Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.

Improved methods to detect GTP-binding proteins from plants.

Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of moss Funaria hygrometrica and coleoptiles of corn (Zea mays) and sorghum (Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer poly clonal anti-Gq (common) antibodies and finally the detection of G-proteins by amplification. In addition to the q-subunit of heterotrimeric G-proteins (M r 41-43 kDa), a small molecular weight class « 30 kDa) was also detected by anti-Gq (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparent K,n of the NAD has been determined to be approximately 1.5 ~ for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2-27-fold. One to three polypeptides representing the q-subunit of heterotrimeric G-proteins of (M,. 37-43 kDa) were ADP-ribosylated in all three plants. The anti-Gp (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.

Screening of monoclonal antibodies to scarce and labile enzymes: A functional immuniassay for isolating monoclonal antibodies to NADPH: nitrate reductase.

A functional immunoassay, that has proved very useful, is described for screening and identifying monoclonal antibodies (McAbs) against scarce and labile enzymes. This method does not require purified enzyme or antigen and it has been successfully applied to isolate three hybridomas secreting McAbs to NADPH:nitrate reductase from the chloronema cells of the moss Funaria hygrometrica. Briefly, the protocol involves: adsorption of murine antibodies from hybridoma supernatants by rabbit antimouse IgG antibody pre-adsorbed to Staphylococcus aureus cells (SAC), reaction with crude extract for 15 min, sedimentation of the SAC complex by centrifugation and measurement of residual enzymatic activity in the supernatant. A depletion indicates the presence of antibodies that bind to the active enzyme. The method is rapid, sensitive and versatile enough to be used to isolate McAbs with exquisite specificities. The three isolated McAbs recognized nitrate reductase protein in a conformation-independenat nd/or a conformation-dependenmt anner.