Juan Cuello

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and qL in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k 3 values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H2O2. The results also showed that sites for monolignol β‐O‐4 cross‐coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.

Comparison of the thylakoidal NAD(P)H dehydrogenase complex and the mitochondrial complex I separated from barley leaves by blue-native PAGE

Plastids contain an NAD(P)H dehydrogenase (NAD(P)H DH) complex preferentially located in the stroma thylakoids, which is homologous to the mitochondrial complex I (EC 1.6.5.3). However, until now, the two complexes have not been simultaneously studied in the same plant species. In this work we initiate a comparative study between both complexes from Hordeum vulgare L. The mitochondrial complex I and the stroma thylakoid NAD(P)H DH complex from barley, when separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by NADH nitroblue tetrazolium staining, showed different sizes and electrophoretic mobilities. Complex I had an apparent molecular mass of 740 kDa and the thylakoidal NAD(P)H DH complex a mass of 690 kDa. The two complexes were identified by immunoblotting assays, since antibodies raised against the NdhA, NdhH and NdhK thylakoidal polypeptides recognized three polypeptides of the barley thylakoidal complex, and antibodies raised against the B12 and B15 bovine complex I subunits reacted with two polypeptides of the barley mitochondrial complex I. Additional immunoblotting revealed that the thylakoidal NAD(P)H DH complex contains the three polypeptides (24, 56, 76 kDa) from the NADH-oxidizing unit of the complex, similar in size and antigenicity to the mitochondrial subunits. Both complexes have at least one more homologous subunit (29 kDa) of the complex minimal structure, which was recognized by the anti-TYKY antibody.

From Zinnia to Arabidopsis: approaching the involvement of peroxidases in lignification

Zinnia elegans constitutes one of the most useful model systems for studying xylem differentiation, which simultaneously involves secondary cell wall synthesis, cell wall lignification, and programmed cell death. Likewise, the in vitro culture system of Z. elegans has been the best characterized as the differentiation of mesophyll cells into tracheary elements allows study of the biochemistry and physiology of xylogenesis free from the complexity that heterogeneous plant tissues impose. Moreover, Z. elegans has emerged as an excellent plant model to study the involvement of peroxidases in cell wall lignification. This is due to the simplicity and duality of the lignification pattern shown by the stems and hypocotyls, and to the basic nature of the peroxidase isoenzyme. This protein is expressed not only in hypocotyls and stems but also in mesophyll cells transdifferentiating into tracheary elements. Therefore, not only does this peroxidase fulfil all the catalytic requirements to be involved in lignification overcoming all restrictions imposed by the polymerization step, but also its expression is inherent in lignification. In fact, its basic nature is not exceptional since basic peroxidases are differentially expressed during lignification in other model systems, showing unusual and unique biochemical properties such as oxidation of syringyl moieties. This review focuses on the experiments which led to a better understanding of the lignification process in Zinnia, starting with the basic knowledge about the lignin pattern in this plant, how lignification takes place, and how a sole basic peroxidase with unusual catalytic properties is involved and regulated by hormones, H2O2, and nitric oxide.

Hormonal regulation of the basic peroxidase isoenzyme from Zinnia elegans

Xylem differentiation in plants is under strict hormonal regulation. Auxins and cytokinins, together with brassinosteroids (BRs), appear to be the main hormones controlling vascular differentiation. In this report, we study the effect of these hormones on the basic peroxidase isoenzyme from Zinnia elegans (ZePrx), an enzyme involved in lignin biosynthesis. Results showed that auxins and cytokinins induce ZePrx, similarly to the way in which they induce seedling secondary growth (in particular, metaxylem differentiation). Likewise, the exogenous application of BR reduces the levels of ZePrx, in a similar way to their capacity to inhibit seedling secondary growth. Consistent with this notion, the exogenous application of BR reverses the auxin/cytokinin-induced ZePrx expression, but has no effect on the auxin/cytokinin-induced secondary growth. This differential hormonal response is supported by the analysis of the ZePrx promoter, which contains (a) cis-elements directly responsive to these hormones and (b) cis-elements targets of the plethora of transcription factors, such as NAC, MYB, AP2, MADS and class III HD Zip, which are up-regulated during the auxin- and cytokinin-induced secondary growth. Taken together, these results suggest that ZePrx is directly and indirectly regulated by the plethora of hormones that control xylem differentiation, supporting the role of ZePrx in xylem lignification.

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP+ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.

Fractionation of thylakoid membranes into grana and stroma thylakoids.

The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids.

Localization of the chloroplast NAD(P)H dehydrogenase, complex in stroma thylakoids from barley

Abstract Plastids contain an NAD(P)H dehydrogenase (NAD(P)H DH) complex which is homologous to the mitochondrial complex I (EC 1.6.5.3). We describe the isolation and partial characterization of the chloroplast NAD(P)H DH complex of Hordeum vulgare L. When chloroplasts were fractionated the stromal thylakoids had a specific NADH-ferricyanide oxidoreductase (NADH-FeCNR) activity seven times higher than that of granal thylakoids and contained abundant polypeptide which reacted with the antibody raised against barley NdhA polypeptide. Native PAGE resolved three NADH-nitroblue tetrazolium (NBT)-staining enzyme bands from detergent-solubilized stroma thylakoids and one from the stroma fraction of chloroplasts. The two major thylakoid enzymes and the stromal enzyme showed NAD(P)H-FeCNR and ferredoxin-NADP oxidoreductase (FNR) (EC 1.18.1.2) activities, and the presence of FNR protein was confirmed by Western blotting. Only the major thylakoid enzyme of high molecular weight was found to contain the NdhA polypeptide, a core subunit of the NAD(P)H DH complex. Analysis by SDS–PAGE of the enzyme complex revealed nine polypeptides with molecular masses ranging from 18 to 63 kDa.

Differential effects of abscisic acid and methyl jasmonate on endoproteinases in senescing barley leaves

Endoproteinase activity was analyzed in chloroplasts isolated from barley leaf segments incubated in the dark with various hormonal senescence effectors. As a control, the endoproteinase activity of the supernatant fraction obtained during chloroplast preparation was also analyzed. Measured against azocaseine as substrate, the endoproteinase activity in chloroplasts increased 18 fold during the induction of senescence. This rise in activity was inhibited by kinetin (the activity increased only 10 fold) and very strongly stimulated by abscisic acid (ABA) (117 fold) and methyl jasmonate (Me-JA) (57 fold). Although less so, the endoproteinase activity of the supernatant fraction, mainly vacuolar and with acid pH optimum, was affected in the same way by all three effectors. Among the five endoproteinases (EC) found in chloroplasts, EC2 and EC4 were induced after incubation in water. ABA increased the levels of EC2 and EC4 (5 fold), and induced the development of EC3 and EC5, while Me-JA totally inhibited EC2 and EC4, and induced the development of EC1. At least one of the endoproteinases, EC2, is synthesized in chloroplasts. Among the six endoproteinases found in the supernatant fraction (E), E1, E2, E3 and E5, which are very probably extrachloroplastic endoproteinases, are stimulated by ABA to varying degrees. However, Me-JA stimulates E1 to a greater extent and totally inhibits E3. The differential effects of ABA and Me-JA on chloroplast and supernatant fraction endoproteinases suggest different action mechanisms for both senescence promotors.

Isolation of an NADH dehydrogenase complex not associated to ferredoxin-NADP oxidoreductase from oat stroma thylakoids

Summary With the aim of isolating for further characterisation the chloroplast NADH dehydrogenase (NADH DH) complex of low abundance, we investigated a plant species of comparatively high complex activity. The chloroplast NAD(P)H-ferricyanide oxidoreductase (NAD(P)H-FeCNR) activities of barley ( Hordeum vulgare L.), wheat ( Triticum aestivum L.), oat ( Avena sativa L.) and pea ( Pisum sativum L.) were studied. Oat chloroplasts have approximately 50 percnt; higher NADH-FeCNR activity than barley and pea chloroplasts, and 70 percnt; higher than wheat chloroplasts, and the highest NADH-FeCNR/NADPH-FeCNR activity ratio. The NADH DH complex isolated from the stroma thylakoids of oat is NADH-specific and its NADH-FeCNR specific activity is double than that found in the barley chloro plast complex. Immunoblotting revealed the presence of the NdhA (35 kDa), NdhH (50 kDa), NdhK (29 kDa) and NADH-binding (56 kDa) polypeptides, which are four subunits of the three subcomplexes into which the plastidial complex can be fractionated. The oat complex has a molecular mass of 587 kDa and is composed of at least 15 different polypeptides with molecular masses ranging from 12 to 80 kDa. Additional immunoblotting revealed that the isolated complex is not associated with ferredoxin-NADP oxidoreductase enzyme (EC 1.18.1.2). The results presented here might be the starting point for further purification and characterisation of the chloroplast NADH DH complex.