ALBERT

Notizen: The strategy of ‘complementation by functional sufficiency’ was used to isolate XvVHA-c′′1, a vacuolar adenosine triphosphatase (V-ATPase) proteolipid subunit c′′ homologue from Xerophyta viscosa. XvVHA-c′′1 rescued Escherichia coli srl::Tn10 mutants that were subjected to a 1.2 M sorbitol osmotic stress. Bioinformatics analyses conducted on XvVHA-c′′1 revealed all signature characteristics that are common amongst subunit c homologues, which include the four transmembrane domain motifs and a conserved glutamate residue in the fourth transmembrane domain. XvVHA-c′′1 shares 90.96% identity with the Oryza sativa (japonica) subunit c homologue and 86.67% identity with a putative vacuolar ATP synthase proteolipid subunit c′ from Arabidopsis thaliana, at the amino acid level. Southern hybridization analysis conducted on X. viscosa genomic DNA confirmed the presence of XvVHA-c′′1 in the X. viscosa genome. Northern hybridization analysis was conducted on X. viscosa tissue subjected to NaCl stress, dehydration and − 20°C shock, in response to which upregulated transcript levels of XvVHA-c′′1 were seen. XvVHA-c′′1's functional relevance was established through complementation using a Saccharomyces cerevisiae vma3 knockout.

Notizen: Resurrection plants have the unique capacity to revive from an air-dried state. In order to tolerate desiccation they have to overcome a number of stresses, mechanical stress being one. In leaves of the Craterostigma species, an extensive shrinkage occurs during drying as well as a considerable cell wall folding. Our previous microscopically analysis using immunocytochemistry on the resurrection plant Craterostigma wilmsii, has shown an increase in labelling of xyloglucan and unesterified pectins in the cell wall during drying. In this study, we have undertaken a biochemical approach to separate, quantify and characterize major cell wall polysaccharides in fully hydrated and dry leaves of C. wilmsii. Our results show that the overall cell wall composition of C. wilmsii leaves was similar to that of other dicotyledonous plants with respect to the pectin content. However, the structure of the hemicellulosic polysaccharide xyloglucan was characterized to be XXGG-type. The data also demonstrate marked changes in the hemicellulosic wall fraction from dry plants compared to hydrated ones. The most conspicuous change was a decrease in glucose content in the hemicellulosic fraction of dry plants. In addition, xyloglucan from the cell wall of dry leaves was relatively more substituted with galactose than in hydrated walls. Together these findings show that dehydration induces significant alteration of polysaccharide content and structure in the cell wall of C. wilmsii, which in turn might be involved in the modulation of the mechanical properties of the wall during dehydration.

Notizen: To test the hypothesis that desiccation sensitivity increases with storage time, recalcitrant seeds of Avicennia marina (Forssk.) Vierh. were dehydrated by soaking in polyethylene glycol solutions after inreasing periods of storage. Germination characteristics and the ultrastructure of root primorida were assessed before and after dehydration. Short-term storage enhanced the apparent rate of germination, consistent with the hypothesis that these seeds commence germination in storage. Root primordia of stored seeds initially showed enhanced subcellular activity, including cell division and vacuolation. Increased storage time resulted in the onset and progression of deleterious changes.Newly shed seeds and seeds stored up to the stage of cell division were comparatively resistant to desiccation. As storage time increased, subsequent dehydration caused increasing subcellular damange and consequent reduction in the rates of germination relative to non-dehydrated controls. Ultrastructural results suggest that after the initiation of cell division of seeds in storage, there is a requirement for additional water for the germination process to continue. A model for the behaviour of recalcitrant seeds is proposed.

Notizen: Leaf tensile strength was measured for the drought-tolerant grass Eragrostis curvula and the desiccation-tolerant grass E. nindensis when fully hydrated, partially dehydrated, naturally air-dried, and flash-dried. Leaf tensile strength increased in intact, air-dried leaves of E. curvula but not for similarly treated leaves of E. nindensis. Examination of leaf cross-sections by light microscopy and histochemical staining for lignins failed to show any significant structural differences between the two species in the hydrated state. When leaves were flash-dried, the tensile strength of E. curvula remained unchanged from leaves dried naturally, while there was a marked increase in the tensile strength of flash-dried leaves of E. nindensis. Proton NMR indicated that the desiccation-tolerant E. nindensis retained mobile water when leaf relative water content was less than 20% if dried naturally but not if flash-dried, whereas no mobile water was detected in leaves of E. curvula when dried either naturally or with flash-drying to below 20% relative water content. This behaviour suggests a fundamental difference in strategy for surviving water loss in vegetative tissues between desiccation-tolerant species and drought-tolerant species.

Notizen: Sucrose accumulated during dehydration is a major potential energy source for metabolic activity during rehydration. The objective of the present study was to investigate aspects of leaf sucrose metabolism during the rehydration of desiccation-tolerant Sporobolus stapfianus Gandoger (Poaceae) over a 10-day period. Comparison was then made to sucrose metabolism during the rehydration of both desiccation-tolerant excised leaf material (dehydrated attached to the parent plant) and desiccation-sensitive leaf material (dehydrated detached from the parent plant to prevent the induction of tolerance) over a 48-h period. The pattern of sugar mobilization and glycolytic enzyme activity during the rehydration of the desiccation-tolerant excised leaves was similar to that in leaves attached to the parent plants. Significant breakdown of sucrose was not apparent in the initial phase of rehydration, suggesting the utilization of alternate substrates for respiratory activity. The desiccation-tolerant excised tissues provided a suitable control to compare the metabolism of rehydrating desiccation-sensitive material. In contrast to the tolerant tissues, sucrose breakdown in the sensitive leaves commenced immediately after watering and the accumulation in hexose sugars was inversely proportionate to the decrease in sucrose content. Hexokinase (EC 2.7.1.1), PFK (ATP phosphofructokinase, EC 2.1.7.11), aldolase (EC 4.1.2.13), enolase (EC 4.2.1.11), and PK (pyruvate kinase, EC 2.7.1.40) activity levels were significantly lower in the desiccation-sensitive material during rehydration.

Notizen: Xerophyta humilis (Bak.) Dur and Schinz is an indigenous Southern African resurrection plant which is able to protect itself from the stresses associated with extreme dehydration. For the purpose of analysing global patterns of gene expression in response to desiccation and recovery on rehydration, we have generated a normalized 10 900 library, representing genes from root and leaf tissue that are expressed during the dehydration-rehydration cycle. For the small-scale microarray analysis described here, 424 cDNAs were sequenced, annotated, arrayed and hybridized with hydrated and dehydrated, leaf-specific RNA. Reverse Northern blots were used as an alternative method to compare the expression results. A total of 55 dehydration-inducible cDNAs were identified combining the results of both methods. Northern blot analysis of 14 of the 55 the dehydration-upregulated cDNAs verified the expression status of all 14 genes. Dehydration-upregulated cDNAs included those homologous to known dehydration stress-responsive genes encoding metallothioneins, galactinol synthases, an aldose reductase and a glyoxalase. A large number of genes encoding late embryonic abundant proteins (LEAs), dehydrins and desiccation-related proteins were also identified, suggesting that proteins that provide mechanical and antioxidant protection against water loss dominate the mRNA population in desiccated X. humilis leaf tissue. Dehydration-upregulated genes identified in this study, and not previously implicated in the dehydration response, include cDNAs encoding a putative chloroplast RNA-binding protein and a protein containing SNF2/helicase domains. Comparisons with microarray data, which profile the dehydration response in desiccation-sensitive plants, reveal differences in expression patterns between X. humilis and Arabidopsis and rice that could provide clues as to the mechanisms underlying desiccation-tolerant and -sensitive phenotypes. The X. humilis library promises to be a useful resource for transcript profiling the dehydration and rehydration response in a desiccation-tolerant plant.

Notizen: The coleoptiles of wheat (Triticum aestivum L.) seedlings of cultivar Trémie are desiccation tolerant when 3 days old, although the roots are not. Cutting some of the coleoptiles open prior to dehydration rapidly increased the drying rate. This rendered the coleoptiles sensitive to desiccation, providing a useful model with which to study desiccation tolerance. Both sensitive and tolerant seedlings were dehydrated to 0.3 g H2O g−1 dry mass (g.g) and thereafter rehydrated. Sensitive tissues accr- ued the lipid peroxidation products H2O2and MDA, and substantial subcellular damage was evident in dry tissues. H2O2 and MDA accumulated slightly only in dry tolerant coleoptiles and no subcellular damage was evident. The activity of antioxidant enzymes glutathione reductase (EC1.6.2.4), superoxide dismutase (EC 1.14.1.1) and catalase (EC 1.11.1.6) increased on drying in both tolerant and sensitive tissues, but were sustained on rehydration in only the tolerant tissues. It is proposed that free radical damage sustained during rapid drying exceeded the ameliorating capacity of antioxidant systems, allowed accrual of lethal subcellular damage. Slow drying enabled sufficient detoxification by antioxidants to minimize damage and allow tolerance to drying. Three LEA- (p11 and Asp 52) and dehydrin- (XV8) like proteins were detected by western blots in tolerant coleoptiles dried to 3.0 g.g and below. Only one (Asp 52) was induced at low water content in rapidly dried sensitive coleoptiles. None were present in root tissues. XV8 RNA (northern analyses) was induced on drying only in tolerant coleoptiles and correlated with protein expression. These stress-putative protein protectants (and XV8 transcripts) appear to be down-regulated during germination but wheat seedlings temporarily retain the ability to reproduce them if drying is slow. Sucrose accumulation during dehydration was similar for both sensitive and tolerant tissues, suggesting that this sugar has little role, or is not effective in isolation, in protecting against desiccation damage in wheat seedlings. In summary, the slower rate at which tolerant coleoptiles were dried allowed for the mobilization of protection mechanisms with which to survive desiccation. Rapid drying of tissues precluded induction of some putative stress protein protectants and caused damage in excess of the ameliorating capacity of the antioxidant protection systems.

Notizen: Changes in ultrastructural, biochemical and biophysical characteristics of embryonic axes of Aesculus hippocastanum during development are related to changing levels of desiccation tolerance. Histodifferentiation was complete 30 days after flowering (DAF) and fruits were shed about 120 DAF. During this period, the dry mass of embryonic axes increased from about 0.5 to 4 mg and the water content decreased from 10.2 to 2.0 g H2O g−1 dry mass (g g−1). In spite of the large changes in water content, water potentials of freshly harvested material declined slightly during development from −0.65 to −2.0 MPa. Tolerance of desiccation increased as embryos matured. If evaluated on the basis of critical water contents for survival, tolerance appeared to increase continuously, maximum tolerance being achieved at 120 DAF when embryos survived water contents as low as 0.30 g g−1. When evaluated from critical water potentials, three distinct levels of desiccation tolerance could be recognized at −1.8 MPa (30-40 DAF), −4 M Pa (48-90 DAF) and −12 MPs (100-120 DAF). During development, total dry matter increased while sugar content (g g−1' dry mass) and osmotically active material (mmol g−1 dry mass) decreased. The subcellular organisation of axes was always typical of metabolically active tissues. Maximum tolerance (−12 MPa) was associated with a reduced amount of monosaccharides and the appearance of water with unusual calorimetric behaviour. Our data are consistent with several of the current hypotheses regarding the mechanisms of desiccation tolerance. Accumulation of dry matter reserves, reduced levels of monosaccharides, presence of dehydrin-like proteins and ability to form glasses appear to be associated with the changes in desiccation tolerance. However, none of these factors allow A. hippocastanum embryos to achieve the extreme level of desiccation tolerance typical of orthodox seeds. This may be because A. hippocastanum embryos do not reach physiological maturity and remain metabolically active even after they are shed from the parent plant. Also, embryos may acquire incompetent protectants or lack as yet unidentified protective mechanisms.

Notizen: Abstract. An aldose reductase homologue (ALDRXV4) was cloned from the resurrection plant Xerophyta viscosa Baker using complementation by functional sufficiency in Escherichia coli. A cDNA library constructed from X. viscosa leaves dehydrated to 85%, 37% and 5% relative water contents (RWC) was converted into an infective phagemid library. Escherichia coli (srl::Tn10) cells transformed with ds-pBluescript phagemids were selected on minimal medium plates supplemented with 1 mM isopropyl β-d-thiogalactopyranoside and 1.25 M sorbitol. Nine cDNA clones that conferred tolerance to the osmotically stressed E. coli cells were selected. The phagemid from one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of tolerating the osmotic stress, whereas control cultures were not. The ALDRXV4 insert contained an open reading frame that can code for 319 amino acids, and the predicted protein had a calculated Mr of 35,667. Amino acid sequence comparisons revealed significant similarity to several aldose reductases, with the highest similarity to aldose reductase proteins from Hordeum vulgare, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respectively. Northern blot analysis revealed that ALDRXV4 was expressed only under dehydration conditions in X. viscosa leaves. Western blot analysis detected a protein of 36 kDa under dehydration conditions only. Aldose reductase activity levels in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sporobolus stafianus as the leaf RWC decreased.