Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed.     

Gene note. Isolation of a cDNA corresponding to a low temperature- and ABA-responsive gene encoding a putative glycine-rich RNA-binding protein in Solanum commersonii

A cDNA encoding a putative RNA-binding glycine-rich protein, SCRGP-1, was isolated from the wild potato species Solanum commersonii. The amino acid sequence of the deduced protein revealed that the protein contains a consensus RNA-binding domain and has a glycine-rich carboxy-terminal domain. The corresponding gene is induced by low temperature, ABA, wounding, and drought in both Solanum commersonii and Solanum tuberosum. The response of this putative RNA-binding protein gene to low temperature and ABA treatments in Solanum sp. suggests that the SCRGP-1 protein might participate in the adaptation process leading to increased freezing tolerance.     

Molecular cloning and expression analysis of a RGA-like gene responsive to plant hormones in Brassica napus

DELLA proteins are negative regulators of GA-induced growth. DELLA protein family is characterized by a DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. A full-length cDNA encoding a putative DELLA protein with high sequence homology to Arabidopsis thaliana RGA (AtRGA), designated as BnRGA, was isolated from Brassica napus. The full-length cDNA of BnRGA contained a 1,740 bp open reading frame (ORF) encoding a precursor protein of 579 amino acid residues. Comparative and bioinformatics analyses revealed that BnRGA showed a high degree of homology with DELLA proteins and contained the DELLA domain, TVHYNP domain, VHIID domain and RVER domain. Using real-time PCR, the expression patterns of BnRGA and two our previously isolated genes, BnGID1a and BnSLY1 in B. napus, were analyzed by adding exogenous gibberellins acid-3 (GA₃), GA biosynthetic inhibitor paclobutrazol (PAC) and abscisic acid (ABA). The results showed that the expression of BnGID1a and BnSLY1 was down-regulated after treated by GA₃ and induced by PAC and ABA. These results suggest that the expression of BnGID1a and BnSLY1 may be negatively regulated by the level of endogenous GA in B. napus. Moreover, BnRGA was not significantly regulated by GA₃, PAC and ABA in the low concentrations. These suggest that GA-GID1-SCF-DELLA complex may have a mechanism of self-regulation, thereby preserving the stability of the expression level of BnRGA in B. napus.     

Molecular cloning of BPL1, a pectate lyase-like gene in Brassica napus

Pectate lyase (EC 4.2.2.2) is an enzyme involved in the maceration and soft rotting of plant tissue via degradation of cell wall in organisms. Plants as well as bacteria and fungi are capable of producing pectate lyases. Here we report the cloning of a novel full-length cDNA of pectate lyase gene, designated BPL1, from Brassica napus by rapid amplification of cDNA ends. BPL1 cDNA is 1787 bp containing a 1503 bp ORF encoding a 500 amino acid protein precursor. The protein precursor has a potential signal peptide with 22 amino acids. Alignment of sequences shows that there are some extremely conserved amino acids among pectate lyase-like proteins from different plant species, and novel C-terminal domains are found in Arabidopsis and Brassica. Phylogenetic analysis of 50 pectate lyase-like proteins from various species demonstrates the obvious distinction among pectate lyase-like proteins from plants, bacteria and fungi, which are subsequently clustered into three groups. The cloning of BPL1 enables us to explore its diverse roles in higher plants and potential application in crop improvement.     

A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.     

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.