RESUMEN
KEY MESSAGE: We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ' subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ' subunit in the PSII complex. We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ' extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ' protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ' is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.
Asunto(s)
Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Rhodophyta/genética , Rhodophyta/metabolismoRESUMEN
MAIN CONCLUSION: Three species chosen as representatives of NADP-ME C4 subtype exhibit different sensitivity toward photoinhibition, and great photochemical differences were found to exist between the species. These characteristics might be due to the imbalance in the excitation energy between the photosystems present in M and BS cells, and also due to that between species caused by the penetration of light inside the leaves. Such regulation in the distribution of light intensity between M and BS cells shows that co-operation between both the metabolic systems determines effective photosynthesis and reduces the harmful effects of high light on the degradation of PSII through the production of reactive oxygen species (ROS). We have investigated several physiological parameters of NADP-ME-type C4 species (e.g., Zea mays, Echinochloa crus-galli, and Digitaria sanguinalis) grown under moderate light intensity (200 µmol photons m-2 s-1) and, subsequently, exposed to excess light intensity (HL, 1600 µmol photons m-2 s-1). Our main interest was to understand why these species, grown under identical conditions, differ in their responses toward high light, and what is the physiological significance of these differences. Among the investigated species, Echinochloa crus-galli is best adapted to HL treatment. High resistance of the photosynthetic apparatus of E. crus-galli to HL was accompanied by an elevated level of phosphorylation of PSII proteins, and higher values of photochemical quenching, ATP/ADP ratio, activity of PSI and PSII complexes, as well as integrity of the thylakoid membranes. It was also shown that the non-radiative dissipation of energy in the studied plants was not dependent on carotenoid contents and, thus, other photoprotective mechanisms might have been engaged under HL stress conditions. The activity of the enzymes superoxide dismutase and ascorbate peroxidase as well as the content of malondialdehyde and H2O2 suggests that antioxidant defense is not responsible for the differences observed in the tolerance of NADP-ME species toward HL stress. We concluded that the chloroplasts of the examined NADP-ME species showed different sensitivity to short-term high light irradiance, suggesting a role of other factors excluding light factors, thus influencing the response of thylakoid proteins. We also observed that HL affects the mesophyll chloroplasts first hand and, subsequently, the bundle sheath chloroplasts.
Asunto(s)
Digitaria/fisiología , Echinochloa/fisiología , Luz , Malato Deshidrogenasa/metabolismo , Fotosíntesis/efectos de la radiación , Zea mays/fisiología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico/efectos de la radiación , Carotenoides/metabolismo , Respiración de la Célula/efectos de la radiación , Clorofila/metabolismo , Clorofila A , Digitaria/enzimología , Digitaria/efectos de la radiación , Echinochloa/enzimología , Echinochloa/efectos de la radiación , Transporte de Electrón/efectos de la radiación , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Peróxido de Hidrógeno/metabolismo , Malondialdehído/metabolismo , Células del Mesófilo/metabolismo , Células del Mesófilo/efectos de la radiación , Metaboloma , Fosforilación/efectos de la radiación , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/efectos de la radiación , Tilacoides/metabolismo , Tilacoides/efectos de la radiación , Zea mays/enzimología , Zea mays/efectos de la radiaciónRESUMEN
MAIN CONCLUSION: Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.
Asunto(s)
Arabidopsis/efectos de la radiación , Cloroplastos/efectos de la radiación , Luz , Tilacoides/efectos de la radiación , Zea mays/efectos de la radiación , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Tilacoides/metabolismo , Zea mays/metabolismoRESUMEN
Metabolic responses to Pb(NO3)2 (Pb) ions of excised leaves of metallicolous (MPs) and nonmetallicolous populations (NMPs) of Armeria maritima, cultivated on normal soil, were examined. Detached leaves were exposure to Pb for 24 h, and metabolic parameters were investigated. Pb decreased the photosynthesis (Pn) rate and photosystem II (PSII) activity, whereas the photochemical efficiency of PSII remained unchanged. In both populations, Pb ions caused increase in O2 uptake of dark-treated leaves; however, respiration after Pn was not affected. Pb increased superoxide dismutase activity in MP leaves and malondialdehyde content in NMP leaves. Other metabolites after Pb treatment were increased (proline or H2O2) or decreased (malate). Ascorbate peroxidase activity and adenosine triphosphate content decreased more in MP than in NMP leaves. Our results indicate that A. maritima is well adapted to heavy metal-contaminated soils, and we discuss potential causes of the stimulation of respiration by Pb ions and possible reasons for the tolerance to oxidative stress of plants growing in a metal-rich habitat.
Asunto(s)
Plomo/toxicidad , Contaminantes del Suelo/toxicidad , Tracheophyta/metabolismo , Respiración de la Célula/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Plomo/metabolismo , Malondialdehído/metabolismo , Estrés Oxidativo , Fotosíntesis/efectos de los fármacos , Hojas de la Planta/metabolismo , Contaminantes del Suelo/metabolismoRESUMEN
C4 photosynthesis includes several anatomical and biochemical modifications that allow plants to concentrate CO2 at the site of Rubisco. The photorespiratory pathway is repressed in C4 plants, since the rates of photosynthesis and biomass production are increased. This is an adaptation to high light intensities, high temperatures and dryness. C4 plants contain two distinct types of photosynthetic cells, mesophyll and bundle sheath. The processes of assimilation and reduction of CO2 are separated spatiality and catayzed by two different enzymes. Only the bundle sheath chloroplasts perform the reactions of the Calvin-Benson cycle with the help of the Rubisco enzyme present exclusively in this cell type. The primary CO2 fixation occurs in mesophyll cells through the action of the phosphoenolpyruvate carboxylase. The light-dependent reactions of the photosynthesis occur exclusively in the latter cell type. These differences in photochemistry lead to distinct redox profiles in both types of cells. C4 plants are divided into three biochemical subtypes on the basis of differences in the mechanisms of decarboxylation of the C4 acids. C4 plants will provide the main source of food for humans and animals in the nearest decade.
Asunto(s)
Fotosíntesis/fisiología , Hojas de la Planta/metabolismo , Plantas/clasificación , Plantas/metabolismo , Adaptación Biológica , Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Células del Mesófilo/metabolismo , Oxidación-Reducción , Fosfoenolpiruvato Carboxilasa/metabolismoRESUMEN
In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns. LC-MS/MS with alternating CID and electron transfer dissociation sequencing of peptide ions mapped 15 protein phosphorylation sites. Phosphorylated D2, CP29, CP26, Lhcb2 proteins, and ATPsynthase were found only in M membranes. A previously unknown phosphorylation site was mapped in phosphoenolpyruvate carboxykinase from the BS cells. Phosphorylation stoichiometry was calculated from the ratios of normalized ion currents for phosphorylated to nonphosphorylated peptide pairs from the D1, D2, CP43, and PbsH proteins of photosystem II (PSII). Every PSII in M thylakoids contained on average 1.5 ± 0.1 or 2.3 ± 0.2 phosphoryl groups in plants grown under either low or high light, while in BS membranes the corresponding numbers were 0.25 ± 0.1 or 0.7 ± 0.2, respectively. It is suggested that the phosphorylation level, as well as turnover of PSII depend on the structure of thylakoids.
Asunto(s)
Fosfoproteínas/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Zea mays/metabolismo , Secuencia de Aminoácidos , Cloroplastos/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/análisis , Fosforilación , Complejo de Proteína del Fotosistema II/análisis , Proteínas de Plantas/análisis , ProteómicaRESUMEN
The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b (6) protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Luz , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacología , Caseínas/metabolismo , Proteínas de Unión a Clorofila/genética , Proteínas de Unión a Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Citocromos b/genética , Citocromos b/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fotoquímica , Fotosíntesis , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Mapeo de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/farmacología , Especificidad por Sustrato , Proteínas de las Membranas de los Tilacoides/genética , Proteínas de las Membranas de los Tilacoides/metabolismo , Tilacoides/efectos de los fármacos , Tilacoides/metabolismoRESUMEN
Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream. The photochemical efficiency of PSII, measured as an Fv/Fm ratio, in the maize leaves treated with Pb was only 10% lower than in control leaves. The PSII activity was not affected by Pb ions in mesophyll thylakoids, whereas in bundle sheath it was reduced. Protein phosphorylation in mesophyll and bundle sheath thylakoids was analyzed using mass spectrometry and protein blotting before and after lead treatment. Both methods clearly demonstrated increase in phosphorylation of the PSII proteins upon treatment with Pb(2+), however, the extent of D1, D2 and CP43 phosphorylation in the mesophyll chloroplasts was clearly higher than in bundle sheath cells. We found that in the presence of Pb ions there was no detectable dephosphorylation of the strongly phosphorylated D1 and PsbH proteins of PSII complex in darkness or under far red light. These results suggest that Pb(2+) stimulates phosphorylation of PSII core proteins, which can affect stability of the PSII complexes and the rate of D1 protein degradation. Increased phosphorylation of the PSII core proteins induced by Pb ions may be a crucial protection mechanism stabilizing optimal composition of the PSII complexes under metal stress conditions. Our results show that acclimation to Pb ions was achieved in both types of maize chloroplasts in the same way. However, these processes are obviously more complex because of different metabolic status in mesophyll and bundle sheath chloroplasts.