Ayumu Kondo

Clumping and dispersal of chloroplasts in succulent plants

Plants have evolved various photoprotective mechanisms to mitigate photodamage. Here we report the diurnal movement of chloroplasts in the leaves of succulent crassulacean acid metabolism (CAM) plants under combined light and water stress. In leaves of water-stressed plants, the chloroplasts became densely clumped in one or sometimes two areas in the cytoplasm under light and dispersed during darkness. The chloroplast clumping resulted in leaf optical changes, with a decrease in absorptance and an increase in transmittance. The plant stress hormone abscisic acid induced chloroplast clumping in the leaf cells under light. We suggest that the marked chloroplast movement in these CAM plants is a photoprotective strategy used by the plants subjected to severe water stress.

Structural and Biochemical Dissection of Photorespiration in Hybrids Differing in Genome Constitution between Diplotaxis tenuifolia (C3-C4) and Radish (C3)

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C3-C4 intermediate species Diplotaxis tenuifolia (DtDt) and the C3 species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO2 compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C3-C4 intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C3-C4 intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study.

Abstract. In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalanchoë species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.

Diurnal changes in photosynthesis in sugarcane leaves: II. Enzyme activities and metabolite levels relating to sucrose and starch metabolism.

Summary Diurnal changes in carbohydrate contents, assimilate export, enzyme activities and metabolite levels associated with sucrose and starch synthesis in field-grown sugarcane (Saccharum sp. cv. NiF4) leaves were investigated during a natural 24 h day-night cycle. Eighty percent of the carbon fixed in sugarcane leaves at midday was exported immediately. Of the total carbon fixed during the day, 82% was exported in the daytime and 17% was accumulated as leaf starch which was exported at night. The activities of sucrose phosphate synthase (SPS), cytosolic fructose-1, 6-bisphosphatase (FBPase), UDP-glucose pyrophosphorylase (UDPG-PPase) and ADP-glucose pyrophosphorylase (ADPG-PPase), and the levels of triose phosphates (triose-P) and fructose-1,6-bisphosphate (FBP) showed distinct diurnal fluctuations during the day-night cycle. The levels of hexose phosphates fluctuated at smaller magnitudes compared with those in triose-P and FBP. The diurnal change in triose-P level was highly related to the changes in carbon exchange rate and sucrose content, while the sucrose content was closely related to the changes in activities of SPS, cytosolic FBPase and UDPG-PPase in leaves of sugarcane. The present results suggest that the availability of triose-P is a key factor in determining the rate of sucrose synthesis in sugarcane leaves.

Diurnal Changes in Photosynthesis in Sugarcane Leaves : I. Carbon dioxide exchange rate, photosynthetic enzyme activities and metabolite levels relating to the C4 pathway and the Calvin cycle

Abstract Diurnal changes in carbon dioxide exchange rate (CER), stomatal conductance (gs), photosynthetic enzyme activities and metabolite levels relating to the C4 pathway and the Calvin cycle in sugarcane (Saccharum sp. cv. NiF4) leaves were characterized during a natural 24 h day-night cycle. The activities of phosphoenolpyruvate carboxylase (PEPcase), NADP-malic enzyme (NADP-ME), pyruvate, P1 dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chloroplast fructose-1,6-bisphosphatase (FBPase) all exhibited distinct diurnal changes. The levels of C4 metabolites, malate, phosphoenolpyruvate (PEP) and pyruvate, showed large diurnal fluctuations, but the oxaloacetate levels were extremely low and exhibited no significant changes in the day-night cycle. Diurnal changes in CER and gs paralleled the changes in radiation of sunlight. The results in this study suggest that at midday when CER is in steady-state and saturated by full sunlight, CER is limited by PPDK activity ; under lower radiation of sunlight, i.e., before CER is saturated in the morning and during the period when CER is decreasing in the afternoon, CER is possibly limited by PEPcase activity or gs.

Pyruvate, Pi Dikinase of Cam Plants: Species Variation in the Subcellular Localization as Revealed by Immunoelectron Microscopy

CAM plants can be divided into two groups, PCK (phosphoenolpyruvate carboxykinase)-CAM and ME (malic enzyme)-CAM plants, based on the difference in decarboxylation process. In ME-CAM plants, pyruvate, Pi dikinase (PPDK) activity is found, and it is involved in the conversion of pyruvate to PEP. PCK-CAM plants lack PPDK activities (1). Recently, we have revealed that in some ME-CAM species, PPDK is present in the cytosol as well as in the chloroplasts of the mesophyll cells (2), although it was conventionally thought to be localized in the chloroplasts (3,4). In Mesembryanthemum crystallinum PPDK was present only in the chloroplasts, whereas in Kalanchoe pinnata and K. daigrenumtiana it was found in both chloroplasts and cytosol (2). ME-CAM plants occur in various families, such as Aizoaceae, Cactaceae, and Crassulaceae. In this study, we investigated the subcellular localization of PPDK for ME-CAM species from various taxonomic groups.