Exploring barley mutants for molecular dissection of chlorophyll biosynthetic enzymes and chloroplast development
Plant Biochemistry and Biophysics
Plant Breeding and Plant Pathology
Final Report Abstract
3 Summary (max. 1 A4 page) - A generally understandable presentation of the most important scientific advances and, if applicable, their application aspects. - "Surprises" in the course of the project and in the results. - References to possible success reports in the public media. The aim of my stay, during my DFG research fellowship, in Prof. Hansson’ Lab at Biology Department, Lund University was to learn gene hunting focusing on barley mutants and learn protein work focusing on the complex enzymes Mg-chelatase and cyclase. In his previous studies, Prof. Hansson has studied a handful of the barley chlorophyll mutants in detail and revealed that Xantha-f, -g and -h encodes subunits of Mg-chelatase and Xantha-l encodes the cyclase. Mg-chelatase and cyclase are chlorophyll biosynthetic enzymes. My plan to work with the molecular basis of chlorophyll biosynthesis especially on protein level in barley is perfectly in line with my future scientific career aims and objectives. I have taken this step in the direction of establishing my own research group, performing research towards engineering a plant architecture for temperate cereal crops, especially wheat and barley. My fellowship funding provided me a great opportunity to expand my knowledge and skills and I can follow the concepts and methodologies outlined in my proposal and apply that to other cereal crops. Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Most of the reactions have been characterized using recombinant proteins. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus. , which are photosynthetic bacteria. In my work, I established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH. On the other hand, the formation of the isocyclic E-ring characteristic of chlorophylls is not yet known. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. During my work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. The results showed that, the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions. In chloroplasts, the most abundant ferredoxin accepts electrons from photosystem I and shuttles electrons via FNR to generate NADPH or directly to ferredoxin dependent enzymes. In addition, plants contain other isoforms of ferredoxins. Two of these, named FdC1 and FdC2 in Arabidopsis, have C-terminal extensions and functions that are poorly understood. During my fellowship work we identified disruption of the orthologous FdC2 gene in barley mutants at the Viridis-k locus which are deficient in the aerobic cyclase reaction of chlorophyll biosynthesis. The Mg-protoporphyrin IX monomethyl ester cyclase is one of the least characterized enzymes of the chlorophyll biosynthetic pathway and its electron donor has long been sought. Agroinfiltrations showed that the viridis-k phenotype could be complemented in vivo by Viridisk but not by canonical ferredoxin. VirK could drive the cyclase reaction in vitro and analysis of cyclase mutants showed that in vivo accumulation of VirK is dependent on cyclase enzyme levels. The chlorophyll deficient phenotype of viridis-k mutants suggests that VirK plays an essential role in chlorophyll biosynthesis that cannot be replaced by other ferredoxins, thus assigning a specific function to this isoform of C-type ferredoxins. In addition, I gained experience in guiding master and bachelor students, and occasionally also interact with undergraduate students at the courses given by Prof. Hansson at the Department of Biology in Lund (such as BioR31 Molecular Biotechnology, BIOR76 Plant Function). This experience will help me in establishing my own group in the near future as well as getting professor ship in one of the German universities. This information can be published on the Internet, provided that the recipient of the license does not object.
Publications
- (2019) Crosstalk among hormones in barley spike contributes to the yield. Plant Cell Rep. 2019 Aug; 38(8):1013-1016
Helmy M. Youssef, Mats Hansson
(See online at https://doi.org/10.1007/s00299-019-02430-0) - (2020) Aerobic barley Mg-protoporphyrin IX monomethyl ester cyclase is powered by electrons from ferredoxin. Plants
David Stuart, Malin Sandström, Helmy M. Youssef, Shakhira Zakhrabekova, Poul Erik Jensen, David W. Bollivar, Mats Hansson
(See online at https://doi.org/10.3390/plants9091157) - (2020) Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits. The Protein Journal
Rabab Mahdi, David Stuart, Mats Hansson, Helmy M. Youssef
(See online at https://doi.org/10.1007/s10930-020-09913-0)
