The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.     

Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus

Nitrate is a major environmental factor in the inhibition of nodulation. In a model legume Lotus japonicus, a CLV1-like receptor kinase, HAR1, mediates nitrate inhibition and autoregulation of nodulation. Autoregulation of nodulation involves root-to-shoot-to-root long-distance communication, and HAR1 functions in shoots. However, it remains elusive where HAR1 functions in the nitrate inhibition of nodulation. We performed grafting experiments with the har1 mutant under various nitrate conditions, and found that shoot HAR1 is critical for the inhibition of nodulation at 10 mM nitrate. Combined with our recent finding that the nitrate-induced CLE-RS2 glycopeptide binds directly to the HAR1 receptor, this result suggests that CLE-RS2/HAR1 long-distance signaling plays an important role in the both nitrate inhibition and the autoregulation of nodulation.     

Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Identification of maize silicon influx transporters

Maize (Zea mays L.) shows a high accumulation of silicon (Si),but transporters involved in the uptake and distribution havenot been identified. In the present study, we isolated two genes(ZmLsi1 and ZmLsi6), which are homologous to rice influx Sitransporter OsLsi1. Heterologous expression in Xenopus laevisoocytes showed that both ZmLsi1 and ZmLsi6 are permeable tosilicic acid. ZmLsi1 was mainly expressed in the roots. By contrast,ZmLsi6 was expressed more in the leaf sheaths and blades. Differentfrom OsLsi1, the expression level of both ZmLsi1 and ZmLsi6was unaffected by Si supply. Immunostaining showed that ZmLsi1was localized on the plasma membrane of the distal side of rootepidermal and hypodermal cells in the seminal and crown roots,and also in cortex cells in lateral roots. In the shoots, ZmLsi6was found in the xylem parenchyma cells that are adjacent tothe vessels in both leaf sheaths and leaf blades. ZmLsi6 inthe leaf sheaths and blades also exhibited polar localizationon the side facing towards the vessel. Taken together, it canbe concluded that ZmLsi1 is an influx transporter of Si, whichis responsible for the transport of Si from the external solutionto the root cells and that ZmLsi6 mainly functions as a Si transporterfor xylem unloading.     

Characterization of Cd translocation and identification of the Cd form in xylem sap of the Cd-hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a Cd hyperaccumulator; however, the mechanismsinvolved in the root to shoot translocation of Cd are not wellunderstood. In this study, we characterized Cd transfer fromthe root medium to xylem in this species. Arabidopsis halleriaccumulated 1,500 mg kg^–1 Cd in the shoot without growthinhibition. A time-course experiment showed that the releaseof Cd into the xylem was very rapid; by 2 h exposure to Cd,Cd concentration in the xylem sap was 5-fold higher than thatin the external solution. The concentration of Cd in the xylemsap increased linearly with increasing Cd concentration in theexternal solution. Cd transfer to the xylem was completely inhibitedby the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone(CCCP). Cd concentration in the xylem sap was decreased by increasingthe concentration of external Zn, but enhanced by Fe deficiencytreatment. Analysis with ¹¹³Cd-nuclear magnetic resonance (NMR)showed that the chemical shift of ¹¹³Cd in the xylem sap wasthe same as that of Cd(NO₃)₂. Metal speciation with Geochem-PCalso showed that Cd occurred mainly in the free ionic form inthe xylem sap. These results suggest that Cd transfer from theroot medium to the xylem in A. halleri is an energy-dependentprocess that is partly shared with Zn and/or Fe transport. Furthermore,Cd is translocated from roots to shoots in inorganic forms.     

Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation

Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot.     

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import

We have previously demonstrated that the targeting signal ofpumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targetingsignal 1)-like sequence, QKL, located at –13 to –11from the C-terminus, which is different from the typical PTS1SKL motif located in the C-terminus. Here we show that Cat1import into peroxisome is dependent on the cytosolic PTS receptor,Pex5p, in Arabidopsis, similar to typical PTS1 import, and thatother components for transport of peroxisomal matrix proteinssuch as Pex14p, Pex13p, Pex12p and Pex10p also contribute tothe import of Cat1. Interestingly, however, we found that Cat1interacts with the N-terminal domain of Pex5p, but not the C-terminaldomain for interaction with the typical PTS1, revealing thatPex5p recognizes Cat1 in a manner distinct from typical PTS1.     

Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo.     

Involvement of Polypyrimidine Tract-Binding Protein (PTB)-Related Proteins in Pollen Germination in Arabidopsis

The pollen grains of most angiosperms contain stores of RNAsand their translation products required for pollen germinationand subsequent early elongation of pollen tubes. Polypyrimidinetract-binding protein (PTB), which is involved in the regulationof pre-mRNA alternative splicing, internal ribosomal entry site(IRES)-mediated translation and mRNA localization/sorting, isknown to act as a bridging molecule between RNAs and a varietyof cellular factors to fulfill cellular functions in both thenucleus and cytoplasm. Moreover, it has been reported that PTBplays roles in the differentiation and development of animalcells and tissues. In the Arabidopsis genome, there are twoPTB-related genes, tentatively termed AtPTB1 and AtPTB2. Inthe present study, the physiological functions of AtPTBs wereinvestigated using genetic and cytological approaches. The AtPTBpromoter was highly active in vegetative cells of mature pollengrains, and AtPTB was localized in the nucleus and cytoplasmof these vegetative cells. Mutations in the AtPTB genes resultedin decreased germination efficiency, and this effect was rescuedby introduction of the AtPTB2 promoter::AtPTB2–GFP. Takentogether, these findings suggest that AtPTB is involved in pollengermination through possible RNA metabolism processes in late-maturingand mature pollen grains.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation

By means of functional screening using the cadmium (Cd)-sensitiveycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1,from Digitaria ciliaris growing in a former mining area in northernJapan, and have shown that it confers Cd tolerance to the yeastcells, which accumulated almost 2-fold lower Cd levels thancontrol cells. The 521 bp DcCDT1 cDNA contains an open readingframe of 168 bp and encodes a deduced peptide, DcCDT1, thatis 55 amino acid residues in length, of which 15 (27.3%) arecysteine residues. Five DcCDT1 homologs (here termed OsCDT1–OsCDT5)have been identified in rice, and all of them were up-regulatedto varying degrees in the above-ground tissues by CdCl₂ treatment.Localization of green fluorescent protein fusions suggests thatDcCDT1 and OsCDT1 are targeted to both cytoplasmic membranesand cell walls of plant cells. Transgenic Arabidopsis thalianaplants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerantphenotype and, consistent with our yeast data, accumulated loweramounts of Cd when grown on CdCl₂. Collectively, our data suggestthat DcCDT1 and OsCDT1 function to prevent entry of Cd intoyeast and plant cells and thereby enhance their Cd tolerance.     

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in theregulation of shikonin formation in Onosma paniculatum cells.NO generated after cells were inoculated into shikonin productionmedium reached the highest level after 2 d of culture, whichwas 16 times that at the beginning of the experiment, and maintaineda high level for 6 d. A nitric oxide synthase (NOS) inhibitor,N-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor,sodium azide (SoA), consistent with their inhibition of NO biosynthesis,decreased shikonin formation significantly. This reduction couldbe alleviated or even abolished by exogenous NO supplied bysodium nitroprusside (SNP), suggesting that the inhibition ofNO biosynthesis resulted in decreased shikonin formation. However,when endogenous NO biosynthesis was up-regulated by the elicitorfrom Rhizoctonia cerealis, shikonin production was enhancedfurther, showing a dependence on the elicitor-induced NO burst.Real-time PCR analysis showed that NO could significantly up-regulatethe expression of PAL, PGT and HMGR, which encode key enzymesinvolved in shikonin biosynthesis. These results demonstratedthat NO plays a critical role in shikonin formation in O. paniculatumcells.     

Role of the Aquaporin PIP1 Subfamily in the Chilling Tolerance of Rice

Although an association between chilling tolerance and aquaporinshas been reported, the exact mechanisms involved in this relationshipremain unclear. We compared the expression profiles of aquaporingenes between a chilling-tolerant and a low temperature-sensitiverice variety using real-time PCR and identified seven genesthat closely correlated with chilling tolerance. Chemical treatmentexperiments, by which rice plants were induced to lose theirchilling tolerance, implicated the PIP1 (plasma membrane intrinsicprotein 1) subfamily member genes in chilling tolerance. Ofthese members, changes in expression of the OsPIP1;3 gene suggestedthis to be the most closely related to chilling tolerance. AlthoughOsPIP1;3 showed a much lower water permeability than membersof the OsPIP2 family, OsPIP1;3 enhanced the water permeabilityof OsPIP2;2 and OsPIP2;4 when co-expressed with either of theseproteins in oocytes. Transgenic rice plants (OE1) overexpressingOsPIP1;3 showed an enhanced level of chilling tolerance andthe ability to maintain high OsPIP1;3 expression levels underlow temperature treatment, similar to that of chilling-tolerantrice plants. We assume that OsPIP1;3, constitutively overexpressedin the leaf and root of transgenic OE1 plants, interacts withmembers of the OsPIP2 subfamily, thereby improving the plants’water balance under low temperatures and resulting in the observedchilling tolerance of the plants.