1. Genomic Characterisation of Nitrogen Assimilation Genes in Cassava (Manihot esculenta Crantz) T.G. Chabikwa, M.E Rauwane, and D.A Odeny ARC-Biotechnology Platform, Private Bag X5, Onderstepoort, 0110, Pretoria, South Africa Introduction Cassava is a woody perennial plant (Fig. 1) grown mainly for its edible starchy roots although the leaves are also eaten in some parts of Africa. The low root protein content of cassava increases the risk of protein malnutrition in communities relying on cassava as a staple (Stephenson et al., 2010). Whilst relatively successful interspecific crosses between M. esculenta and its wild relatives have been made in an effort to improve root protein content (Akinbo et al., 2012 ), the molecular basis of protein metabolism in cassava is poorly understood. Nitrogen assimilation is the first step of protein metabolism and is catalyzed by enzymes glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (glutamine-2-oxoglutarate aminotransferase; GOGAT; EC 1.4.7.1) (Masclaux-Daubresse et al., 2006). The GS gene family consists of cytosolic GS1 and chloroplastic GS2 while the GOGAT gene family consists of NADH-dependent GOGAT (GLT) and Ferrodoxin-dependent GOGAT (GLU). The current work was undertaken to develop a more comprehensive understanding of the molecular features of GS and GOGAT gene families in cassava. References 1.Akinbo, O. et al., 2012. Increased storage protein from interspecific F 1 hybrids between cassava (Manihot esculenta Crantz) and its wild progenitor (M. esculenta ssp. flabellifolia). Euphytica 185(2): 1-9. 2. Masclaux, C. et al., 2000. Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211(4): 510-18. 3. Stephenson, K. et al., 2010. Consuming cassava as a staple food places children 2–5 years old at risk for inadequate protein intake, an observational study in Kenya and Nigeria. Nutrition Journal 9: 9. Fig. 3. Transcript abundance of (a) cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) genes in leaves, stems and roots as determined by quantitative real-time PCR. High expression levels of GS genes was observed in stem tissues. Gene expression is given relative to Ef1 mRNA levels. Data are the means of three technical replicates ± SD. Fig. 4. Transcript abundance of (a) NADH-glutamate synthetase (GLT) and (b) Ferrodoxin dependent-glutamate synthetase (GLU) genes in in leaves, stems and roots as determined by quantitative real-time PCR. Gene expression is given relative to Ef1 mRNA levels. Data are the means of three technical replicates ± SD. Fig. 5. Promoter analysis of the (a) GS Gene Family and (b) the GOGAT gene family as determined on the PlantCARE database. Scaffold numbers shown correspond to numbers retrieved from Phytozome b b b a a a Fig. 2. In vitro cassava plants in a temperature and light controlled growth room Methodology Expressed sequence tags (EST) and amino acid sequences of GS1, GS2, GLT and GLU for Arabidopsis thaliana were retrieved from http://www.arabidopsis.org/ and used to query public databases of cassava, rice (Oryza sativa), poplar (Populus trichocarpa), castor bean (Riccinus communis), soybean (Gycine max) and potato (Solanum tuberosum) for homologues. Multiple alignments of the homologous sequences were done using MAFFT 6.864 and phylogenetic analysis of amino acid sequences was performed using the online phylogenetic platform http://www.phylogeny.fr/version2_cgi/index.cgi. Promoter analysis was done using 600 bp sequences upstream from the ATG codon of cassava GS/GOGAT genes. Primers were designed for selected genes and tested for amplification on cassava genotypes TMS 60444, P1/19 and AR9-2. Total RNA was extracted from 3 month old in vitro cassava plants (Fig. 2), reverse transcribed and used to test the expression of GS/GOGAT genes in leaves, stems and roots. Transcript abundance was determined using quantitative RT- PCR with Ef1 as the reference gene. http://www.arabidopsis.org/ http://www.phylogeny.fr/version2_cgi/index.cgi Fig. 1. A picture of a healthy cassava plant grown in a pot in a glasshouse Results Phylogenetic analysis revealed clustering of GS and GOGAT genes of cassava with those from other plants. Cassava MeGLU, however, failed to cluster with other GLU genes from Arabidopsis, rice, poplar, castor bean and soybean (data not shown). Transcript abundance suggested tissue specific expression of the various classes of genes tested (Fig. 3). There was remarkably high expression levels of the GS1 and GS2 genes in stem tissues and low leaf specific expression of GS1 and GS2 genes. Comparatively higher expression levels of the GLT gene in root tissues and high expression levels of the GLU gene in aerial tissues was observed (Fig. 4). Promoter analysis identified over representation of light induced cis-regulatory elements (Fig. 5). Conclusions This study confirms the tissue-specific expression of GS/ GOGAT genes in cassava, which has been well documented in Arabidopsis (Arabidopsis thaliana). Low expression of the chloroplastic glutamine synthetase GS2 genes in leaf tissues conflicts results obtained in Arabidopsis. This could be due to the physiological age of the cassava seedlings. The experiment may need to be repeated using older plants in a soil or peat based growth media as roots are exposed to light in the agarose-based tissue culture media thus distorting gene expression patterns. The structural differences in MeGLU genes will need to be studied further to determine how the observed difference would affect protein metabolism in cassava as compared to other crops.