The role of the co-chaperone HOP in plant homeostasis during development and stress.

Proteins need to acquire their native structure in order to become fully functional. In specific cases, the active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 organizing protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. Here, we provide an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyse whether these aspects are conserved in plants. In addition, we highlight the HOP clients described in plants and the role of HOP in plant development and stress response.

Evolutionary Implications of a Peroxidase with High Affinity for Cinnamyl Alcohols from Physcomitrium patens, a Non-Vascular Plant.

Physcomitrium (Physcomitrella) patens is a bryophyte highly tolerant to different stresses, allowing survival when water supply is a limiting factor. This moss lacks a true vascular system, but it has evolved a primitive water-conducting system that contains lignin-like polyphenols. By means of a three-step protocol, including ammonium sulfate precipitation, adsorption chromatography on phenyl Sepharose and cationic exchange chromatography on SP Sepharose, we were able to purify and further characterize a novel class III peroxidase, PpaPrx19, upregulated upon salt and H2O2 treatments. This peroxidase, of a strongly basic nature, shows surprising homology to angiosperm peroxidases related to lignification, despite the lack of true lignins in P. patens cell walls. Moreover, PpaPrx19 shows catalytic and kinetic properties typical of angiosperm peroxidases involved in oxidation of monolignols, being able to efficiently use hydroxycinnamyl alcohols as substrates. Our results pinpoint the presence in P. patens of peroxidases that fulfill the requirements to be involved in the last step of lignin biosynthesis, predating the appearance of true lignin.

Overexpression of ZePrx in Nicotiana tabacum Affects Lignin Biosynthesis Without Altering Redox Homeostasis.

Class III plant peroxidases (Prxs) are involved in the oxidative polymerization of lignins. Zinnia elegans Jacq. Basic peroxidase (ZePrx) has been previously characterized as capable of catalyzing this reaction in vitro and the role in lignin biosynthesis of several of its Arabidopsis thaliana homologous has been previously confirmed. In the present work, ZePrx was overexpressed in Nicotiana tabacum to further characterize its function in planta with particular attention to its involvement in lignin biosynthesis. Since Prxs are known to alter ROS levels by using them as electron acceptor or producing them in their catalytic activity, the impact of this overexpression in redox homeostasis was studied by analyzing the metabolites and enzymes of the ascorbate-glutathione cycle. In relation to the modification induced by ZePrx overexpression in lignin composition and cellular metabolism, the carbohydrate composition of the cell wall as well as overall gene expression through RNA-Seq were analyzed. The obtained results indicate that the overexpression of ZePrx caused an increase in syringyl lignin in cell wall stems, suggesting that ZePrx is relevant for the oxidation of sinapyl alcohol during lignin biosynthesis, coherently with its S-peroxidase nature. The increase in the glucose content of the cell wall and the reduction of the expression of several genes involved in secondary cell wall biosynthesis suggests the occurrence of a possible compensatory response to maintain cell wall properties. The perturbation of cellular redox homeostasis occurring as a consequence of ZePrx overexpression was kept under control by an increase in APX activity and a reduction in ascorbate redox state. In conclusion, our results confirm the role of ZePrx in lignin biosynthesis and highlight that its activity alters cellular pathways putatively aimed at maintaining redox homeostasis.

More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited.

The Major Facilitator Superfamily (MFS) is ubiquitous in living organisms and represents the largest group of secondary active membrane transporters. In plants, significant research efforts have focused on the role of specific families within the MFS, particularly those transporting macronutrients (C, N, and P) that constitute the vast majority of the members of this superfamily. Other MFS families remain less explored, although a plethora of additional substrates and physiological functions have been uncovered. Nevertheless, the lack of a systematic approach to analyzing the MFS as a whole has obscured the high diversity and versatility of these transporters. Here, we present a phylogenetic analysis of all annotated MFS domain-containing proteins encoded in the Arabidopsis thaliana genome and propose that this superfamily of transporters consists of 218 members, clustered in 22 families. In reviewing the available information regarding the diversity in biological functions and substrates of Arabidopsis MFS members, we provide arguments for intensified research on these membrane transporters to unveil the breadth of their physiological relevance, disclose the molecular mechanisms underlying their mode of action, and explore their biotechnological potential.

Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism.

Class III peroxidases are glycoproteins with a major role in cell wall maturation such as lignin formation. Peroxidases are usually present in a high number of isoenzymes, which complicates to assign specific functions to individual peroxidase isoenzymes. Arabidopsis genome encodes for 73 peroxidases, among which AtPrx72 has been shown to participate in lignification. Here, we report by using knock out peroxidase mutants how the disruption of AtPrx72 causes thinner secondary walls in interfascicular fibres but not in the xylem of the stem. This effect is also age-dependent, and AtPrx72 function seems to be particularly important when lignification prevails over elongation processes. Finally, the suppression AtPrx72 leads to the down-regulation of lignin biosynthesis pathway, as well as genes and transcription factors involved in secondary wall thickening.

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units.

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl and sinapyl) alcohols in a reaction mediated by peroxidases (EC 1.11.1.7) and laccases (EC 1.10.3.2), yielding H, G and S units, respectively. Although both acidic and basic peroxidases can oxidize p-coumaryl and coniferyl alcohol, only basic peroxidases are able to oxidize sinapyl alcohol. The AtPrx52 from Arabidopsis is a basic peroxidase that has been reported to be highly homologous to the basic peroxidase of Zinnia elegans, the only peroxidase which has been unequivocally linked to lignin formation. Here, we show how the suppression of AtPrx52 causes a change in lignin composition, mainly at the level of stem interfascicular fibers. Quantification of lignins in two different atprx52 knock-out mutants revealed a decrease of lignin amount compared with wild type. The S/G ratio, obtained by both nitrobenzene oxidation and thioacidolysis, indicated a decrease in S units in the atprx52 mutants. As deduced from Wiesner and mainly Mäule staining, this reduction in S unit content appears to be restricted to the interfascicular fibers. Moreover, quantitative polymerase chain reaction analysis in atprx52 plants showed a general downregulation of genes involved in lignin biosynthetic pathway, as well as genes related to secondary cell wall. On the other hand, other routes from phenylpropanoid metabolism were induced. Taken together, our results indicate that AtPrx52 is involved in the synthesis of S units in interfascicular fibers at late stages of the lignification process.

Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana.

Syringyl lignins result from the oxidative polymerization of sinapyl alcohol in a reaction mediated by syringyl (basic) peroxidases. Several peroxidases have been identified in the genome of Arabidopsis thaliana as close homologues to ZePrx, the best characterized basic peroxidase so far, but none of these has been directly involved in lignification. We have used a knock-out mutant of AtPrx4, the closest homologue to ZePrx, to study the involvement of this basic peroxidase in the physiology of the plant under both long- and short-day light conditions. Our results suggest that AtPrx4 is involved in cell wall lignification, especially in syringyl monomer formation. The disruption of AtPrx4 causes a decrease in syringyl units proportion, but only when light conditions are optimal. Moreover, the effect of AtPrx4 disruption is age-dependent, and it is only significant when the elongation process of the stem has ceased and lignification becomes active. In conclusion, AtPrx4 emerges as a basic peroxidase regulated by day length with an important role in lignification.

A proteomic approach to Physcomitrella patens rhizoid exudates.

The interaction between plants and the surrounding environment has been widely studied, specially the defence reactions and the plant-plant interactions. One of the most remarkable metabolic features of plant roots is the ability to secrete a vast array of compounds into the rhizosphere, not only of low molecular weight but also polysaccharides and proteins. Here, we took advantage of proteomics to study the rhizoid exudates of Physcomitrella patens at early and late development stages (7 and 28 days of culture in liquid medium). Samples were extracted, separated and detected with nanoLC-MALDI-TOF/TOF MS/MS, identifying 47 proteins at the development stage of 7 days, and 66 proteins at 28 days. Moreover, 21 proteins were common to the two analyzed periods. All the identified proteins were classified into 8 functional categories: response to stress, response to stimulus, oxido-reduction, cell wall modification, photosynthesis and carbohydrate metabolism, transport, DNA metabolic process and regulation/signalling. Our results show important differences in the protein expression profile along the development of P. patens, mainly at the level of regulation- and senescence-related proteins. Defence-related proteins, such as chitinases, thaumatins and peroxidases have a major role in the interaction of P. patens with the environment.

Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba.

BACKGROUND AND AIMS: Peroxidase isoenzymes play diverse roles in plant physiology, such as lignification and defence against pathogens. The actions and regulation of many peroxidases are not known with much accuracy. A number of studies have reported direct involvement of peroxidase isoenzymes in the oxidation of monolignols, which constitutes the last step in the lignin biosynthesis pathway. However, most of the available data concern only peroxidases and lignins from angiosperms. This study describes the molecular cloning of two novel peroxidases from the 'living fossil' Ginkgo biloba and their regulation by salt stress and salicylic acid. METHODS: Suspension cell cultures were used to purify peroxidases and to obtain the cDNAs. Treatments with salicylic acid and sodium chloride were performed and peroxidase activity and gene expression were monitored. KEY RESULTS: A novel peroxidase was purified, which preferentially used p-hydroxycinnamyl alcohols as substrates and was able to form dehydrogenation polymers in vitro from coniferyl and sinapyl alcohols. Two peroxidase full-length cDNAs, GbPrx09 and GbPrx10, were cloned. Both peroxidases showed high similarity to other basic peroxidases with a putative role in cell wall lignification. Both GbPrx09 and GbPrx10 were expressed in leaves and stems of the plant. Sodium chloride enhanced the gene expression of GbPrx09 but repressed GbPrx10, whereas salicylic acid strongly repressed both GbPrx09 and GbPrx10. CONCLUSIONS: Taken together, the data suggest the participation of GbPrx09 and GbPrx10 in the developmental lignification programme of the cell wall. Both peroxidases possess the structural characteristics necessary for sinapyl alcohol oxidation. Moreover, GbPrx09 is also involved in lignification induced by salt stress, while salicylic acid-mediated lignification is not a result of GbPrx09 and GbPrx10 enzymatic activity.

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses.

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p≤0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.

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.

Ophiobolin A, a sesterterpenoid fungal phytotoxin, displays higher in vitro growth-inhibitory effects in mammalian than in plant cells and displays in vivo antitumor activity.

Ophiobolin A, a sesterterpenoid produced by plant pathogenic fungi, was purified from the culture extract of Drechslera gigantea and tested for its growth-inhibitory activity in both plant and mammalian cells. Ophiobolin A induced cell death in Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells at concentrations ≥10 µM, with the TBY-2 cells showing typical features of apoptosis-like cell death. At a concentration of 5 µM, ophiobolin A did not affect plant cell viability but prevented cell proliferation. When tested on eight cancer cell lines, concentrations <1 µM of ophiobolin A inhibited growth by 50% after 3 days of culture irrespective of their multidrug resistance (MDR) phenotypes and their resistance levels to pro-apoptotic stimuli. It is, thus, unlikely that ophiobolin A exerts these in vitro growth-inhibitory effects in cancer cells by activating pro-apoptotic processes. Highly proliferative human keratinocytes appeared more sensitive to the growth-inhibitory effects of ophiobolin A than slowly proliferating ones. Ophiobolin A also displayed significant antitumor activity at the level of mouse survival when assayed at 10 mg/kg in the B16F10 mouse melanoma model with lung pseudometastases. Ophiobolin A could, thus, represent a novel scaffold to combat cancer types that display various levels of resistance to pro-apoptotic stimuli and/or various MDR phenotypes.

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.

Purification and kinetic characterization of two peroxidases of Selaginella martensii Spring. involved in lignification.

Two cationic peroxidases from Selaginella martensii Spring. (SmaPrx2 and SmaPrx3) were purified using a three-step protocol which includes ammonium sulfate precipitation, adsorption chromatography on phenyl sepharose and cationic exchange chromatography on SP sepharose. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3 kDa and 45.6 kDa, respectively, according to MALDI-TOF/TOF. The isoelectric points were estimated in 9.2 and 9.5 for SmaPrx2 and SmaPrx3, respectively, according to isoelectrofocusing. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403 nm for SmaPrx2 and 404 nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the in vitro capacity of using sinapyl alcohol as substrate and the low K(m) in the µM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignins biosynthesis.

Relationship between hydroxycinnamic acid content, lignin composition and digestibility of maize silages in sheep.

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R2 value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R2 value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.

The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression.

BACKGROUND: Verticillium dahliae is a fungal pathogen that infects a wide range of hosts. The only known genes for resistance to Verticillium in the Solanaceae are found in the tomato (Solanum lycopersicum) Ve locus, formed by two linked genes, Ve1 and Ve2. To characterize the resistance response mediated by the tomato Ve gene, we inoculated two nearly isogenic tomato lines, LA3030 (ve/ve) and LA3038 (Ve/Ve), with V. dahliae. RESULTS: We found induction of H2O2 production in roots of inoculated plants, followed by an increase in peroxidase activity only in roots of inoculated resistant plants. Phenylalanine-ammonia lyase (PAL) activity was also increased in resistant roots 2 hours after inoculation, while induction of PAL activity in susceptible roots was not seen until 48 hours after inoculation. Phenylpropanoid metabolism was also affected, with increases in ferulic acid, p-coumaric acid, vanillin and p-hydroxybenzaldehyde contents in resistant roots after inoculation. Six tomato PAL cDNA sequences (PAL1 - PAL6) were found in the SolGenes tomato EST database. RT-PCR analysis showed that these genes were expressed in all organs of the plant, albeit at different levels. Real-time RT-PCR indicated distinct patterns of expression of the different PAL genes in V. dahliae-inoculated roots. Phylogenetic analysis of 48 partial PAL cDNAs corresponding to 19 plant species grouped angiosperm PAL sequences into four clusters, suggesting functional differences among the six tomato genes, with PAL2 and PAL6 presumably involved in lignification, and the remaining PAL genes implicated in other biological processes. An increase in the synthesis of lignins was found 16 and 28 days after inoculation in both lines; this increase was greater and faster to develop in the resistant line. In both resistant and susceptible inoculated plants, an increase in the ratio of guaiacyl/syringyl units was detected 16 days after inoculation, resulting from the lowered amount of syringyl units in the lignins of inoculated plants. CONCLUSIONS: The interaction between the tomato and V. dahliae triggered a number of short- and long-term defensive mechanisms. Differences were found between compatible and incompatible interactions, including onset of H2O2 production and activities of peroxidase and PAL, and phenylpropanoid metabolism and synthesis of lignins.

Digestibility of silages in relation to their hydroxycinnamic acid content and lignin composition.

BACKGROUND: The effectiveness of the analysis of cell wall-bound hydroxycinnamic acids and the composition of lignin to evaluate the in vivo digestibility of a silage collection with unknown botanical composition was evaluated. RESULTS: Syringyl units content and total etherified phenols showed the highest correlation coefficients with in vivo dry matter digestibility (IVDMD) (r = - 0.792 and r = - 0.703, respectively), while guaiacyl units and total phenols showed the highest correlation coefficients with in vivo organic matter digestibility (IVOMD) (r = - 0.871 and r = - 0.817, respectively). Using the above-mentioned chemical parameters, 10 equations were also developed to predict in vivo digestibility. The prediction of IVDMD produced a high adjusted R(2) value (0.710) using syringyl, total lignin, etherified total phenols, esterified ferulic acid and total phenol content as predictors. The prediction of IVOMD produced a higher adjusted R(2) value (0.821) using guaiacyl, total phenols, total ferulic acid and etherified p-coumaric acid content as predictors. CONCLUSION: Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, syringyl and guaiacyl content and etherified phenols emerge as good predictors of digestibility.

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.