Rie Tomioka

iTRAQ Analysis Reveals Mechanisms of Growth Defects due to Excess Zinc in Arabidopsis

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H+-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role.

A Mutant Strain Arabidopsis thaliana that Lacks Vacuolar Membrane Zinc Transporter MTP1 Revealed the Latent Tolerance to Excessive Zinc

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

Characteristics of a root hair-less line of Arabidopsis thaliana under physiological stresses

Summary Root hairs of Arabidopsis play significant roles in the absorption of water and several minerals, secretion of acid phosphatases and organic acids, and anchoring of roots.

A hydrophilic cation‐binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N‐myristoylation and interacts with calmodulin and the phosphatidylinositol phosphates PtdIns(3,4,5)P3 and PtdIns(3,5)P2

A hydrophilic cation‐binding protein, PCaP1, was found to be stably bound to the plasma membrane in Arabidopsis thaliana. PCaP1 was quantified to account for 0.03–0.08% of the crude membrane fractions from roots and shoots. Its homologous protein was detected in several plant species. We investigated the mechanism of membrane association of PCaP1 by transient expression of fusion protein with green fluorescent protein. The amino‐terminal sequence of 27 residues of PCaP1 had a potential to localize the fusion protein with green fluorescent protein to the plasma membrane, and the substitution of Gly at position 2 with Ala resulted in the cytoplasmic localization of PCaP1. When PCaP1 was expressed in the in vitro transcription/translation system with [3H]myristic acid, the label was incorporated into PCaP1, but not into a mutant PCaP1 with Gly2 replaced by Ala. These results indicate that PCaP1 tightly binds to the plasma membrane via N‐myristoylation at Gly2. We examined the binding capacity with phosphatidylinositol phosphates (PtdInsPs), and found that PCaP1 selectively interacts with phosphatidylinositol 3,5‐bisphosphate and phosphatidylinositol 3,4,5‐triphosphate. Competition assay with the N‐terminal peptide and mutational analysis revealed that PCaP1 interacts with these two PtdInsPs at the N‐terminal part. Interaction of PCaP1 with the membrane and PtdInsPs was not altered in the presence of Ca2+ at physiological concentrations. Furthermore, calmodulin associated with PCaP1 in a Ca2+‐dependent manner, and its association weakened the interaction of PCaP1 with PtdInsPs. These results indicate that the N‐terminal part is essential for both N‐myristoylation and interaction with PtdInsPs, and that PCaP1 may be involved in intracellular signalling through interaction with PtdInsPs and calmodulin.

Common functions or only phylogenetically related? The large family of PLAC8 motif-containing/PCR genes

PLAC8 motif-containing proteins form a large family and members can be found in fungi, algae, higher plants and animals. They include the PCR proteins of plants. The name giving PLAC8 domain was originally found in a protein residing in the spongiotrophoblast layer of the placenta of mammals. A further motif found in a large number of these proteins including several PCR proteins is the CCXXXXCPC or CLXXXXCPC motif. Despite their wide distribution our knowledge about the function of these proteins is very limited. For most of them two membrane-spanning α-helices are predicted, indicating that they are membrane associated or membrane intrinsic proteins. In plants PLAC8 motif-containing proteins have been described to be implicated in two very different functions. On one hand, it has been shown that they are involved in the determination of fruit size and cell number. On the other hand, two members of this family, AtPCR1 and AtPCR2 play an important role in transport of heavy metals such as cadmium or zinc. Transport experiments and approaches to model the 3_D structure of these proteins indicate that they could act as transporters for these divalent cations by forming homomultimers. In this minireview we discuss the present knowledge about this protein family and try to give an outlook on how to integrate the different proposed functions into a common picture about the role of PLAC8 motif-containing proteins.

PCaPs, possible regulators of PtdInsP signals on plasma membrane.

In plants, Ca2+, phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca2+ channel, mediate the signaling. Two new Ca2+-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca2+(cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP2s. Here we propose a model for the switching mechanism of Ca2+-signaling mediated by PtdInsPs. Ca2+ forms a complex with CaM (Ca2+-CaM) when there is an increase in the cytosol free Ca2+. The binding of PCaPs with Ca2+-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.

Root growth enhancement by rhizospheric aluminum treatment in Quercus serrata Thunb. seedlings

This article discusses the effect of aluminum on root growth of Querucus serrata Thunb. In a 9-week hydroponics experiment, the effects of various concentrations (0 to 5.0 mM) of Al on root growth were examined. Results revealed that root biomass increases with the increase in Al concentration up to 2.5 mM, and then it tended to decrease. In the next experiment, the effects of H+ and Al3+ on roots were compared in a 4-week hydroponics experiment using three treatment solutions: control (pH 6.0), −Al (without Al, pH 3.5), and +Al (with 2.5 mM Al, pH 3.5). No clear difference in the biomass and root length between the control and −Al treated roots was observed, and root and shoot biomass were increased by Al treatment. These results confirmed that the H+ concentration level, at a pH of 3.5, is not toxic for Q. serrata and the Al-induced increase in root biomass is not caused by the amelioration of H+ toxicity by Al. In the third experiment, roots were exposed to an Al solution (pH 3.6) intermittently. This treatment clarified that Al stimulated rooting and root elongation. In the fourth experiment, the effect of 1 mM Al on root growth during a 15-month period in a sand culture were examined. This experiment confirmed that Al stimulated good growth and development of root systems at appropriate concentrations. Therefore, it is considered that Al-induced root growth enhancement occurs as a long-term and short-term phenomenon.

Cesium absorption through bark of Japanese cedar (Cryptomeria japonica)

Absorption of radiocesium (137Cs and 134Cs) through bark, and its subsequent translocation into wood and needles, has been suggested as a potential source of tree contamination, but the process is not well understood. Field experiments were conducted to confirm whether Cs could enter a Japanese cedar tree through the bark and how Cs moves within a tree. Stable Cs (133Cs) was applied to the bark at 1.2-m height on 10- and 26-year-old Japanese cedars. The 133Cs concentrations were determined in the bark, sapwood, and heartwood (for 26-year-old cedar only) of stem disks from several heights, as well as in current-year needles from the canopy. The 133Cs concentrations were considerably higher in the sapwood and heartwood of stem disks from 1.2-m height in treated trees than in untreated trees, suggesting that 133Cs penetrated the bark to enter the wood. The average 133Cs concentrations were higher in the heartwood than the sapwood, indicating 133Cs accumulation in the heartwood. High 133Cs concentrations in the needles of treated trees implied acropetal movement of 133Cs to actively growing organs. Our results demonstrate that Cs can enter Japanese cedar trees through the bark and that Cs is transported radially to the heartwood and vertically to the apex.

Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

Effects of calcium on cadmium uptake and transport in the tree species Gamblea innovans

The effect of calcium (Ca) on cadmium (Cd) accumulation in plants was investigated using Gamblea innovans Sieb. & Zucc., a deciduous tree species that is an accumulator plant for Cd and zinc (Zn). Saplings of G. innovans were grown for 3 months and fed with solutions containing only Ca (+Ca), both Ca and Cd (Ca+Cd), or only Cd (+Cd). The Ca concentration in roots was higher in both treatments containing Cd alone (+Cd) and Ca+Cd compared to roots treated with Ca alone (+Ca). In addition, the Cd concentration in roots was higher in the Ca+Cd treatment than the Cd treatment. This showed that the presence of Ca2+ in the rhizosphere relates with Cd uptake into roots. The result that the transport of Cd from roots through stem to leaves was suppressed by Ca treatment indicates that the presence of Ca regulates Cd transport from the roots. A clear correlation between Cd and Zn concentrations in leaves suggests a possibility that the Cd treatment accelerates the transport of Zn into leaves via the same protein transporter in this species.