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«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»

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Tissue-specific expression of the At2g30210 gene Clues to the physiological function(s) of a gene can sometimes be inferred from its pattern of expression in different tissues. RT-PCR was used to detect the expression of the At2g30210 gene in different Arabidopsis tissues. Figure 2.7A shows that At2g30210 is expressed primarily in roots. Several genome-scale transcriptional profiling datasets for Arabidopsis

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Parallel Signature Sequencing (MPSS) (http://mpss.udel.edu/at/, Meyers et al. 2004), and the Genevestigator (https://www.genevestigator.ethz.ch, Zimmermann et al. 2004).

Figures 2.7B and 2.

7C show At2g30210 gene expression patterns in different Arabidopsis tissues as obtained from the MPSS and Genevestigator databases, respectively. Although a previous study (McCaig et al. 2005) showed At2g30210 gene expression was specific to young root tissues, these other techniques found low level expression in additional tissues. Notably, expression was seen during seed development (Figure 2.7C). As none of these tissues are rich in lignin, this data suggests that the gene product may be involved in physiological roles other than lignification. In general, the expression patterns observed using these different approaches agreed with each other, although the exact tissue samples used in the various studies were not identical. Attempts at RT-PCR analysis of At2g30210 expression in siliques failed due to problems with RNA isolation from this tissue, so we were unable to confirm the data for this tissue obtained from online sources.

Discussion The completed genome sequence for Arabidopsis and advanced genomic techniques that can provide mass data by analysis of the

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great resource for plant biologists, but investigation of the in vivo function for every gene in the organism still requires detailed studies specific to each target gene. There are an estimated 25,500 genes in Arabidopsis (ARABIDOPSIS GENOME INITIATIVE 2000), but only a small proportion of them have been well-characterized with respect to function. Within the Arabidopsis genome, 17 genes have been annotated as LMCOs, but none has yet been experimentally characterized, although most have been included in commercial microarray chips for genomescaled studies. Here we report isolation of the first fulllength Arabidopsis LMCO cDNA, and show that the gene structure and enzyme activity are similar to those reported for other plant LMCOs. The completed Arabidopsis genome sequence, as well as the diversity of bioinformatics tools and numerous mutant resources available for the species, makes Arabidopsis an excellent model in which to investigate the physiological function(s) of LMCOs in plants.

Combining 5’ and 3’ RACE, a PCR-based strategy allowed us to obtain the full-length cDNA sequence of At2g30210. The fulllength DNA supports the computationally predicted intron-exon boundaries annotated in the TAIR database, adding only the 5’ UTR and giving the precise sequence of the 3’UTR with polyadenyl tails. A consensus TATA box located at position -25 upstream of

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transcription occurs at position -47 from the coding sequence.

There were 11 ESTs matching At2g30210 in GenBank and only one (BX839990) contained the 5’UTR extending to 30 bp in front of ATG start codon. However, this sequence was not used to predict a 5’UTR for the At2g30210 gene model in the database. The predicted secondary structure of the 5’UTR contains stem-loop structures similar to IREs in animal genes, suggesting possible regulatory significance. Although some members of the LMCO family play roles in iron metabolism, we did not find evidence to support such a function for At2g30210p. Further study to compare the expression level of transcripts and mature protein would be necessary to determine whether the 5’UTR functions in the regulation of this LMCO.

Compared to the 5’UTR, the length of the 3’UTR seems to be more flexible. In general, a consensus polyadenylation signal (AAUAAA) was found 10 to 30 bp upstream from where the primary transcript is cleaved to generate a new 3’-OH end. There were 3 different lengths of 3’UTR with a variety of polyadenyl tails ranging in length from 15 to 23 A residues. Sequence analysis of this region showed a consensus sequence (AAUAAA) for poly (A)+ addition 14 bp upstream of the poly(A)+ tail in the longest 3’UTR sequence (230 bps). Interestingly, a second sequence (AAUAGA), similar to the poly(A)+ signal sequence, was found 12

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shorter than the one having the longest 3’UTR, suggesting that AAUAGA may serve as an alternative signal for poly(A)+ addition.

Additional support for alternate polyadenylation comes from three ESTs in the Arabidopsis EST database (AV538466, AV547158 and AV538596), which terminated their 3’ UTRs 24 to 39 bp downstream of the AAUAGA signal, although the poly(A)+ tails were not identified. In another case, an EST (Z34656) containing a longer 3’UTR was identified amongst cDNAs generated from seven-day-old etiolated Versailles-VB seedlings. This longer 3’UTR was 60 bp longer than the longest we identified, extending 74 bp beyond the poly (A)+ addition site. Further study will be required to understand whether ecotype or the etiolation treatment may be responsible for the unusual 3’UTR.

The function of plant LMCOs has been frequently associated with lignification due to the ability of these enzymes to oxidize monolignols in vitro and their expression in lignifying tissues (Dean et al. 1994). However, the expression of multiple isoenzymes (gene products) with overlapping substrate specificities within specific tissues or organs has been a major obstacle to elucidating the function of individual plant LMCOs (Ranocha et al. 1999). Moreover, lignin content and composition were not changed in transgenic aspen (Ranocha et al. 2002) or

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LMCO expression. In other cases, the high levels of plant LMCO proteins expressed in non-lignifying tissues, such as suspension-cultured Acer cells and Rhus sap, as well as global expression patterns of Arabidopsis LMCOs (McCaig et al. 2005), suggest that plant LMCOs must play multiple roles beyond lignification.

Heterologous expression allowed us to characterize the At2g30210 LMCO with minimal interference from endogenous LMCOs.

The zymogram using a laccase-specific substrate, DAN, gave strong indication that the gene encoded an active LMCO. Plant LMCOs have been considered for the most part exclusively extracellular enzymes, being purified from the spent medium of sycamore maple cell cultures (Driouich et al. 1992, Sterjiades et al. 1993 et al.), from extracts of loblolly pine and poplar xylem cell walls treated with high-salt buffers (Bao et al.

1993, Ranocha et al. 1999), and secretions from transgenic Arabidopsis roots expressing a cotton LMCO (Wang et al. 2004), as well as the sap released by lacquer trees (Keilin and Mann 1939). However, the At2g30210 gene product could only be released from transformed tobacco cells by cell disruption. The same phenomenon was also observed when yellow-poplar LMCOs were expressed in tobacco cells (LaFayette et al. 1999, Hoopes et al.

2004). The authors postulated that a specialized glycochaperonin

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transport of LMCOs to the apoplasm during differentiation, and the factors for the proper processing were not present in suspension-cultured cells (LaFayette et al. 1999). On the other hand, although the At2g30210 gene product was predicted using two different algorithms (Predotar V1.03 and TargetP V1.01) to possess a secretory pathway signal peptide and destined for secretion via the endomembrane system, the At2g30210 protein was predicted to have a nuclear localization signal by SubLoc, and a transmembrane domain within the signal peptide made it difficult to conclude whether this specific LMCO would be secreted.

Efforts are under way to determine more precisely the intracellular localization site of the At2g30210 LMCO in BY2 cells, as well as in transgenic Arabidopsis plants.

Histochemical approaches have previously demonstrated that laccase-like phenoloxidase activities are spatially and temporally associated with lignifying xylem tissue in various herbaceous and woody species. The At2g302120 gene is expressed in root tissue, specifically young roots, which contain little xylem tissue. A study of transcriptional regulation of secondary growth in A. thaliana showed that At2g30210 gene expression is up-regulated in bark compared to xylem tissues (Oh et al. 2003).

These results suggest that the At2g30210 LMCO may not be involved in ligninfication of xylem tissue. However, several

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suberin in endodermal and hypodermal cell walls of developing roots, where the modified cell walls, called the Casparian strip, serve as diffusion barriers that play important roles in response to stress (Zeier et al. 1999; Degenhardt and Gimmler 2000; Yokoyama and Karahar 2001; Ma and Peterson 2003; Karahara et al. 2004).

Another possible function for LMCOs expressed in roots is detoxification of phenolic compounds. LMCOs are one of several extracellular enzymes capable of degrading chemical compounds in their immediate vicinity (Boyajian and Carreira 1997). Wang et al. (2004) showed that transgenic Arabidopsis over-expressing a secretory LMCO that is normally expressed in roots of cotton (Gossypium arboretum), exhibited enhanced resistance to several phenolic allelochemicals and 2,4,6-trichlorophenol. However, Arabidopsis plants in the study showed limited laccase activity in root tissues and concentrated culture medium, and since wildtype plants survived in media containing the same toxic phenolic compounds, the endogenous LMCOs cannot be ruled out as being involved in the detoxification mechanism.

Recent evidence for ferroxidase activity in a LMCO from yellow-poplar suggested another possible function for the Arabidopsis enzymes. Plant LMCOs might have the capacity to act in iron-uptake systems similar to the FET3 system in

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oxidizing activity associated with a LMCO from the pathogenic fungus, Cryptococcus neoformans (Liu et al, 1999). An essential role for the LMCO homolog, ceruloplasmin, in cellular iron efflux is critical to maintaining iron homeostasis in vertebrate animals (Harris et al. 1999). Also, haphaestin, a ceruloplasmin homolog, is involved in releasing iron from intestinal enterocytes into the circulatory system (Vulpe et al. 1999).

More recently, the ferroxidase activities of LMCOs have also been demonstrated in E. coli (Kim et al. 2001), and green algae (Herbik et al. 2002). The result that the At2g30210 gene product could not oxidize ferrous iron in vitro, however, implies that it is likely not involved in iron metabolism. It should be noted that a single LMCO from yellow-poplar is the only one from plants that so far shows ferroxidase activity. Phylogenetic analysis (McCaig et al. 2005) showed that the yellow-poplar LMCO segregates from LMCOs shown not to harbor ferroxidase activity.

Further study to test the ferroxidase activity in other LMCOs grouped with the yellow-poplar LMCO will help us determine the possible functions of different groups of plant LMCOs.

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ARABIDOPSIS GENOME INITIATIVE (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.

Nature 408:796-815 Askwith C, Eide D, Van Ho A, Bernard PS, Li L, Davis-Kaplan S, Sipe DM, Kaplan J (1994) The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake. Cell 76:403-410 Bao W, O'Malley DM, Whetten R, Sederoff RR (1993) A laccase associated with lignification in loblolly pine xylem.

Science 260:672-674 Berks BC, Sargent F, Leeuw ED, Hinsley Ap, Stanley NR, Jack RL, Buchanan G, Palmer T (2000) A novel protein transport system involved in the biogenesis of bacterial electron transfer chains. Biochimica et Biophysica Acta 1459:325-330 Boyajian, G.E., Carreira, L.H. (1997) Phytoremediation: a clean transition from laboratory to marketplace? Nat Biotechnol 15:127−128 Cristobal S, de Gier JW, Nielsen H, von Heijne G (1999) Competition between sec- and tat- dependent protein translocation in Escherichia coli. EMBO J. 18:2982-8990 Dean JFD, Eriksson K-EL (1994) Laccase and the deposition of lignin in vascular plants. Holzforschung 48:21-33 Dean JFD, LaFayette PR, Rugh C, Tristram AH, Hoopes JT, Merkle SA, Eriksson K-EL (1998) Laccases associated with lignifying tissues. In “Lignin and Lignan Biosynthesis”, Lewis NG and Sarkanen S (eds.), ACS Symp Ser 697:96-108 Degenhardt B and Gimmler H (2000) Cell wall adaptations to multiple environmental stresses in maize roots. J Exp Bot 51:595-603 Driouich A, Lainé AC, Vian B, Faye L (1992) Characterization and localization of laccase forms in stem and cell cultures of sycamore. Plant J 2:13–24 Durbin ML, McCaig B, Clegg MT (2000) Molecular evolution of the chalcone synthase multigene family in the morning glory genome. Plant Mol Biol 42:79-92

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Herbik A, Haebel S, Buckhout TJ (2002) Is a ferroxidase involved in the high affinity iron uptake in Chlamydomonas reinhardtii? Plant Soil 241:1-9 Hoopes JT, Dean JF (2001) Staining electrophoretic gels for laccase and peroxidase activity using 1,8diaminonaphthalene. Anal Biochem 293:96-101 Hoopes JT, Dean JFD (2004) Ferroxidase activity in a laccaselike multicopper oxidase from Liriodendron tulipifera.

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