Showing papers in "Plant Molecular Biology in 2003"

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

Abstract: The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response.

Abstract: WRKY proteins are a recently identified class of DNA-binding proteins that recognize the TTGAC(C/T) W-box elements found in the promoters of a large number of plant defense-related genes. With oligo molecules containing the W-box sequences as probes, we detected a number of WRKY DNA-binding activities in Arabidopsis that were induced by salicylic acid (SA). Search of the Arabidopsis genome identifies 72 genes encoding proteins characteristic of WRKY DNA-binding transcription factors that can be divided into three groups based on the number and structures of their WRKY zinc-finger motifs. Northern blotting analysis revealed that 49 of the 72 AtWRKY genes were differentially regulated in the plants infected by an avirulent strain of the bacterial pathogen Pseudomonas syringae or treated by SA. These pathogen- and/or SA-regulated WRKY genes can be further categorized into groups based on their expression patterns in both wild-type plants and mutants defective in defense signaling pathways. Inspection of the 5' sequences upstream of the predicated translation start sites revealed a substantial enrichment of W boxes in the promoters of pathogen- and/or SA-regulated Arabidopsis WRKY genes. These results suggest that defense-regulated expression of WRKY genes involves extensive transcriptional activation and repression by its own members of the transcription factor superfamily.

Cassava biology and physiology

Abstract: Cassava or manioc (Manihot esculenta Crantz), a perennial shrub of the New World, currently is the sixth world food crop for more than 500 million people in tropical and sub-tropical Africa, Asia and Latin America. It is cultivated mainly by resource-limited small farmers for its starchy roots, which are used as human food either fresh when low in cyanogens or in many processed forms and products, mostly starch, flour, and for animal feed. Because of its inherent tolerance to stressful environments, where other food crops would fail, it is often considered a food-security source against famine, requiring minimal care. Under optimal environmental conditions, it compares favorably in production of energy with most other major staple food crops due to its high yield potential. Recent research at the Centro Internacional de Agricultura Tropical (CIAT) in Colombia has demonstrated the ability of cassava to assimilate carbon at very high rates under high levels of humidity, temperature and solar radiation, which correlates with productivity across all environments whether dry or humid. When grown on very poor soils under prolonged drought for more than 6 months, the crop reduce both its leaf canopy and transpiration water loss, but its attached leaves remain photosynthetically active, though at greatly reduced rates. The main physiological mechanism underlying such a remarkable tolerance to drought was rapid stomatal closure under both atmospheric and edaphic water stress, protecting the leaf against dehydration while the plant depletes available soil water slowly during long dry periods. This drought tolerance mechanism leads to high crop water use efficiency values. Although the cassava fine root system is sparse, compared to other crops, it can penetrate below 2 m soil, thus enabling the crop to exploit deep water if available. Leaves of cassava and wild Manihot possess elevated activities of the C4 enzyme PEP carboxylase but lack the leaf Kranz anatomy typical of C4 species, pointing to the need for further research on cultivated and wild Manihot to further improve its photosynthetic potential and yield, particularly under stressful environments. Moreover, a wide range in values of Km (CO2) for the C3 photosynthetic enzyme Rubisco was found among cassava cultivars indicating the possibility of selection for higher affinity to CO2, and consequently higher leaf photosynthesis. Several plant traits that may be of value in crop breeding and improvement have been identified, such as an extensive fine root system, long leaf life, strong root sink and high leaf photosynthesis. Selection of parental materials for tolerance to drought and infertile soils under representative field conditions have resulted in developing improved cultivars that have high yields in favorable environments while producing reasonable and stable yields under stress.

Identification of a copper transporter family in Arabidopsis thaliana.

Abstract: Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.

Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis

Abstract: Osmotic stress-induced accumulation of proline, an important protective osmolyte in higher plants, is dependent on the expression of delta1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) enzymes that catalyze the rate-limiting steps of proline biosynthesis and degradation, respectively. Proline metabolism is modulated by differential regulation of organ specific expression of PDH and duplicated P5CS genes in Arabidopsis. Stimulation of proline synthesis by abscisic acid (ABA) and salt stress correlates with a striking activation of P5CS1 expression. By contrast, P5CS2 is only weakly induced, whereas PDH is inhibited to different extent by ABA and salt stress in shoots and roots of light-grown plants. Proline accumulation and light-dependent induction of PSCS1 by ABA and salt stress is inhibited in dark-adapted plants. During dark adaptation P5CS2 is also down-regulated, whereas PDH expression is significantly enhanced in shoots. The inhibitory effect of dark adaptation on PSCS1 is mimicked by the steroid hormone brassinolide. However, brassinolide fails to stimulate PDH, and inhibits P5CS2 only in shoots. Proline accumulation and induction of P5CS1 transcription are simultaneously enhanced in the ABA-hypersensitive prl1 and brassinosteroid-deficient det2 mutants, whereas P5CS2 shows enhanced induction by ABA and salt only in the det2 mutant. In comparison, the prl1 mutation reduces the basal level of PDH expression, whereas the det2 mutation enhances the inhibition of PDH by ABA. Regulation of P5CS1 expression thus appears to play a principal role in controlling proline accumulation stimulated by ABA and salt stress in Arabidopsis.

Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress.

Abstract: Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.

Transgene integration, organization and interaction in plants

Abstract: It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana.

Abstract: In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al, 2000) Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development

Regulated expression of Arabidopsis shaker K+ channel genes involved in K+ uptake and distribution in the plant.

Abstract: Potassium is the most abundant cation in the cytosol, where it plays a role in basal functions. Rapid uptake and distribution of K+ is therefore required for plant growth. Three members of the so-called Shaker K+ channel gene family (nine genes identified in Arabidopsis) play a role in these transports: AKT1, SKOR and AKT2. The encoded proteins are involved in K+ uptake by the root, K+ secretion into the xylem sap and K+ transport in the phloem tissues, respectively. Using the GUS reporter strategy, we have found that another Shaker channel gene, AtKC1, is expressed in epidermal and cortical cells in roots (supporting the hypothesis of a role in K+ uptake from the soil, together with AKT1), and in trichomes and hydathodes in leaves. These four genes were selected for expression studies, and two-hybrid experiments were performed for channels displaying overlapping expression patterns. The data support the hypothesis that physical interactions could occur in planta between AtKC1 and AKT1, and between AKT1 and AKT2. Potassium deficiency, salt stress and hormonal treatments (ABA, BA, 2,4-D) were found to differentially affect channel mRNA levels, each channel displaying its own regulation pattern. The most prominent effects were (1) a strong induction of AtKC1 transcript accumulation in leaves (hydathodes, trichomes and leaf epidermis) in response to NaCl treatment, suggesting a key role of the protein in adaptation to saline conditions, and (2) a strong decrease in SKOR transcript levels by hormones, supporting the hypothesis that K+ secretion into the xylem sap is under tight hormonal control.

Genome-wide gene expression in an Arabidopsis cell suspension.

Abstract: Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method.

Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism.

Abstract: The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.

Heat-tolerant basmati rice engineered by over-expression of hsp101.

Abstract: Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp101) in imparting thermotolerance to cells, we introduced Arabidopsis thalianahsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T1 lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T2 transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance.

Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana).

Abstract: In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.

Mapping quantitative physiological traits in apple (Malus x domestica Borkh.).

Abstract: Efficient breeding and selection of high-quality apple cultivars requires knowledge and understanding of the underlying genetics The availability of genetic linkage maps constructed with molecular markers enables the detection and analysis of major genes and quantitative trait loci contributing to the quality traits of a genotype A segregating population of the cross between the apple varieties 'Fiesta' (syn 'Red Pippin') and 'Discovery' has been observed over three years at three different sites in Switzerland and data on growth habit, blooming behaviour, juvenile period and fruit quality has been recorded QTL analyses were performed, based on a genetic linkage map consisting of 804 molecular markers and covering all 17 apple chromosomes With the maximum likelihood based interval mapping method, the investigated complex traits could be dissected into a number of QTLs affecting the observed characters Genomic regions participating in the genetic control of stem diameter, plant height increment, leaf size, blooming time, blooming intensity, juvenile phase length, time of fruit maturity, number of fruit, fruit size and weight, fruit flesh firmness, sugar content and fruit acidity were identified and compared with previously mapped QTLs in apple Although 'Discovery' fruit displayed a higher acid content, both acidity QTLs were attributed to the sweeter parent 'Fiesta' This indicated homozygosity at the acidity loci in 'Discovery' preventing their detection in the progeny due to the lack of segregation

Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage.

Abstract: The NR72.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-beta-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips.

Traumatic resin defense in Norway spruce (Picea abies): Methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase

Abstract: Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae.

A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters.

Abstract: A promoter-trap screen allowed us to identify an Arabidopsis line expressing GUS in the root vascular tissues. T-DNA border sequencing showed that the line was mutated in the caffeic acid O-methyltransferase 1 gene (AtOMT1) and therefore deficient in OMT1 activity. Atomt1 is a knockout mutant and the expression profile of the AtOMT1 gene has been determined as well as the consequences of the mutation on lignins, on soluble phenolics, on cell wall digestibility, and on the expression of the genes involved in monolignol biosynthesis. In this mutant and relative to the wild type, lignins lack syringyl (S) units and contain more 5-hydroxyguaiacyl units (5-OH-G), the precursors of S-units. The sinapoyl ester pool is modified with a two-fold reduction of sinapoyl-malate in the leaves and stems of mature plants as well as in seedlings. In addition, LC-MS analysis of the soluble phenolics extracted from the seedlings reveals the occurrence of unusual derivatives assigned to 5-OH-feruloyl malate and to 5-OH-feruloyl glucose. Therefore, AtOMT1 enzymatic activity appears to be involved not only in lignin formation but also in the biosynthesis of sinapate esters. In addition, a deregulation of other monolignol biosynthetic gene expression can be observed in the Atomt1 mutant. A poplar cDNA encoding a caffeic acid OMT (PtOMT1) was successfully used to complement the Atomt1 mutant and restored both the level of S units and of sinapate esters to the control level. However, the over-expression of PtOMT1 in wild-type Arabidopsis did not increase the S-lignin content, suggesting that OMT is not a limiting enzyme for S-unit biosynthesis.

Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control

Abstract: The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin when exposed to various stress situations such as salt stress and high light intensities. Here, the light regulation of Haematococcus carotenoid biosynthesis was examined. Isolation and characterization of the lycopene β cyclase gene involved in carotenoid biosynthesis was carried out using a functional complementation approach. Subsequently, gene expression of lycopene cyclase, phytoene synthase, phytoene desaturase and carotenoid hydroxylase was analysed in green flagellate cells. All four genes revealed higher transcript levels in response to increased illumination. Not only the induction of astaxanthin biosynthesis but also carotenoid gene expression was found to be correlated with the redox state of the photosynthetic electron transport. In accordance with this result, increased transcript levels for carotenoid biosynthesis genes were detected under both blue and red light conditions. The application of different inhibitors of the photosynthetic electron flow indicated that the photosynthetic plastoquinone pool functions as the redox sensor for the up-regulation of carotenoid biosynthesis genes. These results suggested that in Haematococcus not only the specific astaxanthin pathway but also general carotenoid biosynthesis is subject to photosynthetic redox control.

Salinity stress-tolerant and -sensitive rice (Oryza sativa L.) regulate AKT1-type potassium channel transcripts differently.

Abstract: In the indica rice (Oryza sativa L.) a cDNA was characterized that encoded OsAKT1 homologous to inward-rectifying potassium channels of the AKT/KAT subfamily. Transcript analysis located OsAKT1 predominantly in roots with low abundance in leaves. Cell-specificity of OsAKT expression was analyzed by in situ hybridizations. In roots, strongest signals were localized to the epidermis and the endodermis, whereas lower transcript levels were detected in cells of the vasculature and the cortex. In leaves, expression was detected in xylem parenchyma, phloem, and mesophyll cells. Transcriptional regulation and cell specificity of OsAKT1 during salt stress was compared in rice lines showing different salinity tolerance. In the salt-tolerant, sodium-excluding varieties Pokkali and BK, OsAKT1 transcripts disappeared from the exodermis in plants treated with 150 mM NaCl for 48 h but OsAKT1 transcription was not repressed in these cells in the salt-sensitive, sodium-accumulating variety IR29. Significantly, all lines were able to maintain potassium levels under sodium stress conditions, while sodium concentrations in the leaves of IR29 increased 5–10-fold relative to the sodium concentration in BK or Pokkali. The divergent, line-dependent and salt-dependent, regulation of this channel does not significantly affect potassium homeostasis under salinity stress. Rather, repression in Pokkali/BK and lack of repression in IR29 correlate with the overall tolerance character of these lines.

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

Abstract: T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5'-upstream regions, coding sequences and 3'-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5'-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.

Allopolyploidy alters gene expression in the highly stable hexaploid wheat

Abstract: Hexaploid wheat (Triticum aestivum) contains triplicated genomes derived from three distinct species. To better understand how different genomes are coordinated in the same nucleus of the hexaploid wheat, we globally compared gene expression of a synthetic hexaploid wheat with its diploid (Aegilops tauschii) and tetraploid (T. turgidum) parents by cDNA-AFLP display. The results suggested that the expression of a significant fraction of genes was altered in the synthetic hexaploid; most appeared to be diminished and some were activated. We characterized nine cDNA clones in details. Cytogenetic as well as genomic sequence analyses indicated that the gene silencing was not due to chromosome/DNA loss but was caused by gene regulation. Northern and RT-PCR divided these genes into three groups: (I) four genes were down-regulated nonspecifically, likely involving both parental orthologues; (II) four genes were down-regulated in an orthologue-dependent manner; (III) one gene was activated specifically in the synthetic hexaploid wheat. These genes were often altered non-randomly in different synthetic hexaploids as well as natural hexaploid wheat, suggesting that many of the gene expression changes were intrinsically associated with polyploidy.

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2(2) from Lycopersicon esculentum.

Abstract: In tomato, infections by tomato mosaic virus are controlled by durable Tm-22 resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-22 resistance gene and the susceptible allele, tm-2. The Tm-22 gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-22 gene survive. The Tm-22 locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-22 sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-22 allele. Between the tm-2 and the Tm-22 polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-22 gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-22 resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-22-encoded resistance is aimed at the Achilles' heel of the virus.

Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Abstract: Putative phosphate transporters have been identified in a barley (Hordeum vulgare L) genomic library by their homology to known phosphate transporters from dicot species The genes designated HORvu;Pht1;1 and HORvu;Pht1;6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Pht1 family of phosphate transporters HORvu;Pht1;1 is expressed exclusively in roots and is strongly induced by phosphate deprivation HORvu;Pht1;6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves In situ hybridization showed that HORvu;Pht1;6 is expressed in the phloem of vascular bundles in leaves and ears In order to study the biochemical properties of HORvu;Pht1;1 and HORvu;Pht1;6, the genes were expressed in transgenic rice (Oryza sativa L) plants under the control of the rice actin promoter and suspension cell cultures were generated Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters The estimated Km for phosphate for cells expressing HORvu;Pht1;1 was 906±082 μM, which is characteristic of a high-affinity transporter The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu;Pht1;1 operates as a H+/H2PO4− symporter In contrast, the estimated Km for phosphate for cells expressing HORvu;Pht1;6 was 385±61 μM, which is characteristic of a low-affinity transporter Taken together, the results suggest that HORvu;Pht1;1 functions in uptake of phosphate at the root surface, while HORvu;Pht1;6 probably functions in remobilization of stored phosphate from leaves

Isoflavonoid biosynthesis and accumulation in developing soybean seeds

Abstract: Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [14C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed.

Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree).

Abstract: Latex of Hevea brasiliensis (Willd. ex A, Juss.) Mull. Arg. (Brazilian rubber tree) contains 30-50% (w/w) of natural rubber (cis-1,4-polyisoprene), which is an important raw material for many industrial uses. In order to gain insights into the molecular events occurring in latex, we analyzed more than 20,000 cDNA-AFLP-based TDFs (transcription-derived fragments) and 1176 ESTs. The results revealed several novel features of the latex transcriptome. First, the repertoire of the genes expressed in latex is unique. Only seven gene families accounted for more than 51% of the latex transcriptome. Among them, two of the most abundant ESTs were the genes encoding rubber particle proteins REF (rubber elongation factor) and SRPP (small rubber particle protein), comprising 29% of the total ESTs. Unexpectedly, several genes involved in the rubber biosynthesis were expressed at low levels in the latex. In fact, genes encoding cis -prenyltransferase (CPT), a potential candidate for rubber polymerase, were not present in the EST pool because of their low expression level. However, we were able to clone four full-length cDNAs by screening the same latex cDNA library used in the EST analysis and confirmed their enzyme activity in vitro. The second most abundant transcripts were defense- or stress-related genes, suggesting that defense is one of the functions of laticifers. Finally, the presence of the non-mevalonate DXP/MEP pathway for IPP synthesis in latex was noted by up-regulation of the 1-deoxy-D-xylulose 5-phosphate synthase gene.

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells.

Abstract: A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the beta-glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the -1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the -1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

A novel male-sterile mutant of Arabidopsis thaliana, faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine.

Abstract: A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, flp1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis

Abstract: The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.

Profiling ethylene-regulated gene expression in Arabidopsis thaliana by microarray analysis.

Abstract: Ethylene-regulated gene expression in leaves of Arabidopsis thaliana was investigated with an expressed sequence tag-based microarray containing about 6000 unique genes. Comparing expression profiles of the ethylene-insensitive mutant etr1-1, the ethylene-constitutive mutant ctr1-1, ethylene-treated wild-type and untreated wild-type plants identified ca. 7% of the investigated genes as ethylene-regulated. Exogenous ethylene treatment and ctr1-1 had similar changes in gene expression, but differences were noted. Ethylene-regulated genes involved in its own biosynthesis and signal transduction pathway were identified. A large number of transcription factors and some putative signaling components were highly regulated by ethylene. Chloroplast structural protein and photosynthetic genes were generally down-regulated. Ethylene appeared to regulate other primary metabolic genes. Plant defense and PR protein genes were differentially regulated, with some genes within this class highly up-regulated. Other ethylene-regulated genes identified were known sugar-, auxin-, wounding- and jasmonic acid-related genes, suggesting the existence of coordinated interactions between ethylene and other hormonal and defense signaling pathways. Although hundreds of potentially important transcriptome changes were identified, the functions of many ethylene-regulated genes remain unknown.

MADS-box genes expressed during tomato seed and fruit development.

Abstract: MADS-box genes in plants are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers. Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development. We have identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development. According to comparisons of their deduced amino acid sequences, they were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12). With the exception of TAGL12, which is expressed near uniformly in every tissue, the other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs. In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity. Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role.