Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo 1.) have been localized, first by measuring activities of key enzymes in infected and uninfected regions of the cotyledon, and second by histochemical techniques applied to tissue prints of the infected region. A series of progressive metabolic changes occurs within the expanding infected lesion. Virus replication and the synthesis of viral protein at the periphery creates a strong sink demand associated with increased activities of anaplerotic enzymes, increased photosynthesis, and starch accumulation. lnside the lesion, when the synthesis of virus has declined, photosynthesis is reduced, starch is mobilized, and the emphasis of metabolism is shifted toward glycolysis and mitochondrial respiration. These changes are associated spatially with the onset of chlorosis. A decrease in total protein synthesis in this inner zone could be instrumental in some or all of these changes, leading to symptoms of viral infection.Our recent studies of the interaction between CMV and the cotyledons of the marrow plant (Cucurbita pepo L.) have demonstrated the complexity of the metabolic responses to infection (Técsi et al., 1994a(Técsi et al., , 1994b. The first symptoms of CMV infection are circular chlorotic lesions 4 d after infection. We showed that such lesions are not homogeneous but constitute a dynamic structure composed of zones of cells of diverse physiology. Although particles accumulate throughout the lesion, circular zones of cells can be identified within the infected region. From the outside these are (a) a zone (i) of infected cells that do not accumulate starch, (b) a zone (ii) consisting of cells with increased photosynthetic activity, resulting in the accumulation of starch, (c) a zone (iii) of largely starchless cells that appear to have a low photosynthetic activity, and (d) a zone (iv) of cells surrounding the initial point of infection that retain high photosynthetic activity and a high starch content for the first 4 to 6 d postinfection. As the lesions expand during the course of infection, after 10 d the inner region (zones iii and

Phosphoenolpyruvate carboxykinase (PEPCK) was shown to be present in a range of developing seeds by measurement of its activity and by immunoblotting. Its function was investigated during grape (Vitis vinifera L.) seed development. The maximum abundance of PEPCK coincided with the deposition of storage reserves. At this stage of development, immunohistochemistry showed that PEPCK was very abundant in a layer of cells located at the boundary of developing storage tissues and in the chalaza (close to the termination of the vascular supply to the seed) and was also present in the palisade layer of the seed coat (the inner layer of the outer integument). Earlier in development PEPCK was also present in the developing palisade layer and in the inner region of the nucellus which surrounds the developing endosperm. At later stages of development, PEPCK was located in the outer region of the endosperm. However, PEPCK was present in the phloem of the seed at all stages of development. Feeding of asparagine to developing grape seeds led to a strong induction of PEPCK. We suggest that, in developing grape seeds, both the chalaza and palisade tissue may distribute imported assimilates from the vasculature to the developing storage tissues and that PEPCK may play a role in the metabolism of nitrogenous assimilates during their delivery from the vasculature to the storage tissues.

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

organic acids) were largely present in the parenchyma cells of the flesh, their distribution was extremely hetThe compartmentation of key processes in sugar, erogeneous. This study shows that when considering organic acid and amino acid metabolism was studied the metabolism of complex structures such as fruit, it during the development of the flesh and seeds of grape is essential to consider how metabolism is compart-(Vitis vinifera L.) berries. Antibodies specific for mentalized between and within different tissues, even enzymes involved in sugar (cell wall and vacuolar when they are apparently structurally homogeneous. invertases, pyrophosphate5fructose 6-phosphate phosphotransferase, aldolase, NADP-glyceraldehyde-

The biochemical basis for photosynthetic plasticity in tropical trees of the genus Clusia was investigated in three species that were from contrasting habitats and showed marked dierences in their capacity for crassulacean acid metabolism (CAM). Physiological, anatomical and biochemical measurements were used to relate changes in the activities/amounts of key enzymes of C 3 and C 4 carboxylation to physiological performance under severe drought stress. On the basis of gasexchange measurements and day/night patterns of organic acid turnover, the species were categorised as weak CAM-inducible (C. aripoensis Britt.), C 3 -CAM intermediate (C. minor L.) and constitutive CAM (C. rosea Jacq. 9.). The categories re¯ect genotypic dierences in physiological response to drought stress in terms of net carbon gain; in C. aripoensis net carbon gain was reduced by over 80% in drought-stressed plants whilst carbon gain was relatively unaected after 10 d without water in C. rosea. In turn, genotypic dierences in the capacity for CAM appeared to be directly related to the capacities/amounts of phosphoenolpyruvate carboxylase (PEPCase) and phosphoenolpyruvate carboxykinase (PEPCK) which increased in response to drought in both young and mature leaves. Whilst measured activities of PEPCase and PEPCK in well-watered plants of the C 3 -CAM intermediate C. minor were 5±10 times in excess of that required to support the magnitude of organic acid turnover induced by drought, close correlations were observed between malate accumulation/ PEPCase capacity and citrate decarboxylation/PEPCK capacity in all the species. Drought stress did not aect the amount of ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) protein in any of the species but Rubisco activity was reduced by 35% in the weak CAMinducible C. aripoensis. Similar amounts of glycine decarboxylase (GDC) protein were present in all three species regardless of the magnitude of CAM expression. Thus, the constitutive CAM species C. rosea did not appear to show reduced activity of this key enzyme of the photorespiratory pathway, which, in turn, may be related to the low internal conductance to CO 2 in this succulent species. Immuno-histochemical techniques showed that PEPCase, PEPCK and Rubisco were present in cells of the palisade and spongy parenchyma in leaves of species performing CAM. However, in leaves from well-watered plants of C. aripoensis which only performed C 3 photosynthesis, PEPCK was localized around latex-producing ducts. Dierences in leaf anatomy between the species suggest that the association between mesophyll succulence and the capacity for CAM in these hemi-epiphytic stranglers has been selected for in arid environments.Key words: Crassulacean acid metabolism ± Clusia ± Phosphoenolpyruvate carboxylase ± Phosphoenolpyruvate carboxykinase ± Ribulose 1,5-bisphosphate carboxylase/oxygenase Abbreviations and de®nitions: CAM crassulacean acid metabolism; d 13 C carbon-isotope ratio, & relative to Pee Dee Belemnite (vs. PDB); GDC glycine decarboxylase; PEPCase ph...