Monika A. Wimmer

Rapid response reactions of roots to boron deprivation

Upon B removal from the nutrient solution, several response reactions of root cells can be measured within minutes. These include: reduction of cell wall elasticity modulus e, increase of hydraulic conductivity, reduced activity of plasmalemma-bound inducible (NADH) reductase, (smaller) changes of the membrane potential, and liberation of Ca2+ (apoplastic and membrane-bound). The B most demanding (root) tissues are epidermal and outer cortical cells of the extension zone, xylem vessels, and root hair tips. Deprivation of B leads to morphological changes which can be noticed within hours to days, including browning of tissues, growth inhibition, death of apical meristems, and lack of root hairs. How the primary response reaction(s) lead to the expression of visible symptoms, however, is not yet clear. The present review summarizes rapid responses to B deprivation and shows several possibilities how primary might be linked to secondary reactions, including cytoskeleton-mediated responses. Schnelle Reaktionen von Wurzeln auf Bor-Mangel Nach Umsetzen auf ein B-Mangelmedium konnen verschiedene Reaktionen von Wurzelzellen innerhalb von Minuten beobachtet werden, wie z.B. Verringerung des Zellwandelastizitatsmoduls e, Zunahme der hydraulischen Leitfahigkeit, verringerte Plasmalemma-gebundene induzierbare (NADH) Reduktaseaktivitat, (kleinere) Anderungen des Membranpotenzials und eine Freisetzung von apoplastischem und membrangebundenem Ca2+. Die Wurzelgewebe mit dem hochsten B-Bedarf scheinen die epidermalen und auseren Rindenzellen der Streckungszone, Xylem und Wurzelhaarspitzen zu sein. B-Entzug fuhrt zu morphologischen Anderungen, die innerhalb von Stunden und Tagen sichtbar werden, wie Gewebeverbraunung, Wachstumshemmung und Absterben apikaler Meristeme, sowie Fehlen von Wurzelhaaren. Wie die raschen Reaktionen zur (sekundaren) Auspragung von Mangelsymptomen fuhren, ist jedoch noch nicht klar. In dieser Ubersicht werden rasche Reaktionen auf B-Entzug zusammengefasst und Moglichkeiten zur Verknupfung primarer und sekundarer Reaktionen diskutiert.

Membrane-Associated, Boron-Interacting Proteins Isolated by Boronate Affinity Chromatography

Boron deficiency symptoms point to a role for boron in plant membranes, but the molecular partners interacting with boron have not yet been identified. The objective of the present study was to isolate and identify membrane-associated proteins with an ability to interact with boron. Boron-interacting proteins were isolated from root microsomal preparations of arabidopsis (Arabidopsis thaliana) and maize (Zea mays) using phenylboronate affinity chromatography, subsequently separated by two-dimensional gel electrophoresis and identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) peptide mass fingerprinting. Twenty-six boron-binding membrane-associated proteins were identified in A. thaliana, and nine in Z. mays roots. Additional unidentified proteins were also present. Common to both species were the beta-subunit of mitochondrial ATP synthase, several beta-glucosidases, a luminal-binding protein and fructose bisphosphate aldolase. In A. thaliana, binding of these proteins to boron was significantly reduced after 4 d of boron deprivation. The relatively high number of diverse proteins identified as boron interacting, many of which are usually enriched in membrane microdomains, supports the hypothesis that boron plays a role in plant membranes by cross-linking glycoproteins, and may be involved in their recruitment to membrane microdomains.

Review: Mechanisms for boron deficiency-mediated changes in plant water relations

Boron (B) is an essential microelement for plants and is constantly needed throughout the plant life due to its function as a structural element of the plant cell wall. B deficiency is a wide-spread problem in agricultural areas world-wide, and management of B nutrition is challenged by sudden occurrences of B deficiency or inconsistent effects of foliar B application. The effects of insufficient B supply on different structures relevant for the plant water status have been heavily researched, but the resulting conclusions are contradictory and no clear picture has so far emerged that fully explains the inconsistencies. B deficiency can affect water uptake by inhibition of root and shoot growth and by upregulation of water channels. Structural damage to xylem vessels can limit water transport to arial plant parts, while water loss can be altered by impaired barrier functions of leaf surfaces and reduced photosynthesis. In consequence of all these effects, transpiration is reduced in B-deficient plants under well-watered conditions. Under drought conditions, the responsiveness of stomata is impaired. Possible consequences of damaged vasculature for plant B nutrition include the reduced effectiveness of foliar B fertilization, especially in species with high B phloem mobility. Changes in leaf surface properties can further reduce B uptake after foliar application. In species with low B phloem mobility, weakened xylem vessels may not be able to supply sufficient B to arial parts under conditions of increased B demand, such as during bud development of trees. Since structural damage to vessels is hardly reversible, these effects could be permanent, even if B deficiency was only transient. Another consequence of reduced water status is the higher susceptibility of B-deficient plants to other abiotic stresses, which also impair water relations, especially drought. Since damage to vasculature can occur before visible symptoms of B deficiency appear in shoots, the importance to develop reliable diagnostic tools for detection of sub-acute B deficiency is highlighted.

pH Regulation by NHX-Type Antiporters Is Required for Receptor-Mediated Protein Trafficking to the Vacuole in Arabidopsis

Luminal pH of the Golgi, trans-Golgi network, and prevacuolar compartments is controlled by the Na+/H+ antiporters NHX5 and NHX6 and required for protein processing and trafficking to vacuoles. Protein trafficking requires proper ion and pH homeostasis of the endomembrane system. The NHX-type Na+/H+ antiporters NHX5 and NHX6 localize to the Golgi, trans-Golgi network, and prevacuolar compartments and are required for growth and trafficking to the vacuole. In the nhx5 nhx6 T-DNA insertional knockouts, the precursors of the 2S albumin and 12S globulin storage proteins accumulated and were missorted to the apoplast. Immunoelectron microscopy revealed the presence of vesicle clusters containing storage protein precursors and vacuolar sorting receptors (VSRs). Isolation and identification of complexes of VSRs with unprocessed 12S globulin by 2D blue-native PAGE/SDS-PAGE indicated that the nhx5 nhx6 knockouts showed compromised receptor-cargo association. In vivo interaction studies using bimolecular fluorescence complementation between VSR2;1, aleurain, and 12S globulin suggested that nhx5 nhx6 knockouts showed a significant reduction of VSR binding to both cargoes. In vivo pH measurements indicated that the lumens of VSR compartments containing aleurain, as well as the trans-Golgi network and prevacuolar compartments, were significantly more acidic in nhx5 nhx6 knockouts. This work demonstrates the importance of NHX5 and NHX6 in maintaining endomembrane luminal pH and supports the notion that proper vacuolar trafficking and proteolytic processing of storage proteins require endomembrane pH homeostasis.

A miniaturized curcumin method for the determination of boron in solutions and biological samples

Methods for the determination of boron require either sophisticated and expensive equipment (e.g. ICP-OES or ICP-MS) or a relatively large sample volume (e.g. spectrophotometric methods). A modified and miniaturized spectrophotometric curcumin method is presented which allows the determination of boron in sample volumes of 50—150 μl with a detection limit of 0.010 mg B l1. The calibration curve is linear up to a concentration of 3 mg B l1. Interferences with other ions are eliminated by extraction of boron with 2-ethyl-1,3-hexanediol in chloroform. Simultaneously, enrichment of boron in the sample can be performed within the same extraction step. Results show good reproducibility with a relative standard deviation of 15% at B concentrations between 0.05 and 0.40 mg B l—1. In different types of solutions, recovery of added boron was in the range of 98 to 99%. For several plant materials containing from 18 to 58 μg B (g dw)1, an overall relative standard deviation of 4.5% was established. So far, the method has been successfully applied to determine boron in waters, nutrient solutions, different plant parts (roots, stems and leaves) as well as in phloem sap and apoplastic washing fluid of several plant species. Eine miniaturisierte Curcumin-Methode zur Bestimmung von Bor in Losungen und in biologischen Proben Methoden der Bor-Bestimmung erfordern entweder teure und aufwendige Technik (z. B. ICP-OES oder ICP-MS) oder aber ein relativ groses Probenvolumen (z. B. Spektrophotometrische Methoden). Es wird eine modifizierte und miniaturisierte spektrophotometrische Curcumin-Methode vorgestellt, mit der Bor in Probenvolumina von 50—100 μl mit einer Nachweisgrenze von 0,010 mg B l1 bestimmt werden kann. Die Kalibriergerade ist bis zu einer Konzentration von 3 mg B l1 linear. Storende Einflusse anderer Ionen werden durch Extraktion mit 2-Ethyl-1,3-Hexandiol in Chloroform eliminiert. Zugleich kann mit der Extraktion eine Bor-Anreicherung der Probe erfolgen. Die Ergebnisse sind gut reproduzierbar mit einer relativen Standardabweichung von 1—5% bei B-Konzentrationen zwischen 0,05 und 0,40 mg B l1. In verschiedenen Losungen betrug die Wiederfindung zugegebenen Bors 98 bis 99%. Fur unterschiedliche Pflanzenmaterialien mit einem B-Gehalt zwischen 18 und 58 μg B (g TS)1 wurde in allen Fallen die relative Standardabweichung mit rund 4.5% bestimmt. Die Methode wurde bisher erfolgreich zur B-Bestimmung in Wasser, Nahrlosungen, verschiedenen Pflanzenteilen (Wurzeln, Sprosachsen, Blattern) sowie in Phloemsaft und apoplastischer Waschflussigkeit mehrerer Pflanzenarten angewendet.

Boron response in wheat is genotype-dependent and related to boron uptake, translocation, allocation, plant phenological development and growth rate

Wheat genotypes often differ significantly in their response to low and high boron (B) supply, although the underlying mechanisms for such differences are poorly understood. The stable isotopes 10B and 11B were used to investigate the contribution of root retention, uptake rates, translocation and allocation of B within wheat (Triticum aestivum L.) genotypes known to differ in B response. At high B supply, the tolerant GREEK had reduced B concentrations in main shoot leaves associated with lower uptake rates and increased allocation of B to tillers. The equally tolerant BT-SCHOMBURGK exhibited high uptake rates, but accumulation was low because of rapid development, lower concentrations of soluble B in the cell sap and lower B translocation to the shoot. In WlMMC, high uptake rates, slow development, high translocation and allocation to main shoots resulted in high B accumulation and poor tolerance. Retention in roots was not substantial in any of the genotypes. The results suggest that B tolerance is multi-faceted and genotype specific. Mechanisms contributing to B tolerance include reduced uptake rates and differential translocation and allocation within plants. Additionally, plant growth rate and leaf morphology can influence B response by affecting tissue concentrations and allowing completion of plant maturation before B accumulation impairs growth. These mechanisms are expressed to different extents depending on the genotype.

Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection

Boron (B) is an essential nutrient for N(2)-fixing legume-rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate-binding-specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum-Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan-extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B-sufficient, but not from B-deficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B-deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B-deficient nodules. The absence of MAC 203 antigens in B-deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in micro-aerobically grown R. leguminosarum 3841. However, B-deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non-functional nodules, but did not appear to induce oxidative damage in low-B nodules.

Boron Toxicity: the Importance of Soluble Boron

The function of boron in plants has often been associated with cell wall bound insoluble boron. This was mainly due to the fact that B-related nutritional disorders clearly lead to a structural damage of the cell wall, and that the cell wall bound B-RGII-complex has been isolated and characterized in detail. However, more and more evidence indicates that other functions of B are important in plants, which can not be related to cell wall bound B-RGII. Rapid reactions under B deficiency, effects of B toxicity, and, most important of all, the observed essentiality of B for animals (Eckert, 1998, Eckert and Rowe, 1999), can not be explained by a structural function of B in the cell wall. Since animals do not have a pectin-rich cell wall, other B forms, e.g. free or loosely bound B, must be involved in those reactions.

Interaction of salinity and boron toxicity in wheat (Triticum aestivum L.)

Salinity and B toxicity are closely associated, however, available data on B toxicity under saline conditions are limited and contradictory. Although salinity usually reduces shoot B concentrations, B toxicity symptoms can be either reduced or enhanced. Since whole tissue B concentration is a poor indicator of B tolerance, we investigated B/salinity interactions by assessing tissue B distribution and subcellular ion compartmentation in wheat. Under saline conditions the total B concentration increased in leaf tips, decreased in roots, and was not affected in basal leaf parts. However, soluble B concentrations in basal leaf parts significantly increased in the combined salt/high B treatment compared to high B treatment alone. We propose that salinity interacts with B toxicity by a combined effect on B and water uptake and B partitioning within the plant.

Rapid Responses of Plants to Boron Deprivation

B deficiency has been described to affect a wide range of processes in plants, ranging from metabolism of nucleic acids, protein synthesis, metabolism and transport of carbohydrates, chemistry and physics of cell walls, synthesis and transport of plant hormones (especially IAA), regulation of plasma membrane-bound ATPase and oxido-reductase activities, as well as synthesis and metabolism of phenolics and incidence of oxidative damage of membranes, (for reviews see e.g. Goldbach, 1997; Blevins and Lucaszewski, 1998; Bell et al., 2001, this volume). Considering that many observations started at least several hours or even days and weeks after B deprivation, and taking into account that recent reports point to very rapid reactions within minutes (Findeklee and Goldbach, 1996; Findeklee et al., 1997; Muhling et al., 1998; Wimmer and Goldbach, 1999), it is intriguing to explore the sequence of responses to B deprivation. Below, we summarise effects which have been observed within the first minutes to few hours of B deficiency and discuss how these early responses may be linked to the expression of secondary reactions or symptoms in the light of recent observations.