Phi thickenings in roots: novel secondary wall structures responsive to biotic and abiotic stresses.

Phi thickenings are specialized secondary walls found in root cortical cells. Despite their widespread occurrence throughout the plant kingdom, these specialized thickenings remain poorly understood. First identified by Van Tieghem in 1871, phi thickenings are a lignified and thickened cell wall band that is deposited inside the primary wall, as a ring around the cells' radial walls. Phi thickenings can, however, display structural variations including a fine, reticulate network of wall thickenings extending laterally from the central lignified band. While phi thickenings have been proposed to mechanically strengthen roots, act as a permeability barrier to modulate solute movement, and regulate fungal interactions, these possibilities remain to be experimentally confirmed. Furthermore, since temporal and spatial development of phi thickenings varies widely between species, thickenings may perform diverse roles in different species. Phi thickenings can be induced by abiotic stresses in different species; they can, for example, be induced by heavy metals in the Zn/Cd hyperaccumulator Thlaspi caerulescens, and in a cultivar-specific manner by water stress in Brassica. This latter observation provides an experimental platform to probe phi thickening function, and to identify genetic pathways responsible for their formation. These pathways might be expected to differ from those involved in secondary wall formation in xylem, since phi thickening deposition in not linked to programmed cell death.

Thlaspi arvense binds Cu(II) as a bis-(L-histidinato) complex on root cell walls in an urban ecosystem.

Root cell walls accumulate metal cations both during acquisition from the environment and removal from the protoplast to avoid toxicity, but molecular forms of the metals under field conditions remain elusive. We have identified how copper is bound to cell walls of intact roots of native Thlaspi arvense by combining synchrotron X-ray fluorescence and absorption techniques (XANES and EXAFS) at the nano-, micro-, and bulk scales. The plants grew naturally in sediment in a stormwater runoff basin at copper concentrations typical of urban ecosystems. About 90% of acquired copper is bound in vivo to cell walls as a unique five-coordinate Cu(II)-bis(L-histidinato) complex with one L-histidine behaving as a tridentate ligand (histamine-like chelate) and the other as a bidentate ligand (glycine-like chelate). Tridentate binding of Cu(II) would provide thermodynamic stability to protect cells against copper toxicity, and bidentate binding may enable kinetic lability along the cell wall through protein-protein docking with the non-bonded imidazole group of histidine residues. EXAFS spectra are provided as ESI to facilitate further identification of Cu-histidine and distinction of Cu-N from Cu-O bonds in biomolecules.

Differential regulation of serine acetyltransferase is involved in nickel hyperaccumulation in Thlaspi goesingense.

When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense.

Cadmium sorption, influx, and efflux at the mesophyll layer of leaves from ecotypes of the Zn/Cd hyperaccumulator Thlaspi caerulescens.

Differential sorption and transport characteristics of the leaf mesophyll layer of the Prayon and Ganges ecotypes of the hyperaccumulator Thlaspi caerulescens were examined. (109)Cd influx and efflux experiments were conducted with leaf sections, and X-ray absorption near edge structure (XANES) data were collected from leaves as a general comparison of in vivo cadmium (Cd) coordination. There were modest differences in cell wall sorption of Cd between ecotypes. There were obvious differences in time- and concentration-dependent Cd influx, including a greater V(MAX) for Prayon but a lower K(M) for Ganges for concentration-dependent Cd uptake and a notably greater Cd uptake by Ganges leaf sections at 1000 microm Cd. Leaf sections of Prayon had a greater Cd efflux than Ganges. The XANES spectra from the two ecotypes suggested differences in Cd coordination. The fundamental differences observed between the two ecotypes may reflect differential activity and/or expression of plasma membrane and tonoplast transporters. More detailed study of these transporters and the in vivo coordination of Cd are needed to determine the contribution of these processes to metal homeostasis and tolerance.

Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines.

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.

Spatial distribution of cadmium in leaves of metal hyperaccumulating Thlaspi praecox using micro-PIXE.

* Localization of cadmium (Cd) and other elements was studied in the leaves of the field-collected cadmium/zinc (Cd/Zn) hyperaccumulator Thlaspi praecox from an area polluted with heavy metals near a lead mine and smelter in Slovenia, using micro-PIXE (proton-induced X-ray emission). * The samples were prepared using cryofixation. Quantitative elemental maps and average concentrations in whole-leaf cross-sections and selected tissues were obtained. * Cd was preferentially localized in the lower epidermis (820 microg g(-1) DW), vascular bundles and upper epidermis, whereas about twice the lower concentrations were found in the mesophyll. * Taking into account the large volume of the mesophyll compared with the epidermis, the mesophyll is indicated as a relatively large pool of Cd, possibly involved in Cd detoxification/dilution at the tissue and cellular level.