The plant cell

Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists.

Publication Date: 2012 May 15 PMID: 22589468
Authors: Kandasamy, M. K. - McKinney, E. C. - Roy, E. - Meagher, R. B.
Journal: Plant Cell

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

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Nramp5 Is a Major Transporter Responsible for Manganese and Cadmium Uptake in Rice.

Publication Date: 2012 May 15 PMID: 22589467
Authors: Sasaki, A. - Yamaji, N. - Yokosho, K. - Ma, J. F.
Journal: Plant Cell

Paddy rice (Oryza sativa) is able to accumulate high concentrations of Mn without showing toxicity; however, the molecular mechanisms underlying Mn uptake are unknown. Here, we report that a member of the Nramp (for the Natural Resistance-Associated Macrophage Protein) family, Nramp5, is involved in Mn uptake and subsequently the accumulation of high concentrations of Mn in rice. Nramp5 was constitutively expressed in the roots and encodes a plasma membrane-localized protein. Nramp5 was polarly localized at the distal side of both exodermis and endodermis cells. Knockout of Nramp5 resulted in a significant reduction in growth and grain yield, especially when grown at low Mn concentrations. This growth reduction could be partially rescued by supplying high concentrations of Mn but not by the addition of Fe. Mineral analysis showed that the concentration of Mn and Cd in both the roots and shoots was lower in the knockout line than in wild-type rice. A short-term uptake experiment revealed that the knockout line lost the ability to take up Mn and Cd. Taken together, Nramp5 is a major transporter of Mn and Cd and is responsible for the transport of Mn and Cd from the external solution to root cells.

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The Recombinases DMC1 and RAD51 Are Functionally and Spatially Separated during Meiosis in Arabidopsis.

Publication Date: 2012 May 15 PMID: 22589466
Authors: Kurzbauer, M. T. - Uanschou, C. - Chen, D. - Schlogelhofer, P.
Journal: Plant Cell

Meiosis ensures the reduction of the genome before the formation of generative cells and promotes the exchange of genetic information between homologous chromosomes by recombination. Essential for these events are programmed DNA double strand breaks (DSBs) providing single-stranded DNA overhangs after their processing. These overhangs, together with the RADiation sensitive51 (RAD51) and DMC1 Disrupted Meiotic cDNA1 (DMC1) recombinases, mediate the search for homologous sequences. Current models propose that the two ends flanking a meiotic DSB have different fates during DNA repair, but the molecular details remained elusive. Here we present evidence, obtained in the model plant Arabidopsis thaliana, that the two recombinases, RAD51 and DMC1, localize to opposite sides of a meiotic DSB. We further demonstrate that the ATR kinase is involved in regulating DMC1 deposition at meiotic DSB sites, and that its elimination allows DMC1-mediated meiotic DSB repair even in the absence of RAD51. DMC1's ability to promote interhomolog DSB repair is not a property of the protein itself but the consequence of an ASYNAPTIC1 (Hop1)-mediated impediment for intersister repair. Taken together, these results demonstrate that DMC1 functions independently and spatially separated from RAD51 during meiosis and that ATR is an integral part of the regular meiotic program.

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Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with Phospholipase Ddelta to Transduce Hydrogen Peroxide Signals in the Arabidopsis Response to Stress.

Publication Date: 2012 May 15 PMID: 22589465
Authors: Guo, L. - Devaiah, S. P. - Narasimhan, R. - Pan, X. - Zhang, Y. - Zhang, W. - Wang, X.
Journal: Plant Cell

Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane-associated phospholipase D (PLDdelta) to transduce the ROS hydrogen peroxide (H(2)O(2)) signal in Arabidopsis thaliana. Genetic ablation of PLDdelta impeded stomatal response to abscisic acid (ABA) and H(2)O(2), placing PLDdelta downstream of H(2)O(2) in mediating ABA-induced stomatal closure. To determine the molecular link between H(2)O(2) and PLDdelta, GAPC1 and GAPC2 were identified to bind to PLDdelta, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H(2)O(2) promoted the GAPC-PLDdelta interaction and PLDdelta activity. Knockout of GAPCs decreased ABA- and H(2)O(2)-induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDdelta rendered plants less responsive to water deficits than the wild type. The results indicate that the H(2)O(2)-promoted interaction of GAPC and PLDdelta may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.

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Grass MicroRNA Gene Paleohistory Unveils New Insights into Gene Dosage Balance in Subgenome Partitioning after Whole-Genome Duplication.

Publication Date: 2012 May 15 PMID: 22589464
Authors: Abrouk, M. - Zhang, R. - Murat, F. - Li, A. - Pont, C. - Mao, L. - Salse, J.
Journal: Plant Cell

The recent availability of plant genome sequences, combined with a robust evolutionary scenario of the modern monocot and eudicot karyotypes from their diploid ancestors, offers an opportunity to gain insights into microRNA (miRNA) gene paleohistory in plants. Characterization and comparison of miRNAs and associated protein-coding targets in plants allowed us to unravel (1) contrasted genome conservation patterns of miRNAs in monocots and eudicots after whole-genome duplication (WGD), (2) an ancestral miRNA founder pool in the monocot genomes dating back to 100 million years ago, (3) miRNA subgenome dominance during the post-WGD diploidization process with selective miRNA deletion complemented with possible transposable element-mediated return flows, and (4) the miRNA/target interaction-directed differential loss/retention of miRNAs following the gene dosage balance rule. Together, our data suggest that overretained miRNAs in grass genomes may be implicated in connected gene regulations for stress responses, which is essential for plant adaptation and useful for crop variety innovation.

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A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development.

Publication Date: 2012 May 15 PMID: 22589463
Authors: Li, R. - Liu, P. - Wan, Y. - Chen, T. - Wang, Q. - Mettbach, U. - Baluska, F. - Samaj, J. - Fang, X. - Lucas, W. J. - Lin, J.
Journal: Plant Cell

Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was approximately 100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

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Phosphorylation of FAR-RED ELONGATED HYPOCOTYL1 Is a Key Mechanism Defining Signaling Dynamics of Phytochrome A under Red and Far-Red Light in Arabidopsis.

Publication Date: 2012 May 11 PMID: 22582101
Authors: Chen, F. - Shi, X. - Chen, L. - Dai, M. - Zhou, Z. - Shen, Y. - Li, J. - Li, G. - Wei, N. - Deng, X. W.
Journal: Plant Cell

Emerging plants have to adapt to a high ratio of far-red light (FR)/red light (R) light in the canopy before they reach the R-enriched direct sunlight. Phytochrome A (phyA) is the single dominant photoreceptor in young Arabidopsis thaliana seedlings that initiates photomorphogenesis in response to a FR-enriched environment and transduces increasing R signals to early responsive genes. To date, how phyA differentially transmits FR and R signals to downstream genes remains obscure. Here, we present a phyA pathway in which FAR-RED ELONGATED HYPOCOTYL1 (FHY1), an essential partner of phyA, directly guides phyA to target gene promoters and coactivates transcription. Furthermore, we identified two phosphorylation sites on FHY1, Ser-39 and Thr-61, whose phosphorylation by phyA under R inhibits phyA signaling at each step of its pathway. Deregulation of FHY1 phosphorylation renders seedlings colorblind to FR and R. Finally, we show that the weaker phyA response resulting from FHY1 phosphorylation ensures the seedling deetiolation process in response to a R-enriched light condition. Collectively, our results reveal FHY1 phosphorylation as a key mechanism for FR/R spectrum-specific responses in plants and an essential event for plant adaption to changing light conditions in nature.

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A Vacuolar beta-Glucosidase Homolog That Possesses Glucose-Conjugated Abscisic Acid Hydrolyzing Activity Plays an Important Role in Osmotic Stress Responses in Arabidopsis.

Publication Date: 2012 May 11 PMID: 22582100
Authors: Xu, Z. Y. - Lee, K. H. - Dong, T. - Jeong, J. C. - Jin, J. B. - Kanno, Y. - Kim, D. H. - Kim, S. Y. - Seo, M. - Bressan, R. A. - Yun, D. J. - Hwang, I.
Journal: Plant Cell

The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via beta-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different beta-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered beta-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific beta-glucosidases in response to abiotic stresses.

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Assembly of Synthetic Locked Phycocyanobilin Derivatives with Phytochrome in Vitro and in Vivo in Ceratodon purpureus and Arabidopsis.

Publication Date: 2012 May 11 PMID: 22582099
Authors: Yang, R. - Nishiyama, K. - Kamiya, A. - Ukaji, Y. - Inomata, K. - Lamparter, T.
Journal: Plant Cell

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.

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Mechanisms and Concepts Paving the Way towards a Complete Transport Cycle of Plant Vacuolar Sorting Receptors.

Publication Date: 2012 May 8 PMID: 22570446
Authors: Lousa, C. D. - Gershlick, D. C. - Denecke, J.
Journal: Plant Cell

Delivery of proteins to the lytic vacuole in plants is a complex cascade of selective interactions that specifically excludes residents of the endoplasmic reticulum and secreted proteins. Vacuolar transport must be highly efficient to avoid mistargeting of hydrolytic enzymes to locations where they could be harmful. While plant vacuolar sorting signals have been well described for two decades, it is only during the last 5 years that a critical mass of data was gathered that begins to reveal how vacuolar sorting receptors (VSRs) may complete a full transport cycle. Yet, the field is far from reaching a consensus regarding the organelles that could be involved in vacuolar sorting, their potential biogenesis, and the ultimate recycling of membranes and protein machinery that maintain this pathway. This review will highlight the important landmarks in our understanding of VSR function and compare recent transport models that have been proposed so that an emerging picture of plant vacuolar sorting mechanisms can be drawn.

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Inflorescence Meristem Identity in Rice Is Specified by Overlapping Functions of Three AP1/FUL-Like MADS Box Genes and PAP2, a SEPALLATA MADS Box Gene.

Publication Date: 2012 May 8 PMID: 22570445
Authors: Kobayashi, K. - Yasuno, N. - Sato, Y. - Yoda, M. - Yamazaki, R. - Kimizu, M. - Yoshida, H. - Nagamura, Y. - Kyozuka, J.
Journal: Plant Cell

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.

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Operon flv4-flv2 Provides Cyanobacterial Photosystem II with Flexibility of Electron Transfer.

Publication Date: 2012 May 8 PMID: 22570444
Authors: Zhang, P. - Eisenhut, M. - Brandt, A. M. - Carmel, D. - Silen, H. M. - Vass, I. - Allahverdiyeva, Y. - Salminen, T. A. - Aro, E. M.
Journal: Plant Cell

Synechocystis sp PCC 6803 has four genes encoding flavodiiron proteins (FDPs; Flv1 to Flv4). Here, we investigated the flv4-flv2 operon encoding the Flv4, Sll0218, and Flv2 proteins, which are strongly expressed under low inorganic carbon conditions (i.e., air level of CO(2)) but become repressed at elevated CO(2) conditions. Different from FDP homodimers in anaerobic microbes, Synechocystis Flv2 and Flv4 form a heterodimer. It is located in cytoplasm but also has a high affinity to membrane in the presence of cations. Sll0218, on the contrary, resides in the thylakoid membrane in association with a high molecular mass protein complex. Sll0218 operates partially independently of Flv2/Flv4. It stabilizes the photosystem II (PSII) dimers, and according to biophysical measurements opens up a novel electron transfer pathway to the Flv2/Flv4 heterodimer from PSII. Constructed homology models suggest efficient electron transfer in heterodimeric Flv2/Flv4. It is suggested that Flv2/Flv4 binds to thylakoids in light, mediates electron transfer from PSII, and concomitantly regulates the association of phycobilisomes with PSII. The function of the flv4-flv2 operon provides many beta-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving PSII.

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Arabidopsis Microtubule-Associated Protein MAP65-3 Cross-Links Antiparallel Microtubules toward Their Plus Ends in the Phragmoplast via Its Distinct C-Terminal Microtubule Binding Domain.

Publication Date: 2012 May 8 PMID: 22570443
Authors: Ho, C. M. - Lee, Y. R. - Kiyama, L. D. - Dinesh-Kumar, S. P. - Liu, B.
Journal: Plant Cell

Plant cytokinesis is brought about by the phragmoplast, which contains an antiparallel microtubule (MT) array. The MT-associated protein MAP65-3 acts as an MT-bundling factor that specifically cross-links antiparallel MTs near their plus ends. MAP65 family proteins contain an N-terminal dimerization domain and C-terminal MT interaction domain. Compared with other MAP65 isoforms, MAP65-3 contains an extended C terminus. A MT binding site was discovered in the region between amino acids 496 and 588 and found to be essential for the organization of phragmoplast MTs. The frequent cytokinetic failure caused by loss of MAP65-3 was not rescued by ectopic expression of MAP65-1 under the control of the MAP65-3 promoter, indicating nonoverlapping functions between the two isoforms. In the presence of MAP65-3, however, ectopic MAP65-1 appeared in the phragmoplast midline. We show that MAP65-1 could acquire the function of MAP65-3 when the C terminus of MAP65-3, which contains the MT binding site, was grafted to it. Our results also show that MAP65-1 and MAP65-3 may share redundant functions in MT stabilization. Such a stabilization effect was likely brought about by MT binding and bundling. We conclude that MAP65-3 contains a distinct C-terminal MT binding site with a specific role in cross-linking antiparallel MTs toward their plus ends in the phragmoplast.

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A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis.

Publication Date: 2012 May 8 PMID: 22570442
Authors: Dangoor, I. - Peled-Zehavi, H. - Wittenberg, G. - Danon, A.
Journal: Plant Cell

The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.

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The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus.

Publication Date: 2012 May 8 PMID: 22570441
Authors: Gao, C. - Yu, C. K. - Qu, S. - San, M. W. - Li, K. Y. - Lo, S. W. - Jiang, L.
Journal: Plant Cell

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.

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The ARP2/3 Complex Mediates Guard Cell Actin Reorganization and Stomatal Movement in Arabidopsis.

Publication Date: 2012 May 8 PMID: 22570440
Authors: Jiang, K. - Sorefan, K. - Deeks, M. J. - Bevan, M. W. - Hussey, P. J. - Hetherington, A. M.
Journal: Plant Cell

Guard cell actin reorganization has been observed in stomatal responses to a wide array of stimuli. However, how the guard cell signaling machinery regulates actin dynamics is poorly understood. Here, we report the identification of an allele of the Arabidopsis thaliana ACTIN-RELATED PROTEIN C2/DISTORTED TRICHOMES2 (ARPC2) locus (encoding the ARPC2 subunit of the ARP2/3 complex) designated high sugar response3 (hsr3). The hsr3 mutant showed increased transpirational water loss that was mainly due to a lesion in stomatal regulation. Stomatal bioassay analyses revealed that guard cell sensitivity to external stimuli, such as abscisic acid (ABA), CaCl(2), and light/dark transition, was reduced or abolished in hsr3. Analysis of a nonallelic mutant of the ARP2/3 complex suggested no pleiotropic effect of ARPC2 beyond its function in the complex in regard to stomatal regulation. When treated with ABA, guard cell actin filaments underwent fast disruption in wild-type plants, whereas those in hsr3 remained largely bundled. The ABA insensitivity phenotype of hsr3 was rescued by cytochalasin D treatment, suggesting that the aberrant stomatal response was a consequence of bundled actin filaments. Our work indicates that regulation of actin reassembly through ARP2/3 complex activity is crucial for stomatal regulation.

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Identification of a Novel Maize Protein Important for Paramutation at the purple plant1 Locus.

Publication Date: 2012 May 4 PMID: 22562612
Authors: Mach, J.
Journal: Plant Cell

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Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism.

Publication Date: 2012 May 4 PMID: 22562611
Authors: Nechushtai, R. - Conlan, A. R. - Harir, Y. - Song, L. - Yogev, O. - Eisenberg-Domovich, Y. - Livnah, O. - Michaeli, D. - Rosen, R. - Ma, V. - Luo, Y. - Zuris, J. A. - Paddock, M. L. - Cabantchik, Z. I. - Jennings, P. A. - Mittler, R.
Journal: Plant Cell

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

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required to maintain repression2 Is a Novel Protein That Facilitates Locus-Specific Paramutation in Maize.

Publication Date: 2012 May 4 PMID: 22562610
Authors: Barbour, J. E. - Liao, I. T. - Stonaker, J. L. - Lim, J. P. - Lee, C. C. - Parkinson, S. E. - Kermicle, J. - Simon, S. A. - Meyers, B. C. - Williams-Carrier, R. - Barkan, A. - Hollick, J. B.
Journal: Plant Cell

Meiotically heritable epigenetic changes in gene regulation known as paramutations are facilitated by poorly understood trans-homolog interactions. Mutations affecting paramutations in maize (Zea mays) identify components required for the accumulation of 24-nucleotide RNAs. Some of these components have Arabidopsis thaliana orthologs that are part of an RNA-directed DNA methylation (RdDM) pathway. It remains unclear if small RNAs actually mediate paramutations and whether the maize-specific molecules identified to date define a mechanism distinct from RdDM. Here, we identify a novel protein required for paramutation at the maize purple plant1 locus. This required to maintain repression2 (RMR2) protein represents the founding member of a plant-specific clade of predicted proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24-nucleotide RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants.

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The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis.

Publication Date: 2012 Apr 30 PMID: 22547783
Authors: Knoll, A. - Higgins, J. D. - Seeliger, K. - Reha, S. J. - Dangel, N. J. - Bauknecht, M. - Schropfer, S. - Franklin, F. C. - Puchta, H.
Journal: Plant Cell

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

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Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase-Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis.

Publication Date: 2012 May 4 PMID: 22547782
Authors: Cobessi, D. - Dumas, R. - Pautre, V. - Meinguet, C. - Ferrer, J. L. - Alban, C.
Journal: Plant Cell

Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.

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CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II.

Publication Date: 2012 Apr 30 PMID: 22547781
Authors: Hajheidari, M. - Farrona, S. - Huettel, B. - Koncz, Z. - Koncz, C.
Journal: Plant Cell

Phosphorylation of conserved Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats in the C-terminal domain of largest subunit of RNA polymerase II (RNAPII CTD) plays a central role in the regulation of transcription and cotranscriptional RNA processing. Here, we show that Ser phosphorylation of Arabidopsis thaliana RNAPII CTD is governed by CYCLIN-DEPENDENT KINASE F;1 (CDKF;1), a unique plant-specific CTD S(7)-kinase. CDKF;1 is required for in vivo activation of functionally redundant CYCLIN-DEPENDENT KINASE Ds (CDKDs), which are major CTD S(5)-kinases that also phosphorylate in vitro the S(2) and S(7) CTD residues. Inactivation of CDKF;1 causes extreme dwarfism and sterility. Inhibition of CTD S(7)-phosphorylation in germinating cdkf;1 seedlings is accompanied by 3'-polyadenylation defects of pre-microRNAs and transcripts encoding key regulators of small RNA biogenesis pathways. The cdkf;1 mutation also decreases the levels of both precursor and mature small RNAs without causing global downregulation of the protein-coding transcriptome and enhances the removal of introns that carry pre-microRNA stem-loops. A triple cdkd knockout mutant is not viable, but a combination of null and weak cdkd;3 alleles in a triple cdkd123* mutant permits semidwarf growth. Germinating cdkd123* seedlings show reduced CTD S(5)-phosphorylation, accumulation of uncapped precursor microRNAs, and a parallel decrease in mature microRNA. During later development of cdkd123* seedlings, however, S(7)-phosphorylation and unprocessed small RNA levels decline similarly as in the cdkf;1 mutant. Taken together, cotranscriptional processing and stability of a set of small RNAs and transcripts involved in their biogenesis are sensitive to changes in the phosphorylation of RNAPII CTD by CDKF;1 and CDKDs.

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The Gatsby Plant Science Summer School: Inspiring the Next Generation of Plant Science Researchers.

Publication Date: 2012 May 8 PMID: 22534129
Authors: Levesley, A. - Jopson, J. - Knight, C.
Journal: Plant Cell

We provide evidence from a 5-year study to show that a single concerted effort at the start of undergraduate study can have a clear and lasting effect on the attitudes of students toward plant science. Attendance at a week-long residential plant science summer school in the first year of an undergraduate degree resulted in many students changing courses to include more plant science and increased numbers of graduates selecting plant-based PhDs. The evidence shows that the Gatsby Plant Science Summer School has increased the pool of high-quality plant science related PhD applicants in the UK and has had a positive impact on students' career aspirations. The results are discussed within the context of enhancing the pipeline of future plant scientists and reversing the decline of this vulnerable and strategically important subject relevant to addressing food security and other major global challenges. We have shown that a single well-designed and timely intervention can influence future student behavior and as such offers a framework of potential use to other vulnerable disciplines.

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Lotus japonicus E3 Ligase SEVEN IN ABSENTIA4 Destabilizes the Symbiosis Receptor-Like Kinase SYMRK and Negatively Regulates Rhizobial Infection.

Publication Date: 2012 Apr 24 PMID: 22534128
Authors: Den Herder, G. - Yoshida, S. - Antolin-Llovera, M. - Ried, M. K. - Parniske, M.
Journal: Plant Cell

The Lotus japonicus SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. Here, we identified members of the SEVEN IN ABSENTIA (SINA) E3 ubiquitin-ligase family as SYMRK interactors and confirmed their predicted ubiquitin-ligase activity. In Nicotiana benthamiana leaves, SYMRK-yellow fluorescent protein was localized at the plasma membrane, and interaction with SINAs, as determined by bimolecular fluorescence complementation, was observed in small punctae at the cytosolic interface of the plasma membrane. Moreover, fluorescence-tagged SINA4 partially colocalized with SYMRK and caused SYMRK relocalization as well as disappearance of SYMRK from the plasma membrane. Neither the localization nor the abundance of Nod-factor receptor1 was altered by the presence of SINA4. SINA4 was transcriptionally upregulated during root symbiosis, and rhizobia inoculated roots ectopically expressing SINA4 showed reduced SYMRK protein levels. In accordance with a negative regulatory role in symbiosis, infection thread development was impaired upon ectopic expression of SINA4. Our results implicate SINA4 E3 ubiquitin ligase in the turnover of SYMRK and provide a conceptual mechanism for its symbiosis-appropriate spatio-temporal containment.

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NAP1 Family Histone Chaperones Are Required for Somatic Homologous Recombination in Arabidopsis.

Publication Date: 2012 Apr 24 PMID: 22534127
Authors: Gao, J. - Zhu, Y. - Zhou, W. - Molinier, J. - Dong, A. - Shen, W. H.
Journal: Plant Cell

Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR.

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Alternative Oxidases (AOX1a and AOX2) Can Functionally Substitute for Plastid Terminal Oxidase in Arabidopsis Chloroplasts.

Publication Date: 2012 Apr 24 PMID: 22534126
Authors: Fu, A. - Liu, H. - Yu, F. - Kambakam, S. - Luan, S. - Rodermel, S.
Journal: Plant Cell

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.

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Arabidopsis Annexin1 Mediates the Radical-Activated Plasma Membrane Ca2+- and K+-Permeable Conductance in Root Cells.

Publication Date: 2012 Apr 20 PMID: 22523205
Authors: Laohavisit, A. - Shang, Z. - Rubio, L. - Cuin, T. A. - Very, A. A. - Wang, A. - Mortimer, J. C. - Macpherson, N. - Coxon, K. M. - Battey, N. H. - Brownlee, C. - Park, O. K. - Sentenac, H. - Shabala, S. - Webb, A. A. - Davies, J. M.
Journal: Plant Cell

Plant cell growth and stress signaling require Ca(2+) influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH(*). In root cells, extracellular OH(*) activates a plasma membrane Ca(2+)-permeable conductance that permits Ca(2+) influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca(2+)-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH(*)-activated Ca(2+)- and K(+)-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca(2+) in response to OH(*). An OH(*)-activated Ca(2+) conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca(2+)-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca(2+) in plants.

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Disruption of OPR7 and OPR8 Reveals the Versatile Functions of Jasmonic Acid in Maize Development and Defense.

Publication Date: 2012 Apr 20 PMID: 22523204
Authors: Yan, Y. - Christensen, S. - Isakeit, T. - Engelberth, J. - Meeley, R. - Hayward, A. - Emery, R. J. - Kolomiets, M. V.
Journal: Plant Cell

Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects.

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Emergent Decarboxylase Activity and Attenuation of alpha/beta-Hydrolase Activity during the Evolution of Methylketone Biosynthesis in Tomato.

Publication Date: 2012 Apr 20 PMID: 22523203
Authors: Auldridge, M. E. - Guo, Y. - Austin, M. B. - Ramsey, J. - Fridman, E. - Pichersky, E. - Noel, J. P.
Journal: Plant Cell

Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the alpha/beta-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional alpha/beta-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids.

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Secretory Pathway Research: The More Experimental Systems the Better.

Publication Date: 2012 Apr 20 PMID: 22523202
Authors: Denecke, J. - Aniento, F. - Frigerio, L. - Hawes, C. - Hwang, I. - Mathur, J. - Neuhaus, J. M. - Robinson, D. G.
Journal: Plant Cell

Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.

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