ROS-mediated vascular homeostatic control of root-to-shoot soil Na delivery in Arabidopsis

Sodium (Na) is ubiquitous in soils, and is transported to plant shoots via transpiration through xylem elements in the vascular tissue. However, excess Na is damaging. Accordingly, control of xylem-sap Na concentration is important for maintenance of shoot Na homeostasis, especially under Na stress conditions. Here we report that shoot Na homeostasis of Arabidopsis thaliana plants grown in saline soils is conferred by reactive oxygen species (ROS) regulation of xylem-sap Na concentrations. We show that lack of A. thaliana respiratory burst oxidase protein F (AtrbohF; an NADPH oxidase catalysing ROS production) causes hypersensitivity of shoots to soil salinity. Lack of AtrbohF-dependent salinity-induced vascular ROS accumulation leads to increased Na concentrations in root vasculature cells and in xylem sap, thus causing delivery of damaging amounts of Na to the shoot. We also show that the excess shoot Na delivery caused by lack of AtrbohF is dependent upon transpiration. We conclude that AtrbohF increases ROS levels in wild-type root vasculature in response to raised soil salinity, thereby limiting Na concentrations in xylem sap, and in turn protecting shoot cells from transpiration-dependent delivery of excess Na.     

Drought-induced proline synthesis depends on root-to-shoot communication mediated by light perception

Proline accumulation in roots and shoots is one of the most evident responses to environmental stresses such as drought, which is currently one of the main threats for agriculture. Based on this response, in this work, we hypothesize that proline accumulation is dependent on root-to-shoot communication through light perception. Thus, we used exaggerated light response (hp1) and phytochrome-deficient (au) mutants of tomato, which were combined through self-grafting and reciprocal grafting and subjected to drought stress, for posterior determination of shoot and root growth and proline content. Light-affected proline metabolism, as hp1, had the highest accumulation, while au presented the lowest proline values. Reciprocal grafting showed that hp1 and MT as scion or rootstock improved MT and au proline content, respectively, indicating shoot-to-root and root-to-shoot communication modulate the metabolism of this compatible osmolyte. Dry weight, leaf area, and root area presented similar patterns to proline content, indicating the importance of this compound for plant growth under stress conditions. These results provide a new perspective on light mediation of long-distance proline translocation in stressed plants.     

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.     

Visualization of MicrotubuleOrganization and Dynamics in Living Arabidopsis Embryonic Cells

Dear Editor, Embryogenesis is a critical developmental stage during the life cycle of flowering plants. During embryogenesis, the first round of asymmetric cell division in the zygote is followed by a series of cellular events, including cell division （symmetric or asymmetric） and directional cell expan- sion to generate the apical-basal axis, radial and lateral symmetry, and patterns of different cell fates for initiation of different organ primordia, which lay the foundation for post-embryonic development （Jurgens, 2001; Wendrich and Weijers, 2013）.     

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1–green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backward. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1–GFP-labeled plastids, thus validating the use of FtsZ1–GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.     

Cell-to-cell communication via plasmodesmata during Arabidopsis embryogenesis

In Arabidopsis embryogenesis, positional information establishes the overall body plan and lineage-dependent cell fate specifies local patterning. Position-dependent gene expression and responses to the plant hormone auxin are also crucial. Recently, another mechanism that delivers positional information has been uncovered. This pathway utilizes cell-to-cell communication via plasmodesmata. Plasmodesmata span the walls between neighboring plant cells. Groups of cells that allow intercellular transport of biotic and abiotic tracers form symplastic domains of shared communication. Initially, cells of the embryo form one symplast. As development proceeds, symplastic sub-domains that correspond to the major morphological regions of the plant (i.e. shoot apex, cotyledons, hypocotyl, and root) are formed. These sub-domains further resolve into tissue-specific domains of communication (such as protodermal and vascular regions). Cell-to-cell communication via plasmodesmata between embryonic and maternal tissues ceases as development proceeds.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic β-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Xylem exudate composition and root-to-shoot nickel translocation in Alyssum species

Aims

An improved understanding of the Ni root-to-shoot translocation mechanism in hyperaccumulators is necessary to increase Ni uptake efficiency for phytoextraction technologies. It has been presumed that an important aspect of Ni translocation and storage involves chelation with organic ligands. It has been reported that exposing several Ni hyperaccumulator species of Alyssum to Ni elicited a large increase in the histidine level of the xylem sap. In later studies it was shown that as time progressed the histidine:Ni ratio dropped considerably. Moreover, previous studies analyzed the relationship between Ni and ligands in plants that were exposed to Ni only for a few hours and therefore obtained results that are unlikely to represent field soils where plants are at steady-state Ni uptake. The aim of this study was to understand the quantitative relationship between Ni and organic ligands in the xylem sap of various Alyssum genotypes or species that reached steady-state Ni uptake after being exposed to Ni in either nutrient solution or serpentine soil for up to 6 weeks.

Methods

Total Ni concentration, 17 amino acids, 9 organic acids, and nicotianamine were measured in xylem sap of 100-day old plants of Alyssum.

Results

Results showed that the concentration of Ni in xylem sap of various Alyssum genotypes was 10–100 fold higher than the concentration of histidine, malate, citrate, and nicotianamine, which were the predominant Ni ligands measured in the sap.

Conclusion

When the physiology of the whole plant is taken into account, our results indicate that the concentration of organic chelators is too low to account for the complexation of all the Ni present in the xylem sap of Alyssum at steady-state Ni hyperaccumulation, and suggest that most of the Ni in xylem sap of this species is present as the hydrated cation.     

Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii

Sedum alfredii (Crasulaceae) is the only known Cd-hyperaccumulating species that are not in the Brassica family; the mechanism of Cd hyperaccumulation in this plant is, however, little understood. Here, a combination of radioactive techniques, metabolic inhibitors, and fluorescence imaging was used to contrast Cd uptake and translocation between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of S. alfredii. The K(m) of (109)Cd influx into roots was similar in both ecotypes, while the V(max) was 2-fold higher in the HE. Significant inhibition of Cd uptake by low temperature or metabolic inhibitors was observed in the HE, whereas the effect was less pronounced in the NHE. (109)Cd influx into roots was also significantly decreased by high Ca in both ecotypes. The rate of root-to-shoot translocation of (109)Cd in the HE was >10 times higher when compared with the NHE, and shoots of the HE accumulated dramatically higher (109)Cd concentrations those of the NHE. The addition of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) resulted in a significant reduction in Cd contents in the shoots of the HE, and in the roots of the NHE. Cd was distributed preferentially to the root cylinder of the HE but not the NHE, and there was a 3-5 times higher Cd concentration in xylem sap of the HE in contrast to the NHE. These results illustrate that a greatly enhanced rate of root-to-shoot translocation, possibly as a result of enhanced xylem loading, rather than differences in the rate of root uptake, was the pivotal process expressed in the Cd hyperaccumulator HE S. alfredii.     

Volatile profiling of Arabidopsis thaliana - putative olfactory compounds in plant communication

Arabidopsis thaliana from the Brassicaceae family has arisen as the model organism in plant biology research. The plant's genome has been characterized and worldwide studies are conducted at the genetic, protein and metabolic level to unravell the function of genes involved in growth, reproduction, biosynthesis, and plant communication. As part of the multidisciplinary project BIOEMIT at NTNU, metabolomic studies of Arabidopsis T-DNA knock-out mutants and ecotypes have been carried out. Volatile profiles of autolyzed, intact plants and single plant organs were obtained by solid-phase microextraction coupled with gas chromatography-mass spectrometry. The studies were aimed at the diversity of defense-related compounds from the glucosinolate-myrosinase system - the isothiocyanates and nitriles. Metabolites from methionine, leucine and phenylalanine-derived glucosinolates were most abundant (4-methylthiobutyl, 4-methylpentyl, 2-phenylethyl). In addition, 24 monoterpenes, 26 sesquiterpenes and 12 aromatic structures, predominantly observed in inflorescenses, are described. Excluding the vast group of straight chain aliphatic structures, a total of 102 volatile compounds were detected, of which 59 are reported in Arabidopsis thaliana for the first time, thus emphasizing the sensitivity and applicability of solid-phase microextraction for volatile profiling of plant secondary metabolites.     

Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans

The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H₂O₂ challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiating TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicate that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response.Dedicated to Dr Joseph Elmer Varner.     

A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling

Abscisic acid (ABA) conjugates, predominantly their glucose esters, have recently been shown to occur in the xylem sap of different plants. Under stress conditions, their concentration can rise substantially to levels that are higher than the concentration of free ABA. External ABA conjugates cannot penetrate apoplastic barriers in the root. They have to be hydrolysed by apoplastic enzymes in the root cortex. Liberated free ABA can then be redistributed to the root symplast and dragged directly across the endodermis to the stele. Endogenous ABA conjugates are formed in the cytosol of root cells, transported symplastically to the xylem parenchyma cells and released to the xylem vessels. The mechanism of release is unknown; it may include the action of ABC-transporters. Because of its extremely hydrophilic properties, ABA-GE is translocated in the xylem of the stem without any loss to the surrounding parenchyma. After arrival in the leaf apoplast, transporters for ABA-GE in the plasmalemma have to be postulated to redistribute the conjugates to the mesophyll cells. Additionally, apoplastic esterases can cleave the conjugate and release free ABA to the target cells and tissues. The activity of these esterases is increased when barley plants are subjected to salt stress.     

Omics of root-to-shoot signaling under salt stress and water deficit

Maximizing crop yield depends on the leaves receiving an optimal supply of water, mineral nutrients, small organic molecules, proteins, and hormones from the root system via the xylem. Soil drying and salinization alter these xylem fluxes, and modern omics techniques offer unparalleled opportunities to understand the complexity of these responses. Although absolute xylem concentrations of any constituent depend on the genotype and xylem sap sampling methodology, analysis of the relative changes in concentrations has revealed some conserved behavior. Typically, these stresses increase xylem concentrations of the plant hormone abscisic acid (ABA) that limits crop water loss, but decrease the concentrations of certain cytokinins that stimulate expansive growth and prevent premature leaf senescence. Further understanding of the ionic and biophysical alterations in the rhizosphere environment that cause increased xylem concentrations of the ethylene precursor (ACC) is needed. Interactions of these plant hormones with plant nutrient status and xylem nutrient delivery may be important in tuning plant responses to their environment. Xylem proteomics is an emerging area that will help understand mechanisms of plant stress adaptation. Using omics techniques to underpin rootstock-mediate plant improvement is likely to improve crop yields in dry or saline soil.     

Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

Visualization, documentation, analysis, and communication of large-scale gene regulatory networks

Genetic regulatory networks (GRNs) are complex, large-scale, and spatially and temporally distributed. These characteristics impose challenging demands on software tools for building GRN models, and so there is a need for custom tools. In this paper, we report on our ongoing development of BioTapestry, an open source, freely available computational tool designed specifically for building GRN models. We also outline our future development plans, and give some examples of current applications of BioTapestry.     

Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere.

Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing N-acyl-L-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of Pseudomonas putida IsoF and Serratia liquefaciens MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negative P. putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.