MTP1 mops up excess zinc in Arabidopsis cells

The MTP1 protein of Arabidopsis thaliana belongs to a ubiquitous family of transition metal transporters that extrude metal ions from the cytoplasm. In a recent publication, Yoshihiro Kobae and colleagues show that AtMTP1 localizes to the vacuolar membrane in Arabidopsis roots and suspension-cultured cells. They demonstrate that the mtp1-1 mutant is hypersensitive to zinc and complement the mutant by transforming it with the MTP1 coding sequence downstream of a constitutive (35S) promoter.     

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.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

The extracellular EXO protein mediates cell expansion in Arabidopsis leaves

Background  

The EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described.     

Response of cultured tomato cells subjected to excess zinc: role of cell wall in zinc compartmentation

The aim of this preliminary study was to evaluate the role of the cell wall in Zn accumulation and tolerance by tomato suspension-cultured cells. Growth parameters, Zn distribution and accumulation by tomato cells were determined in function of zinc concentration. A particular attention was paid to the variations of the total cell wall material (cell wall carbohydrates, proteins, and exopolymers) in relation to extracellular levels of Zn. Cells treated with 0.5–5 mM Zn showed typical symptoms of heavy metal toxicity as testified by various growth parameters. Fresh and dry weights as well as total cell volume per vial decreased with increasing Zn concentration in the culture medium. Concurrently, the cell wall biomass increased, as well as the Zn amount retained by cell wall polymers. Cell wall appeared to assume important roles in Zn fixation and could therefore limit Zn influx into the cell. Our results also suggested that zinc fixation by cell wall was not only due to an increase in cell wall biomass but also to an improvement of its binding capacity.     

The Arabidopsis EIN2 restricts organ growth by retarding cell expansion

The growth of plant organ to its characteristic size is a fundamental developmental process, but the mechanism is still poorly understood. Plant hormones play a great role in organ size control by modulating cell division and/or cell expansion. ETHYLENE INSENSITVE 2 (EIN2) was first identified by a genetic screen for ethylene insensitivity and is regarded as a central component of ethylene signaling, but its role in cell growth has not been reported. Here we demonstrate that changed expression of EIN2 led to abnormity of cell expansion by morphological and cytological analyses of EIN2 loss-of-function mutants and the overexpressing transgenic plant. Our findings suggest that EIN2 controls final organ size by restricting cell expansion.     

Regulation of the cell expansion gene RHD3 during Arabidopsis development

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.     

Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation

Arabidopsis thaliana leaves were infiltrated with DanePy (3-( N -diethylaminoethyl)- N -dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1 H -pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.     

Trichosanthin inhibits antigen-specific T cell expansion through nitric oxide-mediated apoptosis pathway

Trichosanthin (TCS) has been found to exhibit inflammation-suppressing effect but the underlying mechanisms are not clear. In this study, we found that TCS inhibited OVA-specific T cell proliferation in a dose-dependent manner. Such inhibition was correlated with enhanced cell death. At the same time, inducible nitric oxide synthase (iNOS) mRNA expression and protein levels were found increased in cells treated with TCS, and nitric oxide (NO) production by cells was elevated in the presence of TCS. When L-NIL, the specific inhibitor of iNOS, was added to suppress NO production induced by TCS, OVA-specific cell death was significantly inhibited, meanwhile, thymidine incorporation of cells was rescued towards normal levels. These results indicate that TCS could inhibit antigen-specific T cell activation via NO-mediated apoptosis pathway.     

Green fluorescent protein-mTalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

Conservation,diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

Background  

The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking.     

Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion

? Hyponastic growth is an upward petiole movement induced by plants in response to various external stimuli. It is caused by unequal growth rates between adaxial and abaxial sides of the petiole, which bring rosette leaves to a more vertical position. The volatile hormone ethylene is a key regulator inducing hyponasty in Arabidopsis thaliana. Here, we studied whether ethylene-mediated hyponasty occurs through local stimulation of cell expansion and whether this involves the reorientation of cortical microtubules (CMTs). ? To study cell size differences between the two sides of a petiole in ethylene and control conditions, we analyzed epidermal imprints. We studied the involvement of CMT orientation in epidermal cells using the tubulin marker line as well as genetic and pharmacological means of CMT manipulation. ? Our results demonstrate that ethylene induces cell expansion at the abaxial side of the- petiole and that this can account for the observed differential growth. At the abaxial side, ethylene induces CMT reorientation from longitudinal to transverse, whereas, at the adaxial side, it has an opposite effect. The inhibition of CMTs disturbed ethylene-induced hyponastic growth. ? This work provides evidence that ethylene stimulates cell expansion in a tissue-specific manner and that it is associated with tissue-specific changes in the arrangement of CMTs along the petiole.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.