L. van Rensburg

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.

Vermicomposting of industrially produced woodchips and sewage sludge utilizing Eisenia fetida.

Adult Eisenia fetida were used to vermicompost woodchips (WC) and sewage sludge (SS) that are produced as waste product by platinum mines. The aims of the study were to examine the growth and reproductive success of the worms over 84 days to determine long-term feasibility of large-scale implementation and monitor the bioconcentration of heavy metals and the effects of microorganisms inoculation to quantify possible environmental implications. Results revealed that there were no effects on growth (P>0.05), reproductive success decreased (P<0.05), and aluminum (Al), copper (Cu), and nickel (Ni) were bioconcentrated (P<0.05) in the treatment groups without an inoculate. Earthworms in the treatment group with the microorganism inoculate manifested no effects on growth or reproductive success and did not accumulate Al, Cu, and Ni. It is concluded that the only economically feasible way to bioconvert WC and SS to a potential ameliorant of platinum mine tailings would be with the addition of a microorganism inoculate.

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.

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.

Bioconversion of sewage sludge and industrially produced woodchips

Platinum mines produce large amounts (750 m3 per month) of woodchips as by-product during ore extraction, posing environmental hazards but also a possible source of organic material to be applied in revegetating tailing dams. The aims of this study were to assess the effectiveness of different bioconversion strategies viz. composting, vermicomposting and a commercial micro-organism inoculant (EM). Woodchips (WC) and sewage sludge (SS) with a mixing ratio of 3:1 were composted and vermicomposted for 112 days and the generated products evaluated in terms of their chemical and physical characteristics. Despite low temperatures (33 °C) in all the treatments, no Escherichia coli or Salmonella was detected in any of the end-products. No significant (P ≥ 0.05) difference in the mean percentage change of C in the different bioconversion strategies was observed and it was only in the SS+WC and SS+WC+EM groups that the soil available P (P-Bray 1) increase was statistically significant (P ≤ 0.05). Treatments containing SS showed significant (P ≤ 0.05) decreases in NH4+, whereas NO2- and NO3-increased significantly (P ≤ 0.05). The NH4+:NO3- ratios in the treatments containing SS were lower than 0.16, ranging from 0.011-0.0016, which is an indication of the maturity of the compost. Total solids and ash contents showed an increase, while the volatile solids and the lignin decreased, but it was only in the vermicomposted treatments that these changes were statistically significant (P ≤ 0.05). The percentage neutral detergent fibre (%NDF) and %cellulose decreased significantly (P ≤ 0.05) in all the treatments containing SS and particle size analysis indicated higher reductions in the vermicomposted treatments. It is concluded that vermicomposting of industrially produced woodchips and sewage sludge is more effective than composting and that the addition of a microbial inoculant did not have a significant effect on the decomposition process.

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.

An investigation into the problems associated with revegetating chrysotile tailings

The present investigation was initiated because of the limited success achieved in the past at attempts to revegetate chrysotile tailings with their associated inherent environmental risk factors. The current set of experiments and surveys, as with several previous studies, were conducted at Msauli chrysotile mine, Mpumalanga Province, South Africa. All soil and leaf samples were collected, and the total percentage canopy cover, species frequency, percentage cover per species, percentage alive biomass, and phenology per species quantified. This was done in nine randomly selected 1m2 areas, on both the upper (steeper) and lower (less steep) slopes of previously revegetated slopes, with comparable samples from areas within the native vegetation serving as controis. Nine composite (consisting on average of three 500g samples) replicate soil samples per slope gradient, as well as leaf samples (ca. 5g of dried above ground leaf material) representative of the species composition, were sampled in the same plots...

Rehabilitation of co-disposed kimberlite tailings: Quantifying physical and chemical properties from a rehabilitation perspective

Mine rehabilitation is a complex process and the potential of revegetating kimberlite tailings (after being co-disposed) was evaluated in this study, with regard to physical and chemical properties. High water soluble Na concentrations (8.22 mmol -1) represent the most serious challenge for successful vegetation establishment, as well as low Ca (0.27 mmol l-1) and Mg (0.44 mmol l-1). Potentially chelatable manganese Mn (0.43 mmol l-1) poses slight limitations that can be overcome by means of inorganic ameliorant application. The co-disposed material does not pose any serious health risks, despite slightly elevated Al (2.89 mmol l-1), Se (1.56 mmol l-1) and As (1.15 mmol l-1) concentrations. Another concern is the expansive and shrinking properties of the high smectite clay fraction (81%) of the tailings. It was concluded that, with the exception of certain chemical difficulties that can be addressed, the tailings material constitutes a growth medium that can be rehabilitated to sustain vegetation over the long term.

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...

Determination of Epidermal Transpiration in Four Cultivars of Nicotiana Tabacum L. Using Epidermal Strips in a Quasi-Steady State System

A quasi-steady state method is presented for quantifying epidermal transpiration of epidermal strips where simple relations between transmembrane fluxes and parameters of diffusibility of penetrating compounds hold. Contrary to most permeability studies, we did not use astomatous, enzymatically isolated, or dried cuticular membranes, because these procedures are largely responsible for the problems cited in the literature. Instead, we used freshly harvested stomatous epidermal strips, thus avoiding the sorption of lipids by the cuticular membranes during enzymatic isolation. Our approach allowed estimation of amounts and composition of intracuticular soluble lipids. Diffusion coefficients (D-values) were calculated with smaller associated standard deviations and an order of magnitude lower than previously reported; the fresh material sorption of the diffusing compound by the membrane and hydration of the cuticular pores was greatly reduced. In the present study the hold-up time (te) ranged from 66.2+/-0.3 to 110.3+/-0.9 sec. Furthermore, 0.1 microm thick membranes were used, contrary to previous studies of water permeability that used cuticles more than 2 microm thick. Because a small but constant flow of penetrant could be detected during the first half of the steady flow to te, small holes probably did not influence the reported permeability. Permeability coefficients (Pd) in the order of 0.65 x 10(-9) ms(-1) were calculated. Pd values in the order of 5.68 x 10(-3) ms(-1) were calculated when incomplete stomatal closure occurred, while when areas of mass flow were detected, Pd values in the order of 1.26 x 10(-2) ms(-1) were calculated. The degree of contamination of the epidermal strips by cellular debris was quantified and expressed as the total chlorphyll content per exposed surface area of the epidermal strip, and an average of 8.7% contamination was observed compared to the total leaf chlorophyll content. Leakage from the system was calculated to be approximately 0.18 x 10(-10) ms(-1), which represents an average 2.7% experimental variability. These results are discussed in terms of the limitations associated with using composite membranes that are stomatous and have trichomes, for possible application in drought tolerance selection.