读书报告,英文

硕士研究生读书报告

姓

名：

郜志栋

学科、专业：

果树学

研

究

方

向

：

果树生物技术

指

导

教

师

：

温鹏飞

201 2

年

12

月

1 2

日

Advances in the transcriptional regulation

of the flavonoid biosynthetic pathway

Abstract:

Flavonoids are secondary metabolites involved in several aspects of plant development

and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to

plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks.

Because

they

affect

the

quality

of

flowers

(for

horticulture),

fruits

and

vegetables,

and

their

derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore,

these compounds possess pharmaceutical properties extremely attractive for human health. Thanks

to

easily

detectable

mutant

phenotypes,

such

as

modification

of

petal

pigmentation

and

seeds

exhibiting

transparent

testa,

the

enzymes

involved

in

the

flavonoid

biosynthetic

pathway

have

been characterized in several plant species. Conserved features as well as specific differences have

been described. Regulation of structural gene expression appears tightly organized in a spatial and

temporal

way

during

plant

development,

and

is

orchestrated

by

a

ternary

complex

involving

transcription

factors

from

the

R2R3-MYB,

basic

h elix

–

loop

–

helix

(bHLH),

and

WD40

classes.

This MYB

–

bHLH

–

WD40 (MBW) complex regulates the genes that encode enzymes specifically

involved

in

the

late

steps

of

the

pathway

leading

to

the

biosynthesis

of

anthocyanins

and

condensed tannins.

Key words

: bHLH, flavonoids, MYB, transcription factors, WD40.

Introduction

Flavonoid compounds are secondary metabolite s widely accumulated in vascular plants and

to

a

lesser

extent

in

mosses.

They

accumulate

in

all

organs

and

tissues,

at

different

stage

s

of

development, and depending on the environmental conditions. Beside their multiple roles in plant

development and adaptation to the environment, these molecules are of major interest for human

nutrition and health . Indeed, they contribute to the organoleptic quality of plant-derived products

(colour,

taste,

flavour,

etc.),

and,

in

addition,

they

have

been

shown

to

be

beneficial

to

human

health

and

in

prevention

of

cell

ageing.

In

grape

(

Vitis

vinifera

L.)

berries

for

instance,

the

flavonoid

composition

is

essential

for

wine

quality

and

conservation.

Moreover,

the

regular

consumption

of

red

wine

is

thought

to

explain

the

‘French

pa

radox’,

whereby

the

French

population

suffers

a

relatively

low

incidence

of

coronary

heart

disease

in

spite

of

a

diet

rich

in

saturated fat . The mechanisms involved have long been related to the presence of flavonoids and

stilbenes in red wine.

Work achieved on model plants pinpointed the tight regulation of the flavonoid biosynthetic

pathway during plant development. It is now established that the transcriptional regulation of the

structural gene s is controlled by MYB and basic helix

–

–

helix (bHLH ) transcription factors,

together

with

WD40

proteins.

Special

attention

has

hitherto

been

devoted

to

MYB,

as

demonstrated by the reported publications. Herein, the recent advances in the knowledge of the

transcriptional

regulation

of

the

flavonoid

pathway

are

discussed,

with

a

particular

focus

on

bHLH transcription factors.

The MYB transcription factors

The first MYB transcription factors regulating the flavonoid pathway were identified in 1987

in maize, and comprised C1 and Pl1 (Purple leaf 1), in addition to P1. At that time, identification

of

C1

indicated

that

plant

transcription

factors

were

closely

related

to

those

of

mammals,

constituting

a

milestone

in

plant

molecular

biology.

Indeed,

C1

showed

a

significant

homology

with the vertebrate c-MYB proto- oncogene, derived from avian myeloblastosis virus and known

to control cell proliferation and differentiation. MYB transcription factors are characterized by the

so-called

N-terminal

MYB

domain,

consisting

of

1

to

3

imperfect

repeats

of

almost

52

amino

acids (R1, R2, and R3). While the MYB domain is involved in DNA binding and dimerization, the

C-terminal

region

regulates

target

gene

expression

(i.e.

activation

or

repression).

Plant

MYB

transcription

factors

bind

different

cis-elements,

called

MYB-binding

sites

(MBSs),

and

some

MYB transcription factors show a certain flexibility of recognition. However, MYB transcription

factors belonging to different species and regulating the same pathway, such as PA biosynthesis

for

instance,

seem

to

bind

the

same

motif.

MYB

transcription

factors

regulating

the

flavonoid

pathway

have

been

widely

investigated

and

identified

in

crop,

ornamental,

and

model

plants

(Table 1). Most of them present two R repeats (R2R3 MYB proteins), and belong to subgroups 1-7

of

the

classification

of

Stracke

et

al.

(2001).

Regulators

of

the

PA

and

anthocyanin

pathways

display

the

[D/E]Lx2[R/K]x3Lx6Lx3R

motif

necessary

for

interaction

with

bHLH

transcription

factors

in

their

R3

repeat,

while

MYB

transcription

factors

governing

flavonoidl

biosynthesis

exhibit

the

SG7

[K/R][R/x][R/K]xGRT[S/x][R/

G]xx[M/x]K

and

the

SG7-2

([W/x][L/x]LS)

motifs in their C-terminal end. Nevertheless all regulators of the flavonoid pathway do not fit this

classification perfectly. In potato, a single domain MYB protein, similar to soybean MYB73, is 44

times more expressed in purple flesh compared with white flesh, suggesting a role in the control of

anthocyanin biosynthesis.

Most of the MYB transcription factors characterized to date control only one branch of the

flavonoid pathway. Specific regulators of the anthocyanin pathway have been identified in petunia,

Arabidopsis, strawberry, grapevine, tomato, potato, tobacco, and pear, to name a few. Among them,

the

R3

AtMYBL2

is

an

anthocyanin

repressor,

and

the

R2R3

AtMYB60

inhibits

anthocyanin

synthesis

in

lettuce.

Extensive

protein

sequence

alignments

of

134

MYB

transcription

factors

regulating the anthocyanin pathway revealed conserved residues in the R3 repeat (arginine, valine,

and

alanine)

of

dicots,

as

well

as

a

short

conserved

motif

ANDV

.

In

addition,

the

[R/K]Px[P/A/R]xx[F/Y]

motif

has

been

identified

in

the

C-terminal

region

of

these

anthocyanin-regulating MYBs.

Regulators

of

PA

biosynthesis

have

been

identified

in

Arabidopsis,

grapevine,

leguminous

plants,

persimmon,

and

poplar.

More

recently,

MYBs

regulating

the

flavonol

branch

have

also

been

identified

in

Arabidopsis

and

grapevine.

As

already

mentioned

above,

MYBs

generally

regulate only one branch of the flavonoid pathway. In grapevine for instance, overexpression of

VlMYBA1-2

in

hairy

roots

induced

only

expression

of

structural

genes

related

to

anthocyanin

biosynthesis

and

transport.

Likewise,

ectopic

expression

of

VvMYBPA1

and

VvMYBPA2

in

grapevine hairy roots exclusively activated genes encoding enzymes of the PA pathway such as

anthocyanidin reductase and leucoanthocyanidin reductase. Despite this highly specific function,

some

MYB

transcription

factors

may

play

different

roles.

Over-expression

of

VvMYB5b

in

tomato

affected

both

phenylpropanoid

and

carotenoid

metabolism.

The

single

R3

repeat

CAPRICE

(CPC)

is

known

to

regulate

epidermal

cell

fates

such

as

trichome

and

root

hair

formation

in

Arabidopsis.

Furthermore,

CPC

inhibits

anthocyanin

accumulation

in

homologous

and

heterologous

hosts,

by

competing

with

R2R3

MYB

transcription

factors

regulating

the

flavonoid

pathway.

Since

CPC

does

not

bind

to

DNA,

it

is

likely

that

this

transcription

factor

interferes by interacting with bHLH partners, as demonstrated by yeast two-hybrid assays.

In summary, many recent studies, together with the analysis of new plant genomes, suggest

that primary protein structures and biological functions are correlated within MYB subgroups that

are

conserved

between

divergent

species.

This

is

especially

true

for

MYB

transcription

factors

regulating

the

flavonoid

pathway,

where

specific

motifs

and

conserved

residues

have

been

identified

in

anthocyanin

(Lin-Wang

et

al.,

2010)

and

flavonoid

regulators.

However,

the

biological

functions

of

the

consensus

motifs

present

in

the

C-terminus

of

the

proteins

are

just

beginning to be investigated. It would be of great interest to determine if these specific motifs can

provide the specificity for a MYB transcription factor to regulate a given branch of the flavonoid

pathway, by modulating interactions with DNA and/or with protein partners such as bHLH and/or

WD40 proteins.

The WD40 proteins

WD40

or

WDR

(WD

repeat)

proteins

are

involved

in

many

eukaryotic

cellular

processes

including

cell

division,

vesicle

formation

and

trafficking,

signal

transduction,

RNA

processing,

and

regulation

of

transcription.

They

notably

participate

in

chromatin

remodelling,

through

modifications of the histone proteins, and can thus influence transcription.

WD40 proteins are characterized by a peptide motif of 44-60 amino acids, typically delimited

by

the

GH

dipeptide

on

the

N-terminal

side

(11-24

residues

from

the

N-terminus)

and

the

WD

dipeptide on the C-terminus). This motif can be tandemly repeated 4-16 times within a protein,

with

a

large

majority

of

Arabidopsis

WD40

proteins

exhibiting

4

or

more

WD

repeats.

WD40

proteins are not thought to have any catalytic activity (DNA binding or regulation of expression of

a target gene), but rather seem to be a docking platform, as they can interact with several proteins

simultaneously. Only Arabidopsis TTG1 (Transparent Testa Glabra 1) was clearly demonstrated,

using chromatin immunoprecipitation, to bind the promoter of AtTTG2, a gene encoding a WRKY

transcription

factor

mainly

involved

in

trichome

patterning.A

small

number

of

WD40

proteins

involved

in

the

regulation

of

the

flavonoid

pathway

have

been

identified

so

far

(Table

1),

and

include petunia AN11, Arabidopsis TTG1, perilla PFWD, maize ZmPAC1, Medicago trunculata

MtWD40-1,

and

grapevine

WDR1

and

WDR2.

These

WD40

proteins

appear

to

be

highly

conserved among species. Indeed, PFWD and PhAN11 show 81.3% identity, whereas PFWD and

AtTTG1 share 77.8% identity. The WD40 protein family seems to be less expanded than the the

MYB or bHLH families, since MtWD40-1, AN11, and PAC1, are single-copy genes.

WD40

proteins,

regulating

the

flavonoid

pathway,

such

as

TTG1,

can

control

many

other

physiological

processes,

such

as

trichome

and

root

hair

determination

and

seed

mucilage

production,

and

are

accordingly

expressed

in

tissues

both

accumulating

and

not

accumulating

flavonoids.

In

petunia,

an11

mutants

show

a

reduced

anthocyanin

content

in

the

corolla.

Disturbance

of

petal

coloration

is

attributed

both

to

a

reduction

in

the

expression

of

flavonoid

structural genes and to a modifica?

tion of the vacuolar pH, indicating that AN11 is involved at

least in the regulation of these two metabolic events. In Medicago truncatula, MtWD40-1 mutants

are deficient in accumulation of mucilage, and the synthesis of PAs, flavonols, anthocyanins, and

benzoic acid in seeds, but only in PA synthesis in flowers, and finally they show no modification

of epidermal cell fate. MtWD40-1 mutants show a strong reduction of the expression of flavonoid

structural

genes,

whereas

overexpression

of

MtWD40-1

in

M.

truncatula

hairy

root

does

not