«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»
At2g30210 knockout mutants showed increased sensitivity to salt compared to wild-type plants. To evaluate the impact of salt on At2g3021 gene expression, two-week-old Arabidopsis seedlings grown on MS medium containing NaCl were compared to plants grown on standard medium. QPCR results showed that the At2g30210 gene was slightly, but not significantly, repressed by salt (Figure 4.3), which fits with available microarray data. A contrasting observation was reported for tomato, where a LMCO cDNA that was not detectable under control conditions, was strongly up-regulated in salt-treated or salt/ABA-treated seedlings (Wei et al. 2000). A full-length cDNA is not available for the gene. The partial sequence, when aligned with other plant LMCOs, showed strongest similarity to the Acer LMCO, which does not occur in the same phylogenetic group as the At2g30210 LMCO (McCaig et al. 2005).
These results together support the idea that plant LMCOs play multiple functions in plants. It is not surprising to see functions other than lignin biosynthesis associated with plant LMCOs based on their broad substrate ranges, numerous family members, and varied expression profiles in a single species.
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2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5
Responses of Arabidopsis LMCOs to metal treatment.
Transcript levels of three LMCOs gene were analyzed in samples from plants grown in the presence of excess copper (+Cu), iron deficiency (-Fe), and excess iron (+Fe). IRT, an iron transporter gene, was used as a positive control for the iron response (Vert et al. 2002). Y-axis indicates the fold change in transcription compared to control. Error bars represent standard deviation (S.D.).
144 4 3.5 3 2.5 2 1.5 1 0.5 0 0% 1% 4.5% 1 2 3 Figure 4.2. Responses of Arabidopsis LMCOs to sucrose. At2g30210 (blue) and At2g58910 (green) gene expression levels were analyzed in samples from plants grown in the presence of different sucrose concentrations in MS medium. Y-axis indicates the fold change of transcription compared to control (1% sucrose). Error bars represent S.D.
145 1.4 1.2 1 0.8 0.6 0.4 0.2
Responses of At2g30210 gene expression to salt.
At2g30210 was analyzed from plants grown on different sodium chloride concentrations in MS medium. Y-axis indicates the fold change in transcription compared to control. Error bars represent S.D.
Laccase or laccase-like Multicopper oxidases (LMCOs) are blue copper proteins that catalyze oxidation reactions coupled to the four-electron reduction of molecular oxygen to water. Because of their wide range of potential substrates and difficulty of purification, the physiological functions of the plant LMCOs, in particular, have been difficult to determine. Early studies of plant LMCOs were focused primarily on woody plants due to the abundance of the enzyme in certain species and industrial interests. However, well-founded physiological roles for these LMCOs were difficult to identify because of lack of basic genetic information and tools to manipulate genes in these species. Because of its completed genome sequence and simple techniques for manipulating genes, Arabidopsis provides a good system in which to study the physiological function of LMCOs.
The work described in this dissertation was directed toward understanding of the physiological function of one particular Arabidopsis LMCO gene, At2g30210, by measuring its enzyme activities, patterns of tissue-specific expression, subcellular localization, effects of loss-of-function mutations, and regulation by external factors.
encoded LMCO in a heterologous expression system. The predicted amino acid sequence revealed that the At2g30210 protein possesses the four conserved copper-binding domains that characterize the multicopper oxidase, as well as a glycosyl hydrolase family 1 domain near the C-terminus. Amino acid sequence analyses showed that the At2g30210 protein is closest to another Arabidopsis LMCO, At5g07130, with 73% identity.
Although we were unable to produce active At2g30210 enzyme using E. coli, transgenic tobacco cells allowed us to recover sufficient enzyme for biochemical analyses. Heterologously expressed At2g30210 LMCO showed phenoloxidase activity, but no ferroxidase activity. These results argue against our initial hypothesis, that the At2g30210 gene product might be involved in iron metabolism, which was based on its expression pattern, primarily in roots, and the fact that other LMCOs having ferroxidase activity function in iron homeostasis in a variety of eukaryotes. Further analyses showed that transcript levels from the At2g30210 gene were not regulated by external iron availability, and that mutants exposed to different iron conditions did not show significant phenotypic changes in gene expression. Altogether the results strongly suggest that the At2g30210 gene product is not involved in iron metabolism.
transcripts, a promoter-reporter gene fusion was introduced into wild-type Arabidopsis. Histochemical staining for GUS activity revealed that At2g30210 transcripts were primarily expressed in developing endodermis of the root elongation zone and newly matured roots. The result suggested a new function for Arabidopsis LMCOs in modification of the endodermal walls.
Indeed, endodermal walls of roots are characterized by the Casparian strip, a specialized cell wall structure containing suberin and lignin that functions to seal the perimeter of endodermal cells and, thereby, create an apoplastic transport barrier to water and solutes between the root cortex and stele (Sattlemacher 2001). Changes in structure or composition of the Casparian strip are known to impact hydraulic conductivity and ion flow (Schreiber et al. 2005).
The endodermis is one of the most important adaptations of terrestrial plants. In the absence or malfunction of an endodermis plants would not be able to regulate water uptake by the roots or maintain the water balance of the plant. Soil water enters the root through root hairs and then travels via the symplast (cell-to-cell) and/or apoplast (cell wall and intercellular space). However, the endodermis is impervious to water because of a suberized cell wall matrix called the Casparian strip. Therefore, to enter the stele, apoplastic water must
pass by plasmodesmata into the cells of the stele (Kramer and Boyer 1995). Although the water movement is thought to be driven by transpiration, root pressure also plays a role in the transport of water in the xylem in some plants under certain conditions. Root pressure is created by the osmotic potential of xylem sap, which is, in turn, kept high by dissolved minerals and sugars that have been actively transported into the apoplast of the stele (Patrick 1997, Ma and Peterson 2001). One familiar example of the importance of root pressure is seen in the sugar maple where, in early spring, it hydrolyzes starches stored in the roots to produce simple sugars. The increased levels of sugar in these root tissues draws water through the endodermis and into the xylem ducts where the continuous inflow forces sap to rise up the ducts into the above ground portions of the tree where it can be tapped (harvested) (Kimball 2004).
Plant growth and development requires transport of photoassimilates (e.g. sucrose) from source tissues into developmental sinks. The transport of sugars through the differentiated phloem is explained well by Mϋnch’s pressure flow hypothesis (Ross and Tyree 1980, Bret-Harte and Silk 1994). In the growing root, the phloem transport system ends in roots at the point where differentiation of protophloem sieve tubes is initiated. This is the same region where the most rapid root
pathway for solute transport from the phloem is thought to be apoplastic in mature stem (Hayes et al. 1985) and mature roots (Wyse 1979). Thus, the endodermal cell walls with Casparian strip as apoplastic barriers become critical to insure the delivery of the photoassimilates to root tips for growth. A loss-of-function At2g30210 mutant displayed phenotypic responses to sugar availability and salt levels consistent with functional alteration of the Casparian strip such that sugars destined for the growing root tip diffuse from the stele before reaching the zone of rapid growth at the root tip.
Suberin is a plant polymer containing a core of polyphenolic material resembling lignin. Work done by Kolattukudy (1981) showed that the inclusion of atypical phenolic subunits, such as various hydroxycinnamic acids, distinguishes the “lignin” core of suberin from true lignin (Bernards 2002). Although peroxidases have been implicated most frequently in the polymerization events related to suberization (Quiroga et al.
2000, Bernards and Razem 2001), no one has previously associated LMCOs with suberizing cell walls. Our results are the first to suggest a possible association between an Arabidopsis LMCO and deposition of the lignin-like core associated with suberin.
Combining the results of biochemical analyses showing phenoloxidase activity, cell-specific expression pattern in
mutant analyses, we conclude that the At2g30210 gene is unlikely to be involved with iron-uptake, but is very possibly involved with early lignification of the root endodermis in the process giving rise to the Casparian strip, a structure of some significance to root function.
Further study will focus on uncovering addition details of At2g30210 function. Recognition of the glycosyl hydrolase family 1 domain in the At2g30210 protein C-terminus has stimulated new ideas on LMCO function. Rogers and Campbell (2004) proposed a model in which monolignols are transported from cytoplasm to the cell wall as beta-glucoside intermediates. Once the glucosides have been transported to the cell wall, they must be cleaved, before the resulting aglycones can be polymerized. Perhaps the novel glucosyl hydrolase domain found near the C-terminus of 8 Arabidopsis LMCOs functions to hydrolyze the monolignol glucosides. In addition to phenoloxidase active, such a glucosidase activity would fit well with the previously proposed role for these enzymes in the early stages of lignin polymerization. Further study to demonstrate such glucosidase activity will help to clarify this new puzzle.
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