AtKP1, a kinesin-like protein, mainly localizes to mitochondria in Arabidopsis thaliana

Kinesins and kinesin-like proteins （KLPs） constitute a large family of microtubule-based motors that play important roles in many fundamental cellular and developmental processes. To date, a number of kinesins or KLPs have been identified in plants including Arabidopsis thaliana. Here, a polyclonal antibody against AtKP1 （kinesin-like protein 1 in A. thaliana） was raised by injection the expressed AtKP1 specific C-terminal polypeptides in rabbits, and immunoblot analysis was conducted with the affinity-purified anti-AtKP1 antibody. The results indicated that this antibody recognized the AtKP1 fusion proteins expressed in E. coli and proteins of -125 kDa in the soluble fractions of Arabidopsis extracts. The molecular weight was consistent with the calculated molecular weight based on deduced amino acids sequence of AtKP1. To acquire the subcellular localization of the protein, AtKP1 in Arabidopsis root cells was observed by indirect immunofluorescence microscopy. AtKP1 was localized to particle-like organelles in interphase or dividing cells, but not to mitotic microtubule arrays. Relatively more AtKP1 was found in isolated mitochondria fraction on immunoblot of the subcellular fractions. The AtKP1 protein could not be released following a 0.6 M KI washing, indicating that AtKP1 is tightly bind to mitochondria and might function associated with this kind of organelles.     

Epigenetic variation contributes to environmental adaptation of Arabidopsis thaliana

Epigenetic variation is frequently observed in plants and direct relationships between differences in DNA methylation and phenotypic responses to changing environments have often been described. The identification of contributing genetic loci, however, was until recently hampered by the lack of suitable genome wide mapping resources that specifically segregate for epigenetic marks. The development of epi-RIL populations in the model species Arabidopsis thaliana has alleviated this obstacle, enabling the accurate genetic analysis of epigenetic variation. Comprehensive morphological phenotyping of a ddm1 derived epi-RIL population in different environments and subsequent epi-QTL mapping revealed a high number of epi-QTLs and pleiotropic effects of several DMRs on numerous traits. For a number of these epi-QTLs epistatic interactions could be observed, further adding to the complexity of epigenetic regulation. Moreover, linkage to epigenetic marks indicated a specific role for DNA-methylation variation, rather than TE transposition, in plastic responses to changing environments. These findings provide supportive evidence for a role of epigenetic regulation in evolutionary and adaptive processes.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.     

Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana

The initial step in the de novo biosynthesis of cytokinin in higher plants is the formation of isopentenyladenosine 5'-monophosphate (iPMP) from AMP and dimethylallylpyrophosphate (DMAPP), which is catalyzed by adenylate isopentenyltransferase (IPT). Although cytokinin is an essential hormone for growth and development, the nature of the enzyme for its biosynthesis in higher plants has not been identified. Herein, we describe the molecular cloning and biochemical identification of IPTs from Arabidopsis thaliana. Eight cDNAs encoding putative IPT, designated as AtIPT1 to AtIPT8, were picked up from A. thaliana. The Escherichia coli transformants expressing the recombinant proteins excreted cytokinin species into the culture medium except for that expressing AtIPT2 that is a putative tRNA IPT. A purified recombinant AtIPT1 catalyzed the formation of iPMP from DMAPP and AMP. These results indicate that the small multigene family contains both types of isopentenyltransferase, which could synthesize cytokinin and mature tRNA.     

A glycolate dehydrogenase in the mitochondria of Arabidopsis thaliana

The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants.     

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1 . The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae . The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.     

Cell wall composition contributes to the control of transpiration efficiency in Arabidopsis thaliana

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7(irx3-5) involved in the control of TE. Leaves of the AtCesA7(irx3-5) mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7(irx3-5) mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7(irx3-5) when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency.     

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.     

Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana

Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) from guanosine 5'-triphosphate (GTP). While many cGMP-mediated processes in plants have been reported, no plant molecule with GC activity has been identified. When the Arabidopsis thaliana genome is queried with GC sequences from cyanobacteria, lower and higher eukaryotes no unassigned proteins with significant similarity are found. However, a motif search of the A. thaliana genome based on conserved and functionally assigned amino acids in the catalytic center of annotated GCs returns one candidate that also contains the adjacent glycine-rich domain typical for GCs. In this molecule, termed AtGC1, the catalytic domain is in the N-terminal part. AtGC1 contains the arginine or lysine that participates in hydrogen bonding with guanine and the cysteine that confers substrate specificity for GTP. When AtGC1 is expressed in Escherichia coli, cell extracts yield >2.5 times more cGMP than control extracts and this increase is not nitric oxide dependent. Furthermore, purified recombinant AtGC1 has Mg(2+)-dependent GC activity in vitro and >3 times less adenylyl cyclase activity when assayed with ATP as substrate in the absence of GTP. Catalytic activity in vitro proves that AtGC1 can function either as a monomer or homo-oligomer. AtGC1 is thus not only the first functional plant GC but also, due to its unusual domain organization, a member of a new class of GCs.     

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

The galactolipid monogalactosyldiacylglycerol (MGDG) contributes to photosynthesis-related processes in Arabidopsis thaliana

Processes putatively dependent on the galactolipid monogalactosyldiacylglycerol (MGDG) were recently studied using the knockdown monogalactosyldiacylglycerol synthase 1 (mgd1-1) mutant (∼40% reduction in MGDG). Surprisingly, targeting of chloroplast proteins was not affected in mgd1-1 mutants, suggesting they retain sufficient MGDG to maintain efficient targeting. However, in dark-grown mgd1-1 plants the photoactive to photoinactive protochlorophyllide (Pchlide) ratio was increased, suggesting that photoprotective responses are induced in them. Nevertheless, mgd1-1 could not withstand high light intensities, apparently due to impairment of another photoprotective mechanism, the xanthophyll cycle (and hence thermal dissipation). This was mediated by increased conductivity of the thylakoid membrane leading to a higher pH in the thylakoid interior, which impaired the pH-dependent activation of violaxanthin de-epoxidase (VDE) and PsbS. These findings suggest that MGDG contribute directly to the regulation of photosynthesis-related processes.Key words: conductivity, galactolipid, light stress, photosynthesis, plastid, xanthophyllThe galactolipid monogalactosyldiacylglycerol (MGDG), the major lipid in plastids,¹ is mainly synthesised in inner plastid envelopes,² where monogalactosyldiacylglycerol synthase 1 (MGD1) catalyses the last step of its production.³ Two MGDG-deficient mutants are known: the knockdown mgd1-1 mutant, which accumulates ∼40% less MGDG than wild type,⁴ and the null mutant mgd1-2, which displays extremely severe defects in chloroplast and plant development.⁵ Thus, the mgd1-1 mutant is more suitable for assessing putative roles of MGDG in processes such as protein targeting and photoprotection.There are conflicting indications regarding the involvement of galactolipids in chloroplast protein targeting: some suggest they play an important role,^6–10 but not all.^11,12 The data recently collected for mgd1-1 do not support MGDG''s involvement in protein targeting, since (inter alia) the level of MGDG in mgd1-1 mutants is clearly sufficient for efficient targeting.¹³ Further, the galactolipid associated with the TOC complex¹² is digalactosyldiacylglycerol (DGDG) and the digalactosyldiacylglycerol synthase 1 (dgd1) mutant,¹⁴ which has ∼10% of wild-type levels of DGDG, has impaired import efficiency.^15,16 Hence, this may indicate that DGDG is relatively more important for chloroplast import than MGDG.The prolamellar bodies (PLBs) of etioplasts have high lipid-to-protein ratios compared to thylakoids. Their major lipid and protein are MGDG and NADPH:Pchlide oxidoreductase (POR), respectively,¹⁷ and MGDG putatively plays an important role, interactively with POR, in the formation of PLBs.^18–20 The transformation of PLBs into thylakoids involves phototransformation of photoactive Pchlide (F656), a precursor of chlorophyll. Non-photoactive Pchlide (F631) is susceptible to photooxidative damage, but POR is believed to suppress this.^21,22 After excitation at 440 nm, mgd1-1 mutants display distinctly higher fluorescence emission peaks corresponding to photoactive Pchlide than wild type counterparts and (hence) higher photoactive:non-photoactive Pchlide ratios.¹³ These changes may be photoprotective responses that favour formation of photoactive Pchlide and optimize the plants'' opportunities to use light for chlorophyll production, enabling the transformation of etioplasts into chloroplasts.Interestingly,the xanthophyll cycle, another photoprotective mechanism, is impaired in mgd1-1.¹³ Normally, the xanthophyll cycle pigment violaxanthin is de-epoxidized into antheraxanthin, and then into zeaxanthin, by the enzyme VDE (Fig. 1), which is dependent on MGDG.²³ MGDG is also an integral component of photosynthetic complexes.^24–26 Thus, since mgd1-1 mutants have reduced total amounts of xanthophyll and chlorophyll pigments, but increased chlorophyll a/b ratios, their photosynthesis capacity is unsurprisingly reduced, even though the organization of their electron transport chains is not strongly affected by the MGDG deficiency.¹³Open in a separate windowFigure 1Reactions of the xanthophyll cycle (adapted from ref. ²⁹). VDE, violaxanthin de-epoxidase; ZE, zeaxanthin epoxidase.During short-term high light stress, antheraxanthin and zeaxanthin are thought to facilitate dissipation of excess light energy in the PSII antenna bed by non-photochemical quenching.^27,28 Upon high light stress the pH decreases, triggering photoprotective mechanisms via changes in the PSII antenna system. The PsbS protein, which is involved in thermal dissipation, is protonated and initiates a conformational change in the PSII antenna bed. This change is further stabilized by the de-epoxidation of violaxanthin to zeaxanthin by the luminal VDE.²⁸ However, the thermal dissipation is impaired in mgd1-1 mutants at high light intensities (>1000 µmol m⁻² s⁻¹) making them more susceptible to light stress. Surprisingly, this is not mediated by direct effects on VDE and PsbS activities, but by changes in the proton conductivity of the thylakoid membrane.¹³The steady-state capacity of the xanthophyll cycle is reduced in mgd1-1 mutants, due to a ∼40% reduction in the proton motive force (pmf) across their thylakoid membranes, indicating that they have impaired capacities to energize these membranes. Nevertheless, the pmf is more or less equal to wild type under light-limited conditions (200 µmol m⁻² s⁻¹ light); it is only the increase in pmf in high light intensities that is impaired in the mutants.¹³ This leads to the thylakoid lumen being less acidic in mgd1-1 than in wild type, hampering full activation of VDE and PsbS. Thus, the thylakoid lumen pH is above the threshold level required for full activation of PsbS and VDE under steady-state conditions and so de-epoxidation rates are retarded and the equilibrium between zeaxanthin and violaxanthin starts to shift slightly towards violaxanthin (Fig. 2).¹³ Thus, increased conductivity of the thylakoid membranes is probably responsible for the diminished non-photochemical quenching in mgd1-1, and the findings strongly indicate that MGDG is required for efficient photosynthesis and photoprotection, in addition to being a physical membrane constituent.Open in a separate windowFigure 2Schematic diagram illustrating the normal mode of action of the xanthophyll cycle. In standard light conditions, V is bound to the photosynthetic complexes and harvests light. In strong light, V is released from the complexes and converted to Z by VDE, which is unable to access V when it is associated with the photosynthetic complexes. The newly formed Z then binds to the photosynthetic complexes (at the PsbS protein), where it dissipates excess energy through NPQ. V, violaxanthin; A, antheraxanthin; Z, zeaxanthin; VDE, violaxanthin de-epoxidase; ZE, zeaxanthin epoxidase. Arrows indicate the directions of reactions.     

Auxin-induced inhibition of lateral root initiation contributes to root system shaping in Arabidopsis thaliana

The hormone auxin is known to inhibit root elongation and to promote initiation of lateral roots. Here we report complex effects of auxin on lateral root initiation in roots showing reduced cell elongation after auxin treatment. In Arabidopsis thaliana, the promotion of lateral root initiation by indole-3-acetic acid (IAA) was reduced as the IAA concentration was increased in the nanomolar range, and IAA became inhibitory at 25 nM. Detection of this unexpected inhibitory effect required evaluation of root portions that had newly formed during treatment, separately from root portions that existed prior to treatment. Lateral root initiation was also reduced in the iaaM-OX Arabidopsis line, which has an endogenously increased IAA level. The ethylene signaling mutants ein2-5 and etr1-3, the auxin transport mutants aux1-7 and eir1/pin2, and the auxin perception/response mutant tir1-1 were resistant to the inhibitory effect of IAA on lateral root initiation, consistent with a requirement for intact ethylene signaling, auxin transport and auxin perception/response for this effect. The pericycle cell length was less dramatically reduced than cortical cell length, suggesting that a reduction in the pericycle cell number relative to the cortex could occur with the increase of the IAA level. Expression of the DR5:GUS auxin reporter was also less effectively induced, and the AXR3 auxin repressor protein was less effectively eliminated in such root portions, suggesting that decreased auxin responsiveness may accompany the inhibition. Our study highlights a connection between auxin-regulated inhibition of parent root elongation and a decrease in lateral root initiation. This may be required to regulate the spacing of lateral roots and optimize root architecture to environmental demands.     

Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana

Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.     

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.