Transcript PPT - GCP21

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
Methodology
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.
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 RTPCR with Ef1 as the reference gene.
Fig. 1. A picture of a healthy cassava plant
grown in a pot in a glasshouse
Fig. 2. In vitro cassava plants in a temperature and light controlled growth room
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).
a
b
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.
a
b
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.
a
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.
b
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
References
1.Akinbo, O. et al., 2012. Increased storage protein from interspecific F1 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.