Characterization and diversity of selected Actinorhizal haemoglobin genes and proteins with reference to Alnus-Frankia symbiosis Thesis submitted to the University of North Bengal For the Award of Doctor of Philosophy in Botany By Sanghati Bhattacharya Supervisor Prof. Arnab Sen Department of Botany University of North Bengal Raja Rammohunpur, Siliguri September, 2017 This work is dedicated to my parents Declaration I declare that the thesis entitled “Characterization and diversity of selected Actinorhizal haemoglobin genes and proteins with reference to Alnus-Frankia symbiosis” has been prepared by me under the guidance of Prof. Arnab Sen, Department of Botany, University of North Bengal. No part of the thesis has formed the basis for the award of any degree or fellowship previously. [Sanghati Bhattacharya] Department of Botany, University of North Bengal, RajaRammohunpur, Siliguri-734013 Date: 28.08.2017 i ACCREDITED BY NAAC WITH GRADE A Visit us at: UNIVERSITY OF NORTH BENGAL Department of Botany ENLIGHTENMENT TO PERFECTION RajaRammohunpur Siliguri 734013 West Bengal INDIA Phone: +91 353 2699118 FAX: +91 353 2699001 www.nbu.ac.in Ref. No. ………………………………………………. Date: ………………………………………… Prof. Arnab Sen Professor & Head Certificate I certify that Miss Sanghati Bhattacharya has prepared the thesis entitled “Characterization and diversity of selected Actinorhizal haemoglobin genes and proteins with reference to Alnus- Frankia symbiosis”, for the award of Ph. D. degree of the University of North Bengal, under my guidance. She has carried out the work at the Department of Botany, University of North Bengal. The results incorporated in this thesis have not been submitted for any other degree elsewhere. Place: Siliguri [Prof. Arnab Sen] Supervisor Date: 28/08/2017 Department of Botany University of North Bengal RajaRammohunpur, Siliguri-734013. INDIA Abstract Nitrogen, one of the most essential (actinorhizal plant Hbs and nutrients used for survival of living actinobacterial Hbs) to elucidate the organism and it makes up four-fifths of inherent functional diversity and the the atmosphere, but being inert, cannot underlying evolutionary mechanism among intra and inter-specific be available directly to the higher organisms. Some microorganisms help members of actinoHbs. in fixation of atmospheric nitrogen into A. nepalensis, the host plant of this its reactive forms by making study is an excellent example of association with legume or actinorhizal successional plant as found to be early plants. The process of fixing the inert visitors of the landslide regions. molecular nitrogen into usable form by Present study includes a through the enzyme nitrogenase is called survey of host plant i.e. A. nepalensis Biological Nitrogen Fixation (BNF). in sub Himalayan West Bengal and Nitrogenase enzyme is known to be Sikkim, with its population genetics oxygen labile and needs to get study. The total area was divided into protection from free oxygen to three populations keeping in mind facilitate reduction of molecular about the vehicle-route of Darjeeling nitrogen into ammonia. So as a and Gangtok. The population genetics consequence haemoglobin (Hb) gene study revealed that entire population of comes in the scenario, which is found A. nepalensis has segregated depending to be involved in this aspect. on their geographical distribution and The present study deals with the in this respect river Teesta act as molecular analysis and expression geographical barrier for dispersal of germplasm. Soil from the bottom of study of Alnus nepalensis Hbs, along with in-silico analysis of actinoHbs collected A. nepalensis plants were ii taken during field visit to estimate the non-symbiotic Hbs (nsHbs) showed a soil nutrients present within it. remarkable resemblance with class II However extremely variable soil symbiotic Hb (sHb)/legHbs (Lhbs) and nutrient conditions with little high in those properties differ them from that soil carbon was found to favor the of class I Hbs and ptHbs. growth of A. nepalensis in studied Motif annotation revealed two region. functionally active stretches of 50 and Further, we carried forward an in-silico 21 amino acid sequence containing analysis of actinorhizal Hbs along with “YjbI” like activity in ptHbs, which is different types of plant Hbs (pHbs) and responsible for inorganic ion transport actinobacterial Hbs (bHb) available in and metabolism., similar to bHbs but public domain. PHb constitute a totally absent in other pHbs. This diverse group of haem proteins and finding suggests that ptHb share evolutionarily belong to three different similar characteristics with bHbs, classes - symbiotic, non-symbiotic and which made it distinct from other truncated. Since truncated pHbs pHbs. This finding suggested the (ptHbs) have a 2-on-2 structure, they further analysis of bHbs in detail. For are structurally different from the other this, the codon level analysis and two Hb groups. functional annotation of bHbs were To analyze the various aspects of Hbs, performed. The whole genome two fundamental levels i.e. sequence sequences along with Hb genes of type level and structure level analysis was strain of actinobacteria were retrieved. performed. Codon usage pattern using parameters like GC, GC3, Nc, etc and percentile In sequence level analysis, a thorough calculation revealed that codon sequence analysis of 96 pHbs along biasness of Frankia Hbs are expressed with identified 121 bHbs from 72 type in a moderate to high manner and strain of actinobacteria currently depending upon 1) GC compositional available in public domain were done. constrains and 2) natural selection on Physiochemical properties and motif their transitional efficiency. annotation of actinoHbs was performed A function based phylogenetic by using various software's and approach was undertaken to understand algorithms, which revealed that class II the ancient lineage of the protein iii family which subsequently revealed the homology modeling technique and function of bHbs are depending on 1) peeped into their structural properties. their different functions within same Structure based studies revealed that genera, 2) host specificity and 3) eco- the C. glauca sHb and D. glomerata geographical habitat. Functional ptHb shows distinct stereo-chemical annotation was also revealed that properties from that of the other C. single Hb of Frankia alni contain NOD glauca, M. gale and A. firma nsHb factor which afterward develop a proteins. unique pathway to synthesize chitine To analyse the structural similarity, the based signal molecule and help in the backbone of actinoHbs followed by interaction of host and microsymbiont structure based phylogeny was to facilitate symbiosis. considered. The result revealed that the Sequence comparison was done to backbone structures of pHbs (including identify the conservation level amongst sHb, nsHb and ptHb) and bHbs were actinoHbs. This revealed that the totally different, though the side chain conservation of sHbs and nsHbs of modifications were much more similar actinorhizal Hbs are quiet similar with for ptHbs and bHbs. This result that of other pHbs, whereas ptHbs supports the hypothesis that ptHbs showed high conservation level with have a similar structural arrangement bHbs, which further supported the with bHbs. result found from physiochemical To analyze functional divergence, we property test and motif annotation. It have performed assessment of land was also found that the actinoHb pHbs along with bHb and have done proteins were might be structurally not altered evolutionary rates among all related to the ferredoxin-NADP+ type types of member proteins. The reductase and involve in a different assessment revealed that ptHb was mechanism to reduction of oxidized functionally diverged from the other haem iron into ferrus form. pHbs (class I & II) while some Further, we constructed 3D structures properties are similar with class I of actinorhizal Hb proteins from Alnus nsHbs. firma, Casuarina glauca, Myrica gale Quantitative RT-PCR was performed and Datisca glomerata through to study the expression level of Hb iv genes in different plant parts of A. root region. nepalensis. A partial mRNA Hb gene This might be due to its search to find from A. nepalensis was identified, out their microsymbiont Frankia for which showed 96% similarities with interaction. Present study also suggests class I nsHb of A. firma. The that divergence may occur amongst expression study of Hb genes depicted plant and bacterial haemoglobin that Hb expresses in an elevated proteins where ptHbs have some linker manner in nodules, when inoculated values between them. with Frankia, but the expression level is significantly high in untreated plant v Preface This Ph.D. thesis contains the result of Special thanks to Dr. Malay my research work undertaken at the Bhattacharya, Assistant professor, Department of Botany, University of Department of Tea Science, University North Bengal, from January, 2013 to of North Bengal for his valuable February, 2017. During this course of suggestions, enormous encouragement time I had gone through various and support. He has devoted his favorable and unfavorable mental precious time and helped me a lot to states. Without help, support, finish this research work. motivation & inspiration by others, I I am also thankful to the Head of the would have never been able to finish Department and all the teachers and my research work properly. Therefore I stuff of the Department of Botany, would like to thank those people who University