&..9. . . V . . 1 o .
. V . . . .. V V V . o . . A . V . f . .V . . . . . . .. .
3.3. V . ......1 "M...“ . ... L§.1WW'VOOM...JH,.O.V11..V -. . _ V. . . . . . V . . _ 10 o . . .... N17... .1. .
. 0 y a ~‘ I‘. I. . .. IV ,
1 . ......V 2.. u.l.1.1. -. 7 2 .ro. . . "fl
.. . . .
1.

V . . . . 7.. V. y a o . .. 1 V
. . ...): “...“: ..u ....hhiuugm33$3fiorn.u.n..g4¢. . IJOJJB‘V .. t!
V 0
II .1.

.. ..V....1. ......

c... " 1.....)‘ on. IO...
..‘loéflo 1.... ..I‘. o . . V

21...... .1 are ...11‘. . . 3 ..9 120.41..

.u. u .. . . .... 3.1.1, .s: ......
..1.‘...v..‘
. 0 . 1...

.Q s 1 ......1.
.1 5 .9. .01..

. ..1 1 ..2.
1.111- 1O..?.1

. 11.1.... .01 .1 .u A “Conny...” 9.... 3.7...01v... V
V . 1 .... I .4. . . . V
. V . 1?...an Jun... .1811...” ... ...” .49.. . . . . . .
V . . Iza-Plf! ...—0.! a. .3'1301‘. . . . V. V . .
“(111”? . V . 21 . . ... $.11... 9!. . . V . . V . . V. .
V .11.. ‘11.: V . . . . V V . . . .V. V V . .
. . . .. V .. . . . . .. . V . - V . . ..O . .
. V . . V . . V .V . . . . ..Ip.o.‘kt . .
V 21.1 .1‘101 1);... .15
. 11.!— .I 16.5‘.,.
£29.31 01.91.. .
1.. l I

5J..

.1

. I
V 5 IV. . .N... C ‘37-... .11..~1‘| . .
.Wt. .3. 1...!0 ..‘f 1.. ...... 61...... .39 .3'31 . o to. .
3.1... 1" 7... 13%! ....'9.. 1.. .3 1...: .' c... 0‘3".an0 . L. .4: 2......
. . 5.. 1.5.1.111 :1 1. .1 1.91.7...‘ .... .11... .0101. V _ .
. . . 1...... 1.1.11.1; ... ... c 1.. .9 0.11. V . . .. . .
1.0101. .... 1101 . V . . . . V . . .V . . V V . . .. . . .V
01331.0.112.. . 3:... 1.3.... V . . V H . .. .. . V . . V . . . .
V. V .. V. . 1.1 31"... .SVIgurfio . 1 31.0.... . V V . V. V. V. . . V . . H . V l 1 1 V. . . . V . . A... 311...)”,
.. V . . . . V V V . V. . . 1. 5-11.. ...
111,9... .1031. 7’“? 1. V V . . V. . V. V . . 1.61....09u . . .khfl.f1 . . . V V ....‘...b.1l.§11 ...4.’b.).h| 10
. nip 1 1...... u: . . V . V . . V V 11.11... .0.- a. ....1 ‘Io.9. . V. V . . V . V. \ ....1111 ~VV‘V..~’
.3.- .T. . V 1’... O: . . . . . . I ...VIVVVLFV: 11.! ... . . . ..uh.‘ V
.11....‘3rni . . .. .. . V 1.. 1.1... . . . .
an. . . V .
v ....Q
I11...‘.-‘..1:. ’1 1...].
n o .101. u "341 :1

...s..........~

‘. It‘.‘ 01......I.v. 1 O
o ... 11.... 1.?3‘2-‘I..n 1...
1.21,... lio...1."..l.1. 1.1 .
. ..I V . . . . . V . . . . V V. .
Elie-’1? . . . V. . . .. V V V. H. V . A. , .V .
. . . . . . I .1
V . . .V .’ .. .....1, . . .
. V . . . .V .V 1...... 0.”. . 1.0.. v
. .0..- . 1101..- ...”.on‘l 43’. V V .....11.§
.....JJ’...... . .
Q 0...! V

.. Q. VII... ..I.VVV.
15..) O .....‘wr; .313...-
...1..D§V II 0.10... 1.

v_"

n o ‘o‘:‘bo-1unouu...x. . V

.! ($.11 00......1011‘. 1 . . . . . ...»? . 1.
1.1.9.. .-.. . . . . . . . . V . . . . . . ’1. 1 I . 1. ‘1

00...... ' . . . . . , . V . 3.9‘nhnu...0. I . .5 . . . . . . . . 1 car'ssi’.‘ .. Q. 4 9“.
.. 1....3 . V . . . . . . . . . V . u. 11.“?! ...... . _ . . . . . . . V. .. . V . .. 3...“: 1.

V . . . . V I . 9D I . V . . . . V V . . . . . V . V

. . W1...‘11.OW1_~W.I . V . . . I .I. V. . . V . . . V V . V . 1P1‘

. . . . 0.. ...-1V9. ... . . V V’t’O1...
. V . V 1 1‘. .
.1. . V . Jr. 0.
. . .

’13-.

 

 

I. 1.1.."0...

nun

 

 

... £1.
a.-. 1......
.1 1.. . .. V1.1.V01V. ...
. .l. o o. 1‘ '-

 

1.1.0.”..w0. v1. . V . V . . . . .
‘1... ‘10 . .n-V'O.1.uu 11.1.... 1.11 on. 1 .. V . V . . V. . . . . . . .
V . 1 . ' 1 .. ~ . . . .. V V . V V . V
.I.‘.ov1..o.’loon1019331wvcvuo’...u . ... Q ...... 1 -..:111v .35.... 0 if; . . V. V . A. . V .. . . . . 050.... .1... _
1!:3.1.12f..'..o.$!. 111 81.. V:zl .1 ~ .1 .21.... ......190 V . _ . V . . . . . . CM 1.: . ...1.'..
......1.1.bo..0.1 11.1... 1.. ..1 ’1. . ..V§,I. 1.23.. .70. ... V V . . . . . . .. I}... .’ .0‘.:'..

)ou'p.o‘.. x 3.... - c ‘. I‘lv. I.Y1l.‘9. V .. ”uwrb. v...‘13 .91.,‘I .0 A ‘ . V . . V . V . . 1. V . V. . V . V I. 1. .1. 1.1Hccop.¢ .0 V V . . 5 .0! 1"...9L'.
...: ... '7’ .1... ,1 .1 V . V . o. - V .V .. . .. . V. . . . V . V . H. n V . _. . V . . . .. . . .. J 1:1 1.. V . V '1 . 4211-1...

8|..L 5:83.11... 0%- a, V 1T“!!! . . V . . V Vow’u...ml.-Vo..plo 3 t: “11’ . vutm ....1.‘n.1‘
V V V .V V . . . . . 1.1 (.7. ... V 1. V 10.1...
V . . .. . V .. V V1.1.3f17...

.l"._".1-

. 0‘..1V.11 0 191.19! 1 .
i 1.. V1.1, 0‘19... .-Ioc.l . . .
5.9.1 1.
D . 11...

. . . iii-1.4.1.1 . .11.:Inn'. .
1 0.1 0' V . .. ‘1. ..‘o ’10.,7 V .
.12.. ... ..“a.:..‘.1.3! . . 1 I... Idofu‘u OX 1l',._\.1h:1.:
. 2'. .11 ....v.. u V'X"OO V V . 1'0! Ola. 8.. Q '.li-A.'1-O1-O..1.-O. . . .91. 6 o,
.03.:1 ..1'11.‘A....Vr.o. its ...I‘...V 1011....19 . V . V,
. V . . 4 "Q ... 1.0.
V A . V V 131001.-.

012M

:01, Q 1. .
... 1.0....
. . C151... .st‘.’1."'¥..
fi‘-’. . . I. .11...‘. ...
’3’.

’33".
w

b .

- :4
V \10. 1 .1 ‘10,..11‘0..Vol ,.l... 11“:
c): ... rititlcul 31,11... I-1..2.....‘.1 ...
11\V" .0 ‘0 avg. 1.1911 J, 1 I... 1 .01 ‘1 :r‘..1111.'..uAi1-0 ‘. . V. V
. 11.1.... .. \..11 ‘. .1... {101.1,}! 9.6.. I to 1.6.... V . . .

711.010....- 9.$.~31.(‘.h“.. 0’111119’ . V . V . V V . . . V . V. .
o‘cufitg . . . . V V . . . . . .
a V. V . . V

’ . . .V ..111....”.o'1.. 1.-...‘1
o .. 339...... '5... I

an

I}.

3.111.231 1.1.. .1133... 1. . ...

11.0.1.1 . . 0 .1.‘ Y1 .... (I.
. 1’\... O . V .‘1.&:Al ......" ..VWWEXI .3, 0.0.10-
. ....1?‘ ....VV..‘1.|1 .11..-. 3.1..31... .1351 II.’ 1.

0’0?

..32...’ V v 1.. 0 .I. . .. V- 9.. I".. 1
911:5. QI. ! . 0‘ 01”“...11'1. .IlQ . ...
A 1 .371.1.!. 1 ....

.’ V " 1‘13). 1
91...! ....s.

thoo..3LmV . v. . V

a. 0" ‘..~ f.‘ IV. 111.! 1:30... . . . V

.1 .o I 1‘11! If ... 02's . ...Qo...V 9'!-

.«n« I ...: .. ......x... u q ... .3”. . V V . . . . . n.............:.1v.:

. . . . . . V: . V V

.. V . . . . V . . ... ..Ir .1 11 ..........U...ou.uo.1 .1. .1I‘Ot1VO

V V ...)O, . 1 11" f}. In ..VO‘).V.1.1V . . . ... 4,111.}: 9.....38|.!‘.!... ...-’21.. .‘ 01

11V...11.1oo: . 311.00} gut..rl..l.l\£lin , .1». .IIIQ‘ J....".Vb....... ..Y. ..12’1.?V

V. I 2.1.110..- 11...}. 1‘ 1.6.0.1. Q. 1.1111711 .. . V . .) I1 .. 1.1. . Q. ’I1V....VXI.!

I 1111-18... 101.. 11“.:11191.p . 1.37. 11 VV.0....11..‘o. .. , 1..’..10V\11...I:..O!‘). 1‘53 . 1 1 ..
o. . ....)S’: V... . .3711. .

.“: ...

.4.11I.o'1.". 1:1 1 .
.1751 53.73.33. V V
r... ...-1.30“... .-. 4.1:."— S’o‘ 1.3011 (31...-.1 V.
.... .1 ........$.I1.. a... t“. .31.. ... . . 42.1.34).-. w . . V V
’1 {31.01.811.1' ... .1 .3015... 1...... .91.. .1 9%... V . . . . . . . V . 1. 111...,
OI. 1.117.- 7‘1110391. C 1.... .. . . . . . .. V V . . . . .. ...I, . . .
. .. V . . . . V. . V . . V V V .V . ... . . . V V . 4 .31 1‘91?‘
V . . . . . . .V .. . . . V Sir... ...,ch..1.ol|.
. . . . V. V V V V . . .V ... v.5 1! ‘3.-
. V. V 1 v ......1 ‘1.
.. . ....» .13.. .0

€3.17.“ 18.. . . . .... .11.... b...) . .
1.1.1.9. .l..... V V O 1’. 1.10,)
1: . .I. .. .....fio.’f:..oo... .

 

.1. O...’_

...V v.

1.»... .
31.... . V . .
. . . . . . . 1:1I1 .....313:
.o’“1V1 ... 3.
y. V1...-

11,.

..1...O 1'1, ’1I101'.v.l. V . . . . .
. ‘1t013 I111..t.111-.1‘.3~.11 .. . ., . . V. V 1. 0..
1. 11. . .. . V V V V V ..
. . V . V ..lu . ‘3. 0"?-
. V ...-‘0.) .91:- V
V . . .. .V

T’

.1 l

Hut

2

.
...-.1.

u. .0." 1., .
.1 ‘1...’.ur 3,...-
’.§\JQVI--.0 .’.
3| v|1 .
.le. .1... I. 7’.
V .....ZI.

1‘ .01.! ..
.1 I. 0,11 iv‘ a.
r V Vol.9...

 

I.-...4 1 16.1 10;. 4
. . . «0. .....I1 11....
19 11‘ . ... ...:V. V ......
. .. . . . 1 V v . ..
. .. VV .. V .4...

......ul I '

A.V.o.v.Vl .
a\ .V.... ...-.’v. 1.01
.1.‘ ...V.-..

. . .... .....V. V. VV V .V .
. 3.0.01 . . V. , . . V . . 1. 0..
. tsp;

1.....l. . V . V . . V
$111.1. 1. 2 . . . .1717... .51.
I!!! 04. . V V V . .3
0.0.1.3. 19...:
2.1.1 o .... ...lv 0
......1 1' Vtotl ya..-3V.
.. -1} .0. V .
. 1.- o’.l{ .3...
01-. . . s ‘Q . .
... .Vl‘ou ....o . V V V 1.. 1'11 ....10.
v 1t. 11.3,. ...1. . .V . . ...31' . .013. 1 ... 1) 1 . .V
1 V . V. V. ‘ 5.11.1.1 .l..1.....t&..b'...o ..
. .1 .‘I'. I.-:.

...

.1VA..O- ..l11a..V.a . V
V '01 11....11.|1012.1v..! 1’...Oo "3:.’1...A§.L.
. . . 1-... 3.1.190 ‘. 1‘.-

“watt:

... np‘lb‘io . f .
. $11.31.. . V . V. . . V .. V . ...!.....’..~1VV
O.Vro.rl. 1D... .1‘..11I.q ....w» . . . . .. V V .‘t v.
. ’3‘. .06... .1111.v.:b I. 1 I. .V V . V . _ . .
1.2....3. ... . V. . V. . V . . . .
. .1! V . . . V V . . . V ...‘1 IOI.110 . .
. . . . . . t . V ... ... . ‘Q.’.

W“?

M.”
.'.

‘1:.A:J

. .. l ....b n
.1 V 2'3. .V ' ._
. 1.1 .0; -....v-.llu
. V. .... .
O. Onto-s: . 06. I I
... u . v11.-.

. . .l . . . II1....2...... .. . . V
.1.!I..O| O. V. . . . . V. .. . .. ch ‘3 V .V .. .V . , 1 11. A...V.9‘
I! bla1vlo' o.fil1|1 . V.n.. . ... . V. . . V . . 011.: ... o. . . . I ...... I}... V . . V .. V. V . . . . ....‘1‘l'.us.’1.
..19. \.11 11011111021531... V. .‘-.1..0. '1 V V . V I... v. .71... av .. . V. . . . . . . . V. . V V.... .TID“. 0.1‘0."
1 . .' .-....131...6. ... .....lu.41- .. Va... . ..V II. . 1.... .V . .I-OD.1..L' .' A
. 15 . 5‘:l.. § ... 1.9 .. . . ...... 1 . . . . O... 1 1 . '0‘ 01.,
. _ v. 0111 .v 11 “v . v: .1 u ..1 . . z 31.... O I . ...}.
.111. 10.0"'1V..1|>o“'. 1. .r . . . V . . . .
15...). 3 .l‘...|1..1”.1. ..1 V V. .. . .0- . . . . . . V .
. . 1" O 1311. 3.0.01.1 .... . V V _ . .1... 23......1. ... . V. . V . V . V .
. . .. ... .. .1.. Cl . 5....-. .....u... 1 1 ... 3.... ... . . V . .V . V . o .10.: $114111...D0
. 1. .§..V-‘.‘..Ir. .
v n .

it”

.i,

l.1l.. V
-V 10“u‘
...-..I.L‘\‘..

 

 

' Ofiosfgflitq

    

 
 
 

 

 

I‘
1.1.1. I‘1 .I
.0 ‘l 1 III. 17 ,1 . . : o . . . . ..
1.- ... -03 . . 001‘. ”rho“ 141‘.0.11u ..‘12I‘V‘ I... l. A 1 ..[IV - . . . . . .... V l . . t
V 01’. 0'1 I. Is 911... ., 11.1L0 V.f. . ...... .0. . ...... . .. . . . . - ... 1 . .
C. .. V O. ‘ .11 1.1 x .V 1.01.. 6. .4~.-1CV. . .. ...... . 7*...7. ...... ...... vl ’ _ I... ...1. ...‘A‘..... 11”.. ’1 I .1 .... . v . . . ,. Q . v 1... VV
.. v w -l I. of § ”.1 Q3 — "Mufb 3 V . in V . ul . V. 1. 3.2. . . 1.7. .. .1. 5 .r3 1': ... .1 .111 . A. F 1. 1| 1 fi . 1 .0 o .
10v ._ . J .' V .fi. ...1 V: .. r.t&1~.“ k..0|lm1111 .. ”toqowhvbut 11.179‘r1" O ftfivruuto‘ro‘oé WK amuQ’Jva‘. . no. wacwm ...... 1...“?! v-7..13 "\P; 2 ». .1158..'O.‘v “.1.1lu.c.\ ”11.1“..11 uh”. 9145.:OP.‘ .‘ofib a‘/. I E‘s; ant. auozuikt.’ ...-1.91.. ...... I III. . 01! 1.11 1‘ 1 1
I .19 1 .u I .. ... V. . o» . . . 1 A! .. . ..-.- ..1.t.V.. 1 n 5F «:0 1,11... 9. <1. 1.. I. . ‘fl........z... V ... .... 15 1.lv . .01.-. (.... )0 ....O 4.. V1. 5...... .1. .. n... I. ‘.l..1... .1. U V V ‘1. L1... '1. . . 1.5.0.. ’4.‘ .19... VJ .5.‘ 5‘ 1 4...“. ’ ”$9" 9‘11‘0 o .5 O . n I -
.. ... V. V :0. n 1. . .v v -1 ...n. u...1 V. .. . 1. V .1 V V. V I .Oo.|‘-‘I Av...‘ .|\1...1.O .. ... K?- I01. . 1.0.31.1}- . 1 u
' . l I v . 1 .. .. 1 '. O... 1.. 9‘ l. . I .0 O 1...! O
a! " .
[it ...-II...

 

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

[WOlQCik-a V‘ CMaVac‘kviQu-‘Lioh .6 0~ €6MSs3rb'Qr9 HM.
AWAD‘WQKY’SLS (Same Imuolulcl M Cf u («L-)0.“ Sf“ 3‘3

presented by

wa ‘
ZLULO L‘ONS W3

has been accepted towards fulfillment
of the requirements for

NS degree in BOJRWS

 

Major professor

Dateflji/ ‘35”

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

——-—. +.——- —_-

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c-JCIRCJDateDuo.pes-p.15

MOLECULAR CHARACTERIZATION OF A CONSERVED

ARABIDOPSIS GENE INVOLVED IN CELL WALL SYNTHESIS

By

Zhaohong Wang

A THESIS

Submitted to
Michigan State University
in partial fulfilment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1995

ABSTRACT

MOLECULAR CHARACTERIZATION OF A CONSERVED
ARABIDOPSIS GENE INVOLVED IN CELL WALL SYNTHESIS

By

Zhaohong Wang

Although the plant cell wall plays a critical role in plant cells, very little is
known about cell wall synthesis. Xyloglucan is the major hemicellulose in
the primary cell wall of dicots, comprising about 20% of its dry weight.
Xyloglucan consists of on extended chain of B-1,4-linked glucosyl residues to
which xylosyl and xylosyl-galactosyl-fucosyl side chains are attached at regular
intervals. Xyloglucan hydrogen bonds to and forms cross bridges between
neighboring cellulose microfibrils. Xyloglucan cleavage is thought to allow

cellulose microfibrils to separate during cell growth.

Xyloglucan biosynthesis is known to occur in Golgi membranes. Glucan
synthase I is a glucosyl transferase also present in the Golgi; one of its possible
functions is the synthesis of xyloglucan backbone. Dhugga et. a1. (Dhugga KS
Ulvskov P, Gallagher SR, Ray PM [1991] J. Biol. Chem. 266: 21977-21984) have
purified to homogeneity a 40 kD protein reportedly involved in glucan
synthase I function. The 40 kD protein can be reversibly glycosylated by UDP-
Glc, UDP-Gal, UDP-Xyl, but not UDP-Man. Based on this evidence, they

proposed that this protein could play a role in xyloglucan synthesis.We used

their peptide sequence information to search the dBEST and found 5
Arabidopsis cDNAs that encode these peptides. Restriction mapping of these
clones revealed that the peptides are encoded by two similar, but not identical,
genes. Two cDNA clones, ATAI and ATBl were sequenced. The complete
nucleotide sequence of ATAl was used to search dBEST. Fifteen Arabidopsis
cDNA clones and seven rice cDN A clones were found which are similar to
ATAI as of the last search on June 15. Some of the Arabidopsis clones, were
mapped with restriction enzymes and found to have restriction maps
identical to either the ATA and ATB genes. Although the deduced amino
acid sequences of the rice and Arabidopsis proteins are very similar, they have
little similarity to other proteins in the databases. I postulate that these
proteins play an important role that is unique to plants. Cell wall synthesis is

one possibility.

Based on the work of Dhugga et. al. plus what we learned about this gene
family, we propose two possible functions for this protein. A) It may function
as an monosacchride intermediate. Monosaccharides are transferred from
nucleotide sugar to the protein and then to the growing xyloglucan chain. B)
It works as an oligosaccharide intermediate. Several sugar residues are
transferred to the protein to form an oligosaccharide repeat unit, which is
transferred to the growing xyloglucan chain. Further work will be needed to

evaluate the possibilities.

ACKNOWLEDGMENTS

I wish to express my sincerest thanks to the people that have shared their
knowledge, advice, experience, and time with me. My sincerest thanks to my
advisor Dr. Ken Keegstra for his guidance, help and support for the
experiments described in this thesis, and for his understanding and

encouragment.

I thank Dr. Jon Walton and Dr. Michael Bagadasarian for serving on my

guidance committee.

I also thank members of Keegstra lab, Pat, Amy, John, Eric, Jenny, Arun,
Mitsru and Karen, and my friends at MSU for their kindly help.

And thanks my family for their forever love and support.

iv

TABLE OF CONTENTS

LIST OF FIGURES ....................................................................................................... vi
CHAPTER 1: Introduction ............................................................................................. 1
CHAPTER 2: Materials and Methods ........................................................................ 10
CHAPTER 3: Results ..................................................................................................... 14
CHAPTER 4: Conclusion and Discussion ................................................................. 39
LITERATURE CITED .................................................................................................... 47

LIST OF FIGURES

Figure 1-1. The basic structure of xyloglucan ........................................................ 3
Figure 3-1. The restriction map of Arabidopsis ATA and ATB genes ............ 17
Figure 3-2. Nucleotide and predicted amino acid sequences of ATA1 ........... 19
Figure 3-3. Nucleotide and predicted amino acid sequences of ATB1 ............ 22
Figure 3-4. Aligment of ATA1 nucleotide sequence with that of ATB] ......... 24

Figure 3-5. Aligment of the deduced ATA1 amino acid sequence with that of
ATB] ................................................................................................................................. 27

Figure 3-6. Hydropathy plots of the deduced amino acid sequences of ATA1
and ATB] ......................................................................................................................... 29

Figure 3-7. The nucleotide sequence of ATA1 with that of pea ........................ 31

Figure 3-8. Aligment of the deduced ATA1 amino acid sequence with that of
pea ..................................................................................................................................... 33

vi

Figure 3-9. Aligment of Arabidopsis nucleotide sequences with that of rice

group ................................................................................................................................. 35

Figure 3-10. Aligment of the the deduced Arabidopsis amino acid sequences

with that of rice group .................................................................................................. 37
Figure 3-11. The construct of ATA1 in pET22b(+) vector .................................. 38
Figure 4-1. Scheme for the biosynthesis of the Salmonella O-antigen ........ 42
Figure 4-2. Proposed functions of PSI protein .................................................... 45

vii

CHAPTERI

INTRODUCTION

The wall is an important and unique part of a plant cell. It not only
provides mechanical support to help a plant to maintain form and structural
integrity, but it also contains components involved in signaling,
communication and pathogen defense. The cell walls of higher plants can be
divided into two types: primary walls and secondary walls. Primary walls are
produced by growing cells and can elongate during growth; secondary walls
are less flexible and almost inelastic. Once the primary wall is formed, the
wall continues to thicken after elongation stops, as layers of cellulose and
lignin are deposited to form the secondary wall. This brief review will
consider only the primary wall; it consists of cellulose, hemicellulose, pectins,

and proteins.

Cellulose constitutes 20%-30% of the dry weight of primary walls.
Chemically, cellulose is a linear B-1-4-linked D-glucan. The glucan chains of
cellulose are aggregated together to form structures called microfibrils.
Xyloglucan, a hemicellulose, is about 20% of the dry weight of the primary
wall in dicots. Although it is present both in dicots and monocots, it is a
minor component in most monocot cell walls. The basic structure of this cell
wall polymer consists of a backbone of l3-4-linked-D-glucosyl residues, with D-
xylosyl side chains a-linked to 0-6 of some the glucosyl residues. Some
xylosyl side chains have D-galactosyl or L-fucosyl-a-Z-D-galactosyl B-linked to
the O-2 of the xylosyl residues (see Figure 1-1). In the wall, most of the
xyloglucans are tightly hydrogen bonded to cellulose microfibrils. Pectins

and proteins comprise the remainder (approximately 50%) of the

I fucose

galactose

 

glucose

. xylose

 

 

 

 

Figure 1-1. The basic structure of xyloglucan

primary cell wall (McNeil et al., 1984). Although they are important
components of primary cell walls, they will not be considered further in this

brief review.

How are these different polymers assembled and altered to form an
extensible wall? The pioneering work of Albersheim’s group provided an
early comprehensive model. In their model, all non-cellulosic polymers are
covalently linked except for hydrogen bonding of the xyloglucans to the
cellulose microfibrils (Keegstra et al., 1973; Talmadge et al., 1973) The concept
that primary wall coherence is based on a covalently cross-linked meshwork
of matrix molecules has influenced thinking about wall growth for a long
time. Another new model was later provided by Peter Ray’s group (Talbott
and Ray, 1992). They describe a model in which noncovalent associations,
such as among pectic polyuronides, between them and hydroxyl proline-rich
wall glycoproteins (extensins) and between XG and cellulose, are principally

responsible for the mechanical coherence of the growing wall.

Considerable progress has been made in recent years in understanding the
structure of wall components and the ways in which these components are
arranged in the wall. However, there has been little progress in
understanding the molecular details of the biosynthesis and assembly of the
plant cell wall and its components. This information is also essential for a

complete description of the physiology of plant growth.

Polysaccharides, which are the major components of cell wall, will be the
focus of this brief review of the synthesis of wall components. Their

synthesis can be divided into three steps: prepolymerization, polymerization,

post polymerization (Stoddart, 1984). The prepolymerization steps are
synthesis and activation of monomers. The source of monosaccharide
building units for wall polysaccharides is carbohydrate translocated from
photosynthesis or storage tissues to the site of synthesis. The immediate
donors of monosaccharides for polymerization are the nucleoside
diphosphate monosaccharides (NDP-monosaccharides). These are generated
from the cellular pool of hexose phosphates by soluble nucleoside
triphosphate pyrophosphorylases. In tissues showing active wall synthesis,
total NDP-monosaccharides are estimated to be present in up to millimolar
concentrations in the cytoplasm with UDP-Glc (75%) being by far the most
abundant, the next most abundant being UDP-Gal (Carpita and Delmer, 1981).

The enzymes generating nucleoside precursors have been found both in
soluble and membrane fractions (Fincher and Stone, 1981). Although it’s not
clear in which subcellular compartment they occur, this is a crucial point to
understanding the control of precursor supply to the sites of polysaccharide
synthesis. Current available evidence points to the endomembrane system as
the site of polymerization of all of the wall matrix, like polysaccharides and
glycoproteins (Northcote, 1985). In higher plants, cellulose is simultaneously
polymerized and deposited in the wall by a plasma-membrane-localized
cellulose synthase complex. This latter important process will not be described

here.

The process of polymerization can be divided into three steps: chain
initiation, chain elongation and chain termination. It seems likely that the
first sugar transfer must differ in mechanism from the succeeding transfers
and may involve use of a lipid or protein as acceptor (Maclachlan, 1981;
Stoddart, 1984). Phosphorylated polyprenols have a well-established role
as carrier of mono-, di-, and oligosaccharides in the synthesis of bacterial
wall polymers such as peptidoglycan, teichoic acids, and the O-antigen portion
of lipopolysaccharide (Stoddart, 1984) and in the glycosylation of asparagine-
linked glycoproteins in both plants and animals (Elbein, 1979; Kornfeld et al.,
1985). Such carriers might well be expected to play a role in plant cell wall
polysaccharide synthesis either as chain initiators or in subsequent
elongation. However, to date, there is no solid evidence that any of these

compounds are involved in wall polysaccharide biosynthesis.

Xyloglucan, a branched polysaccharide, has a regular repeat in its structure
(Figure 1-1). However, the pathway whereby the repeat is synthesized is
unknown. A Mn2+-stimulated (1-4)-B-glucan synthase and an associated
xylosyltransferase are thought to be involved in synthesis of xyloglucan; both
enzymes are Golgi located (Hayashi and Matsuda, 1981; Delmer et al., 1985).
The extension of the xyloglucan chain of soybean preparations involves
cooperative transfer since the addition of xylosyl residues is apparently
essential for extension of the glucosyl chain. Thus the soybean xyloglucan
pentasaccharide unit is converted to the heptasaccharide unit and by addition
of a nonreducing glucosyl residue and its substitution by a xylosyl residue

and this conversion is dependent on the UDP-Xyl concentration

(Hayashi et al., 1984) . However in peas (Ray, 1980) the formation of the
glucan backbone was not stimulated by UDP-Xyl but UDP-Glc stimulates

xylose transfer.

Completion of the soybean xyloglucan structure by addition of galactose and
fucose residues to the xylose substituents on the xyloglucan appears to be
independent of chain growth since enzymically synthesized xyloglucan which
lacks these residues has a similar molecular weight to the xyloglucan from
soybean walls (Hayashi et al. 1984). A considerable amount of work on the pea

fucosyl transferase has been done by Maclachalan’s group (Maclachalan 1985).

Construction of the cell wall requires that substances produced inside the
cell migrate through the plasmalemma to the wall. The matrix wall
components are delivered to the plasmalemma by a process similar to
secretion in animal cells. The cell wall proteins, which are synthesized on the
rough endoplasmic reticulum and cell wall polysaccharides, hemicelluloses,
and pectic substances produced in the Golgi apparatus, seem to be
incorporated into secretory vesicles that ”bud” of the Golgi apparatus or the
endoplasmic reticulum and then fuse with the plasmalemma in such a
manner that the products are released to the wall area (Devin and Witham,

1983).

Considering the complex composition and organization of the plant cell
wall, its synthesis should be highly regulated. It could be controlled at the
level of the supply of monosaccharide, at the level of their activation and
transport, at polymerization steps, by control of the amounts of activity of the

polymerase and glycosyltransferases, and by control of precusor

polysaccharide transport to the cell wall surface and their secretion into the
wall. The phytohormones, auxin, gibberellins, cytokinins, abscisic acid and
ethylene also have direct or indirect effects on wall metabolism and could

regulate some or all of these steps.

During their study of cell wall biosynthesis enzymes glucan synthase I (GS-
I) and glucan synthase II (GS-II), Dhugga et. al. found that two closely spaced
polypeptides (doublet) of ~40 kD became reversibly glycosylated by UDP-Glc
under glucan synthase-I (GS-I) assay conditions (Dhugga et al., 1991) . In
addition to being Golgi-associated, these polypeptides also occur in a soluble
form. The 40 kD polypeptides, which can be labeled with [14C]UDP-Glc or
[MCIUDP-Xyl under GS-I assay conditions. The labeling can be inhibited by
including unlabeled UDP-Glc or UDP-Xyl. These polypeptides do not possess
GS-I activity by themselves, but rather appear to be primary acceptors for
sugars (Glc, Gal, Xyl, but not Man) from UDP-sugars (Dhugga et al., 1991) . In
our work, we refer to this protein as polysaccharide synthesis intermediate

(PSI) protein.

Based on this evidence, they proposed that this protein could play a role in
xyloglucan synthesis. Subsequently they have purified the soluble 40 kD
polypeptides to homogeneity by ammonium sulfate precipitation and affinity
chromatography on UDP-Agarose. After passing the ammonium sulfate
concentrate through the affinity column in the presence of Mn“, the bound
polypeptides were eluted with EDTA. They also sequenced 3 peptide
fragments from this 40 kD protein (Dhugga et al., 1994). We compared these
sequences with dBEST database and identified 5 Arabidopsis cDNAs that

could encodes these peptides. Within these 5 clones, there are 2 related but
not identical cDNAs. I characterized these clones, and used ATA1 clone,
which we believe is full length to further search dBEST. This search yielded
several more Arabidopsis and rice clones. All these clones are highly similar.
But searching the protein data bases, we don’t find any similar proteins. All
this suggests that this kind of protein may play an important and essential
role unique to plants, though its function is not known now. And my work is

to do some initial molecular characterization of these Arabidopsis clones.

CHAPTER 2

MATERIALS AND METHODS

11

Restriction Mapping

All of the cDNA clones described here were from the Columbia wild type
of Arabidopsis thaliana (L.) Heynh. They were constructed in a kZipLox
library designated PRL2. For the PRL2 library, equal amounts of poly-A(+)
mRNA from the four tissue types ( seedlings, roots, stems, flowers) were
converted to cDN A using a SuperScript Kit from Gibco BRL. The cDN As were
ligated into the SalI-NotI sites of AZipLox, packaged, and plated on Escherichia
coli Y1090 (pZIP) (Newman et al. 1994) . All of the cDNA clones (38C4T7,
105K4T7, 109C5T7, 86BIOT7, 108D6T7, 114H5T7, 139A8T7 )were kindly

provided by Arabidopsis Biological Resource Center at Ohio State University.

The E. coil Y1090 containing the cDNA clones were grown at 37 0C in Sml
cultures of LB medium plus Ampicillin (100mg/ ml) and plasmids were

extracted using Magic Minipreps from Promega (Madison, WI).

The plasmids were digested with restriction enzymes SalI, EcoRI, AvaII,
HindIII, BamHI, XbaI, NciI and their combinations at 37°C for 4 hours. The
DNA amount is 1.5 pg per aliquot. The result of restriction mapping is shown

in Figure 3-1.

Sequencing and Data analysis

The ATA1 (38C4T7) and ATBI (86B10T7) were digested with restriction
enzymes and the fragments were subcloned into pBluescript II SK(+) vector
(Stratagene) to make constructs for sequencing. The automated fluorescent
sequencing reactions were performed by our Plant Biochemistry Facility at
Michigan State University by T3, T7, SP6 dye primers using the ABI catalyst
800 for Taq cycle sequencing and the ABI 373A Sequencer for the analysis of
products. Both strands of ATA1 and ATBl were sequenced to generate the
results shown in Figure 3-2 and Figure 3-3. The complete nucleotide sequence
of pea clone was kindly provided by Dr. Dhugga. The complete nucleotide
sequence of ATA1 was used to search dBEST and GenBank. The deduced
amino acid sequence of ATA1 was used to search GenBank data. The GCG,
Sequencer, DNASIS, PROSIS program were used to analysis data. The result
were shown in Figure 3-4, Figure 3-5, Figure 3-6, Figure 3-7, Figure 3-8,
Figure 3-9, Figure 3-10.

13

The chimeric protein overexpressed in E. coli and purification

An BamHI/HindIII fragment containing the last two thirds of the coding
sequence from the cDNA clone ATA1 was inserted into corresponding sites
of the pET22b(+) expression vector (Novagen) (see Figure 3—11). A single
colony of E.coli strain BL21(DE3), harbouring the expression plasmid was
grown in 50ml LB medium containing 50ug/ ml ampicillin and 34ug/ml
chloramphenicol at 37°C for about 3 hours until the OD600 reached 0.6-1.
Then IPTG was added to a final concentration of 0.4mM and the expression
was achieved under the control of the T7 promoter for 2-3 hours. The
bacterial was harvested, resuspended in SOmM Tris-HCI, 2mM EDTA pH 8.0,
and disrupted by several cycles of sonication, each 20-30 s with an interval on
ice. The total lysate was separated into soluble and ”inclusion body” fractions

by centrifugation for 20 min at 4°C and at 10,000xg.

The samples were prepared in SDS gel loading buffer containing 0.2% 2-
mer-captoethanol and denatured by heating for 10 min at 100°C. Equal
volumes of extracts were loaded on SDS gel to check the location of fusion
protein. The ATA1 fusion protein was founded in ”inclusion body”. The His-
Tag affinity column (Novagen) was uesd to purify the fusion protein under

denaturing condition suggested by the supplier.

CHAPTER3

RESULTS

Restriction Mapping indicates at least two genes

The restriction map (see Figure3-1) of seven different Arabidopsis cDNA
clones indicated that they can be divided into two gene groups: ATA and
ATB. The ATA group includes ATA1(38C4T7), ATA3(109C5T7). ATA1 has
AvaII, BamHI, NciI, HincII and HindIII restriction sites found by mapping.
ATA3 also has AvaII, NciI and HindIII sites. Its restriction pattern is same as
ATA1, suggesting it may be a partial clone of ATA1. The ATB group includes
ATB](86B10T7), ATB2(139A8T7), ATB3(114H5T7), ATB4(105K4T7),
ATB5(108D6T7). The ATB1 has EcoRI, AvaII, BamHI, AvaII, HindIII sites
found by mapping. The ATB2, ATB3, ATB4 and ATBS has the same

restriction pattern as ATB1.

Structures of the ATA1 and ATB1 genes

The ATA1 was sequenced, showing that it is 1405bp long with ATG start
codon in front of a putative open reading frame and Poly(A) tail. Figure 3-2
shows the nucleotide and deduced amino acid sequence of ATA1. ATA1
potentially encodes a 364 amino acid polypeptide. Approximately 400bp 3’
from the coding region have been sequenced, allowing for the identification
of poly(A) signal AATAAA, located at 27bp upstream of poly(A) addition site
of the cDNA clone. The calculated molecular mass of the ATA1 polypeptide

is 40kD. The amino acid sequence does not contain a typical signal sequence,

16

Legend to Figure 3-1. The restriction map of Arabidopsis ATA and ATB genes.
Seven different ArabidOpsis cDNA clones can be divided into two groups:
ATA and ATB. The ATA group includes ATA1 (38C4T7), ATA3 (109C5T7).
ATA1 has AvaII, BamHI, NciI, HincII, HindIH restriction sites. The ATA3
also has AvaII, NciI, HindHI sites. Its restriction pattern is same as ATA1. The
ATB group has ATBI (86BIOT7), ATBZ (139A8T7), ATBB (114H5T7), ATB4
(105K4T7), ATBS (108D6T7). The ATBl has EcoRI, AvaII, BamHI, AvaII,
HindIII sites. The ATBZ, ATB3, ATB4, ATBS have the same restriction pattern
as ATBl. A stands for AvaII. H stands for Hind 1H.

ATA1

ATA3

ATB1

ATBZ

ATB3

ATB4

ATBS

17

 

 

 

 

 

 

Ava II BamHI N ci I Hinc II Hind III
L m I l 1 1 1 1
Nci I Hinc II Hind III
I J l l l I
EcoR I A H BamHI Ava II Hind III
I I] l l l l l
EcoR I A H BamHI Ava II Hind III
I II I I 11 l
EcoR I A H BamHI Ava II Hind III
I II I I l l l
Ava II Hind HI
I ll 1 I 1
Ava II Hind III
I l l L

if?

 

Figure 3-1. The restriction map of Arabidopsis ATA and ATB genes

18

Legend to Figure 3-2. The nucleotide and deduced amino acid sequences of
the ATA1 gene. Numbers on the right refer to the amino acid sequence.
ATA1 is 1405bp long with ATG start codon and Poly(A) tail. It potentially
encodes a 364 amino acid polypeptide. The amino acid sequences matching
Dhugga's pea peptide fragments are underlined. The DNASIS program was

used to generate this figure.

GAC

AGC

GAG

CCA
P

TTT
CTT
TGG
TAC
GCA

CCA

ACT

CTC

CCA

ATT

TGC

GTT

CTC

GAT

GGT

AGG

TGT

ATG

GTG

N E <

GAG

m

GAC
D

GCA
A

TGG
GCT
TTT
TAT
ATT

CGC

CCG

GAG

TCG

AAC

TTT

GCC

TTC

1

ATC

TAC
Y

AAG
K

GCC
A

TTA
TTT
TTG
TTT
AAA

GTC

TTG

ATG

AAG

CGA

GGG

AAG

CCA

GTC

TGG

ACC

ATG

GAT

TGT

AGC

ATT

ATT

CTT

AGT
3

TTT
TTT
CAT
CTG
AAA

CGG TAA

CTG

"E

TGG AGG

AAG ATC

ATC CTC

TAC ATG

GAT CCA

TCG TCT

CGT GGA

CTC AAC
LJ

AGG TAT
R Y

AAC TTG

GGT CAG

GAC CAC

AAC CCT

CCG TTC

GAG CTC
E L

GCA GAT
A D

GGC
G K

TTA GCT
ACA CAT
TTG GTT
AGT TTT
AAA AAA

ACC

GAT

CCT

CAT

CCC

TAC

ATC

GTG

“’8

CCT

W a P a v

”E

TCA
S

GCC
A

AGC
S

CAA
TTG
TAA
TTT
AAA

ATG

GAG

"’3

GTC

<

CCT

TCT

GGC

TTT

CCT

CCT

GAT

TTT

ATT

AGC

GTT

CAG

AAG

ATG

TTG
L

ATT
AGT
ATA
CTT
GGG

GTT

CTC

CTT

CCT

AAG

AAG

N E X

N

TTC

GAC

GCT

GAC

GGT

TTG

AAC

AAC

ATG

GTT

V

AGA
R

ATC
AGT
TCA
TGT
C

GAG

GAT

CAG

GAA

TTC

AGT

TAC

GTC

CGT

CGT

ACA
T

GCA
A

GTT
TTT
CCG
TTC

19

CCG

P

ATC

CCT

GGT

TCT

AAG

GTG

AAC

CTC

GAT

ATG

GAT

TAC

GTG

AAG

AAG

AAG

TGG

GTA

ACT

CAT

GTT
ATT

GCG

A

GTG

TAC

TAC

TGT

TAT

AAC

ACC

CGT

GCC

ACC

TTG

GAC

AAG

AAG

CTA

GAG

ATT

TGA

CAT
TAT
AAT

AAT

ATT

CAT

GAC

ATC

ATC

GCT

CCG

AAC

ATT

GAT

ACC

GAA

TCG

S

AAG
K

GAA
E

GCC

AAA
CAG
TTA
AAA

ACT

CCG

CTG

TAC

TCG

TTC

CTT

TAT

GGT

ACC

CCA

AAA

TTT
TAC
TAA
AAG

GTT

ACT

ATC

GAG

TTT

ACC

GAG

GAT

GTT

CAA

AAG

CCG

TGG

TTA

AAG

GAA

AGC

TGG

AAG

CTG

TTT

ACA
GCC

GGT

ATC

ATC

CTC

AAG

ATT

CAA

CCT

TCC

CTC

“’fi ”’5 “‘9’

CCG

p g Q g m

TCC

GAT
D

AAA

ATT
CTC
GTG
TTT

CTT

AGA

R

GTC

TAC

GAT

GAT

CAC

TAC

ACT

GTG

ACA

ATG

GGT

TAT

ATC

GTA

TTA

GAG

E

AAG

TTA
ATT
GTG
TCA

CCG GTG
P V

AAC CTC

AAC
N

GAT

N___JL__J1.

CAG GAC
Q D

AAC AGG
N R

TCT GCT
S A

GAC GAT
D D

ATC AAG
I K

CGT GAA
R E

GCT GTT
A V

AAG CCT
K P

CTT

TAC

TGG

”ii "‘3 ”El

ATC TAC

TTC

2 g N

ACT GTT

GAC CCG

CTT AAC
L N

CCA CCA
CGA
GTC

GAT
TAG

ACC
CGG
TAG
GTG

Figure 3-2. Nucleotide and Predicted Amino Acid Sequences of ATA1

GGA
G

AAC

N

TGT

TGC

AAC

GGT

TCC

AAG

CCA

GTT

ATC

CAC

CAG

CAG

TAC

CCA

AAG

TTT

TTA

TAG
TTT

13

33

53

73

93

113

133

153

173

193

213

233

253

273

293

313

333

353

365

20

ER retention signal, or N-glycosylation signal, suggesting that the ATA1

polypeptide does not enter the secretory pathway.

The sequence of ATB] shows that it is 1398bp long and also has an ATG
start codon and Poly(A) tail. The 3’ end of ATB4 has been sequenced and it
matches the 3’ end of ATBI. The 5’ ends of ATB2 and ATB3 are the same as
ATBl. Figure 3-3 shows the nucleotide and deduced amino acid sequence of
ATB1. Similar to ATA1, ATB] potentially encodes a 357 amino acid amino
acid polypeptide. Also the polyA signal AATAAA can be identified at 17bp
upstream of poly(A) addtition site of the cDNA clone. The calculated
molecular mass of ATBl is 39.3 kD. The amino acid sequence of ATBl also

does not contain a typical signal sequence.

The nucleotide sequences of ATA1 and ATB1 were aligned as shown in
Figure 3-4. Comparison at the nucleotide level, demonstrated that the ATA1
and ATB1 sequences are highly similar, are about 85% identical. The 3’ non-
coding region of ATA1 and ATB1 gene show little similarity. In contrast, the
sequences are highly similar within the coding region. As Shown in Figure 3-
5, the deduced amino acid sequences are 93% identical. The peptide fragment
”NLDFLEMXRPFFEQY” and ”EGVPTAVSHGLXLNI” of the 40 kD pea protein
purified by Dhugga et al. (Dhugga et al., 1994) were found in both ATA1 and
ATB] with a little variation. For the first fragment, F, E, Q were substituted by
L, Q ,P in ATA1; E, Q were substituted by Q, P in ATB1. For the second
fragment, P was substitued by S, both in ATA1 and ATBl. And comparing
the size of the deduced amino acid peptide of ATA1 and AT B1

21

Legend to Figure 3-3. The nucleotide and deduced amino acid sequences of
the ATBl gene. Numbers on the right refer to the amino acid sequence. ATBl
is 1398bp long with ATG start codon and Poly(A) tail. It potentially encodes a
357 amino acid polypeptide. The amino acid sequences matching Dhugga's
pea peptide fragments are underlined. The DNASIS program was used to

generate this figure.

GA
ATC

CCA

GTC

TGG

ACA

ATG

GAT

TGT

AGC

ATT

ATG

CCC
AAT

TTG

TGG

ACG
CCA

AAG

AGG

ACC ATT

ATC CTT

TAC ATG

GAT CCA

TCA ACT

CGT GGA

CTC AAC
L J

AGG TAT
R Y

AAC TTG

GGT CAG

GAC CAT

AAC CCG

CCT TTC

GAG CTG
E L

GCA GAT
A D

TGA
*

CTC
TAA
CTC
AAA

GCA

CGT
GTT
TCT
AAA

CGT
ATC

GAT

CCT

GCT

GGT

GTC

TCT

CCA

TAC

ATC

GTG

GCC

CCT

TTG

t‘

TTC

M

TCC

GCT

GCA

TTT
GCC
TTT
AAA

CCG
ATG

GAG

TTT

GTC

CCT

TCC

GGA

Q

CCT

'0

CCT

GAT

TAT

ATT

GGA

GTG

CAG

AAG

ATG

M

AAA

GTT
GGA
GAG
AAG

CTC
GTT

<

CTC

I."

"’3

CCT

N E N E N E m

TTC

'11

TTC

GAT

GCT

GAC

GGT

TTG

AAC

AGC

TTG

GTC

V

AAA

TTT
GTT
GTT
GGC

GAG

GAT

CAG

TAC

GTC

CGT

CGC

GGA

TTG

CCA

GTG

ACT

CCA

GTT

TAT
TTT

TCT CCA
CCG GCG
P A

ATC GTG
I V

CCT TAC
P, 417

GGG TTC
G P

TCC TGC
S C

TAC

AAC

ACC

CGT

GCC

ATG ACC

GAG CTC

TAC GAC

GTG AAG

AAG AAG

AAG CTC

AAG GAG

TGG ATT

CCA CCG
TTG
TTA
CGT

TCT
AAT
TTT

22

CAA
AAC
N

ATC

CAT

TCT
ACC
T

CCC

CTG

GAT TAC

ATT TCC
ATC TTC

GCT CTT

>
P

TAC

GAG GGT

CCT ACC

ATC CCA

ATT GGT

GAT ATG

ACA GGT

GAG TAC

ACG

AAG CTA

GAA GCT

CAG TTT
TTT
AGT
TGT

CTC
GAA
TTC

TTT
GTT

ACG

ATT

GAA

TTC

ACT

GAG

GAC

GTT

CAA

AAG

CCG

TGG

TTG
L

AAG
K

GAA
E

AGC
S

TGG
W

TGG

AAA

TGG
AAT

CTC

ATC

ATC

CTC

AAG

ATT

CAA

CCA

TCC

CTT

GCT

GCT

CCC

GGA

GCT

CCC

GAT

TTA

TTT

TGT
AAT

TCC
ATT

CGT

GTC

CAA GAT

TAC AAC

GAC TCT

GAT GAC

CAC ATC

TAC CGT

ACC GCT
T 44A

GTG AAG
V K

ACT CTT

ATG TAC

GGA TGG

TAC ATT

ATC TTC

GTG ACA

ATT GAT

GAG CTT
E L

TTA GCT
TCC

AGT
AAA

GGC
TCT
AAA

D

AGG

GCT

GAT

AAG

GAA

GTT

M g U

"’3

TGT

0

TAC

TGG

GTT

CCT

P

AAC
N

CAA

GAT

ATT
TCA

TCT
AAC

GAT

GGA

AAC

TGT

TGC

AAC

GGT

TCC

ATC

CAC

CAG

CAA

TAC

CCA

P

CAT

TCT
TAT
TAT

TCT
CAC

TTC
GAT
GAC
CGT
TTC
CTT
GCT
CAC
GAA
ATG
CTC
AAG
AGC

GAG
E
CAA
Q
TTT
F

CCC
P

ATC
TCA

GAC
CCC

Flgure 3-3. Nucleotide and Predicted Amino Acid Sequences of ATB1

CTC
ATC

CTG

CCA

ATC

GTT

CTC

GAC

GGT

AGG

TGT

ATG

GTG

N E <

GAT

U

GAC

ACT

ATC

GTT
CGA
TTA

TGA
CCA

GAG

TCG

AAC

TTC

GCT

TGC

TTC

CTG

ATC

TAC

AAG

N

TAT

CGT
GAC

16

36

56

76

96

116

136

156

176

196

216

236

256

276

296

316

336

356

358

23

Legend to Figure 3-4. Aligment of ATA1 nucleotide sequence with that of
ATBl. The ATA1 and ATB1 sequences are highly similar, are about 85%
identical. The 3' non-coding region of ATA1 and ATBl gene show little
similarity. The two sequences are highly similar within the coding region.
ATG start codons and TGA stop codons are underlined. The GCG GAP

program was used to generate this figure.

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

ATA1

ATBl

18

68

68

118

118

168

168

218

218

268

268

318

318

368

368

418

418

468

468

518A

518

568

568

618

618

668

668

718

718

768

768

818

24
AACCAIQGTTGAGCCGGCGAATACTGTTGGTCTTCCGGTGAACCCGACTC

AATCAIEGTTGAGCCGGCGAACACCGTTGGAATTCCGGTGAACCACATCC

CGTTGCTGAAAGATGAGCTCGATATCGTGATTCCGACTATCAGAAACCTC

CATTGTTGAAGGATGAGCTCGATATCGTGATCCCCACGATCCGTAACCTC

GATTTCCTCGAGATGTGGAGGCCTTTTCTTCAGCCTTACCATCTGATCAT

GATTTCCTGGAGATGTGGAGGCCTTTTTTTCAGCCTTACCATCTGATTAT

CGTCCAGGACGGAGATCCATCGAAGAAGATCCATGTCCCTGAAGGTTACG

CGTCCAAGATGGAGATCCATCGAAGACCATTGCTGTCCCTGAAGGGTTCG

ACTACGAGCTCTACAACAGGAACGACATTAACCGAATCCTCGGACCTAAG

ATTACGAACTCTACAACAGGAACGACATCAACCGTATCCTTGGTCCTAAA

GCTTCTTGTATCTCGTTTAAGGATTCTGCTTGTCGATGCTTTGGGTACAT

GCTTCCTGCATTTCCTTCAAGGACTCTGCTTGTCGTTGCTTCGGCTACAT

GGTGTCTAAGAAGAAGTATATCTTCACCATTGATGACGATTGCTTCGTTG

GGTCTCCAAGAAGAAGTACATCTTCACTATTGATGACGATTGCTTCGTTG

CCAAGGATCCATCAGGCAAAGCAGTGAACGCTCTTGAGCAACACATCAAG

CTAAGGATCCATCTGGAAAAGCTGTGAACGCTCTTGAGCAACACATCAAG

AACCTTCTCTGCCCATCGTCTCCCTTTTTCTTCAACACCTTGTATGATCC

AACCTTCTCTGCCCATCAACTCCATTTTTCTTCAACACCTTGTACGACCC

TTACCGTGAAGGTGCTGATTTCGTCCGTGGATACCCTTTCAGTCTCCGTG

ACCGTGAAGGTGCTGACTTCGTCCGTGGATACCCTTTCAGTCTCCGTG

AAGGTGTTTCCACTGCTGTTTCCCATGGTCTTTGGCTCAACATCCCTGAC

AGGGTGTTTCCACCGCTGTTTCCCACGGTCTGTGGCTCAACATCCCTGAT

TACGATGCCCCGACCCAACTCGTGAAGCCTAAGGAGAGGAACACCAGGTA

TACGATGCCCCTACCCAACTTGTGAAGCCTAAGGAAAGGAACACAAGGTA

TGTGGATGCTGTCATGACCAACCCAAAGGGAACACTTTTCCCAATGTGTG

TGTGGATGCTGTCATGACCATCCCAAAGGGGACTCTTTTCCCTATGTGTG

GTATGAACTTGGCTTTTGACCGTGATTTGATTGGCCCGGCTATGTACTTT

GTATGAACTTGGCCTATGACCGTGAGCTCATTGGTCCGGCTATGTACTTT

GTTCTCATGGGTGATGGTCAGCCTATTGGTCGTTACGACGATATGTGGGC

GGTCTCATGGGTGATGGTCAGCCTATTGGTCGCTACGACGATATGTGGGC

TGGTTGGTGCATCAAGGTGATCTGTGACCACTTGAGCTTGGGAGTGAAGA

TGGATGGTGTATCAAGGTGATCTGTGACCATTTGGGATTGGGAGTGAAGA

67

117

117

167

167

217

217

267

267

317

317

367

367

417

417

467

467

517

517

567

567

617

617

667

667

717

717

767

767

817

817

867

25

ATA1 818 CCGGTTTACCGTATATCTACCACAGCAAAGCGAGCAACCCTTTTGTTAAC 867
| ||||| || || || |||||||||||||| |||||||| ||||| |||
ATBl 868 CAGGTTTGCCCTACATTTACCACAGCAAAGCCAGCAACCCGTTTGTGAAC 917
ATA1 868 CTGAAGAAGGAATACAAGGGAATCTTCTGGCAGGAGGAGATCATTCCGTT 917
|||||||||| |||||||||||||||||||||||||l |||||||| II
ATBl 918 TTGAAGAAGGAGTACAAGGGAATCTTCTGGCAGGAGGATATCATTCCTTT 967
ATA1 918 CTTCCAGAACGCAAAGCTATCGAAAGAAGCAGTAACTGTTCAGCAATGCT 967
|||||||| | ||||||| |||||||||| II II ||||| |||||||
ATB1 968 CTTCCAGAGCCCAAAGCTCACGAAAGAAGCTGTGACAGTTCAACAATGCT 1017
ATA1 968 ACATTGAGCTCTCAAAGATGGTCAAGGAGAAGTTGAGCTCCTTAGACCCG 1017
|||| ||||| II III |||| |||l||||| I III II | II II
ATBl 1018 ACATGGAGCTGTCCAAGTTGGTGAAGGAGAAGCTAAGCCCCATTGATCCT 1067
ATA1 1018T TAC CTTTGACAAGCTTGCAGATGCCATGGTTACATGGATTGAAGCTTGGGA 1067
|||l||||||||||||||||||| ||||| || |||||||||||||||||
ATBl 1068T mTTTGACAAGCTTGCAGATGCTATGGTCACTTGGATTGAAGCTTGGGA 1117
ATA1 1068 TGAGCTTAACCCACCAGCAGCCAGTGGCAAAAGCTTGAGAGCAGTAIQAG 1117
||||||||||||||| | |||||| ||||
ATB1 1118 TGAGCTTAACCCACC .......... CACTAAAGCT ........... IGAG 1146
ATA1 1118 CCAAAAAGAAAAAGCCACCAAAGTTTTGGTTATTTTTAGCTCAAATTATC 1167
| =||| ||| ||||| ||||||l|||| ||||||||| ||||
ATBl 1147 CAGCAAAAAAACCACCACCGCAGTTTTGGTTA...TTAGCTCAACATATC 1193
ATA1 1168 GT.TACTTTTAAATTTCTGATTTTACGAACCTTTCTTGCTTTTTTTACAC 1216
| || || || ||=||||
ATB1 1194 ATCTATCTTCTCCGTTTTGTTTTT .......................... 1217
ATA1 1217 ATTTGAGTAGTTTTCATCATCAGTACTTTCTC..ATTGTCCGGTTATGGT 1264
||||| ll |||||| ||| ||||| |
ATBl 1218 ......... GTTTT ........ GTCTTTTCTCAAATTTTCCGG....CGA 1246
ATA1 1265 TTTTGCATTTGGTTTAAATATCACCGGTTTATTTATAAACAGTGGTGGAT 1314
|| I II II |||| | | |||| | | | |||| |||| | |
ATB1 1247 TTCTTCAGTTCGTTTTTAAGTTGCCGGAGTTTATTTAAATAGTGAATGGT 1296
ATA1 1315 TAGTAGTACTATT .................... TTCTGAGTTTTTT..TC 1342
||||| ||||| || |||| ||||| |
ATBl 1297 ..GTAGTTCTATTTATGACCGAGACAATCTCTCTTTTGAGGTTTTTCGTT 1344

ATA1 1343 TTTGTTTCATTAATAAAAAGGCCTTTTCATAGGTGTTTGCAATTAAAAAA 1392

ATBl 1345 TTTGTTTCAATAATAAAAAATCATATCCCT .......... TAAAAAAAAA 1384
ATA1 1393 AAAAAAAAAGGGC 1405

ATB1 1385 AAAAAAAAAGGGC 1397

Flgure 3-4. Aligment of ATA1 Nucleotide Sequence with That of ATB1

26

Legend to Figure 3-5. Aligment of the deduced ATA1 amino acid sequence
with that of ATB1. The ATA1 encodes a 364 amino acid long protein and
ATBl encodes a 357 amino acid long protein. Their sequences similarity is

93%. The GCG GAP program was used to generate this figure.

ATA1 MVEPANTVGLPVNPTPLLKDELDIVIPTIRNLDFLEMWRPFLQPYHLIIV 50

ATB1 MVEPANTVGIPVNHIPLLKDELDIVIPTIRNLDFLEMWRPFFQPYHLIIV

ATA1 QDGDPSKKIHVPEGYDYELYNRNDINRILGPKASCISFKDSACRCFGYMV 100

ATB1 QDGDPSKTIAVPEGFDYELYNRNDINRILGPKASCISFKDSACRCFGYMV

ATA1 SKKKYIFTIDDDCFVAKDPSGKAVNALEQHIKNLLCPSSPFFFNTLYDPY 150

ATB1 SKKKYIFTIDDDCFVAKDPSGKAVNALEQHIKNLLCPSTPFFFNTLYDPY

ATA1 REGADFVRGYPFSLREGVSTAVSHGLWLNIPDYDAPTQLVKPKERNTRYV 200
||||||||||||||||ll|||||||ll|||||||||||||||||||||||
ATB1 REGADFVRGYPFSLREGVSTAVSHGLWLNIPDYDAPTQLVKPKERNTRYV
ATA1 DAVMTNPKGTLFPMCGMNLAFDRDLIGPAMYFVLMGDGQPIGRYDDMWAG 250
||||| |||||||||lllll=||=||||||||~|||||||||||||||||
ATB1 DAVMTIPKGTLFPMCGMNLAYDRELIGPAMYFGLMGDGQPIGRYDDMWAG

ATA1 WCIKVICDHLSLGVKTGLPYIYHSKASNPFVNLKKEYKGIFWQEEIIPFF 300

ATBl WCIKVICDHLGLGVKTGLPYIYHSKASNPFVNLKKEYKGIFWQEDIIPFF

ATA1 QNAKLSKEAVTVQQCYIELSKMVKEKLSSLDPYFDKLADAMVTWIEAWDE 350

ATB1 QSPKLTKEAVTVQQCYMELSKLVKEKLSPIDPYFDKLADAMVTWIEAWDE
ATA1 LNPPAASGKSLRAV*

ATB1 LNPPT ...... KA*

Figure 3-5. Aligment of the Deduced ATA1 Amino Acid Sequence with That of ATB1

28

with Dhugga’s 40 kD pea protein, it suggested that ATA1 and ATB1 may

encode a protein similar to this 40 kD protein.

The hydropathy analysis (Figure 3-6) of ATA1 and ATB1 in Figure 3-6
indicated that both ATA1 and ATB1 protein are relatively hydrophilic with

no obvious membrane-spanning domains.

Highly conserved in different species

The nucleotide sequences and the deduced amino acid sequences of ATA1
and pea clones (Dhugga personal communication) were aligned in Figure 3-7
and Figure 3-8. Comparision at the nucleotide level, the ATA1 and pea
sequences are very similar with, about 71% identical. The amino acid

sequence identity is 84%.

The complete nucleotide sequence of ATA1 was used to search dBEST.
Fifteen Arabidopsis clones and seven rice clones were found highly similar to
ATA1 as of June 15,1995. Some of the Arabidopsis clones(ATA1, ATA2, ATBl,
ATBZ, ATB3) and four rice clones (081, 082, 053, 054) were used to do

computer alignment analysis.

The nucleotide sequences alignment (Figure 3-9) shows that within the

Arabidopsis group the identity is 83%, and they have 75% identity with the

 

I I I
f I l'-] I“ n l-"
I l 5

29

 

 

a 72‘ 144 216 238 364
Index

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

 

 

' I I
f I r") I 4' D I“ I“) fl '
I I I I I

B ii 142 219; — 234 357'

Figure 3-6. Hydropathy plots of the deduced amino acid sequences of ATA1 and ATB1.
Both ATA1 and ATB1 are relatively hydrophilic. PROSIS program was used to generate
this figure.

30

Legend to Figure 3-7. The nucleotide sequence of ATA1 with that of pea.
Comparision at the nucleotide level, the ATA1 and pea clone sequences are
very similar, about 71% identical. The pea cDNA clone sequence is from
personal communication with Dhugga. The GCG GAP program was used to

generate this figure.

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

31

GACCCACGCGTCCGGTAAACCATGGTTGAGCCGGCGAATACTGTTGGTCT

..................... GCACGAGCGCACTTCTCAACAATGGCTTC

TCCGGTGAACCCGACTCCGTTGCTGAAAGATGAGCTCGATATCGTGATTC

GTTACCCAAACCAACTCCACTCTTGAAAGACGAACTCGACATCGTCATCC

CGACTATCAGAAACCTCGATTTCCTCGAGATGTGGAGGCCTTTTCTTCAG

CTACGATCCGTAACCTTGATTTCCTGGAGATGTGGAGACCCTTTTTTGAA

CCTTACCATCTGATCATCGTCCAGGACGGAGATCCATCGAAGAAGATCCA

CAGTACCATCTCATCATTGTTCAAGATGGTGACCCTTCTAAGGTTATCAA

TGTCCCTGAAGGTTACGACTACGAGCTCTACAACAGGAACGACATTAACC

GGTTCCTGAAGGTTTCGATTATGAACTGTATAATCGGAATGATATCAATA

GAATCCTCGGACCTAAGGCTTCTTGTATCTCGTTTAAGGATTCTGCTTGT

GGATCTTGGGTCCTAAAGCTTCGTGTATCTCCTTCAAGGATTCGGCTTGT

CGATGCTTTGGGTACATGGTGTCTAAGAAGAAGTATATCTTCACCATTGA

CGTTGCTTTGGGTATATGGTTTCGAAGAAGAAGTATATCTACACCATTGA

TGACGATTGCTTCGTTGCCAAGGATCCATCAGGCAAAGCAGTGAACGCTC

TGATGATTGCTTTGTTGCTAAAGACCCAACTGGGCATGAAATCAATGCAC

TTGAGCAACACATCAAGAACCTTCTCTGCCCATCGTCTCCCTTTTTCTTC

TTGAGCAGCACATTAAGAATCTCCTTAGTCCATCCACTCCATTTTTCTTC

AACACCTTGTATGATCCTTACCGTGAAGGTGCTGATTTCGTCCGTGGATA

AACACCCTTTACGATCCATACAGAGAAGGTACTGATTTCGTCCGTGGATA

CCCTTTCAGTCTCCGTGAAGGTGTTTCCACTGCTGTTTCCCATGGTCTTT

CCCTTTCAGTCTTCGTGAAGGTGTCCCCACTGCCGTTTCTCACGGCCTTT

GGCTCAACATCCCTGACTACGATGCCCCGACCCAACTCGTGAAGCCTAAG

GGCTCAACATACCTGATTACGATGCTCCAACTCAGCTTGTCAAGCCCCAT

GAGAGGAACACCAGGTATGTGGATGCTGTCATGACCAACCCAAAGGGAAC

GAGAGGAACACTAGGTTTCTTGATGCTGTTCTGACCATTCCCAAAGGAAG

ACTTTTCCCAATGTGTGGTATGAACTTGGCTTTTGACCGTGATTTGATTG

TCTGTTCCCCATGTGCGGTATGAATCTGGCATTTAACCGTGAACTGATTG

50

29

100

79

150

129

200

179

250

229

300

279

350

329

400

379

450

429

500

479

550

529

600

579

650

629

700

679

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

ATA1

PEA

32

GCCCGGCTATGTACTTTGTTCTCATGGGTGATGGTCAGCCTATTGGTCGT

GACCTGCAATGTACTTCGGACTCATGGGTGATGGTCAGCCTATTGGACGC

TACGACGATATGTGGGCTGGTTGGTGCATCAAGGTGATCTGTGACCACTT

|||||||l|||||||||||| ||l||||| ||||| ||||||||||| ll

TACGACGATATGTGGGCTGGATGGTGCATAAAGGTTATCTGTGACCATTT

GAGCTTGGGAGTGAAGACCGGTTTACCGTATATCTACCACAGCAAAGCGA

I l | |||||||| ||||| |||| II III ||||||||||||

GGGATATGGAGTGAAAACCGGACTACCTTACATTTGGCACAGCAAAGCAA

GCAACCCTTTTGTTAACCTGAAGAAGGAATACAAGGGAATCTTCTGGCAG

||||||| ||||l||| lllll ||||| |||l| ll ||ll||l||||

GCAACCCATTTGTTAATCTGAAAAAGGAGTACAAAGGTATCTTCTGGCAA

GAGGAGATCATTCCGTTCTTCCAGAACGCAAAGCTATCGAAAGAAGCAGT

||||l||||||l|||||||| |l|| |||||||||

GAAGAGATCATTCCATTTTTCCAAGCTGCAACCCTTTCAAAAGATTGCAC

AACTGTTCAGCAATGCTACATTGAGCTCTCAAAGATGGTCAAGGAGAAGT

Ililllll ||||||||||||| ||||| ||| |||l|||||||

CTCTGTTCAGAAATGCTACATTGAACTCTCCAAGCAAGTCAAGGAGAAAC

TGAGCTCCTTAGACCCGTACTTTGACAAGCTTGCAGATGCCATGGTTACA

| l I III II II II III || || ||||||l|||| ||

TTGGAACTATTGATCCCTATTTCATCAAACTCGCCGATGCCATGGTCACT

TGGATTGAAGCTTGGGATGAGCTTAA ........... CCCACCAGCAGCC

l||l|||||||||||l||||||l| l i II |

TGGGTTGAAGCTTGGGATGAGATTAATAACAACAAATCTGAAGAGACAAC

AGTGGCAAAAGCTTGAGAGCAGTATGAGCCAAAAAGAAA.AAGCCACCAA

llllllll III II III |||| II I

TTCAACCAAAGCTTCTGAGGTTGCTGCTACCAAGTGAAACAACTTATAGT

AGTTTTGGTTATTTTTAGCTCAAATTATCGTTACTTTTAAATTTCTGATT

|||| |l|||| |||||||l||l

TGATGAGGAAGAGAGTAGTTTTCAATCAGTTTTATTATTGTTATCATATT

TTACGAACCTTTCTTGCTTTTTTTACACATTTGAGTAGTTTTCATCATCA

l |||| |||| |||| ||||l|| | |

TGTTAGCATTATATTATGATTCTTGTTGATTTTGCTAGATTCCAGAACAA

GTACTTTCTCATTGTCCGGTTATGGTTTTTGCATTTGGTTTAAATATCAC

I II I NIH | || llll |||||| |

TTTATTGAT ........ ATTTATGTTATTAATATTTATATTAAAAAAAAA

CGGTTTATTTATAAACAGTGGTGGATTAGTAGTACTATTTTCTGAGTTTT

AAAAAAAAAAAAAACCTCGAGGGGGGG .......................

Figure 3-7 The Nucleotide Sequence of ATA1 with that of Pea

750

729

800

779

850

829

900

879

950

929

1000

979

1050

1029

1089

1079

1138

1129

1188

1179

1238

1229

1288

1271

1338

1298

33

ATA1 MVEPANTVGLPVNPTPLLKDELDIVIPTIRNLDFLEMWRPFLQPYHLIIV 50

--||||||||||||||||||||||||||||==-||||||
PEA ....... MASLPKPTPLLKDELDIVIPTIRNLDFLEMWRPFFEQYHLIIV 43
ATA1 QDGDPSKKIHVPEGYDYELYNRNDINRILGPKASCISFKDSACRCFGYMV 100
||||||| |-||||=|||||||||||||||||||||||||||||||||||
PEA QDGDPSKVIKVPEGFDYELYNRNDINRILGPKASCISFKDSACRCFGYMV 93

ATA1 SKKKYIFTIDDDCFVAKDPSGKAVNALEQHIKNLLCPSSPFFFNTLYDPY 150

PEA SKKKYIYTIDDDCFVAKDPTGHEINALEQHIKNLLSPSTPFFFNTLYDPY 143

ATA1 REGADFVRGYPFSLREGVSTAVSHGLWLNIPDYDAPTQLVKPKERNTRYV 200

PEA REGTDFVRGYPFSLREGVPTAVSHGLWLNIPDYDAPTQLVKPHERNTRFV 193

ATA1 DAVMTNPKGTLFPMCGMNLAFDRDLIGPAMYFVLMGDGQPIGRYDDMWAG 250

PEA DAVLTIPKGSLFPMCGMNLAFNRELIGPAMYFGLMGDGQPIGRYDDMWAG 243

ATA1 WCIKVICDHLSLGVKTGLPYIYHSKASNPFVNLKKEYKGIFWQEEIIPFF 300
||||||||||=-|||||||||=||||||||||||||||||l|||||||||

PEA WCIKVICDHLGYGVKTGLPYIWHSKASNPFVNLKKEYKGIFWQEEIIPFF 293

ATA1 QNAKLSKEAVTVQQCYIELSKMVKEKLSSLDPYFDKLADAMVTWIEAWDE 350
|-|-|||=---||-||||||| |||||=-=|||| |||||||||=|||||

PEA QAATLSKDCTSVQKCYIELSKQVKEKLGTIDPYFIKLADAMVTWVEAWDE 343

ATA1 L.NPPAASGKSLRAV ...... 364

PEA INNNKSEETTSTKASEVAATK 364

Figure 3-8 Aligment of ATA1 Deduced Amino Acid Sequence with that of Pea.
Both ATA1 and pea amino acid sequences are 364 a.a. long. The identity is 84%.
The GCG GAP program was used to generate this figure.

Legend to Figure 3-9. Aligment of Arabidopsis nucleotide sequences with that
of rice group. The OS stands for rice group. The complete nucleotide sequence
of ATA1 was used to search dBEST. Fifteen Arabidopsis clones and seven rice
clones were found highly similar to ATA1 as of June 15, 1995. Both the
Arabidopsis and rice clone showed here are longer than ATA1 at the 5’ end..
The identity within the Arabidopsis group is 83%, and they have 75% identity
with the rice group. The GCG PILEUP program was used to generate this
figure.

OS 1

O S 2

033

OS 4

ATB2

ATBl

ATB3

ATA2

ATA1
CW.

0 S l

O S 2

OS 3

OS 4

ATB2

ATB 1

ATB3

ATA2

ATA1
CM“.

OS 1

OS 2

053

OS 4

ATBZ

ATBl

ATB3

ATA2

ATA1
CW.

0 S 1

052

083

054

ATBZ

ATBl

ATB3

ATA2

ATA1
Con-mus

OS 1

O S 2

O S 3

084

ATBZ

ATBl

ATB3

ATA2

ATA1
cm.

Figure 3.9. Aligment of Arabidopsis Nucleotide Sequences with That of Rice group

cCaAa tm'rN
COIL: t'rC‘l'C
calla tm'rc
..... chNC

gtOccgtTm
gtazcgtNm
gtazcgtcm
gtkcgtTfl
LCcGfl'OGae
ECWaa
ECMa
mtmtc
Etmtc
W

am a Q? t
amen-t
TAM!!!

CW
CMCCA'I'C
cMGlCCATC
cflGlCCA'l‘C
mama-rt
mecca-rt
egmccht
Wgflc
masque
anemone

@681de
MGaGgQ
gmaOgGI
gfllGaGgGl
ttctNtNtO
ttctCtC ta
ttctCtC ta
tcagCttht
. GacCcAcd:
-GGA--O-Gl

cgtmc
cgtmc
cgtmc
cgtmc
new
TchaaTm
mecca-en
TTCCGaNm
W

mGACtA'l‘Ca
tha

CGOOTCCC O
COCGI'CCCCO
CGCOI'CCCCO
CGC arcccco
gc'l'G‘l'CCCTG
chl‘CCCTO
gcmmmNG
Camccc're
Cam
cam-e

35

Glgdfllfllgl
GAgGfllfllgL
GAgGGNGAgL
Glgflfllflhgl
etcaatccll
atcaatccll
atcaatccll
cAttthlll
Gtcchtlll
Gl-Oalfllll

gtm
gtm
gtoucacca
gtmlCGCCG
mackcma
OCaCAtcxa
OCacltcma
CCCOACtmG
CCCGACtmO
CCCGICOCCG

Mg“
Mg“
Mg“
ocnccrgm
GtAACCTCGA
012m
Gt “CGC“
GaAACCTCGA
Gum

CECHCATCO
GCHCATCG
CI‘CAI'CA'I‘CG
CI‘CA'I'CA'I'CG
Cl'NA‘l'tATNG
CTGATtATCO
CI'GNI'tATNG
CIGA‘I‘CATCO
CIGAI‘CATCG
CT-A'PCA'ICO

moon-can
Agaocncan
Agooc'rrcen
ngoocn'cen
“Gaga-roe;
mm
Mmggtcg
neat-ram
method:
mean

mmc gag
mm gag
mm gag
mm gGg
mm
mm
mm
c cam
c cum
mm

once-rm
once-ream
CHOW
Cm

memo

TTTCC‘I'C GIG
MGM

19W
'1ng
Tgm
MW
TCCAaGltW
mat”
gCEAaGlt NO
NW
W
W

anaemic-2c
(HAW
C'I'ACIAGCTC
CTACGAGC‘IC
t M a CI‘C
unequal-c
g‘l't d ......
CIACGAGC‘I'C
cucemc'rc
02W

50
0:000th0
monotone
GQNGtQCO
GaCGGtGch

1 5 0
Am WC
1% ”C
Am “C
AMOOC WC
AW
AW
AW
1mm
norm
AW

2 0 0
CG . ccccac
CO . cocoac
CG. emcee
CGA . CCCGac
Au . TxA'lic
Am. Twine
NOItNQIN'm
AG . chmc
Am . Tamale
-Gl- -CC-!c

250

36

rice group. The deduced amino acid sequences (Figure 3-10) shows that it is
93% identity within the Arabidopsis group and they have 69% identity with
the rice group. Comparing the variation at nucleotide sequence level and
deduced amino acid sequence level, it can be found that there are a lot of
codon degeneration variation which will not effect sequence. It may indicate
that this gene has some change during evolution and use codon degeneration

to keep its basic function which might be important to the higher plants.

Although the deduced amino acid sequences of the rice and Arabidopsis
clones are very similar, no homologous protein can be found in the data base.
Thus we conclude that these proteins are both highly conserved and
abundant in plants, yet unique to plants, as similar sequences have not been

observed in yeast, animals, etc.
ATA1 fusion protein was overexpressed

We sought to prepare chemical quantities of the 40 kD protein so that it
could be used as antigen for production of specific antiserum. It was decided
to attempt this via expression of a fusion protein in E. coli. For this purpose,
the ATA1 gene was subcloned into pET22b(+) vector(Figure 3-11). The
chimeric construct was transformed into E.coli strain BL21(DE3), and was
IPTG induced and ovexpressed. The overexpressed protein, which was in
"inclusion bodies", was isolated by centrifugation, washed with buffers
solubilized in 6M guanidine HCl, and purifed by nickel affinity column. The
protein has been injected into rabbit and immune serum will be available

80011.

37

1 50
ATB3 ........ AQ ISQSFLSKFS SL*INPIMVE PANTVGIPVN HIPLLKDELD
ATBZ ........ AQ X*QSFLSKXX SX*INPIMVE PANTVGIPVN HIXLLKDELD
OS4 ..... HLLLL LLFLRAREIQ GEGEGEIMAG TVTVPLASVP STPLLKDELD
031 ..HHHHLLLL LLFLRAREIQ GEGEGEIMAG TVTVPLASVP STPLLKDELD
OS2 ..... HLLLL LLFLRAREIQ GEGEGEIMAG TVTVPXASVP STPLLKDELD
OS3 TPHHHHLLLL LLFLRAREIQ GEGEGEIMAG TVTVPSASVP STPLLKDELD
ATA2 .................. RX QLSHFETMVE PANTVGLPXN PTPLLKDELD
ATA1 ........................... MVE PANTVGLPVN PTPLLKDELD
ATB1 ........................... MVE PANTVGIPVN HIPLLKDELD

51 100
ATB3 IVIPTIRNLD FLXMWRXFF* PYHLIXGQDX XXXGDHCXXX RGSV ......
ATBZ IVIPTIRNLD FXEMWRPXFQ PYHLIXVQD. ...GDPSKTI AVPEGFDYGL
OS4 IVIPTIRNLD FLEMWRPFFQ PYHLIIVQD. ...GDPTKTI RVPEGFDYEL
OSl IVIPTIRNLD FLEMWRPFFQ PYHLIIVQD. ...GDPTKTI RVPEGFDYEL
052 IVIPTIRNLD FLEMWRPFFQ PYHLIIVQD. ...GDPTKTI RVPEGFDYEL
OS3 IVIPTIRNLD FLEMWRPFFQ PYHLIIVQD. ...GDPTKTI RVPEGFDYXL
ATA2 IXIPTIRNLD FLEMWRPFLQ PYHLIIVQD. ...GDPSKKI HVPEGYDYEL
ATA1 IVIPTIRNLD FLEMWRPFLQ PYHLIIVQD. ...GDPSKKI HVPEGYDYEL
ATBl IVIPTIRNLD FLEMWRPFFQ PYHLIIVQD. ..GDPSKTI AVPEGFDYEL

Figure 3-10. Alignment of the Deduced Arabidopsis Amino Acid Sequences with That
of Rice Group. The deduced amino acid sequences shows that it is 93% identity within the
Arabidopsis group and they have 69% identity with the rice group. The GCG PILEUP
program was used to generate this figure.

38

His-Tag

  
 

T7 lac promoter

F1 ori

ATA1 in pET-22b(+) vector
Amp

Lacl

‘Ori

Figure 3-11. The construct of ATA1 in pET22b(+) vector.
The Barn HI/ HindIII fragment of ATA1 was subcloned into corresponding
sites in pET22b(+) expression vector.

CHAPTER4

DISCUSSION AND CONCLUSION

Based on my initial work, several conclusions can be drawn. 1) From the
restriction map, sequences and the aligment, there are at least two genes in
Arabidopsis, ATA and ATB gene. 2) Similar genes that are highly conserved
at amino acid sequence level are found in other plants. 3) They are
moderately abundant in different species of plants, eg. Arabidopsis and rice,
because fifteen Arabidopsis cDNA clones and seven rice clones have been
found by using ATA1 to do dBEST search as of June 15, 1995. 4) No sequence
similarity has been found in animals, yeast, etc. All these suggest that this
protein probably plays an important role unique to plants. Based on Dhugga’s

work, one likely possibility is that it is involved in cell wall synthesis.

From Dhugga’s observation, this 40 kD doublet protein can be reversibly
labeled by UDP—[14C]Glc under the conditions of the GS-I assay (Dhugga et al.,
1991). A possible function of GS-I is to form the (1,4)-B-glucan backbone of
xyloglucan which is synthesized in the Golgi system. Both GS-I activity and
labeling of the 40 kD protein are inhibited by UDP-xylose or UDP-Gal, but not
by UDP-Man. Xylose and galactose, but not mannose, are components of
xyloglucan. The above observation suggest that the 40 kD protein participates
in GS-I activity.

Dhugga’s experiment also shows that glucose was attached to the protein by
a covalent bond (Dhugga et al., 1991). This evidence indicates that the 40 kD

polypeptide is not a sugar nucleotide transmembrane translocase, instead it

41

could be a glycosyl transfer intermediate. It was observed that a saturating
glycosylation of approximately one glucose/ 40 kD polypeptide. The 40 kD
protein was found both associated with Golgi membranes and in the soluble
fraction. The soluble form of the 40 kD protein appears to be auto-

glycosylated. This is probably also true of the membrane bound form.

By comparing what is known regarding the synthesis of complex
polysaccharides in other systems with the situation in plants, we may be able
to gain new insight into the role of the 40kD protein in plants. The synthesis
of bacteria lipopolysaccharide (O-Antigen) is one process that may serve as a
model for investigating plant cell wall polysaccharide biosynthesis.
Lipopolysaccharide is a combination of polysaccharide attached to a lipid core
that forms a large part of the outer membrane of gram-negative enteric
bacteria. During its synthesis, first the monosaccharides are transferred
sequentially from their nucleotide carriers to the phosphomonoester of a
specific lipid component of the membrane, known as antigen carrier lipid
(ACL). (Figure 4-1, Robbins and Wright 1967), The trisaccharide repeat, eg. .
mannosylrhamnosylgalactose in Fig 4-1, is formed, then polymerized, and
finally it is transferred to lipopolysaccharide core acceptor. It is possible that
the 40 kD protein is a substitute for ACL, playing the role during xyloglucan

biosynthesis that ACL performs during lipopolysaccharide biosynthesis.

Another process that may provide insight into the possible role of P81 is
glycogen biosynthesis. Glycogen is a storage form of sugar in animals. Its
synthesis is primed via a protein called glycogenin. Glycogenin is the core

protein of glycogen proteoglycan and is, at the same time, a self-glucosylating

42

iii-qr- AI r—fi
tor-m Ll 0F}

 

A A
Gal-FF-ACL Fh-Gel-PP-ACL
1
UMD \ GDP-Man
UDP-Gcl VIC“?
7
-p ,. .... ...
AFL} VIOH'RD“JOi‘t‘I“l-‘~Lt_
[911A ..
[ACL't’F
z. i ,.
(Manh-Gclln-PP-Acl.
7
’ I II it II it
(Mcn-Rh-Gciln- Core Core

Figure 4-1. Scheme for the biosynthesis of the Salmonella O-antigen.
(Robbins and Wright, 1967)

43

enzyme which catalyses early glucosyl transfer steps in the biosynthesis of
glycogen (Manzella et al. 1995). It’s not a single reaction, but with several
consecutive glycosyl transfer steps, in which the product of one reaction
becomes the acceptor and enzyme in the subsequent step. The end product of
glycogenin action is an oligosaccharide composed of 8—11 glucose residues and
the fully glucosylated form of glycogenin serves as the oligosaccharide primer
for further chain elongation by proglycogen synthase and glycogen synthase
(Pitcher et al. 1987; Lomako et al. 1988). Glycogenin can also be considered as a
homologous protein of P51 ,though there are no obvious amino acid

sequence similarity between glycogenin and PSI protein.

From Dhugga’s work, the function of ACL in bacteria cell wall biosynthesis
and the role of glycogenin in glycogen biosynthesis, we propose two possible
functions of PS1 protein. Model A) Monosaccharide intermediate.
Monosaccharide are transferred from nucleotide sugar to PSI, then from PSI
to the growing xyloglucan chain. Model B) Oligosaccharide Intermediate.
Several sugar residues are transferred to PSI to form a oligosaccharide repeat
unit, which is transferred to the growing xyloglucan chain (Figure 4-2).
Where these reactions occurs within a cell is not clear at present. Dhugga
claims that all the 40 kD protein is associated with Golgi ( Dhugga et al., 1991;:
Dhugga personal communication). However it remains to be determined on

which side of the Golgi membrane this protein is located.

Legend to Figure 4-2. The proposed functions of polysaccharide synthesis
intermediate (PSI) protein. Model A) Monosaccharides intermediate.
Monosaccharide are transferred from nucleotide sugar to PSI, then from PSI
to the growing xyloglucan chain. B) Oligosaccharide intermediate. Several
sugar residues are transferred to PSI to form a oligosaccharide repeat unit,

which is transferred to the growing xyloglucan chain.

45

A) Monosaccharide Intermediate

UDP-sugar

\

>—<

Xyloglucan -sugar

B) Oligosaccharide Intermediate

a)
UDP-sugar

\

 

/

-G-G-G

l

b)

XG chain

\
/

-G—G-G-G
l l

XXX

 

Figure 4-2 . Proposed functions of PSI protein

-sugar
Xyloglucan
UDP
/
G-G-G
l l
X X

XG chain+Oligosaccharide

/
\

£6

As noted above, futher work can be done to investigate the role of the PSI
protein in plants. Considering its function, this protein should be Golgi-
localized. Antibody against ATA1 can be used to do cellular
immunolocalization to confirm this hypothesis. The antibody can also be
used to against the crude protein extracts of Arabidopsis and other plants to

detect the protein expression level in different species.

This cDNA clone can be constructed into an expression vector to over
express this protein in E. coli. The overexpressed protein can be UDP[14C]Glc

labelled to detect its function in an in vitro xyloglucan synthesis system.

The Agrobacterium transformation can be used to overexpress this protein
in vivo in Arabidopsis to see how the transgenic plant will function, or to
create a mutation that eliminate the function of this gene. The antisense
technology or gene-disruption techniques can be used to creat a mutant
plant to block the expression of this gene to see how this will affect the

function of the plant.

LITERATURE CITED

48

Carpita NC and Delmer D (1981) Concentration and metabolic turnover of

UDP-glucose in developing cotton fiber. J. Biol. Chem. 256: 308-315

Carpita NC and Gibeaut DM (1993) Structure models of primary cell walls in
flowering plants: consistency of molecular structure with the physical

properties of the walls during growth. The Plant Journal 3: 1-30
Cosgrove DJ (1993) How do plant cell walls extend. Plant Physiol. 102: 1-6

Delmer DP, Cooper G, Alexander D, Cooper J, Hayashi T, Nitsche C, Thelen M
(1985) New approaches to the study of cellulose biosynthesis. J. Cell Sci. Suppl.
2: 33-50

Delmer DP and Stone BA (1988) Biosynthesis of plant cell wall. The
biochemistry of plants. Vol. 14 pp. 373-416. Academic Press, Inc.

Devlin RM and Withman PH (1983) Plant cells: structure and function. Plant
physiology. Fourth Edition. pp. 1-25. PWS Publishers.

Dhugga KS, Ray PM (1994) Purification of the reversibly glycosylated
polypeptides from pea: purified polypeptides exhibit the same properties as
the Golgi-bound form. Plant Physiol. Suppl. 105: 126

Dhugga KS, Ulvskov P, Gallagher SR, Ray PM (1991) Plant polypeptides
reversibly glycosylated by UDP-glucose. Possible components of Golgi B-
glucan synthase in pea cells. J. B. Chem. 266: 21977-21984

49

Driouich A, Faye L, Staehelin A (1993) The plant Golgi apparatus: a factory for
complex polysaccharides and glycoproteins. TIBS 18: 210-214

Elbein AD (1979) The role of lipid-linked saccharides in the biosynthesis of
complex carbohydrates. Annu. Rev. Plant Physiol. 30: 239-272

Fincher GB and Stone BA (1981) Encycl. Plant Physiol. New Ser. 13B: 68-132

Hayashi T (1989) Xyloglucans in the primary cell wall. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 40: 139-68

Hayashi T, Maclachlan G ( 1984) Biosynthesis of pentosyl lipids by pea
membrane. Biochem. J. 217: 791-803

Hayashi T, Matsuda K (1981) Biosynthesis of xyloglucan in suspension-
cultured soybean cells. Evidence that the enzyme system of xyloglucan
synthesis does not contain B-1,4-glucan 4-B-D-glucosyltransferase activity.

Plant Cell Physiol. 22: 1571-1584

Hayashi T, Nakajima T, Matsuda K (1984) Biosynthesis of xyloglucan in
suspension-cultured soybean cells. Processing of the oligosaccharide building

blocks. Agric. Biol. Chem. 48: 1023-1027

Hirschberg CB (1987)Topography of glycosylation in the rough endoplasmic

reticulum and Golgi apparatus. Annu. Rev. Biochem. 56: 63-87

50

Keegstra K, Talmadge K, Bauer WD, Albersheim P (1973) The structure of
plant cell walls. 111. A model of the walls of suspension-culture sycamore cells
based on the interconnection of the macromolecular components. Plant

Physilo 51: 188-196

Kornfeld R and Kornfeld S (1985) Assembly of asparagine-linked
oligosaccharide. Annu. Rev. Biochem. 54: 631-664

Lomaco J, Lamaco WM, Whelan WJ (1988) A self-glucosylating protein is the
primer for rabbit muscle glycogen biosynthesis. FASEB J. 2: 3097-3103

Maclachlan G (1985) Are lipid-linked glycosides required for plant
polysaccharide biosynthesis? In Biochemistry of plant cell wall. pp. 199-220.
Cambridge Univ. Press. Cambridge

Manzella SM, Roden L, Meezan E (1995) Dodecyl-B-D-maltoside as a substrate

for glucosyl and xylosyl transfer by glycogenin. Glycobiology 5: 263-271

McNeil M, lDarvill AG, Fry SC, Albersheim P (1984) Structure and function of

the primary cell walls of plants. Ann. Rev. Biochem. 53: 625-63

Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L,Ohlrogge
John, Raikhel N, Somerville S, Thomashow M, Retzel E, Somerville C (1994)
genes galore: a summary of methods for accessing results from large-scale
partial sequencing of anonymous Arabidopsis cDN A clone. Plant Physiol.

106: 1241-1255

51

Northcote DH (1985) Cell organelles and their function in biosynthesis of cell
wall components: control of cell wall assembly during differentiation.
Biosynthesis and biodegradation of wood components. pp. 87-108. Academic

press. Orlando, Florida

Pitcher J, Symthe C, Campbell DG, Cohen P (1987) Identification of the 38-kD
subunit of rabbit skeletal muscle glycogen synthase as glycogenin. Eur. J.

Biochem. 169: 497-502

Ray PM (1980) Cooperative action of beta-glucan synthetase and UDP-xylose
xylosyl transferase of Golgi membranes in the synthesis of xyloglucan-like

polysaccharide. Biochim. Biophys. Acta 629: 431-444

Robbins PW, Bray D, Dankert M, Wright A (1967) Direction of the chain
growth in polysaccharide synthesis. Work on a bacterial polysaccharide

suggests that elongation can occur at the ”reducing” end of the growthing

chain. Science 158: 1536-1542

Robbins PW and Wright A (1971) Biosynthesis of O-antigens. Microbial
Toxins. pp. 351-367 Academic Press, Inc., New York and London

Talbott LD, Ray PM (1992) Molecular size and separability features of pea cell
wall polysaccharides. Implication for models of primary wall structure. Plant

Physiol. (1992) 98: 357-368

52

Talmadge K, Keegstra K, Bauer WD, Albersheim P (1973) The structure of
plant cell walls. I. The macromolecular components of the walls of
suspension-cultured sycamore cells with a detailed analysis of the pectic

polysaccharides. Plant Physi0151: 158-173

Stoddart RW (1984) Glycosylation in bacteria. The biosynthesis of

polysaccharides. pp 58-87 Macmillan, New York

Varner JE, Lin LS (1989) Plant cell wall architecture. Cell 56: 231-239