photosynthetic
performance which might be explained due to the that in the present study the plants
were not pre-treated with SNP.
Different kinds of stress produce an increase in ROS, thus resulting in oxidative
stress as a consequence of loss of oxidant/antioxidant balance. Increased MDA is a
characteristic feature of oxidative membrane damage that has been reported as a
common response to stress conditions. In this study, water stress withought SNP
increased MDA of about 231% (up to 3 times) when compared with well watered plants
(Figure 1d). Suppression (35%) of MDA
accumulation in sunflower leaves was observed with foliar application of 10 μM SNP
in comparison with water stressed and 0 μM SNP. As low MDA level has been found to
be a characteristic feature of plants tolerant to drought (Sairam & Srivastava, 2001Sairam, R. K., & Srivastava, G. C. (2001). Water stress
tolerance of wheat ( L.): variations in hydrogen peroxide accumulation and
antioxidant activity in tolerant and susceptible genotypes. Triticum
aestivumJournal Agronomy & Crop Science, 186, 63-70.
http://dx.doi.org/10.1046/j.1439-037x.2001.00461.x.
http://dx.doi.org/10.1046/j.1439-037x.20...
; Yildiz-Aktas et al., 2009Yildiz-Aktas, L., Dagnon, S., Gurel, A., Gesheva, E., & Edreva,
A. (2009). Drought tolerance in cotton: involvement of non-enzymatic
ROS-scavenging compounds. Journal Agronomy & Crop Science, 195, 247-253.
http://dx.doi.org/10.1111/j.1439-037X.2009.00366.x.
http://dx.doi.org/10.1111/j.1439-037X.20...
), it seems that in the present study
the cell membrane of sunflower plants was in some way protected against the
oxidative stress induced by water stress under foliar application of 10 μM SNP.
Plants respond to stress through changes in gene expression that might lead to the
production of antioxidants and allowing recovery of growth (Xiong & Zhu, 2002Xiong, L., & Zhu, J. K. (2002). Molecular and genetic aspects of
plant responses to osmotic stress. Plant, Cell & Environment, 25, 131-139.
http://dx.doi.org/10.1046/j.1365-3040.2002.00782.x.
PMid:11841658
http://dx.doi.org/10.1046/j.1365-3040.20...
). The elimination of ROS is achieved
mainly by antioxidant compounds and by ROS-scavenging enzymes. Plants contain
substantial amounts of carotenoids that serve as non-enzymatic scavengers of active
oxygen species and the high carotenoid levels have been suggested to be a measure of
drought tolerance (Chandrasekar et al.,
2000Chandrasekar, V., Sairam, R. K., & Srivastava, G. C. (2000).
Physiological and biochemical responses of hexaploid and tetraploid wheat to
drought stress. Journal Agronomy & Crop Science, 185, 219-227.
http://dx.doi.org/10.1046/j.1439-037x.2000.00430.x.
http://dx.doi.org/10.1046/j.1439-037x.20...
). In this study, the level of carotenoids was not altered by neither
water stress nor water stress plus SNP applications (Table 1).
Proline was found to be the major non-enzymatic antioxidant metabolite under water
stress conditions, resulting in stress tolerance as a consequence of its
ROS-scavenging ability (Guha et al., 2012Guha, A., Sengupta, D., Rasineni, G. K., & Reddy, A. R. (2012).
Non-enzymatic antioxidant defence in drought-stressed mulberry ( L.) genotypes.
Morus indicaTrees, 26, 903-918.
http://dx.doi.org/10.1007/s00468-011-0665-4.
http://dx.doi.org/10.1007/s00468-011-066...
).
It has also been shown that water stress tolerance can be induced by exogenous NO
which was attributed to high accumulation of proline in plants (Lei et al., 2007Lei, Y., Yin, C., & Li, C. (2007). Adaptive responses of to
drought stress and SNP application. Populus przewalskiiActa
Physiologiae Plantarum, 29, 519-526.
http://dx.doi.org/10.1007/s11738-007-0062-1.
http://dx.doi.org/10.1007/s11738-007-006...
), the high accumulation of
proline was attributed to exogenous NO on the activity of some key enzymes involved
in the synthesis of proline (Zhang et al.,
2008Zhang, L. P., Mehta, S. K., Liu, Z. P., & Yang, Z. M. (2008).
Copper-induced proline synthesis is associated with nitric oxide generation in .
Chlamydomonas reinhardtiiPlant & Cell Physiology, 49,
411-419. http://dx.doi.org/10.1093/pcp/pcn017. PMid:18252734
http://dx.doi.org/10.1093/pcp/pcn017...
). Restriction of water supply for four days resulted in an increase
in proline content of about 76% when compared with well watered plants withought SNP
(Figure 1a). A further increase in proline
accumulation of about 39% was observed in response to application of 10 μM SNP in
comparison with water stressed plant plus 0 and 1 μM SNP. At 100 μM SNP the content
of proline was decreased to values similar to water stress without or with 1 μM SNP.
Although it was demonstrated that NO reduces hydrogen peroxide accumulation under
water stress (Sang et al., 2008Sang, J., Jiang, M., Lin, F., Xu, S., Zhang, A., & Tan, M.
(2008). Nitric oxide reduces hydrogen peroxide accumulation involved in water
stress-induced subcellular anti-oxidant defense in maize plants. Journal of
Integrative Plant Biology, 50, 231-243.
http://dx.doi.org/10.1111/j.1744-7909.2007.00594.x.
PMid:18713446
http://dx.doi.org/10.1111/j.1744-7909.20...
), it is well
known that NO action can lead to opposite effects depending on its concentration
(Tian & Lei, 2006Tian, X., & Lei, Y. (2006). Nitric oxide treatment alleviates
drought stress in wheat seedlings. Biologia Plantarum, 50, 775-778.
http://dx.doi.org/10.1007/s10535-006-0129-7.
http://dx.doi.org/10.1007/s10535-006-012...
). Plants control
the level of ROS by several antioxidant enzymes such as pirogalol peroxidase
(PG-POD) and superoxide dismutase (SOD) which are responsible for scavenging
accumulated ROS. This ability to control the level of ROS is important in drought
tolerance in arid and semiarid regions (Ghahfarokhi
et al., 2015Ghahfarokhi, M. G., Mansurifar, S., Taghizadeh-Mehrjardi, R.,
Saeidi, M.; Jamshidi, A. M., & Ghasemi, E. (2015). Effects of drought stress
and rewatering on antioxidant systems and relative water content in different
growth stages of maize ( L.) hybrids. Zea maysArchives of
Agronomy and Soil Science, 61, 493-506.
http://dx.doi.org/10.1080/03650340.2014.943198
http://dx.doi.org/10.1080/03650340.2014....
). Water stress withought SNP did not affect the activity of
SOD nor did the application of SNP under water stress (Figure 1c). The Figure 1b
demonstrates that water stress withought SNP significantly decreased the activity of
PG-POD, while the application of 10 μM SNP under water stress increased the activity
of this enzyme. Thus, NO can effectively protect plants from damage probably by
enhancing the activities of antioxidant enzymes (Boogar et al., 2014Boogar, A. R., Salehi, H., & Jowkar, A. (2014). Exogenous nitric
oxide alleviates oxidative damage in turfgrasses under drought stress. South
African Journal of Botany, 92, 78-82.
http://dx.doi.org/10.1016/j.sajb.2014.02.005.
http://dx.doi.org/10.1016/j.sajb.2014.02...
) or acting as a potent antioxidant in plants (Beligni & Lamattina, 2002Beligni, M. V., & Lamattina, L. (2002). Nitric oxide interferes
with plant photo-oxidative stress by detoxifying reactive oxygen species. Plant,
Cell & Environment, 25, 737-748.
http://dx.doi.org/10.1046/j.1365-3040.2002.00857.x.
http://dx.doi.org/10.1046/j.1365-3040.20...
). In addition,
it is clear in the present study that NO also can protect the plants by enhancing
the levels of proline.
4 CONCLUSION
This study show that the adverse effects of water stress on sunflower plants are dependent on the external NO concentration. A single application of 10 μM SNP as NO donor at the beginning of water stress proved to be beneficial in alleviating the negative effects of water stress on membrane integrity as seen by lower levels of MDA. The action of NO may be explained by its ability to increase the levels of antioxidant compounds and to increase the activity of ROS-scavenging enzymes.
ACKNOWLEDGEMENTS
The authors thank the Brazilian agencies FAPESP and FUNDUNESP for Financial support and Agronomic Institute of Campinas (IAC) that supplied the seeds.
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» http://dx.doi.org/10.1046/j.1365-3040.2002.00782.x - Yang, W., Sun, Y., Chen, S., Chen, F., Fang, W., & Liu, Z. (2011). The effect of exogenously apllied nitric oxide on photosynthesis and antioxidant activity in heat stressed chrysanthemum. Biologia Plantarum, 55, 737-740. http://dx.doi.org/10.1007/s10535-011-0178-4.
» http://dx.doi.org/10.1007/s10535-011-0178-4 - Yildiz-Aktas, L., Dagnon, S., Gurel, A., Gesheva, E., & Edreva, A. (2009). Drought tolerance in cotton: involvement of non-enzymatic ROS-scavenging compounds. Journal Agronomy & Crop Science, 195, 247-253. http://dx.doi.org/10.1111/j.1439-037X.2009.00366.x.
» http://dx.doi.org/10.1111/j.1439-037X.2009.00366.x - Zhang, L. P., Mehta, S. K., Liu, Z. P., & Yang, Z. M. (2008). Copper-induced proline synthesis is associated with nitric oxide generation in . Chlamydomonas reinhardtiiPlant & Cell Physiology, 49, 411-419. http://dx.doi.org/10.1093/pcp/pcn017. PMid:18252734
» http://dx.doi.org/10.1093/pcp/pcn017
Publication Dates
-
Publication in this collection
June 2015
History
-
Received
08 Oct 2014 -
Accepted
26 Jan 2015