Patterns of Codon Usage in Plastidial Genomes of Ancient Plants

Provide Insights into Evolution

Manju Yadav

, Suresh Babu

and Gitanjali Yadav

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India

School of Human Ecology, Ambedkar University of Delhi, New Delhi, India

Keywords: Codon Bias, Molecular Evolution, Biostatistics, Organellar Genomics.

Abstract: Basal angiosperms are the first flowering plants that diverged from ancestral angiosperms, while magnoliids

represent the oldest known angiosperms and are considered to retain the characteristics of more primitive

angiosperms. Availability of the plastidial genomes from several members of both these classes of plants

provides an opportunity to identify and understand large-scale genomic patterns in organelles of early

angiosperms. In this work, chloroplast genomes from nine AT-rich basal angiosperm and magnoliid species

were analyzed to unearth patterns, if any, in terms of codon bias and to identify factors responsible for the

detected patterns. We were able to distinguish nine optimal codons in basal angiosperm chloroplasts and 18

in case of magnoliids. Our findings suggest mutational bias as the most predominant factor shaping codon

usage patterns among the genomes examined, while gene expression, hydrophobicity and aromaticity, were

found to have a limited but important effect on pattern determination.

1 INTRODUCTION

Chloroplasts, initially originated by the process of

endosymbiosis from cyanobacteria about 1-1.5

billion years ago, are the most important cellular

organelles of autotrophs. On account of their small

size, high copy number, conservation and extensive

characterization at the molecular level, a large

number of completely sequenced plastid genomes

are now publicly available.

The angiosperms, or flowering plants, are one of

the major groups of extant seed plants and arguably

the most diverse major extant plant group on the

planet, with at least 260,000 living species classified

in 453 families. Basal angiosperms represent the

first flowering plants that branched off from

ancestral angiosperms at successive occasions before

the appearance of the "true" dicots Eudicots, and

comprise of distinct evolutionary groups, of which

the first three to diverge were Amborellales,

Nymphaeales and Austrobaileyales (Soltis & Soltis

2004). Magnoliids, on the other hand, are the oldest

known angiosperms, represented by a heterogenous

group that are neither eudicotyledons nor

monocotyledons, and are considered to retain the

characteristics of more primitive angiosperms.

Economically important products derived from

magnoliids include edible fruits, spices such as black

and white pepper Piper nigrum, cinnamon

Cinnamomum verum, and camphor Cinnamomum

camphora (Soltis et al., 2005). The magnoliid clade

contains most of those lineages that were typically

referred to as "primitive angiosperms" in earlier

classification schemes (Cronquist, 1988).

This work was undertaken with the aim of

conducting a genome-wide survey of codon usage

patterns across the available chloroplast genomes of

Basal Angiosperms and Magnoliids. The term

‘codon usage bias’ describes the unbalanced usage

of synonymous codons during translation of a given

genome. Codon usage can vary between species and

also between different genomic regions of the same

species, so there is much fluctuation observed in

genes and genomes. Several factors support this

phenomenon, such as genome composition bias

(Bernardi and Berbardi, 1986), natural translation

selection (Ikemura, 1985), hydrophobhicity and

aromaticity.

Previous codon usage studies demonstrate that

codon usage bias is a complex phenomenon, which

involves various biological factors such as gene

expression level, gene length, gene translation

initiation signal, protein amino acid composition,

protein structure, tRNA abundance, mutation

144

Yadav M., Babu S. and Yadav G..

Patterns of Codon Usage in Plastidial Genomes of Ancient Plants Provide Insights into Evolution.

DOI: 10.5220/0005246301440149

In Proceedings of the International Conference on Bioinformatics Models, Methods and Algorithms (BIOINFORMATICS-2015), pages 144-149

ISBN: 978-989-758-070-3

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

frequency and patterns and GC composition (Sharp

et al., 1993). Ikemura and colleagues found that

some specific codons of highly expressed genes are

best recognized by the most abundant tRNA

isoacceptors (Ikemura, 1985). It is believed that

codon usage pattern in chloroplast genomes is

similar to that of E. coli as the translational

machinery in both cases has its own genomic

environment that resembles prokaryotes.

Composition bias is the predominant factor

responsible for codon bias in chloroplast genome of

plants. High A+T content, which matches the

composition bias of noncoding regions, is rich in

degenerate positions (Morton, 1996), but this fact is

not accepted in case of psbA gene, which has high C

content at the third position of specific synonymous

groups. Selection is thought to act strongly on the

codon usage of psbA such that it has a noticeably

unique codon usage pattern, and at a very weak

intensity on the codon usage of some other highly

expressed genes of the plant chloroplast genomes

(Morton, 1996).

The main purpose of this study is to gain an

understanding of the selection factors that are

responsible of codon usage bias by focusing on the

data from nine chloroplast genomes belonging to

magnoliids and basal angiosperms. We have tried to

address questions regarding the change in codon

usage pattern during evolution in chloroplast

genomes, an event that has not previously been

explored in depth. We also attempt to compare our

findings with published literature although earlier

studies have all been restricted to individual or very

few species in plants.

2 MATERIALS & METHODS

2.1 Dataset

Complete chloroplast genomes and collection of

coding sequences of nine species of plants

representing two major taxa, namely Basal

Angiosperms and Mangnoliids were obtained from

GenBank, NCBI:http://www.ncbi.nlm.nih.gov.

The nine genomes used as dataset for this study

comprised of three basal angiosperms, one each

from Amborellales, Nymphaeales and

Austrobaileyales, namely Amborella trichopoda,

Nymphaea alba and Nuphar advena respectively.

The six remaining chloroplast genomes

encompassed the magnoliids, namely Calycanthus

floridus var. glaucus, Liriodendron tulipifera,

Drimys granadensis, Piper cenocladum,

Chloranthus spicatus and Illicium oligandrum.

Details of these nine genomes are provided in

Table1.

Only those sequences were included which

comprised appropriate start and stop codons and

were of full length. To minimize stochastic

variation, a threshold of 100 codons was defined,

since there is a negative correlation between codon

usage bias and gene length.

Table 1: Summary of Organisms.

Species code

Accession No.

# protein

coding

genes

# Filtered

genes

used

GC %

BASAL ANGIOSPERMS

A.tr NC_005086 84 54 38

N.al NC_006050 85 58 39

N.ad NC_008788 85 55 39

MAGNOLIIDS

C.f.g NC_004993 86 55 39

L.tu NC_008326 84 53 39

D.gr NC_008456 85 55 38

P.ce NC_008457 85 53 38

C.sp NC_009598 86 56 38

I.ol: NC_009600 83 56 39

2.2 Codon Usage Indices & Parameters

A number of codon usage indices were calculated

for this study using the program CodonW 1.4.4

http://codonw.sourceforge.net/. All statistical

analysis was performed using SPSS version 16.0.

The effective number of codons Nc, independent

of gene length is a simple measure of bias in codon

usage (Wright, 1990). Equation for the calculation

of Enc plot is: ENc = 2 + S + 29/S2 + 1-S2 where S

is the frequency of G+C i.e. GC3s. A plot of Enc

against GC3s NC-plot was effectively used to detect

the codon usage variation among genes, for

example, if GC3s is zero, then only codons ending in

A and T will be used, thus restricting the number of

codons used to 20 out of the 61 sense codons.

Wright 1990 argued that if a particular gene is

subject to G+C compositional constraint, it would lie

on or just below the expected curve, as against a

gene subject to selection for transitionally optimal

codons, that would lie considerably below the

expected curve.

Relative synonymous codon usage RSCU value

for a codon is simply the observed frequency of that

codon divided by the frequency expected under the

assumption of uniform usage H0* of the

synonymous codons for an amino acid (Sharp,

1986). RSCU values close to 1.0 indicate a lack of

codon bias. RSCU values are largely independent of

PatternsofCodonUsageinPlastidialGenomesofAncientPlantsProvideInsightsintoEvolution

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amino acid composition and are particularly useful

in comparing codon usage among genes, or sets of

genes that differ in their size and amino acid

composition. The formula for RCSU is given by:

RSCU



j1



where X

is the number of occurrences of the j

codon for the i

amino acid, and n

is the number

from one of six of alternative codons for the i

amino acid. Relative adaptive-ness of a codon, w

, is

the frequency of use of that codon compared to the

frequency of the optimal codon for that amino acid,

and it is given by:

 RSCU

RSCU

i max

 X

i max

where RSCU

imax

and X

imax

are the RSCU and X

values for that codon which is used most frequently

for the i

amino acid.

Codon Adaptation Index CAI measures the

relative adaptation of a gene of the codon usage of

highly expressed genes. CAI uses a reference set of

highly expressed genes from a species to assess the

relative merits of codon and identifies the role of

selective pressure in modeling the patterns of codon

usage (Sharp and Li, 1987). To calculate CAI, the

first step is to construct a reference table of relative

synonymous codon usage RSCU values from very

highly expressed genes of the organism in question.

The CAI values are calculated in relation to the

psbA gene of the same genome.

The psbA gene demonstrates atypical codon

usage and its codon bias is a remnant of the ancestral

bias degrading toward the compositional bias

(Morton and Levin, 1997). A CAI values close to

1.0 reflects strong bias in codon usage and

potentially high-expression level of the considered

gene (Sharp and Li, 1987).

The most commonly used characteristic is the

pattern of codon usage itself, defined in terms of

optimal codons. An optimal codon is any codon

whose frequency of usage is significantly higher

than its synonymous codons in putatively highly

expressed genes. Significance is estimated using a

two-way chi-squared contingency test, with a cut-off

at p<0.01. Codon usage was composed using chi-

square contingency test of the groups, and codons

whose frequency of usage were significantly higher

p-value < 0.01 in highly expressed genes than in

genes with low level of expression would be defined

as the optimal codons.

GC content is calculated as the fraction of

nucleotides in a sequence, that are guanine or

cytosine. The index GC3s is the frequency of G or C

nucleotides present at the third position of

synonymous codons i.e. excluding Met, Trp and

termination codons.

Hydrophobicity is measured in terms of gravy

score, while aromaticity denotes the frequency of

aromatic amino acids Phe, Tyr and Trp in the

translated sequences (Kyte and Doolittle, 1982).

To normalize and identify intra-genomic

variation with differing amino acid compositions,

relative synonymous codon usage RSCU was

analyzed for correspondence analysis COA for the

59 informative codons excluding Met, Trp, and the

three stop codons (Greenacre, 1984). This analysis

partitions the variation along 59 orthogonal axes,

with 41 degrees of freedom. The first axis is the one

that captures most of the variation in the codon

usage, with each subsequent axis explains a

diminishing amount of the variance. The

correspondence analysis also reflects the

corresponding distribution of synonymous codons.

RSCU values are close to 1.0 when all synonymous

codons are used equally without any bias. In

subsequent part of this work, the terms axis 1 RSCU

and axis 2 RSCU will be used to represent first-and

second-major axis of COA.

3 RESULTS

3.1 Detection of Codon Usage Patterns

As described in methods, the pattern of synonymous

codons usage across the codons in each genome was

investigated by the Nc-plot between ENc value and

GC3s value. The values range from 20 extremely

biased to 61 no bias (Wright, 1990), and the

respective plots are shown in Figure 1 for the basal

angiosperms, and Figure 2 for magnoliids. Nc-plots

of basal angiosperm chloroplast genomes follow a

trajectory path, i.e majority of points are on and just

below the Nc-plot.

Table 2 lists the Nc and GC3 values for all

species investigated and it can be seen that basal

angiosperms have very low GC3s values and their

Nc values range from about 38 to 61, the lowest

being 38.39 GC3s is 0.232 in case of the rps18 gene

of Amborella trichopoda.

Overall, the majority of genes follow a parabolic

line of trajectory indicating G+C mutational bias as

the predominant factor for variation in codon usage,

although some genes lie well below the expected

curve, hinting at additional factors responsible for

codon bias in basal angiosperms.

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Figure 1: Nc-plots (Nc values vs GC3s) for the three basal

Angiosperms. Nc was plotted against GC content at the

third codon position. The expected ENc from GC3s are

shown as a solid line.

Table 2: Genes with highest bias per taxon (by Nc-Plots).

Species code

Accession No.

Gene NC value

(GC3s)

BASAL ANGIOSPERMS

A.tr NC_005086 Rps18 38.39 (0.232)

MAGNOLIIDS

D.gr NC_008456 Rps14 34.86 (0.309)

Although the mean Nc values of basal

angiosperms and magnoliids are close to each other

as shown in Table 3, and both sets of Nc plots

display a parabolic trend, it can be seen from Figure

2 that the magnoliid Nc plots exhibit a wider

scattering of points as compared to basal

angiosperms, and there are more magnoliid genes

lying well below the expected curve.

As can be seen from Table 2, least Nc-value is

displayed by Drimys granadensis i.e. 34.86 GC3s is

0.309 on rps14 gene. These observations suggest

that in case of magnoliids, G+C mutational bias is

the predominant factor for codon usage bias but

translational selection may also be an important

factor.

3.2 Optimal Codons

Table 4 lists the results from optimal codon

identification and the data shows nine significantly

preferred optimal codons for basal angiosperms p<

0.01, while in case of magnoliids, 18 codons were

identified as being used more frequently p< 0.05

Table 4. These optimal codons are optimal for genes

at higher expression level, as estimated from CAI

analysis. Only four optimal codons were found to be

common between the two taxa.

Table 3: Means of Nc value in each taxon.

No. of Genomes # Genes Mean NC

BASAL ANGIOSPERMS

3 367 7.5394

MAGNOLIIDS

6 367 7.2954

Figure 2: Nc-plots (Nc values vs GC3s) for six magnoliids

used in this study. Parameters same as in Figure 1.

As shown in Table 5, plants from both taxa show a

higher occurrence of A/U at the third position of

their optimal codons. This result is consistent with

the AT richness of the third-codon position in

chloroplast genes.

Table 4: Occurrence of Optimal Codons (out of 64).

Codon Basal Ang Magnolid

AGU (Ser) 1 1

GGA (Gly) - 2

GCC (Ala) 1 1

GAA (Glu) - 1

GAC (Asp) - 1

GAU (Asp) - 1

ACA (Thr) 1 -

ACU (Thr) - 1

AUA (Ile) - 1

AUU (Ile) - 1

CGA (Arg) 1 -

CGU (Arg) 1 1

CAA (Gln) - 1

CAU (His) 1 1

CCA (pro) 2 -

UUA (Leu) - 3

UUG (Leu) - 1

UGU (Cys) - 1

UAC (Tyr) 1 -

3.3 Correspondence and Correlation

Analysis

Previous studies have shown a significant variation

in the codon usage among genes from different

species (Ikemura, 1985; Sharp et al., 1988). Thus, in

order to understand the variations and trends in

codon usage among genes in basal angiosperms and

magnoliids, a series of orthogonal axes were

generated by performing COA of RSCU.

PatternsofCodonUsageinPlastidialGenomesofAncientPlantsProvideInsightsintoEvolution

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Coordinates of each gene on the four axes reflected

the variation in codon usage. Axis 1 COA/RSCU

possesses the maximum variation that diminished

with axes 2, 3 and 4 respectively.

Spearman's rank correlation analyses were

performed among different indices of codon usage

and amino acid composition such as CAI, GC

content, Nc, GC3s, hydrophobicity, aromaticity and

data from the first four axes are presented in Table 6.

Table 5: Top Ranked Optimal Codons by Species.

Species code

Accession No.

BASAL ANGIOSPERMS

A.tr NC_005086 CCA (Pro) CCA(Pro) CCA

(Pro)

N.al NC_006050 UAC (Tyr) UAC

(Tyr)

UAC

(Tyr)

N.ad NC_008788 CAU (His) CAU

(His)

CAU

(His)

MAGNOLIIDS

C.f.g NC_004993 UUA Leu)

L.tu NC_008326 ACU (Thr) ACU

(Thr)

ACU

(Thr)

D.gr NC_008456 UUA(Leu) UUA

(Leu)

UUA

(Leu)

P.ce NC_008457 UGU(Cys) UGU

(Cys)

UGU

(Cys)

C.sp NC_009598 CGU (Arg) CGU

(Arg)

CGU

(Arg)

I.ol: NC_009600 UUG(Leu) UUG

(Leu)

UUG

(Leu)

As can be seen from this table, basal angiosperms

possess more correlation significant values with Nc

than magnoliids. Genes in all three basal

angiosperms are correlated with Nc with first,

second and third axes. Distribution of genes in all

magnoliids is correlated with first three axes, except

in case of I. oligandrum. In basal angiosperms,

Nyphea alba at axis 4 is correlated with CAI with

value r = -.389, p – value < 0.01, and Nuphar

advena is correlated at axis 4 with value r = .477, p –

value 0.01. Magnoliids also showed a significant

correlation with CAI at axes 3 and 4.

The distribution of genes on third axis is

correlated with CAI in all magnoliids all r < -.282,

p-value < 0.05; all r < -.251, p-value < 0.01. GC is

significantly correlated with different axes in both

taxa except for Piper cenocladum of magnoliids.

Hydrophobicity showed significant correlation in

case of four genomes out of nine.

Table 6: Correlation analysis between codon usage and

amino acid usage indices in plastidial genomes.

Species code

Accession No.

Axis1 Axis 2 Axis3

CAI Values

BASAL ANGIOSPERMS

A.tr NC_005086 0.041 0.082 0.130

N.al NC_006050 -0.089 0.114 0.176

N.ad NC_008788 0.009 0.073 0.088

MAGNOLIIDS

C.f.g NC_004993 0.233 0.150 0.148

L.tu NC_008326 -0.011 -0.093 -0.350*

D.gr NC_008456 -0.054 0.211 -0.537**

P.ce NC_008457 -0.200 0.094 -0.351**

C.sp NC_009598 -0.065* 0.276* -0.318*

I.ol: NC_009600 -0.090 0.232 0.285*

*Represents significance at P < 0.05; **at P < 0.01

4 CONCLUSIONS

Our results strongly suggest mutational bias, gene

expression, compositional constraint and

hydrophobicity as the selective forces in shaping the

variation in the codon usage among genes of these

organisms. We analyzed the putative optimal codons

and hypothesize that frequencies of preferred codons

in genes seem to be correlated with the gene

expression, majority of which end with U and may

be useful in the detection of gene expression of those

genes where this is unexplored.

According to our results codon bias is

significantly correlated with gene expression. Our

data provide evidence that natural selection can also

play an important role in shaping the codon usage in

chloroplast genomes. Correlation results strongly

support the hypothesis that besides mutation bias,

there are some other factors that direct the change in

the codon usage frequency in chloroplast genomes.

Among other factors, aromaticity and

hydrophobicity have played an important role in

shaping codon usage in many chloroplast genomes.

This study has provided a basic understanding of the

mechanisms for codon usage bias, which could be

useful in further studies of their molecular evolution,

gene transfer and heterologous expression of these

chloroplast genomes from basal angiosperms and

magnoliids.

AUTHOR CONTRIBUTIONS

MY and GY developed the analysis pipeline. SB

assisted with the statistics. All authors coordinated

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148

to draft, read and approve the final manuscript.

ACKNOWLEDGEMENTS

This work was funded by the SERB project of Dept.

of Science & Technology (DST) Govt. of India

(Grant No. BT/PR12919/AGR/02/676/2009) and

Young Scientist Grant by Indian Natl Science

Academy (INSA) to GY. MM was recipient of the

senior research fellowship (SRF) of the DBT, Govt.

of India during the period of work. Facilities

provided under the Biotechnology Information

System Network (BTISNET) grant of the NIPGR

Sub-Distributed Information center (Sub-DIC) of the

DBT, Govt. of India, are gratefully acknowledged

(Grant No. BT/BI/04/069/2006).

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