The Tomato FRUITFULL Homologs

TDR4/FUL1 and MBP7/FUL2 Regulate

Ethylene-Independent
Aspects of Fruit Ripening
Marian Bemer, Rumyana Karlova, Ana Rosa Ballester, Yury
M. Tikunov, Arnaud G. Bovy, Mieke Wolters-Arts, Priscilla de
Barros Rossetto, Gerco C. Angenent, and Ruud A. de Maagdb

The Plant Cell, Vol. 24: 4437

4451

Paper Seminar
By
AVI RAIZADA

Introduction and literature

the gene regulatory networks
involved in the development of both
dry and fleshy fruits may be similar,
although the outcomes are
morphologically very different.
Arabidopsis thaliana - Fruitfull(ful)
silique mutants short and bumpy
poor development of zone of
dehiscence- defect in seed dispersal.

Research on fleshy fruit formation has primarily

focused on the model system tomato, which
produces a true berry derived from the ovary
Autoinhibitory ethylene production system 1
(ACS1A), ACS2, ACS4,
(ACO1), ACO3, ACO4
Autocatalytic production system 2

Ripening

The NAM, ATAF1, CUC2 (NAC)-domain family protein

NONRIPENING (NOR), the SQUAMOSA promoter
binding protein (SBP) COLORLESS NON-RIPENING
(CNR), and the MADS domain protein RIPENING
INHIBITOR (RIN) - upstream ripening regulators
Corresponding mutants, nor, Cnr, and rin, are impaired
in many aspects of ripening
The MADS domain transcription factor RIN belongs to
the
SEPALLATA (SEP) class of MADS domain proteins, of
which the
members have been found to interact with members
from several other classes, mainly involved in
flowering, floral organ
formation, and reproduction. As such, the SEP proteins
are
thought to function as cofactors that allow higher order

Pleiotropic phenotype- rin mutantdisturbances of different MADS complexes

TAGL1, TAGL11, FUL1, FUL2 interact with

RIN
Binding of rin to promoters depend on
cnr activity
TAGL1 knockdown yellow orange
friuts- decreased expression of RIN
target ACS2.

Tomato contains 5 MADS Box genes

that belong to SQUAMOSA/AP1 clade
Arabidopsis
tomato
Expression
AGL79
MBP10,MBP20
vegetative
tissues
AP1
MADS-MC
inflorescences
FUL
TDR4, MBP7
fruit
Because of sequence similarity between At FUL and SI TDR4 (75%) - FUL1
At FUL and SI MBP7 (77%) - FUL2

The Tomato FUL Genes Are

Predominantly Expressed during Fruit
Development
Relative expression profiles of FUL1 and FUL2 in different MT
tissues obtained by qRT-PCR. FL,.flower; I, 5-mm fruits; II, 1-cm
fruits; L, leaf; FL, flower; I, 5mm fruits; II, 1cm fruits; MG,
mature green; Br, breaker; Br+7, 7 days after breaker stage
(red ripe).

The Tomato FUL Genes Are Predominantly

Expressed during Fruit Development
(B) Relative expression profiles of SlFUL1 and SlFUL2 in
. fruits. S 5 seeds; Pu 5
separated fruit tissues of red ripe
pulp; C 5 columella; Pl 5 Placenta; Sp 5 septa; M 5
mesocarp; E+P 5 exocarp and peel.

Objectives
To understand the gene regulatory
network involved in tomato ripening
To know the function of FUL1 and
FUL2 homologs
To compare dry and fleshy fruit
development network conservation
and divergence

Sequence of study
Function of FUL1 and FUL2 homologs
ful mutants
Relation of ful homologs with CNR,
RIN and TAGL1
Position of ful homologs in regulatory
network
Discussion
conclusion

Function of ful homologs

Double knockdowns- ful1/2 RNAi
plants

Single knockdowns ful1 and ful2

RNAi plants

Double knockdowns
stable transgenic MT lines
35S:FUL1 RNA interference (RNAi)
construct
coding sequences- 78% identical,
the RNAi fragmentwas amplified from
the 3 end of FUL1, including a part of
the 3 untranslated region (UTR), where
the identical stretches did not exceed
20 nucleotides

Phenotypes
(A) Fruits of FUL1
RNAi lines R1-10
and R1-25 and
wild-type (WT) MT
at stage Br + 7.

(C) Binocular
microscopy
pictures of
handmade
pericarp sections
of wild-type and
R1-10 fruits at
stage Br + 7.

(B) Comparison of the carotenoid levels in wild-type and

R1-10 fruits at stage MG, B+7 (Br + 7 d), and B+10 (Br
+ 10 d). AU, absorbance units; FW, fresh
weight.

Phenotypes
(D) Water loss in wildtype and R1-10 and R129 fruits after harvesting
at stage Br + 7. The y
axis depicts the
percentage of weight
loss since the day of
harvesting.
(F) Cuticle thickness in
wild-type and R1-10 Br +
7 fruits, measured from
the top of the epidermal
cell to the surface

Phenotypes
(E) Cuticle
thickness of wildtype and R1-10 Br
+ 7 fruits is visible
in light microscopy
pictures of thin
pericarp sections
stained with Sudan
IV. The side panels
show the
desiccated
tomatoes 40 d
after harvesting.

strongest
phenotypes shown
by these

(D)
Downregulation of
FUL1 and FUL2 in
the FUL1 RNAi
lines R1-10, R125, R1-29, and
R1-30 compared
with the wild type
(WT). The relative
expression levels
were obtained by
qRT-PCR.

Single knockdowns
- FUL2RNAi construct 3UTR and part of 3ORF
The plants all exhibited an orange-ripe fruit
phenotype similar to the fruits of the FUL1 RNAi
lines
- specific FUL1RNAi only 3UTR
- variable levels of FUL1 and no downregulation of
FUL2
- specific FUL2 RNAi only 3 UTR
- FUL1 is reduced to 60% of WT.

Down regulation
of FUL1 and FUL2
in the FUL2RNAi
and specific FUL1
and FUL2 lines.

Phenotypes

I. Jelly of red
ripe fruits
from lines

Conclusion
FUL1 and
FUL2 act
reduntantly

Functions of FUL homologs

Caroteinoid biosynthesis- lycopene
reduced and its precursor phytoene
decreases
Lutein remain same
Beta carotene increases at br + 10
stage of R1-10
Normal pericarp
Decreased glutamate content

Relation with CNR, RIN and

TAGL1
MICROARRAY analysis of R1-10 and
R1-30 from br stage
Confirms quantitative RTPCR results
and shows several differentially
expressed genes, which gives clues
for relation between FUL homologs
and upstream ripening regulators.

Microarray results
Gene/enzy
me

FUL1/2

PSY1

cnr

rin

DXS1

PG2A

XYL

PAT

GAD2, GAD3

TAGL1
Silenced
-

 Tagl1 rnai and rin plants - ripening

deficient
- ACS2, ACS4 and ACO1 downregulated
Cnr mutants ripening deficient
- ACO downregulated
FUL1/2 RNAi plants no downregulation
of these genes.(microarray analysis)

To confirm
thisBr or Br + 3 stage
fruits from
transgenics and
wild type are
harvested and
ethylene was
measured

Position of FUL paralogs in

ripening regulatory network

Expression of all three FUL1,

FUL2 and TAGL1
downregulated

Also get affected in rin

mutants

 Rin and tagl1 upregulated 2-3 times

negative feedback
Cnr remains unchanged

DISCUSSION

Strength and weakness of

study
Use of microarray analysis to confirm
qRTPCR results
Doesnt clarify that why there is no
downregulation (even slight) of FUL2
in specific FUL1 RNAi plants.
However, FUL1 is slightly
downregulated in FUL2 RNAi plants

Future prospectives

References

Acknowledgement
I thank Dr. Marian Bemer and his colleagues who
did this wonderful work.
I am highly thankful to Dr. H.S. Misra and other
committee members who selected this paper for
me.
I gratefully acknowledge all the examiners for their
critical evaluation.
I thank my friends and colleagues who helped me to
prepare for the presentation.

Thank you