G.H.J. Krüger

Proline Accumulation as Drought-tolerance Selection Criterion: its Relationship to Membrane Integrity and Chloroplast Ultrastructure in Nicotiana tabacum L.

Summary The value of proline accumulation as a criterion in selecting for drought-tolerance, was evaluated in four tobacco cultivars of differing drought-tolerance. Proline determination, measurement and calculation of a membrane integrity index (MII), and ultrastructural observations were conducted simultaneously under controlled environmental conditions during the stress period. Water stress of increasing severity (0.2-0.3 MPad-1) that ranged from light (-0.52MPa) to severe (-2.51 MPa) was induced by withholding water. A substantial accumulation of proline was observed in all four cultivars, the extent of which correlated positively with their individual drought-tolerance. Ultrastructural observation indicated that the drought-tolerant cultivars mobilized the, more than adequate, store of starch to a greater extent than the drought-sensitive cultivars during stress. Water stress-induced membrane damage occurred earlier and was much more severe in the drought-sensitive cultivars, as their MII-values already rose to between 20.4% and 23.4% at a ΨL of -1.27 MPa, while those of the drought-tolerant cultivars only reached values of 18.5% and 20.9% at a ΨL of -1.67 MPa. The ΨL at which proline levels start to increase dramatically (the shorter the response time the better) and the end concentrations of proline accumulated, are advocated as criteria to be used in selecting for drought-tolerant tobacco genotypes as early as the F1- or F2-generations.

Evaluation of components of oxidative stress metabolism for use in selection of drought tolerant cultivars of Nicotiana tabacum L.

Summary Effect of drought stress on lipid peroxidation, chlorophyll stability and several antioxidant enzyme activities was evaluated under controlled environmental conditions, for possible use as drought tolerance selection criteria, in four tobacco ( Nicotiana tabacum L.) cultivars of different, but known, drought tolerance. A progressive highly significant (p L of -1.5 MPa), on average, the superoxide dismutase (SOD, EC 1.15.1.1) activity of the drought-tolerant cultivars increased by 244% while that of the drought-sensitive cultivars only increased by 161 %. Contrary to the other enzyme activities monitored, only a moderate increase in catalase (EC 1.11.1.6) activity (p L of -1.5 MPa) in the drought-tolerant cultivars under stress, but was also more pronounced than the increase in catalase activity. This indicated that ascorbate peroxidase rather than catalase might be mainly responsible for scavenging drought-stress produced H 2 O 2 . On reaching a Ψ L of -2.5 MPa, the glutathione reductase activity of the drought-sensitive cultivars increased by only 159% (TL33) and 187% (CDL28), opposed to the 233% (GS46) and 250% (ELSOMA) in the drought-tolerant cultivars. A differential (significantly higher in the drought-tolerant cultivars at aΨL of -2.5 MPa) drought stress-induced increase in the level of lipid peroxidation occurred in all cultivars which decreased faster in the drought-tolerant cultivars upon rehydration. The latter was also observed for catalase and glutathione reductase activity, but of which the activity returned to levels comparable with that of their respective controls. The levels of SOD and ascorbate peroxidase activity on the other hand remained higher (p a/b ratio. These results are discussed in relation to the potential drought tolerance adaptive advantage of an effective antioxidant system. In this regard the use of monitoring the capacity to increase ascorbate peroxidase activity and/ or glutathione reductase activity as possible drought tolerance selection criteria in tobacco is advocated.

Comparative Analysis of Differential drought Stress-induced Suppression of and Recovery in Carbon Dioxide Fixation: Stomatal and Non-stomatal Limitation in Nicotiana tabacum L.

Summary Observed differences in drought tolerance in C 3 plants have in the past been incorrectly ascribed to differences in water use efficiency (WUE) and low CO 2 compensation points (Γ). When these parameters thus were applied as screening procedures for cultivars with higher net photosynthetic rates (A) and/or lower photorespiration rates, they have proved to be invalid. To clarify this discrepancy, the stomatal and non-stomatal limitations to photosynthesis were evaluated in four tobacco cultivars of different, but known, drought tolerance. Experiments were carried out under controlled environmental conditions at increasing drought stress and upon rewatering. A drought stress-induced decrease in the carboxylation efficiency (∂A/a∂c i ) ;which was less pronounced in the drought-tolerant cultivars, was observed in all four cultivars. This coincided with a slower stress-induced increase in the Γ and intercellular CO 2 concentration (c i ) in the drought-tolerant cultivars, which was due to the maintenance of higher A rates. All cultivars showed a decrease in WUE and hence an increase in the marginal cost in terms of water used to carbon gained (∂E/∂A). These changes occurred more slowly in the drought-tolerant cultivars, which was due to the maintenance of higher A values, as stomatal conductance (g) decreased more slowly in spite of higher transpiration rates (E). As the relative degree of stomatal limitation (l) did not increase by much, (ca. 35 %) nor differ significantly among the cultivars, the increase in c i and Γ was interpreted as indicating that mesophyllic rather than stomatal factors were responsible for the drought stress-induced decrease in A, because of the decrease in the ∂A/∂c i component of the mesophyllic photosynthetic capacity. Upon rewatering all the parameters monitored recovered to a greater or lesser extent, though the recovery time needed by the drought-sensitive cultivars were much longer. We conclude that in all cultivars decreasing leaf water potential (Ψ L ) caused simultaneous reductions in g and the biochemical capacity for photosynthesis. The ∂A/∂c i component of the biochemical capacity, however, proved to be the most sensitive to drought stress. Thus, in tobacco, during drought stress diffusion itself only marginally limits A, a fact not widely appreciated. Drought tolerance in the four tobacco cultivars investigated may therefore be resistance to stress-induced decreases in ∂A/∂c i , which in turn could result in the maintenance of higher A rates, which is vital for fast recovery upon rewatering.

Differential Inhibition of Photosynthesis (in vivo and in vitro) and Changes in Chlorophyll a Fluorescence Induction Kinetics of four Tobacco Cultivars under Drought Stress

Summary This study was conducted in an attempt to explain the observed difference in drought tolerance of four cultivars of Nicotiana tabacum L., by monitoring plant water status and several aspects of photosynthesis, including chlorophyll a fluorescence, under controlled environmental conditions. Drought stress of increasing intensity, ranging from light to severe, was induced over a twelve-day period by withholding water. Photosynthetic oxygen evolution was monitored in vivo at a CO 2 concentration exceeding 5 % to exclude any stomatal limitation and was expressed as quantum efficiency (Φ). Partial photochemical reactions were determined polarographically with isolated thylakoids of the drought-stressed tissue. From the Kautsky curves the I/P-ratio and ΔF-values were determined at various decreasing leaf water potentials (Ψ L ). All four cultivars exhibited a drought stress-induced decline in Φ, PSI-, PSII- and PSI + PSII-activity, but the extent and nature differed among the different cultivars. The Φ-values of the drought-tolerant cultivars were characterized by a fast initial decline that stabilized as the Ψ L declined, whereas the Φ-values of drought-sensitive cultivars did not show a similar response, but declined at a slow continuous rate as the drought stress intensified. The PSII-activity and PSI- + PSII-activity were influenced in much the same way. PSII-activity was found to be more sensitive than PSI-activity and even more so in the case of the drought-sensitive than in the drought-tolerant cultivars. It was concluded that the observed stress-induced decline in Φ and PSI- + PSII-activity could largely be ascribed to an inhibition of PSII-activity. This conclusion is corroborated by both the fluorescence data regarding the I/P-ratio and ΔF-values, of which the former was found to increase earlier and reach higher end-values in the drought-sensitive cultivars, and the latter, which indicated that the drought-tolerant cultivars were characterized by a higher photosynthetic efficiency at low Ψ L . A mechanism is proposed which might retard or reverse drought stress induced stromal acidification and might explain the apparently higher photosynthetic dehydration tolerance of the drought-tolerant cultivars. The possible involvement of ABA is discussed.

Drought tolerance, traditional crops and biotechnology: breeding towards sustainable development

Germplasm of traditional vegetable crops were screened for drought tolerance. Different physiological, morphological and biochemical traits of drought tolerance were investigated, including enzymes of the antioxidative pathway (SOD, AP and GR), turgor maintenance (LWP, RWC), membrane stability (CMS, TTC), osmoprotection (proline), productivity (photosynthesis), rooting (root architecture), early drought tolerance and leaf area. Different in vitro screening techniques were carried out. Useful traits of drought tolerance were identified in Amaranthus tricolor, A. hybridus, A. hypochondriacus, Vigna unguiculata and V. subterranea, and are currently being applied in a breeding programme in an attempt to develop tolerant genotypes of neglected vegetable and seed crops that might contribute to secure food production in rural areas in Africa.

Immunogold Localization and Quantification of Cellular and Subcellular Abscisic Acid, Prior to and During Drought Stress

An immunogold labeling procedure and experimental data are presented, which demonstrate that antibodies produced against a bovine serum albumin-abscisic acid conjugate can be used both to characterize the cellular and subcellular localization of abscisic acid (ABA), and to permit quantitative comparisons of this hormone in the subcellular compartments prior to and at times of drought stress. At the control leaf water potential (approximately -0.45 MPa), a quantitatively similar positive labeling pattern was observed in the chloroplasts and apoplast. A twofold drought stress-induced increase in the apoplastic ABA concentration was observed in the drought stressed leaf tissue (i.e., at a leaf water potential of approximately -1.55 MPa), while the ABA concentration in the chloroplasts did not differ from that of the controls. Three histochemical controls and the physiological observations validated the specificity of the procedure. Based on the labeling patterns we observed and literature cited, the validity of the hypothesis that drought stress induces a release of chloroplastic ABA is questioned. We interpreted our results as providing indirect evidence for a drought stress-induced root source origin for the increased apoplastic ABA concentrations.

Protein turnover: possible contributing factor in determining drought tolerance in Nicotiana tabacum L.

Drought stress-induced changes in the water-soluble protein concentration and total number of sulphydryl (-SH) groups were monitored under controlled environmental conditions in four tobacco cultivars (Nicotiana tabacum L.) with different drought tolerances. The drought stress ranged from light (-0.52 MPa) to severe (-2.5 MPa). A statistically significant decline in both water-soluble protein concentration and total number of -SH groups already occurred at a leaf water potential (ψL of −0.77 MPa. The initial rate of decline in both the concentration of water-soluble protein and the number of -SH groups, as well as the end-values reached by both these parameters, differed significantly between the drought-tolerant (GS46 and ELSOMA) and drought-sensitive (TL33 and CDL28) cultivars. In contrast with the fast initial decline observed in GS46 and ELSOMA at light stress levels, which stabilized and occurred more gradually as the drought stress intensified, the concentration of water-soluble protein and the numb...

Seasonal performance of Quercus robur L. along an urbanization gradient

Some seasonal time-course water relation and gas exchange parameters were monitored under natural conditions in three groups of 80-year-old Quercus robur L. trees growing along an urbanization gradient, in order to explain the positive correlation observed between the degree of die-back and urbanization exhibited by these trees. All three groups of trees exhibited the same general pattern of seasonal variation in preciawn (ψpd) and midday (ψmd) leaf water potential, net photosynthesis (A), transpiration (E) and stomata! conductance (g). i.e. a decrease as the season progressed (from spring to winter). The extent of these decreases were, however, more pronounced in the trees growing at the edge of town (group b) and the urban trees (group c). Unlike the rural trees {group a), which were able to maintain their A/E ratios within 50% of that during spring and were typified by both lower A/g and E/g ratios, the trees of groups b and c were unable to maintain their seasonal A/E ratios and had higher A/g and E/g ratios. The trees of group a maintained higher A rates (accompanied by higher E) and could intercept more radiation. We propose that the trees of groups b and c were dying back because of a reduced water supply, resulting in the cost to gain carbon (because of smaller leaf area, reduced light interception, insect herbivory, stomatal and non-stomatal inhibition) to exceed the cost in terms of available water.

Intercellular space variation among air-cured Nicotiana tabacum L. genotypes and its relation to their water use efficiency

The possible causal relationship between the percentage intercellular spaces in the leaf palisade parenchyma and water use efficiency (WUE) was investigated in related Nicotiana tabacum L. genotypes grown under controlled environmental conditions. Experimental observations included the initial quantification of the percentage intercellular spaces, and the measurement of gas exchange by infra-red gas analysis prior to, during, and after recovery from drought stress. Statistically significant differences in the percentage intercellular spaces per surface area existed between the respective genotypes. The percentage intercellular spaces correlated positively with the pre-stress photosynthetic and transpiration rates, as well as with the WUE of drought-stressed plants, but negatively with the WUE of recovered plants and the rate of stomatal conductance during and after drought stress. It is concluded that in air-cured tobacco, high pre-stress photosynthetic rates are accompanied by high transpiration rates in genotypes with a high percentage of intercellular spaces. This discourages use of the former in drought tolerance selection programmes, as used in the past, while selection on the basis of a smaller percentage intercellular spaces would seem to be a more valid screening parameter. This contention is supported by data which indicate that the genotypes with a high percentage intercellular spaces maintain a higher degree of WUE when drought-stressed by sharply decreasing their stomatal conductance, but, contrary to the genotypes with a lower percentage intercellular spaces, they are unable to reachieve or exceed their pre-stress WUE when rewatered.