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Biochemical and Physiological Characterization of Nonsymbiotic Plant Hemoglobins

Leiva, Nélida LU (2014)
Abstract (Swedish)
Popular Abstract in English

People normally associate hemoglobin with blood. It is true that the iron in the center of the hemoglobin molecule makes it excellent at carrying oxygen in the bloodstream from the lungs to every part of the body. However, seventy five years ago a hemoglobin was found in a plant. And plants do not have blood. So what is hemoglobin doing in plants? What are its functions in these sessile, oxygen-producing organisms? The first plant hemoglobin was found in the root nodules of soybean, a legume plant. Since legumes form symbiosis with soil-living bacteria, the hemoglobins found in these plant species became known as symbiotic hemoglobins. Their role is to deliver oxygen to bacteria to stimulate... (More)
Popular Abstract in English

People normally associate hemoglobin with blood. It is true that the iron in the center of the hemoglobin molecule makes it excellent at carrying oxygen in the bloodstream from the lungs to every part of the body. However, seventy five years ago a hemoglobin was found in a plant. And plants do not have blood. So what is hemoglobin doing in plants? What are its functions in these sessile, oxygen-producing organisms? The first plant hemoglobin was found in the root nodules of soybean, a legume plant. Since legumes form symbiosis with soil-living bacteria, the hemoglobins found in these plant species became known as symbiotic hemoglobins. Their role is to deliver oxygen to bacteria to stimulate their growth. In return, the bacteria give nitrogen to the plant. About forty five years later, other types of hemoglobins were found in non-legume plants and hence they became known as nonsymbiotic hemoglobins. These new hemoglobins are present in all plants, including legumes. After analyzing their DNA sequence they were divided into three groups, class-1, class-2, and class-3. So, in total four different types of hemoglobins can be found in plants

Since plants neither have blood nor a system to transport oxygen in the same way as in humans, why do plants need all these different types of hemoglobin? Researchers have been trying to answer this question for many years and some advances have been done. For example, it is clear that all plant species have the class-3 nonsymbiotic hemoglobin, which is very similar to the hemoglobins found in bacteria. Also, it is well accepted that symbiotic hemoglobins evolved from the class-2 group of nonsymbiotic hemoglobin, and that class-2 evolved from the class-1 group. Regarding their role, it seems like nonsymbiotic hemoglobins do not transport oxygen. They hold on to it too strong and do not release it easily. Instead, it has been suggested that these hemoglobins bind nitric oxide, a signaling molecule produced by the plant; for instance, when it is under stress.

In this thesis we have mainly studied the two first classes of nonsymbiotic hemoglobins in sugar beet. We chose this plant because of its economical importance in Sweden and Europe. Also, because it belongs to a family where hemoglobins have not been studied at all. We started by identifying all the hemoglobin genes present in its genome. We found four different hemoglobins, two from class-1 and one from each of class-2 and class-3. Once identified, the gene expression of both class-1 and the class-2 hemoglobins in sugar beet plants was determined. Thanks to the information about their DNA sequence, the three hemoglobin proteins were produced in the lab to be able to characterize them. Their capacity to bind both oxygen and carbon monoxide was analyzed. Additionally, a bacterial hemoglobin and two class-2 nonsymbiotic hemoglobins were expressed in seeds of Arabidopsis thaliana and Lepidium campestre in order to evaluate the effect on their oil content. Finally, a class-1 nonsymbiotic hemoglobin from aspen was characterized and its role as a NO scavenger determined.

Even though some preferential roles have been given to both class-1 and class-2 nonsymbiotic hemoglobins, we conclude that a specific function can not be given to them. A specific role can not be given to a specific class either, as their role can either complement or overlap depending on the plant species. Since the diversification of plant hemoglobins has followed the evolution of plants they need to be studied in a diverse variety of species. Only in this way it will be possible to determine their role in plants. On the other side, the variety of roles these proteins may have open up great research and biotechnological possibilities as they may play key roles within a plant cell. Hemoglobins are a clear example of successful protein adaptation which allowed them to keep on with the evolution of species all the way from bacterial ancestors to humans. (Less)
Abstract
Hemoglobins (Hb) are usually associated with blood in humans. However, these proteins are widely distributed among living organisms. In plants the most known group are the leghemoglobins. Still, other Hbs that not participate in symbiosis are also found. They are known as nonsymbiotic Hbs (nsHbs). NsHbs are divided into class-1 and class-2. In this thesis three nsHbs from sugar beet (BvHb1-1, BvHb1-2, and BvHb2) and one class-1 nsHb from poplar (PttHb1) have been studied. Additionally, the possibility of using nsHbs for biotechnological application is explored. A holistic expression study of the three BvHbs was done. Following the recombinant production of the three BvHbs, their kinetics and binding affinities to oxygen and CO were... (More)
Hemoglobins (Hb) are usually associated with blood in humans. However, these proteins are widely distributed among living organisms. In plants the most known group are the leghemoglobins. Still, other Hbs that not participate in symbiosis are also found. They are known as nonsymbiotic Hbs (nsHbs). NsHbs are divided into class-1 and class-2. In this thesis three nsHbs from sugar beet (BvHb1-1, BvHb1-2, and BvHb2) and one class-1 nsHb from poplar (PttHb1) have been studied. Additionally, the possibility of using nsHbs for biotechnological application is explored. A holistic expression study of the three BvHbs was done. Following the recombinant production of the three BvHbs, their kinetics and binding affinities to oxygen and CO were determined. Concerning PttHb1, its role as a NO scavenger was investigated by expressing it in roots and mutant yeast. We conclude that, even though differential roles have been given to both class-1 and class-2 nsHbs, a specific function can not be given to each group. On the contrary we found that for some nsHbs their roles may be complementary and overlapping. However, some class-1 nsHbs seem to have a clear NO dioxygenase function. In this study we demonstrate that PttHb1 alleviates NO toxicity in cells when expressed together with PtthFNR, a reductase. To finalize, given that nsHbs have a variety of potential roles, the possibility of being interesting biotechnological targets is studied. Early results regarding the expression of class-2 nsHbs in the model plant A. thaliana and in a potential crop Lepidium campestre are presented. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Hebelstrup, Kim, University of Aarhus, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Hemoglobin, Non-symbiotic hemoglobin, Sugar beet, Ligand binding, Nitric oxide, Gene expression, Gene overexpression, Poplar, Arabidopsis, Lepidium campestre.
categories
Higher Education
pages
54 pages
publisher
Lund University
defense location
Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund University Faculty of Engineering
defense date
2014-09-29 10:15
ISBN
978-91-7422-370-5
language
English
LU publication?
yes
id
8ba39c24-50f0-45b2-a4c7-c746db1f2e38 (old id 4616605)
date added to LUP
2014-09-04 09:53:08
date last changed
2016-09-19 08:45:07
@misc{8ba39c24-50f0-45b2-a4c7-c746db1f2e38,
  abstract     = {Hemoglobins (Hb) are usually associated with blood in humans. However, these proteins are widely distributed among living organisms. In plants the most known group are the leghemoglobins. Still, other Hbs that not participate in symbiosis are also found. They are known as nonsymbiotic Hbs (nsHbs). NsHbs are divided into class-1 and class-2. In this thesis three nsHbs from sugar beet (BvHb1-1, BvHb1-2, and BvHb2) and one class-1 nsHb from poplar (PttHb1) have been studied. Additionally, the possibility of using nsHbs for biotechnological application is explored. A holistic expression study of the three BvHbs was done. Following the recombinant production of the three BvHbs, their kinetics and binding affinities to oxygen and CO were determined. Concerning PttHb1, its role as a NO scavenger was investigated by expressing it in roots and mutant yeast. We conclude that, even though differential roles have been given to both class-1 and class-2 nsHbs, a specific function can not be given to each group. On the contrary we found that for some nsHbs their roles may be complementary and overlapping. However, some class-1 nsHbs seem to have a clear NO dioxygenase function. In this study we demonstrate that PttHb1 alleviates NO toxicity in cells when expressed together with PtthFNR, a reductase. To finalize, given that nsHbs have a variety of potential roles, the possibility of being interesting biotechnological targets is studied. Early results regarding the expression of class-2 nsHbs in the model plant A. thaliana and in a potential crop Lepidium campestre are presented.},
  author       = {Leiva, Nélida},
  isbn         = {978-91-7422-370-5},
  keyword      = {Hemoglobin,Non-symbiotic hemoglobin,Sugar beet,Ligand binding,Nitric oxide,Gene expression,Gene overexpression,Poplar,Arabidopsis,Lepidium campestre.},
  language     = {eng},
  pages        = {54},
  publisher    = {ARRAY(0x8ffcaf8)},
  title        = {Biochemical and Physiological Characterization of Nonsymbiotic Plant Hemoglobins},
  year         = {2014},
}