Keywords: Vaccinium macrocarpon, gas exchange, chlorophyll, salt, stomatal conductance, CO2 assimilation, intercellular CO2. Abstract. Photosynthetic activity ...
Gas Exchange and Chlorophyll Content of Cranberry under Salt Stress P. Jeranyama and C.J. DeMoranville University of Massachusetts-Amherst Cranberry Station East Wareham, MA 02538 USA Keywords: Vaccinium macrocarpon, gas exchange, chlorophyll, salt, stomatal conductance, CO2 assimilation, intercellular CO2 Abstract Photosynthetic activity decreases when plants are grown under saline conditions leading to reduced growth and productivity. Leaf gas exchange and chlorophyll content of ‘Stevens’ cranberry exposed to increasing sodium chloride (NaCl) concentration were measured. Cranberry was grown in a sand based culture under greenhouse conditions. The NaCl concentrations in irrigation water were 0 (control), 82, 164 and 246 mg.L-1. In addition to salt solutions, plants were fertilized with a slow release NPK fertilizer. Intercellular CO2 concentration in leaves increased with increase in salinity (r=0.72, p ≤ 0.01). Net CO2 assimilation rate decreased by 43%, as exposure rates increased from 0 to 164 mg.L-1NaCl and stomatal conductance relative to the control was reduced by 68% at 164 mg.L-1 NaCl. Stomatal conductance had a negative correlation with intercellular CO2 concentration (r=-0.53, p ≤ 0.1). The ratio of chlorophyll a to chlorophyll b was 1:2 except at 164 mg.L-1NaCl where the ratio was 1:1. The insignificant correlation between stomatal conductance and net CO2 assimilation suggests hat physiological factors other than stomatal conductance were responsible for limiting photosynthesis under salt stress. INTRODUCTION Salinity is one of the most important abiotic stresses limiting crop production in areas with high salt content soils. Cranberry (Vaccinium macrocarpon Ait.) is an important economic fruit in the north coastal regions of the United States as well as in Wisconsin and parts of Canada. The commercial cranberry is indigenous to acidic soils and is found naturally in marshes and river banks of the coastal regions (Kumudini, 2004). When cranberry bogs are constructed near highways there is a concern regarding salt runoff from surfaces treated with de-icing salts into bogs or water holding ponds. Visible damage attributed to road salt from direct deposition or vehicle overspray has been observed along the edges of cranberry bogs bordered by highways. Cranberry damage from salt water incursion during hurricanes has also been documented (Chandler and DeMoranville, 1959). Salinity effects on plants include ion toxicity, osmotic stress, mineral deficiencies, morphological, physiological and biochemical perturbations, and combinations of these stresses (Munns, 1993; Hasegawa et al., 2000). The relative growth rate (RGR), leaf area ratio, or net assimilation rate, an index of the photosynthetic capacity and gas exchange, can be used at the whole plant level to study variations in salt tolerance. Photosynthesis is an important parameter used to monitor plant response to abiotic stress. There is a close association between growth and photosynthetic rate in wheat (Triticum aestivum L.) genotypes differing in salt tolerance (El-Hendawy et al., 2005). Salinity stress results in the reduction of plant photosynthesis through stomatal and nonstomatal factors, although the latter are not yet fully understood (Dionisio-Sese and Tobita, 2005; Sharma and Hall, 1991). In a wheat study, El-Hendawy et al. (2005) found that the reduction in stomatal conductance of salt-sensitive cultivars under saline conditions was significantly greater than that of salt tolerant cultivars. Salinity causes high Na+ concentration in plants but also influences the uptake of essential nutrients such as K+ and Ca2+ because of the effect of ion selectivity (Marschner, Proc. IXth IS on Vaccinium Eds.: K.E. Hummer et al. Acta Hort. 810, ISHS 2009
753
1995). Crop plants use K+, rather than Na+, as an important component of osmotic adjustment and an essential macronutrient. However, K+ and Na+ compete to enter plant cells because they are similar in ionic radius and ion hydration energies (Schachtman and Liu, 1999). Consequently, crops growing in saline conditions may suffer Na+ toxicity and K+ deficiency. Measurement of chlorophyll provides quantitative information about photosynthesis. Previous reports of NaCl effects on chlorophyll in diverse organ, tissue and cell preparations have been inconsistent (Bongi and Loreto, 1989; Misra et al., 2001). There are limited data available on salt effects in cranberry. It is still an open question as to whether salinity directly affects functioning of photosystem II (PSII) or whether stomatal closure is the main factor inhibiting photosynthesis and biomass production under NaCl stress. The objective of this study was to evaluate the effect of increasing NaCl concentrations in irrigation water on cranberry gas exchange, stomatal conductance, chlorophyll concentration and net assimilation rates. MATERIALS AND METHODS ‘Stevens’ cranberry was grown in a sand based culture in the greenhouse. Eight pots of ‘Stevens’ constituted a treatment unit. Each experimental unit was subjected to irrigation water with one of the four levels of NaCl concentrations (dissolved in tap water) in a factorial design replicated five times. The four levels of NaCl concentrations were 0 (control), 82, 164 and 246 mg.L-1. Pots were irrigated between 9am and 10am with prepared salt solutions for six days and on the seventh day all pots received water (no added salt). Cranberry plants were irrigated throughout the growing season with the salt solutions (i.e., at all growth stages). Fruit yield for each treatment was determined (data not reported here) and gas exchange and stomatal conductance were measured at fruit set using an LCi Portable Photosynthesis System (ADC Biosciences, UK). A conifer cuvette (chamber) was clamped onto the top 10 cm of non fruiting cranberry upright, and on fruited uprights, the chamber was clamped immediately above the fruit (about 10 cm). Temperature and vapor pressure deficit (VPD) in the chamber were not controlled. Greenhouse temperature ranged between 21 to 30oC during the day and 12 to 16oC at night. Air flow and CO2 in the greenhouse were maintained at ambient levels. The chamber was clamped around the upright for 5 min, and then measurements (stomatal conductance, intercellular CO2 and net CO2 assimilation) were taken every 15 s until a steady rate (±0.2 µmol.m-2.s-1) was reached. The part of the upright enclosed in the chamber was sampled and used to determine the leaf area that was enclosed and to adjust measurement based on actual leaf area. Leaf area removed per upright measurement was quantified using a leaf area meter (LI-3100; LI-COR, Lincoln, NE). Photosynthesis measurements were replicated six times. To determine chlorophyll concentration, a leaf sample of 50 mg was extracted with 10 ml di-methyl sulfoxide (DMSO) in the dark. A slurry of this extract was filtered and absorbance was determined at 645 and 663 nm. Concentrations of chlorophyll a (Chl a) and chlorophyll b (Chl b) were estimated using the equations of Arnon (1949). Total chlorophyll concentration and the ratio of Chl a to Chl b were also determined. Data were subjected to analyses of variance in Proc GLM of SAS (SAS Institute, Cary, NC) and correlation analysis using Proc CORR of SAS. RESULTS AND DISCUSSION Photosynthesis and Stomatal Conductance Intercellular CO2 concentration increased with increase in NaCl concentration, although the only significant increase was between control and 246 mg.L-1NaCl (Fig. 1A). This significant increase represented a 23% rise in intercellular CO2 concentration in plant cells. Intercellular CO2 concentration was correlated positively (r=0.79, p ≤ 0.01) with NaCl. Net CO2 assimilation rate (photosynthesis rate) decreased by 43% as exposure 754
rates increased from 0 to 164 mg.L-1 NaCl, but increased at 246 mg.L-1NaCl by 42% relative to the control (Fig. 1B). Cranberry plants treated with 246 mg.L-1 NaCl produced higher vegetative biomass (data not shown) compared with other rates, however, this did not necessarily translate into higher fruit yield. The soil water in a bog with an area of dying vines had approximately 700 mg.L-1 Cl in the injured area, but the adjacent ditch water contained only 37 mg.L-1 Cl (Chandler and DeMoranville, 1959). Stomatal conductance had a negative correlation with intercellular CO2 concentration (r=-0.53, p ≤ 0.1). Stomatal conductance relative to the control was reduced by 68% at 164 mg.L-1 NaCl (Fig. 1C). Since there was a significant reduction in photosynthesis (up to 164 mg.L-1) and stomatal conductance under salt stress, it was likely that the decrease in photosynthesis can be attributed to NaCl effects on stomatal closure. Other researchers have reported decreasing photosynthesis and stomatal conductance with NaCl (Netondo et al., 2004; Zhao et al., 2007). They found a positive correlation between stomatal conductance and CO2 assimilation rate under salinity stress, suggesting that stomatal conductance was a primary factor limiting photosynthesis in their test crops. However, in the present study we could not attribute stomatal conductance to be the sole cause for photosynthesis limitation, particularly for the highest salt rate. It is possible that at exposure up to 164 mg.L-1 NaCl, stomatal factors are important. We propose that beyond 164 mg.L-1 nonstomatal factors were responsible for limiting photosynthesis. The high photosynthetic rates at 246 mg.L-1 are expected to be a result of new vegetative growth, although this did not result in high fruit yield (data not shown). Non-stomatal effects are biochemical in nature and are often analyzed in terms of ribulose bisphophate carboxylase oxygenase (RuBP) activity and the regeneration of RuBP. For example, Faver et al., (1996), working with cotton (Gossypium hirsutum L.), concluded that non-stomatal factors were largely responsible for small reductions in CO2 assimilation rate under mild stress, but stomatal factors became more important than nonstomatal factors as stress became more severe. In contrast, Flexas and Medrano (2002) concluded that stomatal closure was the earliest plant response to mild stress and that with increased stress, the progressive down-regulation or inhibition of metabolic processes or non-stomatal effects, lead to decreased RuBP regeneration and inhibition of CO2 assimilation rate. Thus the relative importance of stomatal vs. non-stomatal limitations remain unclear and the discussion has become polarized (Lawlor, 2002). Chlorophyll Concentration Our data did not show a clear trend in chl a or chl b, but at 164 mg.L-1 NaCl, concentration of chl a and chl b were similar resulting in chl a/b ratio of 1:1 (Table 1). Most plant species have a chl a:b ratio in the range of 3:1 or 4:1. In our study the highest chl a:b ratio was almost 2:1. Chlorophyll measurements in this experiment were conducted near the time of harvest, possibly affecting levels of leaf pigments. Cranberry leaves usually turn red after harvest, which might be an indication of active accumulation of anthocyanins and may attenuate the quality and quantity of light captured by chlorophyll and carotenoids. The major physiological activity at this point is nutrient resorption into the stems and roots (Matile, 2000; Field et al., 2001). The small changes in chlorophyll indicate little reorganization in chlorophyll and the chlorophyll-protein complexes in the thylakoids (Wells, 2001). CONCLUSION The lack of correlation between stomatal conductance and CO2 assimilation rate suggests that other physiological or biochemical factors were responsible for limiting photosynthesis under salt stress in cranberry. The small changes in chlorophyll concentrations under salt stress indicated little reorganization in chlorophyll and the chlorophyll-protein complexes in the thylakoids. It is also proposed that the low chlorophyll concentration in the study was as a result of active synthesis of anthocyanins.
755
At levels below 164 mg.L-1 NaCl we do not expect major changes in cranberry photosynthesis rate. ACKNOWLEDGEMENTS Funding by the Cape Cod Cranberry Growers Association (CCCGA) and the UMASS Amherst, NRE Start-up Funds is sincerely acknowledged. Literature Cited Arnon, D.I. 1949. Copper enzyme in isolated chloroplasts. 1. Poplyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15. Bongi, G. and Loreto, F. 1989. Gas exchange properties of salt-stressed olive (Olea europea L.) leaves. Plant Physiol. 90:1408-1416. Chandler, F.B. and DeMoranville, I.E. 1959. The harmful effect of salt on cranberry bogs. Cranberries 24:6-9. Dionisio-Sese, M.L. and Tobita, S. 2005. Effects of salinity on sodium content and photosynthetic responses of rice seedlings differing in salt tolerance. J. Plant Physiol. 157:54-58. El-Hendawy, S.E., Hu, Y. and Schmidhalter, U. 2005. Growth, ion content, gas exchange, and water relations of wheat genotypes differing in salt tolerances. Aust. J. Agric. Res. 56:123-134. Faver, K.L., Gerik, T.J., Thaxton, P.M. and El-Zik, K.M. 1996. Late season water stress in cotton: II. Leaf gas exchange and assimilation capacity. Crop Sci. 36:922-928. Field, T.S., Lee, D.W. and Holbrook, N.M. 2001. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiol. 127:566-574. Flexas, J. and Medrano, H. 2002. Drought-inhibition of photosynthesis in C3 plant: Stomatal and non-stomatal limitations revisited. Ann. Bot (Lond.) 89:183-189. Hasegawa, P.M., Bressan, R.A., Zhu, J.K. and Bohnert, H.J. 2000. Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Mol. Biol. 51:463499. Kumudini, S. 2004. Effect of radiation and temperature on cranberry photosynthesis and characterization of diurnal change in photosynthesis. J. Amer. Soc. Hort. Sci. 129:106111. Lawlor, D.W. 2002. Limitation to photosynthesis in water-stressed leaves: Stomata vs. metabolism and the role of ATP. Ann. Bot. (Lond.) 89:871-885. Marschner, H. 1995. Mineral nutrient of higher plants. Academic press, London. Matile, P. 2000. Biochemistry of Indian summer: physiology of autumn leaf coloration. Exp. Gerontology 35:145-158. Misra, A.N., Srivastava, A. and Strasser, R.J. 2001. Utilization of fast chlorophyll fluorescence technique in assessing the salt/ion sensitivity of mung bean and Brassica seedlings. J. Plant Physiol. 158:1173-1181. Munns, R. 1993. Physiological processes limiting plant growth in saline soils. Some dogmas and hypotheses. Plant Cell Environ. 25:239-250. Netondo, G.W, Onyango, J.C. and Beck, E. 2004. Sorghum and salinity: Gas exchange and chlorophyll fluorescence of sorghum under salt stress. Crop Sci. 44:806-811. Schachtman, D.P. and Liu, W.H. 1999. Molecular pieces to the puzzle of the interaction between potassium and sodium uptake in plants. Trends Plant Sci. 4:281-287. Sharma, P.K. and Hall, D.O. 1991. Interaction of salt stress and photoinhibition on photosynthesis in barley and sorghum. J. Plant Physiol. 138:614-619. Wells, R. 2001. Leaf pigment and canopy photosynthetic response to early flower removal in cotton. Crop Sci. 41:1522-1529. Zhao, G.Q., Ma, B.L. and Ren, C.Z. 2007. Growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. Crop Sci. 47:123131.
756
Tables
Table 1. Effects of increasing NaCl concentration in irrigation water on Chl a, Chl b and ratio of Chl a:b in cranberry. NaCl Mg.L-1 0 82 164 246 LSD 5%
Chl a Chl b ------------ mg.g-1 FW ---------0.49 0.29 0.60 0.35 0.16 0.16 0.51 0.34 0.24 0.12
Ratio of Chl a/b 1.67 1.72 1.00 1.50 0.56
757
Figures
A
B
C
Fig 1. Effects of increasing NaCl concentration in irrigation water on intercellular CO2 concentration (A), CO2 assimilation rate (B) and stomatal conductance (C) in cranberry.
758
