Planta (2013) 237:1599–1612 DOI 10.1007/s00425-013-1865-5
ORIGINAL ARTICLE
Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis Joaquı´n Herrero • Francisco Ferna´ndez-Pe´rez • Tatiana Yebra • ´ ngeles Pedren˜o • Juan Cuello Esther Novo-Uzal • Federico Pomar • Ma A • Alfredo Gue´ra Alberto Esteban-Carrasco • Jose´ Miguel Zapata
•
Received: 14 November 2012 / Accepted: 21 February 2013 / Published online: 19 March 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract 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
F. Fernandez-Perez and T. Yebra contributed equally to this work. In memoriam of Prof. Alfonso Ros Barcelo´, to be able to continue with your work is an honor. J. Herrero (&) T. Yebra A. Gue´ra J. M. Zapata Department of Plant Biology, University of Alcala´, 28871 Alcala´ de Henares (Madrid), Spain e-mail:
[email protected] ´ . Pedren˜o J. Cuello F. Ferna´ndez-Pe´rez E. Novo-Uzal M. A Department of Plant Biology, University of Murcia, 30100 Murcia, Spain F. Pomar Department of Animal Biology, Plant Biology and Ecology, University of La Corun˜a, 15071 La Corun˜a, Spain A. Esteban-Carrasco Department of Plant Biology, Complutense University, 28040 Madrid, Spain
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 UPSII 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. Keywords Arabidopsis AtPrx72 Bioinformatics Chlorophyll a fluorescence Histochemistry Lignification Peroxidase Xylem vessels ZePrx Zinnia Abbreviations AtPrx72 Basic peroxidase 72 from Arabidopsis thaliana Chl Chlorophyll G Guaiacyl ORF Open reading frame S Syringyl ZePrx Basic peroxidase isoenzyme from Zinnia elegans
Introduction Lignins are three-dimensional phenolic heteropolymers covalently associated with polysaccharides in plant cell walls (Anterola and Lewis 2002). They are mainly located
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in the impermeable water-transport conduits of the xylem and other supporting tissues, such as phloem fibers, and are produced by oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction that can be mediated by both peroxidases (Ros Barcelo´ 1997) and laccases (Berthet et al. 2011). The process of sealing plant cell walls through lignin deposition is known as lignification, and this provides mechanical strength to the stems, protecting cellulose fibers from chemical and biological degradation. Also, lignification is a metabolically costly process that requires large quantities of carbon skeletons and reducing equivalents (Amthor 2003). Since plants do not possess a mechanism to degrade lignins, any carbon invested in lignin biosynthesis is not recoverable so that plants must carefully balance the synthesis of lignin polymers against the availability of resources, meaning that lignification is a strongly regulated process (Patzlaff et al. 2003). The participation of class III plant peroxidases in lignin biosynthesis has been studied in many plant species and tissue culture systems (Østergaard et al. 2000; Marjamaa et al. 2009). Although many studies on anionic peroxidases have been reported, e.g., anionic AtPrx53 (ATPA2) (Østergaard et al. 2000), evidence of cationic peroxidases involved in lignification has also been reported, e.g., a cationic peroxidase of tomato with pI 9.6 (Quiroga et al. 2000), and more importantly, the cationic peroxidase from Zinnia elegans (ZePrx) has been shown to be involved in lignification (Gabaldo´n et al. 2005). In this latter case, the ZePrx is the only one to have recently been molecularly and functionally characterized (Gabaldo´n et al. 2005, 2006), and it is considered solely and unequivocally responsible for the process of lignification in Zinnia plants (Gabaldo´n et al. 2006). This basic peroxidase is highly conserved, probably due to the fact that lignification has been an important process during plant evolution, has high capacity to oxidize both coniferyl and sinapyl alcohols and has been located in lignifying secondary cell walls in Zinnia hypocotyls and cell suspension cultures (Gabaldo´n et al. 2005). However, Z. elegans is recalcitrant to transformation processes and its genome has not been sequenced yet. In contrast, arabidopsis cell cultures resemble differentiating xylem cells and constitute an exceptionally useful model for monitoring the expression of enzymes from the lignin biosynthetic pathway (Demura et al. 2002). Arabidopsis has 73 peroxidases and it would be reasonable to expect that at least one of them is orthologous to ZePrx. Bibliographical evidence has indicated that the arabidopsis peroxidase 72 (AtPrx72) could have an important role in lignification (Lebedeva et al. 2003; Armengaud et al. 2004; Vale´rio et al. 2004; Irshad et al. 2008). AtPrx72 is a protein which has been identified in the
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cell walls of etiolated hypocotyls of 11-day-old arabidopsis plants, corresponding to a phase of hypocotyl growth arrest, and it has been classified as a protein involved in cell wall expansion (Irshad et al. 2008). Furthermore, it has been reported that AtPrx72 is expressed in roots and stems of arabidopsis (Vale´rio et al. 2004). The localization can be explained by peroxidases having two modes of action, one being the production of ROS, which can break cell wall polysaccharides down nonenzymatically, favoring the extension of the cell wall, and the other being intervention in the cross-linking of cell wall components in the same way as ZePrx. In their studies on potassium nutrition, Armengaud et al. (2004) described AtPrx72 as a cell wall peroxidase. In addition, Lebedeva et al. (2003) found that AtPrx72 had an acceptable level of expression in the plants studied. Also important was the study conducted by Kubo et al. (2005) who, based on the model system of transdifferentiating mesophyll cells from Zinnia, established an in vitro transdifferentiating system in arabidopsis suspension cell cultures, to obtain an expression profile of xylem cell differentiation-related genes. In this study, microarray analysis indicated that 1,705 genes showed a more than an eightfold change in expression over time. These genes were clustered and AtPrx72 was present in a set that showed up-regulated expression when the xylem vessel elements were actively forming. This evidence, together with its basic nature similar to ZePrx, indicates that AtPrx72 plays a role in the lignification process. Therefore, in an attempt to establish an arabidopsis ortholog to ZePrx, we performed a bioinformatic analysis of AtPrx72 in comparison with ZePrx. One way to study secondary xylem development is to determine the function of candidate genes by reverse genetics or by the identification of knock-out mutants. We conducted histochemical, photosynthetic, and phenotypical analyses of an arabidopsis knock-out mutant of peroxidase 72 with the aim of characterizing the changes which occurred as a result of the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. Our results demonstrated the fundamental nature of this peroxidase in plant cell wall formation and showed that the plant responded plastically to a deficit or absence of lignification.
Materials and methods Bioinformatic analysis and molecular modeling The BLASTP program was used to determine the homology of AtPrx72 with ZePrx. The Arabidopsis Ensembl (http://atensembl.arabidopsis.info), National Center for
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Biotechnology Information (NCBI) (http://www.ncbi.nlm. nih.gov/), UniProt (http://www.uniprot.org/uniprot/), PeroxiBase (http://www.peroxibase.isb-sib.ch/) and The Arabidopsis Information Resource (TAIR) (http://www.arabid opsis.org/) databases were used to locate information about the Arabidopsis thaliana peroxidase 72. Sequence homology analysis was carried out using CLUSTALW (http://www.ebi.ac.uk), and PROSITE (http:// www.expasy.org/prosite) algorithms. Protein sequences were retrieved from the NCBI and TAIR databases and the PeroxiBase database, a class III plant peroxidase database. Peroxidase models were constructed from the primary structure of the corresponding mature proteins, using the automated protein modeling server Fold and Function Assignment System (FFAS) (http://ffas.burnham.org/) (Jaroszewski et al. 2011), according to the similarities of the modeled sequences to the known structures, available in the Protein Data Bank (PDB). AtPrx72 was modeled using 1PA2 (Arabidopsis thaliana peroxidase A2) as the template structure. From the 3D structures, the molecular surface charges were computed using simple Coulomb interactions. The protein was considered to be at pH 7.0, with a default protonation state for all residues. As default, only charged residues (R, K, E, and D) were taken into account and the charges were located at the corresponding (non-H) atom positions. Net surface charges in the images ranged from -4.0 C (red) to ?4.0 C (blue). Surface electrostatic potentials were mapped using the Coulomb computation method, assuming a dielectric constant (solvent) of 80,000 (Gabaldo´n et al. 2007). Plant materials Seeds of Arabidopsis thaliana Columbia 0 (Col-0) and AtPrx72 Salk (Salk Institute Genome Analysis Laboratory, SIGnAL), line Salk_136893.44.70.x, were obtained from the Nottingham Arabidopsis Stock Centre (NASC) OnLine Catalog at http://nasc.nott.ac.uk/home.html. Seeds were sterilized, washed and kept in cold conditions (4 °C) for 3 days (Harrison et al. 2006) before in vitro culture. Plants were grown under controlled conditions with a day/ night temperature regime of 22 °C/20 °C, 60 % relative humidity and under a 16-h photoperiod of white light (135 lmol m-2 s-1) in an Ing. Climas 450 Growth Chamber (Ing. Climas, Barcelona, Spain). In vitro culture and mutant selection AtPrx72 Salk seeds were placed on solid MS medium supplemented with 0.1 % (w/v) sucrose with 50 lg ml-1 of kanamycin (Sigma-Aldrich, Madrid, Spain) as selection agent. Next, cultured seeds were exposed to 6 h light to
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induce germination, and then maintained for 2 days in darkness at 24 °C as described by Harrison et al. (2006). One week after germination, plants were transferred to soil. Col-0 was used as negative control, and AtPrx72 Salk and Col-0 seeds grown in the absence of kanamycin were used as positive control. DNA isolation and amplification The isolation of plant genomic DNA from leaves of at least 2- to 3-week-old seedlings/plants was carried out as described by Edwards et al. (1991) for rapid extraction of DNA suitable for PCR analysis. Screening of homozygous mutant plants was carried out by PCR amplification in accordance with protocols from SIGnAL (http://signal.salk.edu/tdnaprimers.html). Primer design was performed using the primer design tool from SIGnAL. Briefly, one PCR amplification with three primers was conducted to determine the genotype. Left and right genomic primers were used to detect wild allele, and T-DNA border primer to detect the insertion (Østergaard and Yanofsky 2004). Histochemical assays for detecting lignins Lignins were detected in the inflorescence stem sections of WT and atprx72 mutant plants using the toluidine blue and Ma¨ule and Wiesner staining. Plants collected at development stage 6.3 (Boyes et al. 2001) corresponding to a plant height of 15 cm were used, and cross sections were made from the first 5 cm of the basal parts of stems. Stems were fixed in Mc Dowell during 24 h and post-fixed with 1 % OsO4 in the same conditions. Following post-fixation, samples were stained with uranyl acetate, dehydrated and embedded in Spurr as described by Go´mez-Ros et al. (2007). Ultrathin sections (60 nm) were examined, after staining with toluidine blue with a Leica DMRB optical microscopy. Wiesner staining was performed by soaking 500 lm thick sections in 1.0 % (w/v) phloroglucinol–HCl in 25:75 (v/v) HCl/ethanol for 10–15 min (Pomar et al. 2002). Lignins were also stained with the Ma¨ule test, a specific reaction for syringyl moieties (Pomar et al. 2002). Phenotypic analysis Development analyses of Col-0 and AtPrx72 Salk mutant plants were carried out as described by Boyes et al. (2001). At least 25 plants of each type were used to perform the analyses. Lignin analyses Cell walls were prepared with a Triton X-100 washing procedure in which the final steps include washing with
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ethanol (3 times) and diethyl ether (also 3 times) (Espin˜eira et al. 2011). Lignin quantification was performed using acetyl bromide, as described by Iiyama and Wallis (1988). Alkaline nitrobenzene oxidation of lignifying cell walls and HPLC analyses were performed essentially as described elsewhere (Espin˜eira et al. 2011). Quantitation of p-hydroxybenzaldehyde, vanillin and syringaldehyde was performed at 290 nm using the corresponding standards. Thioacidolysis of lignifying cell walls, which solubilizes the b-O-4 lignin core, and GC–MS analyses were performed (Novo-Uzal et al. 2009) using a Thermo Finnigan Trace GC gas chromatograph, a Thermo Finnigan Polaris Q mass spectrometer and a DB-XLB, J&W (60 mm9 0.25 mm I.D.) column. Mass spectra were recorded at 70 eV. Fourier transform infrared spectra (FTIR) of finely ground cell wall samples were recorded on a Bruker Vector 22 FTIR spectrophotometer (Bruker Optics, Madrid, Spain). Chlorophyll a fluorescence Chlorophyll a fluorescence was measured in vivo with a modulated light fluorometer (PAM-2000, Walz, Effeltrich, Germany). Samples were kept in the dark for 15 min before fluorescence was measured. The minimum (dark) fluorescence yield (Fo) was obtained after excitation of the plant with a weak measuring beam from a light-emitting diode. The maximum fluorescence yield (Fm) was determined with an 800 ms saturating pulse of white light (8,000 lmol photons m-2 s-1). Maximum variable fluorescence (Fv) was calculated as Fm - Fo. The effective quantum efficiency of PSII 0 0 photochemistry (UPSII) was calculated as ðFm Ft Þ=Fm , where Ft is the level of fluorescence during actinic illumination (Kramer et al. 2004). Non-photochemical quenching (NPQ) was calculated according to Bilger and 0 0 Bjo¨rkman (1990) as ðFm Fm Þ=Fm . The parameter qL gives an estimation of the proportion of quinone reduced or closed reaction centers (Kramer et al. 2004) and is 0 0 calculated as qL ¼ qP Fo0 =Ft , where qP ¼ Fm Ft =Fm Fo0 . UPSII, UNPQ, and UNO are, respectively, the proportion of light energy converted in the PSII in photochemistry, dissipated as heat by down-regulating non-photochemical reactions or by other energy losses (Kramer et al. 2004). Statistic method The Student’s t test for independent replicates of each ecotype was performed to compare the mean of Chl fluorescence parameters, and the Kruskal–Wallis one way ANOVA (P \ 0.05) was used.
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Results and discussion Bioinformatic analysis The databases searched indicated that the ORFs of the fulllength cDNA of AtPrx72 corresponded to one polypeptide of 336 amino acids (Fig. 1). Like other class III plant peroxidases, the predicted mature polypeptide, which putatively starts at SKA (Fig. 1, typed in green), contains a peroxidase active site signature (64-PASLLRLHFHDC) (Fig. 1, underlined in black), a peroxidase proximal hemeligand signature (192- DLVSLSGSHTI) (Fig. 1, underlined in blue), and eight conserved cysteines (C42, C75, C80, C122, C128, C207, C329, C339) (Fig. 1, typed in red), which probably yield the four disulfide bridges (C42– C122, C75–C80, C128–C329, C207–C339) common to all class III plant peroxidases (Welinder et al. 2002). AtPrx72 contains also two putative N-glycosylation sites (173NNTF and 216-NQSG) (Fig. 1, shaded in yellow), three putative protein kinase C phosphorylation sites (94-SeK, 104-SaR, 209-SfR), four putative casein kinase II phosphorylation sites (127-ScaD, 165-SnnD, 242-SggD, 275-SsdE), six putative N-myristoylation sites (79-GCdaSI, 90-GTiiSE, 159-GTiiSE, 203-GNsrCT, 243-GGdqTL, 272-GLlsSD) and two Ca2? binding sites (Fig. 1 typed in blue), one distal (D74 and D81) and the other proximal (D252 and D260), characteristic of all the active class III peroxidases (Welinder et al. 2002). The N-terminus of a protein is an active area for cotranslational and post-translational modifications, and it is frequently subject to co-translational proteolytic cleavage. N-terminal peptidases reveal penultimate residues, which (depending on their nature) might later become substrates for a set of transferases that modify the amino acid or side chains at the N-terminal residues. N-terminal transferases might acetylate, myristoylate, arginylate, deaminate or cyclize residues at the N-terminus of proteins (Walling 2006), modifications which can influence protein stability, localization or activity. From the N-terminal sequence SKAYGSGGY, obtained from the databases, it would be reasonable to expect that the polypeptide of AtPrx72 will be co-translationally processed by proteolytic cleavage. In fact, a signal peptide cleavage site was predicted between 23C and 24S (C|| SKA), which is in accordance with the usual cleavage sites described for eukaryotes (Nielsen et al. 1997), and with the N-terminal sequence of mature AtPrx72 (SKAYGSGGY). Establishment of the polypeptide cleavage site at the above position also suggests that the immature polypeptides contain a signal peptide (N-terminal pro-peptide) of 23 amino acids (MAKSLNILIAALSLIAFSPFCLC), which directs the polypeptide chains to the ER membrane, the mature polypeptides therefore showing 313 amino acids.
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Fig. 1 Structural alignment and secondary structural elements of ZePrx and AtPrx72. The secondary structural elements of AtPrx72 (Fig. 2) consist of 11 a-helices (shaded in purple): helix A (P43-H59), helix B (A65-D74), helix C (F108-E121), helix D (C128-T143), helix E (F176-Q186), helix F (L190-L196), helix F0 (Q229-R238), helix G (N261-M269), helix H (D277-F281), helix I (K285-E296) and helix J (Q298-N314), and four short b-strands (shaded in blue): the b-strand I (S147-V150), the b-strand II (S197-G198), the
b-strand III (L248-F250) and the b-strand IV (E324-R327). Start of mature polypeptide: typed in green. Peroxidase active site signature: underlined in black. Peroxidase proximal heme–ligand signature: underlined in blue. Ca2? binding sites are typed in blue. Conserved cysteines are typed in red. N-glycosylation sites are shaded in yellow. Basic amino acids (K and R) are represented by blue wavy underline. Acidic amino acids (E and D) are represented by red wavy underline
The AtPrx72 lacks a C-terminal pro-peptide extension susceptible to protease cleavage. In fact, the proximity (7 amino acids) of the conserved 8th C to the end codon (Fig. 1) suggests that this polypeptide has no C-terminal pro-peptide extensions to target the protein for vacuolar transport, which (when present in class III plant peroxidases) (Welinder et al. 2002) usually adds a 20–30 amino acid tail downstream of the conserved 8th C. Since the polypeptide (Fig. 1) did not contain any of the ER motifs for retrograde transport, it can be concluded that the protein follows the default pathway, which ends in the cell wall. In the internal core of AtPrx72, six cryptic N-myristoylation sites were deduced. Since these potential N-myristoylation sites also begin at internal amino acid positions, it would not normally be myristoylated in vivo unless post-translational cleavage occurred, unmasking the N-terminal glycines, as described above. AtPrx72 contains eight conserved cysteines (Fig. 1, typed in red). Heme proteins containing free cysteines are susceptible to inhibition by thiol reagents (De Gara 2004). It has been suggested that the eight conserved cysteines form the four disulfide bridges common to all class III plant peroxidases (Welinder et al. 2002), thus distinguishing
them from other plant peroxidases, such as ascorbate peroxidases. With some exceptions, the majority of plant peroxidases carry one or two putative N-linked glycans (Welinder et al. 2002), and this also appears to be the case for AtPrx72, where two putative N-glycosylation sites (Fig. 1, shaded in yellow) may be predicted. The quadruplet 173-NNTF contains an adjacent proline; however, it does not occur in 216-NQSG, where it is thought that this glycosylation site in AtPrx72 may be completely filled. To ascertain the spatial localization of glycans in the 2D/3D structure of the proteins, AtPrx72 was modeled by means of the FFAS (http://ffas.burnham.org/) using arabidopsis ATP A2 peroxidase (Østergaard et al. 1998) as the template structure. The secondary structural elements of AtPrx72 (Fig. 2) consist of 11 a-helices and four short b-strands (Østergaard et al. 1998). Common features that AtPrx72 shared with ZePrx (Fig. 1) included the invariable positioning of the helices A, B, C, D, E, F, F0 , G, H, I, and J. Helix D0 (hD0 ) was absent from ZePrx, AtPrx72 (Fig. 1) and from all the syringyl peroxidases of basal land plants described earlier (Go´mez-Ros et al. 2007). This observation might be important because helix D0 relies on the heme prosthetic
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Fig. 2 Predicted 3D structures and surface charge map of ZePrx and AtPrx72. a-Helices are numbered according to Fig. 1. Surface structure and mapping of the electrostatic potential of ZePrx and AtPrx72 according to the predicted 3D structure (above). The molecular surface charges were computed using simple Coulomb interactions. The protein was considered to be at pH 7.0, with a default protonation state for all residues. As default, only charged residues (R, K, E and D) were taken into account and the charges were located at the corresponding (non-H) atom positions. Net surface charges in the images ranged from -4.0 C (red) to ?4.0 C (blue). Areas of glycan influence are circled. The common features that AtPrx72 shared with ZePrx included the invariable positioning of the helices A, B, C, D, E, F, F0 , G, H, I, and J. Helix D0 (hD0 ) was absent from ZePrx, AtPrx72 and from all the basal S peroxidases described earlier. Special attention should be paid to the b-strand sited upstream of the proximal histidine in ZePrx and AtPrx72, which probably influences the catalytic center of the enzymes
group. The absence of helix D0 in S peroxidases represents a relaxation factor for the heme crevice and enables the docking of S moieties because helix D0 fixes the IPS motif, which determines the conformation and hydrophobicity of the substrate-binding site (Østergaard et al. 1998). As with helix D0 , the helix F00 was found to be absent from AtPrx72. Helix F00 is located before helix F0 , because it does not appear in all peroxidases. The real importance in catalysis is not yet known, but these differences in topology are likely to be of relevance in determining the branching direction in a polymerization reaction and/or the orientation of a polymeric substrate (Henriksen et al. 2001). As for helix D0 and F00 , special attention should be paid to the b-strand sited upstream of the proximal histidine in ZePrx and AtPrx72, which probably influences the catalytic center of the enzymes, although its real importance in catalysis requires confirmation by crystallographic data and site-directed mutagenesis (Ros Barcelo´ et al. 2007).
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The two N-glycosylation sites (173-NNTF and 216-NQSG) (Fig. 1, shaded in yellow) in AtPrx72 are located in an immediately upstream loop region of helix E (173-NNTF) and an immediately upstream loop region of helix F0 (216-NQSG). Since the glycan attached to the N-glycosylation site, 216-NQSG, is close to the proximal histidine (Fig. 1), this glycosylation probably affects reaction dynamics, as it is strengthened by the differential catalytic properties of ZePrx (Gabaldo´n et al. 2006). Furthermore, since helix F0 is located in the surface region of AtPrx72 (Østergaard et al. 1998), and by analogy in the surface region of ZePrx (Gabaldo´n et al. 2007), it is also probable that glycans attached to the N-glycosylation site, 216-NQSG, may participate in protein recognition and/or protein membrane (protein/cell wall) interactions, which determine the cellular trafficking of AtPrx72, and their final localization (compartmentalization). In fact, the singular surface properties of AtPrx72 could determine the nature of these interactions. The mature
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Table 1 Annotation of the A. thaliana peroxidase 72 (AtPrx72) and the Z. elegans peroxidase (ZePrx) involved in lignification Name
TAIR gene number
Protein accession number
C-term/target prediction
pI/MW
Protein length (AA)
E-value
ZePrx
–
Q4W1I8
No/SP
8.47/34,245
321
–
AtPrx72
AT5G66390
NP_201440.1/Q9FJZ9
No/SP
8.54/35,004
336
9e-84
The TAIR number, the UniProt number and some other names are reported. The C-terminal extension (C-term) was obtained from Valerio et al. (2004). The isoelectric point (pI) and molecular weight (MW) of AtPrx72 and ZePrx were calculated from the deduced amino acid sequence of the mature protein using the IEP program in the EMBOSS Package. EMBOSS version 2.0.1
protein of AtPrx72 (Fig. 1) contains 45 basic amino acids (H, K, and R), constituting 14.37 %, and 33 acidic amino acids, (D and E), constituting 10.54 %. Therefore, the mature protein is of a strongly basic nature and has a theoretical pI of 8.54 (Table 1). After the correction (DpI * ?2.0) for the two calcium ions and the heme (Welinder et al. 2002), the theoretical pI for AtPrx72 fits well with 10.54. Surface charge characteristics became most evident when the molecular surface charge of AtPrx72 was computed using simple Coulomb interactions (Fig. 2). The results of mapping the electrostatic potential indicated a characteristic distribution of charges on the surface, with the almost total predominance of ?4.0 C net surface charges (Fig. 2, shaded in blue). In fact, at pH 7.0 and excluding the two H, AtPrx72 contains 43 positively charged amino acids against 33 negatively charged amino acids. Bearing this in mind, mapping of the electrostatic potential (Fig. 2) clearly demonstrated that negatively charged amino acids of AtPrx72 are coated by positively charged amino acids on the surface, resulting in a positivenet surface electrostatic charge pattern. From the high isoelectric point (about 10.54), and from the electrostatic surface properties (Fig. 2), it can be deduced that AtPrx72 is firmly bound to the pectin matrix of the primary plant cell wall. Pectin-basic protein interactions may be attenuated by coating basic proteins with glycans, the best example being extensin (Cassab and Varner 1988), and in this way, AtPrx72 glycans might reduce the electrostatic interactions with pectins, allowing AtPrx72 to move freely in the cell wall matrix and reach the secondary cell wall layer where, after having started in the primary cell walls, lignin deposition progressively finalizes (Smith et al. 1994). In support of this conclusion, it is noteworthy that the N-glycosylation site (216-NQSG) in AtPrx72 is located in a surface region with a high predominance of ?4.0 C net surface charges (Fig. 2), induced by the presence of seven basic amino acids (K and R) (Fig. 1, wavy underline in blue) but only two acidic amino acids (E and D) (Fig. 1, wavy underline in red), this region being the best surface domain of AtPrx72 for interactions with pectins (Fig. 2). This is especially relevant for the glycans attached to the 216-N, which is flanked by seven basic amino acids (Fig. 1). undoubtedly, the location of glycans in this
positive-net surface region attenuates the electrostatic interactions of AtPrx72 with pectins. At this point, it is interesting to remember that AtPrx72 does not contain the motif of four clustered arginine residues (R117, R262, R268, and R271) responsible for interactions with pectins. Following our approach from the protein to the transcript, the efficiency of translation initiation from an eukaryotic mRNA was to a large extent determined from the length and secondary structure of the 50 -UTR (Kozak 1991). Eukaryotic mRNAs have 50 -UTRs, which range from three to several hundred nucleotides with an average length of about 90 (Kozak 1991). It seems that a moderately long ([20 nt) and unstructured 50 -UTR is necessary and sufficient for efficient initiation of translation, whereas a heavily structured leader is a major obstacle to the scanning 40S ribosomal subunit (Kozak 1991). mRNAs codifying stress-induced proteins, as is the case of some peroxidases (Welinder et al. 2002), are characterized by presenting 50 -UTRs containing 40–71 % adenine (Østergaard et al. 2000). The A-rich quality of these 50 -UTRs may affect transcription, translation, and/or mRNA stability. For example, it has been found that more than 80 % of the 50 -UTRs from heat shock proteins from invertebrates and plants are AU rich (Østergaard et al. 2000). Thus, the A-rich peroxidase 50 -UTRs presumably have very little potential to form stable secondary structures and so the protein turnover rate may be affected, being higher for those proteins with a high decay rate. This was the case of AtPrx72, since the transcript had a 50 -UTR adenine content of about 47 %. In the light of these results, it can be deduced that AtPrx72 has optimal expression levels. Histochemical analysis of syringyl lignins in the atprx72 mutant xylem Taking into account that peroxidases catalyze the last step of lignin biosynthesis and the reported cell wall localization of AtPrx72 protein, structure, and composition of cell walls were evaluated to test whether AtPrx72 may be crucial for cell wall formation. Toluidine blue staining revealed thinner cell walls in the mutant, both in interfascicular fibers and vascular bundles (Fig. 3a, b). The reduction of cell wall thickness was especially marked in interfascicular fibers.
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Fig. 3 Toluidine blue staining in WT (a) and atprx72 mutant (b) cinnamyl aldehydes end groups in WT (c) and atprx72 mutant (d) revealed by the phloroglucinol–HCl reagent and syringyl moieties in WT (e) and atprx72 mutant (f) revealed with the Ma¨ule reaction. The arrows indicate the xylem vessels. Bars 100 lm
Phloroglucinol staining revealed that the presence of lignins was restricted to vascular bundles and interfascicular fibers (Fig. 3c, d), with slightly lower intensity in the knock-out mutant. Moreover, some xylem vessels appeared partially collapsed, which may be due to thinner cell walls with less lignin. Since AtPrx72 is a basic enzyme which putatively oxidizes sinapyl alcohol, the Ma¨ule test, which is specific for syringyl groups, was performed (Pomar et al. 2002). Xylem vessels were brown-stained, which point out lignins that are mainly composed of guaiacyl units, whereas red staining in the interfascicular fibers indicated the presence of syringyl groups, both in WT and mutant plants (Fig. 3e, f). This staining revealed that cell walls were thinner in the interfascicular fibers, as shown with toluidine blue staining. Moreover, the reduction of cell wall thickness was accompanied by a decrease in the intensity of the red staining, which suggests a reduction in the content of syringyl lignins, restricted to the interfascicular
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fibers. It has been previously reported that S lignins are mainly deposited in the interfascicular fibers, whereas xylem is mainly composed of G units (Zhong et al. 2000), which would explain why xylem vessels were equally stained in both WT and mutant plants. Phenotypic analysis A biometric analysis of the atprx72 mutant and Col-0 plants was carried out. Phenotypic differences are usually small and typical methodologies are unable to detect them. In an attempt to rapidly discover the function of a gene, we used a slightly modified version of the methodology described by Boyes et al. (2001) to highlight the differences between the mutant and control plants. The results showed that atprx72 mutant plants grow more slowly than WT plants, are unable to retain an upright growth habit, and exhibit altered leaf morphology (Fig. 4).
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Fig. 4 Phenotypical differences between WT and atprx72 mutant plants. Differences in vegetative development of both types of plants. Small window differences in number of seeds germinated and seedling development 12 days after planting (seeds were planted the same day in MS medium without kanamycin)
Over the course of their life cycle, the number of branches as well as principal stems and number of siliques diverge significantly (Fig. 5). In fact, atprx72 mutant plants had a decreased production of stems (Fig. 5a), lateral branches (Fig. 5b) and siliques (Fig. 5c). Similar results have been achieved using the AtPrx72 SAIL_891_H09 mutant (data not shown). The large initial differences decreased over time and at the end of their growth cycle, mutant plants reached similar values to those of Col-0 (Fig. 6). This may represent a strategy of plant adaptation to low lignin production. The smaller size of the mutant compared with the wild type is probably related to its earlier flowering time (data not shown). Flowering time can be an indirect marker reflecting plant growth conditions and overall plant health (Passardi et al. 2007). The differences between Col-0 and atprx72 mutants could be due to a change in the balance of hormones in relation to variations in the expression of certain class III peroxidases. It is known that peroxidases are able to degrade auxin in vitro, and thus, a change in auxin balance would influence the rate of tissue formation (Passardi et al. 2007). Lignin analyses Peroxidases are known to be involved in the last step of lignin biosynthesis, the oxidation of monolignols in the cell wall. As mentioned above, AtPrx72 is a secreted peroxidase that may have a role in the lignification of cell walls. The manipulation of different enzymes participating in the
lignin biosynthesis pathway to understand the many modifications of the route has been widely reported in the literature. The down-regulation of these enzymes is commonly followed by a reduction in lignin content, but a total suppression of lignin biosynthesis has not been achieved yet, which indicates that plants have developed alternative enzymes to yield a functional lignin polymer (Chabannes et al. 2001; Sibout et al. 2005). Quantification of lignins from inflorescence stem cell walls using acetyl bromide, which solubilizes lignins through bromination and acetylation reactions and cleavage of a-ether linkages (Lu and Ralph 1996), revealed a reduced amount of lignin in the knock-out atprx72 mutant compared to WT (Table 2). These results agree with the lower intensity of phloroglucinol staining found in the atprx72 mutant (Fig. 3c). Lignin levels were about 60 % of those found in WT plants (Table 2). Similar levels of lignin content decrease were found in cad-c cad-d mutants (Sibout et al. 2005), in which expression of the CAD encoding gene, the precedent enzyme of lignin biosynthesis that leads to the formation of hydroxycinnamyl alcohols, was suppressed. A reduction in the total amount of lignin has also been described by Li et al. (2003b), where PrxA3a, an acidic peroxidase, was down-regulated in transgenic aspen. In the latter study, the decrease in lignin content was accompanied by changes in lignin composition. Nitrobenzene oxidation provides information on lignin monomer composition. WT arabidopsis plants showed an H/G/S proportion of 1/64/35, as reported previously
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Fig. 5 Number of principal stems (a), number of branches from the principal stem (b), and number of total siliques (c) analyzed in WT (gray bars) and atprx72 mutants (black bars)
Fig. 6 Total area of rosette (a), rosette radius measure (b), and length of principal stem (c) between 22 and 36 days of growth of Arabidopsis thaliana plants. WT (black symbols), atprx72 mutants (white symbols)
(Rogers et al. 2005; Sibout et al. 2005). The atprx72 mutant and WT lignins showed no differences in monomer composition (Table 2). Thioacidolysis analysis allows the study of both the composition and structure of the lignin polymer by solubilizing lignins through the cleavage of b-ether linkages (Lapierre et al. 1995). Results were similar to those obtained with nitrobenzene oxidation, although the S % of Col-0 lignins was higher with thioacidolysis (Table 2). This datum is consistent with the fact that, unlike nitrobenzene oxidation, thioacidolysis only solubilizes the uncondensed (b-O-4) linked lignin fraction, which is especially enriched in S units (Lapierre et al. 1995). The S/G ratio indicated a decrease in S units in the atprx72 mutant (0.7 in WT and 0.5 in the mutant, Table 2). As deduced from the Ma¨ule staining (Fig. 3f), this reduction in
S unit content seems to be restricted to the interfascicular fibers, as the intensity of xylem vessel staining showed no difference with WT plants. Li et al. (2003b) reported a decrease in G units when the acidic peroxidase PrxA3a was down-regulated. Nevertheless, the suppression of an acidic peroxidase in tobacco did not produce any change in lignin quantity (Lagrimini et al. 1997). The theoretical ability of the AtPrx72 protein to oxidize sinapyl alcohol, based on the absence of any steric restriction on the catalytic site, explains why a suppression of AtPrx72 expression leads to a reduction in S unit content. Regarding lignin structure, thioacidolysis revealed a much higher Rb-O-4/RO-4 end ratio for the atprx72 mutant than for WT (60.6 and 25.3, respectively, Table 3), providing a more linear structure to the lignin macromolecule (Durbeej and Eriksson 2003).
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All the spectra obtained by FTIR spectroscopy of the inflorescence stem cell walls were similar, with slight but significant differences between WT and the atprx72 mutant cell walls (Fig. 7). an alteration was found in the 1,596 cm-1 band, corresponding to vibration in the aromatic skeleton and a C=O stretch (Rana et al. 2010). Moreover, the peak at 1,421 cm-1, corresponding to inplane bending in the C–H group (Rana et al. 2010), was higher in WT plants than in mutant plants. Interestingly, in the atprx72 mutant, there was a shift in the band at 1,330 cm-1, referring to the syringyl ring breathing with CO stretching (Fengel and Wegener 2003). This change in the 1,330 cm-1 band, together with another shift in the band at 1,505 cm-1, which represents vibrations in the aromatic skeleton mainly referring to G units, confirmed the data obtained by thioacidolysis, with a lower proportion of S units in the mutant than in control lignins. At 1,103 and 1,154 cm-1, a shift in the characteristic peaks of cellulose and carbohydrates (McCann et al. 2007) was found in the mutant cell walls. To confirm the differences in cellulose content shown by FTIR, a detailed analysis of carbohydrates was carried out. It has been previously reported that a reduction in total lignin levels can be accompanied by an increase in both quantity and quality of cell wall carbohydrates. This shift in composition may reinforce cell walls with lower lignin content (Li et al. 2003a). By means of acid hydrolysis followed by HPLC analysis, levels of different carbohydrates were measured, but no differences in comparison with the WT plants were found (data not shown). In a recent study, the down-regulation of 4-coumarate:coenzyme A ligase induced a reduction in lignin content, but no change in cellulose content was observed (Voelker et al. 2010). Chlorophyll a fluorescence Chlorophyll a fluorescence provides information about the efficiency of photosynthetic light reactions at PSII. The parameter Fv/Fm reflects the maximum quantum efficiency of PSII (Schreiber 2004) and is used as a sensitive indicator of plant photosynthetic performance, with optimal values
Table 3 Monomeric degradation products obtained by thioacidolysis of WT and atprx72 mutant plants P P P P Sample b-O-4 O-4 b-O-4/ O-4 WT
620.7
24.5
atprx72
281.2
4.6
25.3 60.6 -8
Values are given in total ionic current (TIC) 9 10 mg-1 CW. SD values were within 5 % P b-O-4, aryl-glycerol-b-aryl ether structures derived from theP b-O-4 cross coupling of p-hydroxycinnamyl alcohols and aldehydes; O-4end, the amount of O-4-linked end monomers
Fig. 7 FTIR spectra of cell walls. Mean FTIR spectra of cell walls from WT (red line) and atprx72 mutant (blue line) plants, in the wave number range from 1,800 to 800 cm-1. Each spectrum is a mean of spectra from five individual samples
around 0.83 in healthy, unstressed plants (Schreiber 2004); a decrease in this value is indicative of photoinhibition. Our results showed that atprx72 mutants did not show significant variations in Fv/Fm in comparison with WT (Fig. 8). However, a small but significant decrease in the photosynthetic quantum yield (UPSII) was observed in the atprx72 mutant plants (Fig. 9). There is a linear relationship between UPSII, which represents the actual efficiency of photochemistry in PSII, and the efficiency of carbon fixation (Genty et al. 1989). In the absence of photoinhibition, a decrease in UPSII after a period of illumination is
Table 2 Lignin content and cell wall monomer composition of cell wall of WT and atprx72 mutant plants Sample
Lignin (mg g-1 CW)
H/G/S
S/G
NBO
Thioacidolysis
NBO
Thioacidolysis
WT
159 ± 16
1/64/35
1/60/39
0.5
0.7
atprx72
104 ± 17
2/63/35
1/64/35
0.5
0.5
Lignin content, measured by the acetyl bromide method, and monomer composition, expressed as the molar H/G/S and S/G ratios, determined by nitrobenzene oxidation (NBO) and thioacidolysis, in cell walls of WT and atprx72 mutant CW cell wall
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Fig. 8 Chlorophyll a fluorescence parameters. a Fo, minimum fluorescence yield in dark-adapted samples (white bars); Fv/Fm, maximum quantum yield of PSII photochemistry (gray bars); Fm, maximum fluorescence intensity in dark-adapted samples (black bars). b UNO, yield of other non-photochemical losses (white bars); UNPQ, yield for dissipation by down-regulation (photoprotection) (gray bars); UPSII, yield of photochemistry (black bars)
usually a consequence of the activation of photoprotective mechanisms. The magnitude of light energy dissipation as heat is estimated by NPQ of chlorophyll fluorescence (Krause and Jahns 2004), but no significant differences between the atprx72 mutant and WT plants were observed (Figs. 8, 9). Therefore, the lower steady-state values registered for atprx72 cannot be attributed to an increase in dissipative processes in this mutant. On the other hand, the rate of quinone re-oxidation during illumination (indicated by the parameter qL) was much lower in atprx72 than in WT plants (Fig. 9). This effect can be attributed to several processes that directly or indirectly affect the PSII acceptor pool size such as, for instance, a decrease in reducing equivalent consumption. In conclusion, AtPrx72 mutation does affect the primary photosynthetic processes, but the effects are indirect and probably related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. Our current research will try to clarify this apparent and unexpected pleiotropic effect of the suppression of peroxidase expression in A. thaliana. In summary, we have demonstrated the high homology between AtPrx72 and ZePrx at 1D, 2D, and 3D levels as
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Fig. 9 Chlorophyll a fluorescence analysis. Non-photochemical quenching (NPQ) (a), quantum yield of PSII during exposure to light (UPSII) (b), and fraction of open reaction centers (qL) (c) in WT (black symbols) and atprx72 mutants (white symbols). Data are mean ± SE (n = 5). Significantly different from WT at P \ 0.05, by Student’s t test
well as the cell wall localization of AtPrx72. Use of atprx72 knock-out mutant plants allowed us to study the effect of absence of this protein on plant physiology and morphology. The atprx72 mutant plants possess a lower amount of lignin and S units, lower growth and smaller rosette and principal stem than WT plants, together with a significant decrease in UPSII that could be related to the efficiency of carbon fixation. Therefore, our results have shown that atprx72 mutant plants present an alteration in the synthesis of lignin in the
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xylem vessels. In this case, the results indicated not only structural and phenotypic differences, but also a possible adaptation in response to a peroxidase deficit. This response, consistent with the strategy that plants typically adopt under stress conditions, provides an indication of the importance of AtPrx72. Acknowledgments This work has been funded with support from the Spanish MICINN and the European Commission FEDER (BFU2006-11577 and BFU2009-08151) and from the Fundacio´n Se´neca, Agencia de Ciencia y Tecnologı´a de la Regio´n de Murcia en el marco de II PCTRM 2007-10 (08610/PI/08). Joaquı´n Herrero and Francisco Ferna´ndez-Pe´rez hold a fellowship (FPU) from the MICINN and Esther Novo-Uzal holds a JdC grant from MICINN (Spain).
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