Tadahiko Mae

Physiological nitrogen efficiency in rice: Nitrogen utilization, photosynthesis, and yield potential

Characteristics of rice (Oryza sativa) as a crop plant are briefly introduced, and the relationship between formation of yield potential and nitrogen (N) nutrition is described on the basis of studies using 15N as a tracer. In addition, the relationship between the leaf photosynthetic capacity and leaf N, and the factors limiting leaf photosynthesis under different growth conditions are reviewed. Finally, targets for improving rice yield potential are discussed with a focus on the role of increased photosynthesis efficiency in relation to leaf N status and the photosynthetic components in the leaves.

Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by an ATG Gene-Dependent Autophagic Process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H+-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.

The effect of elevated partial pressures of CO2 on the relationship between photosynthetic capacity and N content in rice leaves

The effects of growth CO2 levels on the photosynthetic rates; the amounts of ribulose-1,5-bisphosphate carboxylase (Rubisco), chlorophyll (Chl), and cytochrome f; sucrose phosphate synthase activity; and total N content were examined in young, fully expanded leaves of rice (Oryza sativa L.). The plants were grown hydroponically under two CO2 partial pressures of 36 and 100 Pa at three N concentrations. The light-saturated photosynthesis at 36 Pa CO2 was lower in the plants grown in 100 Pa CO2 than those grown in 36 Pa CO2. Similarly, the amounts of Rubisco, Chl, and total N were decreased in the leaves of the plants grown in 100 Pa CO2. However, regression analysis showed no differences between the two CO2 treatments in the relationship between photosynthesis and total N or in the relationship between Rubisco and Chl and total N. Although a relative decrease in Rubisco to cytochrome f or sucrose phosphate synthase was found in the plants grown in 100 Pa CO2, this was the result of a decrease in total N content by CO2 enrichment. The activation state of Rubisco was also unaffected by growth CO2 levels. Thus, decreases in the photosynthetic capacity of the plants grown in 100 Pa CO2 could be simply accounted for by a decrease in the absolute amount of leaf N.

Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective Km and Vmax values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the Vmax values between the two species were greater. When the net activity of CO2 exchange was calculated at the physiological CO2-O2 concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO2-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO2-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO2 pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO2 assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.

Responses of Ribulose-1,5-Bisphosphate Carboxylase, Cytochrome f, and Sucrose Synthesis Enzymes in Rice Leaves to Leaf Nitrogen and Their Relationships to Photosynthesis

The photosynthetic gas-exchange rates and various biochemical components of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco) content, cytochrome (Cyt) f content, and the activities of two sucrose synthesis enzymes, were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically in different nitrogen concentrations. The light-saturated rate of photosynthesis at an intercellular CO2 pressure of 20 Pa (CO2-limited photosynthesis) was linearly dependent on leaf nitrogen content, but curvilinearly correlated with Rubisco content. This difference was due to a greater than proportional increase in Rubisco content relative to leaf nitrogen content and the presence of a CO2 transfer resistance between the intercellular air spaces and the carboxylation sites. CO2-limited photosynthesis was proportional to Cyt f content, one of the key components of electron transport, but was not proportional to the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, the two regulatory enzymes of sucrose synthesis. Light-saturated photosynthesis above an intercellular CO2 pressure of 60 Pa (CO2-saturated photosynthesis) was curvilinearly dependent on leaf nitrogen content. This CO2-saturated photosynthesis was proportional to Cyt f content in the low- and normal-nitrogen leaves, and correlated better with the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase in the high-nitrogen leaves. The increase in the activities of these two enzymes with increasing leaf nitrogen was not as great as the increase in Cyt f content. Thus, as leaf nitrogen increased, the limitation caused by the activities of sucrose synthesis enzymes came into play, which resulted in the curvilinear relationship. However, this limitation by sucrose synthesis enzymes did not affect photosynthesis under normal ambient air.

Photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase in rice leaves from emergence through senescence. Quantitative analysis by carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate

Changes in gas-exchange rates during the life span of the leaves of rice (Oryza sativa L.) were analyzed quantitatively by measuring changes in the carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate (RuBP) at photon fluence rates of 2000 (saturating) and 500 (subsaturating) μmol quanta·m-2·s-1 under ambient air conditions. The RuBP levels were always higher than the active-site concentrations of RuBP carboxylase (EC 4.1.1.39), irrespective of the irradiance supplied. Analysis of the CO2-assimilation rate as a function of intercellular CO2 concentration indicated that RuBP regeneration does not limit CO2 assimilation. The estimated RuBP-carboxylase/oxygenase activity in vivo was linearly correlated to the rate of CO2 assimilation at each level of irradiance. This enzyme activity was just enough to account for the rate of CO2 assimilation at the saturating irradiance and was 35% more than the rate of CO2 assimilation at the subsaturating irradiance. Analysis of the assimilation rate at subsaturating irradiance as a function of intercellular CO2 concentration indicated that a limitation caused by enzyme activation comes into play. The results indicate that the rate of CO2 assimilation in rice leaves under ambient air conditions is limited during their entire life span by the RuBP-carboxylation/oxygenation capacity.

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100–115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.

Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves

Abstract. The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471–479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN3, which is a potent inhibitor of active oxygen-scavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU.

Leaf photosynthesis, plant growth and nitrogen allocation in rice under different irradiances

Abstract. The photosynthetic rates and various components of photosynthesis including ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), chlorophyll (Chl), cytochrome (Cyt) f, and coupling factor 1 (CF1) contents, and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically under two irradiances, namely, 1000 and 350 μmol quanta · m−2 · s−1, at three N concentrations. The light-saturated rate of photosynthesis measured at 1800 μmol · m−2 · s−1 was almost the same for a given leaf N content irrespective of growth irradiance. Similarly, Rubisco content and SPS activity were not different for the same leaf N content between irradiance treatments. In contrast, Chl content was significantly greater in the plants grown at 350 μmol · m−2 · s−1, whereas Cyt f and CF1 contents tended to be slightly smaller. However, these changes were not substantial, as shown by the fact that the light-limited rate of photosynthesis measured at 350 μmol · m−2 · s−1 was the same or only a little higher in the plants grown at 350 μmol · m−2 · s−1 and that CO2-saturated photosynthesis did not differ between irradiance treatments. These results indicate that growth-irradiance-dependent changes in N partitioning in a leaf were far from optimal with respect to N-use efficiency of photosynthesis. In spite of the difference in growth irradiance, the relative growth rate of the whole plant did not differ between the treatments because there was an increase in the leaf area ratio in the low-irradiance-grown plants. This increase was associated with the preferential N-investment in leaf blades and the extremely low accumulation of starch and sucrose in leaf blades and sheaths, allowing a more efficient use of the fixed carbon. Thus, morphogenic responses at the whole-plant level may be more important for plants as an adaptation strategy to light environments than a response of N partitioning at the level of a single leaf.

Effects of Growth Temperature on the Responses of Ribulose-1,5-Biphosphate Carboxylase, Electron Transport Components, and Sucrose Synthesis Enzymes to Leaf Nitrogen in Rice, and Their Relationships to Photosynthesis

Effects of growth temperature on the photosynthetic gas-exchange rates and their underlying biochemical properties were examined in young, fully expanded leaves of rice (Oryza sativa L.). The plants were grown hydroponically under day/night temperature regimes of 18/15[deg]C, 23/18[deg]C, and 30/23[deg]C and all photosynthetic measurements were made at a leaf temperature of 25[deg]C and an irradiance of 1800 [mu]mol quanta m-2 s-1. Growth temperature affected the photosynthetic CO2 response curve. The relative ratio of the initial slope to the CO2-saturated photosynthesis increased with rising growth temperature. This was caused mainly by an increase in CO2-limited photosynthesis for a given leaf nitrogen content with rising growth temperature. However, there was no difference in ribulose-1,5-bisphosphate carboxylase (Rubisco) content at any given leaf nitrogen content among temperature treatments. In addition, the activation state and catalytic turnover rate of Rubisco were not affected by growth temperature. The increase in CO2-limited photosynthesis with rising growth temperature was the result of an increase in the CO2 transfer conductance between the intercellular airspaces and the carboxylation sites. The amounts of total chlorophyll and light-harvesting chlorophyll a/b protein II increased for the same leaf nitrogen content with rising growth temperature, but the amounts of cytochrome f and coupling factor 1 and the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were the same between plants grown at 23/18[deg]C and those grown at 30/23[deg]C. Similarly, CO2-saturated photosynthesis was not different for the same leaf nitrogen content between these treatments. For the 18/15[deg]C-grown plants, a slight decrease in the amounts of cytochrome f and coupling factor 1 and an increase in the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were found, but these were not reflected in CO2-saturated photosynthesis.