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Page 1: Evolution of chloroplast matK genes among lower land plants

Evolution of chloroplast Evolution of chloroplast matKmatK genes among lower land plants genes among lower land plants

Shu-Lan Chuang (莊樹嵐 ) and Jer-Ming Hu (胡哲明 )Institute of Ecology and Evolutionary Biology, National Taiwan University

The introns of chloroplast trnKUUU contain an open reading frame denoted as matK. The putative gene product MatK is the only one having maturase function in chloroplasts.

However, only those chloroplasts of land plants and higher green algae such as Characeae have introns in their trnK genes, but not in other green algae examined. Chloroplast matK genes are indispensable since in nonphotosynthetic parasitic plant, Epifagus virginiana, the chloroplast matK is functional even being a free-standing from with dismissed trnK exons.

The chloroplasts of Psilotum, moss and liverworts all have trnK5’-matK-trnK3’ structure, but it is found that matK is a pseudogene in hornwort Anthoceros formosae. We found a clear trnK5’-matK-trnK3’ structure in Ophioglossum petiolatum, Lycopodiella cernua and Selaginella doederleinii. RT-PCR results showed matK genes are expressed in Ophioglossum petiolatum and Lycopodiella cernua, but no signal detected in Selaginella doederleinii. So the function and expression of matK are not consistent in lower land plants.

Codon usage analysis showed that the use of codons in matK is in congruent with the average use of chloroplast genomes, showing a bias that can be explained by constraints on GC contents. The result of correspondence analysis suggests the codon usage of chloroplast matK has some properties that is correlated with their evolutionary relationship.

Lycopodiella cernua and Selaginella doederleinii are placed into different group in matK phylogenetic analysis, but the incongruence is likely due to the disputable sequence alignment, which causes long branch attraction that will affect phylogenetic inference. Nonetheless, the result showed that Pinus, Ginkgo, Cycas from a monophyletic group, which is sistered to angiosperms. Together they form a clade that is sistered to Gnetales.

M M R R I I M M R R I I

L O L O L O L O L O L O

RT-PCR Genomic DNA

500bp

Fig. 3. Dot blot hybridization, indicating that matK is likely present in all of the samples examined.

Lycopodiella S

elaginella

Ophioglossum A

diantum

rbcL

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22

1. Anthoceros formosae

2. Marchantia polymorpha

3. Equisetum ramosissimum

4. Isoetes taiwanensis

5. Selaginella doederleinii

6. Selaginella delicatula

7. Selaginella tamariscina

8. Selaginella involuens

9. Selaginella tamariscina

10.Lycopodiella cernua

11.Lycopodium pseudoclavatum

12.Ophioglossum petiolatum

13.Angiopteris palmiformis

14.Osmunda banksiifolia

15.Adiantum capillus-veneris

16.Dicranopteris linearis

17.Lygodium japonicum

18.Sphenomeris biflor

19.Nicotiana sylvestris

20.plasmid sd4

21.plasmid lyco8

22.plasmid ophio11

Fig 4. Codon usage analysis. (A, B) Correspondence analysis of codon usage. Major groupings are indicated. (C) Nc-plot shows the bias of matK codon usage is correlated to GC contents.

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0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350

GC3s

NictmatK

AratmatK

AtrbmatK

SpiomaK

OenematK

LotjmatK

CalfmatK

MaggmatK

AmbtmatK

EpivmatK

OrysmatK

TriamatK

ZeammatK

PintmatK

PinkmatK

CycpmatK

GinbmatK

GnepmatK

Gnemmat

EphsmatK

AdicmatK

OphpmatK

SeldmatK

LyccmatK

PsinmatK

PhypmatK

MarpmatK

ChagmatK

ChacmatK

(A) Maximum parsimony tree

NictmatK

AratmatK

TroamatK

AmbtmatK

NymomatK

MaggmatK

OrysmatK

TriamatK

ZeammatK

CycrmatK

CycpmatK

GinbmatK

PintmatK

PinkmatK

GnegmatK

GnepmatK

GnemmatK

WelmmatK

EphsmatK

OphpmatK

PsinmatK

SeldmatK

LyccmatK

PhypmatK

MarpmatK

Strict

100

Core eudicots

Basal angiosperms

Basal eudicots

Monocots

Ginkgo

Lycophytes and ferns

Bryophytes

Cycads

Gnetophytes

Conifers

(B) Bayesian inference tree

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0.99

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1.001.00

0.61

1.00

0.52

1.00

0.56

1.00

NictmatK

AratmatK

TroamatK

OrysmatK

TriamatK

ZeammatK

MaggmatK

AmbtmatK

NymomatK

PintmatK

PinkmatK

CycrmatK

CycpmatK

GinbmatK

GnegmatK

GnepmatK

GnemmatK

WelmmatK

EphsmatK

OphpmatK

PsinmatK

SeldmatK

LyccmatK

PhypmatK

MarpmatK

Core eudicots

Basal eudicots

Basal angiosperms

Monocots

Cycads

Lycophytes and ferns

Bryophytes

Conifers

Ginkgo

Gnetophytes

Kishino-Hasegawa test

Parsimony criteria Likelihood criteria

Tree Length Diff. P* -ln L Diff. P*

TreeBayesian 5596 42 0.0034* 23093.72952 (best)

TreeParsimony 5554 (best) 23137.92301 44.19349 0.000*

P<0.05 *

AbstractAbstract

Fig. 6. Phylogenetic analyses of matK showed Gnetales is sister to other seed plants.

ResultsResults

Selaginella doederleinii

M R I1 I 2 M R I1 I2

500bp

RT-PCR Genomic DNA

Fig. 2. Detection of chloroplast matK expression by RT-PCR. (A) Results from Lycopodiella cernua (L) and Ophioglossum petiolatum (O). On the right showing a PCR of genomic DNA as controls. (B) Results from Selaginella doederienii, and a PCR of genomic DNA is on the right. Chloroplast matKs are expressed in L. cernua and O. petiolatum, but not in S. doederienii. Abbreviations: M (matK), R (rbcL), I and I1 (intergenetic spacer: rbcL/atpB), I2 (trnL intron).

(A)

(B)

Table 1. The results of Kishino-Hasegawa test show that the TreePars

imony is preferred in parsimony criteria, but the TreeBayesian is favored by likelihood criteria, and both the alternative tree topologies are rejected.

The matK is present in the chloroplasts of lower land plants, but trnK5’-matK-trnK3’ structure may be lost in ferns due to chloroplast genome rearrangement.

Chloroplast matKs are expressed in Ophioglossum and Lycopodiella, but not expressed in Selaginella.

Chloroplast matK follows chloroplast average codon usage and the bias is influenced by GC content.

Codon usage of matK does have evolutionary properties.

Phylogenetic analysis of matK showed Gnetales is sister to other seed plants.

Nicotiana tabacum

Atropa belladonna

Epifagus virginiana

Spinacia oleracea

Arabidopsis thaliana

Oenothera elata

Lotus corniculatus

Zea mays

Oryza sativa

Triticum aestivum

Calycanthus floridus

Amborella trichopoda

Pinus koraiensis

Pinus thunbergii

Adiatum capillus-veneris

Psilotum nudum

Physcomitrella patens

Marchantia polymorpha

Anthoceros formosae

Chaetosphaeridium globosum

trnVclpP rpl2 rpl12 rpoC1 rps16ycf3 ycf66 trnH trnGndhBatpF ndhA ndhH petB petD rpl6 rpl16 rps12 ycf2 ycf10 trnA trnI trnK trnTtrnL rrn23

Fig. 7. Distribution of chloroplast introns. Arrow A indicates the presence of trnK/matK in Chaetosphaeridium + land plant chloroplasts. Arrow B indicates matK being a pseudogene in Anthoceros.

A B

DiscussionDiscussion

Chara vulgaris

(C)

Intron

No intron

Free-standing matK

pseudogene

(A)

EpifagusAxis 1

Axis

Green algae