Final Report Summary - ABC TRANSPORTER (Biochemical characterization of the Arabidopsis ABC transporter AtABCB14 and exploring why it exhibits an import activity)
An executive summary
The ABC (ATP-Binding Cassette) protein superfamily is one of the largest protein families known, and conserved from bacteria to human (Higgins et al., 1986). Arabidopsis thaliana contains about 130 ABC proteins, which are likely to contribute to the transport of diverse materials, including toxic substances (Martinoia et al., 2001). Among them, AtABCB14 has been reported to play a role in stomatal movement under high CO2 conditions by transporting malate into the cell (Lee et al, 2008). For further functional analysis of AtABCB14, I tried to find interaction partner of it by yeast two hybrid. By using only the NBD domain of ABC transporter, this experiment revealed one candidate gene that has a similar tissue specificity and cellular localization as AtABCB14, phospholipase C2 (PLC2) (Otterhag et al, 2001). Furthermore, it was reported that some of the PLC genes are involved in guard cell signal transduction pathways (Staxen et al., 1999). However, it is still needed to be confirmed whether these two proteins really interact by retransformation of them. After confirming this likely interaction, I will perform further experiments to analyze what domain is important for the interaction of the two proteins by mutation or deletion of amino acid of them. I am confident that using this study we will get more some information about the function and regulation of AtABCB14.
Within this project I also tried to find other ABC transporters which can transport weak acids to get a clue about the important amino acid of AtABCB14 for substrate specificity based on similarity of the amino acid sequence of two different genes. I found two genes which confer acetic acid resistance in the yeast cell, these are AtABCC7 and 8. Especially, AtABCC8 transports 3H-IAA which is another candidate substrate of AtABCB14 in the yeast cell. The possibility that AtABCC8 is involved in the auxin transport was supported by reduced free IAA content in the two different AtABCC8 knockout Arabidopsis compared to wild type although no growth difference was observed between wild type and AtABCC8 knockout Arabidopsis plants in media containing auxin or auxin transport inhibitors. Interestingly and in accordance with our observations that ABC transporters have more than one substrate, AtABCC8 knockout Arabidopsis had a lower coniferin, monolignol glucoside content, than that of wild type. Together with this, tissue specific expression of AtABCC8 in the reproductive organ and wounding site suggest that AtABCC8 could participate both in weak acid as well as in monolignol glucoside transport. The important thing is that AtABCC8 transport IAA which is another candidate substrate of AtABCB14. The analysis of the amino acid sequence of AtABCB14 and AtABCC8, may be helpful to find which amino acid is important for substrate specificity and direction of transport in further studies.
During my period to perform this project, I could not analyze the biochemical characteristics of AtABCB14 well. After finding a more efficient transformation method for AtABCB14 in BY2 cell, I can continue to analyze the biochemical characteristics of AtABCB14. However, I found two genes that will be helpful to learn more about AtABCB14. Concerning the PLC2, there is no report about the interaction between phospholipases and ABC transporter yet. This will therefore be a fascinating topic to follow and to elucidate the signalling events of ABC transporters by PLCs. In the case of AtABCC8, it is valuable to first study its function in detail to understand its function in plant cell. If we know more about this aspect and if we see that there is indeed an overlap in the substrate specificity, an analysis of the common part of sequence with AtABCB14 may reveal new and interesting aspects how substrate specificity is establishes in ABC transporters.
Potential impact, use and any target group.
Firstly, result of this project will contribute to research of medical science by supporting the basic information of the function of ABC transporter. In eukayotic systems ABC transporters have been identified as proteins conferring resistance to anticancer drugs in the cancer cells. In addition, there are some human diseases which are caused by disruption of ABC transporters function such as cystic fibrosis (Higgins CF, 1992). Because of that, ABC transporters are of major importance for medical science. It has been reported that some ABC transporters exhibit functional similarity based on the sequence homology. Thus, analysis of amino acid sequence between AtABCB14 and AtABCC8 will be helpful to identify the function and substrate specificity of ABC transporters, a knowledge which is likely to be transferred into the human system and which could help to identify new pharmaceutical targets.
Secondly, looking more from the plant side, finding candidate ABC proteins involved auxin, weak acid and/or monolignol glucoside, such as it can be hypothesized from our results on AtABCC8, will be useful to understand basic mechanism of transport of central compounds for plant development, growth and stress resistance.
Thirdly, knowledge of auxin transport is also a prerequisite for modern breeding. In case of auxin, some hormone transport defective mutant showed abnormal shape or growth pattern. It should be mentioned that several among the shorter crop cultivars are impaired in baso-lateral auxin transport due to a mutation in an AtABCB transporter. Furthermore, transporters play a role in the downstream of hormone signalling, therefore, it can change hormone concentrations within a given cell. Based on this information, we can breed plants of different shapes. Such knowledge may also allow to produce plants producing larger biomass which can be used for bio energy to solve green house effect.
Fourthly, in case of the possibility of monolignol-glucoside transport, it will be helpful for research of bio-energy. Now, lignin is a well known barrier to produce bio ethanol with high efficiency. Monolignol-glucosides are a storage form of monolognol which is a component to make lignin. Further studies for the identification of the function of AtABCC8 related to monolignol transport, may be helpful to understand the mechanism of lignin accumulation and in future also allow to produce plants with a reduced lignin content suitable for bioenergy production.
Project public website:
There is no public website for project.
The ABC (ATP-Binding Cassette) protein superfamily is one of the largest protein families known, and conserved from bacteria to human (Higgins et al., 1986). Arabidopsis thaliana contains about 130 ABC proteins, which are likely to contribute to the transport of diverse materials, including toxic substances (Martinoia et al., 2001). Among them, AtABCB14 has been reported to play a role in stomatal movement under high CO2 conditions by transporting malate into the cell (Lee et al, 2008). For further functional analysis of AtABCB14, I tried to find interaction partner of it by yeast two hybrid. By using only the NBD domain of ABC transporter, this experiment revealed one candidate gene that has a similar tissue specificity and cellular localization as AtABCB14, phospholipase C2 (PLC2) (Otterhag et al, 2001). Furthermore, it was reported that some of the PLC genes are involved in guard cell signal transduction pathways (Staxen et al., 1999). However, it is still needed to be confirmed whether these two proteins really interact by retransformation of them. After confirming this likely interaction, I will perform further experiments to analyze what domain is important for the interaction of the two proteins by mutation or deletion of amino acid of them. I am confident that using this study we will get more some information about the function and regulation of AtABCB14.
Within this project I also tried to find other ABC transporters which can transport weak acids to get a clue about the important amino acid of AtABCB14 for substrate specificity based on similarity of the amino acid sequence of two different genes. I found two genes which confer acetic acid resistance in the yeast cell, these are AtABCC7 and 8. Especially, AtABCC8 transports 3H-IAA which is another candidate substrate of AtABCB14 in the yeast cell. The possibility that AtABCC8 is involved in the auxin transport was supported by reduced free IAA content in the two different AtABCC8 knockout Arabidopsis compared to wild type although no growth difference was observed between wild type and AtABCC8 knockout Arabidopsis plants in media containing auxin or auxin transport inhibitors. Interestingly and in accordance with our observations that ABC transporters have more than one substrate, AtABCC8 knockout Arabidopsis had a lower coniferin, monolignol glucoside content, than that of wild type. Together with this, tissue specific expression of AtABCC8 in the reproductive organ and wounding site suggest that AtABCC8 could participate both in weak acid as well as in monolignol glucoside transport. The important thing is that AtABCC8 transport IAA which is another candidate substrate of AtABCB14. The analysis of the amino acid sequence of AtABCB14 and AtABCC8, may be helpful to find which amino acid is important for substrate specificity and direction of transport in further studies.
During my period to perform this project, I could not analyze the biochemical characteristics of AtABCB14 well. After finding a more efficient transformation method for AtABCB14 in BY2 cell, I can continue to analyze the biochemical characteristics of AtABCB14. However, I found two genes that will be helpful to learn more about AtABCB14. Concerning the PLC2, there is no report about the interaction between phospholipases and ABC transporter yet. This will therefore be a fascinating topic to follow and to elucidate the signalling events of ABC transporters by PLCs. In the case of AtABCC8, it is valuable to first study its function in detail to understand its function in plant cell. If we know more about this aspect and if we see that there is indeed an overlap in the substrate specificity, an analysis of the common part of sequence with AtABCB14 may reveal new and interesting aspects how substrate specificity is establishes in ABC transporters.
Potential impact, use and any target group.
Firstly, result of this project will contribute to research of medical science by supporting the basic information of the function of ABC transporter. In eukayotic systems ABC transporters have been identified as proteins conferring resistance to anticancer drugs in the cancer cells. In addition, there are some human diseases which are caused by disruption of ABC transporters function such as cystic fibrosis (Higgins CF, 1992). Because of that, ABC transporters are of major importance for medical science. It has been reported that some ABC transporters exhibit functional similarity based on the sequence homology. Thus, analysis of amino acid sequence between AtABCB14 and AtABCC8 will be helpful to identify the function and substrate specificity of ABC transporters, a knowledge which is likely to be transferred into the human system and which could help to identify new pharmaceutical targets.
Secondly, looking more from the plant side, finding candidate ABC proteins involved auxin, weak acid and/or monolignol glucoside, such as it can be hypothesized from our results on AtABCC8, will be useful to understand basic mechanism of transport of central compounds for plant development, growth and stress resistance.
Thirdly, knowledge of auxin transport is also a prerequisite for modern breeding. In case of auxin, some hormone transport defective mutant showed abnormal shape or growth pattern. It should be mentioned that several among the shorter crop cultivars are impaired in baso-lateral auxin transport due to a mutation in an AtABCB transporter. Furthermore, transporters play a role in the downstream of hormone signalling, therefore, it can change hormone concentrations within a given cell. Based on this information, we can breed plants of different shapes. Such knowledge may also allow to produce plants producing larger biomass which can be used for bio energy to solve green house effect.
Fourthly, in case of the possibility of monolignol-glucoside transport, it will be helpful for research of bio-energy. Now, lignin is a well known barrier to produce bio ethanol with high efficiency. Monolignol-glucosides are a storage form of monolognol which is a component to make lignin. Further studies for the identification of the function of AtABCC8 related to monolignol transport, may be helpful to understand the mechanism of lignin accumulation and in future also allow to produce plants with a reduced lignin content suitable for bioenergy production.
Project public website:
There is no public website for project.