Expression and evolution of functionally distinct haemoglobin genes in plants

Haemoglobin genes have been found in a number of plant species, but the num ber of genes known has been too small to allow effective evolutionary infer ences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including class 1 haemoglobins from cotton, Citrus and tomato, class 2 haemoglobins from cotton, tomato, sugar beet and canola and two hae moglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of sym biotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both cl ass 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we ha ve detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst th e symbiotic haemoglobin from Parasponia groups with the class 1 non-symbiot ic genes. Probably, there have been two independent recruitments of symbiot ic haemoglobins. Although the functions of the two non-symbiotic haemoglobi ns remain unknown, their patterns of expression within plants suggest diffe rent functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functi onally distinct. The class 2 haemoglobin gene (GLB2) is expressed in the ro ots, leaves and inflorescence and can be induced in young plants by cytokin in treatment in contrast to the class 1 gene (GLB1) which is active in germ inating seedlings and can be induced by hypoxia and increased sucrose suppl y, but not by cytokinin treatment.