Adaptive significance of photosynthetic and metabolic regulation in Nicotiana tabacum L. plants during drought stress

Abstract

The subject matter covered in this thesis has close affinities with that variously known as wholeplant physiology, environmental plant physiology and crop physiology, although each of these terms mean different things to different people. Emphasis was placed on integration and trying to assess quantitatively the importance of each physiological trait identified which may contribute to the drought tolerance of a tobacco (Nicotiana tabacum L.) cultivar, in the context of the whole plant-environmental system. Four tobacco cultivars, TL33, CDL28, GS46 and ELSOMA, in sequence of increasing drought tolerance, were subjected to intensifying drought stress under controlled environmental conditions by withholding watering. Both photosynthesis related and other drought stress-induced metabolic changes, were comparatively monitored with changes in the severity, duration and time course of the stress. This was done in order to gain more insight into the underlying biochemical mechanisms, which may bestow differential degrees of drought tolerance on tobacco cultivars and which could in practice lead to more clearlydefined quantitative parameters being implemented in drought tolerance selection programmes. When the stomata! and non-stomata! limitations to photosynthesis were evaluated, a drought stress-induced decrease in the carboxylation efficiency (oA/oc) which was less pronounced in the drought-tolerant cultivars, was observed. This coincided with a slower stress- induced increase in the CO 2 compensation point (r) af\d intercellular CO 2 concentration ( c) in the drought-tolerant cultivars, which was due to the maintenance of higher net photosynthetic rates (A). All cultivars showed a decrease in water use efficiency (WUE) and hence an increase in the marginal cost in terms of water used to carbon gained (8E/8A). These changes occurred more slowly in the drought-tolerant cultivars, which was due to the maintenance of higher A values, as stomata! conductance (g) decreased more slowly in spite of higher transpiration rates (E). The relative degree of stomata! limitation (1) did not increase much (ca. 35%), nor differ significantly among the cultivars. The increase in Ci and r was interpreted as indicating that mesophyllic rather than stomata! factors were responsible for the drought stress-induced decrease in A, because of the decrease in the 8A/8 ci component of the mesophyllic photosynthetic capacity. From the results obtained when the partial photochemical reactions were determined polarographically with isolated thylakoids, it became clear that in all four cultivars drought stress induced a decline in the quantum efficiency (<1>), PSI-, PSII-, and PSI + PSII-activit y. The nature and extent of this decline, however, differed between the respective cultivars. The <t> , P SII- and PSI + PSII- activity of the drought-tolerant cultivars were characterised by a fast initial decline, which subsequently declined at a slower continuous rate as the drought stress intensified. PSII- 4 activity was found to be more drought sensitive than PSI-activity, and even more so in the case of the drought-sensitive cultivars. The observation that PSII-activity was most affected by drought stress is corroborated by the fluorescence data which indicated that the I/P-ratio and M- values of the drought-sensitive cultivars increased earlier and reached higher end values. This indicated that the drought-tolerant cultivars were characterised by a higher photosynthetic efficiency at low 'I' L · On evaluating some of the more biochemical drought stress-induced changes, a progressive highly significant (p < 0.01), but differential, increase in glutathione reductase activity was detected in all four cultivars as their leaf water potential ('I' J decreased. On reaching 'I' L of -2. 51 MP a, the glutathione reductase activity of the drought-sensitive cultivars increased by 159% (TL33) and 187% (CDL28), as opposed to the 233% (GS46) and 250% (ELSOMA) in the drought-tolerant cultivars. In spite of an initial lag (up to a 'I' L of ca.-1.5 MP a), on average, the superoxide dismutase (SOD) activity of the drought-tolerant cultivars increased by 244% while that of the drought-sensitive cultivars only increased by 161%. Contrary to all other enzyme activities monitored, the moderate increase (doubled at most) in catalase activity was more pronounced (p < 0.05) in the drought-sensitive cultivars. Increased ascorbate peroxidase activity was not only observed to be ca. 300-400% higher in the drought-tolerant cultivars under stress, but was also more pronounced than the increase in catalase activity. This seem to indicate that ascorbate peroxidase rather than catalase may be mainly responsible for scavenging drought stress-produced H 20 2 . A statistically significant (p < 0.01) decline in both water soluble protein concentration and total number of -SH groups already occurred at a 'l'L of ca.-0.77 MPa . The initial rate of decline in both the concentration of water-soluble protein and number of -SH groups, as well as the end-values reached by both these parameters, differed significantly between the drought- tolerant and drought-sensitive cultivars. In contrast with the fast initial decline observed in GS46 and ELSOMA at light stress levels, which stabilised and occurred more gradually as the drought stress intensified, the concentration of water-soluble protein and number of -SH groups declined at a slower but continuous rate in TL33 and CDL28 as 'I' L decreased. At a 'I' L of -2 . 51 MP a, the number of -SH groups present in GS46 and ELSOMA respectively, was 63 .6% and 65 .2%, and that of TL33 and CDL28 39.9% and 46.8% of their respective control values. Furthermore, the concentration of water-soluble protein of ELS OMA was still 75 .5% and that of GS46, 65.4% of their controls, even at a 'I' L of ca.- 2. 51 MP a, which contrasts with the corresponding values of 46.6% and 55 .3% for TL33 and CDL28, respectively. The tobacco plants adjusted largely to drought stress by the accumulation of solutes. 5 This resulted in a decrease in osmotic potential at full turgor (\JI It 100 ), which was more pronounced in the drought-tolerant cultivars. Drought stress-induced an increase in the proportion of bound water (B) and the volumetric bulk modulus of elasticity ( E), but did not alter the relative water content at incipient plasmolysis (RWC0). In spite of the latter response, due to the lower E of the drought-sensitive cultivars, the \JI L at which incipient plasmolysis (\JI L0) occurred was consistently less negative in these cultivars. Furthermore, because of their higher E (ca.0.23 MPa for GS46, and 0.28 MPa for ELSOMA) the threshold \JIL values at which both ABA and proline accumulate rapidly occurred earlier in these cultivars. A substantial accumulation of both ABA and proline was observed in all four cultivars, the extent of which in the case of the proline, but not necessarily the ABA, correlated positively with the drought tolerance of the four cultivars. When, with the aid of antibodies produced against a bovine serum albumin -(±)- ABA conjugate, quantitative comparisons of the ABA concentrations in the sub-cellular compartments of GS46 were made prior to and when the plants were drought stressed, at \JI L of ca . -0.45 MPa, a quantitatively similar positive immunogold labelling pattern was observed in both chloroplasts and apoplast. However, at a \JI L of ca. -1. 5 5 MP a a twofold drought stress-induced increase in the apoplastic ABA concentration was observed while the ABA concentration in the chloroplasts did not differ from that of the controls. Results are also presented, which emphasise the adaptive significance of effective leaf movements and indicate that drought stress specifically influence the carotenoid composition. A strong quantitative correlation existed between the formation of zeaxanthin and the type of fluorescence quenching indicative of non-radioactive energy dissipation, which occurred to a lesser extent in GS46 and ELSOMA . Ultrastructural observations, indicated that the drought- tolerant cultivars mobilised a more than adequate starch reserve to a greater extent. Anatomical differences in terms of differences in resistance to water flow and the percentage intercellular spaces exist between the respective cultivars. Both these traits correlated positively with the slower decrease in WUE and faster recovery upon rehydration in the drought-tolerant cultivars of several of the physiological parameters monitored . From the results presented and extensive literature search regarding the physiological changes monitored, which are discussed in relation to their ph ysi ological cost and potential drought tolerance adaptive advantage, it is clear that drought tolerance in tobacco, as is the case in other plants, is the complex consequence of many physiological, biochemical and morphological factors . Furthermore, as each of the traits examined forms part of an integrated plant response, it would seem as if plant breeders should be selecting the best sets of physiologically based adaptive traits for particular environments. It is in this regard that the following general conclusions (the details of which and 6 potential selection value are given in each paper) drawn from this investigation, may be of value: Drought tolerance may be: (i) resistance to (a) stress induced decreases in 8A/c ci, that could result in the maintenance of higher A rates, which is vital for fast recovery upon rewatering, or (b) -SH oxidation or -SH/-SS- interchange as best explained by a combination of the -SH/-SS- hypothesis and the concept of protein turnover, (ii) the consequence of regulatory mechanisms such as , (a) PSII-regulation as reflected by fluorescence changes, or (b) an effective antioxidant system, (iii) a capability to (a) predict (ABA accumulation), (b) postpone (by osmoregulation), or (c) to limit the severity (by pro line accumulation) of the stress, and/or finally (iv) the result of environmentally driven anatomical changes such as (a) differences in vessel length and diameter, or (b) differences in the percentage intercellular spaces.