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 O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O2after oxygenation. Thus, it is possible that theinvivofunction of nsHbs-1 is other than O2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however theinvivoactivity of rice nsHbs-1 has been poorly analyzed. Aninvivoanalysis 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 recombinantEscherichiacoliTB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O2are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarlyinvivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinantE.coliTB1 growth. Effect of the synthesis of the O2-carrying soybean leghemoglobina, cowpea leghemoglobin II andVitreoscillaHb in the recombinantE.coliTB1 growth was also analyzed as an O2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII andVitreoscillaHb inhibits the recombinantE.coliTB1 growth and that growth inhibition was stronger when recombinantE.coliTB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differentlyin vivo.
Highlights
Non-symbiotic hemoglobins are O -binding proteins widely 2 distributed in land plants, including rice[1]
Electrophoretic analysis of the PCR reaction and EcoRI- and NcoI-double digestions showed that plasmids isolated from recombinant E. coli TB1 contained inserts corresponding to the rice Hb1, rice Hb2, soybean leghemoglobin a (Lba), cowpea leghemoglobin II (LbII) and Vitreoscilla Hb cDNAs (Figure 1A)
Analysis by SDS-PAGE showed that rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb existed in the soluble extracts of recombinant E. coli TB1 (Figure 1B)
Summary
Non-symbiotic hemoglobins (nsHbs) are O -binding proteins widely 2 distributed in land plants, including rice[1]. The O2-affinity of nsHbs-2 is moderate mostly because of a moderate to high koff rate constant for O2, apparently nsHbs-2 release O2 after oxygenation[2,3,6]. 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 O2-transport, O2-sensing, NOscavenging and redox-signaling[6,11]. 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 Hb212 are nsHbs-1 that have been generated in recombinant E. coli TB1
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