Tissue distribution of primary metabolism between epidermal, mesophyll andparenchymatous bundle sheath cells in barley leaves

In order to investigate the roles of different cell types, metabolite compa rtmentation in barley (Hordeum vulgare L.) leaf tissue was mapped at the si ngle-cell level, using single-cell sampling and analysis (SiCSA) techniques . The partitioning of recently fixed photoassimilate was investigated for t he first time at single- cell resolution, using BAMS (biological accelerato r mass spectroscopy) for precise measurement of C-14 in femtomole quantitie s. The data obtained by BAMS qualitatively reflect concentrations of sugars in different cell types measured by SiCSA. Calculation of C-14-specific ac tivities showed that the radioactive label saturated the mesophyll and pare nchymatous bundle sheath (PBS) pools within the 45-min labelling period. Du ring the photoperiod, sucrose concentration increased to 200 mM in mesophyl l cells. The concentration of malate also increased during the photoperiod in mesophyll and PBS cells. Epidermal cells contained very low concentratio ns of sugar but high concentrations of malate (120-180 mM) and did not show significant diurnal changes. Accumulation of sugars and fructan synthesis could be induced in mesophyll and PBS cells by reduced export of sugars fro m leaves or, alternatively, when sugars were supplied from excised leaf bla de bases immersed in a sucrose solution in the dark. The epidermis accumula ted additional malate in step with the accumulation of sugar by the mesophy ll/PBS cells during the long-term reduction of export. Immunolocalisation o f Rubisco and cytochrome oxidase proteins was used to analyse the distribut ion of enzymes of photoassimilation and respiration between functionally di fferent cells in mature leaves of barley.