The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.     

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.     

Spiralizations and tropisms in Arabidopsis roots.

When Arabidopsis seedlings are grown on a hard-agar plate, their primary roots show characteristic spiralling movements, apparent as waves, coils and torsions, together with a slanting toward the right-hand side. All these movements are believed to be the result of three different processes acting on the roots: circumnutation, positive gravitropism and negative thigmotropism. The basic movement of the roots is described as that of a growing right-handed helix, which, because of the root tip hitting the agar plate, is continuously switched from the right-hand to the left-hand of the growth direction, and vice versa. This movement also produces a slanting root-growth direction toward the right-hand because of the incomplete waves made by the right-handed root to the left-hand. By contrast, the torsions seen in the coils and waves are interpreted as artefacts that form as an adaptation of the three-dimensional root helix to the flat two-dimensional agar surface.     

Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori

The isovaleric acid-emanating silkworm mutant skunk (sku) was first studied over 30?years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsible for the sku mutant. Because of its specific features and symptoms similar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer-monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects.     

Wrinkly-Spreader fitness in the two-dimensional agar plate microcosm: maladaptation, compensation and ecological success

Bacterial adaptation to new environments often leads to the establishment of new genotypes with significantly altered phenotypes. In the Wrinkly Spreader (WS), ecological success in static liquid microcosms was through the rapid colonisation of the air-liquid interface by the production of a cellulose-based biofilm. Rapid surface spreading was also seen on agar plates, but in this two-dimensional environment the WS appears maladapted and rapidly reverts to the ancestral smooth (SM)-like colony genotype. In this work, the fitness of WS relative to SM in mixed colonies was found to be low, confirming the WS instability on agar plates. By examining defined WS mutants, the maladaptive characteristic was found to be the expression of cellulose. SM-like revertants had a higher growth rate than WS and no longer expressed significant amounts of cellulose, further confirming that the expression of this high-cost polymer was the basis of maladaptation and the target of compensatory mutation in developing colonies. However, examination of the fate of WS-founded populations in either multiple-colony or single mega-colony agar plate microcosms demonstrated that the loss of WS lineages could be reduced under conditions in which the rapid spreading colony phenotype could dominate nutrient and oxygen access more effectively than competing SM/SM-like genotypes. WS-like isolates recovered from such populations showed increased WS phenotype stability as well as changes in the degree of colony spreading, confirming that the WS was adapting to the two-dimensional agar plate microcosm.     

Mutation in a novel gene required for photomixotrophic growth leads to enhanced photoautotrophic growth of Synechocystis sp. PCC 6803

In the glucose-tolerant strain of Synechocystis sp. PCC 6803, we found two types of cells with distinct growth properties. Under photoautotrophic conditions at any light intensity, one type gave larger colonies (designated WL) than the other (designated WS). Notably, the WL cells produced much larger colonies than the WS cells at higher light intensity. In contrast, growth of the WL cells was severely suppressed under mixotrophic conditions with glucose and light, while the WS cells grew normally. A gene which could complement the WL phenotype was obtained from a wild-type genomic library. The gene, designated pmgA, coded for a 23 kDa polypeptide of 204 amino acid residues with no apparent homology to known genes. In the WL genome, the base substitution of T for C at position 193 of pmgA caused replacement of Leu with Phe at position 65 of the product. The phenotype of pmgA disruption mutants was similar to that of the WL cells, indicating that the WS cells expressed a functional pmgA product. By direct sequencing of polymerase chain reaction-amplified pmgA from genomic DNA, it was revealed as an example of microevolution that WL had expelled WS from the photoautotrophic culture of wild-type in our laboratory for a year or so. Mixed culture in liquid also demonstrated that the WL cells increased gradually under photoautotrophic conditions, while they decreased rapidly under photomixotrophic conditions. These results suggest that pmgA product is essential for photomixotrophic growth, whereas it represses photoautotrophic growth. To our knowledge, the WL cells and pmgA-disrupted mutants are the first in cyanobacteria, which shows much improved photosynthetic growth than wild-type especially at high light intensity.     

Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae)

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. We tested the hypothesis that wild-type Arabidopsis would acquire more phosphorus under phosphorus-limiting conditions than mutants that do not have the root hair response. The growth and phosphorus acquisition of wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under eight phosphorus treatments ranging from 0.4 mmol/m to 54 mmol/m phosphorus. At the lowest phosphorus treatment, all plants were small and showed severe phosphorus stress symptoms. At 1.5 mmol/m phosphorus, WS plants had greater shoot biomass, absolute growth rate, total phosphorus, and specific phosphorus absorption than the two root hair mutants. At the highest phosphorus treatment, there was no difference between genotypes in any of the parameters measured. We conclude that the response of increased root hair growth under low phosphorus availability in Arabidopsis is important in increasing phosphorus acquisition under phosphorus-limiting conditions.     

The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. In addition, plants with the root hair response acquire more phosphorus than mutants that have root hairs that do not respond to phosphorus limiting conditions. The purpose of this experiment was to determine the efficiency of root hairs in phosphorus acquisition at high- and low-phosphorus availability. Root hair growth, root growth, root respiration, plant phosphorus uptake, and plant phosphorus content of 3-wk-old wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under high (54 mmol/m) and low (0.4 mmol/m) phosphorus availability. A cost-benefit analysis was constructed from the measurements to determine root hair efficiency. Under high-phosphorus availability, root hairs did not have an effect on any of the parameters measured. Under low-phosphorus availability, wild-type Arabidopsis had greater total root surface area, shoot biomass, phosphorus per root length, and specific phosphorus uptake. The cost-benefit analysis shows that under low phosphorus, wild-type roots acquire more phosphorus for every unit of carbon respired or unit of phosphorus invested into the roots than the mutants. We conclude that the response of root hairs to low-phosphorus availability is an efficient strategy for phosphorus acquisition.     

Elongation rates and endogenous indoleacetic acid levels in roots of pea mutants differing in internode length

Growth of the primary root of 12 genotypes of peas ( Pisum sativum ) differing in their stem height was recorded for 14 days. The growth rate of roots of wild-type tall, gibberellin (GA)-deficient le dwarf or slender mutants (with la cry^s ) was similar (3 cm day⁻¹); that of severely GA-deficient nana ( na-1 ) plants was 50% of wild-type but elongation ceased after 8 days; moderately severe dwarf GA-deficient lines ls-1 and lh-1 had a 15% reduction in elongation rate but displayed no time-dependent slowing of the growth rate and brassinosteroid-insensitive and -deficient dwarfs lka and lkb showed slightly decreased root elongation. GA (levels reported in Yaxley et al. 2001 ) is not substantially limiting to root growth until it is severely deficient. The terminal 3 cm of roots of tall plants contained about 25 or 35 ng g⁻¹ fresh weight indole-3-acetic acid (IAA), depending on the genetic background, and le-1 dwarfs were similar. Nana ( na-1 ) had less than 50% the level of IAA of tall, all the moderately severe dwarfs had reductions of about 30% and the slender plants had about 40% more IAA than the corresponding wild-type. With the exception of slender plants, IAA level in the root tips correlated with root elongation. Root growth seems to be promoted by IAA within the range of the internal concentrations detected. Nana plants had a reduced amount of IAA and a lower root-growth rate. Whereas external application of IAA always inhibits root growth, even at very low concentrations, root growth is not similarly inhibited by internal IAA as slender plants had the highest IAA level and growth rate similar to wild-type, regardless of the shoot GA content.     

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.     

The Arabidopsis WAVY GROWTH 2 protein modulates root bending in response to environmental stimuli

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an alpha/beta-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.     

WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana

Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione-S-transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules.     

Adenovirus type 12 gene 401 function and maintenance of transformation.

Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.     

Lateral root frequency decreases when nitrate accumulates in tobacco transformants with low nitrate reductase activity: consequences for the regulation of biomass partitioning between shoots and root1

Accumulation of nitrate in the shoot of low-nitrate reductase tobacco transformants leads to an increase of the shoot:root ratio to higher values than in nitrogen-sufficient wild-type plants, even though the transformants are severely deficient in organic nitrogen. In the present paper, wild-type plants and low- nitrate reductase transformants were grown on vertical agar plates to investigate whether this inhibition of root growth by internal nitrate (i) can be reversed by adding sugars to the roots and (ii) is due to slower growth of the main roots or to a decreased number of lateral roots. When grown with a low nitrate supply, the transformants resembled wild-type plants with respect to amino acid and protein levels, shoot-root allocation, lateral root frequency, and rates of growth. When the transformants were grown with a high nitrate supply in the absence of sucrose they grew more slowly and had lower levels of amino acids and protein than wild-type plants, but accumulated more nitrate and developed a high shoot:root ratio. Root length was not affected, but the number of lateral roots per plant decreased. The slower root growth was accompanied by an increase of the concentration of sugars in the roots. Addition of 2% sucrose to the medium partially reversed the high shoot:root ratio in the transformants, but did not increase the frequency of lateral roots. It is concluded that nitrate accumulation in the plant leads to decreased root growth via (i) changes in carbon allocation leading to decreased allocation of sugars to root growth, and (ii) a decrease in the number of lateral roots and a shift in the sensitivity with which root growth responds to the sugar supply. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.     

A simple method to isolate biofilm-forming Bacillus subtilis and related species from plant roots

A novel method was developed to isolate pure cultures of wild-type Bacillus subtilis and related species from plant roots, even roots washed free of adhering soil. The method uses casein digest-mannitol agarose (CM) media that promote rapid dendritic growth (low K+ ion) or profuse surface film formation (high K+ ion) of Bacillus species at 40 degrees C. Inoculation from the tips of surface growth on agarose leads to self-purification and streaking on CM agar plates (hard agar and high K+) leads to characteristic colony morphology. Phenotypic and 16S rDNA analysis revealed that most root isolates obtained by this method are spore-forming Bacillus species, with enrichment for B. subtilis and its close relatives. Of particular interest is the finding that the majority of these Bacillus isolates and the B. subtilis Marburg strain also form adhering biofilms on inert surfaces. Thus the methods presented may be useful in isolation of biofilm-forming Bacillus and investigation of their role on plant roots.     

Two homologous ATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis photomorphogenesis and root development by mediating polar auxin transport

Light and auxin control many aspects of plant growth and development in an overlapping manner. We report here functional characterization of two closely related ABC (ATP-binding cassette) transporter genes, AtMDR1 and AtPGP1, in light and auxin responses. We showed that loss-of-function atmdr1 and atpgp1 mutants display hypersensitivity to far-red, red, and blue-light inhibition of hypocotyl elongation, reduced chlorophyll and anthocyanin accumulation, and abnormal expression of several light-responsive genes, including CAB3, RBCS, CHS, and PORA, under both darkness and far-red light conditions. In addition, we showed that the atmdr1-100 and atmdr1-100/atpgp1-100 mutants are defective in multiple aspects of root development, including increased root-growth sensitivity to 1-naphthalene acetic acid (1-NAA), and decreased sensitivity to naphthylphthalamic acid (NPA)-mediated inhibition of root elongation. Consistent with the proposed role of AtMDR1 in basipetal auxin transport, we found that expression of the auxin responsive DR5::GUS reporter gene in the central elongation zone is significantly reduced in the atmdr1-100 mutant roots treated with 1-NAA at the root tips, compared to similarly treated wild-type plants. Moreover, atmdr1-100, atpgp1-100, and their double mutants produced fewer lateral roots, in the presence or absence of 1-NAA or NPA. The atmdr1-100 and atmdr1-100/atpgp1-100 mutants also displayed enhanced root gravitropism. Genetic-epistasis analysis revealed that mutations in phyA largely suppress the randomized-hypocotyl growth and the short-hypocotyl phenotype of the atmdr1-100 mutants under far-red light, suggesting that phyA acts downstream of AtMDR1. Together, our results suggest that AtMDR1 and AtPGP1 regulate Arabidopsis (Arabidopsis thaliana) photomorphogenesis and multiple aspects of root development by mediating polar auxin transport.