PERAN BIOKIMIA DALAM

MENDUKUNG
PERKEMBANGAN
BIOTEKNOLOGI TANAMAN
Yekti Asih Purwestri

Biochemistry Laboratory, Faculty of Biology, Universitas Gadjah Mada,

Yogyakarta, Indonesia

Kuliah Pendahuluan Biokimia S2

2013
 Elucidation of Hd3a Florigen
Activation Complex in rice
flower signaling

Yekti Asih Purwestri

Biochemistry Laboratory, Faculty of Biology, Universitas Gadjah Mada,

Yogyakarta, Indonesia

Kuliah Pendahuluan Biokimia S2

2013
 Hd3a protein is a major component of florigen

meristem
leaf SD
Hd3a
phloem

Hd3a OsGI

Hd1

Hd3a
Hd3a::Hd3a-GFP

Hd3a Hd3a protein

flowering
Hd3a
protein
movement (Hayama et al. 2003; Tamaki et al. 2007)
 The objectives and approaches of study

Searching Hd3a interacting proteins

to explore Hd3a function by using yeast two-hybrid screening
Z
X
Hd3a
Putative proteins in the Hd3a
Y protein complex : X, Y, Z

Analysis of Localization of the Phenotypic analysis

proteins binding putative mRNA & of mutants
proteins

yeast two-hybrid assay, subcellullar localization overexpression /

in vitro pull-down assay, Tissue specific expression knock-down/knock-
co-imunoprecipitation, (RT PCR) out genes
BiFC
 Putative Hd3a interacting proteins

Name of proteins Name of proteins

Carbohydrate metabolism Signaling

1. Ribulose 1,5-bisphosphate 10. GF14-c protein (14-3-3 homolog)
carboxylase (RuBisCO)small subunit 11. BRI1-KD interacting protein 116b
2. RuBisCO activase large isoform (BIP116b)
precursor protein 12. OsKANADI1
13. Putative
Unknowns endonuclease/phosphatase
3. Unknown protein 14. Beta cyanoalanine synthase
4. Unknown protein 15. Putative Anthranilate
phosphoribosyltransferase
5. OSJNBb0012E24.15
16. Putative NADPH- dependent
retinol dehydrogenase/reductase
Protein synthesis/ DNA & RNA 17. Putative SF16 protein
binding proteins 18. Putative heat shock 70 KD protein
6. Putative RNA helicase (HSP70)
7. Putative 40S ribosomal protein 19. Ser/Thr kinase
8. Translation initiation factor 5A/eIF5A 20. Putative glutathione S-transferase
9. Linker histon 21. Thioredoxin H-type (TRX-H)
 Role of GF14c in Hd3a signaling in rice
flowering
Hd3a
flowering
Cells of the SAM
Hd3a
nucleus

leaf Companion cells

Hd3a
meristem

Hd3a

Hd3a
GF14c

Hd3a Hd3a
Purwestri et al. 2009
Florigen Activation Complex (FAC)
Hd3a :: Hd3a -GFP transgenic rice is early flowering

Hd3a promoter Hd3a :GFP nosT

(Day)
70

60
Days to flowering

50

40

30

20
Panicle
10

0 Hd3a :: Hd3a::
WT WT
Hd3a -GFP Hd3a-GFP
rolC::Hd3a-GFP chrysanthemum show flowering
under non-inductive conditions

rolC promoter Hd3a :GFP nosT

WT rolC
Hd3a-GFP

460 bp
(Hd3a)

（Asao et al., unpublished data）

potato show tuber formation under short day
conditions

Short Day (SD) Long Day (LD)

potato:
Short day plant
 rolC::Hd3a-GFP potato lines show tuber
formation under non-inductive conditions (LD)

rolC promoter Hd3a :GFP nosT

WT rolC::Hd3a-GFP
（Tamaki et al., unpublished data）
Overexpression of Hd3a:GFP affects multiple
developmental processes in rice

RPP16::Hd3a:GFP rice showed

panicle
• Early flowering
• Semi-dwarfism
• High branching (tillering)
• Altered panicle architecture

WT RPP16::Hd3a:GFP

Chika Tachibana, Shojiro Tamaki

 rolC::Hd3a:GFP potato lines showed
high branching

mild severe

Wild-type RPP16::Hd3a:GFP

Shojiro Tamaki
Collaboration: Dr. Salomé Prat, Centro Nacional de Biotecnología- CSIC
 Hd3a/FT can have a more general role in biological
prosesses

Trifoliate
Poplar Tomato
orange

35S::CiFT Wild-type 35S::PtFT1 Wild-type 35S::SFT

High branching Loss of short-day induced Altered sympodial growth

dormancy Early release of lateral shoots

Endo et al., 2005 Bohlenius et al., 2006 Lifschits et al., 2006

 Hd3a/FT protein may have multiple functions in growth
and development in plants

Flowering Tuber
formation
(all plants?) (potato)

Hd3a/FT

Leaf shape Branching

(Arabidopsis, tomato, chrysanthemum) (rice, tomato, potato,
trifoliate orange)
Gene-specific regions of Hd3a and RFT1 were used for the
Hd3a and RFT1 RNAi constructs.5UTR of RFT1 and 3UTR of
Hd3a were used for the double RFT1-Hd3a constructs
（Komiya et al., 2009）
 Genetic Modification (GM) is
another breeding method
Recombinant DNA (or
GM) allows a single
gene to be introduced
into a genome. This
method can be faster
than conventional
breeding

Elite tomato Poor tomato but

disease resistant

Elite, disease resistant tomato

 Source of gene Once a gene is
(disease-resistant introduced into the
plant) plant genome it
functions like any
Gene of interest other gene

Isolate gene of
interest using
molecular Recombine into
biology methods recipient plant DNA
Why are GM methods used sometimes
and molecular breeding others?
Molecular breeding

1. Desired trait must be 2. Genetic resources 3. Plant should be

present in population must be available propagated sexually

Photo credits: Gramene.org

 Why are GM methods used
sometimes and molecular breeding
others?
Molecular breeding

1. Desired trait must be 2. Genetic resources 3. Plant should be

present in population must be available propagated sexually
GM

1. Gene can come from 3. Plant can be

2. Genetic resources not propagated vegetatively
any source required
Photo credits: Gramene.org ETH Life International
 GM Example: Disease resistant
banana by introduction of a gene
from pepper

Resistant Susceptible
Banana bacterial wilt is destroying
plants in eastern Africa. Transgenic
plants carrying a resistance gene from
pepper are resistant to the disease
Tripathi, L., Mwaka, H., Tripathi, J.N., and Tushemereirwe, W.K. (2010). Expression of sweet pepper Hrap gene in banana
enhances resistance to Xanthomonas campestris pv. musacearum. Molecular Plant Pathology 11: 721-731.
 GM Example: Insect resistance
through introduction of the Bt gene
Wild-type peanut plant Peanut plant expressing the Bt gene

Photo by Herb Pilcher USDA

Bacillus thuringiensis (Bt) bacteria
produce insecticidal proteins
Bacillus thuringiensis
expressing insecticidal Bt
Bacillus toxin can be sprayed onto
thuringiensis
expressing Bt plants
toxin

Plant cell
expressing Bt
toxin

Or the plants can be

engineered to express the
Bt gene coding for Bt toxin
 The effect of Bt toxin is highly
specificIntestine
Bacillus
thuringiensis
expressing Bt
toxin

Processing

Plant cell Receptor

expressing Bt binding
toxin Intestinal
lumen

The Bt toxin affects only some insects

because to be effective it has to be
processed and bind to a specific
receptor protein
 The effect of Bt toxin is highly
specificIntestine
Bacillus
thuringiensis
expressing Bt
toxin

Plant cell
expressing Bt
toxin

Pore
After binding, the insecticidal proteins assembly
assemble to form a pore in the lining of
the insect intestine which kills the insect
 Makanan yang dibiofortifikasi secara genetis
Iron-enriched rice

()

Wild-type (top) and

antioxidant-enriched
tomatoes
Vitamin A–enriched rice
Photo credits: Golden Rice Humanitarian Board © 2007; Credit: ETH Zurich / Christof Sautter; Reprinted by permission
from Macmillan Publishers, Ltd: Butelli, E., et al., Nature Biotechnology 26, 1301 - 1308 copyright (2008).
Artemisia annua ,tumbuhan yang
mengandung senyawa antimalaria

Artemisinin

Artemisia telah digunakan oleh herbalis Cina selama

ribuan tahun. Pada tahun 1972 bahan aktif, artemisinin,
dimurnikan.
Photo credit: www.anamed.net
Pengembangan produksi tinggi
Artemisia

Photo credit: www.york.ac.uk/org/cnap/artemisiaproject/

 Perubahan satu gen tunggal dapat
meningkatkan toleransi tanaman
terhadaap kekeringan
Drought-resistant

Wild-type
Well-watered 10 days drought 20 days drought After re-watering

Yu, H., et al. Plant Cell 2008;20:1134-1151

Sistem perakaran yang lebih besar
berpengaruh terhadap toleransi thd
kekeringan
Drought Drought
Wild-type tolerant Wild-type tolerant

Pemuliaan
tanaman untuk
sistem akar yang
lebih besar
dapat
membantu
mereka tumbuh
di daerah rawan
kekeringan.
Seedlings Mature plants
Yu, H., et al. Plant Cell 2008;20:1134-1151