Mol Gen Genet (1992) 234:275-284 © Springer-Verlag 1992

Homologous genes for the C4 isoform of phosphoenolpyruvate carboxylase in a C3 and a C4 Flaveria species Jiirgen Hermans and Peter Westhoff Institut ftir Entwicklungs- und Molekularbiologieder Pflanzen, Heinrich-Heine-Universitfit, Universit/itsstrasse 1, W-4000 Diisseldorf 1, FRG Received January 20, 1992

Summary. The C4 isoform of phosphoenolpyruvate carboxylase (PEPCase) in Flaveria trinervia is encoded by the ppeA subgroup of the PEPCase gene family and is abundantly expressed in the mesophyll cells of leaves. The homologous ppcA genes in the C3 plant F. pringlei are only weakly expressed and their transcripts do not show the strictly leaf-specific accumulation pattern observed for the F. trinervia genes. Two representative members of the ppcA subfamilies of F. trinervia (C4) and F. pringlei (C3) - named ppcA1 - were characterized by Southern blotting, nucleotide sequencing and primer extension analysis. Comparison of the deduced amino acid sequences reveals a close similarity between C4 and C3 isoforms. Only few C4-specific positions can be detected when all known plant PEPCases are included in the comparison. A regulatory domain involved in lightdependent phosphorylation/dephosphorylation of the C4 and crassulacean acid metabolism (CAM) isoforms is present in the ppcA1 gene products of both the C3 and C4 Flaveria. The 5' flanking regions are essentially homologous. The putative promoter regions share several identical sequence motifs (CCAAT, AT-1 and GT-1 box III/III* elements). Additionally, alterations in elements that could contribute to differences in expression rates and light regulation are found. The significance of these findings is discussed with respect to the molecular evolution of C4 photosynthesis in Flaveria. Key words: Phosphoenolpyruvate carboxylase - C4 plants - Flaveria - Gene family

Introduction It is commonly accepted that C4 plants have evolved from C3 ancestor species and that this transition has occurred independently several times during the evoluCorrespondence to: P. Westhoff

tion of angiosperms (Smith et al. 1979; Moore 1982). This polyphyletic origin of C4 plants suggests that the changes required to convert a C3 into a C4 species must occur quite readily in evolutionary terms. Not surprisingly, each enzyme reported to be typical for the C4 cycle (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase and NADP-malic enzyme) is also found in different metabolic pathways of C3 plants (Moore 1982; Cockburn 1983). By implication, these C3 ancestor genes will have served as the basis for the evolution of the C4 isoform genes. What changes might be required to convert a C3 into a C4 isoform gene? For phosphoenolpyruvate carboxylase and NADP-malic enzyme it is well documented that the C4 and C3 isoforms differ in their kinetic and regulatory properties (Andreo et al. 1987; Fathi and Schnarrenberger 1990). Thus mutations in the coding regions of the C3 ancestor genes must have occurred to bring about the necessary adaptations. Wilson et al. (1977) have pointed out that the acquisition of a new metabolic pathway usually requires that the activities of rate-limiting proteins be increased. Consistent with this view is the observation that the levels of the C4 and C3 isozymes and their corresponding mRNAs differ drastically (Hermans and Westhoff 1990; Rosche and Westhoff 1990; Rajeevan et al. 1991). Therefore, mutations altering the effectiveness of gene expression should have played an important additional role in shaping the genes towards their function in the C4 syndrome. Finally, cellspecific expression patterns of the C4 isoform genes had to be developed, since a strict compartmentation of the corresponding enzymes is imperative for a functional C4 cycle (Langdale and Nelson 1991). To gain an insight into the molecular changes which have occurred during the evolution of C4 plants, the phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) genes of the genus Flaveria (Asteraceae) were chosen as a model system. Flaveria contains C3 as well as C4 plants (Powell 1978) and offers the opportunity to compare closely related plants that differ in their photosynthetic properties. Additionally, this genus en-

276 compasses a wide spectrum of plants with intermediate photosynthetic characteristics, suggesting that the evolution of C, photosynthesis is still in progress (Monson and Moore 1989). PEPCase catalyzes the primary fixation of CO2 in the leaf mesophyll cells of C4 plants. Multiple additional roles of PEPCase activity in the basic metabolism of C4 as well as Ca plants have been identifed (Ting and Osmond 1973; O'Leary 1982; Latzko and Kelly 1983). Hence, it has been found in Sorghum bieolor, Zea mays and Flaveria that PEPCase is encoded by gene families whose members exhibit differential developmental and tissue-dependent accumulation patterns (Hudspeth et al. 1986; Hermans and Westhoff 1990; Cr6tin et al. 1991). Recently, the PEPCase gene families of the C3 plant F. pringlei and the C4 species F. trinervia have been isolated (Hermans and Westhoff 1990). Analysis of their expression profiles and quantitative hybridization experiments revealed a similar organisation of the gene families within both species. At least four distinct classes of PEPCase genes could be identified (named ppeA-ppcD), each of which contains several members. In contrast to the monocotyledonous C4 plants Z. mays and S. bicolor, even the C4-specific PEPCase isoform of F. trinervia is encoded by a separate subfamily of closely related genes, named ppcA (Poetsch et al. 1991). The ppeA genes of F. trinervia are abundantly expressed in the leaf mesophyll cells. The homologous ppeA genes of F. pringlei are also preferentially expressed in the leaf, but transcript levels are quite low as compared to their counterparts in F. trinervia (Hermans and Westhoff 1990). In the present paper, one representative of each of the ppeA genes from F. trinervia (C4) and F. pringlei (C3), named ppeA1, has been characterized in detail at the molecular level to provide a basis for identifying key changes which may have occurred during the evolution of C3 from C4 plants. Materials and methods

Standard methods. Growth of plant material, DNA and RNA isolation, Southern analysis, construction and screening of genomic libraries and DNA sequencing have been described previously (B6rsch and Westhoff 1990; Hermans and Westhoff 1990). Primer extension mapping of RNA 5' ends. A sequencespecific oligonucleotide (5'-TCGATCGATGCTAATTTCTCCAAATTCCGGTTAGCC-3') was synthesized, uncapped, detritylated and finally purified by polyacrylamide gel electrophoresis (Boorstein and Craig 1989). 5'-Labelling was performed in a 20gl assay (120ng primer; 50raM TRIS-HC1; 10mM MgC12; 0.1 mM EDTA; 5 mM D/T; 50 gCi [7-32p] ATP, 5000 Ci/mmol; pH 7.6) by incubation with 8 units T4 polynucleotide kinase at 37° C for 45 min. Unincorporated nucleotides were separated by chromatography on Sephadex G-10. The primer was annealed to the RNA in a volume of 10 gl (300 mM NaC1; 10 mM TRIS-HC1; 2 mM EDTA; pH 7.5) for 2 h (for amounts of nucleic

acids and temperatures see Results). For the extension reaction, the volume was brought to 50 gl (final concentrations 50mM TRIS-HC1; 60raM KC1; 10raM MgCI2; 1 mM dNTPs; 1 mM DTT; 50 ng/ml actinomycin D; 1 unit RNasin; pH 7.6) and incubated with 25 units reverse transcriptase (M-MuLV) at 37° C for 1 h. In the next step, the RNA template was removed by incubation with 0.5 units RNase for 30 rain at 37° C. The extension products were concentrated by alcohol precipitation, dissolved in 3 pl formamide loading buffer (90% formamide; 10 mM EDTA; pH 8.0) and analyzed by electrophoresis in a denaturing polyacrylamide gel (6% acrylamide; 8.3 M urea). Computer analysis. Phylogenetic trees were constructed by the programme PAUP (Phylogenetic Analysis Using Parsimony, Version 3.0i; from D.L. Swofford, Illinois Natural History Survey). Sequence elements were searched for by the programme MATSCAN of the PC/GENE software package (Genofit, Geneva, Switzerland). The statistical occurrence of a sequence element containing a given number of mismatches within a DNA fragment (e.g. the promoter) was calculated using the following formulae: PE ----

I~ Wj~ I~ (1--Wj~)

k=l

k=l

(1)

The probability P of a matching sequence element, which has a length of E base pairs and contains N mismatches, is calculated with formula 1. Wj are the probabilities for the occurrence of a particular nucleotide (A, C, G or T) at the position j in the DNA fragment (WjA -~-Wjc ~- WjG 4- WjT : 1). OF = (L-E) PE

(2)

This formula defines the probability for the occurrence (OF) of the sequence element within a DNA fragment with length L.

Results

Sequence analysis of the ppcA1 genes of F. trinervia and F. pringlei Lambda-DASH genomic clones of the ppcA1 genes from F. trinervia (lnFtppc8) and F. pringlei (lnFppc65; Hermans and Westhoff 1990) were divided into overlapping plasmid subclones (pBluescript) that covered the whole coding and the 5' and 3' flanking regions. For sequence analysis, successive deletion clones were produced by the exonuclease III method (Henikoff 1984). The detailed sequencing strategies, physical maps and the exon/intron structures of the ppcA1 genes are shown in Fig. 1. The two genes exhibit the same exon/intron structure as the PEPCase genes from Z. mays and Mesembryanthemum crystallinum (Hudspeth and Grula 1989; Cushman et al. 1989). The coding regions of the ppcA1 genes of F. trinervia and F. pringlei have the same size (2901 bp)

277

A

F. trinervia ppcA1

.=_.o "rX

8

I

I

I/

~

"~'~

-

~Z

~.c_ XI

X

I

II

_

uJ

I

I

5"

3"

I

II

III

IV

V Vl VII

VIII

IX

X

L

B F. pringlei ppcA1 _--

E:

_--~

8

8

•- r .-r

UJ

I

I

W

I I

,,o, ~-

,',

x-r

I

X

\I

I 3"

5"

I

II

Ill

IV

V VI Vll

Vlll

IX

I

I

I

I

I

I

I

I

I

I

0

1000

2000

3000

4000

5000

6000

7000

8000

9000 bp

Fig. 1 A, B. Sequencing strategy and exon/intron structure of the ppeA1 genes of Flaveria trinervia (C4) and F. pringlei (C3). Exons I-X ofF. trinervia (A) and F. pringlei (B) are represented by stipping, the intervening sequences by open bars. The thinner bars reflect the

A E

c

o w

23

F. trinervia

i23

I

i23

5' and 3' non-coding regions. Putative positions of R N A 3' ends are deduced from c D N A clones (Poetsch et al. 1991), the R N A 5' ends were determined by primer extension analysis (see below). Individual sequencing reactions are marked by arrows

B ~ ×

i23

o

F. pringlei

-7~:--

X

123

rn

LU

-r

(0

X

X

23,1 23,1 9,42 9,42 6,68 -

~ O o 6,68 -

4,36 4,36 -

3,53 -

3,53 2,031,61 -

,

2,03 -

1,61 -

iili ii ¸ !~i;

.....

Fig. 2A, B. Genomic southern blot analysis of the phosphoenolpyruvate carboxylase (PEPCase) genes in F. trinervia and F. pringlei. D N A from three individual F. trinervia plants (A : 1-3) or from cuttings of F. pringlei (B) were digested by BarnHI, EcoRI, HindIII, XbaI and SaLI only F. pringlei). The full-length c D N A pcFtppel-1 was used as hybridization probe. Hybridization was carried out at 68 ° C (125 mM NaHPO4, 7% SDS, pH 7.2); washings were at 62 ° C, 0.1 x SSC

278 Ser(P} ....

F. t r i n .

C4

MANRNV

F.prin.

c~

.....

N. t a b a c . M. c r y s t .

C~

M. c r y s t .

C3

S. b i c o l .

C3

.PE.H ......

S.bicol.

C4

. .S . . . . . . .

'---v.rR~l. . . . . A . A . . . . . . E - - . I Q . . . . . V . R . . . . . . . . . .

PS.R.F..E...V..D

Z. m a y s

C4

. .S T K A P G P G

. . .w.~ ILl/ . . . . . Q . . . . . . . . . . . .

PS.R.F.

F. ~ r i n .

L A E EVQ I A Y R R R I - K L K S G D F A D E A N A T T E S D I E E T F I ( R L V H K L N K S PE E V F D A L K N Q T V E L V L T A H P T Q S I RRS L L Q K H G R I R ~ Q L Y A K D

F.prin.

C4 C3

N. t a b a c .

C3

. . . . . . . . . . . . .

M. cryst.

CAM

.......

EK L ~

L .......

~.SL

.....

DAQLRLLVPGKVS

l i a r

..................

-'~//

.....

• .- . . . . . . . . . .

.

A .....................

.......

I LeD LHGEDLKEAVQQCYE

I ...........................

.........

--~.~

E DDKLVEYDALLLDKFLD

A .................

R ............

V.R .......... I ...... V.R.

PKKLD E LG S LLTS LD PGDS I VIAKAFS ~ ....

T..E .................

~ ...............

A .................

LSAEYEGKHD

S .................

IR.T..E...R

PH.R.F.

.N .......

E

~V

..................

.E .........

........................

V .....

NDR. EAR . G . . . .

A .....

K ....

.........

V..S

P ......

V.SS .........

.... K.TS..G...AK..G.A.A.A.LV.

.E. . .V. . D . . . . G . T T .

HMLNLAN

V ........................

.G. . . A K . . G . A . A . A . L V .

SSIL

.......

SSIL

.......

Cys .................

R ....................

0

o

R

......

.

K..L

Ko 0 0 K 0

. . . . . . . . . . . . .

SR.K.VK.V.K...M..NT.M

..................

....... V ............

l~.r . . . . . . . . . . . . . . . d

C3

.............

C3

......

C4

..... EL.H.

Z. m a y s

C4

........

F. ~ r i n .

C, C3

E I QAAFRTDE IRRTPPTPQDEMRAGMSYFHETIWKGvPKFLRRv~TALE~IGINERFPYNAPLIQFSSWMGGDRDGKHPR~PEVTRD~S~

.

.......

s ,

........

. ....

. .H. - - - . . . S . . . . . . . . . .

..... I .........

D..F

. . . . . . . V . . . . . . . . . . . . . . . ,. ,. . . . . . . . . . . . . . . . . .

0ooo. . . . . . . . . . . . .

0 o ,

.......

L .... S- .G.T.A.

H. . .NS. . .K.G .... GS ..........

L .... SEVG

v ............

. .E ..... S.D. .F ....... A .......

.......

Q

" . . . . . Q"

..

S .bicol.

.GTS.

. ......

. . . . . . . . . . . . . . . . . . . . .

S.blcol.

.....

AE.G

............

M. cryst.

v 0

...............

...... D..F

ITPDDKQE - LDEALHR

V .............................

........... 0 .......... v

.... M...LR..IVD.K...Q.I.ET

K...K..LS..NS

D ..........

........

N A . . . .I

E ....... D. .F ....... A .......

NA. . . ~.

T..N

Q.

A ....

I mT . .S. . . V . V E .

.K.- .......

. . . . . D . . . . . - . . . . . Q.

Cys F.prln.

FSQI ED~I

........................................................

V .....................................

m

C3

..............

L ....................

~.~ ..... ~ . L Y

M. crys~.

C3

........................................................

V ...................

- N . . . . . . . . . . . . .I.I. .

S.bicol.

...........................................

L ...................

-N ..........

S.bicol.

C3 C4

.........

Z. m a y s

C4

.............

N. tabac.

A .........................................

N .... L .....

m

A ............

F

E

F

....

M. crys~.

DF..AQ

........

Y .... I...V.N

AQ .... A...Y

.... I...V

Cys C4

EMSM~SELRVRAE

F .prin.

C3

. . . . . . .~ . .m. . . . . . . . . . .

L..DV...K.C

.......................

..........

L...VS..R

...........

N ..........

--.

..........

....

M . c ~ ' y s ~.

CAM .L...

M. crys~.

~3

, - , ~ _

," r"-...E /Irn/Ir~

.....

S.blcol.

C3

. L . . . .l hnD. . .I. H =

S .bicol. Z. m ~ y s

~,

.,....~.Lo...~

C4

.L...

F. t r i n . F.p~in.

C4 3

D FLRQVSTFGLSLVKLD

N. t a b a c .

~

E FWKQI P P N Q P Y R V I L G D V R D K L ~ R S R H L L V D G K S D V..TZ

........................

........

. . . . . . . . . . . . . .

HKYS...r.l.r Sdgl/| ...........

SSE ......

A .......

0

-N ..........

I~ . I

.D.

. H . S S - - ~ D h ~[_ =

.........

.... v Q ~ . , ~ . ~ . ~

...........

F .........

....

, .....

....

E..V..TF.

0..s.v

Y .........

. .E ..............

F

IN.V .... F

..... A hA n.L.ID...E..F

.... E .........

Q..ASEV.

........ . .... , ..................... V.

AH.I

Y ......

.~s~--~

F

.........

Cys

I PDEAVYTNVEQLLEPLE~S~SGDHVIADGSLL

....

o

.........

, l ~4 . S.. .~. A A . L . ~

Cys

ELYRTA-- ~VIEMYI

.

i

..... F ...............

~.~ ~ . . . A A . L

C4

F. ~ i n . N. ~abac.

I ............

~A A . L

SS.I.E.

~'.,

........ .......

EID. F .....

.... , o ~

C..R

Ih//r.l.r_./hl"l"C" II

.RPV ......

..... 4 ~ o 4 ~ c

.E. . T F . . . . . F . . . . . . .=I.I.=. .

~

~lhc

.... •

KP .......

, .......

A ...........

A .... ~.,.,.~,~..,.~.~,

.....

E .......

H ...........

A .... AS.V.E.SA.SSF.SI.EF

..... {L~K. ~C..KA

~.L~.4.~..~

.......

i

~ D

.........

HSSSGS~T.

Y ...........

.......

C4 I RQE SDRHTEVLDAITQHLG

.......................... ............ F.R ...........

IGSYREWS

D ......... D .........

EEKRQEWLLAELSGKRP

~PDL

E ........................... E .......... R ...... S .......

PKTEEVKDCLDTFNVLAELP

SDCFGAY

M. c r y s t .

C3 CAM

M. c r y s t .

C3

...... F ....... R ...........

D.M .......

S.bicol.

C3

...... FN. . .A ..............

D . . .S. . T . . . . . . . A . . . . . . . .

S. b l c o l .

C4

.L . . . . F . . . . . . . . . . . . . . .

E. . . D . I . . . . T . . . . . . . .

Z. m a y s

C4

.L ....

E...D.I

F.trin.

C4 C3

LLQREYHI I~PLRVVPLFEKLADLEAAPAAMTRLFSMDWYRNR IDGKQEVMIGYSDS~AGRFSAAWQLYKTQEQIVKIAKEFGVKLy~GR~R~L

• .M. . . A . . . . C . . . . . . . . . .

E. . . D . M . . . . T . . . . . . . .

F ...............

....

~.

D.T ..... D. . .S. .R ..........

E .... K ............ S.P.D.

T ..........

S .......

.M. . W - C R V K

S..R

R.D. IA.V.

~.S.

D . . . S . . R . . . . ...0

P.D .......

F.p~in.

..... C.V .......................

. .LH ....................

S . . .Q. . . T A . V . G . . H

...... A ............

..... C.V.Q

...........

M. c r y s t .

CAM

..... CKV.K

...................

M. cryst.

C3

.....

. . . . . . . . . . . . . . . . . . . .

...... IES. .P ..... C.AP

....

......

bb~ ....

Q.D. IA.VIGA.H

P.S..P

S .blcol.

C3

..... C.V. o . .T .................

S.bicol.

C4 C4

..... .....

Z. m a y s

. . . . . . . . . . . .

VA .... I...M...N

.............

R .... e..RPSVEK...T... I.H.N...Q..V R....QS...SVE .... V...MD..K...Q..V

MWHSPAVP . . . . . . CGVRQ..P ......

A..E.I.V

,

,

.

....../I,.,...L

C4

SQP PDTINGSLRVTVQGE%rI

F.prin.

C3

.......

N. t ~ b a c . M. c~yst.

C3

.......

.......

P I S PRPEWRELMDQMAVVATEEYR~KE

PRFVEYFRLATPELE

H ................................................................................ "l"r Q ................... .4., ...... T ........ H..V..K .... A...EI..I...K.. |]~.. . . . . . . . .

M. c~yst. S. b i c o l .

C3

S.blcol.

c~

~ .........

~,. . . . . . . . . .

~

Z.mays

C4

..........

I ..........

F C . . . . .I I ~ 2 Q . . . . . T . . . . . . . .

F. t r l n .

C4

G I ESLRAI

F.prin.

C3

.................................

N. t a b a c .

C3

...........

M. c r y s ~ . M. c r y s t .

CAM C3

........... i~]~T. . . . . . . . . . . . .............................

, .............

, .....

.....

YT ...........

K..K

T ........

L~.~

. . . . . . . . . . . . .

, , ~ lm~ . . . . . . . . . . . . . .

,o.i Ii....I.

C4

EQS FGEE~RTLQRFCAATLEHGMNP

CAM A . . A E . . G ...... I ........... O I. I l . r I. I .... E.V ........................... C3 .......

ELI .V..Hy.

V A . . E M A . V . . K Y . . . . v'~l__.l........... V. "~'''/ . I R R A . . E M A Q V . . R Y . . . . . ,r.T/.1. . . . . . . . . . . . . . / ~ . . . . I .

Cys F. t r i n .

£

....

..L . . . . . . .

I.

.... T~ ............. .l....I. . . . . . .,,4 . . . . . . . . . . . . . . . . . . .I I.

....

| ] rr ' " . L C S M - . M . . A . .

....../~.,.1

.......

AP .......

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .B .

. . . . r E . . . . . . N . . . . . . V . . . . . "l.I . . . . . . . . . . . . A..ELI.V...H S . . . . . . V . . . K . . . . . . . . . . . . . . . . . . . .I.I L ..... M..V. .ELL.VS.K ....

.......

PEP binding

D...S.S..VA

w

P ....... AP .......

.... |L~ .... M. . .I A . V I ~

A .... I ..................................

C3

P ....... AP .......

. .I. .I . . . . . . S . . . . V . . . . . A P . . . . . . .

. .Q. . . I A . V .

Lys N. ~ a b a c .

I I SMATSTSDVLAV~

S .......... V ......................... . . . . R. . .I A . V . . . L H . I . . . . . . . . . . . . . . . . .

.....

~

........

. . . . ~,~,~.~os . . . . • . . . . . .

....

A.L ............

H ..... K .... A.L.EI , .....

~....v

~..~...,

.......

H..V..K

,~...~..~

T.Y ................

.........

Y ................

, .... ~.~

N-.,v,.~...,