Keitaro Tanoi

Leaf senescence in rice due to magnesium deficiency mediated defect in transpiration rate before sugar accumulation and chlorosis.

Magnesium (Mg) is an essential macronutrient supporting various functions, including photosynthesis. However, the specific physiological responses to Mg deficiency remain elusive. In this study, 2-week-old rice seedlings (Oryza sativa. cv. Nipponbare) with three expanded leaves (L2-L4) were transferred to Mg-free nutrient solution for 8 days. In the absence of Mg, on day 8, L5 and L6 were completely developed, while L7 just emerged. We also studied several mineral deficiencies to identify specific responses to Mg deficiency. Each leaf was analyzed in terms of chlorophyll, starch, anthocyanin and carbohydrate metabolites, and only absence of Mg was found to cause irreversible senescence of L5. Resupply of Mg at various time points confirmed that the borderline of L5 death was between days 6 and 7 of Mg deficiency treatment. Decrease in chlorophyll concentration and starch accumulation occurred simultaneously in L5 and L6 blades on day 8. However, nutrient transport drastically decreased in L5 as early as day 6. These data suggest that the predominant response to Mg deficiency is a defect in transpiration flow. Furthermore, changes in myo-inositol and citrate concentrations were detected only in L5 when transpiration decreased, suggesting that they may constitute new biological markers of Mg deficiency.

OsHKT1;4-mediated Na+ transport in stems contributes to Na+ exclusion from leaf blades of rice at the reproductive growth stage upon salt stress

BackgroundNa+ exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K+ transporter (HKT) family have been demonstrated to mediate leaf blade-Na+ exclusion upon salinity stress via Na+-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na+ exclusion mechanism in rice remain to be elucidated.ResultsHere, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na+ transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na+ selectivity among cations tested, including Li+, Na+, K+, Rb+, Cs+, and NH4+, in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na+ overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, 22Na+ tracer experiments using peduncles of RNAi and WT plants suggested xylem Na+ unloading by OsHKT1;4.ConclusionsTaken together, our results indicate a newly recognized function of OsHKT1;4 in Na+ exclusion in stems together with leaf sheaths, thus excluding Na+ from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.

Development of real-time radioisotope imaging systems for plant nutrient uptake studies

Ionic nutrition is essential for plant development. Many techniques have been developed to image and (or) measure ionic movement in plants. Nevertheless, most of them are destructive and limit the analysis. Here, we present the development of radioisotope imaging techniques that overcome such restrictions and allow for real-time imaging of ionic movement. The first system, called macroimaging, was developed to visualize and measure ion uptake and translocation between organs at a whole-plant scale. Such a device is fully compatible with illumination of the sample. We also modified fluorescent microscopes to set up various solutions for ion uptake analysis at the microscopic level. Both systems allow numerical analysis of images and possess a wide dynamic range of detection because they are based on radioactivity.

The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation

Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 μm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters.

Characterization of rapid intervascular transport of cadmium in rice stem by radioisotope imaging

Participation of the intervascular transport system within the rice stem during cadmium (Cd) partitioning was investigated by characterizing 109Cd behaviour in the shoot. In addition, 45Ca, 32P, and 35S partitioning patterns were analysed for comparison with that of 109Cd. Each tracer was applied to the seedling roots for 15min, and the shoots were harvested either at 15min (i.e. immediately after tracer application) or at 48h. Distribution patterns of each element at 15min were studied to identify the primary transport pathway before remobilization was initiated. 32P was preferentially transported to completely expanded leaf blades having the highest transpiration rate. The newest leaf received minimal amounts of 32P. In contrast, the amount of 35S transported to the newest leaf was similar to that transported to the other mature leaf blades. Preferential movement towards the newest leaf was evident for 109Cd and 45Ca. These results directly indicate that elemental transport is differentially regulated in the vegetative stem as early as 15min before the elements are transported to leaves. Cd behaviour in the stem was investigated in detail by obtaining serial section images from the bottom part of shoots after 109Cd was applied to a single crown root. At 30min, the maximum amount of 109Cd was distributed in the peripheral cylinder of the longitudinal vascular bundles (PV) and, interestingly, some amount of 109Cd was transported downwards along the PV. This transport manner of 109Cd provides evidence that Cd can be loaded on the phloem at the stem immediately after Cd is transported from the root.

Expression and Functional Analysis of the CorA-MRS2-ALR- Type Magnesium Transporter Family in Rice

Maintenance of an appropriate magnesium ion (Mg(2+)) concentration is essential for plant growth. In Arabidopsis thaliana, the CorA-MRS2-ALR-type proteins, named MRS2/MGT family proteins, are reportedly localized in various membranes and they function in Mg transport. However, knowledge of this family in other plant species is extremely limited. Furthermore, differential diversification among dicot and monocot plants suggested by phylogenetic analysis indicates that the role of the Arabidopsis MRS2/MGT family proteins is not the same in monocot plants. For a further understanding of this family in higher plants, functional analysis and gene expression profiling of rice MRS2/MGT family members were performed. A phylogenetic tree based on the isolated mRNA sequences of nine members of the OsMRS2 family confirmed that the MRS2/MGT family consists of five clades (A-E). A complementation assay in the yeast CM66 strain showed that four of the nine members possessed the Mg(2+) transport ability. Transient green fluorescent protein (GFP) expression in the isolated rice protoplast indicated that OsMRS2-5 and OsMRS2-6, belonging to clades D and A, respectively, localized in the chloroplast. Expression levels of these genes were low in the unexpanded yellow-green leaf, but increased considerably with leaf maturation. In addition, diurnal oscillation of expression was observed, particularly in OsMRS2-6 expression in the expanded leaf blade. We conclude that OsMRS2 family members function as Mg transporters and suggest that the genes belonging to clade A encode the chloroplast-localized Mg(2+) transporter in plants.

Application of 28Mg for characterization of Mg uptake in rice seedling under different pH conditions

The magnesium (Mg) uptake of rice seedlings treated with different pH solutions was investigated using tracer of 28Mg. The application of 28Mg enabled the determination of Mg uptake amount within 15xa0min of the different pH treatments. The uptake amount of 28Mg and its distribution in rice plants were determined by using the imaging plate. The result demonstrated that the Mg uptake amount was approximately 3xa0μmol/15xa0min per plant in 0.27xa0mMxa0Mg solution at pH 5.6, and this uptake decreased by 30xa0% at lower pH conditions (pH 4.5). On the contrary, the pH 6.5 condition induced the Mg uptake activity to 4.4xa0μmol/15xa0min. The inverse relationship found between proton concentration in nutrient solution and the Mg uptake amount within 15xa0min of treatment demonstrated that the low pH condition could inhibit the Mg uptake activity as a consequence of the competition between Mg2+ and proton. It is concluded that under low pH conditions, regardless of other factors, there is a decrease of Mg uptake activity in rice roots.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below μm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

Nondestructive real-time radioisotope imaging system for visualizing 14C-labeled chemicals supplied as CO2 in plants using Arabidopsis thaliana

We have developed a real-time radioisotope imaging system (RRIS) that can nondestructively trace 14C-labeled chemicals in plants. In an experiment, after feeding 14CO2 to a plant, the plant was fixed inside a box where lighting was regulated, and beta rays emitted from the 14C in the plant were intermittently imaged using the developed system. As a first step, using a series of standard sources of 14C, the data depth and detection limits of the 14C images captured by the RRIS were evaluated for various integral times. As a result, the linearity between the 14C activity and signal intensity was determined for the range 103. Next, the linearity was validated using plant (Arabidopsis thaliana) organs, resulting that the linearity was shown in the case of young leaf, but was not maintained in the thick organs, such as a flower, mature leaf, siliques, and stem. Considering the good correlation between the intensity by RRIS and the PSL value by an imaging plate (IP) as well as the relative low energy of beta rays emitted from 14C, the thickness of the organs would easily affect the quantitativity of the RRIS as well as an IP. Our findings prove that sequential images of 14C in a living plant sample in a regulated light and air environment can be nondestructively analyzed using the developed system, whose quantitativity is similar to that of an IP.

Different magnesium uptake and transport activity along the rice root axis revealed by 28Mg tracer experiments

We characterized magnesium (Mg) uptake and transport along the primary root of six-day-old rice seedlings (Oryza sativa L. cv. Nipponbare) using 28Mg as a radioactive tracer. We used a multi-compartment transport box, which separated the root region every 1u2009cm in order to apply 28Mg to a particular root region. Then, the root region was defined as R-A, R-B, and R-C in order from the root tip region to the lateral root-developing area. The amount of 28Mg taken up in the R-C region in 15u2009min, 1u2009h, and 3u2009h was nearly three times as much as that of R-A and R-B. Thirty percent to 50% of 28Mg absorbed from R-C was transported toward the lower part of the root. Furthermore, when 28Mg was absorbed from R-C, it was detected only in the root part lower than R-C after 5u2009min. In contrast, less than 5% of 28Mg absorbed from R-B was transported downward. This characteristic uptake and transport behavior was not observed with 32P(phosphorus)-phosphate or 45Ca (calcium). Our results demonstrate that Mg uptake and transport activity differ between root regions and further suggest that phloem loading of Mg occurs at R-C within minutes after uptake from the culture solution. Then, Mg is transported toward the root tip directly without going up the shoot.