Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants

Abstract

Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16% reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1 and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b synthesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to 72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.

Full text

Light-dependent regulation of chlorophyll b biosynthesis
in chlorophyllide a oxygenase overexpressing tobacco plants
Gopal K. Pattanayak
a
, Ajaya K. Biswal
a
, Vanga S. Reddy
b
, Baishnab C. Tripathy
a,*
a
School of Life Sciences, Jawaharlal Nehru University, New Delhi 11067, India
b
International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
Received 8 November 2004
Available online 20 November 2004
Abstract
Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase
chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low
and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16%
reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The
increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was
accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1
and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b syn-
thesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to
72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.
2004 Elsevier Inc. All rights reserved.
Keywords: Chlorophyllide a oxygenase; Chlorophyll b; Chlorophyll a/b ratio; Sun and shade plants; Chlorophyll biosynthesis; Light-harvesting
chlorophyll protein complex II
Plants are adapted to live in extremely different light
environments such as the forest floor, deep oceans, and
in open fields. There are substantial differences in spec-
tral distribution of light in these habitats. Different
fluences of light regulate growth and development of
plants. Chlorophyll (Chl) biosynthesis requires light
and its biosynthetic intermediates are involved in the
regulation of chloroplast biogenesis [1–4]. The amount
and composition of the Chl-protein complexes are vari-
able in thylakoid membranes [5]. The variation in light-
harvesting chlorophyll protein complexes depends
mainly on the incident irradiance during plant growth
[6]. Photosynthetic organisms acclimatize to various
light intensities by adjusting the size of the Chl antenna
associated with each photosystem [7,8]. Plants grown in
shade habitat have a bigger antenna size and reduced
Chl a/b ratio. Conversely, plants grown in Sun habitat
have smaller antenna size and increased Chl a/b ratio.
The flexibility of the antenna size helps the plant to
adapt itself to changing light regimes [9,10].
The Chl a/b ratio is mostly governed by the enzyme
chlorophyllide a oxygenase (CAO) [11–13] that is
responsible for the conversion of methyl group on the
D ring of the porphyrin molecule to a formyl group at
that position. The CAO that converts chlorophyllide a
to chlorophyllide b is a mononuclear iron containing
protein and has one Fe–S center and a tyrosine radical
[11,12,14]. As CAO does not convert protochlorophyl-
lide (Pchlide) a to protochlorophyllide b there may be
another enzyme that could catalyze Pchlide a to Pchlide
b[15–17]. The CAO is localized on the chloroplast enve-
lope and thylakoid membranes of mature chloroplast
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.11.049
*
Corresponding author. Fax: +91 11 26717558.
E-mail address: bctripathy@mail.jnu.ac.in (B.C. Tripathy).
www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 326 (2005) 466–471
BBRC

and mostly in the inner envelope membrane of dark-
grown Chlamydomonas reinhardtii cells [14].
In the present study, CAO is overexpressed in tobacco
to increase the Chl b content and modulate its Chl a/b
ratio. The effect of low and high light intensities on
Chl contents, Chl a/b ratio, and gene and protein expres-
sion of CAO and light-harvesting chlorophyll–protein
complex is studied to understand the interrelationship.
Materials and methods
Reagents and supplies. The enzymes and chemicals used for DNA
manipulation were purchased from Promega, USA. The oligonucleo-
tides were obtained from Microsynth, Switzerland. DNA purification
kit, plasmid isolation kit, and nick translation kit were obtained from
Amersham–Pharmacia, USA. Plant hormones, antibiotics, Hepes,
Mops, acrylamide, etc were procured from Sigma Chemical, USA, and
rest of the chemicals were from Qualigens, India.
Construction of binary vector. A binary vector pCAMBIA 2301 was
digested with EcoRI and SalI, end-filled by Klenow, and then re-ligated
using the standard procedure [18]. A CaMV 35 S promoter cassette was
taken out from pRT101 [19] and inserted into the HindIII site of the
binary vector. For the amplification of a CAO cDNA fragment
(1611 bp), cDNA library of Arabidopsis thaliana was used. PCR was
carried out with a pair of primers 50-gc gaa ttc atg aac gcc gcc gtg ttt ag-
30and 50-gc gaa ttc tta gcc gga gaa agg tag-30. In both the primer EcoRI
restriction sites were introduced (underlined). The amplified cDNA
fragment was ligated to pGEMT-Easy, then EcoRI digested CAO
cDNA fragment was taken out from cloned pGEMT-Easy and inserted
into the modified pCAMBIA 2301 plant transformation vector. The
recombinant plasmid (pCAMBIA 2301-CAO) was transformed into
competent Agrobacterium tumefaciens (LBA4404) cells.
Plant transformation and PCR analysis. The LBA4404 strain of
Agrobacterium tumefaciens, carrying the gene-construct p35S: CAO:
nos poly(A) was used to transform tobacco (Nicotiana tabacum cv.
Petit Havana) by leaf disc method [20]. Transformed plantlets regen-
erated on MS-medium containing kanamycin (300 mg/L) along with
the untransformed tobacco leaf discs as control. A number of primary
transformants were selected and allowed to grow to flower and set
seeds. In the next generation (T1) genomic DNA was isolated by
CTAB method [21]. Presence of CAO gene in the plants was confirmed
by PCR using 35S forward internal primer (50-ccc act atc ctt cgc aag
ac-30) and CAO internal reverse primer (5 0-gc gaa ttc tta gcc gga gaa
agg tag-30). Kanamycin (nptII) gene specific primers (50-tcg acc atg ggg
att gaa caa gat gg-30and 5 0-att cga gct ctc aga aga act cgt caa gaa ggc-
30) were also used for PCR analysis to ensure the incorporation of the
whole cassette. PCR was carried out in a Perkin–Elmer thermal cycler
as described before [22]. Plantlets selected for kanamycin resistance
were potted in agropit soil and maintained in the greenhouse. Gradual
hardening of these potted plants was done to acclimatize them with the
normal greenhouse environmental condition.
Plant material and growth conditions. Wild-type and transgenic
tobacco seeds (Nicotiana tabacum cv. Petit Havana) were grown in
agropit soil and watered daily. Both wild-type (WT) and transgenic
sense plants (S1 and S2) were grown in greenhouse in natural photo-
period for 25–30 days under light intensity of 70–80 lmolm
2
s
1
at
25 C±2C. They were shifted to low (70–80 lmol m
2
s
1
) or high
(700–800 lmolm
2
s
1
) light for additional 20–25 days in green house.
Low light intensity was obtained by covering the plants with velum
paper that uniformly cuts off the photosynthetic active radiation. Light
intensity was measured with a quantum sensor (LI-COR, USA). Plant
material was harvested at indicated time and either immediately ana-
lyzed or stored at 80 C. Leaf numbers were always counted from the
top of the shoot.
Chlorophyll estimation. Total Chl, Chl a, and Chl b contents were
estimated after 80% acetone extraction [23].
Isolation of thylakoids. Thylakoids were isolated from tobacco
leaves by homogenizing about 5 g of tissue in 40 ml of isolation buffer
containing 50 mM Hepes, pH 7.5, 400 mM sucrose, 1 mM EDTA,
1 mM MgCl
2
, and 2 mM isoascorbate at 4 C[24].
Northern blot analysis. Total RNA was isolated [25] and 30 lgof
total RNA was fractionated in a 1.2% agarose–formaldehyde gel,
blotted on Nylon membrane (Hybond NX) by capillary flow method
[18], and UV cross-linked. The blot was stained with methylene blue to
check for equal loading and even transfer. Probes for hybridization
were PCR amplified using appropriate primers. For CAO, Northern
blots were hybridized with [a-
32
P]dCTP-labeled (nick translated), gel-
purified DNA probes prior to washing the blots, and exposure of
autoradiograms and quantification of the hybridization signals were
detected by Fluor-S-Multi Image (Bio-Rad).
The AtCAO probe was a 1.6 kb fragment obtained by PCR
(primers were 50-gc gaa ttc atg aac gcc gcc gtg ttt ag-3 0and 50-gc gaa ttc
tta gcc gga gaa agg tag-30) under standard conditions (94 C, 30 s;
55 C 30 s; and 72 C 1 min 30 s; 30 cycles) with cloned CAO cDNAs
as template, the Lhcb1 probe was (702 bp) also obtained by PCR
(94 C, 30 s; 55 C 30 s; and 72 C 1 min; 30 cycles). The primers were
50-gg atg gcc gcc tcc aca atg gc-30and 50-tca ctt tcc ggg aac aaa gtt gg-
30. The Pisum sativum chlorophyll a/b-binding probe was produced by
PCR using T7 and T3 primer from pBSK
+
cloned chl a/b cDNA as
template. The final wash was of 60 C for AtCAO and 62 C for Chl a/b
and Lhcb1 in 0.1·SSC containing 0.1% SDS.
SDS–PAGE and Western blot. Protein content of the plastid was
determined [26]. Thirty micrograms of proteins were loaded in each
lane and were resolved in 12.5% SDS–PAGE [27]. For immunoblot
analysis, thylakoid proteins were separated on SDS–PAGE and were
electrophoretically transferred to nitrocellulose membrane [1,28] using
the semidry Transblot apparatus (ATTO, Japan). Alkaline phospha-
tase conjugated antibodies were used for detection at dilution specified
by manufacturer.
Results and discussion
Construction of plant transformation vector and
transformation of tobacco plant
The full-length cDNA sequence of CAO was ampli-
fied by PCR from cDNA library of A. thaliana using
the gene specific primers. The cDNA sequence was in-
serted into the modified (as described in Materials and
methods) binary vector pCAMBIA 2301 under the
control of CaMV 35S-promoter in sense orientation
(Fig. 1A). Nicotiana tabacum leaves were transformed
by A. tumefaciens LBA 4404 carrying the gene construct.
The transformed tissue was selected on modified MS
medium (RM) containing high level of kanamycin
(300 mg/L). Randomly selected few sense transformants
were used for further studies. Control plants were grown
(without antibiotic) along with the transformed ones
under similar conditions.
PCR analysis of selected plants
The CAO gene integration along with the CaMV 35S-
promoter into the nuclear genome of tobacco plants was
confirmed by PCR analysis. An expected size of 1.7 kb
G.K. Pattanayak et al. / Biochemical and Biophysical Research Communications 326 (2005) 466–471 467

DNA fragment was observed when PCR amplified using
35S-promoter internal forward primer and CAO inter-
nal reverse primer, indicating the stable transformation
of CAO into the genome (Fig. 1B). The restriction anal-
ysis of the PCR product was carried out by using an
internal enzyme site XbaI to ensure the authenticity of
the amplified product. XbaI digestion yielded 1.17 and
0.53 kb fragments as expected (Fig. 1C). To strengthen
the above results PCR analysis of genomic DNA using
specific primers for nptII gene was also carried out and
a desirable amplification product of 0.8 kb was ob-
tained only in transformed plants (Fig. 1D).
Chlorophyll contents and chlorophyll a/b ratio of wild-
type and chlorophyllide a oxygenase-overexpressing
transformants of tobacco plants grown in low and high
light intensities
Both wild-type and transgenic tobacco lines over-
expressing CAO were grown in low (70–80 lmol m
2
s
1
)
and high (700–800 lmol m
2
s
1
) light intensities in nat-
ural photoperiod in greenhouse as described in Materials
and methods. In low light, the total Chl contents of
leaves of both the sense lines (S1 and S2) overexpressing
the CAO increased 6–7% (Table 1). However, the
amount of Chl b in S2 line increased 20%. This resulted
in 16% reduction in the Chl a/b ratio in the transgenic S2
line. The Chl a change in the transgenic S1 line was only
marginal. Chl a/b ratio did not change in S1 line.
In high light the changes in Chl contents and Chl a/b
ratio among wild-type and transgenic lines were more
pronounced. As compared to low-light-grown plants to-
tal Chl contents decreased in wild-type and the trans-
genic lines (S1 and S2) grown in high light. It is in
agreement with a previous report that shade acclimated
leaves contain more Chl than Sun leaves that is consis-
tent in an increased allocation of resources to light-har-
vesting functions as compared to those involved in
photochemical reactions, i.e., electron transport [9].In
high-light-grown wild-type plants the Chl a/b ratio
Fig. 1. Transformation of tobacco using CAO gene. (A) Schematic representation of the construct used to overexpress CAO in tobacco plant. (B)
The CAO gene integration along with the 35S-promoter into the genome of the tobacco plants was confirmed by PCR analysis. An expected size of
1.7 kb DNA fragment was observed when PCR amplified using 35S-promoter internal forward primer and CAO internal reverse primer. PCR
products were resolved in a 1% agarose gel containing ethidium bromide and visualized by ultraviolet transillumination. Lane 1, 1 kb ladder
(Promega), lanes 2–8, PCR amplified products, and lane 9, genomic DNA of untransformed wild plant was used for PCR as a negative control,
where no amplification product was found. (C) Amplified PCR product using 35S-promoter internal forward primer and CAO gene specific internal
reverse primer was digested with XbaI to ensure the authenticity of the amplified product. Expected sizes of two DNA fragments of 1.17 and 0.53 kb
were observed. Lane 1, 1 kb ladder (Promega), lanes 2–8, digested products. (D) Kanamycin (npt II) gene specific primers were used for PCR
amplification. Lane 1, 1 kb ladder, lane 2, untransformed tobacco DNA was used for PCR analysis as a negative control where no amplification
product was found, and lanes 3–8, PCR amplification product of 0.8 kb from individual transgenic lines using npt II gene specific primers.
468 G.K. Pattanayak et al. / Biochemical and Biophysical Research Communications 326 (2005) 466–471

increased to 3.38 (as compared to 2.88 in low light). It is
in agreement with previous observations where Chl a/b
ratio decreased in shade plants and increased in Sun
plants [29]. The total Chl contents of transgenic S2 line
in high light were 31% higher than those of wild type.
The increase of Chl a was 19% and that of Chl b was
72%. This resulted in the 31% decline of Chl a/b ratio
in S2 line. Overexpression of CAO in A. thaliana re-
sulted in only 7% decline in Chl a/b ratio [30]. As the
S1 line did not show much significant changes in its
Chl a/b ratio further studies were carried out in wild-
type and transgenic S2 line.
Northern blot analysis of CAO, Lhcb1, and Chl a/b
To understand the molecular mechanism of changes
in Chl b content and Chl a/b ratio in the wild-type
and transgenic (S2 line) tobacco plants grown in differ-
ent light regimes the CAO expression was studied (Fig.
2). The increase in Chl b content was accompanied by
enhanced CAO gene expression. As compared to low
light, in high light the wild-type plants had reduced
expression of CAO. This could be due to reduced tran-
scription and mRNA stability in high light [31,32].
As compared to wild type, the CAO message abun-
dance was much higher (8-fold) in low-light-grown
transgenic plants. In high light the CAO expression in
transgenic plants was only 20% of that of low light
(Fig. 2). This could be due to reduced mRNA stability
in high light [32].
In high light the Chl b content significantly increased
resulting in decreased Chl a/b ratio in transgenic plants
(Table 1). The light-harvesting Chl–protein complex II is
the major component of thylakoid membrane proteins
and is enriched in Chl b. To understand if increased
Chl b synthesis was accompanied by enhancement in
Lhcb gene expression, Northern blot analysis of wild-
type and transgenic plants grown in low and high light
was performed (Fig. 2). In low light the message levels
of Lhcb1 and Chl a/b were almost similar in wild-type
and transgenic lines. However, as compared to low light,
in high light their message abundance decreased (35–
60%) in wild type and significantly increased (37–50%)
in transgenic CAO overexpressing plants. This suggests
a close relationship between Chl b synthesis and cab
gene expression. The cab gene products form LHCPII
that is required to accommodate increased amounts of
Chl b synthesized in high-light-grown transgenic plants.
The Chl b is localized in the thylakoid membranes of
chloroplast whereas cab genes are coded by the nuclear
genome. Therefore, it is proposed that Chl b sends a sig-
nal to the nucleus for enhanced expression of Chl a/b
binding LHCP protein. However, the chemical nature
of the message needs to be identified.
Western blot of CAO and LHCb proteins
To study the correlation of gene expression and pro-
tein levels of CAO and LHCP both wild-type and CAO
Table 1
Total chlorophyll contents and chlorophyll a/b ratio of wild-type and CAO overexpressing transgenic tobacco S1 and S2 lines grown in low light
(70–80 lmolm
2
s
1
) and high light (700–800 lmol m
2
s
1
) intensities
Plant lines Light intensity Chlorophyll, mg (g fresh weight)
1
Chl a + b Chl a Chl b Chl a/b
WT Low 2.17 (±.07) 1.61 (±.05) 0.56 (±.01) 2.88 (±.02)
S1 Low 2.31 (±.03) 1.73 (±.02) 0.58 (±.008) 2.98 (±.01)
S2 Low 2.30 (±.03) 1.63 (±.02) 0.67 (±.01) 2.43 (±.007)
WT High 1.58 (±.04) 1.22 (±.03) 0.36 (±.009) 3.38 (±.01)
S1 High 1.59 (±.04) 1.20 (±.02) 0.39 (±.01) 3.07 (±.01)
S2 High 2.07 (±.03) 1.45 (±.01) 0.62 (±.01) 2.33 (±.01)
Fig. 2. Northern blot analysis of CAO,Lhcb1, and Chl a/b of wild-
type (WT) and CAO overexpressing transgenic sense plants (S2) grown
in low light (70–80 lmolm
2
s
1
) (LL) and high light (700–
800 lmolm
2
s
1
) (HL) for 20–25 days. Second leaves from the top
of the shoot of both the plants were harvested at 9 AM in the morning,
total RNA was isolated, and 30 lg of total RNA was loaded in each
lane.
G.K. Pattanayak et al. / Biochemical and Biophysical Research Communications 326 (2005) 466–471 469

overexpressing transgenic line (S2) were grown in low
and high light, and their thylakoid membranes were
isolated. The immunoblot analysis of CAO, Lhcb1,
Lhcb2, Lhcb4, and LHCII was performed (see Fig.
3). In low light in contrast to its gene expression, the
increase in CAO protein abundance in the transgenic
S2 line was only marginal. This could be due to post-
translational degradation of CAO protein. Pulse-chase
experiments need to be performed to substantiate the
proposition.
As compared to wild type, the proteins of light-har-
vesting complex, LHCII, Lhcb1, Lhcb2, and Lhcb4,
partially increased in low-light-grown transgenic plants
(Fig. 3). Wild-type plants grown in high light had re-
duced LHCII, Lhcb1, and Lhcb4 protein expression as
compared to their low-light-grown counterparts. The
transgenic line in high light had a partial increase in
expression of these proteins. This does not correspond
to 71% increase in Chl b contents in transgenic line. This
suggests that LHCPII could bind more amounts of Chl
b. Plants grown in high light usually do not need bigger
antenna [9]. Therefore, the transgenic plants although
contain higher Chl and more Chl b than wild-type
plants, they do not need an extensive light-harvesting
apparatus in high light. These results demonstrate that
increased total Chl and Chl b contents and decreased
Chl a/b ratio may not necessarily result in a larger an-
tenna in high-light-grown plants.
Acknowledgments
We thank Dr. S. Leelavati and Mr. Vijaykanth for
assistance. Antisera against LHCP II were a kind gift
of Dr. A.K. Matoo, Beltsville, USA; Lhcb1 was a kind
gift of Dr. S. Jansson, Umea, Sweden; Lhcb2 was a kind
gift of Dr. N.P. Huner, Western Ontario, Canada; CAO
was a kind gift of Dr. J.A. Brusslan, California, USA.
The work was supported by grants from the Department
of Biotechnology, Government of India (BT/IC2-2/Bel-
arus). G.K.P is a recipient of CSIR fellowship, Govern-
ment of India.
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