Csaba Koncz

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

SummaryA “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.

Brassinosteroids Rescue the Deficiency of CYP90, a Cytochrome P450, Controlling Cell Elongation and De-etiolation in Arabidopsis

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development.

Polyamines: molecules with regulatory functions in plant abiotic stress tolerance

Early studies on plant polyamine research pointed to their involvement in responses to different environmental stresses. During the last few years, genetic, transcriptomic and metabolomic approaches have unravelled key functions of different polyamines in the regulation of abiotic stress tolerance. Nevertheless, the precise molecular mechanism(s) by which polyamines control plant responses to stress stimuli are largely unknown. Recent studies indicate that polyamine signalling is involved in direct interactions with different metabolic routes and intricate hormonal cross-talks. Here we discuss the integration of polyamines with other metabolic pathways by focusing on molecular mechanisms of their action in abiotic stress tolerance. Recent advances in the cross talk between polyamines and abscisic acid are discussed and integrated with processes of reactive oxygen species (ROS) signalling, generation of nitric oxide, modulation of ion channel activities and Ca2+ homeostasis, amongst others.

EIN3-Dependent Regulation of Plant Ethylene Hormone Signaling by Two Arabidopsis F Box Proteins: EBF1 and EBF2

The plant hormone ethylene regulates a wide range of developmental processes and the response of plants to stress and pathogens. Genetic studies in Arabidopsis led to a partial elucidation of the mechanisms of ethylene action. Ethylene signal transduction initiates with ethylene binding at a family of ethylene receptors and terminates in a transcription cascade involving the EIN3/EIL and ERF families of plant-specific transcription factors. Here, we identify two Arabidopsis F box proteins called EBF1 and EBF2 that interact physically with EIN3/EIL transcription factors. EBF1 overexpression results in plants insensitive to ethylene. In contrast, plants carrying the ebf1 and ebf2 mutations display a constitutive ethylene response and accumulate the EIN3 protein in the absence of the hormone. Our work places EBF1 and EBF2 within the genetic framework of the ethylene-response pathway and supports a model in which ethylene action depends on EIN3 protein stabilization.

Methods in Arabidopsis research

One of the ways to make consistent progress in a particular field of biology consists in choosing a good model system on which to focus the experimental efforts of the scientific community. It has taken a long time for scientists interested in various aspects of the life of plants to reach some sort of consensus. With the advent and impact of molecular biology, the small weed Arabidopsis is now the object of rapidly growing scientific attention. Since it is reasonable to assume that the general molecular mechanisms that are responsible for the physiological, cellular and biochemical properties of plants will be essentially conserved in all plants, it follows that these mechanisms should also operate in Arabidopsis and hence that its genome should contain most of the genes that we need to know about if we want to understand the genetic determination of the life processes in plants.Arabidopsis has a small genome and well documented genetic studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous or heterologous genes are available. It is therefore realistic and desirable to aim at exploring the genome of this plant in very great detail. As will be illustrated in this book all the elements for such a grand strategy are in place.More and more scientists are therefore willing to accept the obvious and very real practical disadvantages resulting from its small size when experiments call for the isolation of proteins, membranes, subcellular fractions etc, in order to benefit from its extraordinary experimental advantages as a model system in molecular genetics. One can safely predict that in the next decade studies with Arabidopsis will provide major breakthroughs in our understanding of most aspects of plant physiology and developmental biology. The importance of this knowledge for plant breeding and therefore for a sustainable highly productive agriculture cannot be overestimated. We therefore expect that this book will provide valuable guidelines to all those who are planning experiments aimed at understanding various aspects of plant growth, productivity and interactions with the environment. The book offers a wealth of methodical and theoretical information as well as valuable references. It should be of use to students, teachers, as well as advanced researchers and those breeders who want to use molecular techniques in breeding.

Differential expression of two P5CS genes controlling proline accumulation during salt‐stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.

Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants

The Ri plasmid A4 of Agrobacterium rhizogenes contains within its T‐DNA genetic information able to trigger root formation in infected plants. Tobacco plants regenerated from transformed roots display the hairy root (hr) syndrome. We show that DNA fragments containing the rol B locus alone are able to induce root formation both in tobacco and kalanchoe tissues. The rol A and the rol C loci by themselves are also able to induce root formation in tobacco but not in kalanchoe. This capacity to induce root formation in either host is greatly increased when the rol A and/or C loci are combined with the rol B locus. Root induction is shown to be correlated with the expression of the rol loci. Transgenic plants exhibit all the characteristics of the hairy root syndrome only when all three loci are present and expressed. Although the activity of the rol encoded functions is synergistic, each of them appears to independently influence host functions involved in the determination of root differentiation.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T‐DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T‐DNA‐tagged genomic loci showed that T‐DNA integration resulted in target site deletions of 29–73 bp. In those cases where integrated T‐DNA segments turned out to be smaller than canonical ones, the break‐points of target deletions and T‐DNA insertions overlapped and consisted of 5–7 identical nucleotides. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred‐DNA (T‐DNA)‐mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T‐DNA, was mapped to the position of the ch‐42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch‐42 mutants indicated allelism. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis

Here we report on a lipid‐signalling pathway in plants that is downstream of phosphatidic acid and involves the Arabidopsis protein kinase, AGC2‐1, regulated by the 3′‐phosphoinositide‐dependent kinase‐1 (AtPDK1). AGC2‐1 specifically interacts with AtPDK1 through a conserved C‐terminal hydrophobic motif that leads to its phosphorylation and activation, whereas inhibition of AtPDK1 expression by RNA interference abolishes AGC2‐1 activity. Phosphatidic acid specifically binds to AtPDK1 and stimulates AGC2‐1 in an AtPDK1‐dependent manner. AtPDK1 is ubiquitously expressed in all plant tissues, whereas expression of AGC2‐1 is abundant in fast‐growing organs and dividing cells, and activated during re‐entry of cells into the cell cycle after sugar starvation‐induced G1‐phase arrest. Plant hormones, auxin and cytokinin, synergistically activate the AtPDK1‐regulated AGC2‐1 kinase, indicative of a role in growth and cell division. Cellular localisation of GFP‐AGC2‐1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The agc2‐1 knockout mutation results in a reduction of root hair length, suggesting a role for AGC2‐1 in root hair growth and development.