Abstract
Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O 2-affinity of nsHbs-1 is very high mostly because of an extremely low O 2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O 2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O 2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Es cherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O 2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O 2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O 2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lb a, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.
Highlights
Non-symbiotic hemoglobins are O2-binding proteins widely distributed in land plants, including rice[1]
Electrophoretic analysis of the PCR reaction and EcoRI- and NcoIdouble digestions showed that plasmids isolated from recombinant E. coli TB1 contained inserts corresponding to the rice Hb14, rice Hb24, soybean leghemoglobin a (Lba)[16], cowpea LbII17 and Vitreoscilla Hb18 cDNAs (Figure 1A)
Analysis by SDS-PAGE showed that rice Hb1, rice Hb2, soybean Lba, cowpea leghemoglobin II (LbII) and Vitreoscilla Hb existed in the soluble extracts of recombinant E. coli TB1 (Figure 1B)
Summary
Non-symbiotic hemoglobins (nsHbs) are O2-binding proteins widely distributed in land plants, including rice[1]. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation (koff) rate constant[3,4,5] resulting in that nsHbs-1 apparently do not release. Based on the available information on the properties of rice nsHbs and data from the analysis of other plant and non-plant Hbs, it was proposed that rice nsHbs could exhibit a variety of functions in vivo, including O -transport, O -sensing, NO-scavenging and redox-signaling[6,12,13]. An approach to analyze the in vivo activity of nsHbs is generating knock out rice for individual nshb genes, this is complicated because of the existence of five copies of nshb in the rice genome. An alternative approach to analyze the in vivo activity of rice nsHbs is examining individual rice nsHbs in a heterologous system, such as recombinant Escherichia coli. Rice Hb14 and Hb214 are nsHbs-1 that have been generated in recombinant
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