Plant and Cell Physiology

Identification of a Regulatory Subunit of Protein Phosphatase 1 Which Mediates Blue Light Signaling for Stomatal Opening.

Publication Date: 2012 May 13 PMID: 22585556
Authors: Takemiya, A. - Yamauchi, S. - Yano, T. - Ariyoshi, C. - Shimazaki, K. I.
Journal: Plant Cell Physiol

Protein phosphatase 1 (PP1) is a eukaryotic Ser/Thr protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization, and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping, and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata.

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Engineering Calcium Oxalate Crystal Formation in Arabidopsis.

Publication Date: 2012 May 10 PMID: 22576773
Authors: Nakata, P. A.
Journal: Plant Cell Physiol

Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal accumulating plant, Arabidopsis. The success of this approach hinged on the ability to genetically transform Arabidopsis into a calcium oxalate crystal accumulating plant. To accomplish this transformation, two oxalic acid biosynthetic genes, obcA and obcB, from the oxalate-secreting phytopathogen, Burkholderia glumae, were inserted into the Arabidopsis genome. The co-expression of these two bacterial genes in Arabidopsis not only conferred the ability to produce measurable amount of oxalate but also to form crystals of calcium oxalate. Biochemical and cellular studies of crystal accumulation in Arabidopsis revealed features that are similar to those observed in the cells of crystal-forming plants. Thus, it appears that at least some of the basic components that comprise the calcium oxalate crystal formation machinery are conserved even in non-crystal accumulating plants.

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Mechanistic Insight into Pentatricopeptide Repeat Proteins as Sequence-Specific RNA-Binding Proteins for Organellar RNAs in Plants.

Publication Date: 2012 May 9 PMID: 22576772
Authors: Nakamura, T. - Yagi, Y. - Kobayashi, K.
Journal: Plant Cell Physiol

The pentatricopeptide repeat (PPR) protein family is highly expanded in terrestrial plants. Arabidopsis contains 450 PPR genes, which represents 2% of the total protein-coding genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of organellar genes. Most PPR proteins affect a single or small subset of gene(s), acting in a gene-specific manner. Studies over the last 10 years have revealed the significance of this protein family in coordinated gene expression in different compartments: the nucleus, chloroplast, and mitochondrion. Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.

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Opening the Door to Epigenetics in PCP.

Publication Date: 2012 May PMID: 22566217
Authors: Kinoshita, T. - Jacobsen, S. E.
Journal: Plant Cell Physiol

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Reduced Calcification Decreases Photoprotective Capability in the Coccolithophorid Emiliania huxleyi.

Publication Date: 2012 May 3 PMID: 22555817
Authors: Xu, K. - Gao, K.
Journal: Plant Cell Physiol

Intracellular calcification of coccolithophores generate CO(2) and consumes additional energy for acquisition of calcium and bicarbonate ions, therefore, it may correlate with photoprotective processes by influencing the energetics. To address this hypothesis, a calcifying Emiliania huxleyi strain (CS-369) was grown semi-continuously at reduced (0.1 mM, LCa) and ambient Ca(2+) concentrations (10 mM, HCa) for 150 days (>200 generations). The HCa-grown cells had higher photosynthetic and calcification rates and higher contents of chl a and carotenoids compared to the naked (bearing no coccoliths) LCa-grown cells. When exposed to stress-full levels of PAR, LCa-grown cells displayed lower photochemical yield and less efficient non-photochemical quenching (NPQ). When the LCa or HCa-grown cells were inversely shifted to their counterpart medium, LCa to HCa transfer increased photosynthetic carbon fixation (P), calcification rate (C), C/P ratio, NPQ and pigments contents, while those shifted from HCa to LCa exhibited the opposite performance. Increased NPQ, carotenoids and quantum yield clearly linked with increased or sustained calcification in E. huxleyi. The calcification must have played a role in dissipating excessive energy or as an additional drainage of electrons absorbed by the photosynthetic antennae. This phenomenon was further supported by testing two noncalcifying strains, which showed insignificant changes photosynthetic carbon fixation and NPQ when transferred to LCa condition.

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Root-to-shoot translocation of alkaloids is dominantly suppressed in Nicotiana alata.

Publication Date: 2012 May 2 PMID: 22555816
Authors: Pakdeechanuan, P. - Shoji, T. - Hashimoto, T.
Journal: Plant Cell Physiol

In tobacco (Nicotiana tabacum), nicotine and related pyridine alkaloids are produced in the root, and then transported to the aerial parts where these toxic chemicals function as part of chemical defense against insect herbivory. Although a few tobacco transporters have been recently reported to take up nicotine into the vacuole from the cytoplasm or into the cytoplasm from the apoplast, it is not known how the long-range translocation of tobacco alkaloids between organs is controlled. Nicotiana langsdorffii and N. alata are closely related species of diploid Nicotiana section Alatae, but the latter does not accumulate tobacco alkaloids in the leaf. We show here that N. alata does synthesize alkaloids in the root, but lacks the capacity to mobilize the root-borne alkaloids to the aerial parts. Interspecific grafting experiments between N. alata and N. langsdorffii indicate that roots of N. alata are unable to translocate alkaloids to their shoot system. Interestingly, genetic studies involving interspecific hybrids between N. alata and N. langsdorffii and their self-crossed or back-crossed progenies showed that the non-translocation phenotype is dominant over the translocation phenotype. These results indicate that a mechanism to retain tobacco alkaloids within the root organ has evolved in N. alata, which may represent an interesting strategy to control distribution of secondary products within a whole plant.

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Control of plant cell cycle by developmental and environmental cues.

Publication Date: 2012 May 2 PMID: 22555815
Authors: Komaki, S. - Sugimoto, K.
Journal: Plant Cell Physiol

Plant morphogenesis relies on cell proliferation and differentiation strictly controlled in space and time. As in other eukaryotes, progression through the plant cell cycle is governed by cyclin-dependent kinases (CDKs) that associate with its activator proteins called cyclins (CYCs) and the activity of CYC-CDK is modulated at both transcriptional and post-translational levels. Compared to animals and yeasts, plants generally possess many more genes encoding core cell cycle regulators and it has been puzzling how their functions are specified or overlapped in development or in response to various environmental changes. Thanks to the recent advances in high-throughput, genome-wide transcriptome and proteomic technologies, we are finally beginning to see how core regulators are assembled during the cell cycle and how their activities are modified by developmental and environmental cues. In this review we will summarise the latest progress in the plant cell cycle research and overview some of the emerging molecular interfaces that link upstream signalling cascades and the cell cycle regulation.

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Characterization of the Subdomains in the Amino-terminal Region of Histidine Kinase Hik33 in the Cyanobacterium Synechocystis sp. PCC 6803.

Publication Date: 2012 May 2 PMID: 22555814
Authors: Shimura, Y. - Shiraiwa, Y. - Suzuki, I.
Journal: Plant Cell Physiol

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33 while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.

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Evaluation of Unbound Free Heme in Plant Cells by Differential Acetone Extraction.

Publication Date: 2012 May 2 PMID: 22555813
Authors: Espinas, N. A. - Kobayashi, K. - Takahashi, S. - Mochizuki, N. - Masuda, T.
Journal: Plant Cell Physiol

Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of "free" heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Meanwhile, it was proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely-bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using highly sensitive heme assay, we found that not only basic acetone but also neutral acetone can extract low levels of heme from plant samples. Moreover, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. The level of neutral acetone-extractable heme remained unchanged by precursor (5-minolevulinic acid) feeding but increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to nucleus. Present data demonstrate that the combination of single step acetone extraction following sensitive heme assay is the ideal method for determining total and free heme in plants.

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Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan.

Publication Date: 2012 Apr 25 PMID: 22537759
Authors: Lee, C. - Teng, Q. - Zhong, R. - Ye, Z. H.
Journal: Plant Cell Physiol

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of beta-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that mutations of two Arabidopsis family GT8 genes, GUX1 and GUX2, affect the addition of GlcA and MeGlcA to xylan, but it is not known whether they encode glucuronyltransferases (GlcATs) or indirectly regulate the GlcAT activity. In this report, we performed biochemical and genetic analyses of three Arabidopsis GUX genes for their roles in the GlcA substitution of xylan and secondary wall deposition. The GUX1/2/3 genes were found to be expressed in interfascicular fibers and xylem cells, the two major types of secondary wall-containing cells that have abundant xylan. When expressed in tobacco BY2 cells, the GUX1/2/3 proteins exhibited an activity capable of transferring GlcA residues from the UDP-GlcA donor onto xylooligomer acceptors, demonstrating that these GUX proteins possess xylan GlcAT activity. Analyses of the single, double and triple gux mutants revealed that simultaneous mutations of all three GUX genes led to a complete loss of GlcA and MeGlcA side chains on xylan, indicating that all three GUX proteins are involved in the GlcA substitution of xylan. Furthermore, a complete loss of GlcA and MeGlcA side chains in the gux1/2/3 triple mutant resulted in reduced secondary wall thickening, collapsed vessel morphology, and reduced plant growth. Together, our results provide biochemical and genetic evidence that GUX1/2/3 are GlcATs responsible for the GlcA substitution of xylan, which is essential for normal secondary wall deposition and plant development.

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Molecular and Functional Characterization of Broccoli EMBRYONIC FLOWER 2 Genes.

Publication Date: 2012 Apr 25 PMID: 22537758
Authors: Liu, M. S. - Chen, L. F. - Lin, C. H. - Lai, Y. M. - Huang, J. Y. - Sung, Z. R.
Journal: Plant Cell Physiol

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and similar gene expression pattern as that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower-organ-identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type (WT). Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.

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Characterization Of Mutations In Barley fch2 Encoding Chlorophyllide a Oxygenase.

Publication Date: 2012 Apr 25 PMID: 22537757
Authors: Mueller, A. H. - Dockter, C. - Gough, S. P. - Lundqvist, U. - von Wettstein, D. - Hansson, M.
Journal: Plant Cell Physiol

The barley (Hordeum vulgare L.) mutants fch2 and clo-f2 comprise an allelic group of 14 chlorophyll b-deficient lines. The genetic map position of fch2 corresponds to the physical map position of the gene encoding chlorophyllide a oxygenase. This enzyme converts chlorophyllide a to chlorophyllide b and it is essential for chlorophyll b biosynthesis. The fch2 and clo-f2 barley lines were shown to be mutated in the gene for chlorophyllide a oxygenase. A five-base insertion was found in fch2 and basedeletions in clo-f2.101, clo-f2.105, clo-f2.2800, and clo-f2.3613. In clo-f2.105 and clof2.108 nonsense base exchanges were discovered. All of these mutations lead to a premature stop of translation and none of the mutants formed chlorophyll b. The mutant clo-f2.2807 was transcript deficient and formed no chlorophyll b. Missense mutations in clo-f2.102 (leading to the amino acid exchange D495N) and clo-f2.103 (G280D) resulted in a total lack of chlorophyll b, whereas in the missense mutants clo-f2.107 (P419L), clo-f2.109 (A94T), clo-f2.122 (C320Y), clo-f2.123 (A94T), clof2.133 (A376V), and clo-f2.181 (L373F) intermediate contents of chlorophyll b were determined. The missense mutations affect conserved residues and their effect on chlorophyllide a oxygenase is discussed. The mutations in clo-f2.102, clo-f2.103, clof2.133, and clo-f2.181 may influence electron transfer as illustrated in the active site of a structural model protein. The changes in clo-f2.107, clo-f2.109, clo-f2.122, and clo-f2.123 may lead to chlorophyll b deficiency by interfering with the regulation of chlorophyllide a oxygenase. The correlation of mutations and phenotypes strongly supports that the barley locus fch2 encodes chlorophyllide a oxygenase.

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Activation and Epigenetic Regulation of DNA Transposon nDart1 in Rice.

Publication Date: 2012 May PMID: 22514089
Authors: Eun, C. H. - Takagi, K. - Park, K. I. - Maekawa, M. - Iida, S. - Tsugane, K.
Journal: Plant Cell Physiol

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.

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Transition of Chromatin Status During the Process of Recovery from Drought Stress in Arabidopsis thaliana.

Publication Date: 2012 May PMID: 22505693
Authors: Kim, J. M. - To, T. K. - Ishida, J. - Matsui, A. - Kimura, H. - Seki, M.
Journal: Plant Cell Physiol

Changes in chromatin status are correlated with gene regulation of biological processes such as development and stress responses in plants. In this study, we focused on the transition of chromatin status toward gene repression during the process of recovery from drought stress of drought-inducible genes (RD20, RD29A and AtGOLS2) and a rehydration-inducible gene (ProDH). In response to drought, RNA polymerase II was recruited on the drought-inducible genes and rapidly disappeared after rehydration, although mRNA levels of these genes were maintained to some degree after rehydration, suggesting that the transcriptional activities of these genes were rapidly inactivated by rehydration treatment. Histone H3K9ac was enriched by drought and rapidly removed from these regions by rehydration. In contrast, histone H3K4me3 was gradually decreased by rehydration but was maintained at low levels after rehydration, suggesting that H3K4me3 functions as an epigenetic mark of stress memory. These results show that the transcriptional activity and chromatin status are rapidly changed from an active to inactive mode during the recovery process. Our results demonstrate that histone modifications are correlated with the inactivation of drought-inducible genes during the recovery process by rehydration.

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Epigenetic reprogramming in plant reproductive lineages.

Publication Date: 2012 May PMID: 22505692
Authors: Gutierrez-Marcos, J. F. - Dickinson, H. G.
Journal: Plant Cell Physiol

Monoecious flowering plants produce both microgametophytes (pollen) and megagametophytes (embryo sacs) containing the male and female gametes, respectively, which participate in double fertilization. Much is known about cellular and developmental processes giving rise to these reproductive structures and the formation of gametes. However, little is known about the role played by changes in the epigenome in dynamically shaping these defining events during plant sexual reproduction. This has in part been hampered by the inaccessibility of these structures-especially the female gametes, which are embedded within the female reproductive tissues of the plant sporophyte. However, with the recent development of new cellular isolation technologies that can be coupled to next-generation sequencing, a new wave of epigenomic studies indicate that an intricate epigenetic regulation takes place during the formation of male and female reproductive lineages. In this mini review, we assess the fast growing body of evidence for the epigenetic regulation of the developmental fate and function of plant gametes. We describe how small interfereing RNAs and DNA methylation machinery play a part in setting up unique epigenetic landscapes in different gametes, which may be responsible for their different fates and functions during fertilization. Collectively these studies will shed light on the dynamic epigenomic landscape of plant gametes or 'epigametes' and help to answer important unresolved questions on the sexual reproduction of flowering plants, especially those underpinning the formation of two products of fertilization, the embryo and the endosperm.

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Plant Imprinted Genes Identified by Genome-wide Approaches and Their Regulatory Mechanisms.

Publication Date: 2012 May PMID: 22492232
Authors: Ikeda, Y.
Journal: Plant Cell Physiol

Genomic imprinting is an epigenetic phenomenon found in mammals and flowering plants that leads to differential allelic gene expression depending on their parent of origin. In plants, genomic imprinting primarily occurs in the endosperm, and it is associated with seed development. The imprinted expression is driven by the epigenetic memory programmed in each lineage of female and male germlines. Several imprinted genes have been identified based on genetic studies in maize and Arabidopsis. Recent advances in genome-wide analyses made it possible to identify multiple imprinted genes including many nuclear proteins, such as transcription factors and chromatin-related proteins in different plant species. Some of these genes are conserved in Arabidopsis, rice and maize, but many are species specific. Genome-wide analyses also clarified the regulation mechanism of imprinted genes orchestrated by DNA methylation and histone methylation marks. Additionally, genetic analyses using Arabidopsis revealed new regulatory factors of DNA demethylation and imprinting and shed light on the more precise mechanisms.

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Stress-induced chromatin changes: a critical view on their heritability.

Publication Date: 2012 May PMID: 22457398
Authors: Pecinka, A. - Mittelsten Scheid, O.
Journal: Plant Cell Physiol

The investigation of stress responses has been a focus of plant research, breeding and biotechnology for a long time. Insight into stress perception, signaling and genetic determinants of resistance has recently been complemented by growing evidence for substantial stress-induced changes at the chromatin level. These affect specific sequences or occur genome-wide and are often correlated with transcriptional regulation. The majority of these changes only occur during stress exposure, and both expression and chromatin states typically revert to the pre-stress state shortly thereafter. Other changes result in the maintenance of new chromatin states and modified gene expression for a longer time after stress exposure, preparing an individual for developmental decisions or more effective defence. Beyond this, there are claims for stress-induced heritable chromatin modifications that are transmitted to progeny, thereby improving their characteristics. These effects resemble the concept of Lamarckian inheritance of acquired characters and represent a challenge to the uniqueness of DNA sequence-based inheritance. However, with the growing insight into epigenetic regulation and transmission of chromatin states, it is worth investigating these phenomena carefully. While genetic changes (mainly transposon mobility) in response to stress-induced interference with chromatin are well documented and heritable, in our view there is no unambiguous evidence for transmission of exclusively chromatin-controlled stress effects to progeny. We propose a set of criteria that should be applied to substantiate the data for stress-induced, chromatin-encoded new traits. Well-controlled stress treatments, thorough phenotyping and application of refined genome-wide epigenetic analysis tools should be helpful in moving from interesting observations towards robust evidence.

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Characterization of a Novel Y2K-type Dehydrin VrDhn1 from Vigna radiata.

Publication Date: 2012 May PMID: 22440330
Authors: Lin, C. H. - Peng, P. H. - Ko, C. Y. - Markhart, A. H. - Lin, T. Y.
Journal: Plant Cell Physiol

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to approximately 30% at 1 DAI, and was not detectable at approximately 7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

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High-throughput cryopreservation of plant cell cultures for functional genomics.

Publication Date: 2012 May PMID: 22437846
Authors: Ogawa, Y. - Sakurai, N. - Oikawa, A. - Kai, K. - Morishita, Y. - Mori, K. - Moriya, K. - Fujii, F. - Aoki, K. - Suzuki, H. - Ohta, D. - Saito, K. - Shibata, D.
Journal: Plant Cell Physiol

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 degrees C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

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An Arabidopsis T-DNA Insertion Mutant for Galactokinase (AtGALK, At3g06580) Hyperaccumulates Free Galactose and is Insensitive to Exogenous Galactose.

Publication Date: 2012 May PMID: 22437845
Authors: Egert, A. - Peters, S. - Guyot, C. - Stieger, B. - Keller, F.
Journal: Plant Cell Physiol

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of alpha-d-galactose (Gal) to alpha-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.

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Chloroplast Downsizing Under Nitrate Nutrition Restrained Mesophyll Conductance and Photosynthesis in Rice (Oryza sativa L.) Under Drought Conditions.

Publication Date: 2012 May PMID: 22433461
Authors: Li, Y. - Ren, B. - Yang, X. - Xu, G. - Shen, Q. - Guo, S.
Journal: Plant Cell Physiol

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. 'Shanyou 63' hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Psi(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Psi(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.

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Arginase induction represses gall development during clubroot infection in Arabidopsis.

Publication Date: 2012 May PMID: 22433460
Authors: Gravot, A. - Deleu, C. - Wagner, G. - Lariagon, C. - Lugan, R. - Todd, C. - Wendehenne, D. - Delourme, R. - Bouchereau, A. - Manzanares-Dauleux, M. J.
Journal: Plant Cell Physiol

Arginase induction can play a defensive role through the reduction of arginine availability for phytophageous insects. Arginase activity is also induced during gall growth caused by Plasmodiophora brassicae infection in roots of Arabidopsis thaliana; however, its possible role in this context has been unclear. We report here that the mutation of the arginase-encoding gene ARGAH2 abrogates clubroot-induced arginase activity and results in enhanced gall size in infected roots, suggesting that arginase plays a defensive role. Induction of arginase activity in infected roots was impaired in the jar1 mutant, highlighting a link between the arginase response to clubroot and jasmonate signaling. Clubroot-induced accumulation of the principal amino acids in galls was not affected by the argah2 mutation. Because ARGAH2 was previously reported to control auxin response, we investigated the role of ARGAH2 in callus induction. ARGAH2 was found to be highly induced in auxin/cytokinin-triggered aseptic plant calli, and callus development was enhanced in argah2 in the absence of the pathogen. We hypothesized that arginase contributes to a negative control over clubroot symptoms, by reducing hormone-triggered cellular proliferation.

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Local Root Apex Hypoxia Induces NO-Mediated Hypoxic Acclimation of the Entire Root.

Publication Date: 2012 May PMID: 22422934
Authors: Mugnai, S. - Azzarello, E. - Baluska, F. - Mancuso, S.
Journal: Plant Cell Physiol

Roots are very sensitive to hypoxia and adapt effectively to a reduced availability of oxygen in the soil. However, the site of the root where oxygen availability is sensed and how roots acclimate to hypoxia remain unclear. In this study, we found that the root apex transition zone plays central roles in both sensing and adapting to root hypoxia. The exposure of cells of the root apex to hypoxia is sufficient to achieve hypoxic acclimation of the entire root; particularly relevant in this respect is that, of the entire root apex, the transition zone cells show the highest demand for oxygen and also emit the largest amount of nitric oxide (NO). Local root apex-specific oxygen deprivation dramatically inhibits the oxygen influx peak in the transition zone and simultaneously stimulates a local increase in NO emission. The hypoxia-induced efflux of NO is strictly associated with the transition zone and is essential for hypoxic acclimation of the entire root.

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Molecular Cloning, Characterization and Analysis of the Intracellular Localization of a Water-Soluble Chl-Binding Protein from Brussels Sprouts (Brassica oleracea var. gemmifera).

Publication Date: 2012 May PMID: 22419824
Authors: Takahashi, S. - Yanai, H. - Nakamaru, Y. - Uchida, A. - Nakayama, K. - Satoh, H.
Journal: Plant Cell Physiol

A water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera), hereafter termed BoWSCP, is categorized into the Class II WSCPs (non-photoconvertible WSCPs). Previous studies on BoWSCP have focused mainly on its biochemical characterization. In this study, we cloned the cDNA encoding BoWSCP. Sequence analysis revealed that the BoWSCP gene was composed of a single exon corresponding to 654 bp of an open reading frame encoding 218 amino acid residues, including 19 residues of a deduced signal peptide targeted to the endoplasmic reticulum (ER). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of native BoWSCP revealed that the molecular mass of the subunit was 19,008.523 Da, corresponding to a mature protein of 178 amino acids, indicating the removal of 21 residues in the C-terminal region. Functional BoWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (BoWSCP-His). When BoWSCP-His was mixed with thylakoid membranes in aqueous solution, BoWSCP-His was able to remove Chls from the thylakoid membranes. The absorption spectrum of the reconstituted BoWSCP-His was identical to that of the native BoWSCP. Chl binding analyses of BoWSCP-His revealed that the BoWSCP-His bound both Chl a and Chl b with almost the same affinity in 40% methanol solution, although the native BoWSCP had a higher content of Chl a. To reveal the intracellular localization of BoWSCP, we constructed a transgenic plant expressing the fluorescent protein fused with the N-terminal deduced signal peptide of BoWSCP. The fluorescence emitted from the chimeric protein was detected in the ER body, an ER-derived compartment observed only in Brassicaceae plants.

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Functional interaction between plastidial starch phosphorylase and starch branching enzymes from rice during the synthesis of branched maltodextrins.

Publication Date: 2012 May PMID: 22414443
Authors: Nakamura, Y. - Ono, M. - Utsumi, C. - Steup, M.
Journal: Plant Cell Physiol

The present study established the way in which plastidial alpha-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules.

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ARABIDOPSIS TRITHORAX-RELATED3/SET DOMAIN GROUP2 is Required for the Winter-Annual Habit of Arabidopsis thaliana.

Publication Date: 2012 May PMID: 22378382
Authors: Yun, J. Y. - Tamada, Y. - Kang, Y. E. - Amasino, R. M.
Journal: Plant Cell Physiol

The winter-annual habit of Arabidopsis thaliana requires active alleles of FLOWERING LOCUS C (FLC), which encodes a potent flowering repressor, and FRIGIDA (FRI), an activator of FLC. FLC activation by FRI is accompanied by an increase in specific histone modifications, such as tri-methylation of histone H3 at lysine 4 (H3K4me3), and requires three H3K4 methyltransferases, the Drosophila Trithorax-class ARABIDOPSIS TRITHORAX1 (ATX1) and ATX2, and yeast Set1-class ATX-RELATED7/SET DOMAIN GROUP25 (ATXR7/SDG25). However, lesions in all of these genes failed to suppress the enhanced FLC expression caused by FRI completely, suggesting that another H3K4 methyltransferase may participate in the FLC activation. Here, we show that ATXR3/SDG2, which is a member of a novel class of H3K4 methyltransferases, also contributes to FLC activation. An ATXR3 lesion suppressed the enhanced FLC expression and delayed flowering caused by an active allele of FRI in non-vernalized plants. The decrease in FLC expression in atxr3 mutants was accompanied by reduced H3K4me3 levels at FLC chromatin. We also found that the rapid flowering of atxr3 was epistatic to that of atxr7, suggesting that ATXR3 functions in FLC activation in sequence with ATXR7. Our results indicate that the novel-class H3K4 methyltransferase, ATXR3, is a transcriptional activator that plays a role in the FLC activation and establishing the winter-annual habit. In addition, ATXR3 also contributes to the activation of other FLC clade members, such as FLOWERING LOCUS M/MADS AFFECTING FLOWERING1 (FLM/MAF1) and MAF5, at least partially explaining the ATXR3 function in delayed flowering caused by non-inductive photoperiods.

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DNA Methylation in Plants: Relationship to Small RNAs and Histone Modifications, and Functions in Transposon Inactivation.

Publication Date: 2012 May PMID: 22302712
Authors: Saze, H. - Tsugane, K. - Kanno, T. - Nishimura, T.
Journal: Plant Cell Physiol

DNA methylation is a type of epigenetic marking that strongly influences chromatin structure and gene expression in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate its control mechanisms. These studies have shown that small RNAs are involved in the induction of DNA methylation, that there is a relationship between DNA methylation and histone methylation, and that the base excision repair pathway has an important role in DNA demethylation. Some aspects of DNA methylation have also been shown to be shared with mammals, suggesting that the regulatory pathways are, in part at least, evolutionarily conserved. Considerable progress has been made in elucidating the mechanisms that control DNA methylation; however, many aspects of the mechanisms that read the information encoded by DNA methylation and mediate this into downstream regulation remain uncertain, although some candidate proteins have been identified. DNA methylation has a vital role in the inactivation of transposons, suggesting that DNA methylation is a key factor in the evolution and adaptation of plants.

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An Epigenetic Integrator: New Insights into Genome Regulation, Environmental Stress Responses and Developmental Controls by HISTONE DEACETYLASE 6.

Publication Date: 2012 May PMID: 22253092
Authors: Kim, J. M. - To, T. K. - Seki, M.
Journal: Plant Cell Physiol

Histone acetylation ranks with DNA methylation as one of major epigenetic modifications in eukaryotes. Deacetylation of histone N-terminal tails is intimately correlated with gene silencing and heterochromatin formation. In Arabidopsis, histone deacetylase 6 (HDA6) is a well-studied histone deacetylase that functions in gene silencing. Recently, it has been reported that HDA6 cooperates with DNA methylation on its direct target locus in the gene silencing mechanism. HDA6 has the multifaceted role in regulation of genome maintenance, development and environmental stress responses in plants. Elucidation of HDA6 function provides important information for understanding of epigenetic regulation in plants. In this review, we highlight recent progress in elucidating the HDA6-mediated gene silencing mechanisms and deciphering the biological function of HDA6.

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The effects of heat induction and the siRNA biogenesis pathway on the transgenerational transposition of ONSEN, a copia-like retrotransposon in Arabidopsis thaliana.

Publication Date: 2012 May PMID: 22173101
Authors: Matsunaga, W. - Kobayashi, A. - Kato, A. - Ito, H.
Journal: Plant Cell Physiol

Environmental stress influences genetic and epigenetic regulation in plant genomes. We previously reported that heat stress activated a copia-like retrotransposon named ONSEN. To investigate the heat sensitivity and transgenerational activation of ONSEN, we analyzed the stress response by temperature shift and multiple heat stress treatments. ONSEN was activated at 37 degrees C, and the newly inserted ONSEN was transcriptionally active and mobile to the next generation subjected to heat stress, indicating that the regulation of ONSEN is independent of positional effects on the chromosome. Reciprocal crosses with activated ONSEN revealed that the transgenerational transposition was inherited from both sexes, indicating that the transposition is suppressed independently of gametophytic regulation. We showed previously that ONSEN was transposed in mutants deficient in small interfering RNA (siRNA) biogenesis, including nrpd2 and rdr2, but not dcl3. To define the functional redundancy of Dicer-like (DCL) proteins in Arabidopsis, we analyzed ONSEN activation in mutants deficient in DCL proteins, including dcl2, dcl3 and dcl4. ONSEN was nearly immobile in a single Dicer mutant; however, some transgenerational transpositions were observed in dcl2/dcl3/dcl4 triple mutants subjected to heat stress. This indicated that the Dicer family is redundant for ONSEN transposition. To examine the activation of ONSEN in undifferentiated cells, ONSEN transcripts and synthesized DNA were analyzed in heat-stressed callus tissue. In contrast to vegetative tissue, high accumulation of the transcripts and amplified DNA copies of ONSEN were detected in callus. This result indicated that ONSEN activation is controlled by cell-specific regulatory mechanisms.

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FLC: A Hidden Polycomb Response Element Shows Up in Silence.

Publication Date: 2012 May PMID: 22107881
Authors: Buzas, D. M. - Tamada, Y. - Kurata, T.
Journal: Plant Cell Physiol

A sizeable fraction of eukaryotic genomes is regulated by Polycomb group (PcG) and trithorax group (trxG) proteins, which play key roles in epigenetic repression and activation, respectively. In Drosophila melanogaster, homeotic genes are well-documented PcG targets; they are known to contain cis-acting elements termed Polycomb response elements (PREs), which bind PcG proteins and satisfy three defined criteria, and also often contain binding sites for the trithorax (trx) protein. However, the presence of PREs, or an alternative mode for PcG/trxG interaction with the genome, has not been well documented outside Drosophila. In Arabidopsis thaliana, PcG/trxG regulation has been studied extensively for the flowering repressor gene FLOWERING LOCUS C (FLC). Here we evaluate how PRE-like activities that reside within the FLC locus may satisfy the defined Drosophila criteria, by analyzing four FLC transcription states. When the FLC locus is not transcribed, the intrinsic PcG recruitment ability of the coding region can be attributed to two redundant cis-acting elements (Modules IIA and IIB). When FLC is highly expressed, trxG recruitment is to a region overlapping the transcription start site (Module I). Exposure to prolonged cold converts the active FLC state into a repressed state that is maintained after the cold period finishes. These two additional transcriptional states also rely on the same three modules for PcG/trxG regulation. We conclude that each of Modules I, IIA and IIB partially fulfills the PRE function criteria, and that together they represent the functional FLC PRE, which differs structurally from canonical PREs in Drosophila.

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