Our immunological and immunocyto-logical studies have suggested that GS1 is important for the export of nitrogen from senescing rice leaves.11-13 During natural senescence of a leaf blade of rice plants, the contents of GS2 declined in parallel with other chroloplastic enzymes such as RuBisCO and Fd-GOGAT. In contrast, the GS1 protein remained relatively constant throughout the senescence period.12 When various positions of leaves are tested, the relative content of GS1 protein was found to be highest in the oldest leaf blade on the main stem of rice plants and gradually declined toward the youngest unexpanded blade.14 Tissue print immuno-blots clearly show that GS1 protein is detected in large and small vascular bundles, whereas GS2 protein is in mesophyll cells.11 When cross sections from the lowest position of the attached leaf blade were immunostained with an affinity purified anti-GS1 IgG, strong signals for GS1 protein were detected in companion cells and parenchyma cells of vascular bundles.13 The signals for GS1 were barely detected in mesophyll cells, where the
GS2 protein was mainly located. Companion cell-specific localization of GS1 was also shown using immunogold labeling by a Portuguese group working with tobacco15 and potato16 plants. The spatial localization of these GS isozymes indicates distinct and non-overlapping roles in nitrogen metabolism in rice plants. The companion cells and vascular parenchyma cells are active in the transport of solutes, since they contain abundant mitochondria and endoplasmic reticulum.17 The companion cells are important in the regulation of phloem loading.17,18 The localization of GS1 in these cells of rice leaves further supports our hypothesis11-13 that GS1 is important in the synthesis of glutamine for nitrogen export. Conclusive evidence to support our hypothesis could be obtained either by creating mutants lacking GS1
or transformants having reduced amounts of the GS1 gene product. We are currently working on the reduction of GS1 protein in transgenic rice using techniques to express the antisense RNA driven with a companion cell-specific promoter.
GS1 protein was detected not only in the senescing leaf blades but also in the younger blades of rice plants, although the content in the developing leaf blade was less than that in the senescing blade.14 When the cellular localization of GS1 was tested in the different leaf positions of the blades, the signals in companion cells were less striking in the younger green leaf blades and were hardly detected in the non-green portion of the unexpanded blade.13 In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylem parenchyma cells of vascular bundles.13 Thus, the cellular localization of GS1 in vascular bundles of rice plants changed during leaf development. When the metabolic route for generation of ammonium ions is considered in these cell types, there is no immediate answer as to a function for GS1. Biosynthesis of lignin polymer in these compartments would be one explanation for the generation of ammonium ions by a reaction of phenylalanine ammonia-lyase (EC 220.127.116.11) and GS1 might be important for the assimilation of generated ammonium ions.
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