Photosynthetic carbohydrate metabolism in the resurrection plant Craterostigma plantagineum

The resurrection plant Craterostigma plantagineum (Hochst) is able to survive almost complete tissue dehydration when water is withheld from it, and then can rehydrate rapidly on rewatering. This ability is believed to be the result of the accumulation of sucrose in aerial tissues as a result of metabolism of 2-octulose. In this work the metabolic activity of well-watered Craterostigma plantagineum plants has been investigated. It is shown that Craterostigma makes raffinose series oligosaccharides as a product of photosynthesis and translocates them in the phloem. Evidence is also provided that 2-octulose is a product of photosynthesis and accumulates in the leaves over the light period and is mobilized at night. Thus 2-octulose acts as a temporary storage carbohydrate in leaves during photosynthesis in a similar fashion to starch in most C3 plants. Other potential roles of 2-octulose are discussed. Other than these observations Craterostigma plants are very similar to other C3 plants under these conditions.     

An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst.

Procedures previously established for plant regeneration and Agrobacterium tumefaciens-mediated genetic transformation of the desiccation-tolerant plant, Craterostigma plantagineum, have been further developed. A highly effective tissue culture system was established based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. The undesirable hyperhydricity of Craterostigma tissue in tissue culture was also circumvented by these modifications, which serve as an alternative to the previously described procedures. The high efficiency of plant regeneration from the callus phase provided the basis for optimising genetic transformation in Craterostigma. For gene delivery, both a standard (method A) and a modified protocol (method B), the latter having previously resulted in successful Agrobacterium-mediated transformation of monocot cereals, were applied. Physical and biochemical key variables in transformation were evaluated, such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension cultures into target tissues and the influence of in vitro pre-induction of vir genes. While the physical enhancement of Agrobacterium infection (microwounding, infiltration) had no positive effect, the in vitro pre-induction of vir genes (biochemical enhancement) resulted in a twofold increase in the transformation frequency as compared to the conventional protocol (method A).     

Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco

Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3-flanking region in an Agrobacterium vector and introduced into tobacco. Transgenic plants expressed the encoded Craterostigma proteins at high levels. This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters. In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma. These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests.     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author     

Investigation into the ability of roots of the poikilohydric plant Craterostigma plantagineum to survive dehydration stress

The ability of the root system of the poikilohydric plant Craterostigma plantagineum to survive dehydration was investigated. The data presented here reveal that the root system is capable of surviving dehydration, but shortly after rehydration the root system senesces. Two weeks after rehydration the growth of a complete new root system is initiated. During dehydration sucrose accumulates from 36 to a maximum of 111 micromol g-1 DW in the roots. It is suggested that the accumulation of sucrose protects the root system during dehydration. There are major stores of stachyose in the roots of Craterostigma (making up over 40% of the dry weight of the tissue) and during dehydration these stores are metabolized. It is suggested that these stachyose stores act as carbohydrate reserves for the synthesis of sucrose. However, over 350 micromol g-1 DW stachyose is metabolized in the roots, which is well in excess of that required for the accumulation of sucrose observed. It is likely that the stachyose reserves in the root system are translocated to other regions of the plant to support carbohydrate metabolism during dehydration of the tissue. During rehydration, the stachyose reserves return to their original level within 96 h. There is no change in the elevated sucrose content of the roots over this period. Thus the roots maintain the protective properties of sucrose much longer than they are needed. The maintenance of high sucrose contents in rehydrating roots is discussed as a possible survival strategy against recurrent desiccation events.     

Desiccation Tolerance Studied in the Resurrection Plant Craterostigma plantagineum

This review will focus on the acquisition of desiccation tolerancein the resurrection plant Craterostigma plantagineum. Molecularaspects of desiccation tolerance in this plant will be comparedwith the response of non-tolerant plants to dehydration. Uniquefeatures of C. plantagineum are described like the CDT-1 (Craterostigmadesiccation tolerance gene-1) gene and the carbohydrate metabolism.Abundant proteins which are associated with the desiccationtolerance phenomenon are the late embryogenesis abundant (=LEA)proteins. These proteins are very hydrophilic and occur in severalother species which have acquired desiccation tolerance.     

Investigating the molecular basis of desiccation tolerance using the resurrection plant Craterostigma plantagineum as an experimental system

Conclusion The array of genes isolated and characterised from Craterostigma to date allow many insights to be made into the protection mechanisms underlying the desiccation tolerance phenomenon and drought-induced changes in gene expression. However, more work is necessary to define which gene products positively contribute to stress tolerance and which may be considered as secondary stress metabolites or as gene regulators. It is apparent that the gene regulatory mechanisms involved are complex, but future work with more refined biochemical and genetic approaches, together with the analysis of defined mutants will greatly help to dissect the important area of desiccation perception, perhaps faciliate manipulation of desiccation tolerance in a genetic engineering context, and undoubtedly bring much progress in the coming years.