Actin-based motility of endosomes is linked to the polar tip growth of root hairs

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells. We demonstrate their presence at the growing tip and differential distribution upon cessation of tip growth. We also show that both the presence of endosomes as well as their rapid movements within the tip region depends on an intact actin cytoskeleton and involves actin polymerization. In conclusion, actin-propelled endosomal motility is tightly linked to the polar tip growth of root hairs.     

Spatial and temporal analysis of the local response to wounding

We studied the local response to wounding in Arabidopsis thaliana leaves using a two-step microarray analysis. A microarray containing 3500 cDNA clones was first screened to enrich for genes affected by wounding in the immediate vicinity of the wound (4 h post wounding). 359 non-redundant putative wound responsive genes were then spotted on a smaller wound-response array for detailed analysis of spatial expression (local, adjacent and systemic), timing of expression (0.5, 4, 8, 17 h), and effect of hormone treatments (methyl jasmonate, ethylene and abscisic acid). Our results show that genes that respond early at the site of the wound also respond throughout the plant, with similar kinetics. Early-induced genes which respond systemically encode predominantly signal transduction and regulatory factors (36%), and the expression of many of them is also controlled by methyl jasmonate (about 35% of the 36%). Genes specific to the wound site and the wounded leaf have a slower response to wounding and are mainly metabolic genes. At the wound, many genes of the lignin biosynthesis pathway were induced. In silico analysis of the 5′ promoter regions of genes affected by wounding revealed G-box-related motifs in a significant proportion of the promoters. These results show that the establishment of a systemic response to wounding is a priority for the plant, and that the local response at the wound site is established later. Ethylene and abscisic acid are involved in the local response, regulating repression of photosynthetic genes and expression of drought responsive genes respectively.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.     

Biosynthesis of monolignols. Genomic and reverse genetic approaches

The biosynthesis of monolignols is one of the most studied pathways of plant natural product biosynthesis. However, the pathway has recently undergone considerable revision, and it would appear that our understanding of the exact routes for synthesis of the building blocks of lignin and lignans is still not fully understood. Early studies of in vitro enzyme specificity failed to appreciate the catalytic promiscuity of some of the enzymes of the monolignol pathway, and the evolving model of a metabolic grid for monolignol biosynthesis may fail to appreciate the possible extent of metabolic channeling within the pathway. New approaches to the study of monolignol biosynthesis include genomics, advanced cellular imaging techniques, and transgenic manipulation. This article summarizes the use of these approaches to gain a better understanding of the operation of a complex metabolic pathway.     

Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana

PCR amplification of cDNA prepared from poly(A)⁺ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis.     

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.     

蔗糖合酶基因AtSUS3干涉后对拟南芥角果发育的影响

蔗糖合酶(SuSy)是植物蔗糖代谢关键酶之一,该研究利用反向遗传学手段,采用RNAi技术抑制拟南芥中AtSUS3基因的表达,测定纯系转基因植株的抽苔率,并对酶活性、糖含量等指标以及糖代谢相关基因的表达进行了检测,探讨SuSy在植物发育中的作用。结果显示:(1)转基因拟南芥的抽苔平均早于野生型植株2～3d,且优先3～4d完成抽苔。(2)开花后生长天数对角果蔗糖和葡萄糖含量有显著影响,而对果糖含量影响不显著;开花后5d时,野生型株系的葡萄糖含量显著高于转基因株系SUS3-2,至15d时,两种转基因株系葡萄糖含量均显著低于野生型株系。(3)开花后生长天数对SuSy、SPS、INV的活性均有显著影响,随开花时间延长,野生型株系SuSy活性显著低于转基因株系,而SPS和INV则相反。(4)AtSUS3基因沉默对其他糖代谢基因有不同程度的影响,开花后5d时,转基因植株的角果中AtCesA1、AtCesA7和AtCINV1的表达量较野生型都有所增加;开花后15d时,转基因植株的角果中AtCesA1、AtCesA7的表达量较野生型高,而AtCINV、AtCwINV的表达量比野生型低。研究表明,拟南芥AtSUS3基因沉默后,在正常生长条件下未造成植株发育异常,同时还可能通过同源家族中其他SuSy的表达水平增加,促进了该酶及糖代谢相关基因整体水平的增加,有助于角果成熟。     

Isoenzymes and flow cytometry for the assessment of true-to-typeness of calluses and cell suspensions of barrel medic prior to regeneration

A successful exploitation of in vitro tools for breeding and for the understanding of gene functioning requires the regeneration of true-to-type plants and experiments were therefore performed with several genotypes of Medicago truncatula (J5, TRV25, TR122) to characterize mother plants and regenerating tissues. Each sample was assessed by flow cytometry and, whatever the genotype and regeneration pathway, a divergent phenotype was systematically linked to an abnormal nuclear DNA content. All samples assessed were classed according to their flow cytometry profiles into normal (true-to-type) material, aneuploids, endoreduplicated tissues, tetraploids, mixoploids and senescent tissues. Deviating calluses failed to regenerate or gave rise to infertile, non-viable plants. In turn, all tissues with non true-to-type flow cytometry profiles were examined in terms of isoenzyme banding patterns compared to the mother plants. Esterases, Peroxidases and Leucine aminopeptidase appeared to be the best isozyme systems to show differences between the original genotypes but also between diverging materials and the mother plants. Interestingly, such differences were more often qualitative (presence or absence of bands) than quantitative (i.e. differences in colour intensity of bands) thereby making easier an accurate distinction between genotypes. Peroxidases were prone to variation with culture medium and tissue age. The results stressed the importance of using more than one approach when undertaking the characterisation of materials as, for some of the genotypes analysed, differences compared to the respective mother plants could be shown with flow cytometry that were not reflected in a different banding pattern with isoenzymes.     

Relationship between allelic state of T-DNA and DNA methylation of chromosomal integration region in transformed <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

T-DNA insertions are currently used as a tool to introduce, or knock out, specific genes. The expression of the inserted gene is frequently haphazard and up to now, it was proposed that transgene expression depends on the site of insertion within the genome, as well as the number of copies of the transgene. In this paper, we show that the allelic state of a T-DNA insertion can be at the origin of epigenetic silencing. A T-DNA insertional mutant was characterized to explore the function of AtBP80a′, a vacuolar sorting receptor previously associated with germination. Seeds homozygous for the T-DNA do not germinate, but this can be overcome by a cold treatment and maintained by the following generations. The non-germinating phenotype is only observed in homozygous seed produced by heterozygous plants indicating that it is correlated with the allelic state of the T-DNA in parental lines. Analysis of the region between the T-DNA insertion and the ATG codon of atbp80a′ showed that cytosine methylation is highly enhanced in chromatin containing the T-DNA. Data presented here show that an unpaired DNA region during meiosis could be at the origin of a de novo cytosine methylation mechanism.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Identification of more than 200 glucose-responsive <Emphasis Type="Italic">Arabidopsis</Emphasis> genes none of which responds to 3-O-methylglucose or 6-deoxyglucose

The response of some plant genes to glucose analogues 3-O-methylglucose (3OMG) or 6-deoxyglucose (6DOG) has been cited as evidence for metabolism-independent glucose signalling. To analyse such signalling using a genetic approach, we sought to identify Arabidopsis glucose-responsive genes which also respond to 3OMG and 6DOG in seedlings. Microarray analysis of gene expression in glucose-treated seedlings and RT-PCR analysis of glucose-treated leaf sections identified more than 200 glucose-responsive genes, but none responded to 3OMG or 6DOG. These data together with other published data on individual genes fail to identify any Arabidopsis sugar-responsive genes which also respond to 3OMG or 6DOG.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana

A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced. The cDNA contains the complete reading frame for the precursor of the Pchlide reductase. The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat. The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli. An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A. thaliana.When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined. Similar light effects have been described previously for other angiosperms. In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light.     

Defensin gene family in <Emphasis Type="Italic">Medicago truncatula:</Emphasis> structure,expression and induction by signal molecules

A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research’s (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.     

苜蓿叶绿素合成酶(CHLG)编码基因的表达及对生物量的影响

叶绿素由一系列酶促反应催化生成,其最后一步是叶绿素合成酶(chlorophyll synthase,CHLG)催化合成叶绿素a和叶绿素b.该实验主要以蒺藜苜蓿(Medicago truncatula)生态型R108和两种不同秋眠级紫花苜蓿(Medicago sativa)为材料,采用生物信息学方法对蒺藜苜蓿叶绿素合成酶...