Natural variation in the fast phase of chlorophyll a fluorescence induction curve (OJIP) in a global rice minicore panel.

Photosynthesis can be probed through Chlorophyll a fluorescence induction (FI), which provides detailed insight into the electron transfer process in Photosystem II, and beyond. Here, we have systematically studied the natural variation of the fast phase of the FI, i.e. the OJIP phase, in rice. The OJIP phase of the Chl a fluorescence induction curve is referred to as "fast transient" lasting for less than a second; it is obtained after a dark-adapted sample is exposed to saturating light. In the OJIP curve, "O" stands for "origin" (minimal fluorescence), "P" for "peak" (maximum fluorescence), and J and I for inflection points between the O and P levels. Further, Fo is the fluorescence intensity at the "O" level, whereas Fm is the intensity at the P level, and Fv (= Fm - Fo) is the variable fluorescence. We surveyed a set of quantitative parameters derived from the FI curves of 199 rice accessions, grown under both field condition (FC) and growth room condition (GC). Our results show a significant variation between Japonica (JAP) and Indica (IND) subgroups, under both the growth conditions, in almost all the parameters derived from the OJIP curves. The ratio of the variable to the maximum (Fv/Fm) and of the variable to the minimum (Fv/Fo) fluorescence, the performance index (PIabs), as well as the amplitude of the I-P phase (AI-P) show higher values in JAP compared to that in the IND subpopulation. In contrast, the amplitude of the O-J phase (AO-J) and the normalized area above the OJIP curve (Sm) show an opposite trend. The performed genetic analysis shows that plants grown under GC appear much more affected by environmental factors than those grown in the field. We further conducted a genome-wide association study (GWAS) using 11 parameters derived from plants grown in the field. In total, 596 non-unique significant loci based on these parameters were identified by GWAS. Several photosynthesis-related proteins were identified to be associated with different OJIP parameters. We found that traits with high correlation are usually associated with similar genomic regions. Specifically, the thermal phase of FI, which includes the amplitudes of the J-I and I-P subphases (AJ-I and AI-P) of the OJIP curve, is, in turn, associated with certain common genomic regions. Our study is the first one dealing with the natural variations in rice, with the aim to characterize potential candidate genes controlling the magnitude and half-time of each of the phases in the OJIP FI curve.

Genome-wide association study identifies variation of glucosidase being linked to natural variation of the maximal quantum yield of photosystem II.

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, Fv /Fm ) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in Fv /Fm , which shows low level of variations in plants under non-stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome-wide association study (GWAS) and transgenic analysis in a rice mini-core collection. We report here that four single-nucleotide polymorphisms, located in the promoter region of ß-glucosidase 5 (BGlu-5), are associated with observed variation in Fv /Fm . Indeed, our transgenic analysis showed a good correlation between BGlu-5 and Fv /Fm . Thus, our work demonstrates the feasibility of using GWAS to study natural variation in Fv /Fm , suggesting that cis-element polymorphism, affecting the BGlu-5 expression level, may, indirectly, contribute to Fv /Fm variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.

Changes in the photosynthesis properties and photoprotection capacity in rice (Oryza sativa) grown under red, blue, or white light.

Non-photochemical quenching (NPQ) of the excited state of chlorophyll a is a major photoprotective mechanism plants utilize to survive under high light. Here, we report the impact of long-term light quality treatment on photosynthetic properties, especially NPQ in rice. We used three LED-based light regimes, i.e., red (648-672 nm), blue (438-460 nm), and "warm" white light (529-624 nm), with the incident photon flux density of 300 µmol photons m-2 s-1, the difference in the absorbed photon flux densities by leaves grown under different light quality being less than 7%. Our results show that blue light, as compared to white light, induced a significant decrease in Fv/Fm, a decreased rate of reduction of P700+ after P700 was completely oxidized; furthermore, blue light also induced higher NPQ with an increased initial speed of NPQ induction, which corresponds to the qE component of NPQ, and a lower maximum quantum yield of PSII, i.e., Y(II). In contrast, rice grown under long-term red light showed decreased Y(II) and increased NPQ, but with no change in Fv/Fm. Furthermore, we found that rice grown under either blue or red light showed decreased transcript abundance of both catalase and ascorbate peroxidase, together with an increased H2O2 content, as compared to rice grown under white light. All these data suggest that even under a moderate incident light level, rice grown under blue or red light led to compromised antioxidant system, which contributed to decreased quantum yield of photosystem II and increased NPQ.

Leaf Photosynthetic Parameters Related to Biomass Accumulation in a Global Rice Diversity Survey.

Mining natural variations is a major approach to identify new options to improve crop light use efficiency. So far, successes in identifying photosynthetic parameters positively related to crop biomass accumulation through this approach are scarce, possibly due to the earlier emphasis on properties related to leaf instead of canopy photosynthetic efficiency. This study aims to uncover rice (Oryza sativa) natural variations to identify leaf physiological parameters that are highly correlated with biomass accumulation, a surrogate of canopy photosynthesis. To do this, we systematically investigated 14 photosynthetic parameters and four morphological traits in a rice population, which consists of 204 U.S. Department of Agriculture-curated minicore accessions collected globally and 11 elite Chinese rice cultivars in both Beijing and Shanghai. To identify key components responsible for the variance of biomass accumulation, we applied a stepwise feature-selection approach based on linear regression models. Although there are large variations in photosynthetic parameters measured in different environments, we observed that photosynthetic rate under low light (Alow) was highly related to biomass accumulation and also exhibited high genomic inheritability in both environments, suggesting its great potential to be used as a target for future rice breeding programs. Large variations in Alow among modern rice cultivars further suggest the great potential of using this parameter in contemporary rice breeding for the improvement of biomass and, hence, yield potential.

Rapid stomatal response to fluctuating light: an under-explored mechanism to improve drought tolerance in rice.

Light inside a canopy constantly fluctuates. Under fluctuating light (FL) conditions, stomatal conductance and photosynthetic rate constantly change. In this study, we explored whether this dynamics of stomata movements upon FL influenced the water use efficiency of rice in the field. We used a USDA-curated rice mini-core diversity panel consisting of 204 worldwide distributed accessions. A priori model on dynamic stomatal response to FL was utilised to identify kinetic parameters describing the stomatal delays during the closing (τcl) and the opening (τop) phase. Result showed that τcl had a larger variation than τop across the mini-core panel. τcl was negatively correlated with water use efficiency (WUE) related traits, stem diameter, grain weight per tiller and heading time, but positively correlated with maximum annual temperature, carbon assimilation related traits and biomass (P<0.05). We further showed a strong correlation of τcl with the relative decrease of biomass under drought in 14 accessions with different τcl. We discussed the adjustment of stomatal conductance under fluctuating light in light of the trade-off between optimising CO2 uptake and optimising water saving. This study suggests that stomatal dynamics under fluctuating light is closely related to drought resistance and hence detailed study is needed to enable its application in breeding drought tolerance in rice.

Characterization of the structural changes and photochemical activity of photosystem I under Al(3+) effect.

The photochemical activity of photosystem I (PSI) as affected by Al(3+) was investigated in thylakoid membranes and PSI submembrane fractions isolated from spinach. Biophysical and biochemical techniques such as oxygen uptake, light induced absorbance changes at 820nm, chlorophyll fluorescence emission, SDS-polyacrylamide gel electrophoresis, and FTIR spectroscopy have been used to analyze the sites and action modes of this cation on the PSI complex. Our results showed that Al(3+) above 3mM induces changes in the redox state of P700 reflected by an increase of P700 photooxidation phase and a delay of the slower rate of P700 re-reduction which reveals that Al(3+) exerted an inhibitory action at the donor side of PSI especially at plastocyanin (PC). Furthermore, results of P700 photooxidation monitored in the presence of DCMU with or without MV suggested that the same range of Al(3+) concentrations impairs the photochemical reaction centers (RC) of PSI, as shown by the decline in the amount of active population of P700, and disrupts the charge separation between P700 and the primary electron acceptor A0 leading to the inhibition of electron transfer at the acceptor side of PSI. These inhibitory actions were also accompanied by an impairment of the energy transfer from light harvesting complex (LHCI) to RC of PSI, following the disconnection of LHCI antenna as illustrated by an enhancement of chlorophyll fluorescence emission spectra at low temperature (77K). The above results coincided with FTIR measurements that indicated a conformational change of the protein secondary structures in PSI complex where 25% of α-helix was converted into ß-sheet, ß-antiparallel and turn structures. These structural changes in PSI complex proteins are closely related with the alteration photochemical activity of PSI including the inhibition of the electron transport through both acceptor and donor sides of PSI.

Mechanism of interaction of Al3+ with the proteins composition of photosystem II.

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al 3+, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn4O5Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (Fv/Fm) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F0 and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al 3+ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of ß-sheet and random coil structures in Al 3+-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.

Variations between the photosynthetic properties of elite and landrace Chinese rice cultivars revealed by simultaneous measurements of 820 nm transmission signal and chlorophyll a fluorescence induction.

The difference between the photosynthetic properties of elite and landrace Chinese rice cultivars was studied, using chlorophyll a fluorescence induction (mostly a monitor of Photosystem II activity) and I820 transmission signal (mostly a monitor of Photosystem I activity) to identify potential photosynthetic features differentiating these two groups, which show different degrees of artificial selection and grain yields. A higher fluorescence (related to PSII) IP rise phase and a lower P700(+) (related to PSI) accumulation were observed in the elite cultivars as compared to the landraces. Using these data, together with simulation data from a kinetic model of fluorescence induction, we show that the high IP rise phase and the low P700(+) accumulation can be a result of transient block on electron transfer and traffic jam on the electron acceptor side of PSI under a high [NADPH]/[NADP(+)] ratio. Considering that the ferredoxin NADP(+) reductase (FNR) transcript levels of XS134 (a representative elite cultivars) remains unaffected during the first few minutes of light/dark transition compared to Q4145 (a representative landrace cultivars), which shows a strong decline during the same time range, we propose that the FNR of elite cultivars may take more time to be inactivated in darkness. During this time the FNR enzyme can continue to reduce NADP(+) molecules, leading to initially high [NADPH]/[NADP(+)] ratio during OJIP transient. These data suggested a potential artificial selection of FNR during the breeding process of these examined elite rice cultivars.

Protective action of spermine and spermidine against photoinhibition of photosystem I in isolated thylakoid membranes.

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O2 uptake rates, P700 photooxidation and the accumulation of superoxide anions (O2(-)) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O2(-) generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O2(-) generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.

Destabilization of the oxygen evolving complex of photosystem II by Al3+.

The inhibitory effect of Al(3+) on photosynthetic electron transport was investigated in isolated thylakoid membranes of spinach. A combination of oxygen evolution, chlorophyll fluorescence induction (FI) and decay and thermoluminescence measurements have been used to characterize photosystem II (PSII) electron transport in the presence of this toxic metal cation. Our results show that below 3 mm, Al(3+) already caused a destabilization of the Mn4 O5 Ca cluster of the oxygen evolving complex (OEC). At these concentrations, an increase in the relative amplitude of the first phase (OJ) of FI curve and retardation of the fluorescence decay kinetics following excitation with a single turnover flash were also observed. A transmembrane structural modification of PSII polypeptides due to the interaction of Al(3+) at the OEC is proposed to retard electron transfer between the quinones QA and QB . Above 3 mm, Al(3+) strongly retarded fluorescence induction and significantly reduced Fv /Fm together with the maximal amplitude of chlorophyll fluorescence induced by a single turnover flash. This chlorophyll fluorescence quenching was attributed to the formation of P680(+) due to inhibition of electron transfer between tyrosine 161 of D1 subunit and P680.

Positive charges of polyamines protect PSII in isolated thylakoid membranes during photoinhibitory conditions.

The effects of the positive charges of amines such as spermine (SPM), putrescine (PUT) and methylamine (MET) on the protection of PSII against excessive illumination were investigated in isolated thylakoid membranes. Under photoinhibition conditions, water oxidation, the kinetics of the Chl fluorescence rise and charge recombination in PSII were affected. A low concentration of SPM (1 mM) added before photoinhibition produced a significant improvement of F(v)/F(0), the oxygen yield and the amplitude of the B-band of thermoluminescence compared with the other amines. Amongst the amines studied, only SPM could protect the photosynthetic apparatus under photoinhibition conditions. This protection was probably provided by the polycationic nature of SPM (four positive charges at physiological pH), which can stabilize surface-exposed proteins of PSII through electrostatic interaction.

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II.

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y(Z) and P680(+). As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F(0).