Man Mohan Johri

Occurrence and biosynthesis of auxin in protonema of the moss Funaria hygrometrica

Abstract We have investigated the presence of auxin and the ability of chloronema cells to synthesize indole-3-acetic acid (IAA) in axenic protonema cell cultures of the moss Funaria hygrometrica. The endogenous level of auxin activity was 4 and 7μg-IAA equivalents/kg in caulonema and chloronema cell types, respectively. Based on an indole-α-pyrone fluorometric assay, the level of putative IAA was observed to be 5.0 and 1.9.μg/kg in caulonema and chloronema cells, respectively. [3H]Tryptophan was metabolized into IAA via the indole-pyruvate pathway by intact chloronema cells and also by the cell free homogenates. More [3H]IAA accumulated when homogenates from cells pre-grown at low cell densities ( 0.5 mg/ml) were used. Since the activities of peroxidase and IAA-oxidase are known to be high at high cell densities, the lack of accumulation of radioactivity in IAA at high densities can be attributed to a high level of IAA-oxidizing enzymes. Our results suggest a possible relationship between IAA accumulation and caulonema differentiation.

Ammonium represses NADPH-nitrate reductase in the moss Funaria hygrometrica

In chloronema cell suspension cultures of the Funaria hygrometrica, ammonium addition led to a rapid loss of nitrate reductase (NR) activity and a reduction in the levels of NR protein. The mRNA for NR was absent in ammonium-grown cells. Intracellular nitrate accumulation was also suppressed. Ammonium repression involving a transcriptional regulation has been proposed.

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 mossFunaria 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 ofFunaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in theFunaria 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 fromFunaria. 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 theFunaria gene shows extensive homology with CDPKs from higher plants, 73% identity with theFragaria CDPK and 71% identity with CDPK isoform 7 ofArabidopsis. Phylogenetic analysis revealed that theFunaria CDPK is closer to the CDPKs from higher plants like strawberry andArabidopsis as compared to those from lower plants such as the liverwortMarchantia, the green algaChlamydomonas or another mossTortula. 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.

Ca2+ dPKs from the protonema of the moss Funaria hygrometrica: Effect of indole-acetic acid and cultural parameters on the activity of a 44 kDa Ca2+ dPK

Using the in-gel kinase assay, four polypeptides of Mr 44, 48, 63, and 70 kDa showed calcium-dependent phosphorylation in the crude extract of the moss Funaria hygrometrica Hedw. Analysis by SDS-PAGE suggests that these phosphopolypeptides represent the calcium-dependent protein kinases (PK), which could undergo autophosphorylation and phosphorylate casein. Temporal changes in the activity of PK in cells grown under different conditions were detectable using the in-gel kinase assay. The 44 kDa Ca 2+ dPK showed an increase in activity in cells grown either in the presence of auxin, at pH 5, or when starved of nitrate. This PK is shown to share epitopes with calmodulin suggesting that it could be a PK with calmodulin-like domain. The in-gel kinase assay, its possible applications and the existence of multiple Ca 2+ dPKs in moss are discussed.

Partial purification and characterization of cyclic AMP phosphodiesterases from Funaria hygrometrica

Abstract Cyclic AMP phosphodiesterase activity from chloronema cells of the moss Funaria hygrometrica has been purified 86-fold. This preparation contains more than a single phosphodiesterase activity since it hydrolyzes both 3′, 5′-cAMP and 2′, 3′-cAMP. It shows a high affinity (Km = 8.7 ± 2.8 μM) and a low affinity (Km = 1.38 ± 0.37 mM) component for 3′,5′-cAMP as substrate, but only one component (Km = 0.58 ± 0.11 mM) for 2′,3′-cAMP as substrate. The low Km component is competitively inhibited by 2′,3′-cAMP (Ki = 0.63 ± 0.04 mM). It has a molecular weight of 85,000, its activity is Zn2+ dependent, and is optimal at pH 5.5–6.5. Two peaks of enzyme activity are obtained upon further purification of the low Km component of cAMP phosphodiesterase by affinity chromatography. Peak I activity, eluted by buffer, shows an acidic pH optimum, is insensitive to methylxanthines and imidazole, and copurifies with nucleotidases. Peak II activity binds to the affinity column and is eluted by 100 μ m cAMP. This activity is free from nucleotidases, produces only 5′-AMP from cAMP, is maximally active at pH 7.5–8.0, is inhibited by methylxanthines, and is stimulated by imidazole. Thus peak II activity has properties similar to the cAMP phosphodiesterases present in animal tissues, while peak I resembles the phosphodiesterases from higher plants. The present work is the first clear evidence for the existence of both plant-like and animal-like phosphodiesterases in a plant development system.

Purification and characterization of a Ca2+-dependent/calmodulin-stimulated protein kinase from moss chloronema cells

We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the mossFunaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 (AM free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Underin vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

Screening of monoclonal antibodies to scarce and labile enzymes: A functional immunoassay 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 mossFunaria hygrometrica. Briefly, the protocol involves: adsorption of murine antibodies from hybridoma supernatants by rabbit antimouse IgG antibody pre-adsorbed toStaphylococcus 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-independent and/or a conformation-dependent manner.

Regulation of nitrate reductase by molybdenum in chloronema cell cultures of the moss Funaria hygrometrica

The relationship between NADPH: nitrate reductase (NR) activity and NR protein was investigated in the chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw. When cells were transferred to fresh medium containing nitrate, other major elements and Hellers trace elements, the NR activity was induced. It increased to a maximum on the third day then decreased to undetectable levels by the ninth day. A single polypeptide of about 116 kDa (NR protein) was detected on the immunoblots probed with NR-specific monoclonal antibody. The increase and decrease of NR activity were associated with similar changes in the level of NR protein only during the first 6 days of culture. Subsequently, the NR protein increased again to reach a second peak on the ninth day. This increase seems to be due to lack of adequate molybdenum in the medium. Upon adding molybdenum, the nonfunctional NR did not accumulate and the increase and decrease in NR activity were mediated by the increase and decrease in NR protein. The profiles of partial activities of NR protein and of xanthine dehydrogenase activity also suggest that molybdenum becomes limiting five or six days after culture. The production of non-functional NR is suggested to be due to lack of end product repression.

Cyclic AMP phosphodiesterases of Funaria hygrometrica

Abstract High affinity cAMP phosphodiesterase (PDE) activity has been purified ( ca 474-fold) from the chloronema cells of the moss Funaria hygrometrica . The activity in the 15 000 g supernatant was precipitated at 40–80% saturation with (NH 4 ) 2 SO 4 and separated by DEAE-cellulose chromatography into two peaks of activity A and B with pH optima at pH 5.5 and 7.5, respectively. When rechromatographed on DEAE-cellulose, the PDE B activity redistributed into two peaks (A′and B′). The recovery of PDE activity in the latter peaks suggests a conversion of alkaline PDE into acid PDE. The phosphodiesterase activity B was further resolved by affinity chromatography on a cAMP—agarose column into PDE I and PDE II. The latter was free from nucleotidases, was Ca 2+ -dependent and showed only a high-affinity component towards cAMP as the substrate, the K m being 11.8 ± 4.8 μM.

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 mossFunaria 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 polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (Mr 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentKm of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (Mr 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (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.