Structural and functional analysis of a plant nucleolar RNA chaperone-like protein.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

The Arabidopsis thaliana Kinesin-5 AtKRP125b Is a Processive, Microtubule-Sliding Motor Protein with Putative Plant-Specific Functions.

The formation and maintenance of the mitotic spindle during cell division requires several microtubule-interacting motor proteins. Members of the kinesin-5 family play an essential role in the bipolar organization of the spindle. These highly conserved, homotetrameric proteins cross-link anti-parallel microtubules and slide them apart to elongate the spindle during the equal separation of chromosomes. Whereas vertebrate kinesin-5 proteins are well studied, knowledge about the biochemical properties and the function of plant kinesin-5 proteins is still limited. Here, we characterized the properties of AtKRP125b, one of four kinesin-5 proteins in Arabidopsis thaliana. In in vitro motility assays, AtKRP125b displayed the archetypal characteristics of a kinesin-5 protein, a low velocity of about 20 nm·s-1, and a plus end-directed, processive movement. Moreover, AtKRP125b was able to cross-link microtubules and to slide them apart, as required for developing and maintaining the mitotic spindle. In line with such a function, GFP-AtKRP125b fusion proteins were predominantly detected in the nucleus when expressed in Arabidopsis thaliana leaf protoplasts or Nicotiana benthamiana epidermis cells and analyzed by confocal microscopy. However, we also detected GFP signals in the cytoplasm, suggesting additional functions. By generating and analyzing AtKRP125b promoter-reporter lines, we showed that the AtKRP125b promoter was active in the vascular tissue of roots, lateral roots, cotyledons, and true leaves. Remarkably, we could not detect promoter activity in meristematic tissues. Taken together, our biochemical data support a role of AtKRP125b in mitosis, but it may also have additional functions outside the nucleus and during interphase.

Structural Features of a Full-Length Ubiquitin Ligase Responsible for the Formation of Patches at the Plasma Membrane.

Plant U-box armadillo repeat (PUB-ARM) ubiquitin (Ub) ligases have important functions in plant defense through the ubiquitination of target proteins. Defense against pathogens involves vesicle trafficking and the formation of extracellular vesicles. The PUB-ARM protein SENESCENCE ASSOCIATED UBIQUITIN E3 LIGASE1 (SAUL1) can form patches at the plasma membrane related to tethering multi-vesicular bodies (MVBs) to the plasma membrane. We uncovered the structure of a full-length plant ubiquitin ligase and the structural requirements of SAUL1, which are crucial for its function in patch formation. We resolved the structure of SAUL1 monomers by small-angle X-ray scattering (SAXS). The SAUL1 model showed that SAUL1 consists of two domains: a domain containing the N-terminal U-box and armadillo (ARM) repeats and the C-terminal ARM repeat domain, which includes a positively charged groove. We showed that all C-terminal ARM repeats are essential for patch formation and that this function requires arginine residue at position 736. By applying SAXS to polydisperse SAUL1 systems, the oligomerization of SAUL1 is detectable, with SAUL1 tetramers being the most prominent oligomers at higher concentrations. The oligomerization domain consists of the N-terminal U-box and some N-terminal ARM repeats. Deleting the U-box resulted in the promotion of the SAUL1 tethering function. Our findings indicate that structural changes in SAUL1 may be fundamental to its function in forming patches at the plasma membrane.

PMR4-dependent cell wall depositions are a consequence but not the cause of temperature-induced autoimmunity.

Plants possess a well-balanced immune system that is required for defense against pathogen infections. In autoimmune mutants or necrotic crosses, an intrinsic temperature-dependent imbalance leads to constitutive immune activation, resulting in severe damage or even death of plants. Recently, cell wall depositions were described as one of the symptoms following induction of the autoimmune phenotype in Arabidopsis saul1-1 mutants. However, the regulation and function of these depositions remained unclear. Here, we show that cell wall depositions, containing lignin and callose, were a common autoimmune feature and were deposited in proportion to the severity of the autoimmune phenotype at reduced ambient temperatures. When plants were exposed to reduced temperature for periods insufficient to induce an autoimmune phenotype, the cell wall depositions were not present. After low temperature intervals, sufficient to induce autoimmune responses, cell wall depositions correlated with a point of no return in saul1-1 autoimmunity. Although cell wall depositions were largely abolished in saul1-1 pmr4-1 double mutants lacking SAUL1 and the callose synthase gene GSL5/PMR4, their phenotype remained unchanged compared to that of the saul1-1 single mutant. Our data showed that cell wall depositions generally occur in autoimmunity, but appear not to be the cause of autoimmune phenotypes.

Unrecognized controls on microbial functioning in Blue Carbon ecosystems: The role of mineral enzyme stabilization and allochthonous substrate supply.

Tidal wetlands are effective carbon sinks, mitigating climate change through the long-term removal of atmospheric CO2. Studies along surface-elevation and thus flooding-frequency gradients in tidal wetlands are often used to understand the effects of accelerated sea-level rise on carbon sequestration, a process that is primarily determined by the balance of primary production and microbial decomposition. It has often been hypothesized that rates of microbial decomposition would increase with elevation and associated increases in soil oxygen availability; however, previous studies yield a wide range of outcomes and equivocal results. Our mechanistic understanding of the elevation-decomposition relationship is limited because most effort has been devoted to understanding the terminal steps of the decomposition process. A few studies assessed microbial exo-enzyme activities (EEAs) as initial and rate-limiting steps that often reveal important insight into microbial energy and nutrient constraints. The present study assessed EEAs and microbial abundance along a coastal ecotone stretching a flooding gradient from tidal flat to high marsh in the European Wadden Sea. We found that stabilization of exo-enzymes to mineral sediments leads to high specific EEAs at low substrate concentrations in frequently flooded, sediment-rich zones of the studied ecotone. We argue that the high background activity of a mineral-associated enzyme pool provides a stable decomposition matrix in highly dynamic, frequently flooded zones. Furthermore, we demonstrate that microbial communities are less nutrient limited in frequently flooded zones, where inputs of nutrient-rich marine organic matter are higher. This was reflected in both increasing exo-enzymatic carbon versus nutrient acquisition and decreasing fungal versus bacterial abundance with increasing flooding frequency. Our findings thereby suggest two previously unrecognized mechanisms that may contribute to stimulated microbial activity despite decreasing oxygen availability in response to accelerated sea-level rise.

PP7L is essential for MAIL1-mediated transposable element silencing and primary root growth.

The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.

E3 ligase SAUL1 serves as a positive regulator of PAMP-triggered immunity and its homeostasis is monitored by immune receptor SOC3.

In both plants and animals, intracellular nucleotide-binding leucine-rich repeat proteins (NLRs; or Nod-like receptors) serve as immune receptors to recognize pathogen-derived molecules and mount effective immune responses against microbial infections. Plant NLRs often guard the presence or activity of other host proteins, which are the direct virulence targets of pathogen effectors. These guardees are sometimes immune-promoting components such as those in a mitogen-activated protein kinase cascade. Plant E3 ligases serve many roles in immune regulation, but it is unclear whether they can also be guarded by NLRs. Here, we report on an immune-regulating E3 ligase SAUL1, whose homeostasis is monitored by a Toll interleukin 1 receptor (TIR)-type NLR (TNL), SOC3. SOC3 can associate with SAUL1, and either loss or overexpression of SAUL1 triggers autoimmunity mediated by SOC3. By contrast, SAUL1 functions redundantly with its close homolog PUB43 to promote PAMP-triggered immunity (PTI). Taken together, the E3 ligase SAUL1 serves as a positive regulator of PTI and its homeostasis is monitored by the TNL SOC3.

Top-down control of carbon sequestration: grazing affects microbial structure and function in salt marsh soils.

Tidal wetlands have been increasingly recognized as long-term carbon sinks in recent years. Work on carbon sequestration and decomposition processes in tidal wetlands focused so far mainly on effects of global-change factors such as sea-level rise and increasing temperatures. However, little is known about effects of land use, such as livestock grazing, on organic matter decomposition and ultimately carbon sequestration. The present work aims at understanding the mechanisms by which large herbivores can affect organic matter decomposition in tidal wetlands. This was achieved by studying both direct animal-microbe interactions and indirect animal-plant-microbe interactions in grazed and ungrazed areas of two long-term experimental field sites at the German North Sea coast. We assessed bacterial and fungal gene abundance using quantitative PCR, as well as the activity of microbial exo-enzymes by conducting fluorometric assays. We demonstrate that grazing can have a profound impact on the microbial community structure of tidal wetland soils, by consistently increasing the fungi-to-bacteria ratio by 38-42%, and therefore potentially exerts important control over carbon turnover and sequestration. The observed shift in the microbial community was primarily driven by organic matter source, with higher contributions of recalcitrant autochthonous (terrestrial) vs. easily degradable allochthonous (marine) sources in grazed areas favoring relative fungal abundance. We propose a novel and indirect form of animal-plant-microbe interaction: top-down control of aboveground vegetation structure determines the capacity of allochthonous organic matter trapping during flooding and thus the structure of the microbial community. Furthermore, our data provide the first evidence that grazing slows down microbial exo-enzyme activity and thus decomposition through changes in soil redox chemistry. Activities of enzymes involved in C cycling were reduced by 28-40%, while activities of enzymes involved in N cycling were not consistently affected by grazing. It remains unclear if this is a trampling-driven direct grazing effect, as hypothesized in earlier studies, or if the effect on redox chemistry is plant mediated and thus indirect. This study improves our process-level understanding of how grazing can affect the microbial ecology and biogeochemistry of semi-terrestrial ecosystems that may help explain and predict differences in C turnover and sequestration rates between grazed and ungrazed systems.

Membrane-Associated Ubiquitin Ligase SAUL1 Suppresses Temperature- and Humidity-Dependent Autoimmunity in Arabidopsis.

Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid-dependent and pathogenesis-related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, or Hyaloperonospora arabidopsidis Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell-wall depositions. Confocal analysis on aniline blue-stained leaf sections and cellular universal micro spectrophotometry further showed that these cell-wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4 but not SAG101. Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

MAIL1 is essential for development of the primary root but not of anchor roots.

MAIN-LIKE1 (MAIL1) is a ubiquitously expressed nuclear protein, which has a crucial function during root development. We have recently described loss of function mutants for MAIL1, in which the organization and function of the primary root meristem is lost soon after germination. Moreover cell differentiation is impaired resulting in primary root growth arrest soon after emergence. Here we show that mail1 mutants form several anchor roots from the hypocotyl to root junction. These anchor roots show similar defects in the organization of the stem cell niche as the primary root. In contrast, differentiation processes are not impaired and thus anchor roots seem to be able to compensate for the loss of primary root function. Our data show that MAIL1 is essential for specification of cell fate in the primary root but not in anchor roots.

Plasma membrane-association of SAUL1-type plant U-box armadillo repeat proteins is conserved in land plants.

Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box (PUB) armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

Identification of MAIN, a factor involved in genome stability in the meristems of Arabidopsis thaliana.

Stem cells in the root and shoot apical meristem provide the descendant cells required for growth and development throughout the lifecycle of a plant. We found that mutations in the Arabidopsis MAINTENANCE OF MERISTEMS (MAIN) gene led to plants with distorted stem cell niches in which stem cells are not maintained and undergo premature differentiation or cell death. The malfunction of main meristems leads to short roots, mis-shaped leaves, reduced fertility and partial fasciation of stems. MAIN encodes a nuclear-localized protein and is a member of a so far uncharacterized plant-specific gene family. As main mutant plants are hypersensitive to DNA-damaging agents, expression of genes involved in DNA repair is induced and dead cells with damaged DNA accumulate in the mutant meristems, we propose that MAIN is required for meristem maintenance by sustaining genome integrity in stem cells and their descendants cells.

An IQ domain mediates the interaction with calmodulin in a plant cyclic nucleotide-gated channel.

Cyclic nucleotide-gated channels (CNGCs) form non-selective cation entry pathways regulated by calmodulin (CaM), a universal Ca(2+) sensor in eukaryotes. Although CaM binding has been shown to be important for Ca(2+)-dependent feedback regulation of CNGC activity, the CaM-binding properties of these channels have been investigated in a few cases only. We show that CNGC20 from Arabidopsis thaliana binds CaM in a Ca(2+)-dependent manner and interacts with all AtCaM isoforms but not with the CaM-like proteins CML8 and CML9. CaM interaction with the full-length channel was demonstrated in planta, using bimolecular fluorescence complementation. This interaction occurred at the plasma membrane, in accordance with our localization data of green fluorescent protein (GFP)-fused CNGC20 proteins. The CaM-binding site was mapped to an isoleucine glutamine (IQ) motif, which has not been characterized in plant CNGCs so far. Our results show that compared with the overlapping binding sites for cyclic nucleotides and CaM in CNGCs studied so far, they are sequentially organized in CNGC20. The presence of two alternative CaM-binding modes indicates that ligand regulation of plant CNGCs is more complex than previously expected. Since the IQ domain is conserved among plant CNGCs, this domain adds to the variability of Ca(2+)-dependent channel control mechanisms underlining the functional diversity within this multigene family.

Early senescence and cell death in Arabidopsis saul1 mutants involves the PAD4-dependent salicylic acid pathway.

Age-dependent leaf senescence and cell death in Arabidopsis (Arabidopsis thaliana) requires activation of the transcription factor ORESARA1 (ORE1) and is not initiated prior to a leaf age of 28 d. Here, we investigate the conditional execution of events that regulate early senescence and cell death in senescence-associated ubiquitin ligase1 (saul1) mutants, deficient in the PLANT U-BOX-ARMADILLO E3 ubiquitin ligase SAUL1. In saul1 mutants challenged with low light, the switch of age-dependent cell death was turned on prematurely, as indicated by the accumulation of ORE1 transcripts, induction of the senescence marker gene SENESCENCE-ASSOCIATED GENE12, and cell death. However, ORE1 accumulation by itself was not sufficient to cause saul1 phenotypes, as demonstrated by double mutant analysis. Exposure of saul1 mutants to low light for only 24 h did not result in visible symptoms of senescence; however, the senescence-promoting transcription factor genes WRKY53, WRKY6, and NAC-LIKE ACTIVATED BY AP3/PI were up-regulated, indicating that senescence in saul1 seedlings was already initiated. To resolve the time course of gene expression, microarray experiments were performed at narrow intervals. Differential expression of the genes involved in salicylic acid and defense mechanisms were the earliest events detected, suggesting a central role for salicylic acid in saul1 senescence and cell death. The salicylic acid content increased in low-light-treated saul1 mutants, and application of exogenous salicylic acid was indeed sufficient to trigger saul1 senescence in permissive light conditions. Double mutant analyses showed that PHYTOALEXIN DEFICIENT4 (PAD4) but not NONEXPRESSER OF PR GENES1 (NPR1) is essential for saul1 phenotypes. Our results indicate that saul1 senescence depends on the PAD4-dependent salicylic acid pathway but does not require NPR1 signaling.

The plastid outer envelope protein OEP16 affects metabolic fluxes during ABA-controlled seed development and germination.

Previously, the OEP16.1 channel pore in the outer envelope membrane of mature pea (Pisum sativum) chloroplasts in vitro has been characterized to be selective for amino acids. Isolation of OEP16.2, a second OEP16 isoform from pea, in the current study allowed membrane localization and gene expression of OEP16 to be followed throughout seed development and germination of Arabidopsis thaliana and P. sativum. Thereby it can be shown on the transcript and protein level that the isoforms OEP16.1 and OEP16.2 in both plant species are alternating: whereas OEP16.1 is prominent in early embryo development and first leaves of the growing plantlet, OEP16.2 dominates in late seed development stages, which are associated with dormancy and desiccation, as well as early germination events. Further, OEP16.2 expression in seeds is under control of the phytohormone abscisic acid (ABA), leading to an ABA-hypersensitive phenotype of germinating oep16 knockout mutants. In consequence, the loss of OEP16 causes metabolic imbalance, in particular that of amino acids during seed development and early germination. It is thus concluded that in vivo OEP16 most probably functions in shuttling amino acids across the outer envelope of seed plastids.

C-terminal armadillo repeats are essential and sufficient for association of the plant U-box armadillo E3 ubiquitin ligase SAUL1 with the plasma membrane.

Ubiquitination plays important roles in plant growth and development. Whereas ubiquitin-dependent protein degradation and modulation in the cytoplasm and nucleus are well established in plants, ubiquitination events mediated by E3 ubiquitin ligases at the plasma membrane are largely unknown. Here, it is demonstrated that the suppressor of premature senescence and cell death SENESCENCE-ASSOCIATED UBIQUITIN LIGASE 1 (SAUL1), a plant U-box armadillo repeat (PUB-ARM) E3 ubiquitin ligase, localizes at the plasma membrane. Among the members of the PUB-ARM protein family, this localization is unique to SAUL1 and its two closest homologues. A novel armadillo repeat domain was identified at the SAUL1 C-terminus that directs specific association with the plasma membrane and is crucial for SAUL1 function in vivo. The data suggest that a small subgroup of PUB-ARM proteins including SAUL1 have functions at the plasma membrane probably by modifying target proteins by ubiquitination.

Survey of T-2 and HT-2 toxins by LC-MS/MS in oats and oat products from European oat mills in 2005-2009.

T-2 and HT-2 toxins were analysed in oats (n = 243), oat flakes (n = 529), oat meal (n = 105) and oat by-products (n = 209) from 11 European mills during 2005-2009 by high-performance liquid chromatography with a triple quadrupole mass spectrometer. Limits of quantification were 5 µg kg(-1) for both T-2 and HT-2 toxins in oats. The incidence of T-2 + HT-2 (>5 µg kg(-1)) in oats, oat flakes, oat meal and oat by-products was 93, 77, 34 and 99%, respectively. The mean values of T-2 + HT-2 were 94, 17, 11 and 293 µg kg(-1) for oats, oat flakes, oat meal and oat by-products, respectively. T-2 and HT-2 occurred together and the T-2 level was 52% of HT-2 in oats. Maximal T-2 and HT-2 concentration in oat flakes and oat meal were 197 and 118 µg kg(-1). The toxins were reduced by 82-88% during processing, but increased 3.1 times in oat by-products.

Arabidopsis zinc-finger protein 2 is a negative regulator of ABA signaling during seed germination.

The hormone abscisic acid (ABA) mediates plant development and adaptation to environmental stresses. ABA-dependent transcription factors are central regulators of ABA signaling. Here, we report on the identification of the ABA-induced transcriptional repressor Arabidopsis zinc-finger protein 2 (AZF2) as ABA signaling component. We isolated azf2-1 mutants lacking AZF2 full-length transcripts that were hypersensitive to ABA during seed germination. In line with a function of AZF2 in seed germination and seedling development, AZF2-promoter activity was observed in radicles and young cotyledons of AZF2-promoter:GUS plants. Our results indicate that AZF2 is a negative regulator of ABA signaling in seeds.

An ABA-responsive element in the AtSUC1 promoter is involved in the regulation of AtSUC1 expression.

Abscisic acid (ABA) and sugars regulate many aspects of plant growth and development, and we are only just beginning to understand the complex interactions between ABA and sugar signaling networks. Here, we show that ABA-dependent transcription factors bind to the promoter of the Arabidopsis thaliana AtSUC1 (At1g71880) sucrose transporter gene in vitro. We present the characterization of a cis-regulatory element by truncation of the AtSUC1 promoter and by electrophoretic mobility shift assays that is identical to a previously characterized ABA-responsive element (ABRE). In yeast 1-hybrid analyses we identified ABI5 (AtbZIP39; At2g36270) and AREB3 (AtbZIP66; At3g56850) as potential interactors. Analyses of plants expressing the beta-glucuronidase reporter gene under the control of ABI5 or AREB3 promoter sequences demonstrated that both transcription factor genes are co-expressed with AtSUC1 in pollen and seedlings, the primary sites of AtSUC1 action. Mutational analyses of the identified cis-regulatory element verified its importance for AtSUC1 expression in young seedlings. In abi5-4 seedlings, we observed an increase of sucrose-dependent anthocyanin accumulation and AtSUC1 mRNA levels. This suggests that ABI5 prevents an overshoot of sucrose-induced AtSUC1 expression and confirmed a novel cross-link between sugar and ABA signaling.

Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells.

The genome of Arabidopsis thaliana contains six genes, AtPMT1 to AtPMT6 (Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTER 1-6), which form a distinct subfamily within the large family of more than 50 monosaccharide transporter-like (MST-like) genes. So far, only AtPMT5 [formerly named AtPLT5 (At3g18830)] has been characterized and was shown to be a plasma membrane-localized H(+)-symporter with broad substrate specificity. The characterization of AtPMT1 (At2g16120) and AtPMT2 (At2g16130), two other, almost identical, members of this transporter subfamily, are presented here. Expression of the AtPMT1 and AtPMT2 cDNAs in baker's yeast (Saccharomyces cerevisiae) revealed that these proteins catalyse the energy-dependent, high-capacity transport of fructose and xylitol, and the transport of several other compounds with lower rates. Expression of their cRNAs in Xenopus laevis oocytes showed that both proteins are voltage-dependent and catalyse the symport of their substrates with protons. Fusions of AtPMT1 or AtPMT2 with the green fluorescent protein (GFP) localized to Arabidopsis plasma membranes. Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes). The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (alphaAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2. The physiological roles of the proteins are discussed and related to plant cell wall modifications.