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Autophagy is an intracellular process leading to the vacuolar degradation of cytoplasmic components. Autophagic degradation of chloroplasts is particularly activated in leaves under conditions of low sugar availability. Here, we investigated the importance of autophagy in the energy availability and growth of Arabidopsis (Arabidopsis thaliana). autophagy-deficient (atg) mutants showed reduced growth under short-day conditions. This growth inhibition was largely relieved under continuous light or under short-day conditions combined with feeding of exogenous sucrose, suggesting that autophagy is involved in energy production at night for growth. Arabidopsis accumulates starch during the day and degrades it for respiration at night. Nighttime energy availability is perturbed in starchless mutants, in which a lack of starch accumulation causes a transient sugar deficit at night. We generated starchless and atg double mutants and grew them under different photoperiods. The double mutants showed more severe phenotypes than did atg or starchless single mutants: reduced growth and early cell death in leaves were observed when plants were grown under 10-h photoperiods. Transcript analysis of dark-inducible genes revealed that the sugar starvation symptoms observed in starchless mutants became more severe in starchless atg double mutants. The contents of free amino acids (AAs) increased, and transcript levels of several genes involved in AA catabolism were elevated in starchless mutant leaves. The increases in branched-chain AA and aromatic AA contents were partially compromised in starchless atg double mutants. We conclude that autophagy can contribute to energy availability at night by providing a supply of alternative energy sources such as AAs.

Flavodiiron protein (FLV) mediates photoreduction of O₂ to H₂O. It is conserved from cyanobacteria to gymnosperms but not in angiosperms. The introduction of a moss (Physcomitrella patens) FLV (PpFLV) gene into Arabidopsis (Arabidopsis thaliana) made photosystem I (PSI) resistant to fluctuating light. Here, we used the same strategy with three rice (Oryza sativa) genotypes. PpFLV in the wild-type rice background functioned as an efficient PSI electron sink and increased resistance to PSI photodamage under fluctuating light. The introduction of PpFLV into the PGR5-RNAi mutant [defective in PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron transport around PSI, CET-PSI], the crr6 mutant [defective in chloroplast NAD(P)H-dehydrogenase-like complex (NDH)-dependent CET-PSI], and the PGR5-RNAi crr6 double mutant (double defective in CET-PSI activity) alleviated PSI photodamage under fluctuating light. Furthermore, PpFLV substituted for the function of PGR5- and NDH-dependent CET-PSI without competing for CO₂ assimilation under constant light, as there was no difference in CO₂ assimilation per Rubisco content and biomass production was recovered to the wild-type level. Thus, the exogenous FLV system could act not only as a safety valve under fluctuating light, but also generate a proton motive force for balancing the ATP/NADPH production ratio during steady-state photosynthesis.

Rubisco is composed of eight small subunits coded for by the nuclear RBCS multigene family and eight large subunits coded for by the rbcL gene in the plastome. For synthesis of the Rubisco holoenzyme, both genes need to be expressed coordinately. To investigate this molecular mechanism, the protein synthesis of two subunits of Rubisco was characterized in transgenic rice (Oryza sativa) plants with overexpression or antisense suppression of the RBCS gene. Total RBCS and rbcL messenger RNA (mRNA) levels and RBCS and RbcL synthesis simultaneously increased in RBCS-sense plants, although the increase in total RBCS mRNA level was greater. In RBCS-antisense plants, the levels of these mRNAs and the synthesis of the corresponding proteins declined to a similar extent. The amount of RBCS synthesized was tightly correlated with rbcL mRNA level among genotypes but not associated with changes in mRNA levels of other major chloroplast-encoded photosynthetic genes. The level of rbcL mRNA, in turn, was tightly correlated with the amount of RbcL synthesized, the molar ratio of RBCS synthesis to RbcL synthesis being identical irrespective of genotype. Polysome loading of rbcL mRNA was not changed. These results demonstrate that the availability of RBCS protein up-regulates the gene expression of rbcL primarily at the transcript level in a quantitative manner for stoichiometric assembly of Rubisco holoenzyme.

Photorespiration coupled with CO2 assimilation is thought to act as a defense system against photoinhibition caused by osmotic stress. In the present study, we examined whether such a mechanism is operative for the protection of photosystem I (PSI) in rice ( Oryza sativa L.) including transgenic plants with decreased and increased Rubisco content ( RBCS -antisense and RBCS -sense plants, respectively). All plants were hydroponically grown and moderate osmotic stress was imposed using hydroponic culture solutions containing poly(ethylene glycol) (PEG) at 16% or 20% (w/v) for 2 d. In wild-type plants, the rates of CO2 assimilation ( A ) were significantly decreased by the PEG treatment, whereas the photorespiration activity estimated from the rates of electron transport in photosystem II (PSII) and A were not affected. The maximal quantum efficiency of PSII ( F v/ F m) and the maximal activity of PSI ( P m) were also not affected. In RBCS -antisense plants, A and the estimated photorespiration activity were considerably lower than those in wild-type plants in the presence or absence of the PEG treatment. P m and both F v/ F m and P m decreased in the 16% PEG-treated and 20% PEG-treated RBCS -antisense plants, respectively. Thus, the decrease in Rubisco content led to the photoinhibition of PSI and PSII, indicating the importance of photorespiration coupled with CO2 assimilation for the protection of PSI from moderate PEG-induced osmotic stress. It was also shown that PSI was more sensitive to osmotic stress than PSII. In the PEG-treated wild-type and RBCS -antisense plants, osmotic-stress responses of the photosynthetic electron transport reactions upstream of PSI led to the oxidation of P700, which is thought to prevent PSI from over-reduction. Although such a defense system operated, it was not sufficient for the protection of PSI in RBCS -antisense plants. In addition, there were no large differences in the parameters measured between wild-type and RBCS -sense plants, as overproduction of Rubisco did not increase photorespiration activity.

BackgroundImprovement in photosynthesis is one of the most promising approaches to increase grain yields. Transgenic rice plants overproducing Rubisco by 30% (RBCS-sense rice plants) showed up to 28% increase in grain yields under sufficient nitrogen (N) fertilization using an isolated experimental paddy field (Yoon et al. in Nat Food 1:134–139, 2020). The plant N contents above-ground sections and Rubisco contents of the flag leaves were higher in the RBCS-sense plants than in the wild-type rice plants during the ripening period, which may be reasons for the increased yields. However, some imprecise points were left in the previous research, such as contributions of photosynthesis of leaves below the flag leaves to the yield, and maintenance duration of high photosynthesis of RBCS-sense rice plants during ripening periods.ResultIn this research, the photosynthetic capacity and canopy architecture were analyzed to explore factors for the increased yields of RBCS-sense rice plants. It was found that N had already been preferentially distributed into the flag leaves at the early ripening stage, contributing to maintaining higher Rubisco content levels in the enlarged flag leaves and extending the lifespan of the flag leaves of RBCS-sense rice plants throughout ripening periods under sufficient N fertilization. The higher amounts of Rubisco also improved the photosynthetic activity in the flag leaves throughout the ripening period. Although the enlarged flag leaves of the RBCS-sense rice plants occupied large spatial areas of the uppermost layer in the canopy, no significant prevention of light penetration to leaves below the flag leaves was observed. Additionally, since the CO2 assimilation rates of lower leaves between wild-type and RBCS-sense rice plants were the same at the early ripening stage, the lower leaves did not contribute to an increase in yields of the RBCS-sense rice plants.ConclusionWe concluded that improvements in the photosynthetic capacity by higher leaf N and Rubisco contents, enlarged leaf area and extended lifespan of flag leaves led to an increase in grain yields of RBCS-sense rice plants grown under sufficient N fertilization.

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, CO 2 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 CO 2 assimilation in leaves.

Metabolome analyses have indicated an accumulation of sedoheptulose 7-phosphate in transgenic rice plants with overproduction of Rubisco (Suzuki et al. in Plant Cell Environ 35:1369–1379, 2012 . doi: 10.1111/j.1365-3040.2012.02494.x ). Since Rubisco overproduction did not quantitatively enhance photosynthesis even under CO 2 -limited conditions, it is suspected that such an accumulation of sedoheptulose 7-phosphate hampers the improvement of photosynthetic capacity. In the present study, the gene of transketolase, which is involved in the metabolism of sedoheptulose 7-phosphate, was co-overexpressed with the Rubisco small subunit gene in rice. Rubisco and transketolase were successfully overproduced in comparison with those in wild-type plants by 35–53 and 39–84 %, respectively. These changes in the amounts of the proteins were associated with those of the mRNA levels. However, the rate of CO 2 assimilation under high irradiance and different [CO 2 ] did not differ between co-overexpressed plants and wild-type plants. Thus, co-overproduction of Rubisco and transketolase did not improve photosynthesis in rice. Transketolase was probably not a limiting factor of photosynthesis as overproduction of transketolase alone by 80–94 % did not affect photosynthesis.

We have previously suggested that in rice (Oryza sativa L.) leaves of different ages and N nutrition statuses, photosystems II and I (PSII and PSI, respectively) are regulated depending on N partitioning to Rubisco, which can determine the magnitude of unutilized light energy. The robustness of this mechanism was tested using Rubisco-antisense transgenic rice plants, in which reduced N partitioning to Rubisco markedly increases unutilized light energy. In wild-type plants, N partitioning to Rubisco tended to be smaller in the leaves at lower positions owing to leaf senescence. In the transgenic plants, N partitioning to Rubisco was generally smaller than in the wild-type plants and was relatively constant among leaf positions. The quantum efficiency of PSII [Y(II)] and quantum yield of non-photochemical quenching [Y(NPQ)] correlated positively and negatively, respectively, with N partitioning to Rubisco irrespective of leaf position or genotype. The oxidation levels of the reaction center chlorophyll of PSI (P700) [Y(ND)] negatively correlated with N partitioning to Rubisco. However, in mature and early senescent leaves of the transgenic plants, Y(ND) was markedly lower than expected from N partitioning to Rubisco. These results suggest that in the transgenic plants, the regulation depending on N partitioning to Rubisco is robust for PSII but fails for PSI in mature and early senescing leaves. In these leaves, the magnitudes of P700 oxidation were found to be less than expected from the Y(II) and Y(NPQ) values. The mechanistic reasons and physiological implications of these phenomena are discussed.

It has been reported that overproduction of Rubisco activase (RCA) in rice (Oryza sativa L.) decreased Rubisco content, resulting in declining photosynthesis. We examined the effects of RCA levels on Rubisco content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and Rubisco small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%–20% decrease in Rubisco content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, Rubisco content did not change in RCA-HI lines. Conversely, in plants with 50%–60% increased RCA content (RCA-MI lines), Rubisco levels remained unchanged, regardless of N concentration. Such effects on Rubisco content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, Rubisco levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on Rubisco content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in Rubisco content, depending on the degree of RCA production.