Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice.

Rubisco limits C3 photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of Rubisco is often accompanied by a decline in Rubisco activation, and the protein ratio of Rubisco activase (RCA) to Rubisco (RCA/Rubisco) greatly decreases in Rubisco-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both Rubisco and RCA (RBCS-RCA-ox). Rubisco content in RBCS-RCA-ox plants increased by 23%-44%, and RCA/Rubisco levels were similar or higher than those of wild-type plants. However, although the activation state of Rubisco in RBCS-RCA-ox plants was enhanced, the rates of CO2 assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C-36°C), the rates of CO2 assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO2 levels. The activation state of Rubisco in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated Rubisco content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO2 assimilation against intercellular CO2 pressures (A:Ci) at 36°C. Thus, a simultaneous increase in Rubisco and RCA contents leads to enhanced photosynthesis within the optimal temperature range.

Expression of flavodiiron protein rescues defects in electron transport around PSI resulting from overproduction of Rubisco activase in rice.

Fragility of photosystem I has been observed in transgenic rice plants that overproduce Rubisco activase (RCA). In this study, we examined the effects of RCA overproduction on the sensitivity of PSI to photoinhibition in three lines of plants overexpressing RCA (RCA-ox). In all the RCA-ox plants the quantum yield of PSI [Y(I)] decreased whilst in contrast the quantum yield of acceptor-side limitation of PSI [Y(NA)] increased, especially under low light conditions. In the transgenic line with the highest RCA content (RCA-ox 1), the quantum yield of PSII [Y(II)] and CO2 assimilation also decreased under low light. When leaves were exposed to high light (2000 µmol photon m-2 s-1) for 60 min, the maximal activity of PSI (Pm) drastically decreased in RCA-ox 1. These results suggested that overproduction of RCA disturbs PSI electron transport control, thus increasing the susceptibility of PSI to photoinhibition. When flavodiiron protein (FLV), which functions as a large electron sink downstream of PSI, was expressed in the RCA-ox 1 background (RCA-FLV), PSI and PSII parameters, and CO2 assimilation were recovered to wild-type levels. Thus, expression of FLV restored the robustness of PSI in RCA-ox plants.

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant, Flaveria bidentis.

C4 plants can fix CO2 efficiently using CO2 -concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C4 plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase-like complex (NDH) accumulated in the thylakoid membrane in leaves of C4 plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C4 plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C4 photosynthesis. In this study, PGR5 was overexpressed in a C4 dicot, Flaveria bidentis. In PGR5-overproducing (OP) lines, PGR5 levels were 2.3- to 3.0-fold greater compared with wild-type plants. PGR5-like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5-OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO2 assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves.

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.

NDH-Mediated Cyclic Electron Flow Around Photosystem I is Crucial for C4 Photosynthesis.

C4 photosynthesis exhibits efficient CO2 assimilation in ambient air by concentrating CO2 around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) through a metabolic pathway called the C4 cycle. It has been suggested that cyclic electron flow (CEF) around PSI mediated by chloroplast NADH dehydrogenase-like complex (NDH), an alternative pathway of photosynthetic electron transport (PET), plays a crucial role in C4 photosynthesis, although the contribution of NDH-mediated CEF is small in C3 photosynthesis. Here, we generated NDH-suppressed transformants of a C4 plant, Flaveria bidentis, and showed that the NDH-suppressed plants grow poorly, especially under low-light conditions. CO2 assimilation rates were consistently decreased in the NDH-suppressed plants under low and medium light intensities. Measurements of non-photochemical quenching (NPQ) of Chl fluorescence, the oxidation state of the reaction center of PSI (P700) and the electrochromic shift (ECS) of pigment absorbance indicated that proton translocation across the thylakoid membrane is impaired in the NDH-suppressed plants. Since proton translocation across the thylakoid membrane induces ATP production, these results suggest that NDH-mediated CEF plays a role in the supply of ATP which is required for C4 photosynthesis. Such a role is more crucial when the light that is available for photosynthesis is limited and the energy production by PET becomes rate-determining for C4 photosynthesis. Our results demonstrate that the physiological contribution of NDH-mediated CEF is greater in C4 photosynthesis than in C3 photosynthesis, suggesting that the mechanism of PET in C4 photosynthesis has changed from that in C3 photosynthesis accompanying the changes in the mechanism of CO2 assimilation.

Growth Properties and Biomass Production in the Hybrid C4 Crop Sorghum bicolor.

Hybrid vigor (heterosis) has been used as a breeding technique for crop improvement to achieve enhanced biomass production, but the physiological mechanisms underlying heterosis remain poorly understood. In this study, to find a clue to the enhancement of biomass production by heterosis, we systemically evaluated the effect of heterosis on the growth rate and photosynthetic efficiency in sorghum hybrid [Sorghum bicolor (L.) Moench cv. Tentaka] and its parental lines (restorer line and maintainer line). The final biomass of Tentaka was 10-14 times greater than that of the parental lines grown in an experimental field, but the relative growth rate during the vegetative growth stage did not differ. Tentaka exhibited a relatively enlarged leaf area with lower leaf nitrogen content per leaf area (Narea). When the plants were grown hydroponically at different N levels, daily CO2 assimilation per leaf area (A) increased with Narea, and the ratio of A to Narea (N-use efficiency) was higher in the plants grown at low N levels but not different between Tentaka and the parental lines. The relationships between the CO2 assimilation rate, the amounts of photosynthetic enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase, Chl and Narea did not differ between Tentaka and the parental lines. Thus, Tentaka tended to exhibit enlargement of leaf area with lower N content, leading to a higher N-use efficiency for CO2 assimilation, but the photosynthetic properties did not differ. The greater biomass in Tentaka was mainly due to the prolonged vegetative growth period.

Diurnal and developmental changes in energy allocation of absorbed light at PSII in field-grown rice.

The allocation of absorbed light energy in PSII to electron transport and heat dissipation processes in rice grown under waterlogged conditions was estimated with the lake model of energy transfer. With regard to diurnal changes in energy allocation, the peak of the energy flux to electron transport, J(PSII), occurred in the morning and the peak of the energy flux to heat dissipation associated with non-photochemical quenching of Chl fluorescence, J(NPQ), occurred in the afternoon. With regard to seasonal changes in energy allocation, J(PSII) in the rapidly growing phase was greater than that in the ripening phase, even though the leaves of rice receive less light in the growing phase than in the ripening period in Japan. This seasonal decrease in J(PSII) was accompanied by an increase in J(NPQ). One of the reasons for the lower J(PSII) in the ripening phase might be a more sever afternoon suppression of J(PSII). To estimate energy dissipation due to photoinhibition of PSII, J(NPQ) was divided into J(fast), which is associated with fast-recovering NPQ mainly due to qE, and J(slow), which is mainly due to photoinhibition. The integrated daily energy loss by photoinhibiton was calculated to be about 3-8% of light energy absorption in PSII. Strategies for the utilization of light energy adopted by rice are discussed. For example, very efficient photosynthesis under non-saturating light in the rapidly growing phase is proposed.

Exploiting transplastomically modified Rubisco to rapidly measure natural diversity in its carbon isotope discrimination using tuneable diode laser spectroscopy.

Carbon isotope discrimination (Δ) during C3 photosynthesis is dominated by the fractionation occurring during CO2-fixation by the enzyme Rubisco. While knowing the fractionation by enzymes is pivotal to fully understanding plant carbon metabolism, little is known about variation in the discrimination factor of Rubisco (b) as it is difficult to measure using existing in vitro methodologies. Tuneable diode laser absorption spectroscopy has improved the ability to make rapid measurements of Δ concurrently with photosynthetic gas exchange. This study used this technique to estimate b in vivo in five tobacco (Nicotiana tabacum L. cv Petit Havana [N,N]) genotypes expressing alternative Rubisco isoforms. For transplastomic tobacco producing Rhodospirillum rubrum Rubisco b was 23.8±0.7‰, while Rubisco containing the large subunit Leu-335-Val mutation had a b-value of 13.9±0.7‰. These values were significantly less than that for Rubisco from wild-type tobacco (b=29‰), a C3 species. Transplastomic tobacco producing chimeric Rubisco comprising tobacco Rubisco small subunits and the catalytic large subunits from either the C4 species Flaveria bidentis or the C3-C4 species Flaveria floridana had b-values of 27.8±0.8 and 28.6±0.6‰, respectively. These values were not significantly different from tobacco Rubisco.

Using tunable diode laser spectroscopy to measure carbon isotope discrimination and mesophyll conductance to CO2 diffusion dynamically at different CO2 concentrations.

In C3 leaves, the mesophyll conductance to CO2 diffusion, g(m) , determines the drawdown in CO2 concentration from intercellular airspace to the chloroplast stroma. Both g(m) and stomatal conductance limit photosynthetic rate and vary in response to the environment. We investigated the response of g(m) to changes in CO2 in two Arabidopsis genotypes (including a mutant with open stomata, ost1), tobacco and wheat. We combined measurements of gas exchange with carbon isotope discrimination using tunable diode laser absorption spectroscopy with a CO2 calibration system specially designed for a range of CO2 and O2 concentrations. CO2 was initially increased from 200 to 1000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm, or the sequence was reversed. In 2% O2 a step increase from 200 to 1000 ppm significantly decreased g(m) by 26-40% in all three species, whereas following a step decrease from 1000 to 200 ppm, the 26-38% increase in g(m) was not statistically significant. The response of g(m) to CO2 was less in 21% O2. Comparing wild type against the ost1 revealed that mesophyll and stomatal conductance varied independently in response to CO2. We discuss the effects of isotope fractionation factors on estimating g(m) .

Manganese toxicity disrupts indole acetic acid homeostasis and suppresses the CO2 assimilation reaction in rice leaves.

Despite the essentiality of Mn in terrestrial plants, its excessive accumulation in plant tissues can cause growth defects, known as Mn toxicity. Mn toxicity can be classified into apoplastic and symplastic types depending on its onset. Symplastic Mn toxicity is hypothesised to be more critical for growth defects. However, details of the relationship between growth defects and symplastic Mn toxicity remain elusive. In this study, we aimed to elucidate the molecular mechanisms underlying symplastic Mn toxicity in rice plants. We found that under excess Mn conditions, CO2 assimilation was inhibited by stomatal closure, and both carbon anabolic and catabolic activities were decreased. In addition to stomatal dysfunction, stomatal and leaf anatomical development were also altered by excess Mn accumulation. Furthermore, indole acetic acid (IAA) concentration was decreased, and auxin-responsive gene expression analyses showed IAA-deficient symptoms in leaves due to excess Mn accumulation. These results suggest that excessive Mn accumulation causes IAA deficiency, and low IAA concentrations suppress plant growth by suppressing stomatal opening and leaf anatomical development for efficient CO2 assimilation in leaves.

Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry.

In C(4) plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C(4) dicot Flaveria bidentis grown at a low (LI; 150 micromol quanta m(2) s(-1)) and medium (MI; 500 micromol quanta m(2) s(-1)) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO(2) assimilation per leaf area measured at 1500 micromol quanta m(2) s(-1) were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO(2) (phi) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. phi increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (S(m)) and bundle sheath surface area per unit leaf area (S(b)) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO(2) diffusion were greater in MI compared with LI plants. Despite being a C(4) species, F. bidentis is very plastic with respect to growth irradiance.

Transgenic rice overproducing Rubisco exhibits increased yields with improved nitrogen-use efficiency in an experimental paddy field.

The green revolution's breeding of semi-dwarf rice cultivars in the 1960s improved crop yields, with large increases in the use of nitrogen (N) fertilizer. However, excess N application has caused serious environmental problems, including acid rain and the eutrophication of rivers and oceans. To use N to improve crop yields, while minimizing the associated environmental costs, there is a need to produce crops with higher N-use efficiency and higher yield components. Here we show that transgenic rice overproducing ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco)-the key enzyme of photosynthesis-exhibits increased yields with improved N-use efficiency for increasing biomass production when receiving sufficient N fertilization in an experimental paddy field. This field experiment demonstrates an improvement in photosynthesis linked to yield increase due to a higher N-use efficiency in a major crop.

Light and CO2 do not affect the mesophyll conductance to CO2 diffusion in wheat leaves.

In C(3) plants, diffusion of CO(2) into leaves is restricted by stomata and subsequently by the intercellular airspaces and liquid phase into chloroplasts. While considerable information exists on the effect of environmental conditions on stomatal conductance (g(s)), little is known on whether the mesophyll conductance to CO(2) diffusion (g(m)) changes with respect to photon flux density (PFD) and CO(2) partial pressure (pCO(2)). In this study, the effects of PFD and/or pCO(2) on g(m) were examined in wheat leaves by combining gas exchange with carbon isotope discrimination measurements using a membrane inlet mass spectrometer. Measurements were made in 2% O(2) to reduce the fractionation associated with photorespiration. The magnitude of g(m) was estimated using the observed carbon isotope discrimination (Delta), ambient and intercellular pCO(2), CO(2) assimilation and respiration rates, either from an individual measurement made under one environmental condition or from a global fit to multiple measurements where PFD was varied. It was found that respiration made a significant and variable contribution to the observed discrimination, which associated with the difference in isotopic composition between CO(2) in the greenhouse and that used for gas exchange measurements. In wheat, g(m) was independent of PFD between 200 and 1500 micromol m(-2) s(-1) and was independent of p(i) between 80 and 500 microbar.

Relationships between quantum yield for CO2 assimilation, activity of key enzymes and CO2 leakiness in Amaranthus cruentus, a C4 dicot, grown in high or low light.

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus.

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.

Irradiance and phenotype: comparative eco-development of sun and shade leaves in relation to photosynthetic CO2 diffusion.

The subject of this paper, sun leaves are thicker and show higher photosynthetic rates than the shade leaves, is approached in two ways. The first seeks to answer the question: why are sun leaves thicker than shade leaves? To do this, CO2 diffusion within a leaf is examined first. Because affinity of Rubisco for CO2 is low, the carboxylation of ribulose 1,5-bisphosphate is competitively inhibited by O2, and the oxygenation of ribulose 1,5-bisphosphate leads to energy-consuming photorespiration, it is essential for C3 plants to maintain the CO2 concentration in the chloroplast as high as possible. Since the internal conductance for CO2 diffusion from the intercellular space to the chloroplast stroma is finite and relatively small, C3 leaves should have sufficient mesophyll surfaces occupied by chloroplasts to secure the area for CO2 dissolution and transport. This explains why sun leaves are thicker. The second approach is mechanistic or 'how-oriented'. Mechanisms are discussed as to how sun leaves become thicker than shade leaves, in particular, the long-distance signal transduction from mature leaves to leaf primordia inducing the periclinal division of the palisade tissue cells. To increase the mesophyll surface area, the leaf can either be thicker or have smaller cells. Issues of cell size are discussed to understand plasticity in leaf thickness.