Molecular characterisation of the gene encoding [Delta 1]-Pyrroline-5- Carboxylate Reductase isolated from Arabidopsis thaliana (L.) Heynh.
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Date
1995
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Abstract
In Arabidopsis thaliana (L.) Heyhn, the size of the pool of free proline increases up to 27-fold in response to osmotic stress. The magnitude of this accumulation is dependent upon
the rate of imposition of the stress. Numerous reports have suggested a role for proline
accumulation as a general adaptation to environmental stress. However, controversy
surrounds the beneficial effect of proline accumulation in plants under adverse
environmental conditions.
Stress-induced proline accumulation in plants occurs mainly by de novo synthesis from
glutamate. The final and only committed step of proline biosynthesis in plants is catalysed
by Δ¹-pyrroline-5-carboxylate reductase (P5CR). The sequence of an incomplete 999 bp
cDNA encoding P5CR from A. thaliana was determined. This enabled a preliminary
molecular study of the structure and function of both the gene and the corresponding
enzyme.
The 999 bp cDNA insert in the clone Y AP057 was sequenced on the sense and antisense
strands following subcloning of four sub-fragments in appropriate orientations. Comparison
with known plant P5CR sequences revealed that Y AP057 does not encode the first 23 N-terminal
amino acids of P5CR from Arabidopsis. However, it does encode the remaining
253 amino acid residues of Arabidopsis P5CR The cDNA Y AP057 is complete on the 3'
end as indicated by the presence of a poly(A) tail. The nucleotide sequence determined
shows complete homology to the corresponding exons of the genomic copy of a bona fide
gene encoding P5CR in A. thaliana (Verbruggen et al, 1993). The only difference observed
between the sequence of Y AP057 and that of a cDNA sequenced by these workers is that
polyadenylation was initiated seven nucleotides earlier in Y AP057 than in the sequence of
the published cDNA.
Genomic Southern analysis suggests the presence of only a single copy of the gene
encoding P5CR in Arabidopsis. Restriction mapping and sequencing the ends of another
incomplete Arabidopsis P5CR cDNA clone FAFJ25 (664 bp) indicated that the regions
sequenced were completely homologous to the corresponding portions of Y AP057. Analysis of codon usage in the Arabidopsis gene encoding P5CR revealed it to closely resemble the consensus pattern of codon usage in A. thaliana. This suggests that the gene
is moderately. expressed. Expression of the gene encoding P5CR in Arabidopsis is not likely
to be subject to translational control.
Although P5CR from A. thaliana has a fairly high composition of hydrophobic amino acid
residues, it does not possess any stretches of hydrophobic amino acids of sufficient length
to act as membrane-spanning domains or to anchor the enzyme in a membrane. Neither does
it contain an N- terminal leader sequence capable of directing it to either the plastid or
mitochondrion. The enzyme therefore appears to be cytosolic.
The nucleic acid and deduced amino acid sequences of Arabidopsis P5CR were compared
with those from·eleven other organisms for which P5CR sequences are currently available.
Except among the three different plants examined, P5CR sequences displayed less identity
at the amino acid level than at the nucleotide level.
The deduced amino acid sequence of Arabidopsis P5CR exhibits high similarity to the
corresponding genes and amino acid sequences of P5CR from soybean and pea. Lower but
significant similarity was observed to the amino acid sequences of P5CRs from human,
Saccharomyces cerevisiae and the bacteria Escherichia coli, Pseudomonas aeruginosa,
Thermus thermophilus, Mycobacterium leprae; Treponema pallidum and Methanobrevibacter
smithii. Similarity was also observed to the translational product of a gene from Bacillus
subtilis with high homology to the E. coli proC gene. However, construction of a
phenogram indicating the relatedness of the various P5CR enzymes suggests that sequence
analysis of this enzyme is not a good indicator of evolutionary relatedness of organisms
from different biological kingdoms.
Multiple alignment of the twelve known P5CR sequences indicated homology between the
sequences across their entire lengths. Homology was particularly high in the C-terminal
portions of the P5CRs studied. It is speculated that this region may be of importance in
binding of the substrate Δ¹-pyrroline-S-carboxylate (P5C). Another region displaying high
sequence conservation was found in the central portion of all P5CRs. All P5CRs studied,
with the exception of PSCR from T. pallidum contained an N-terminal domain capable of
binding a nicotinamide dinucleotide cofactor. Comparison of this region with consensus sequences for NADH and NADPH binding sites in proteins suggests that NADPH is the preferred reductant used by P5CRs from plants and human. In contrast, the N-terrninal
domains of P5CRs from S. cerevisiae, M smithii, T. thermophilus and M leprae display
greater similarity to a consensus NADH-binding site. The definite preference of plant P5CRs
for NADPH in comparison with NADH suggests that P5CR may be involved in regulating
the redox potential within plant cells and that this step in proline biosynthesis from
glutamate may be of importance in overall metabolic regulation.
Three amino acid residues are universally conserved in all P5CRs studied. All are found
within blocks of high sequence similarity. These residues are likely to be of importance in
the structure or catalytic mechanism of P5CR. A number of other residues are common to
several of the enzymes examined. These may also be of importance in subsequent
manipulation of Arabidopsis P5CR at the molecular level.
Prediction of the putative secondary structures of A. thaliana, soybean, pea, human and E.
coli indicated a high degree of similarity between the enzymes. This was particularly evident
in the region of the putative P5C-binding domain. Considerable similarity exists in
hydrophobicity profiles of P5CRs from these five organisms.
Proline levels in reproductive organs of unstressed Arahidopsis plants were considerably
higher than those in vegetative tissues. This suggests differential expression of enzymes
involved in proline metabolism in these organs. In situ hybridisation studies indicated an
increase in levels of mRNA transcripts encoding P5CR in stem tissues in response to water
deprivation stress. Regulation of levels of mRNA transcript encoding P5CR in Arabidopsis
therefore appears to be an osmotically sensitive process. Furthermore, this accumulation of
transcript occurred in a tissue-specific manner. In particular, an increase in levels of
transcript encoding P5CR was observed in the cortical parenchyma, phloem, vascular
cambium and pith parenchyma in the vicinity of the protoxylem.
The significance of these findings in contributing to a better understanding of the role of
proline in adaptation to environmental stress is discussed.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.
Keywords
Plants, Effect of environment on., Plants, Effect of stress on., Arabidopsis thaliana., Plant genetics., Plant molecular biology., Proline., Theses--Botany.