Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo

In plants, chloroplast division FtsZ proteins have diverged into two families, FtsZ1 and FtsZ2. FtsZ1 is more divergent from its bacterial counterparts and lacks a C-terminal motif conserved in most other FtsZs. To begin investigating FtsZ1 structure-function relationships, we first identified a T-DNA insertion mutation in the single FtsZ1 gene in Arabidopsis thaliana, AtFtsZ1-1. Homozygotes null for FtsZ1, though impaired in chloroplast division, could be isolated and set seed normally, indicating that FtsZ1 is not essential for viability. We then mapped five additional atftsZ1-1 alleles onto an FtsZ1 structural model and characterized chloroplast morphologies, FtsZ protein levels and FtsZ filament morphologies in young and mature leaves of the corresponding mutants. atftsZ1-1(G267R), atftsZ1-1(R298Q) and atftsZ1-1(Delta404-433) exhibit reduced FtsZ1 accumulation but wild-type FtsZ2 levels. The semi-dominant atftsZ1-1(G267R) mutation caused the most severe phenotype, altering a conserved residue in the predicted T7 loop. atftsZ1-1(G267R) protein accumulates normally in young leaves but is not detected in rings or filaments. atftsZ1-1(R298Q) has midplastid FtsZ1-containing rings in young leaves, indicating that R298 is not critical for ring formation or positioning despite its conservation. atftsZ1-1(D159N) and atftsZ1-1(G366A) both have overly long, sometimes spiral-like FtsZ filaments, suggesting that FtsZ dynamics are altered in these mutants. However, atftsZ1-1(D159N) exhibits loss of proper midplastid FtsZ positioning while atftsZ1-1(G366A) does not. Finally, truncation of the FtsZ1 C-terminus in atftsZ1-1(Delta404-433) impairs chloroplast division somewhat but does not prevent midplastid Z ring formation. These alleles will facilitate understanding of how the in vitro biochemical properties of FtsZ1 are related to its in vivo function.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Ultrastructural characterization of exine development of the transient defective exine 1 mutant suggests the existence of a factor involved in constructing reticulate exine architecture from sporopollenin aggregates

A male-sterile mutant of Arabidopsis thaliana, in which filament elongation was defective although pollen fertility was normal, was isolated by means of T-DNA tagging. Transmission electron microscopy (TEM) analysis revealed that primexine synthesis and probacula formation, which are thought to be the initial steps of exine formation, were defective, and that globular sporopollenin aggregation was randomly deposited onto the microspore at the early uninucleate microspore stage. Sporopollenin aggregation, which failed to anchor to the microspore plasma membrane, was deposited on the locule wall and in the locule at the uninucleate microspore stage. However, visually normal exine with a basic reticulate structure was observed at the middle uninucleate microspore stage, indicating that the exine formation was restored in the mutant. Thus, the mutant was designated transient defective exine 1 (tde1). These results indicated that tde1 mutation affects the initial process of the exine formation, but does not impair any critical processes. Our results also suggest the existence of a certain factor responsible for exine patterning in A. thaliana. The TDE1 gene was found to be identical to the DE-ETIOLATED 2 gene known to be involved in brassinosteroid (BR) biosynthesis, and the tde1 probacula-defective phenotypes were recovered in the presence of BR application. These results suggest that BRs control the rate or efficiency of initial process of exine pattern formation.     

Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.     

The RNAi-mediated silencing of xanthine dehydrogenase impairs growth and fertility and accelerates leaf senescence in transgenic Arabidopsis plants

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.     

The effect of ring size on vibrational spectroscopy and hydrogen bonding properties for the complexes between small ring carbonyl compounds with HF and HCl: Theoretical analysis

The effect of the molecular structure on the properties of C = O…HX (X = F, Cl) bonds was investigated in a set of small cyclic carbonyl compounds, using vibrational spectroscopy and B3LYP/6–311G** calculations. Two main effects were studied: the size of the ring and the inclusion of oxygen atoms in the ring. In these complexes the C = O and H–X participating bonds in the hydrogen–bond are elongated, while others bonds are compressed. The calculated vibrational spectra were interpreted and band assignments were reported. Surface potential energy calculations are carried out with scanning HCl and HF near oxygen atom.     

The localization of PHRAGMOPLAST ORIENTING KINESIN1 at the division site depends on the microtubule-binding proteins TANGLED1 and AUXIN-INDUCED IN ROOT CULTURES9 in Arabidopsis

Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN1_1–132), we assessed the localization and function of TAN1_1–132 in the tan1 air9 double mutant. TAN1_1–132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN1_1–132, which disrupted the POK1–TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN1_1–132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1–POK1 interactions.

Specific amino acids within TANGLED1 are required for its correct localization and function partially through interaction with POK1; both TANGLED1 and AIR9 mediate POK1 division site localization.

IN A NUTSHELL Background: Unlike animal cells, plant cells cannot move due to their semi-rigid cell walls. The correct positioning of the new cell wall is important for overall plant growth and development. Three microtubule-binding proteins are involved in division plane orientation: TANGLED1 (TAN1), AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), and PHRAGMOPLAST ORIENTING KINESIN1 (POK1). These proteins localize as a ring at the edge of the cell where the new cell wall will insert during cell division, at a position called the division site. Here, we focused on how TAN1 and POK1 interactions promote their localization to the division site, and their function in plant growth and division plane positioning. Question: How do TAN1 and AIR9 contribute to POK1 localization, and how does POK1 localization affect new cell wall placement? Findings: TAN1 and AIR9 together maintain POK1 at the division site in Arabidopsis thaliana. When mutant versions of TAN1 that no longer interact with POK1 were transformed into the tan1 air9 double mutant, POK1 and TAN1 localization was partially disrupted and cell wall placement defects occurred. This suggests that POK1 interaction with TAN1 is important for their correct division site localization and new cell wall placement. Next steps: This work strongly suggests that yet unknown proteins mediate TAN1 and POK1 interaction. Discovering what those proteins are, and how AIR9 contributes to division plane positioning are next. Understanding how plants position their division plane will contribute to understanding plant growth and has the long-term potential to contribute to next-generation crop development.     

The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant

In order to test whether an increased export of carbohydrates by leaves and starch mobilization are critical for floral transition in Arabidopsis thaliana, the Columbia ecotype as well as its starchless mutant pgm and starch-in-excess mutant sex1 were investigated. Induction of flowering was achieved by exposure of plants to either one long day (LD) or one displaced short day (DSD). The following conclusions were drawn: (i) Both the pgm and sex1 mutants have a late-flowering phenotype in days shorter than 16 h. (ii) When inductive treatments cause a large percentage of induced plants, there is always a large, early and transient increase in carbohydrate export from leaves. By contrast, when an inductive treatment results in only a low percentage of induced plants (pgm plants exposed to one DSD), the export of carbohydrates from leaves is not increased, supporting the idea that phloem carbohydrates have a critical function in floral transition. (iii) Starch mobilization is not required to obtain an increased carbohydrate export when induction is by one LD (extended period of photosynthesis), but is absolutely essential when induction is by one DSD (period of photosynthesis unaffected). (iv) Floral induction apparently increases the capability of the leaf phloem-loading system. Received: 27 August 1997 / Accepted: 6 March 1998     

The influence of drought on the relationship between leaf and conducting sapwood area in Eucalyptus globulus and Eucalyptus nitens

 Development of the relationship between leaf area (A _l ) and sapwood area (A _s ) was investigated in two important hardwoods, Eucalyptus globulus (Labill) and E. nitens (Deane and Maiden) Maiden, growing in an experimental plantation established in a low rainfall zone (approx. 515 mm year^–1) of Tasmania. The experiment compared irrigated controls and a rainfed treatment which was subjected to cyclical summer droughts from age 1 to 6 years old. Leaf area and sapwood area were determined by destructive sampling at ages 2, 3 and 6 years old. There was no effect of stand age on A _l :A _s when sapwood area was measured at crown break. At age 3 years old A _l :A _s was significantly greater in the rainfed than the irrigated trees. It was concluded that this difference was due to earlier canopy closure in the irrigated trees. When the plantation was 6 years old A _l :A _s was significantly greater in the irrigated than the rainfed treatment. An analysis based on an equation which links A _l :A _s with transpiration and volumetric flow rate (Whitehead et al. 1984) was used to infer a positive correlation between stem hydraulic conductivity (k _h ) and water availability. Independent of water availability E. globulus maintained a higher A _l :A _s than E. nitens at all ages. Received: 20 March 1997 / Accepted: 30 December 1997     

The fruit of their labour: plants and plant processing at EeRb 140 (860±60 uncal B.P. to 160±50 uncal B.P.), a late prehistoric hunter-gatherer-fisher site on the southern Interior Plateau, British Columbia, Canada

Palaeoethnobotanical analysis of Late Period contexts (860±60 uncal B.P. to 160±50 uncal B.P.) at the site of EeRb 140, indicates that it probably served as a seasonal work area, utilised during the mid to late summer by the residents of an adjacent pit-house winter village. Food processing was evidently the primary plant-related activity at this open-air hunter-gatherer-fisher site on the British Columbia Plateau in Canada. One feature appears to have functioned as both an open hearth for the drying and preservation of berries and as a pit-oven, possibly for preparing foods for immediate consumption. Comparisons of the archaeobotany of EeRb 140 with Plateau ethnographies suggest that women's task group activities are represented here. Most significantly, the identification of a specialised plant-processing site in such close proximity to a winter village contrasts strongly with existing ethnoarchaeological models for the British Columbia Plateau and introduces a type of site not previously identified archaeologically in this region. Received August 14, 2001 / Accepted March 13, 2002     

Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg-1 and Asp8

Summary An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu¹⁰ or Asp¹⁸. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg⁻¹ basic moiety and either the Asp⁸ or Glu¹⁰ acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg⁻¹ and Asp⁸ in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.     

Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of ¹³C into metabolic intermediates and end products when cells were labelled with [1‐¹³C], [2‐¹³C], or [¹³C₆]glucose, in combination with ¹⁴C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.     

Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNA^Met _i) by the addition of a 2′-O-ribosyl phosphate group to Adenosine 64. As a result, tRNA^Met _i is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNA^Met _i in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1α (eEF-1α), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1α are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNA^Met _i for elongation. Thus, under conditions in which the components of the ternary met-tRNA^Met _i:GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2′-O-ribosyl phosphate modification in tRNA^Met _i is important for the provision of adequate amounts of tRNA^Met _i for formation of this ternary complex. Received: 20 November 1998 / Accepted: 7 April 1999     

The SOS response is induced by replication fork blockage at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites

A new model system for the study of the SOS response has been developed. In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids. Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter. SOS induction can, therefore, be controlled by arabinose. The extent of the SOS response was monitored by measuring the activity of β-galactosidase, expressed from a lacZ gene fused to the 5′ region of the sfiA gene, a typical SOS-responsive gene. Expression of the fusion gene is completely dependent on recA ⁺ and lexA ⁺ genes. Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction. The properties of this system are discussed. Received: 10 May 1998 / Accepted: 20 May 1999     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.