Arabidopsis thaliana peroxidases involved in lignin biosynthesis: in silico promoter analysis and hormonal regulation.

Phytohormones such as auxins, cytokinins, and brassinosteroids, act by means of a signaling cascade of transcription factors of the families NAC, MYB, AP2 (APETALA2), MADS and class III HD (homeodomain) Zip, regulating secondary growth. When the hormonal regulation of Zinnia elegans peroxidase (ZePrx), an enzyme involved in lignin biosynthesis, was studied, it was found that this peroxidase is sensitive to a plethora of hormones which control xylem lignification. In a previous study we sought Arabidopsis thaliana homologues to ZePrx. Peroxidases 4, 52, 49 and 72 are the four peroxidases that fulfill the restrictive conditions that a peroxidase involved in lignification must have. In the present study, we focus our attention on hormonal regulation in order to establish the minimal structural and regulatory elements contained in the promoter region which an AtPrx involved in lignification must have. The results indicate that of the four peroxidases selected in our previous study, the one most likely to be homologous to ZePrx is AtPrx52. The results suggest that hormones such as auxins, cytokinins and BRs directly regulate AtPrx52, and that the AtPrx52 promoter may be the target of the set of transcription factors (NAC, MYB, AP2 and class I and III HD Zip) which are up-regulated by these hormones during secondary growth. In addition, the AtPrx52 promoter contains multiple copies of all the putative cis-elements (the ACGT box, the OCS box, the OPAQ box, the L1BX, the MYCL box and the W box) known to confer regulation by NO and H2O2.

Looking for Arabidopsis thaliana peroxidases involved in lignin biosynthesis.

Monolignol polymerization into lignin is catalyzed by peroxidases or laccases. Recently, a Zinnia elegans peroxidase (ZePrx) that is considered responsible for monolignol polymerization in this plant has been molecularly and functionally characterized. Nevertheless, Arabidopsis thaliana has become an alternative model plant for studies of lignification, filling the gaps that may occur with Z. elegans. The arabidopsis genome offers the possibility of performing bioinformatic analyses and data mining that are not yet feasible with other plant species, in order to obtain preliminary evidence on the role of genes and proteins. In our search for arabidopsis homologs to the ZePrx, we performed an exhaustive in silico characterization of everything from the protein to the transcript of Arabidopsis thaliana peroxidases (AtPrxs) homologous to ZePrx, with the aim of identifying one or more peroxidases that may be involved in monolignol polymerization. Nine peroxidases (AtPrx 4, 5, 52, 68, 67, 36, 14, 49 and 72) with an E-value greater than 1e-80 with ZePrx were selected for this study. The results demonstrate that a high level of 1D, 2D and 3D homology between these AtPrxs and ZePrx are not always accompanied by the presence of the same electrostatic and mRNA properties that indicate a peroxidase is involved in lignin biosynthesis. In summary, we can confirm that the peroxidases involved in lignification are among AtPrx 4, 52, 49 and 72. Their structural and mRNA features indicate that exert their action in the cell wall similar to ZePrx.

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 q L 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.

Dehydration rate and time of desiccation affect recovery of the lichen alga [corrected] Trebouxia erici: alternative and classical protective mechanisms.

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F'(o)) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q (E)), whereas after fast dehydration it was photoinhibition (q (I)). Although q (E) seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.

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.

Post-translational modifications of the basic peroxidase isoenzyme from Zinnia elegans.

The major basic peroxidase (ZePrx) from Zinnia elegans suspension cell cultures was purified and cloned. The purification resolved ZePrxs in two isoforms (ZePrx33.44 and ZePrx34.70), whose co-translational and post-translational modifications are characterized. Based on the N-terminal sequence obtained by Edman degradation of mature ZePxs, it may be expected that the immature polypeptides of ZePrxs contain a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chains to the ER membrane. These immature polypeptides are co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggested that the processing of the N-terminal pro-peptide of ZePrxs is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases. When the post-translational modifications of ZePrxs were characterized by trypsin digestion, and tryptic peptides were analyzed by reverse phase nano liquid chromatography (RP-nanoLC) coupled to MALDI-TOF MS, it was seen that, despite the presence in the primary structure of the protein of several (disulphide bridges, N-glycosylation, phosphorylation and N-myristoylation) potential post-translational modification sites, ZePrxs are only post-translationated modified by the formation of N-terminal pyroglutamate residues, disulphide bridges and N-glycosylation. Glycans of ZePrxs belong to three main types and conduce to the existence of at least ten different molecular isoforms. The first glycans belong to both low and high mannose-type glycans, with the growing structure Man(3-9)(GlcNAc)(2). Low mannose-type glycans, Man(3-4)(GlcNAc)(2), coexist with the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), in the G(3) and G(4 )sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man(3)Xyl(1)Fuc(3)(GlcNAc)(5), and the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), appear to fill the two putative sites for N-glycosylation. Since the two N-glycosylation sites in ZePrxs are located in an immediately upstream loop region of helix F'' (close to the proximal histidine) and in helix F'' itself, and are flanked by positive-charged amino acids that produce an unusual positive-net surface electrostatic charge pattern, it may be expected that glycans not only affect reaction dynamics but may well participate in protein/cell wall interactions. These results emphasize the complexity of the ZePrx proteome and the difficulties involved in establishing any fine structure-function relationship.

Inactivation of a plastid evolutionary conserved gene affects PSII electron transport, life span and fitness of tobacco plants.

Chloroplasts contain a plastoquinone-NADH-oxidoreductase (Ndh) complex involved in protection against stress and the maintenance of cyclic electron flow. Inactivation of the Ndh complex delays the development of leaf senescence symptoms. Chlorophyll a fluorescence measurements, blue native gel electrophoresis, immunodetection and other techniques were employed to study tobacco (Nicotiana tabacum) Ndh-defective mutants (DeltandhF). The DeltandhF mutants compared with wild-type plants presented: (i) higher photosystem II : photosystem I (PSII : PSI) ratios; (ii) similar or higher levels of ascorbate, carotenoids, thylakoid peroxidase and superoxide dismutase, yield (Phi(PSII)) and maximal photochemical efficiency of PSII levels (F(v)/F(m)) than wild-type plant leaves of the same age; (iii) lower values of nonphotochemical quenching yield (Phi(NPQ)), but not at very high light intensities or during induced leaf senescence; (iv) a similar decrease of antioxidants during senescence; (v) no significant differences in the total foliar area and apical growth rate; and (vi) a production of viable seeds significantly higher than wild-type plants. These results suggest that the Ndh complex is involved in one of the redundant mechanisms that play a safety role in photosynthesis under stress, which has been conserved during evolution, but that its deletion increases fitness when plants are grown under favourable controlled conditions.