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.

The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.

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.