Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

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.


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 -affinity of nsHbs-1 is very high mostly because of an extremely low O -dissociation rate constant resulting in that nsHbs-1 apparently do not release O after oxygenation. Thus, it is possible that the function of nsHbs-1 is in vivo other than O -transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in activity of rice nsHbs-1 has been poorly analyzed. An analysis for vivo in vivo 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 s TB1. The rice Hb1 and Hb2 amino acid E cherichia coli sequence, tertiary structure and rate and equilibrium constants for the reaction of O are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly . As an initial approach to test this hypothesis we analyzed the in vivo effect of the synthesis of rice Hb1 and Hb2 in the recombinant .

Amendments from Version 1
We incorporated some of the references suggested by Dr. Matilla into the revised version of the article and indicated in the legend to Figure 1 that molecular sizes and molecular masses correspond to the Hb cDNAs and proteins analyzed in this work.

Introduction
Non-symbiotic hemoglobins (nsHbs) are O 2 -binding proteins widely distributed in land plants, including rice 1 . The nsHbs are classified into type 1 and type 2 (nsHbs-1 and nsHbs-2, respectively) based on sequence similarity and O 2 -affinity 2,3 . The O 2 -affinity of nsHbs-1 is very high mostly because of an extremely low O 2 -dissociation (k off ) rate constant 3-5 resulting in that nsHbs-1 apparently do not release O 2 after oxygenation 6,7 . In contrast, the O 2 -affinity of nsHbs-2 is moderate mostly because of a moderate to high k off rate constant for O 2 , thus apparently nsHbs-2 easily release O 2 after oxygenation 2,3,6,7 . Hence, it is possible that the in vivo function of nsHbs-1 is other than O 2 -transport and that nsHbs-2 function in vivo as O 2 -carriers.
Five copies (hb1 to 5) of the nshb gene have been detected in the rice genome, which are differentially expressed in embryonic and vegetative organs from plants growing under normal and stress conditions [8][9][10][11] . 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 2 -transport, O 2 -sensing, NO-scavenging and redox-signaling 6,12,13 . However, the in vivo activity of rice nsHbs has been poorly analyzed 12 . An in vivo analysis for rice nsHbs is essential to elucidate the biological function(s) of these proteins. An approach to analyze the in vivo activity of nsHbs is generating knock out rice for individual nshb genes, however 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 Hb1 4 and Hb2 14 are nsHbs-1 that have been generated in recombinant E. coli TB1. The rice Hb1 and Hb2 amino acid sequence 4 , tertiary structure 15 and rate and equilibrium constants for the reaction of O 2 4,14 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. Our results showed that synthesis of rice Hb1 and Hb2 inhibited 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.

Results and discussion
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 Hb1 4 , rice Hb2 4 , soybean Lba 16 , cowpea LbII 17 and Vitreoscilla Hb 18 cDNAs ( Figure 1A). Likewise, 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). This evidence indicated that rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb were synthesized by recombinant E. coli TB1. Figure 2 shows that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb inhibited the recombinant E. coli TB1 growth. This was unexpected for soybean Lba, cowpea LbII and Vitreoscilla Hb because these proteins would promote cell growth due to their O 2 -transport activity 17,19-22 . However, under the conditions tested in this work apparently soybean Lba, cowpea LbII and Vitreoscilla Hb affected some aspects of the recombinant E. coli TB1 metabolism, possibly owed to the constitutive expression of these proteins into the host cells. Synthesis of rice Hb1 inhibited the recombinant E. coli TB1 growth similarly (∼37%) to the synthesis of soybean Lba, cowpea LbII and Vitreoscilla Hb. This observation suggests that rice Hb1 could function in vivo similarly to O 2 -carrying Hbs. Likewise, synthesis of rice Hb2 also inhibited the recombinant E. coli TB1 growth. However, growth inhibition was stronger (∼61%) when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. This observation suggests that rice Hb2 could function in vivo by scavenging O 2 , possibly owing to its extremely low k off rate constant for O 2 14 .

Conclusions
Results presented in this work suggest that in spite of the high similarity between rice Hb1 and Hb2 these proteins could function differently in vivo. In order to elucidate the apparent metabolic effects generated by the synthesis of rice Hb1 and Hb2, future work might focus on the physiological and biochemical characterization of recombinant E. coli TB1. This may include measuring cell respiratory rates and identifying cell proteins and metabolites using oximetry and proteomic and metabolomic approaches, respectively. Results from these analyses could provide valuable information to understand the in vivo function of rice nsHbs.
Author contributions EAS and RAP conceived the study. EAS executed the experiments. RAP prepared the first draft of the manuscript. EAS and RAP revised the draft manuscript and have agreed to the final content.

Competing interests
No competing interests were disclosed.
I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements
Authors wish to express their gratitude to Dr. Dale A. Webster (Illinois Institute of Technology, USA) for kindly providing the pUC18::VHb plasmid. 1.

5.
and Lba (Fig. 1B) are confusing to the reader; (ii) I would eliminate from Fig. 2 the results of LBA, LbII and HBv growth (include as data not shown) because the main importance of this work are the results concerning nb1 and nbII; (iii) "... these proteins would promote cell growth due to their O -transport activity"; this conclusion is based in old results and is very risky; this growth promotion should also be referred to higher plants?; please discuss; and (iv) I repeat, some actual references must be also included into discussion.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.