Expression of the Arabidopsis thaliana Histone Gene AtHTA1 Enhances Rice Transformation Efficiency

We expressed the Arabidopsis thaliana histone AtHTA1 in rice under the control of the maize ubiquitin promoter. Transformation efficiencies of rice plants that constitutively expressed AtHTA 1 were 28-44% higher than calli containing an empty vector control. Furthermore, co-infection of rice calli with a vector containing AtHTA 1 and another vector with the target gene increased transformation by 27-50%. Thus, expression of AtHTA 1 either transiently or in stably transformed cells improved rice transformation efficiency.     

Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana

Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.     

Control of PHERES1 Imprinting in Arabidopsis by Direct Tandem Repeats

Genomic imprinting is an epigenetic phenomenon that causes monoallelic expression of specific genes dependent on the parent-of-origin. Imprinting of the Arabidopsis gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED （FIS） Polycomb group complex as well as a distally located methylated region containing a tandem triple repeat sequence. In this study, we investigated the regulation of the close PHERES1 homolog PHERES2. We found that PHERES2 is also a direct target gene of the FIS Polycomb group complex, but, in contrast to PHERES1, PHERES2 is equally expressed from maternal and paternal alleles. Thus, PHERES2 is not regulated by genomic imprinting, correlating with the lack of tandem repeats at PHERES2. Eliminating tandem repeats from the PHERES1 locus abolishes PHERES1 imprinting, demonstrating that tandem repeats are essential forPHERES1 imprinting. Taking these results together, our study shows that the recently duplicated genes PHERES1 and PHERES2 are both target genes of the FIS Polycomb group complex but only PHERES1 is regulated by genomic imprinting, which is likely caused by the presence of repeat sequences in the proximity of the PHERES1 locus.     

Open and Closed： The Roles of Linker Histones in Plants and Animals

Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications （PTMs） to core histones, but linker histone function is less well understood. In this review, we describe the current understand- ing of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and devel- opment, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

The Translational Apparatus of Plastids and Its Role in Plant Development

Chloroplasts （plastids） possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco （plastid-encoded components） and Arabidopsis （nucleus-encoded components）. This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant develop- ment at various levels, presumably via retrograde signaling pathway（s）. In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.     

Structure variations of TBA G-quadruplex induced by 2＋-O-methyl nucleotide in K＋ and Ca2＋ environments

Thrombin binding aptamer （TBA）, a 15-mer oligonucleo- tide of d（GGTTGGTGTGGTTGG） sequence, folds into a chair-type antiparallel G-quadruplex in the K＋ environ- ment, and each of two G-tetrads is characterized by a syn-anti-syn-anti glycosidic conformation arrangement. To explore its folding topology and structural stability, 2＇-0- methyl nucleotide （OMe） with the C31-endo sugar pucker conformation and anti glycosidic angle was used to selectively substitute for the guanine residues of G-tetrads of TBA, and these substituted TBAs were characterized using a circular dichroism spectrum, thermally differential spectrum, ultra- violet stability analysis, electrophoresis mobility shift assay, and thermodynamic analysis in K＋ and Ca2＋ environments. Results showed that single substitutions for syn-dG residues destabilized the G-quadruplex structure, while single substi- tutions for anti-dG residues could preserve the G-quadruplex in the K＋ environment. When one or two G-tetrads were modified with OMe, TBA became unstructured. In contrast, in Ca2＋ environment, the native TBA appeared to be un- structured. When two G-tetrads were substituted with OMe, TBA seemed to become a more stable parallel G-4 structure. Further thermodynamic data suggested that OMe-substitu- tions were an enthalpy-driven event. The results in this study enrich our understanding about the effects of nucleo- tide derivatives on the G-quadruplex structure stability in different ionic environments, which will help to design G-quadruplex for biological and medical applications.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.     

Paramutation： A Heritable Change in Gene Expression by Allelic Interactions In Trans

Epigenetic gene regulation involves the stable propagation of gene activity states through mitotic, and sometimes even meiotic, cell divisions without changes in DNA sequence. Paramutation is an epigenetic phenomenon involving changes in gene expression that are stably transmitted through mitosis as well as meiosis. These heritable changes are mediated by in trans interactions between homologous DNA sequences on different chromosomes. During these in trans interactions, epigenetic information is transferred from one allele of a gene to another allele of the same gene, resulting in a change in gene expression. Although paramutation was initially discovered in plants, it has recently been observed in mammals as well, suggesting that the mechanisms underlying paramutation might be evolutionarily conserved. Recent findings point to a crucial role for small RNAs in the paramutation process. In mice, small RNAs appear sufficient to induce paramutation, whereas in maize, it seems not to be the only player in the process. In this review, potential mechanisms are discussed in relation to the various paramutation phenomena.