Using immunohistochemistry to study plant metabolism: the examples of its use in the localization of amino acids in plant tissues, and of phosphoenolpyruvate carboxykinase and its possible role in pH regulation.

To understand many aspects of the metabolism of complex plant structures such as leaves, fruit and roots it is important to understand how metabolic processes are compartmentalized between tissues. The aim of this article is to show how immunohistochemistry, in conjunction with biochemical and physiological studies, is useful in understanding both the function of an enzyme in a tissue and metabolic processes occurring in plant tissues. This is illustrated by two examples. Firstly, the use of immunohistochemisty in the localization of amino acids in plant tissues is described. Secondly, the use of immunohistochemistry in understanding the function of an enzyme in a tissue and the metabolic processes occurring within the tissue is described. To illustrate this the example of phosophoenolpyruvate carboxykinase (PEPCK), an enzyme which is present in many plant tissues in which its function is unknown, is used. Evidence is provided that PEPCK may play a role in pH regulation in tissues active in the metabolism of nitrogen.

Are isocitrate lyase and phosphoenolpyruvate carboxykinase involved in gluconeogenesis during senescence of barley leaves and cucumber cotyledons?

The aim of this study was to investigate whether gluconeogenesis catalysed by phosphoenolpyruvate carboxykinase (PEPCK) occurs during leaf senescence. This was addressed by determining changes in the abundance and intercellular location of enzymes necessary for gluconeogenesis during the senescence of barley leaves and cucumber cotyledons. PEPCK was never present in barley leaves, despite the presence of large amounts of isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle, and of its product, glyoxylate. Although PEPCK was present in non-senescent cucumber cotyledons, its abundance declined during senescence. Throughout senescence, PEPCK was only present in the trichomes and vasculature, whereas ICL was located in mesophyll cells. Pyruvate,Pi dikinase (PPDK) which, in concert with NAD(P)-malic enzyme, is also capable of catalysing gluconeogenesis, was present in non-senescent barley leaves and cucumber cotyledons, but in both plants its abundance decreased greatly during senescence. The abundance of ICL was greatly reduced in senescing detached barley leaves by either illumination or by co-incubation with sucrose, and greatly increased in darkened attached barley leaves. These results argue against the large-scale occurrence of gluconeogenesis during senescence catalysed either by PEPCK or PPDK. In cucumber cotyledons, PEPCK may play a role in metabolic processes linked to the export of amino acids, a role in which phosphoenolpyruvate carboxylase may also be involved. The amount of ICL was increased by starvation and during senescence may function in the conversion of lipids to organic acids, which are then utilised in the mobilisation of amino acids from leaf protein.

An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries.

The compartmentation of key processes in sugar, organic acid and amino acid metabolism was studied during the development of the flesh and seeds of grape (Vitis vinifera L.) berries. Antibodies specific for enzymes involved in sugar (cell wall and vacuolar invertases, pyrophosphate: fructose 6-phosphate phosphotransferase, aldolase, NADP-glyceraldehyde-P dehydrogenase, cytosolic fructose 1,6-bisphosphatase), photosynthesis (Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase), amino acid metabolism (cytosolic and mitochondrial aspartate aminotransferases, alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase), organic acid metabolism (phosphoenolpyruvate carboxylase, NAD- and NADP-dependent malic enzyme, ascorbate peroxidase), and lipid metabolism (acetyl CoA carboxylase, isocitrate lyase) were used to determine how their abundance changed during development. There were marked changes in the abundance of many of these enzymes in both the flesh and seeds. The intercellular location of some enzymes was investigated using immunohistochemistry. Several enzymes (e.g. phosphoenolpyruvate carboxylase and those involved in amino acid metabolism) were associated with tissues likely to function in the transport of imported assimilates, such as the vasculature. Although other enzymes (e.g. NADP-malic enzyme and soluble acid invertase, involved in the metabolism of sugars and organic acids) were largely present in the parenchyma cells of the flesh, their distribution was extremely heterogeneous. This study shows that when considering the metabolism of complex structures such as fruit, it is essential to consider how metabolism is compartmentalized between and within different tissues, even when they are apparently structurally homogeneous.

A Spatial Analysis of Physiological Changes Associated with Infection of Cotyledons of Marrow Plants with Cucumber Mosaic Virus.

Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo L.) 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. Inside 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.