The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium -transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo . These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.

Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized. Two highly homologous RAD51 genes were found to be present. Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns. Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless RAD51 genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants.

ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5′ end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40 s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules.

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

Arabidopsis Snf1‐related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD‐protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1‐cullin‐F‐box) ubiquitin ligase subunit, which suppresses the skp1‐4 mitotic defect in yeast, interacts with the PRL1‐binding C‐terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1‐binding proteasomal protein, α4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co‐immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal α‐subunits show nuclear co‐localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co‐purification of epitope‐ tagged SKP1/ASK1 with SnRK, cullin and proteasomal α‐subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and α4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo . The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium -mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

Summary We have used a [3H] thymidine incorporation assay and microscopic observation in order to reassess recently published data dealing with the response of tobacco protoplasts to phytohormones, lipochitooligosaccharides and peptides ( Harling et al . 1997 ; Hayashi et al . 1992 ; Miklashevichs et al . 1996 ; Miklashevichs et al . 1997 ; Röhrig et al . 1995 ; Röhrig et al . 1996 ; van de Sande et al . 1996 ; Walden et al . 1994 ). These proliferation assays reveal that, in contrast to published data, isolated cells of the investigated mutant plant lines axi159 ( Hayashi et al . 1992 ; Walden et al . 1994 ), axi4/1 ( Harling et al . 1997 ) and cyi1 ( Miklashevichs et al . 1997 ), which were generated by activation T‐DNA tagging, were unable to grow in the absence of auxin or cytokinin. Furthermore, lipochitooligosaccharides which play a key role in the induction of nodules on roots of legumes were unable to promote auxin‐ or cytokinin‐independent cell division in tobacco protoplasts as claimed by Röhrig et al . (1995 , 1996 ). The finding of van de Sande et al . (1996 ) that ENOD40 confers tolerance of high auxin concentration to wild‐type tobacco protoplasts was also reinvestigated. The results of our investigations show that we were unable to reproduce the proliferation data presented in this study, which were obtained by counting tobacco protoplast‐derived cells undergoing division. In total, none of the published data on phytohormone‐independent division of tobacco cells could be reproduced.

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro. AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

A protocol for establishment and high-frequency Agrobacterium mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines, The promoter, pOp, consists of lac operators cloned upstream of a minimal promoter, No expression was detected from this promoter when placed upstream of a beta-glucuronidase (GUS) reporter gene in transgenic plants, Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4, This factor comprised transcription-activation domain-II from Gal4 of Saccharomyces cerevisiae fused to a mutant lac-repressor that binds its operator with increased affinity, When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter, The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed, The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention, It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant. cak1At encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At. cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-beta II isoenzyme involved in human insulin signaling.

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, and is responsible for the synthesis of abundant P5CS mRNA in dividing cells. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signaling. Inhibition of protein synthesis by cycloheximide affects the induction of AtP5CS mRNA accumulation. Mutations aba1, abi1 and axr2, affecting ABA synthesis and perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signaling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes. Promoter regions of the AtP5CS genes has been cloned and the sequence of 1 kb. fragments has been determined. Sequence analysis of the promoter regions of the AtP5CS genes revealed the presence of putative conserved cis-acting elements, including a G-box. cDNA of AtP5CS1 has been placed under the control of different plant promoter sequences in binary expression vectors. Genetic transformation of tobacco and alfalfa resulted in the regeneration of transgenic plants containing increased internal proline content. Studies on the effect of proline accumulation on salt and drought tolerance is in progress.

The Arabidopsis CPD gene encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light-grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down-regulated by brassinolide in both dark and light. Steady-state mRNA levels of a CPD promoter-driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C-6 oxidation pathways of brassinolide, carrying C-22 and C-23 side-chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.

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 Δ1-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, and is responsible for the synthesis of abundant P5CS mRNA in dividing cells. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signaling. Inhibition of protein synthesis by cycloheximide affects the induction of AtP5CS mRNA accumulation. Mutations abal, abil and axr2, affecting ABA synthesis and perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signaling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes. Promoter regions of the AtP5CS genes has been cloned and the sequence of 1 kb. fragments has been determined. Sequence analysis of the promoter regions of the AtP5CS genes revealed the presence of putative conserved cis-acting elements, including a G-box. cDNA of AtP5CS1 has been placed under the control of different plant promoter sequences in binary expression vectors. Genetic transformation of tobacco and alfalfa resulted in the regeneration of transgenic plants containing increased internal proline content. Studies on the effect of proline accumulation on salt and drought tolerance is in progress.

A T-DNA tagged Arabidopsis locus, A37, identified by a promoter-trap aph(3′)II reporter gene fusion expressed in calli and roots, encodes an ortholog of evolutionarily conserved SNZ growth arrest response proteins. Gene A37 is located on chromosome 3–35, lacks introns, and shares considerable sequence identity with HEVER1 from rubber tree, SLEXORFA-1 from Stellaria longipes, SNZ1 from yeast, and SNZ-homologs from bacteria and archaebacteria. Southern DNA hybridization and physical mapping data show that A37 is a single copy gene, but sequence similarity to expressed sequence tags (ESTs) suggests that at least two other SNZ-homologs are present in Arabidopsis. The A37 gene is abundantly expressed in cultured callus tissues and at lower levels in leaves, stems and roots. In the promoter-trap locus a37, the T-DNA-linked aph(3′)II reporter gene is transcribed oppositely to the A37 gene by a cryptic promoter located 0.52 kb upstream of the A37 coding region. Promoter deletion studies with uidA-reporter gene constructs show that the cryptic promoter consists of regulatory sequences located in both promoter and transcribed regions of the A37 gene that activate transcription only in roots. The a37 promoter trap is thus controlled by transcriptional regulatory sequences that function as an active promoter only in linkage with a promoterless reporter gene introduced artificially into the Arabidopsis genome by a T-DNA tag.