ABSTRACT
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
Subject(s)
Fungi/genetics , Luminescent Proteins/genetics , Amino Acid Sequence , Animals , Biosynthetic Pathways/genetics , Caffeic Acids , Cell Line , Cell Line, Tumor , Female , Gene Duplication/genetics , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Sequence Alignment , Xenopus laevisABSTRACT
Jasmonates are plant hormones that are involved in the regulation of different aspects of plant life, wherein their functions and molecular mechanisms of action in wheat are still poorly studied. With the aim of gaining more insights into the role of jasmonic acid (JA) in wheat growth, development, and responses to environmental stresses, we have generated transgenic bread wheat plants overexpressing Arabidopsis 12-OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3), one of the key genes of the JA biosynthesis pathway. Analysis of transgenic plants showed that AtOPR3 overexpression affects wheat development, including germination, growth, flowering time, senescence, and alters tolerance to environmental stresses. Transgenic wheat plants with high AtOPR3 expression levels have increased basal levels of JA, and up-regulated expression of ALLENE OXIDE SYNTHASE, a jasmonate biosynthesis pathway gene that is known to be regulated by a positive feedback loop that maintains and boosts JA levels. Transgenic wheat plants with high AtOPR3 expression levels are characterized by delayed germination, slower growth, late flowering and senescence, and improved tolerance to short-term freezing. The work demonstrates that genetic modification of the jasmonate pathway is a suitable tool for the modulation of developmental traits and stress responses in wheat.
Subject(s)
Arabidopsis/metabolism , Arabidopsis/physiology , Freezing , Triticum/metabolism , Triticum/physiology , Arabidopsis/genetics , Cyclopentanes/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Oxylipins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/physiology , Triticum/geneticsABSTRACT
In light of recent climate change, with its rising temperatures and precipitation changes, we are facing the need to increase the valuable crop's tolerance against unfavorable environmental conditions. Emmer wheat is a cereal crop with high nutritional value. We investigated the possibility of improving the stress tolerance of emmer wheat by activating the synthesis of the stress hormone jasmonate by overexpressing two genes of the jasmonate biosynthetic pathway from Arabidopsis thaliana, ALLENE OXIDE SYNTHASE (AtAOS) and OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3). Analyses of jasmonates in intact and mechanically wounded leaves of non-transgenic and transgenic plants showed that the overexpression of each of the two genes resulted in increased wounding-induced levels of jasmonic acid and jasmonate-isoleucine. Against all expectations, the overexpression of AtAOS, encoding a chloroplast-localized enzyme, does not lead to an increased level of the chloroplast-formed 12-oxo-phytodienoic acid (OPDA), suggesting an effective conversion of OPDA to downstream products in wounded emmer wheat leaves. Transgenic plants overexpressing AtAOS or AtOPR3 with increased jasmonate levels show a similar phenotype, manifested by shortening of the first and second leaves and elongation of the fourth leaf, as well as increased tolerance to osmotic stress induced by the presence of the polyethylene glycol (PEG) 6000.
Subject(s)
Arabidopsis , Triticum , Triticum/genetics , Osmotic Pressure , Arabidopsis/metabolism , Plant Leaves/metabolism , Plants, Genetically Modified/geneticsABSTRACT
Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.
Subject(s)
Firefly Luciferin/metabolism , Nicotiana/genetics , Plants, Genetically Modified/metabolism , Caffeic Acids/metabolism , Fungi/genetics , Fungi/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.