In this study adaptations of bryophytes and lichens to desiccation stress were examined. The aim was to test whether desiccation tolerance in the selected species is constitutive or if desiccation tolerance could be induced by various hardening treatments. In addition, some putative tolerance mechanisms were investigated, including the accumulation of sugars, increase in ROS scavenging systems and other mechanisms e.g. energy dissipating processes. To determine if hardening treatments prior to desiccation stress increased desiccation tolerance, mosses and lichens were partially desiccated or treated with ABA. The effect of hardening treatments on the physiology of the moss Atrichum androgynum and lichens Peltigera polydactyla, Ramalina celastri and Telochistes capensis during a desiccation-rehydration cycle was investigated. Photosynthesis, respiration and chlorophyll fluorescence measurements were used as rapid tools to determine the metabolic activities in these lichens and moss species. In A. androgynum partial desiccation following slow drying at 52% RH increased the rate of recovery of net photosynthesis. Net photosynthesis recovered almost completely following slow drying in the material that was partially dehydrated and/or treated with ABA. This suggests that partial dehydration hardens the moss, and that ABA can fully substitute for partial dehydration. In R celastri and P. polydactyla both partial dehydration and ABA treatments displayed some improvement in desiccation tolerance depending on the duration and severity of stress. The reduction in the re-saturation respiration burst in P. polydactyla, although not quite significant, strongly suggests that hardening increases mycobiont tolerance. However, it is more difficult to establish whether the hardening treatments improve photobiont performance. In the moss A. androgynum ABA treatment increased the rate of recovery of photosynthesis and PSII activity, and also doubled non-photochemical quenching (NPQ). Increased NPQ activity will reduce ROS formation, and may explain in part how ABA hardens the moss to desiccation. In ABA treated, but not untreated mosses, desiccation significantly increased the concentration of soluble sugars in A. androgynum. Sugar accumulation may promote vitrification of the cytoplasm and protect membranes during desiccation. Starch concentrations in freshly collected A. androgynum and R. celastri were only c. 40 and 80 mg g ¯¹ dry mass respectively, and slightly rose during desiccation, but were only slightly affected by ABA pretreatment. ABA did not reduce chlorophyll breakdown during desiccation. In P. polydactyla ABA pretreatment had little effect on any of these parameters. Changes in the activities of the free radical scavenging enzymes ascorbate peroxidase, catalase and superoxide dismutase were measured during wetting and drying cycles in the moss A. androgynum and in the lichens P. polydactyla, R. celastri and T capensis. These species normally grow in the understorey of the Afromontane forest, moist, xeric, and extremely xeric miicrohabitats respectively. In A. androgyum, enzyme activity was measured shortly after collection, after 3 d storage following hardening by partial dehydration and/or 1 h treatment with ABA or distilled water and during desiccation and rehydration. In A. androgynum enzyme activities of CAT and SOD in untreated material were always higher than in the hardened treatments, while both partial dehydration and ABA treatments tended to reduce both CAT and the induction of SOD activity, although these effects were not significant between the treatments. This suggests that ABA may not be involved in the induction of free radical scavenging enzymes and probably these enzymes are not important in desiccation tolerance of A. androgynum. In lichens, the enzyme activity was measured shortly after collection, after hydration for 48 hat 100% RH, after desiccation for 14 d and 28 d, and during the first 30 min of hydration with liquid water. Enzyme activities tended to rise or stay the same following rehydration in all the species tested. After desiccation for 14 d, enzyme activities decreased, and then decreased further to very low values after 4 weeks desiccation. In all species, including T capensis from an extremely xeric habitat, the activities of all enzymes remained at very low values during the 30 min following rehydration, and were therefore unavailable to remove any reactive oxygen species accumulating in lichen tissues as a result of desiccation. Results suggests that the enzymic antioxidants are more likely to be involved in removing reactive oxygen species produced during the normal metabolic processes of lichens than having a role in desiccation tolerance. The Afromontane understorey moss Atrichum androgynum displayed an oxidative burst of H₂O₂ during rehydration following desiccation. Maximum rates of H₂O₂ production occur during the first 15 min of rehydration. While the production of H₂O₂ increases with increasing desiccation times, the moss produced significant amounts of H₂O₂ during rehydration after desiccation for times that did not inhibit photosynthesis or cause K⁺ leakage. A. androgynum may produce more H₂O₂ during desiccation than rehydration, because desiccation artificially induced using polyethylene glycol strongly stimulates production. Experiments involving inhibitors and exogenously supplied reductants indicate that peroxidases are responsible for the synthesis of H₂O₂. Factors that influence the rate of H₂O₂ production during rehydration include light and the hormone ABA. Patterns of H₂O₂ production are discussed in terms of their possible role as a defence against pathogenic fungi and bacteria. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

y-Aminonbutyric acid (GABA) is a ubiquitous non-protein amino acid found ill many plants and organisms. GABA accumulation in plants has previously been reported as result of stresses such as water deprivation, high salinity and temperature extremes. It is thought that GABA accumulates as a compatible solute in the cytoplasm where it becomes a major constituent of the free amino acid pool. GABA is synthesised from the decarboxylation of glutamate by glutamate decarboxylase (GDC). In some plants, GDC is activated by the lowering of the cytoplasmic pH and the presence of calmodulin and Ca²+ A calmodulin-induced activation of may be due to the physiological factors and environmental stimuli acting in concert leading to the synthesis and accumulation of GABA. The GABA content of Arabidopsis thaliana var. Columbia (L) Heynh leaves was found to increase by over 130% due to water deprivation. NaCl concentrations of up to 100 mM seemed to cause GABA accumulation due to a decrease in osmotic potential. Concentrations of NaCl above 100 mM probably caused GABA accumulation due to combined hyperosmosis and salt toxicity effects. The high levels of GABA in the leaves were maintained throughout a 24 h stress-application period, consistent with its role as compatible solute. The accumulation of GABA followed by its decline in the dark could be attributed to its rapid metabolism because of an active GABA shunt. This is in contrast to the absence of major variations in the amount of GABA in the light confirming its decreased role as a channel for the glutamate carbon and nitrogen under such conditions. A substantial increase in the GABA content was followed by a dramatic decrease in the last 12 h of incubation. This profile of GABA could support its proposed role as a temporary sink for nitrogen and carbon from glutamate during environmental stress. Glutamate decarboxylase appears to be encoded by a single gene in the genome of Arabidopsis. Sequence analysis reveals that the protein possesses what could be a carboxy-terminal, calmodulin- binding domain, which is consistent with other glutamate decarboxylases. The 30-amino acid peptide contains a TrpLysLys motif found in some calmodulin targets. The secondary structure predictions of this peptide suggest a potential to form an a- helix which is also consistent with proteins known calmodulin- binding domains. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

In Arabidopsis thaliana (L.) Heyhn, the size of the pool of free proline increases up to 27-fold in response to osmotic stress. The magnitude of this accumulation is dependent upon the rate of imposition of the stress. Numerous reports have suggested a role for proline accumulation as a general adaptation to environmental stress. However, controversy surrounds the beneficial effect of proline accumulation in plants under adverse environmental conditions. Stress-induced proline accumulation in plants occurs mainly by de novo synthesis from glutamate. The final and only committed step of proline biosynthesis in plants is catalysed by Δ¹-pyrroline-5-carboxylate reductase (P5CR). The sequence of an incomplete 999 bp cDNA encoding P5CR from A. thaliana was determined. This enabled a preliminary molecular study of the structure and function of both the gene and the corresponding enzyme. The 999 bp cDNA insert in the clone Y AP057 was sequenced on the sense and antisense strands following subcloning of four sub-fragments in appropriate orientations. Comparison with known plant P5CR sequences revealed that Y AP057 does not encode the first 23 N-terminal amino acids of P5CR from Arabidopsis. However, it does encode the remaining 253 amino acid residues of Arabidopsis P5CR The cDNA Y AP057 is complete on the 3' end as indicated by the presence of a poly(A) tail. The nucleotide sequence determined shows complete homology to the corresponding exons of the genomic copy of a bona fide gene encoding P5CR in A. thaliana (Verbruggen et al, 1993). The only difference observed between the sequence of Y AP057 and that of a cDNA sequenced by these workers is that polyadenylation was initiated seven nucleotides earlier in Y AP057 than in the sequence of the published cDNA. Genomic Southern analysis suggests the presence of only a single copy of the gene encoding P5CR in Arabidopsis. Restriction mapping and sequencing the ends of another incomplete Arabidopsis P5CR cDNA clone FAFJ25 (664 bp) indicated that the regions sequenced were completely homologous to the corresponding portions of Y AP057. Analysis of codon usage in the Arabidopsis gene encoding P5CR revealed it to closely resemble the consensus pattern of codon usage in A. thaliana. This suggests that the gene is moderately. expressed. Expression of the gene encoding P5CR in Arabidopsis is not likely to be subject to translational control. Although P5CR from A. thaliana has a fairly high composition of hydrophobic amino acid residues, it does not possess any stretches of hydrophobic amino acids of sufficient length to act as membrane-spanning domains or to anchor the enzyme in a membrane. Neither does it contain an N- terminal leader sequence capable of directing it to either the plastid or mitochondrion. The enzyme therefore appears to be cytosolic. The nucleic acid and deduced amino acid sequences of Arabidopsis P5CR were compared with those from·eleven other organisms for which P5CR sequences are currently available. Except among the three different plants examined, P5CR sequences displayed less identity at the amino acid level than at the nucleotide level. The deduced amino acid sequence of Arabidopsis P5CR exhibits high similarity to the corresponding genes and amino acid sequences of P5CR from soybean and pea. Lower but significant similarity was observed to the amino acid sequences of P5CRs from human, Saccharomyces cerevisiae and the bacteria Escherichia coli, Pseudomonas aeruginosa, Thermus thermophilus, Mycobacterium leprae; Treponema pallidum and Methanobrevibacter smithii. Similarity was also observed to the translational product of a gene from Bacillus subtilis with high homology to the E. coli proC gene. However, construction of a phenogram indicating the relatedness of the various P5CR enzymes suggests that sequence analysis of this enzyme is not a good indicator of evolutionary relatedness of organisms from different biological kingdoms. Multiple alignment of the twelve known P5CR sequences indicated homology between the sequences across their entire lengths. Homology was particularly high in the C-terminal portions of the P5CRs studied. It is speculated that this region may be of importance in binding of the substrate Δ¹-pyrroline-S-carboxylate (P5C). Another region displaying high sequence conservation was found in the central portion of all P5CRs. All P5CRs studied, with the exception of PSCR from T. pallidum contained an N-terminal domain capable of binding a nicotinamide dinucleotide cofactor. Comparison of this region with consensus sequences for NADH and NADPH binding sites in proteins suggests that NADPH is the preferred reductant used by P5CRs from plants and human. In contrast, the N-terrninal domains of P5CRs from S. cerevisiae, M smithii, T. thermophilus and M leprae display greater similarity to a consensus NADH-binding site. The definite preference of plant P5CRs for NADPH in comparison with NADH suggests that P5CR may be involved in regulating the redox potential within plant cells and that this step in proline biosynthesis from glutamate may be of importance in overall metabolic regulation. Three amino acid residues are universally conserved in all P5CRs studied. All are found within blocks of high sequence similarity. These residues are likely to be of importance in the structure or catalytic mechanism of P5CR. A number of other residues are common to several of the enzymes examined. These may also be of importance in subsequent manipulation of Arabidopsis P5CR at the molecular level. Prediction of the putative secondary structures of A. thaliana, soybean, pea, human and E. coli indicated a high degree of similarity between the enzymes. This was particularly evident in the region of the putative P5C-binding domain. Considerable similarity exists in hydrophobicity profiles of P5CRs from these five organisms. Proline levels in reproductive organs of unstressed Arahidopsis plants were considerably higher than those in vegetative tissues. This suggests differential expression of enzymes involved in proline metabolism in these organs. In situ hybridisation studies indicated an increase in levels of mRNA transcripts encoding P5CR in stem tissues in response to water deprivation stress. Regulation of levels of mRNA transcript encoding P5CR in Arabidopsis therefore appears to be an osmotically sensitive process. Furthermore, this accumulation of transcript occurred in a tissue-specific manner. In particular, an increase in levels of transcript encoding P5CR was observed in the cortical parenchyma, phloem, vascular cambium and pith parenchyma in the vicinity of the protoxylem. The significance of these findings in contributing to a better understanding of the role of proline in adaptation to environmental stress is discussed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.