Transcript NbCMT3-2

Developmental- and tissue-specific expression of NbCMT3-2 encoding a chromomethylase in Nicotiana benthamiana
Yu-Ting
1
Lin (林郁婷)
, Huei-Mei
1
Wei ,
Syue-Yu
1
Lyu ,
Yung-I
2
Lee ,
and Shih-Feng
1*
Fu (傅士峰)
1Department
of Biology, National Chunghua University of Education (彰化師範大學生物系)
2Botany Department, National Museum of Natural Science (自然科學博物館植物園)
2. Expression of NbCMT3 and NbCMT3-2 in tissues of
N. benthamiana
Abstract
DNA methylation is a heritable epigenetic process controlling gene expression and
developmental programs in various organisms. The chromomethylase (CMT) protein family A
is unique to plants and controls non-CpG methylation. Here, we investigated the
B
developmental expression of CMT3-2 in Nicotiana benthamiana (NbCMT3-2) and its
significance by analyzing plants with silencing of NbCMT3-2 and analyzing leaf tissues
transiently expressing the N-terminal polypeptide. Alignment of the NbCMT3-2 amino acid
sequence with other plant CMT3s showed a specific N-terminal extension required for
nuclear localization. Transient expression of the N-terminal polypeptide in N. benthamiana
resulted in the formation of chlorotic lesions, which indicates its vital role in leaf development.
NbCMT3-2 was expressed mainly in proliferating tissues such as the shoot apex and
developing leaves. We generated transgenic N. benthamiana harboring a fusion reporter
construct linking the NbCMT3-2 promoter region and -glucuronidase (GUS) reporter
(pNbCMT3-2::GUS) to analyze the tissue-specific expression of NbCMT3-2. NbCMT3-2 was
expressed in shoot and root apical meristem and leaf primordia in young seedlings and highly Fig. 2 Expression of NbCMT3 and NbCMT3-2 in different tissues. (A)
expressed in developing leaves and ovary as well as lateral buds in mature plants. Virus- Diagrams of gene-specific primers corresponding to NbCMT3 and NbCMT3-2
induced gene silencing used to knock down NbCMT3-2 expression led to partial loss of cDNA sequences. Apical shoots (A), young leaves (Y), mature leaves (M),
flowers (F) and roots (R).The tissue culture (T) of mature leaf explants during
genomic CHG DNA methylation. Silencing NbCMT3-2 interfered with leaf development and
shoot regeneration for 2 weeks were collected for gene expression analysis.
the expression of genes involved in jasmonate homeostasis. The differential roles between NbEF1 was a reference gene.
NbCMT3 and NbCMT3-2 were investigated and compared. We reveal the expression patterns
of NbCMT3-2 in proliferating tissues. NbCMT3-2 may play an essential role in leaf
3. Shoot apex- and root tip-specific expression of
development by modulating jasmonate pathways.
NbCMT3 and NbCMT3-2 promoter
Results
1. Nuclear localization of NbCMT3-2 protein
C
F ig . 1 Su b cellu lar lo caliz atio n o f
NbCMT3-2 proteins in N. benthamiana.
(A)Schematic diagram of the constructs
used for transient expression of proteins in
leaf epidermal cells. The unique N-terminal
extension (N) is in yellow, the Bromoadjacent homology (BAH) domain is in
green, and the chromo domain (CD) is in
red. The blue boxes indicate the conserved
methyltransferase catalytic motifs I, IV, VI,
VIII, IX and X. (B) Agrobacterium
carrying the fusion constructs under the
control of Cauliflower mosaic virus 35S
promoter was infiltrated into N.
benthamiana epidermal cells. At 4 days
after agroinfiltration, fluorescence of
NbCMT3-derived proteins was observed
by confocal laser scanning microscopy (C)
Western blot analysis of NbCMT3-derived
protein fusions expressed from pSITEII4C1 vectors in agroinfiltrated leaves
Fig. 3 Histochemical localization of GUS activity in transgenic N.
benthamiana plants expressing GUS gene driven by the NbCMT3 or
NbCMT3-2 promoter region. Cotyledons (Cy), Center zone (CZ),
Peripheral zone (PZ), RZ (Ribzone), PM (Primary meristem), AM
(Apical meristem), RC (Root cap), Co (Cortex), E (Epidermis).
4. Expression of NbCMT3 or NbCMT3-2 promoter
in stigma
6. Suppression of endogenous NbCMT3 or NbCMT3-2
expression by VIGS
Fig. 6 Molecular
Fig. 4 Distribution of pNbCMT3-2::GUS activity in various organs of
mature transgenic N. benthamiana plants. (A) lateral bud, (B) developing
leaves, (C) ovary and (D) stigma.
5. Accumulation of NbCMT3– or NbCMT3-2–GFP-GUS
fusion protein during organogenesis
Fig. 5 Promoter
NbCMT3- or
NbCMT3-2-driven
GFP and GUS
expression during
callus formation and
organogenesis in
vitro. Callusinducing medium
(CIM). Shoot
inducing medium
(SIM) (A-F). (G and
H ) We s t e r n b l o t
analysis of GFP
expressed from
t r a n s g e n i c
p Nb CM T 3 : : G FP GUS or pNbCMT32::GFP-GUS leaf
explants. Wild-type
N. benthamiana
plants were a
negative control.
The star (*) denotes
the non-specific
cross-reaction of
a n t i b o d i e s .
characterization o f
NbCMT3-, NbCMT3-2or NbCMT3/3-2silenced
N.
benthamiana plants .(A)
NbCMT3 or NbCMT32 cDNA structures and
the VIGS construct
containing the cDNA
fragments. (B)
Suppression of
NbCMT3 or NbCMT32 transcripts by virusinduced gene
suppression (VIGS). (C)
Quantification of
g l o b a l D N A
methylation in
NbCMT3-silenced
p l a n t s .
7. Regulation of leaf development by NbCMT3 and
NbCMT3-2
Fig. 7 Alternation
in N. benthamiana
N b C M T 3 - 2
expression led to
abnormality in plant
g row th an d
development. (A)
P h e n o t y p i c
characterization of
N b C M T 3 - ,
NbCMT3-2- and
NbCMT3/3-2s i l e n c e d
N.benthamiana
p l a n t s .
(B)Evaluation of
g row th an d
development using
transient gene
expression from a
PVX-based vector.
Conclusions and future work
NbCMT3-2 and its promoter activity were expressed in
proliferating tissues such as shoot apex and root tips. The unique Nterminal extension of NbCMT3-2 has a role in nuclear localization
and leaf development.
Further investigation into the roles of the unique N-terminal
extension will help elucidate the molecular actions of NbCMT3-2 in
plant epigenetic regulation.