Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3

Adenine phosphoribosyltransferase (APT) catalyzes the conversion of adenine and cytokinin bases to the corresponding nucleotides. An Arabidopsis thaliana mutant lacking the major APT isoform, APT1, is male sterile due to defects soon after meiosis. We have now used electron microscopy to define the effects of APT1 deficiency on pollen development to determine whether the changes might be attributed to adenine or cytokinin metabolism. Changes were observed in mutant anthers in both tapetal and pollen mother cells prior to meiosis with additional defects found at later stages, in both compartments. Principal changes include altered lipid accumulation in the tapetal cells, changes in pollen cell wall development, and a loss of synchrony in the development of the tapetum and microspores. Taken together our results suggest that APT1 deficiency causes a general metabolic decrease in energy metabolism, due to the lack of adenine recycling into adenylate nucleotides, which ultimately leads to pollen abortion. The early onset of meiosis in the mutant may be associated with altered cytokinin metabolism.     

A Heat-Sensitive Arabidopsis thaliana Kinase Substitutes for Human p70s6k Function In Vivo

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70^s6k has been implicated in the selective translational upregulation of 5′TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70^s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70^s6k and identify a new signalling pathway in plants.     

A plant DJ-1 homolog is essential for Arabidopsis thaliana chloroplast development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development.     

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.     

A novel male-sterile mutant of Arabidopsis thaliana,faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.     

Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K_m of 44 μM, and k_cat of 55 s⁻¹ using a synthetic substrate. The enzyme was inhibited by pepstatin A.     

A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent K_m that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDE_cd) at acidic and neutral pH. At neutral pH, VDE_cd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.     

A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS.     

A simple method for the addition of rotenone in Arabidopsis thaliana leaves

A simple and reproducible method for the treatment of Arabidopsis thaliana leaves with rotenone is presented. Rosette leaves were incubated with rotenone and Triton X-100 for at least 15 h. Treated leaves showed increased expression of COX19 and BCS1a, 2 genes known to be induced in Arabidopsis cell cultures after rotenone treatment. Moreover, rotenone/Triton X-100 incubated leaves presented an inhibition of oxygen uptake. The simplicity of the procedure shows this methodology is useful for studying the effect of the addition of rotenone to a photosynthetic tissue in situ.     

A tumor suppressor homolog,AtPTEN1, is essential for pollen development in Arabidopsis

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene (AtPTEN1) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.     

拟南芥RPP1A参与幼苗生长的蛋白质组学分析

核糖体蛋白不仅参与蛋白质合成,而且参与植物生长发育的调控.利用拟南芥核糖体磷酸蛋白P1(ribosomal phosphoprotein P1,RPP1)家族基因RPP1A缺失突变体rpp1a研究RPP1A缺失对幼苗蛋白质表达水平的影响,揭示其参与调控幼苗生长的作用机制.表型分析发现,与野生型WT相比,RPP1A缺失导...     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.     

A role for IAA in the infection of Arabidopsis thaliana by Orobanche aegyptiaca

BACKGROUND: Vascular continuity is established between a host plant and the root parasite broomrape. It is generally accepted that the direction of vascular continuity results from polar flow of auxin. Our hypothesis was that chemical disruptions of auxin transport and activity could influence the infection of the host by the parasite. METHODS: A sterile system for the routine infection of Arabidopsis thaliana seedlings in Nunc cell culture plates by germinated seeds of Orobanche aegyptiaca was developed. This method permitted a quantitative assay of the rate of host infection. The three-dimensional structure of the vascular contacts was followed in cleared tissue. IAA (indole acetic acid) or substances that influence its activity and transport were applied locally to the host root. RESULTS: The orientation of the xylem contacts showed that broomrape grafts itself upon the host by acting hormonally as a root rather than a shoot. Local applications of IAA, PCIB (p-chlorophenoxyisobutyric acid) or NPA (naphthylphthalamic acid) all resulted in drastic reductions of Orobanche infection CONCLUSIONS: Broomrape manipulates the host by acting as a sink for auxin. Disruption of auxin action or auxin flow at the contact site could be a novel basis for controlling infection by Orobanche.