Identification of two chilling-regulated 1-aminocyclopropane-1-carboxylate synthase genes from citrus (Citrus sinensis Osbeck) fruit.

Diurnal change in the temperature below or above 12.5 degrees C hastens the degreening of citrus peel and elicits the phytohormone ethylene production in citrus fruit. Ethylene triggers the degradation of chlorophyll and synthesis of carotenoids in citrus peel. To investigate if ethylene is required for the degreening of citrus peel elicited by low temperatures, we studied the chilling-regulated gene expression of ACC synthase, one of the key enzymes catalyzing ethylene biosynthesis. We isolated and characterized a chilling-inducible 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) gene, CS-ACS1, and a chilling-repressible gene, CS-ACS2, from citrus peel. The CS-ACS1 transcript 1.7 kb in length encodes a polypeptide of 483 amino acids (Mr 54,115, pI 6.63), whereas the CS-ACS2 transcript of 1.8 kb encodes a polypeptide of 477 amino acids (Mr 53,291, pI 6.72). Both genes showed a rapid but transient induction (within 2.4 h) of transcripts upon rewarming after the chilling (4 degrees C) treatment. After 24 h of incubation at room temperature, CS-ACS1 mRNA diminished to an undetectable level, whereas the CS-ACS2 mRNA regained its basal level of expression attained prior to the chilling treatment. Chilling-induced ethylene production and ACC accumulation were also observed upon rewarming. Both genes were also induced by the wound stress (excision). The protein synthesis inhibitor cycloheximide super-enhances the accumulation of both ACS transcripts at room temperature. Molecular analysis of the 3.3 kb genomic DNA of CS-ACS1 revealed that this gene consists of three introns and four exons. The intron 3 is exceptionally large ( 1.2 kb) and shares significant homology with mitochondrial DNA, supporting the intron-late theory.

Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the general phenylpropanoid pathway leading to the production of phenolic compounds with a significant range of biological function. A PAL gene we designated gPAL1, cloned from tobacco, consists of two exons separated by an intron of 1932 bp. Exon I, 398 bp, and exon II, 1747 bp, together encode a polypeptide of 715 amino acids. A putative TATA box and polyadenylation signal are found 144 bp upstream of the initiation codon and 193 bp downstream from the stop codon, respectively. Using various parts of gPAL1 as probes, genomic Southern blots indicated the presence of a small family of PAL genes in the tobacco genome that can be divided into two distinct subfamilies, one consisting of pal1 and pal2 and another of pal3 and pal4. Comparative genomic blot analysis of progenitor species (Nicotiana tomentosiformis and N. sylvestris) indicated that each species contains one PAL gene from each of the subfamilies, suggesting that pal1 and pal3 (or pal2 and pal4) diverged prior to the evolution of N. tabacum. Expression of the PAL gene family was examined using RNA gel blots. PAL transcript levels were significantly higher in flowers and roots than in leaves and stems of mature plants. PAL transcripts accumulate differentially during flower and leaf maturation in that mRNA levels decline during flower maturation but increase during leaf maturation. In leaves, PAL transcripts rapidly accumulated afer wounding.

Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity.

A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs. These wt and mutant ACC synthases were over-expressed in a heterologous E. coli expression system. It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity. Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity. Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely. Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 microM, which is much smaller than that of the corresponding Cdel (280 microM) and closer to that of wt (22 microM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.

Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice.

A receptor-like protein kinase, OsPK10, has been cloned from rice (Oryza sativa). The 2.8 kb cDNA contains an open reading frame capable of encoding a peptide sequence of 824 amino acids. The topological features of the predicted OsPK10 protein include an N-terminal signal peptide, a cysteine-rich extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic domain possessing all the hallmarks of catalytic domains of eukaryotic protein kinases. The cytoplasmic domain was selectively expressed in Escherichia coli and assayed for kinase activity. The results show the protein is capable of autophosphorylation using either ATP or GTP as the phosphate donor. Phosphoamino acid analysis reveals phosphorylation of threonines, consistent with the substrate specificity indicated by sequence motifs in the catalytic core. A single amino acid substitution of Glu for Lys-528 completely abolishes autophosphorylation activity. DNA gel blot analyses suggest that the haploid rice genome contains a single copy of the OsPK10 gene. OsPK10 transcripts appear to be more abundant in shoots than in roots of rice seedlings.

Identification of differentially expressed members of tobacco homeobox families by differential PCR.

As a first step to investigate the functions of homeobox genes in tobacco genetic tumorigenesis, we have used polymerase chain reaction to identify Hot (Homeobox in tobacco) genes that are expressed in tobacco genetic tumors. Five Hot genes that are actively expressed in tobacco genetic tumors are identified. Particularly, Hot1 is profoundly abundant in tumorous tissues, suggesting that it acts as a positive regulator of cell growth and differentiation during genetic tumorigenesis.

Molecular evolutionary analysis of the psbP gene family of the photosystem II oxygen-evolving complex in Nicotiana.

Photosystem II psbP protein of the oxygen-evolving complex is involved in the photosynthetic oxygen evolution in plants. Four psbP polypeptides were detected in Nicotiana tabacum on a two-dimensional gel by immunostaining the proteins with antiserum against the pea psbP Comparison of the protein patterns of psbP from N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, indicated that each of the ancestral parents has contributed a pair of psbP proteins. This was supported by Southern hybridization results, which suggested that psbP in Nicotiana is encoded by a gene family consisting of four members in N. tabacum and two members each in N. glauca, N. langsdorffii, N. sylvestris, and N. tomentosiformis. A scheme of molecular evolution of the psbP genes in Nicotiana is also proposed.

Cloning and characterization of a novel member of protein kinase family from soybean.

The cDNA coding for a novel protein kinase from soybean (Glycine max L.), named GmPK6, was sequenced. The primary sequence of GmPK6 consists of 462 amino acids with an N-terminal sequence similar to the central region of Xenopus U1 snRNP 70K protein, and a C-terminal kinase domain representing structural mosaicism with features diagnostic of both protein serine/threonine and tyrosine kinases in eukaryotic organisms. The GmPK6 gene is expressed as 2.5 kb transcript in a variety of tissues.

Replication of satellite RNA in vitro by homologous and heterologous cucumoviral RNA-dependent RNA polymerases.

The RNA-dependent RNA polymerases (RdRp) of cucumber mosaic virus (CMV) and peanut stunt virus (PSV), members of the cucumovirus group, have been purified from virus infected plants and were used to study RNA synthesis in vitro using different viral RNAs, two cucumoviral satellites, and chimeric satellite cDNA clone transcripts as templates. The results show that solubilized RdRp preparations of CMV and PSV have a high degree of template dependency and catalyze (-) strand synthesis of the homologous cucumoviral RNAs with greater efficiency than the RNAs of heterologous cucumoviruses, although the PSV RdRp exhibits a lesser specificity than the CMV RdRp. On the other hand, both (-) and (+) strands of the satellite RNAs of CMV and PSV are synthesized by their homologous but not by the heterologous viral RdRps, indicating that recognition of satellites by the viral RdRp determines their replicative dependence upon specific helper viruses. Cucumoviral RdRp reactions using chimeric satellite transcripts suggest that the promoter structure for the satellite (-) strand synthesis resides in regions harboring the 3' termini of the two satellites.

Molecular identification of a soybean protein kinase gene family by using PCR.

In this study we report identification of six members of a protein kinase gene family from soybean (Glycine max L.). Two fully degenerate oligonucleotide primers corresponding to two conserved motifs (DLK-PENV and GTHEYLAPE) in the catalytic domains of eukaryotic protein serine/threonine kinases were used in a polymerase chain reaction (PCR) to amplify soybean cDNA. Sequence analysis showed that 28 of the PCR sequences represented six different putative protein serine/threonine kinases. These results not only demonstrate that catalytic domains of protein kinases are highly conserved between plants and other eukaryotes but also suggest that there are multiple genes encoding protein kinases in plants.

Possible repetitive DNA markers for Eusorghum and Parasorghum and their potential use in examining phylogenetic hypotheses on the origin of Sorghum species.

Genomic structures of two major species in section Eusorghum (Sorghum), Sorghum bicolor and Sorghum halepense, and their phylogenetic relationships with a species in section Parasorghum, Sorghum versicolor, were studied by using cloned repetitive DNA sequences from the three species. Of the five repetitive DNA clones isolated from S. bicolor and S. halepense, four produced qualitatively similar hybridization patterns with detectable variations in copy numbers of some of the restriction fragments on the Southern blots of the two genomic DNAs. One clone was shown to be diagnostic for S. halepense. Molecular analysis at the DNA level indicates that S. bicolor and S. halepense have similar but not identical genomes, consonant with differences in karyotypes, meiotic chromosome behaviors, morphology, and physiology of the species. In addition to five repetitive clones isolated from S. bicolor and S. halepense, eight more sequences were cloned from S. versicolor. Nine clones were found to be specific for either S. bicolor and S. halepense or S. versicolor. The remaining four had a moderate to strong homology with sequences present in all Sorghum species studied. We speculate that the genome in the common ancestor of Sorghum has differentiated to give rise to genomes of at least three major chromosome sizes; large, medium, and small, as seen at present. Amplifications, eliminations, rearrangements, and new syntheses of repetitive sequences may have been involved in genome differentiation of these species. The results also suggest that the S. versicolor genome has strongly diverged from the genomes of the two species in section Eusorghum.

Diversity of the protein kinase gene family in rice.

Multiple genes have been found to encode families of protein kinases in animals and yeasts. Little is known of the diversity of protein kinase families in plants. We have used the polymerase chain reaction to identify members of protein kinase gene family in rice. We have cloned eight partial cDNA sequences from which deduced amino acid sequences contained conserved sequences or amino acid residues characteristic of catalytic domains of eukaryotic protein serine/threonine kinases. Our results suggest that there is great complexity in the protein kinase gene family in plants and that protein phophorylation may play an as important role in plants as in in other eukaryotes.