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.     

The FLOWERING LOCUS T/TERMINAL FLOWER 1 family in Lombardy poplar

Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1)family have been shown to be important in the control of theswitch between vegetative and reproductive growth in severalplant species. We isolated nine members of the FT/TFL1 familyfrom Lombardy poplar (Populus nigra var. italica Koehne). Sequenceanalysis of the members of the FT/TFL1 family revealed considerablehomology within their coding regions both among family membersand to the members of the same family in Arabidopsis, tomatoand grapevine. Moreover, members of this family in all fourspecies examined display a common exon–intron organization.Phylogenetic analysis revealed that the genes fall into fourdifferent clades: two into the TFL1 clade; five into the FTclade; and one each into the MOTHER OF FT AND TFL1 and BROTHEROF FT AND TFL1 clades. One gene in the TFL1 clade, PnTFL1, isexpressed in vegetative meristems, and transgenic Arabidopsisthat ectopically expressed PnTFL1 had a late-flowering phenotype.The expression patterns of two genes in the FT clade, PnFT1and PnFT2, suggested a role for them in the promotion of flowering,and transgenic Arabidopsis that ectopically expressed eitherPnFT1 or PnFT2 had an early-flowering phenotype.     

AtAMT1;4, a Pollen-Specific High-Affinity Ammonium Transporter of the Plasma Membrane in Arabidopsis

Pollen represents an important nitrogen sink in flowers to ensurepollen viability. Since pollen cells are symplasmically isolatedduring maturation and germination, membrane transporters arerequired for nitrogen import across the pollen plasma membrane.This study describes the characterization of the ammonium transporterAtAMT1;4, a so far uncharacterized member of the ArabidopsisAMT1 family, which is suggested to be involved in transportingammonium into pollen. The AtAMT1;4 gene encodes a functionalammonium transporter when heterologously expressed in yeastor when overexpressed in Arabidopsis roots. Concentration-dependentanalysis of ¹⁵N-labeled ammonium influx into roots of AtAMT1;4-transformedplants allowed characterization of AtAMT1;4 as a high-affinitytransporter with a K_m of 17 µM. RNA and protein gel blotanalysis showed expression of AtAMT1;4 in flowers, and promoter–genefusions to the green fluorescent protein (GFP) further definedits exclusive expression in pollen grains and pollen tubes.The AtAMT1;4 protein appeared to be localized to the plasmamembrane as indicated by protein gel blot analysis of plasmamembrane-enriched membrane fractions and by visualization ofGFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes.However, no phenotype related to pollen function could be observedin a transposon-tagged line, in which AtAMT1;4 expression isdisrupted. These results suggest that AtAMT1;4 mediates ammoniumuptake across the plasma membrane of pollen to contribute tonitrogen nutrition of pollen via ammonium uptake or retrieval.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

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.     

Cold-Tolerant Crop Species Have Greater Temperature Homeostasis of Leaf Respiration and Photosynthesis Than Cold-Sensitive Species

Some plant species show constant rates of respiration and photosynthesismeasured at their respective growth temperatures (temperaturehomeostasis), whereas others do not. However, it is unclearwhat species show such temperature homeostasis and what factorsaffect the temperature homeostasis. To analyze the inherentability of plants to acclimate respiration and photosynthesisto different growth temperatures, we examined 11 herbace-ouscrops with different cold tolerance. Leaf respiration (R_area)and photosynthetic rate (P_area) under high light at 360 µll^–1 CO₂ concentrations were measured in plants grown at15 and 30°C. Cold-tolerant species showed a greater extentof temperature homeostasis of both R_area and P_area than cold-sensitivespecies. The underlying mechanisms which caused differencesin the extent of temperature homeostasis were examined. Theextent of temperature homeostasis of P_area was not determinedby differences in leaf mass and nitrogen content per leaf area,but by differences in photosynthetic nitrogen use efficiency(PNUE). Moreover, differences in PNUE were due to differencesin the maximum catalytic rate of Rubisco, Rubisco contents andamounts of nitrogen invested in Rubisco. These findings indicatedthat the temperature homeostasis of photosynthesis was regulatedby various parameters. On the other hand, the extent of temperaturehomeostasis of R_area was unrelated to the maximum activity ofthe respiratory enzyme (NAD-malic enzyme). The R_area/P_area ratiowas maintained irrespective of the growth temperatures in allthe species, suggesting that the extent of temperature homeostasisof R_area interacted with the photosynthetic rate and/or thehomeostasis of photosynthesis.     

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.     

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.     

Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulatephotoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 andHd3a were identified as orthologs of GI, CO and FT, respectively,and are also important regulators of flowering. Although GIhas roles in both flowering and the circadian clock, our understandingof its biochemical functions is still limited. In this study,we purified novel OsGI-interacting proteins by using the tandemaffinity purification (TAP) method. The TAP method has beenused effectively in a number of model species to isolate proteinsthat interact with proteins of interest. However, in plants,the TAP method has been used in only a few studies, and no novelproteins have previously been isolated by this method. We generatedtransgenic rice plants and cell cultures expressing a TAP-taggedversion of OsGI. After a two-step purification procedure, theinteracting proteins were analyzed by mass spectrometry. Sevenproteins, including dynamin, were identified as OsGI-interactingproteins. The interaction of OsGI with dynamin was verifiedby co-immunoprecipitation using a myc-tagged version of OsGI.Moreover, an analysis of Arabidopsis dynamin mutants indicatedthat although the flowering times of the mutants were not differentfrom those of wild-type plants, an aerial rosette phenotypewas observed in the mutants. We also found that OsGI is presentin both the nucleus and the cytosol by Western blot analysisand by transient assays. These results indicate that the TAPmethod is effective for the isolation of novel proteins thatinteract with target proteins in plants.     

The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitatesymmetric binary fission and that involve prokaryotic-like stromaldivision factors such as tubulin-like GTPase FtsZ and the divisionsite regulator MinD. In Arabidopsis, a nuclear-encoded prokaryoticMinE homolog, AtMinE1, has been characterized in terms of itseffects on a dividing or terminal chloroplast state in a limitedseries of leaf tissues. However, the relationship between AtMinE1expression and chloroplast phenotype remains to be fully elucidated.Here, we demonstrate that a T-DNA insertion mutation in AtMinE1results in a severe inhibition of chloroplast division, producingmotile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor)plants, dividing chloroplasts possess either single or multipleFtsZ rings located at random intervals and showing constrictiondepth, mainly along the chloroplast polarity axis. The AtMinE1sense plants displayed equivalent chloroplast phenotypes toarc11, a loss-of-function mutant of AtMinD1 which forms replicatingmini-chloroplasts. Furthermore, a certain population of FtsZrings formed within developing chloroplasts failed to initiateor progress the membrane constriction of chloroplasts and consequentiallyto complete chloroplast fission in both AtMinE1 sense and arc11/atminD1plants. Our present data thus demonstrate that the chloroplastdivision site placement involves a balance between the opposingactivities of AtMinE1 and AtMinD1, which acts to prevent FtsZring formation anywhere outside of the mid-chloroplast. In addition,the imbalance caused by an AtMinE1 dominance causes multiple,non-synchronous division events at the single chloroplast level,as well as division arrest, which becomes apparent as the chloroplastsmature, in spite of the presence of FtsZ rings.     

Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall, and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and Phenolics in Their Production

The hydroxyl radical produced in the apoplast has been demonstratedto facilitate cell wall loosening during cell elongation. Cellwall-bound peroxidases (PODs) have been implicated in hydroxylradical formation. For this mechanism, the apoplast or cellwalls should contain the electron donors for (i) H₂O₂ formationfrom dioxygen; and (ii) the POD-catalyzed reduction of H₂O₂to the hydroxyl radical. The aim of the work was to identifythe electron donors in these reactions. In this report, hydroxylradical (·OH) generation in the cell wall isolated frompea roots was detected in the absence of any exogenous reductants,suggesting that the plant cell wall possesses the capacity togenerate ·OH in situ. Distinct POD and Mn-superoxidedismutase (Mn-SOD) isoforms different from other cellular isoformswere shown by native gel electropho-resis to be preferably boundto the cell walls. Electron paramagnetic resonance (EPR) spectroscopyof cell wall isolates containing the spin-trapping reagent,5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO),was used for detection of and differentiation between ·OHand the superoxide radical (O₂^–·). The data obtainedusing POD inhibitors confirmed that tightly bound cell wallPODs are involved in DEPMPO/OH adduct formation. A decreasein DEPMPO/OH adduct formation in the presence of H₂O₂ scavengersdemonstrated that this hydroxyl radical was derived from H₂O₂.During the generation of ·OH, the concentration of quinhydronestructures (as detected by EPR spectroscopy) increased, suggestingthat the H₂O₂ required for the formation of ·OH in isolatedcell walls is produced during the reduction of O₂ by hydroxycinnamicacids. Cell wall isolates in which the proteins have been denaturated(including the endogenous POD and SOD) did not produce ·OH.Addition of exogenous H₂O₂ again induced the production of ·OH,and these were shown to originate from the Fenton reaction withtightly bound metal ions. However, the appearance of the DEPMPO/OOHadduct could also be observed, due to the production of O₂^–·when endogenous SOD has been inactivated. Also, O₂^–·was converted to ·OH in an in vitro horseradish peroxidase(HRP)/H₂O₂ system to which exogenous SOD has been added. Takentogether with the discovery of the cell wall-bound Mn-SOD isoform,these results support the role of such a cell wall-bound SODin the formation of ·OH jointly with the cell wall-boundPOD. According to the above findings, it seems that the hydroxycinnamicacids from the cell wall, acting as reductants, contribute tothe formation of H₂O₂ in the presence of O₂ in an autocatalyticmanner, and that POD and Mn-SOD coupled together generate ·OHfrom such H₂O₂.     

Four TFL1/CEN-Like Genes on Distinct Linkage Groups Show Different Expression Patterns to Regulate Vegetative and Reproductive Development in Apple (Malusxdomestica Borkh.)

Recent molecular analyses in several plant species revealedthat TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS (CEN) homologsare involved in regulating the flowering time and/or maintainingthe inflorescence meristem. In apple (Malusxdomestica Borkh.),four TFL1/CEN-like genes, MdTFL1, MdTFL1a, MdCENa and MdCENb,were found and mapped by a similar position on putatively homoeologouslinkage groups. Apple TFL1/CEN-like genes functioned equivalentlyto TFL1 when expressed constitutively in transgenic Arabidopsisplants, suggesting that they have a potential to complementthe TFL1 function. Because MdTFL1 and MdTFL1a were expressedin the vegetative tissues in both the adult and juvenile phases,they could function redundantly as a flowering repressor anda regulator of vegetative meristem identity. On the other hand,MdCENa was mainly expressed in fruit receptacles, cultured tissuesand roots, suggesting that it is involved in the developmentof proliferating tissues but not in the control of the transitionfrom the juvenile to the adult phase. In contrast, MdCENb wassilenced in most organs probably due to gene duplication bythe polyploid origin of apple. The expression patterns of MdTFL1and MdCENa in apple were also supported by the heterologousexpression of β-glucuronidase fused with their promoterregions in transgenic Arabidopsis. Our results suggest thatfunctional divergence of the roles in the regulation of vegetativemeristem identity may have occurred among four TFL1/CEN-likegenes during evolution in apple.     

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.