Decreasing nitrogen assimilation under drought stress by suppressing DST-mediated activation of Nitrate Reductase 1.2 in rice.

Nitrogen is an essential nutrient for plant growth and development, and plays vital roles in crop yield. Assimilation of nitrogen is thus fine-tuned in response to heterogeneous environments. However, the regulatory mechanism underlying this essential process remains largely unknown. Here, we report that a zinc-finger transcription factor, drought and salt tolerance (DST), controls nitrate assimilation in rice by regulating the expression of OsNR1.2. We found that loss of function of DST results in a significant decrease of nitrogen use efficiency (NUE) in the presence of nitrate. Further study revealed that DST is required for full nitrate reductase activity in rice and directly regulates the expression of OsNR1.2, a gene showing sequence similarity to nitrate reductase. Reverse genetics and biochemistry studies revealed that OsNR1.2 encodes an NADH-dependent nitrate reductase that is required for high NUE of rice. Interestingly, the DST-OsNR1.2 regulatory module is involved in the suppression of nitrate assimilation under drought stress, which contributes to drought tolerance. Considering the negative role of DST in stomata closure, as revealed previously, the positive role of DST in nitrogen assimilation suggests a mechanism coupling nitrogen metabolism and stomata movement. The discovery of this coupling mechanism will aid the engineering of drought-tolerant crops with high NUE in the future.

Genome-Wide Association Study Unravels LRK1 as a Dark Respiration Regulator in Rice (Oryza sativa L.).

Respiration is a major plant physiological process that generates adenosine triphosphate (ATP) to support the various pathways involved in the plant growth and development. After decades of focused research on basic mechanisms of respiration, the processes and major proteins involved in respiration are well elucidated. However, much less is known about the natural variation of respiration. Here we conducted a survey on the natural variation of leaf dark respiration (Rd) in a global rice minicore diversity panel and applied a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with Rd. This rice minicore diversity panel consists of 206 accessions, which were grown under both growth room (GR) and field conditions. We found that Rd shows high single-nucleotide polymorphism (SNP) heritability under GR and it is significantly affected by genotype-environment interactions. Rd also exhibits strong positive correlation to the leaf thickness and chlorophyll content. GWAS results of Rd collected under GR and field show an overlapped genomic region in the chromosome 3 (Chr.3), which contains a lead SNP (3m29440628). There are 12 candidate genes within this region; among them, three genes show significantly higher expression levels in accessions with high Rd. Particularly, we observed that the LRK1 gene, annotated as leucine rich repeat receptor kinase, was up-regulated four times. We further found that a single significantly associated SNPs at the promoter region of LRK1, was strongly correlated with the mean annual temperature of the regions from where minicore accessions were collected. A rice lrk1 mutant shows only ~37% Rd of that of WT and retarded growth following exposure to 35 °C for 30 days, but only 24% reduction in growth was recorded under normal temperature (25 °C). This study demonstrates a substantial natural variation of Rd in rice and that the LRK1 gene can regulate leaf dark respiratory fluxes, especially under high temperature.

Overexpression of maize transcription factor mEmBP-1 increases photosynthesis, biomass, and yield in rice.

Identifying new options to improve photosynthetic capacity is a major approach to improve crop yield potential. Here we report that overexpression of the gene encoding the transcription factor mEmBP-1 led to simultaneously increased expression of many genes in photosynthesis, including genes encoding Chl a,b-binding proteins (Lhca and Lhcb), PSII (PsbR3 and PsbW) and PSI reaction center subunits (PsaK and PsaN), chloroplast ATP synthase subunit, electron transport reaction components (Fd1 and PC), and also major genes in the Calvin-Benson-Bassham cycle, including those encoding Rubisco, glyceraldehyde phosphate dehydrogenase, fructose bisphosphate aldolase, transketolase, and phosphoribulokinase. These increased expression of photosynthesis genes resulted in increased leaf chlorophyll pigment, photosynthetic rate, biomass growth, and grain yield both in the greenhouse and in the field. Using EMSA experiments, we showed that mEmBP-1a protein can directly bind to the promoter region of photosynthesis genes, suggesting that the direct binding of mEmBP-1a to the G-box domain of photosynthetic genes up-regulates expression of these genes. Altogether, our results show that mEmBP-1a is a major regulator of photosynthesis, which can be used to increase rice photosynthesis and yield in the field.

Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL.

Atmospheric carbon dioxide (CO2) is assimilated by the most abundant but sluggish enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here we show that acetylation of lysine residues of the Rubisco large subunit (RbcL), including Lys201 and Lys334 in the active sites, may be an important mechanism in the regulation of Rubisco activities. It is well known that Lys201 reacts with CO2 for carbamylation, a prerequisite for both carboxylase and oxygenase activities of Rubisco, and Lys334 contacts with ribulose-1,5-bisphosphate (RuBP). The acetylation level of RbcL in plants is lower during the day and higher at night, inversely correlating with the Rubisco carboxylation activity. A search of the chloroplast proteome database did not reveal a canonical acetyltransferase; instead, we found that a plant-derived metabolite, 7-acetoxy-4-methylcoumarin (AMC), can non-enzymatically acetylate both native Rubisco and synthesized RbcL peptides spanning Lys334 or Lys201. Furthermore, lysine residues were modified by synthesized 4-methylumbelliferone esters with different electro- and stereo-substitutes, resulting in varied Rubisco activities. 1-Chloroethyl 4-methylcoumarin-7-yl carbonate (ClMC) could transfer the chloroethyl carbamate group to lysine residues of RbcL and completely inactivate Rubisco, whereas bis(4-methylcoumarin-7-yl) carbonate (BMC) improved Rubisco activity through increasing the level of Lys201 carbamylation. Our findings indicate that RbcL acetylation negatively regulates Rubisco activity, and metabolic derivatives can be designed to dissect and improve CO2 fixation efficiency of plants through lysine modification.

Light-harvesting regulation from leaf to molecule with the emphasis on rapid changes in antenna size.

In the sunlight-fluctuating environment, plants often encounter both light-deficiency and light-excess cases. Therefore, regulation of light harvesting is absolutely essential for photosynthesis in order to maximize light utilization at low light and avoid photodamage of the photosynthetic apparatus at high light. Plants have developed a series of strategies of light-harvesting regulation during evolution. These strategies include rapid responses such as leaf movement and chloroplast movement, state transitions, and reversible dissociation of some light-harvesting complex of the photosystem II (LHCIIs) from PSII core complexes, and slow acclimation strategies such as changes in the protein abundance of light-harvesting antenna and modifications of leaf morphology, structure, and compositions. This review discusses successively these strategies and focuses on the rapid change in antenna size, namely reversible dissociation of some peripheral light-harvesting antennas (LHCIIs) from PSII core complex. It is involved in protective role and species dependence of the dissociation, differences between the dissociation and state transitions, relationship between the dissociation and thylakoid protein phosphorylation, and possible mechanism for thermal dissipation by the dissociated LHCIIs.

The reduced plastid-encoded polymerase-dependent plastid gene expression leads to the delayed greening of the Arabidopsis fln2 mutant.

In Arabidopsis leaf coloration mutants, the delayed greening phenomenon is common. Nonetheless, the mechanism remains largely elusive. Here, a delayed greening mutant fln2-4 of FLN2 (Fructokinase-Like Protein2) was studied. FLN2 is one component of Transcriptionally Active Chromosome (TAC) complex which is thought to contain the complete plastid-encoded polymerase (PEP). fln2-4 displayed albino phenotype on medium without sucrose. The PEP-dependent plastid gene expression and chloroplast development were inhibited in fln2-4. Besides interacting with thioredoxin z (TRX z), we identified that FLN2 interacted with another two members of TAC complex in yeast including its homologous protein FLN1 (Fructokinase-Like Protein1) and pTAC5. This indicates that FLN2 functions in regulation of PEP activity associated with these TAC components. fln2-4 exhibited delayed greening on sucrose-containing medium. Comparison of the PEP-dependent gene expression among two complete albino mutants (trx z and ptac14), two yellow mutants (ecb2-2 and ys1) and the fln2-4 showed that fln2-4 remains partial PEP activity. FLN2 and FLN1 are the target proteins of TRX z involved in affecting the PEP activity. Together with the data that FLN1 could interact with itself in yeast, FLN1 may form a homodimer to replace FLN1-FLN2 as the TRX z target in redox pathway for maintaining partial PEP activity in fln2-4. We proposed the partial PEP activity in the fln2 mutant allowed plastids to develop into fully functional chloroplasts when exogenous sucrose was supplied, and finally the mutants exhibited green phenotype.

A mutation of OSOTP 51 leads to impairment of photosystem I complex assembly and serious photo-damage in rice.

Gene expression in chloroplasts is regulated by many nuclear-encoded proteins. In this study, we isolated a rice (Oryza sativa subsp. japonica) mutant osotp51 with significant reduction in photosystem I (PSI). The osotp51 is extremely sensitive to light and accumulates a higher level of reactive oxygen species. Its leaves are almost albino when grown at 40 µmol photons/m(2) per s. However, grown at 4 µmol photons/m(2) per s, osotp51 has a similar phenotype to the wild-type. 77K chlorophyll fluorescence analysis showed a blue shift in the highest peak emission from PSI in osotp51. In addition, the level of PSI and PSII dimer is dramatically reduced in osotp51. OSOTP 51 encodes a pentatricopeptide repeats protein, homologous to organelle transcript processing 51 in Arabidopsis. Loss-of-function OSOTP51 affects intron splicing of a number of plastid genes, particularly the ycf3 coding a protein involved in the assembly of PSI complex. OSOTP51 is functionally conserved in higher plants. The mutation of osotp51 indirectly leads to a widespread change in the structure and functions of PSI, results in severe photoinhibition, and finally dies, even when grown under very low light intensity.

Reversible association of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase with the thylakoid membrane depends upon the ATP level and pH in rice without heat stress.

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) in the thylakoid membrane (TM) has been shown to play a role in protection and regulation of photosynthesis under moderate heat stress. However, the physiological significance of RCA bound to the TM (TM-RCA) without heat stress remains unknown. In this study, it is first shown, using experiments in vivo, that the TM-RCA varies in rice leaves at different development stages, under different environmental conditions, and in a rice mutant. Furthermore, it is shown that the amount of TM-RCA always increased when the Rubisco activation state and the pH gradient across the TM (DeltapH) decreased. It was then demonstrated in vitro that the RCA bound dynamically to TM and the amount of TM-RCA increased during Rubisco activation. A high level of ATP and a high pH value promoted the dissociation of RCA from the TM. Both the RCA association with and dissociation from the TM showed conformational changes related to the ATP level or pH as indicated by the changes in fluorescence intensity of 1-anilinonaphthalene-8-sulphonic acid (ANS) binding to RCA. These results suggest that the reversible association of RCA with the TM is ATP and pH (or DeltapH) dependent; it might be involved in the RCA activation of Rubisco, in addition to the previously discovered role in the protection and regulation of photosynthesis under heat stress.

[Exploring the observation methods of photosynthetic responses to light and carbon dioxide].

Through observing the responses of photosynthesis in leaves of broad bean grown under natural conditions to light and CO(2) using a portable photosynthetic analyzer LI-6400, the following findings were obtained. (1) Observing the response of photosynthesis to light using a leaf without being induced by light might lead to an artifact that photosynthesis was not saturated even under full sunlight. (2) The calculated saturating light intensity of photosynthesis by some empirical equations was much lower than actual value obtained by observing. (3) During observation of photosynthetic response to CO(2), each step of changing CO(2) concentration should be accompanied by a match step of the photosynthetic analyzer, otherwise, there will be substantial deviations in the results obtained. (4) Observing photosynthetic response to CO(2) at non-saturating light might lead to an underestimation of leaf photosynthetic capacity.

In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits.

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

Photosynthetic acclimation in rice leaves to free-air CO2 enrichment related to both ribulose-1,5-bisphosphate carboxylation limitation and ribulose-1,5-bisphosphate regeneration limitation.

Net photosynthetic rates (Pns) in leaves were compared between rice plants grown in ambient air control and free-air CO2 enrichment (FACE, about 200 micromol mol(-1) above ambient) treatment rings. When measured at the same CO2 concentration, the Pn of FACE leaves decreased significantly, indicating that photosynthetic acclimation to high CO2 occurs. Although stomatal conductance (Gs) in FACE leaves was markedly decreased, intercellular CO2 concentrations (Ci) were almost the same in FACE and ambient leaves, indicating that the photosynthetic acclimation is not caused by the decreased Gs. Furthermore, carboxylation efficiency and maximal Pn, both light and CO2-saturated Pn, were decreased in FACE leaves, as shown by the Pn-Ci curves. In addition, the soluble protein, Rubisco (ribulose-1,5-bisphosphate caboxylase/oxygenase), and its activase contents as well as the sucrose-phosphate synthase activity decreased significantly, while some soluble sugar, inorganic phosphate, chlorophyll and light-harvesting complex II (LHC II) contents increased in FACE leaves. It appears that the photosynthetic acclimation in rice leaves is related to both ribulose-1,5-bisphosphate (RuBP) carboxylation limitation and RuBP regeneration limitation.