Arabidopsis Glucan Synthase-Like1 (GSL1) Is Required for Tolerance to Low-Calcium Conditions and Exhibits a Function Comparable to GSL10.

Crops that exhibit symptoms of calcium (Ca) deficiency constitute a major agricultural problem. Molecular breeding of resistant cultivars is a promising method for overcoming this problem. However, the involved genes must first be identified. Here, we show that the glucan synthase-like (GSL) 1 gene is essential for low-Ca tolerance in Arabidopsis thaliana. GSL1 is homologous to GSL10, which we previously showed was essential for low-Ca tolerance. Under low-Ca conditions, gsl1 mutants exhibit reduced growth and the onset of necrosis in new leaves. These symptoms are typical of Ca-deficient crops. A grafting experiment suggested that the shoot genotype, but not the root genotype, was important for the suppression of shoot necrosis. The ectopic accumulation of callose under low-Ca conditions was significantly reduced in gsl1 mutants compared with wild-type plants. Because the corresponding single-mutant phenotypes are similar, we investigated the interaction between GSL1 and GSL10 by testing the gsl1 gsl10 double mutant for sensitivity to low-Ca conditions. The double mutant exhibited a more severe phenotype than did the single mutants, indicating that the effects of GSL1 and GSL10 on low-Ca tolerance are additive. Because GSL genes are highly conserved within the plant kingdom, the GSL loci may be useful for breeding low-Ca tolerant crops.

Callose Synthesis Suppresses Cell Death Induced by Low-Calcium Conditions in Leaves.

Despite the importance of preventing calcium (Ca) deficiency disorders in agriculture, knowledge of the molecular mechanisms underlying plant adaptations to low-Ca conditions is limited. In this study, we provide evidence for a crucial involvement of callose synthesis in the survival of Arabidopsis (Arabidopsis thaliana) under low-Ca conditions. A mutant sensitive to low-Ca conditions, low calcium sensitive3 (lcs3), exhibited high levels of cell death in emerging leaves and had defects in its expanding true leaves under low-Ca conditions. Further analyses showed that the causal mutation was located in a putative ß-1,3-glucan (callose) synthase gene, GLUCAN SYNTHASE-LIKE10 (GSL10). Yeast complementation assay results showed that GSL10 encodes a functional callose synthase. Ectopic callose significantly accumulated in wild-type plants under low-Ca conditions, but at a low level in lcs3 The low-Ca sensitivity of lcs3 was phenocopied by the application of callose synthase inhibitors in wild-type plants, which resulted in leaf expansion failure, cell death, and reduced ectopic callose levels under low-Ca conditions. Transcriptome analyses showed that the expression of genes related to cell wall and defense responses was altered in both wild-type plants under low-Ca conditions and in lcs3 under normal-Ca conditions, suggesting that GSL10 is required for the alleviation of both cell wall damage and defense responses caused by low Ca levels. These results suggest that callose synthesis is essential for the prevention of cell death under low-Ca conditions and plays a key role in plants' survival strategies under low-Ca conditions.

Verification of radiocesium decontamination from farmlands by plants in Fukushima.

The purpose of this study was to verify radiocesium decontamination from Fukushima farmland by plants and to screen plants useful for phytoremediation. Thirteen species from three families (Asteraceae, Fabaceae, and Poaceae) of crops were grown in shallow and deeply cultivated fields (0-8 and 0-15 cm plowing, respectively). To change plowing depth was expected to make different contacting zone between root system and radiocesium in soil. The radioactivity values of the plants due to the radiocesium ¹³4Cs and ¹³7Cs were 22-179 and 29-225 Bq kg dry weight⁻¹, respectively. The ¹³4Cs and ¹³7Cs transfer factors for plants grown in the shallow field ranged from 0.021 to 0.12 and fro 0.019 to 0.13, respectively, with the geometric means of 0.051 and 0.057, respectively. The ¹³4Cs and ¹³7Cs transfer factors for plants grown in the deep field ranged from 0.019 to 0.13 and from 0.022 to 0.13, respectively, with the geometric means of 0.045 and 0.063, respectively. Although a reducing ratio was calculated to evaluate the decrease in radiocesium from contaminated soil during cultivation (i.e., phytoremediation ability), no plant species resulted in a remarkable decrease in radiocesium in soil among the tested crops. These results should be followed up for several years and further analyses are required to evaluate whether the phytoremediation technique is applicable to radioactively contaminated farmlands.

Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley.

The concentration of ions in plant cells and tissues is an essential factor in determining physiological function. In the present study, we established that concentration gradients of mobile ions exist in both xylem exudates and tissues within a barley (Hordeum vulgare) primary leaf. For K(+) and NO3 (-) , ion concentrations generally decreased from the leaf base to the tip in both xylem exudates and tissues. Ion gradients were also found for Pi and Cl(-) in the xylem. The hydathode strongly absorbed Pi and re-translocated it to the rest of the plant, whereas Cl(-) was extruded. The ion concentration gradients developed early during leaf growth, increased as the tissue aged and remained under both high and low transpiration conditions. Measurement of the expression profiles of Pi, K(+) and NO3 (-) transporters along the longitudinal axis of the leaf revealed that some transporters are more expressed at the hydathode, but for most transporters, there was no significant variation along the leaf. The mechanisms by which longitudinal ion gradients develop in leaves and their physiological functions are discussed.

[Multidisciplinary medical care in radiation oncology].

Multidisciplinary medical care is now needed in the clinical setting of cancer treatment. In Japan, radiotherapy technologists and nurses have long played an important role in radiation oncology departments in many hospitals, resulting in spontaneously-arising multidisciplinary medical care teams. Multidisciplinary medical care, in which many medical staff work together on an equal footing, is based on multidisciplinary conferences, interdisciplinary communications, and most of all, doctors' decisions to transfer important daily work to paramedicals. The newly-introduced consultation fee for outpatient radiotherapy is a special charge for radiotherapy patients. To charge this fee, daily observation of patients by technologists or nurses is required, a practice which promotes multidisciplinary medical care in the field of radiation oncology. The number of technologists and nurses who specialize in radiation oncology has been increasing in recent years. When hospitals allow them to devote their specialized knowledge and skills entirely to radiation oncology practice, multidisciplinary medical care will develop within the clinical practice of radiation oncology.

The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments.

Salinity stress can greatly reduce seed production because plants are especially sensitive to salt during their reproductive stage. Here, we show that the sodium ion transporter AtHKT1;1 is specifically expressed around the phloem and xylem of the stamen in Arabidopsis thaliana to prevent a marked decrease in seed production caused by salt stress. The stamens of AtHKT1;1 mutant under salt stress overaccumulate Na+, limiting their elongation and resulting in male sterility. Specifically restricting AtHKT1;1 expression to the phloem leads to a 1.5-fold increase in the seed yield upon sodium ion stress. Expanding phloem expression of AtHKT1;1 throughout the entire plant is a promising strategy for increasing plant productivity under salinity stress.

Establishment of an in planta magnesium monitoring system using CAX3 promoter-luciferase in Arabidopsis.

The direct determination of elemental concentrations in plants is laborious. To overcome this, a novel monitoring system for magnesium (Mg) in plants was established. Mg deficiency-induced genes were identified by microarray analysis and transgenic lines that expressed luciferase (LUC) under the control of the Mg deficiency-inducible CAX3 promoter were established. The transgenic lines showed a clear response under low Mg conditions, and the degree of luminescence reflected the accumulation of endogenous CAX3 mRNA. The CAX3 expression pattern was also examined in a previously characterized low Mg-sensitive mutant, mrs2-7. In mrs2-7 mutant plants, CAX3 expression was more than three times higher than in the wild-type. In addition, CAX3 expression was negatively correlated with the shoot Mg concentration. Together, these results indicate that CAX3 transcription is a quantitative marker of the Mg status in Arabidopsis.

EFFECT OF RICE PLANT ROOT TTC-REDUCING ACTIVITY ON THE CHEMICAL FORM OF IODINE IN CULTIVATED SOIL SOLUTIONS.

This study aimed to evaluate the effect of rice plant root activity on the chemical form of stable iodine (I) in a cultivated soil solution. Concentrations of I-, IO3- and organic-I were analyzed 4 days after exposure I- or IO3- solutions to each of the cultivated soil surface. When exposed to I-, its concentration in the cultivated soil was approximately the same as that in the non-planted soil. When the rhizosphere was exposed to IO3-, the I- concentration in the soil increased under cultivation conditions. IO3- remained undetected in the soil solution. The organic-I concentration in the cultivated soil solution was higher than that in the non-cultivated soil. Concentrations of organic-I increased under IO3- addition compared to I- addition. A weak positive correlation was observed between the TTC-reducing activity of plant roots, and the total and organic-I concentrations in the soil solution. It was suggested that the amount of organic I formed from IO3- was determined by the reducing activity of the roots.

INHIBITORY EFFECT OF CALCIUM ON CAESIUM ABSORPTION IN PLANT ROOTS.

Radiocaesium is released into the environment by accidents in nuclear facilities. One of the major issues related with this is the assessment and reduction of internal exposure of crops grown in contaminated soil. To solve this, we developed a method of inhibiting Cs absorption by plant roots. In the presence of potassium concentrations >100 µM, Cs absorption occurs mainly via voltage-insensitive cation channels (VICC). Since the transport activity of VICC was inhibited by calcium at millimolar concentrations, we hypothesised that Ca would effectively inhibit Cs absorption. We grew Arabidopsis and rice plants in the presence of Cs and Ca in the nanomolar and millimolar range and measured Cs concentrations in the plants using inductively coupled plasma mass spectrometry. The results showed that the Cs concentration in both plants decreased with increasing Ca concentration in the medium. This suggests that Ca has an inhibitory effect on Cs absorption.

Role of GSL8 in low calcium tolerance in Arabidopsis thaliana.

Calcium (Ca) deficiency affects the yields and quality of agricultural products. Susceptibility to Ca deficiency varies among crops and cultivars; however, its genetic basis remains largely unknown. Genes required for low Ca tolerance in Arabidopsis thaliana have been identified. In this study, we identified a novel gene required for low Ca tolerance in A. thaliana. We isolated a mutant sensitive to low Ca concentrations and identified Glucan synthase-like (GSL) 8 as a gene responsible for low Ca tolerance. GSL8 is a paralog of the previously identified low Ca tolerance gene GSL10, which encodes ß-1,3 glucan(callose) synthase. Under low Ca conditions, the shoot growth of gsl8 mutants were inhibited compared to wild-type plants. A grafting experiment indicated that the shoot, but not root, genotype was important for the shoot growth phenotype. The ectopic accumulation of callose under low Ca conditions was reduced in gsl8 mutants. We further investigated the interaction between GSL8 and GSL10 by testing the gsl8 gsl10 double mutant for sensitivity to low Ca concentrations. The double mutant exhibited a more severe phenotype than the single mutant under 0.3 mM Ca, indicating additive effects of GSL8 and GSL10 with respect to low Ca tolerance. These results establish that GSL genes are required for low Ca tolerance in A. thaliana.

Potassium transporter KUP9 participates in K+ distribution in roots and leaves under low K+ stress.

Potassium (K) is a major essential element in plant cells, and KUP/HAK/KT-type K+ transporters participate in the absorption of K+ into roots and in the long-distance transport to above-ground parts. In Arabidopsis thaliana, KUP9 is involved in the transport of K+ and Cs+ in roots. In this study, we investigated KUP9 function in relation to the K+ status of the plant. The expression of KUP9 was upregulated in older leaves on K+-depleted medium, compared to the expression of the other 12 KUP genes in the KUP/HAK/KT family in Arabidopsis. When grown on low K+ medium, the kup9 mutant had reduced chlorophyll content in seedlings and chlorosis in older rosette leaves. Tissue-specific expression of KUP9 determined by KUP9 promoter:GUS assay depended on the K+ status of the plants: In K+ sufficient medium, KUP9 was expressed in the leaf blade towards the leaf tip, whereas in K+ depleted medium expression was mainly found in the petioles. In accordance with this, K+ accumulated in the roots of kup9 plants. The short-term 43K+ tracer measurement showed that 43K was transferred at a lower rate in roots and shoots of kup9, compared to the wild type. These data show that KUP9 participates in the distribution of K+ in leaves and K+ absorption in roots under low K+ conditions.

Arabidopsis thaliana 26S proteasome subunits RPT2a and RPT5a are crucial for zinc deficiency-tolerance.

RPTs (regulatory particle triple-A-ATPase) are components of 26S proteasome. We found novel roles of RPT2a and RPT5a in Zn deficiency-tolerance. Arabidopsis thaliana mutants carrying T-DNA in RPT2a and RPT5a were more sensitive to Zn deficiency than the wild-type. In the rpt mutants, the shoot Zn contents were similar to those of the wild-type. Transcripts of Zn deficiency-inducible genes were highly accumulated in the rpt mutants, suggesting that the rpt mutants suffer from various Zn deficiency symptoms, although the Zn levels are not reduced. Lipid peroxidation levels, known to be increased under Zn deficiency, were higher in the rpt mutants than in the wild-type. Poly-ubiquitinated proteins were accumulated upon exposure to Zn deficiency, especially in the rpt mutants. Overall, this study indicates that RPT2a and RPT5a are involved in Zn deficiency-tolerance, possibly through alleviation of oxidative stresses and/or processing of poly-ubiquitinated proteins.

AtKUP/HAK/KT9, a K+ transporter from Arabidopsis thaliana, mediates Cs+ uptake in Escherichia coli.

Although cesium is known to be absorbed by plants, the pathway by which cesium enters has not been identified. We found that the AtKUP/HAK/KT9 gene from Arabidopsis thaliana was functionally expressed in a potassium transport-deficient Escherichia coli mutant. AtKUP/HAK/KT9 mediated potassium uptake as well as cesium transport. We found that AtKUP/HAK/KT9 might be involved in the pathway in questions.

Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers.

The formation of Casparian strips and suberin lamellae at the endodermis limits the free diffusion of nutrients and harmful substances via the apoplastic space between the soil solution and the stele in roots [1-3]. Casparian strips are ring-like lignin polymers deposited in the middle of anticlinal cell walls between endodermal cells and fill the gap between them [4-6]. Suberin lamellae are glycerolipid polymers covering the endodermal cells and likely function as a barrier to limit transmembrane movement of apoplastic solutes into the endodermal cells [7, 8]. However, the current knowledge on the formation of these two distinct endodermal barriers and their regulatory role in nutrient transport is still limited. Here, we identify an uncharacterized gene, LOTR1, essential for Casparian strip formation in Arabidopsis thaliana. The lotr1 mutants display altered localization of CASP1, an essential protein for Casparian strip formation [9], disrupted Casparian strips, ectopic suberization of endodermal cells, and low accumulation of shoot calcium (Ca). Degradation by expression of a suberin-degrading enzyme in the mutants revealed that the ectopic suberization at the endodermal cells limits Ca transport through the transmembrane pathway, thereby causing reduced Ca delivery to the shoot. Moreover, analysis of the mutants showed that suberin lamellae function as an apoplastic diffusion barrier to the stele at sites of lateral root emergence where Casparian strips are disrupted. Our findings suggest that the transmembrane pathway through unsuberized endodermal cells, rather than the sites of lateral root emergence, mediates the transport of apoplastic substances such as Ca into the xylem.

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1.

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters.

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells.

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.

Addition of a peptide tag at the C terminus of AtHKT1 inhibits its Na+ transport.

Eight transmembrane segments in the Arabidopsis Na+ transporter, AtHKT1, have been proposed to be arranged around the axis of the pore. We report here that the addition of a GFP or a FLAG tag to the C terminus of AtHKT1 impairs the Na+ transport in Xenopus ooytes as detected by electrophysiological measurements.