A novel high efficiency,low maintenance,hydroponic system for synchronous growth and flowering of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Arabidopsis thaliana is now the model organism for genetic and molecular plant studies, but growing conditions may still impair the significance and reproducibility of the experimental strategies developed. Besides the use of phytotronic cabinets, controlling plant nutrition may be critical and could be achieved in hydroponics. The availability of such a system would also greatly facilitate studies dealing with root development. However, because of its small size and rosette growth habit, Arabidopsis is hardly grown in standard hydroponic devices and the systems described in the last years are still difficult to transpose at a large scale. Our aim was to design and optimize an up-scalable device that would be adaptable to any experimental conditions.     

An improved procedure for transformingArabidopsis thaliana (Landsbergerecta) root explant

Agrobacterium tumefaciens-mediated transformation has been widely used in molecular characterization of genes inArabidopsis thaliana. A number of procedures have been developed for transformation ofArabidopsis explants usingAgrobacterium. This paper describes an improved protocol for transformation ofArabidopsis thaliana root explants. Most significantly, using this protocol one can achieve efficient root regeneration of transformation in Landsbergerecta, an ecotype which is widely used in genetic and molecular analyses and which has been difficult to transform in the past. Additional modifications allow easy production of roots for transformation and regeneration of large numbers of transformation t shoots.     

Morphological, physiological and molecular genetic characterization ofArabidopsis himalaica, with reference toA. thaliana

Arabidopsis himalaica (Edgeworth) O.E. Schulz, a poorly characterized species typical of HimalayanArabidopsis, was analyzed in terms of its morphology, physiology, chromosome number and molecular genetics, in comparison withA. thaliana which is the standard species in the genusArabidopsis. From view point of developmental genetics, several features which are specific toA. himalaica seem not to be derived by single-gene mutations inA. thaliana. Phylogenetic analyses based onrbcL sequences suggested that genusArabidopsis is not monophyletic. The detailed characterization ofA. himalaica should provide clues to understand the trait of evolution of particular features of Himalayan species ofArabidopsis and their genetic basis.     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Rapid bioassays to evaluate the plant growth promoting activity of <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> (L.) Le Jol. using a model plant, <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

Ascophyllum nodosum extract products are used commercially in the form of liquid concentrate and soluble powder. These formulations are manufactured from seaweeds that are harvested from natural habitats with inherent environmental variability. The seaweeds by themselves are at different stages of their development life-cycle. Owing to these differences, there could be variability in chemical composition that could in turn affect product consistency and performance. Here, we have tested the applicability of using Arabidopsis thaliana as a model to study the activity of two different extracts from A. nodosum. Three different bioassays: Arabidopsis root-tip elongation bioassay, Arabidopsis liquid growth bioassay and greenhouse growth bioassay were evaluated as growth assays. Our results indicate that both extracts promoted root and shoot growth in comparison to controls. Further, using Arabidopsis plants with a DR5:GUS reporter gene construct, we provide evidence that components of the commercial A. nodosum extracts modulates the concentration and localisation of auxins which could account, at least in part, for the enhanced plant growth. The results suggest that A. thaliana could be used effectively as a rapid means to test the bioactivity of seaweed extracts and fractions.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Ara-Rhizotron: An Effective Culture System to Study Simultaneously Root and Shoot Development of Arabidopsis

Studying Arabidopsis thaliana (L.) Heynh. root development in situ at the whole plant level without affecting shoot development has always been a challenge. Such studies are usually carried out on individual plants, neglecting competition of a plant population, using hydroponic systems or Agar-filled Petri dishes. Those both systems, however, present some limitations, such as difficulty to study precisely root morphogenesis or time-limited culture period, respectively. In this paper, we present a method of Arabidopsis thaliana (L.) Heynh. cultivation in soil medium, named “Ara-rhizotron”. It allows the non-destructive study of shoot and root development simultaneously during the entire period of vegetative growth. In this system, roots are grown in 2D conditions, comparable to other soil cultures. Moreover, grouping several Ara-rhizotrons in a box enables the establishment of 3D shoot competition as for plants grown in a population. In comparison to a control culture grown in pots in the same environmental conditions, the Ara-rhizotron resulted in comparable shoot development in terms of dry mass, leaf area, number of leaves and nitrogen content. We used this new culture system to study the effect of irrigation modalities on plant development. We found that irrigation frequency only affected root partitioning in the soil and shoot nitrogen content, but not shoot or root growth. These effects appeared at the end of the vegetative growth period. This experiment highlights the opportunity offered by the Ara-rhizotron to point out tardy effects, affecting simultaneously shoot development and root architecture of plants grown in a population. We discuss its advantages in relation to root development and physiology, as well as its possible applications.     

一株耐盐甲基杆菌Methylobacterium sp.W-1的分离及促生潜能研究

[背景] 多种甲基杆菌属细菌对寄主植物有促生作用,其分布区域较广。筛选具有耐盐与促生特性的甲基杆菌属菌株可为微生物菌肥的开发提供依据。[目的] 从新疆乌尔禾地区盐渍土壤中筛选耐盐促生菌,对其培养基成分进行优化及促生能力进行研究,为微生物菌肥的开发提供依据。[方法] 采用阿须贝无氮培养基筛选耐盐菌株,对菌株进行基因序列分析及生理生化测定,采用平板试验法初步研究该菌对拟南芥的生长影响。[结果] 筛选出中度耐盐菌株W-1,经鉴定为甲基杆菌属（Methylobacteriumsp.）。菌株生长最佳无机盐为NaCl,最适浓度为1%–3%,最高耐受浓度达7%。最佳氮源为酸水解酪蛋白,产生长素最高达33.53 mg/L。溶磷能力达28.71 mg/L。菌株W-1接种拟南芥幼苗后叶绿素a和叶绿素b含量均高于对照组,同时对其根系发育有显著的促进作用。[结论] 菌株W-1促生性能显著,可为生物肥料制备提供菌种资源。     

Molecules and morphology: comparative developmental genetics of the Brassicaceae

For approximately 20 years Arabidopsis has been a model system to investigate developmental and physiological questions in plant biology, leading to the identification of genes and genetic systems involved in many processes. Extending ideas arising from knowledge of developmental genetic systems in Arabidopsis to other species of the Brassicaceae will require the application of genomics technologies developed in Arabidopsis and the establishment of additional genetic systems and resources in other species. Morphological variation in all plant organs, as well as in growth habit, mating systems, and physiology are represented in the breadth of Brassicaceae species offering ample opportunity to investigate the molecular basis of morphological evolution in this family. In addition, the frequent recent hybridization events in Brassica and Arabidopsis facilitate study of this pervasive force in the evolution of all plants.     

Auxiliary Photosynthetic Functions of Arabidopsis thaliana—Studies in vitro and in vivo

An improved cultivation system for Arabidopsis thaliana was developed, allowing advanced biochemical studies in vitro and in vivo of this important model plant. Highly functional Arabidopsis thylakoids were isolated and used to study both basic and regulatory photosynthetic functions with the aim to create a platform for the characterization of mutants deficient in auxiliary proteins. Light-induced proteolytic degradation of the D1 protein could be followed and shown to be a subsequent event to photoinactivation of electron transport. The phosphorylation and dephosphorylation of thylakoid proteins resembled that seen in spinach leaves although phospho-CP43 revealed an unusual regulatory behavior.     

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.     

Overexpression of AtMHX in tobacco causes increased sensitivity to Mg<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, and Cd<Superscript>2+</Superscript> ions,induction of V-ATPase expression,and a reduction in plant size

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis. Electrophysiological analysis showed that it exchanges protons with Mg²⁺, Zn²⁺, and Fe²⁺ ions. The physiological impact of AtMHX was examined so far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. Here we investigated the impact of AtMHX on growth, response to different metals, and metal accumulation of mature tobacco plants, as well as Arabidopsis plants in which we overexpressed this transporter. The analyses were carried out in hydroponic growth-systems, in which the mineral composition could be effectively controlled, and the metal content of roots could be examined. Transformed tobacco plants showed necrotic lesions and apical burnings upon growth with increased levels of Mg²⁺, Zn²⁺, and Cd²⁺ ions. This suggested that AtMHX can carry in planta not only Mg²⁺ and Zn²⁺ ions, as previously deduced based on observations in tissue-culture, but also Cd²⁺ ions. Transformed plants of both tobacco and Arabidopsis showed a reduction in plant size. However, the overall response of Arabidopsis to AtMHX overexpression was minor. No change was detected in the mineral content of any organ of the transgenic tobacco or Arabidopsis plants. The necrotic lesions in tobacco resembled those seen in plants with perturbed proton balancing, raising the assumption that AtMHX can affect the proton homeostasis of cells. In agreement with this assumption, the transformed tobacco plants had increased expression and activity of the vacuolar H⁺-ATPase. The relative significance of AtMHX for metal and proton homeostasis still has to be elucidated.     

<Emphasis Type="Italic">PCC1</Emphasis>: a merging point for pathogen defence and circadian signalling in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Using a cDNA-array we identified expressed sequence tag 163B24T7 as rapidly up-regulated in Arabidopsis thaliana (L.) Heynh. after pathogen exposure. Detailed expression analysis revealed that the corresponding gene is up-regulated not only after exposure to avirulent Pseudomonas syringae pv. tomato but also to virulent strains. This up-regulation is dependent on functional salicylic acid defence-signalling pathways. Moreover, we found the gene was circadian-regulated, showing peaks of expression at the end of the day. Using plants overexpressing the clock component CCA1, we showed that the PCC1 gene is regulated by the inner clock of Arabidopsis. Accordingly, we named the gene PCC1, for pathogen and circadian controlled. PCC1 is a member of a novel family of six small polypeptides in Arabidopsis. A functional role for PCC1 in plant defence was demonstrated since plants overexpressing PCC1 are resistant against normally virulent oomycetes. Thus, PCC1 demonstrates a potential interrelationship between pathogen and circadian signalling pathways.Abbreviations cfu Colony-forming units - EST Expressed sequence tag - Pst Pseudomonas syringae pv. tomato - TAIR The Arabidopsis information resource     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Rays,intrusive growth,and storied cambium in the inflorescence stems of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

Arabidopsis thaliana is a model plant used in analysis of different aspects of plant growth and development. Under suitable conditions, secondary growth takes place in the hypocotyl of Arabidopsis plants, a finding which helps in understanding many aspects of xylogenesis. However, not all developmental processes of secondary tissue can be studied here, as no secondary rays and intrusive growth have been detected in hypocotyl. However, results presented here concerning the secondary growth in inflorescence stems of Arabidopsis shows that both secondary rays and intrusive growth of cambial cells can be detected, and that, in the interfascicular regions, a storied cambium can be developed.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a Model System for Graft Union Development in Homografts and Heterografts

Homografting of Arabidopsis thaliana scions on stocks of A. thaliana and heterografting on other species were used to study the compatibility and the ontogeny of graft union formation. Highly compatible homografting with scions of young leafy inflorescence stems was obtained on stocks of inflorescence stems growing from large 3-month-old A. thaliana plants. Histologic analysis revealed four developmental stages of graft union formation in Arabidopsis homografting: (1) development of a necrotic layer, (2) callus proliferation in the grafted scion, (3) differentiation of new vascular tissues within the scion, and (4) a full vascular graft union formation between the scion and the stock. Vascular connections were formed within the callus bridge between rootstocks and scions 15 days after grafting. Heterografts of Arabidopsis on two members of Brassicaceae, cabbage (Brassica) and radish (Raphanus), showed partial incompatible interaction with a lower level of vascular differentiation. Arabidopsis grafting on tomato (Solanaceae) rootstock showed complete incompatibility and limited noncontinuous differentiation of new vascular tissues that did not cross the scion/stock boundary. Although lacking scion/stock vascular connections, Arabidopsis scions grafted onto tomato rootstock flowered and produced seeds. This may indicate some nonvascular functional connections between the two plants, probably of parenchyma cells, further emphasizing the usefulness of Arabidopsis as a model plant for studying various levels of the complicated scion/stock relationships expressed in grafting biology. Experiments with dye transport in the xylem showed that although in general there was an agreement between the histologic study and dye transport, in Arabidopsis homografts water transport frequency was lower than functional and histologic compatability. We conclude that homografting and heterografting of Arabidopsis inflorescence stems is a convenient and reproducible method for studying the fundamental cellular genetic and molecular aspects of grafting biology.     

Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.Abbreviations CaMV Cauliflower Mosaic Virus - 2,4D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEG polyethylene glycol     

Heterologous expression of IAP1, a seed protein from bean modified by indole-3-acetic acid,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6× histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC–MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species. Alexander Walz and Claudia Seidel contributed equally to the paper.     

Flavonols are not essential for fertilization in Arabidopsis thaliana

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.Abbreviations CHS chalcone synthase - TLC thin-layer chromatography     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.