Investigation into the relationship between aluminium treatment and the superoxide dismutase (SOD) enzyme system in Lolium perenne (L. perenne cv. Nui) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Plant Biology at Massey University
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Date
2009
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Massey University
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Abstract
Lolium perenne cv. Nui is a cultivar of ryegrass grown throughout New Zealand in
pastures due to favourable traits such as high palatability for livestock and its ability to
withstand intensive grazing. However, the productivity of pastures is reduced when
levels of aluminium and other metals accumulate in soils to toxic levels, a phenomenon
referred to as the ‘acid soil syndrome’. In response to this toxicity, plants activate a
series of antioxidant reactions, with one catalysed by the superoxide dismutase (SOD)
enzymatic system. The enzyme system comprises three isoenzymes, a Cu/ZnSOD,
FeSOD and a MnSOD which catalyse the same reaction but differ in amino acid
sequence, molecular mass and the metal ion co-factor (hence Cu/ZnSOD, FeSOD and
MnSOD). Together these isoenzymes combat the damaging effect of superoxide
radicals which accumulate due to metal toxicity. In this thesis, the isolation of genes
encoding isoenzymes of the SOD enzyme from L. perenne cv. Nui is described. As
well, the growth of L. perenne cv. Nui and changes in expression of the SOD genes
encoding each isoenzyme in response to aluminium treatment (0.2mM AlCl3) is
investigated.
A 1072 bp FeSOD gene sequence and a 705 bp MnSOD gene sequence were isolated
from shoot tissue of L. perenne cv. Nui using a combination of RT-PCR with
degenerate primers and 3'-RACE. The FeSOD gene comprised 572 bp of the coding
sequence and 500 bp of 3'-UTR while the MnSOD gene comprised 508 bp of coding
sequence and a 197 bp 3'-UTR. By alignment of each sequence with the gene from the
database with highest identity it was predicted that the translation start codon (ATG) is
located a further 196 bp upstream for the FeSOD gene (aligned with an Oryza sativa
FeSOD sequence as a reference) and a further 152 bp upstream for the MnSOD
sequence (aligned with a Triticum aestivum MnSOD sequence as a reference). Using
RT-PCR with degenerate primers, a 313 bp CuSOD sequence was predominantly
cloned from shoot tissue of L. perenne cv. Nui, but it was not possible to generate the
3'-UTR using 3'-RACE.
For growth analysis, seedlings of L. perenne cv. Nui were germinated and acclimatised
in Hoagland’s solution, and then subjected to either aluminium treatment (0.2mM
AlCl3) or no treatment to act as a control over a designated time course of 0, 4, 8, or 24
hours. Two growth trials were conducted that differed in the age of seedlings used and
plant tissues were separated into root and shoot tissues. Similar growth trends were
observed in both trials, but the sampling regime in the second growth trial meant that
statistical analysis could be carried out. In this trial, analysis revealed that over a time
course of 24 hours exposure to 0.2mM aluminium, both root and shoot tissue fresh
weight did not significantly differ when compared to the control (no aluminium). A
general trend of an increase in root and shoot fresh weight was observed in plants
treated with aluminium, but this trend was not significant at P=0.05. No significant
change in fresh weight partitioning from shoot to root, or root to shoot in response to
aluminium was also observed.
Using semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqRTPCR)
and primers based around the 3'-UTR with RNA isolated from plants grown in the
second hydroponic trial, it was determined that under the conditions used, expression of
the FeSOD and MnSOD genes isolated in this study were neither up-regulated or downregulated
in response to aluminium treatment in both shoot and root tissue. Further,
using degenerate primers to detect expression of one or more genes encoding the
Cu/ZnSOD isoenzyme, total expression of the Cu/ZnSOD isoenzyme was also
unresponsive to aluminium treatment.
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Keywords
Ryegrasses, Acid soil syndrome, Lolium perenne, Perennial ryegrass