Ashton J. Delauney

Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, Which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.

A Cell Plate–Specific Callose Synthase and Its Interaction with Phragmoplastin

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-β-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate–associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate–specific enzyme is demonstrated by the observations that the green fluorescent protein–CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.

A soybean gene encoding Δ1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated

SummaryWe have isolated several cDNA clones encoding Δ11-pyrroline-5-carboxylate reductase (P5CR, l-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the λ ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encoded a 28 586 Mr polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

Cloned nodulin genes for symbiotic nitrogen fixation

p lant genes encoding nodule-specific proteins (nodulins) are expressed only after the infection of the legume plant with Rhizobium and their expression occurs prior to and independent of the commencement of nitrogen fixation in nodules. Nodulins appear to play three major roles: they participate in the morphogenesis of the nodule including the formation of the endosymbiotic compartment; they are involved in the facilitation of nitrogen fixation and in nodulespecific carbon and nitrogen assimilatory pathways; and they control the compatibility of the endosymbiont bacteria (see Verma & Delauney, 1988; Verma & Nadler, 1984). Since nodulin genes representing these subclasses differ greatly in their temporal and spatial expression, their regulatory mechanisms are not expected to be similar. Nodulin genes were first identified and isolated from soybean (Legocki & Verma, 1980; Verma et al., 1986) and have since been characterized in a number of legumes as listed in Table 1. Some of these genes are common to different legumes while others are species specific (Verma & Nadler, 1984). Since nodulin genes have most likely evolved from pre-existing genes in ancestral plants (Verma & Delauney, 1988; Verma & Nadler, 1984), it is not surprising that some genes that show elevated expression in nodules are also expressed at lower levels in other tissues. Examples include glutamine synthetase in Pisum sativum (Tingey et al., 1987) and possibly Glycine max (Hirel et al., 1987). This enzyme has been shown, however, to be nodule specific in Phaseolus vulgaris (Cullimore et al., 1984) and Medicago sativa (Dunn et al., 1988). A similar situation may exist for the sucrose synthase of G.

A nodule-specific sequence encoding a methionine-rich polypeptide, nodulin-21

Several proteins, nodulins, are specifically expressed in the root nodules of legume plants during symbiosis with Rhizobium species [10]. Genes encoding these proteins have been isolated from many legumes [4], but have to date been most extensively characterized in soybean. Cloned nodulin genes with known functions include leghemoglobin [9], uricase II (nodulin-35 [2]), glutamine synthetase (GS) in Phaseolus vulgaffs [3, 7] and alfalfa [6], and sucrose synthetase (nodulin-100 [12]). Nodulin-26 appears to be an ion channel of the peribacteroid membrane; however, functions for the majority of isolated nodulin genes remain to be elucidated [4], and many cloned nodulin genes are identified solely by numbers based on their molecular weights [ 13, 14]. We recently constructed a soybean nodule cDNA library in a specialized lambda vector, 2 ZaplI [ 11 ] and converted this library into a plasmid (phagemid) library [5] for complementation of specific mutants of Escherichia coli. Several full-length cDNA clones of previously characterized nodulin genes have been isolated from this library (unpublished results). In addition, we isolated a new nodule-specific cDNA clone (pCP2) encoding a 21 kDa nodule-specific polypeptide, nodulin-21, which represents an abundant nodulin. Although this clone was isolated using an E. coli mutant defective in the gene encoding phosphoribosyl pyrophosphate amidotransferase, the first enzyme of the purine biosynthetic pathway, this sequence did not recomplement the mutant and thus may have, in some manner, facilitated the growth of a revertant. The sequence of pCP2 showed no significant homology to other nodulins or any of the sequences in the GenBank data base. As shown in Fig. 1, the pCP2 insert hybridizes strongly to a ca. 1 kb RNA transcript in nodules, but does not hybridize to mRNA from uninfected roots or leaves. The level of hybridization suggests that it is an abundantly transcribed sequence which was not isolated previously by screening of the root nodule libraries based on sequence abundance.

Isolation of plant genes by heterologous complementation in Escherichia coli

The earliest successes in the cloning of plant genes were restricted to those genes whose transcripts represented a high proportion of the mRNA population in particular tissues, such as leghemoglobin transcripts in legume nodules and storage protein transcripts in developing seeds. The subsequent cloning of less abundantly expressed genes has been achieved using a variety of specialized and technically demanding strategies. Generally, it involves purification of the protein of interest. Antibodies raised against the purified protein can then be used to screen cDNA expression libraries directly or to immunoprecipitate polysomal RNA, thereby enriching for the desired sequence before cDNA library construction. Alternatively, microsequencing of the protein may allow the synthesis of corresponding DNA oligomers that may be used to prime the specific synthesis of the appropriate cDNAs, or as hybridization probes to screen cDNA and genomic libraries [2].

Maintenance of a plant line containing an antisense gene and silencing of the target gene following sexual crosses

Abstract Doubly transformed tobacco plants containing both sense and antisense chloramphenicol aceltyltransferase (CAT) genes and showing > 90% inhibition of CAT gene expression were previously produced. One such plant was back-crossed with untransformed tobacco, and progeny were selected in which the CAT and anti-CAT genes were segregated. Progency containing the CAT gene but lacking the antisense construct showed fully restored CAT activity whereas those containing only the antisense genes expressed no CAT activity. The latter were stably maintained through two generations. Recombination of the sense and antisense genes by sexual crossing resulted in efficient antisense RNA-mediated inactivation of the CAT gene.

Internalization of Rhizobium by Plant Cells: Targeting and Role of Peribacteroid Membrane Nodulins

Rhizobium enters the root cells of legume plants through a process resembling endocytosis. To reach appropriate host cells and to avoid any pathogenic reaction, both organisms have evolved an elaborate mechanism involving many signals (Lerouge et al., 1990) that induce specific genes leading to the formation of the infection thread and eventual release of the bacteria inside the plant cell (see for reviews, Verma and Nadler, 1984; Verma and Fortin, 1989; Long, 1989). During this state bacteria remain enclosed in a membrane envelope (peribacteroid membrane, PBM) which is constituted by the components of the plasma membrane of the host cell (Verma et al., 1978) and forms a novel “extracellular compartment”. Physiological “internalization” of this compartment is a key step in acceptance of the foreign organism inside the plant cell. Success of this process allows bacteria to reduce dinitrogen for the benefit of the host plant and establish an endosymbiosis. Rhizobium enclosed in PBM behaves as an “organelle” having many properties in common with mitochondria. The PBM, while keeping bacteria “outside” the host cell cytoplasm and avoiding any pathogenic interaction, allows development of a close contact between the plant cell and bacteria for rapid and efficient exchange of metabolites.