| Summary: | Far from being merely fluid filled sacs, plant vacuoles have multiple fimctions including a hydrolytic pattern of behaviour similar to the mammalian lysosome, whilst also having the unique ability to deposit nitrogen-rich pr~teins for postgerminative growth.To maintain an effective cellular balance, it is of paramount importance to segregate these highly diverse fimctions to prevent an energyinefficient, perpetual arms race between storage and degradation. The tonoplast intrinsic proteins (TIPs) are aquaporins of the major intrinsic protein superfamily for the vacuolar membranes. They have historically been used as markers for the functionally distinct vacuoles. In particular, the alpha (u) isoform has been used as a marker for the protein storage vacuole, the gamma (y) isoform for the lytic vacuole and the delta (0) isoform for the 'vegetative' storage vacuole. These, together with well documented soluble markers, characterised by their peptide sorting determinants, have been employed in attempts to resolve the enigma of fimctional separation. The 'two-vacuole hypothesis' has recently been proposed, largely from immunofluorescence data in pea and barley root tips, in which both storage and lytic vacuoles were assumed to coexist and remain fimctionally separated by virtue of distinct protein transport routes. This thesis examines the two-vacuole hypothesis in the model plant organism Arabidopsis thalialla, by studies involving fluorescent protein tagging of the three major isoforms of the tonoplast intrinsic proteins. Constitutive expression of these isoforms showed no discernible differences in their subcellular localisation in all tissues studied, including roots, leaves, protoplasts and in a detailed time course of seed maturation and germination. When controlled by their native genomic sequences, the expression of the fluorescently tagged TIP isoforms appears to be tightly regulated in terms of developmental and tissue-specificity. These findings suggest that either the TIPs cannot discriminate between coexisting vacuoles, or that their vacuolar specificity is a product of their temporal rather than spatial regulation. The trafficking route of u-TIP to the tonoplast was also investigated.
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