Many antimicrobial peptides (AMPs) have been identified in plants. These peptides are highly divergent at the primary sequence level and vary in their hierarchical structures. Some common biochemical features include ...Many antimicrobial peptides (AMPs) have been identified in plants. These peptides are highly divergent at the primary sequence level and vary in their hierarchical structures. Some common biochemical features include the ability to form disulfide bonds, tandemly repeated amino acid sequences and a net charge at pH 7. Unusual Cysteine containing repeats has been identified in several plant seed storage proteins that may act as AMPs. We identified a Cys repeat within a vicilin (seed storage protein) of a wild legume, Centrosema virginianum. Cleavage of the vicilin protein during germination would generate a vicilin derived Cys peptide (VDCP). We investigated the antimicrobial properties of this VDCP and compared its efficacy as an antimicrobial agent to VDCPs from other species. We developed transgenic tobacco plants that expressed cloned sequences encoding the Cysteine repeat unit from C. virginianum, Theobroma cacao and Gossypium hirsutum. Extracts from fully expanded leaves were tested for antimicrobial activity against a fungal pathogen, Botrytis cinerea. The Cys motif from C. virginianum was also expressed in two E. coli cell lines (reducing or oxidizing cytoplasm) and peptide fusion protein fractions were tested for antimicrobial activity against a battery of fungal strains. The unique Cysteine repeat single unit from C. virginianum exhibited antimicrobial properties greater than or equal to the antimicrobial activity associated with expression of the multiple Cys-repeat VDCPs from G. hirsutum or T. cacao in transgenic tobacco. When expressed in bacteria, a C. virginianum VDCP fusion protein exhibited antifungal activity against 3 of the 4 fungi tested. Although the primary role of seed storage proteins is to provide a pool of amino acids and nitrogen for germinating seeds and developing plantlets, it is likely that seed storage protein proteolytic products also provide beneficial antimicrobial properties during germination and young plantlet development.展开更多
Several potentially practical biochemical processes in plant systems still remain hidden, especially the NADH-glutamate dehydrogenase (GDH) synthesis of nongenetic code-based RNA that optimizes crop nutritious yield b...Several potentially practical biochemical processes in plant systems still remain hidden, especially the NADH-glutamate dehydrogenase (GDH) synthesis of nongenetic code-based RNA that optimizes crop nutritious yield by degrading superfluous genetic code-based RNA. In continued characterization of the biochemistry of cowpea grain yield, GDH was purified by electrophoresis from seeds of cowpea treated with solutions of stoichiometric mixes of mineral salts. The GDH was made to synthesize RNAs in the amination (α-KG/NADH/</span><span><span></span><span style="font-family:""><span style="font-family:Verdana;">) and then in the deamination (L-Glu/NAD</span><sup><span style="font-family:Verdana;">+</span></sup><span style="font-family:Verdana;">) direction. The initial product RNAs were captured and sequenced. The grand challenge was to discover the specific molecular roles of the redox enzyme in the optimization of cowpea grain yields. In the amination direction, the GDH hexamers synthesized plus-RNA, but in the deamination direction</span></span><span style="font-family:Verdana;">,</span><span style="font-family:""><span style="font-family:Verdana;"> they synthesized minus-RNA. The plus-RNAs and minus-RNAs were homologous to about the same numbers of different mRNAs encoding the key enzymes that regulate photosynthesis;saccharide biochemistry and glycolysis;phenylpropanoid biosynthesis;nodulation nitrogen fixing processes;dehydrin drought and glutathione environmental stress resistance processes;purine, pyrimidine, DNA, RNA and essential amino acid biosynthesis;storage protein vicilin accumulation;isoflavone earliness of cowpea maturity;peroxidase synthesis of lignin and sequestration of CO</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;"> to enrich soil organic carbon contents;triglyceride physiology in the biosynthesis of bioactive compounds that render cowpea resistant to insects and fungi;etc</span></span><span style="font-family:Verdana;">.</span><span style="font-family:Verdana;">, all of which constitute the GDH chemical pathways for discrimination of biochemical, physiological, metabolic, genetic reactions;and optimization of cowpea dry grain yields. Each stoichiometric mix of mineral salts produced optimally yielding biochemical variant of purple hull cowpea;the K</span><span style="font-family:""> </span><span style="font-family:Verdana;">+</span><span style="font-family:""> </span><span style="font-family:Verdana;">K</span><span style="font-family:""> </span><span style="font-family:Verdana;">+</span><span style="font-family:""> </span><span style="font-family:Verdana;">K mix was spectacular because it increased the grain yield to 7598 kg from the 3644 kg</span><span style="font-family:""><span style="font-family:Verdana;">·</span><span style="font-family:Verdana;">ha</span><sup><span style="font-family:Verdana;">-1</span></sup> </span><span style="font-family:Verdana;">in the control cowpea. Optimized nutritious staple crop yield buttresses food security. The synthesis of plus-RNA in amination and minus-RNA in deamination is an economic tactical plan in biochemistry for the selection of superfluous mRNAs that would be degraded to assure the survival of cowpea growing under unfavorable environmental conditions.展开更多
The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea (Pisum sativum) is complex, resulting from cleavage (processing) of precursors expressed from multiple copies of genes enco...The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea (Pisum sativum) is complex, resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin, the major storage globulins. Translation in vitro of mRNAs hybrid-selected from mid-maturation pea seed RNAs by denned vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed; the derived polypep tides were of comparable sizes to those observed in vivo. The feasibility of transcribing mENA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone, providing a method for determining polypeptide products of an expressed sequence. This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.展开更多
文摘Many antimicrobial peptides (AMPs) have been identified in plants. These peptides are highly divergent at the primary sequence level and vary in their hierarchical structures. Some common biochemical features include the ability to form disulfide bonds, tandemly repeated amino acid sequences and a net charge at pH 7. Unusual Cysteine containing repeats has been identified in several plant seed storage proteins that may act as AMPs. We identified a Cys repeat within a vicilin (seed storage protein) of a wild legume, Centrosema virginianum. Cleavage of the vicilin protein during germination would generate a vicilin derived Cys peptide (VDCP). We investigated the antimicrobial properties of this VDCP and compared its efficacy as an antimicrobial agent to VDCPs from other species. We developed transgenic tobacco plants that expressed cloned sequences encoding the Cysteine repeat unit from C. virginianum, Theobroma cacao and Gossypium hirsutum. Extracts from fully expanded leaves were tested for antimicrobial activity against a fungal pathogen, Botrytis cinerea. The Cys motif from C. virginianum was also expressed in two E. coli cell lines (reducing or oxidizing cytoplasm) and peptide fusion protein fractions were tested for antimicrobial activity against a battery of fungal strains. The unique Cysteine repeat single unit from C. virginianum exhibited antimicrobial properties greater than or equal to the antimicrobial activity associated with expression of the multiple Cys-repeat VDCPs from G. hirsutum or T. cacao in transgenic tobacco. When expressed in bacteria, a C. virginianum VDCP fusion protein exhibited antifungal activity against 3 of the 4 fungi tested. Although the primary role of seed storage proteins is to provide a pool of amino acids and nitrogen for germinating seeds and developing plantlets, it is likely that seed storage protein proteolytic products also provide beneficial antimicrobial properties during germination and young plantlet development.
文摘Several potentially practical biochemical processes in plant systems still remain hidden, especially the NADH-glutamate dehydrogenase (GDH) synthesis of nongenetic code-based RNA that optimizes crop nutritious yield by degrading superfluous genetic code-based RNA. In continued characterization of the biochemistry of cowpea grain yield, GDH was purified by electrophoresis from seeds of cowpea treated with solutions of stoichiometric mixes of mineral salts. The GDH was made to synthesize RNAs in the amination (α-KG/NADH/</span><span><span></span><span style="font-family:""><span style="font-family:Verdana;">) and then in the deamination (L-Glu/NAD</span><sup><span style="font-family:Verdana;">+</span></sup><span style="font-family:Verdana;">) direction. The initial product RNAs were captured and sequenced. The grand challenge was to discover the specific molecular roles of the redox enzyme in the optimization of cowpea grain yields. In the amination direction, the GDH hexamers synthesized plus-RNA, but in the deamination direction</span></span><span style="font-family:Verdana;">,</span><span style="font-family:""><span style="font-family:Verdana;"> they synthesized minus-RNA. The plus-RNAs and minus-RNAs were homologous to about the same numbers of different mRNAs encoding the key enzymes that regulate photosynthesis;saccharide biochemistry and glycolysis;phenylpropanoid biosynthesis;nodulation nitrogen fixing processes;dehydrin drought and glutathione environmental stress resistance processes;purine, pyrimidine, DNA, RNA and essential amino acid biosynthesis;storage protein vicilin accumulation;isoflavone earliness of cowpea maturity;peroxidase synthesis of lignin and sequestration of CO</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;"> to enrich soil organic carbon contents;triglyceride physiology in the biosynthesis of bioactive compounds that render cowpea resistant to insects and fungi;etc</span></span><span style="font-family:Verdana;">.</span><span style="font-family:Verdana;">, all of which constitute the GDH chemical pathways for discrimination of biochemical, physiological, metabolic, genetic reactions;and optimization of cowpea dry grain yields. Each stoichiometric mix of mineral salts produced optimally yielding biochemical variant of purple hull cowpea;the K</span><span style="font-family:""> </span><span style="font-family:Verdana;">+</span><span style="font-family:""> </span><span style="font-family:Verdana;">K</span><span style="font-family:""> </span><span style="font-family:Verdana;">+</span><span style="font-family:""> </span><span style="font-family:Verdana;">K mix was spectacular because it increased the grain yield to 7598 kg from the 3644 kg</span><span style="font-family:""><span style="font-family:Verdana;">·</span><span style="font-family:Verdana;">ha</span><sup><span style="font-family:Verdana;">-1</span></sup> </span><span style="font-family:Verdana;">in the control cowpea. Optimized nutritious staple crop yield buttresses food security. The synthesis of plus-RNA in amination and minus-RNA in deamination is an economic tactical plan in biochemistry for the selection of superfluous mRNAs that would be degraded to assure the survival of cowpea growing under unfavorable environmental conditions.
文摘The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea (Pisum sativum) is complex, resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin, the major storage globulins. Translation in vitro of mRNAs hybrid-selected from mid-maturation pea seed RNAs by denned vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed; the derived polypep tides were of comparable sizes to those observed in vivo. The feasibility of transcribing mENA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone, providing a method for determining polypeptide products of an expressed sequence. This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.
