<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Twisted growth and organization of cortical microtubules

In plants, directional cell expansion greatly contributes to the final shape of mature cells, and thus to organ architecture. A particularly interesting mode of cell expansion is helical growth in which the growth axis is continuously tilted either to the right or to the left as the cell grows. Fixed handedness of helical growth raises fundamental questions on the possible origin of left–right asymmetry. Twisting mutants of Arabidopsis thaliana offer unique opportunities to study the cellular basis of helical growth. Most of the twisting mutants with fixed handedness have been shown to have defects in microtubule functions, whereas mutants that twist in non-fixed directions appear to be defective in auxin response or transport. Good correlations have been found between the tilted growth direction and alignment of cortical microtubule arrays in twisting mutants with compromised microtubule functions. The present challenge is to understand how particular array patterns are organized during progression of the interphase in rapidly expanding cells. Molecular and cell biological studies on twisting mutants will lead to better understanding on how wild-type plant cells utilize the microtubule cytoskeleton to initiate and rigorously maintain straight growth. An erratum to this article can be found at     

Isolation and identification of blue light-induced growth inhibitor from light-grown <Emphasis Type="Italic">Arabidopsis</Emphasis> shoots

Phototropic stimulation of dark-grown hypocotyls of Arabidopsis thaliana increased a growth inhibitor in the wild-type but not in the non-phototropic nph3-101 mutant. From light-grown wild-type shoots the inhibitor was isolated and identified as indole-3-acetonitrile (IAN) from its ¹H NMR spectrum. The content of endogenous IAN in the hypocotyls of wild-type and mutant unilaterally exposed to blue light was determined using a physicochemical assay. The IAN concentration (28 M) in the phototropically stimulated wild-type hypocotyls was about three times larger than in the dark control. However, its content in the mutant hypocotyls did not change. IAN inhibited the hypocotyl growth of the nph3-101 to the same extent as in the wild-type at concentrations higher than 10 M. These results suggest that IAN plays a role in the phototropism of Arabidopsis thaliana hypocotyls.     

拟南芥ERD15基因缺失突变体的分子鉴定和表型分析

为探究ERD15基因功能,利用反向遗传学,通过PCR及半定量PCR筛选鉴定出拟南芥(Arabidopsis thaliana) ERD15基因的T-DNA插入纯合突变体,并对其表型进行观察分析。结果表明,erd15突变体莲座叶数目显著增多,提前3～4 d开花,突变体比野生型更早从营养生长转向生殖生长。拟南芥野生型植株主茎为圆柱体,平均直径1.29 mm,而erd15突变体主茎扁平,平均直径达到2.27mm,具极显著差异。与野生型相比,erd15突变体果实心皮发育受到影响,隔膜上排列有多排种子,果荚顶端膨大,长度缩短37.67%,但角果平均结籽数升高。因此,ERD15基因参与了调控拟南芥植株的生殖生长过程。     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Plasma membrane depolarization‐activated calcium channels, stimulated by microtubule‐depolymerizing drugs in wild‐type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half‐life in ton 2 mutant cells affected in the organization of cortical microtubules

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch–clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein–protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch–clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

A pleiotropic Arabidopsis thaliana mutant with inverted root chirality

Circumnutation is an oscillating movement of a growing plant organ that is believed to result from an endogenous rhythmic process intrinsic to growth. Circumnutating organs, as they extend, describe a helical trace. In Arabidopsis thaliana (L.) Heynh. circumnutation is particularly evident in primary roots and occurs, as in most plants, in a right-handed direction when viewed from above in the direction of the growing tips. We have discovered a pleiotropic mutant of Arabidopsis with left-handed root circumnutation. Major abnormalities of the mutant are: (i) a reduced size of all organs, mainly due to a defect in cell elongation or expansion; (ii) a zigzagging pattern of stem pith cells, reminiscent of the “erectoides” phenotype of the lk mutant of Pisum; (iii) roots of the mutant are gravitropic but as they grow, they form tight, left-handed coils. Genetically, the mutant depends on the presence of two independent monogenic recessive factors acting additively. The mutant alleles of both factors alter the growth of the aerial organs in a similar manner but differ at the root level: one mainly produces non-circumnutating roots, the other changes the direction of circumnutation from right to left hand. Received: 18 July 1996 / Accepted: 30 November 1996     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

Gravitropism in a starchless mutant of Arabidopsis

The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10^o curvature developed in mutants rotated on a clinostat following a 5 min induction at 1 · g, compared with 14^o in the WT. Curvatures induced during intervals from 2.5 to 30 min were 70% as great in the mutant as the WT. Thus under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Arabidopsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70–80% as much curvature as the WT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the WT. Thus, starch is also unnecessary for gravity reception in these tissues.Abbreviations PAR photosynthetically active radiation - PAS periodic acid-Schiff's reagent - PGM phosphoglucomutase - WT wild-type     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Nutritional suitability and ecological relevance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis> as foodplants for the cabbage butterfly, <Emphasis Type="Italic">Pieris rapae</Emphasis>

The thale cress, Arabidopsis thaliana, is considered to be an important model species in studying a suite of evolutionary processes. However, the species has been criticized on the basis of its comparatively small size at maturity (and consequent limitations in the amount of available biomass for herbivores) and on the duration and timing of its life cycle in nature. In the laboratory, we studied interactions between A. thaliana and the cabbage butterfly, Pieris rapae, in order to determine if plants are able to support the complete development of the herbivore. Plants were grown in pots from seedlings in densities of one, two, or four per pot. In each treatment, one, two, or five newly hatched larvae of P. rapae were placed on fully developed rosettes of A. thaliana. In a separate experiment, the same densities of P. rapae larvae were reared from hatching on single mature cabbage (Brassica oleracea) plants. Pupal fresh mass and survival of P. rapae declined with larval density when reared on A. thaliana but not on B. oleracea. However, irrespective of larval density and plant number, some P. rapae were always able to complete development on A. thaliana plants. A comparison of the dry mass of plants in different treatments with controls (= no larvae) revealed that A. thaliana partially compensated for plant damage when larval densities of P. rapae were low. By contrast, single cress plants with 5 larvae generally suffered extensive damage, whereas damage to B. oleracea plants was negligible. Rosettes of plants that were monitored in spring, when A. thaliana naturally grows, were not attacked by any insect herbivores, but there was often extensive damage from pulmonates (slugs and snails). Heavily damaged plants flowered less successfully than lightly damaged plants. Small numbers of generalist plant-parasitic nematodes were also recovered in roots and root soil. By contrast, plants monitored in a sewn summer plot were heavily attacked by insect herbivores, primarily flea beetles (Phyllotreta spp.). These results reveal that, in natural populations of A. thaliana, there is a strong phenological mismatch between the plant and most of its potential specialist insect herbivores (and their natural enemies). However, as the plant is clearly susceptible to attack from non-insect generalist invertebrate herbivores early in the season, these may be much more suitable for studies on direct defense strategies in A. thaliana.     

Wall architecture in the cellulose-deficientrsw1 mutant ofArabidopsis thaliana: Microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots

Summary Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.Abbreviations DCB 2,6-dichlorobenzonitrile - REGR relative elemental growth rate Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

UV-B预处理诱导拟南芥耐旱性的提高

为了解UV-B提高拟南芥(Arabidopsis thaliana)耐旱性的生理机制,将2周龄的野生型拟南芥(WT)和sto突变体幼苗用不同剂量UV-B预处理1周,再用30%PEG模拟干旱处理24 h,对植株的表型进行统计,并测定类黄酮、脯氨酸和MDA含量。结果表明,低剂量UV-B预处理能够提高拟南芥的耐旱性,植株的类黄酮与脯氨酸含量分别提高了20%～40%和50%～65%,细胞膜受损程度降低,从而提高了保水性。低剂量UV-B提高拟南芥耐旱性的效应在sto突变体中消失,证明这种效应在分子机制上可能与STO蛋白相关。     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.