Choon Hwan Lee

Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions

Photosystem II (PSII) is vulnerable to high light (HL) illumination resulting in photoinhibition. In addition to photoprotection mechanisms, plants have developed an efficient PSII repair mechanism to save themselves from irreversible damage to PSII under abiotic stresses including HL illumination. The phosphorylation/dephosphorylation cycle along with subsequent degradation of photodamaged D1 protein to be replaced by the insertion of a newly synthesized copy of D1 into the PSII complex, is the core function of the PSII repair cycle. The exact mechanism of this process is still under discussion. We describe the recent progress in identifying the kinases, phosphatases and proteases, and in understanding their involvement in the maintenance of thylakoid structure and the quality control of proteins by PSII repair cycle during photoinhibition.

ZEBRA-NECROSIS, a thylakoid-bound protein, is critical for the photoprotection of developing chloroplasts during early leaf development

The zebra-necrosis (zn) mutant of rice (Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness.

Production of superoxide from Photosystem II in a rice ( Oryza sativa L.) mutant lacking PsbS

BackgroundPsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ.ResultsIn studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that.ConclusionThese results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy.

Loss-of-function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa).

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T-DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild-type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair.

Age-dependent changes in the functions and compositions of photosynthetic complexes in the thylakoid membranes of Arabidopsis thaliana

Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (Fv/Fm) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28xa0days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.

Developmental stage-dependent differential gene expression of superoxide dismutase isoenzymes and their localization and physical interaction network in rice ( Oryza sativa L.)

Superoxide dismutase (SOD) isoenzymes are essential for scavenging excess reactive oxygen species in living organisms. So far, expression pattern of SOD isoenzymes genes along leaf development plus their sub-cellular localization and physical interaction network have not yet been clearly elucidated. Using multiple bioinformatics tools, we predicted the sub-cellular localizations of SOD isoforms and described their physical interactions in rice. Using in silico approaches, we obtained several evidences for existence of seven SOD genes and a SOD copper chaperone gene. Their transcripts were differentially expressed along with different developmental stage of rice leaf. Finally, we performed quantitative real time-polymerase chain reaction (qRT-PCR) to validate in silico differential expression pattern of SOD genes experimentally. Expression of two cytosolic cCuZn-SODs was high during the whole vegetative stage. Two plastidic Fe-SODs were found and their expression levels were very low and started to increase from the late vegetative stage. Their expression patterns were very similar to each other, indicating the formation of heterodimer. However, their expression patterns are different from those for ArabidopsisFe-SODs. The expression of pCuZn-SOD was very high in the early developmental stage, but qRT-PCR results were different, which remains for further study. From the results on the differential expression of SOD genes, we can understand the role of each SOD gene and even predict their role under certain circumstances based on in silico analysis.

Identification and differential expression of two dehydrin cDNAs during maturation of Jatropha curcas seeds

Plant dehydrin proteins (DHNs) are known to be important for environmental stress tolerance and are involved in various developmental processes. Two full-length cDNAs JcDHN-1 and JcDHN-2 encoding two dehydrins from Jatropha curcas seeds were identified and characterized. JcDHN-1 is 764 bp long and contains an open reading frame of 528 bp. The deduced JcDHN-1 protein has 175 a.a. residues that form a 19.3-kDa polypeptide with a predicted isoelectric point (pI) of 6.41. JcDHN-2 is 855 bp long and contains an open reading frame of 441 bp. The deduced JcDHN-2 protein has 156 a.a. residues that form a 17.1-kDa polypeptide with a predicted pI of 7.09. JcDHN-1 is classified as type Y3SK2 and JcDHN-2 is classified as type Y2SK2 according to the YSK shorthand for structural classification of dehydrins. Homology analysis indicates that both JcDHN-1 and JcDHN-2 share identity with DHNs of other plants. Analysis of the conserved domain revealed that JcDHN-2 has glycoside hydrolase GH20 super-family activity. Quantitative real time PCR analysis for JcDHN-1 and JcDHN-2 expression during seed development showed increasing gene expression of both their transcript levels along with the natural dehydration process during seed development. A sharp increase in JcDHN-2 transcript level occurred in response to water content dropping from 42% in mature seeds to 12% in dry seeds. These results indicate that both JcDHNs have the potential to play a role in cell protection during dehydration occurring naturally during jatropha orthodox seed development.

Production of superoxide from photosystem II-light harvesting complex II supercomplex in STN8 kinase knock-out rice mutants under photoinhibitory illumination

When phosphorylation of Photosystem (PS) II core proteins is blocked in STN8 knock-out mutants of rice (Oryza sativa) under photoinhibitory illumination, the mobilization of PSII supercomplex is prevented. We have previously proposed that more superoxide (O2(-)) is produced from PSII in the mutant (Nath et al., 2013, Plant J. 76, 675-686). Here, we clarify the type and site for the generation of reactive oxygen species (ROS). Using both histochemical and fluorescence probes, we observed that, compared with wild-type (WT) leaves, levels of ROS, including O2(-) and hydrogen peroxide (H2O2), were increased when leaves from mutant plants were illuminated with excess light. However, singlet oxygen production was not enhanced under such conditions. When superoxide dismutase was inhibited, O2(-) production was increased, indicating that it is the initial event prior to H2O2 production. In thylakoids isolated from WT leaves, kinase was active in the presence of ATP, and spectrophotometric analysis of nitrobluetetrazolium absorbance for O2(-) confirmed that PSII-driven superoxide production was greater in the mutant thylakoids than in the WT. This contrast in levels of PSII-driven superoxide production between the mutants and the WT plants was confirmed by conducting protein oxidation assays of PSII particles from osstn8 leaves under strong illumination. Those assays also demonstrated that PSII-LHCII supercomplex proteins were oxidized more in the mutant, thereby implying that PSII particles incur greater damage even though D1 degradation during PSII-supercomplex mobilization is partially blocked in the mutant. These results suggest that O2(-) is the major form of ROS produced in the mutant, and that the damaged PSII in the supercomplex is the primary source of O2(-).

Activation of mitochondrial respiration in chlorophyll-deficient rice mutant seedlings

Previously we described a knock-out mutant of the riceoschlh gene, which encodes a Mg-chelataseH subunit and is involved in chlorophyll biosynthesis. This mutant exhibits ATP-dependent activities of plasma membrane outward-rectifying K+ channel currents that are supported by mitochondrial activation. Here, we have investigated mitochondrial activity inoschlh mutants. Growth rates were similar between the wild type and the mutant, and were enhanced by the addition of sucrose under darkness, indicating that the mutants have active mitochondrial respiration. Proteomic analyses led to the identification of 41 proteins (P <0.05) involved in a range of functions that differed between the mutant and the wild type. Of these, 15 were up-regulated and 26 were down-regulated by more than 2-fold in the mutant. We hypothesize that loss of functioning in the chloroplasts, mainly ATP production, can be restored via beneficial interactions with other cellular compartments, especially the mitochondria, through the inter-organellar regulation of metabolites. Oxygen consumption is greater during mitochondrial respiration in chlorina mutants than in the wild type, so that those mutants produce large amounts of ATP in the presence of sucrose. These results imply that gene expression of photosynthetic organisms is strongly connected through energy-driven networks of transcriptional regulators that can control factors in other cellular compartments, thus indicating the re-programming of cellular functions.

An algorithm for weighted bilinear regression

Bilinear models in which the expectation of a two-way array is the sum of products of parameters are widely used in spectroscopy. In this paper we present an algorithm called combined-vector successive overrelaxation (COV-SOR) for bilinear models, and compare it with methods like alternating least squares, singular value decomposition, and the Marquardt procedure. Comparisons are done for missing data also.