World Congress on Root and Tuber Crops January 18-22, 2016, Nanning, China

Plenary Session PS15, Jan. 22, 2016 RIKEN Cassava Initiative: 1. Collaboration with ASEAN countries and CIAT 2. Integrated Omic Analysis (Transcriptome, Metabolome, Hormonome and Epigenome)

Motoaki Seki,Yoshio Takei (S09-05),Tetsuya Sakurai,

Tomoko Abe and Yoshinori Utsumi (SP06-22) (RIKEN, Yokohama City Univ., JST CREST)

Cassava is an important crop in worldwide -Usage and application of cassava biomass-

Foods & industrial material Use as food and feed

Indonesia = 23.9 Mt/year

Thailand = 22 Mt/year

China= 4.7 Mt/year

Brazil = 24.5 Mt/year Colombia = 2.4 Mt/year

Paraguay= 2.6 Mt/year Democratic Republic of Congo = 15.0 Mt/year

Ghana = 13.5 Mt/year

Asia = 75.2 Mt/year

Africa = 121.0 Mt/year

South America = 31.7 Mt/year

FAO STAT2010 (http://faostat.fao.org/)

Vietnam = 8.5 Mt/year

Nigeria = 37.5 Mt/year

An Important Tropical Crop for Food Security, Poverty Reduction and industrial material in many Asian and African countries

(an important source for a billion people’s food and income generation).

3

India = 10.0 Mt/year

π

Cassava production：265Mt (2007)→266Mt (2010)

Cassava production:58.2Mt (2004)→85.2Mt (2010)

Cassava production：161Mt (2000)→239Mt (2010)

Japan (RIKEN) 0.66Mt（2014）

Ethanol industry

Cassava Relationship between ASEAN Counties and Japan

Citation Agriculture & Livestock Industries Corporation(http://www.alic.go.jp/）

India

Thailand (Mahidol Univ., DOA, KMUTT etc.; PI: Dr. Naranganjavana)

Vietnam (AGI; PI: Dr. Ham)

Cambodia

Indonesia (LIPI, ILETRI)

China

Laos

Myanmar

Cambodia (Univ. of Battambang))

Cassava:

Mahidol Univ. (Thailand) ・Marker Breeding (HB60XHN) ・CAD, CBB

CIAT (Colombia) ・Useful genetic resources ・Molecular breeding

RIKEN (Japan) ・Identification of candidate genes using microarray ・Production of useful cassava by transformation ・Heavy-ion beam irradiation (with Dr. Abe, RIKEN Nishina) ・Visit of Vietnamese Vice Prime Minister (May 22, 2013) and VAAS President (May 2, 2014) to RIKEN

・Selection of elite lines as pre- commercial variety

AGI (Vietnam) ・Evaluation of newly developed useful cassava candidates in the field

Developing Contribution to Green Innovation

Cassava International Collaborative Research in e-ASIA program (Japan and ASEAN countries)

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

ATG STOP

Genome DNA

mRNA

Partial cDNA

Full-length cDNA

Can’t produce proteins

・Can produce proteins (Applicable to transgenic plants)

Exon Intron

Full-length cDNA is an useful tool for basic and applied sciences.

・Useful for correct genome annotation, CRISPR design and marker development.

Large-scale collection of Cassava full-length cDNAs （Collaboration with CIAT)

・Treatment for isolation of full-length cDNA (abiotic stress) : drought、 heat、PPD、heavy metal pollution（Al） ・EST: 20,000 full length cDNA (BMC Plant Biology 2007)

Treatment for isolation of full-length cDNA Biotic stress : Mealybug, Whitefly, Mite, Bacterial blight and Root rots

Treatment for isolation of full-length cDNA Biotic stress : Whitefly, Green mites, Mealybug, Hornworm, Bacterial blight Other condition : Pesticide, fungicide, auxin, drought, fertilization water

2. MECU72(tolerance to whitefly)

1. MTAI16(elite cultivar in east-Asia）

3. MPER417-003（M. peruviana）（tolerance to whitefly and mealybug ）

4. Huay Bong 60（high yield cultivar）

5. Hanatee（low yield cultivar）

Meaybug treatment

CMC40 MPER417-003

・high yield ・high starch ・high cyanide ・tolerance to CAD

・low yield ・low starch ・low cyanide ・sensitive to CAD

Sakurai et al. (2007) BMC Plant Biol. Fernando et al. (in preparation)

Identification of more than 30,000 genes from cassava.

Cultivars Number of clones

Number of tags Number of mapped

tags

KU50

Sanger

19,968 35,400 34,432

MECU72 29,952 21,364 19,879

MPER417-003 19,968 15,626 15,309

MECU72 Roche/454

- 154,397 104,379

MPER417-003 - 471,934 397,932

MPER417-003

Illumina

- 51,919,340 12,387,186

Hanatee - 13,519,852 6,381,632

Huay Bong 60 - 107,583,892 39,497,396

Number of genes from Phytozome v10.1：30,666

Number of genes from full-length cDNA resources ：27,153

Number of novel genes ：more than 4,000

9

Sakurai et al. (2007) BMC Plant Biol. Fernando et al. (in preparation)

Full-length sequences of about 7,000 KU50 FL-cDNAs (with Dr. Iuchi, RIKEN BRC)

Improving Genome Annotation (with CIAT)

Isolation of about 27,000 cassava Full-length cDNAs.

Development of Cassava Oligoarray (containing about 30,000 genes)

2010~: 30,000 gene Agilent oligoarray

developed

September: >15,000 gene Agilent oligoarray

developed

Agilent 20K oligoarray experiments (in progress)

Agilent 30K oligoarray experiments

were done

2009 2010 2011

2nd custom 30K oligoarray

Futures: •>30,000 gene proves on a chip • More biotic-related genes • Genes from wild species

10

Utsumi et al. (2012) DNA Res. Sojikul et al. (2015) Plant Mol. Biol. Utsumi et al. (revised)

Construction of cassava database based on FL-cDNA information (Cassava Online Archive, http://cassava.psc.riken.jp/)

（Sakurai et al. 2013 PLoS One）

11

Integration with outer database

Collection of FL-cDNA Seq information

Results

BLAST

Top page

Keyword

Genome Browser

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

Huay Bong 60 (HB60) Hanatee (HN) ・High yield

2006

・Resistant to Cassava anthracnose disease (CAD)

・High starch content ・High HCN content

・Low yield

・Sensitive to CAD

・Low starch content ・Low HCN content

Cassava Marker Breeding in Thailand -Development of F1 population (HB60 X HN)-

(Collaboration with Mahidol Univ.)

Linkage map analysis : Results

Fresh root yield Starch content Cyanogen content

Starch properties CAD resistance

Marker Breeding (Collaboration with Mahidol Univ.)

14

・Deep sequencing revealed 595 SNPs in genomic regions including candidate genes involved in CAD resistance and starch content. (in collaboration with Mahidol Univ.) ・Identification of 10,546 SNPs in 3,252 genes （123 SNPs on suar and starch metabolism pathway genes）

17

Identification of candidate genes of CAD tolerance by microarray analysis (Collaboration with Mahidol Univ.)

A

Kunkeaw et al. (2010) Australas Plant Pathol, 39:547-550

B

But resistance mechanism for CAD is still unknown…

HB60 KU50 HN

HN KU50 HB60

Venn diagram analysis of the genes with higher (A) and lower (B) expression in HB60 and HN.

18

Identification of the genes with higher expression in a CAD-tolerant cultivar (HB60) .

Utsumi et al. revised)

GO term of the genes with higher expression in HB60 compared with HN under non-treatment condition

Various immune systems are involved in CAD resistance in HB60.

GO term of the genes with higher expression in HB60 compared with HN at 72 h after CAD infection

Utsumi et al. revised

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

Cassava Breeding by Heavy-Ion Irradiation (non-GM) (Collaboration with AGI, RIKEN Nishina-Center )

Cassava Fruit

Heavy-ion irradiation (50〜150 Gy)

Screening

Collect the embryo Germination

on the media 21

Collect the seeds

About 1,000 heavy ion beam-irradiated cassava KU50 plants have been grown in AGI (Vietnam)

Pandurate leaflobe Bloom in Hanoi

5 leaf lobes 9 leaf lobes

Red petiole Green petiole

Purple apical leaf (parent) Dark green apical leaf Light green apical leaf High starch (30%)

Phenotype variations in about 1,000 ion beam-irradiated lines

Can analyze more than 30,000 gene expression

(3) Preparation of custom oligomicroarray

(1) Isolation and sequencing of about 27,000 Full-Length cDNAs

(2) Cassava database

(4) Marker Breeding

(6) Transgenic Cassava

(5) Heavy-Ion Irradiation Breeding

Development of Cassava Research Platform towards Molecular Breeding

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

Friable embryogenic callus(FEC)

Transgenic Plantlets

Agrobacterium infection

In Vitro mother plantlets

Axillary buds or small leaves

Somatic embryogenic callus

Transformed FEC

Cotyledon fragments

Transgenic shoot initials

Agrobacterium infection

Agrobacterium-mediated cassava transformation of friable embryogenic calli (FEC)

PCR and GFP selection

Control leaf

Regeneration from FEC

Somatic embryogenic callus

List of media for FEC production in cassava Genotype Successful genotype to FEC

prodaction Media for FEC production Sucrose concentration Auxin Reference

1. TMS60444 and M.COL. 1505 TMS60444 and M.COL. 1505 MS, ½MS, ,MS(-NH4), MS(-NH4NO3), SH, N6, NN, GD, WPM and B6 2% 12 mg/l picloram Taylor et al. 1996 (Nat. Biotechnol)

2. TMS60444 TMS60444 GD 2% 10 mg/l picloram Raemakers et al. 1996 (Mol. Breeding)

3. TMS60444 and MCOL22 TMS60444 and MCOL22 GD 2% 12 mg/l picloram Zhang et al. 2000 (Ph.D. thesis in ETH)

4. TMS60444, Adira 4, R60, R90, Thai5, M7, Mcol22, Adira 1, L11 and Gading

TMS60444, Adira 4, R60, R90, Thai5*1 and M7*1 GD 6% 6 mg/l picloram

6 mg/l NAA Raemakers et al. 2001 (Euphytica)

5. TMS60444 TMS60444 GD 2% 12 mg/l picloram Schreuder et al. 2001 (Euphytica)

6. Bujá Preta, Rosinha Bujá Preta, Rosinha GD 2% 12 mg/l picloram Ibrahim et al. 2008 (Afri J Biotechnol)

7. TMS60444 TMS60444 GD 2% 12 mg/l picloram Bull et al. 2009 (Nat Protoc.)

8. T200, AR9-18, MTAI16, CR25-4, CM523-7, BRA1183, MCOL2261 and SM707-17

- MS*2 2% 12 mg/l picloram Rossin et al. 2010 (S. Afr. J. Bot.)

9. TME 3, TME 7 and TME 14 TME 3, TME 7 and TME 14 GD 2% 12 mg/l picloram Zainuddin et al. 2012 (Plant Methods)

10. TMS60444 and T200 TMS60444 and T200 GD 2% 12 mg/l picloram Chetty et al. 2013 (N Biotechnol.)

11. Ebwanatereka, Serere and Kibandameno Ebwanatereka, Serere and Kibandameno

GD with different concentrations of tyrosine (125, 250 and 500 µM) 2% 50 µM picloram Nyaboga et al. 2013 (Frontiers in plant sci.)

12. Aladu, Ebwanateraka, 60444 Aladu, Ebwanateraka, 60444 GD with 500 µM tyrosine 2% 50 µM picloram Apio et al. 2015 (African J Biotechnol)

13. TME14 TME14 GD 2% 12 mg/l picloram Nyaboga et al. 2015 (Frontiers in plant sci.)

*1 Thai5 and M7 were classified as relatively difficult lines to produce FEC. The efficiency of FEC production is about 1%. *2 The production of somatic embryo from all genotypes was reported in this manuscript.

Media

TMS60444 KU50

Number of somatic

embryo

Number of colony of friable

embryogenic callus

Percentage (%)

Number of organized callus

Number of colony of friable

embryogenic callus after

cultured during 4 weeks

Percentage (%)

Murashige and Skoog medium (MS) 13 4 31 27 0 0

MS (×1/100 NO3) medium 12 0 0 25 0 0

MS (×1/100 NH4) medium 12 0 0 25 0 0

Media X exp1 15 15 100 24 0 0

exp2 70 51 73 - - -

exp3 56 29 52 - - -

MS (-Zn) medium 13 1 8 30 0 0

MS (-Br) medium 13 1 8 21 0 0

Gresshoff & Doy basal medium exp1 29 9 31 50 0 0

exp2 60 22 37 - - -

exp3 59 20 34 - - -

McCow'n woody plant medium 16 6 38 25 0 0

Chu N6 medium 14 4 29 28 0 0

Gamborg B5 medium 15 5 33 25 0 0

Optimization of media for FEC production in cassava

Ha, Utsumi et al. in preparation

Workflow of cassava transformation

Ha, Utsumi et al. in preparation

Preparation of in vitro plantlets from Vietnamese local cultivars

Multiplication of in vitro cassava plant

Candidate cassava cultivars 1 KM140 KM98-1 x KM36 2 KM325 SC5 x SC5 3 NA1 MIF 4 KM397 KM108-9-1 x KM219 5 HL2004-28 (GM444-2 x GM444-2) x XVP 6 TaXanhDB Unknown 7 Rayong11 R5 x OMR29-20-118 8 KM987 SM1717 x CM321-188 [AGI, VNM] 9 TCĐB SC5 x SC5 [AGI, VNM]

10 TĐĐB Unknown [AGI, VNM] 11 KU50 Rayong1 x Rayong90 12 TMS60444 Unknown [IITA, NGA]

Selection and screening of Asian cultivars based on efficiency of FEC production

KU50 callus on-going step…

Qualitative and Quantitative Improvement of Cassava Biomass using Agrobacterium-mediated Cassava Transformation System

8 constructs

→Food security and stable biomass supply →Increase of non-food biomass →Industrial material

Quantity Quality

9 constructs

Improvement of photosynthesis Increased tuberous roots Early bulking Early flowering Stress tolerance

32

What is Fructose 1,6-bisphosphate Aldolase (FBA)?

①Fructose 1,6-bisphosphate ⇄ Dihydroxyacetone phosphate ＋Glyceraldehyde 3- phosphate

②Sedoheptulose 1,7-bisphosphate ⇄ Dihydroxyacetone phosphate + erythrose 4-phosphate

Viewpoint ‐ Bypass of the pathway

between Calvin Cycle and Sucrose transport.

‐ One of the key enzyme of photosynthesis

‐ AtFBA3 is localized in Plastid and Cytoplasm

The Plant Journal(2010) 61,1067‐1091 Mark Stitt et al. Arabidopsis and Primary photosynthetic metabolism – more than the icing on the cake.

①

② ①

Reaction

Aldolase is a candidate gene for improving the photosynthetic carbon fixation

Photosynthesis Research 75: 1-10 2003 Christine A. Raines The Calvin cycle revisited

Aldolase is candidate of engineering to improve photosynthetic carbon fixation.

Ald

olas

e ac

tivity

(μ u

nit/μ

g/m

in)

AtFBA3-ox cassava plants has 2.0-5.9-fold higher aldolase activities

than WT.

WT AtFBA3-ox C8

AtFBA3-ox E6

AtFBA3-ox E3

×10

3 Co

py n

umbe

r/ng

tot

al R

NA

Gene expression of AtFBA3 and Aldolase Activity in Leaves

AtFBA3-ox cassava plants has higher gene expression than WT.

Sample is grown in Green house (sun light, 28℃) and collected from 2nd to 4th leaves from top of the stem at 10:00‐12:00 . Mean and error bar indicate the average value and standard deviation with each triplicate experiments using three biological replicates

WT AtFBA3-ox C8

AtFBA3-ox E6

AtFBA3-ox E3

Gene expression Aldolase activity (Takei et al. in prep.)

Red : p<0.05 Blue : p<0.1 by T-test

The tuber yield of FBA OX lines is increased under 400 ppm CO2 condition.

(Takei et al. in prep.)

ＲR RIKEN CSRS has established integrated Omic Analysis Platform. Molecular Analysis to elucidate the mechanism is in progress.

Metabolome

Hormonome

Transcriptome and Epigenome

Agilent Microarray Scanner

Ion Proton

SOLiD5500

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

39

Table 1. The sampling plan during developing stage.

4 weeks 6 weeks 8 weeks 10 weeks 12 weeks

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total

Fibrous* Parenchyma ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ 15

Cortex

Intermediate Parenchyma

- - - - - - ○ ○ ○ ○ ○ ○ ○ ○ ○ 9

Cortex ○ ○ ○ ○ ○ ○ ○ ○ ○ 9

Storage Parenchyma

- - - - - - ○ ○ ○ ○ ○ ○ ○ ○ ○ 9

Cortex ○ ○ ○ ○ ○ ○ ○ ○ ○ 9

Top of leaves ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ 15

Total 66

: Metabolome, Transcriptome : Hormonome

Fibrous root； Diameter <2mm Intermediate root；Diameter 2-5mm Storage root; Diameter > 5mm

Cortex Parenchyma

Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during Tuberous Root Development (with Mahidol Univ.)

(unpublished, Utsumi et al.) 39

Starch content in root tuber samples

Starch biosynthesis has started already in fibrous root from F8 and F12

Star

ch c

onte

nt

(mg/

mg

F.W

.)

(Utsumi et al. in prep.)

Unknown signal

ABA ↑ GA − IAA − IAAsp − iP ↑ tZ ↑ cZ ↑ Sucrose↑ G6P↑ F6P↑ AMP−

ABA ↓ GA ↑ IAA − IAAsp ↓ iP ↑ tZ ↑ cZ ↑ Sucrose↑

G6P↑ F6P↑ AMP↑

Sucrose↑ G6P↑ F6P↑ AMP−

Parencyma (0.02%) (3-5%) (10-18%)

1. Post-embryonic development 2. Response to stimulus 3. Phosphorylation 4. Response to chemical stimulus 5. Reproductive process in a multicellular organism

1. Response to stimulus 2. Post-embryonic development 3. Glucan biosynthetic process 4. Cellular glucan metabolic process 5. Glucan metabolic process

Metabolites

Plant hormones

Transcriptome

Root state (Starch content)

Fibrous Storage Pre-storage

Integrated Omic Analysis during Tuberous Root Development in Cassava (Utsumi et al. in prep.)

Intermediate

Stimulation to plant

hormone and sugar Parencyma

(8-15%)

ABA↓ GA − IAA − IAAsp ↓ iP ↑ tZ ↑ cZ ↓

Cortex (5-9%) Cortex (5-9%)

ABA ↓ GA − IAA − IAAsp ↓ iP ↑ tZ ↑ cZ ↓

Sucrose↑ G6P↑ F6P↑ AMP↑

Sucrose↑ G6P↑ F6P↑ AMP−

(unpublished, Utsumi and Seki et al.) 41

1. Post-embryonic development 2. Photosynthesis 3. Cellular glucan metabolic process 4. Glucan metabolic process 5. Response to stimulus

ABA ↓ GA ↑ IAA − IAAsp ↓ iP ↑ tZ ↑ cZ ↑

1. Response to stimulus 2. Post-embryonic development 3. Glucan biosynthetic process 4. Cellular glucan metabolic process 5. Glucan metabolic process

1. Post-embryonic development 2. Photosynthesis 3. Cellular glucan metabolic process 4. Glucan metabolic process 5. Response to stimulus

G1P

L-kestose

Sucrose F6P

2-1,3-aminopiperidin l-arginine l-ornitine

A

B C D

F

E

G H I J K

L

G6P l-serine 4-aminobutyric acid beta-alanine Maltotriose UMP AMP l-aspartic acid l-glutamic acid Glycolic acid CMP

trans-zeatin

Heatmap of 154 metabolites increasing tuberization process

Oxalic acid l-threonic acid l-cysteine Stigmasterol 5-aminolevulinic acid 3-ureidopropionic acid Threonic acid-1,4-lactone d-gluconic acid

F8

I8_C

I8

_P

M8_

C

M8_

P F1

2 I1

2_C

I1

2_P

M12

_C

M12

_P

d-maltose

Metabolisms involved in sugars and nucleotide increased in all tissues in comparison to fibrous roots at 4 weeks after cutting.

(Utsumi et al. in prep.)

1. Development of an integrative, functional- genomics platform for cassava 2. Transcriptome Analysis (CAD etc.) 3. Heavy-Ion Beam Mutagenesis 4. Transformation 5. Integrated Omic Analysis (Transcriptome, Metabolome and Hormonome) during tuberous root development 6. Epigenetic Regulator (towards development of stress-tolerant cassava)

Contents

44 Area with high-salinity stress-affected damage （Sparks 1995）

High-salinity stress problems have been reported in North East of Thailand and Indonesia.

Area with drought stress-affected damage （Tottori Univ., Arid Region Research Center)

Areas with High-salinity or drought stress-affected damage occur in the world.

Asia = 75.2 Mt/year

Africa = 121.0 Mt/year

South America = 31.7 Mt/year

Indonesia = 23.9 Mt/year

Thailand = 22 Mt/year

China= 4.7 Mt/year

Brazil = 24.5 Mt/year

Colombia = 2.4 Mt/year

Paraguay= 2.6 Mt/year Democratic Republic of Congo = 15.0 Mt/year

Ghana = 13.5 Mt/year

FAO STAT2010 (http://faostat.fao.org/)

Vietnam = 8.5 Mt/year

Nigeria = 37.5 Mt/year

India = 10.0 Mt/year

Cassava Production

We need to develop the cassava plants tolerant to the abiotic stresses!

45

Salinity-accumulating Soil : 28,970 km2 Area with salt damage : 2,830 km2 鉱床岩塩 : 約 18 兆t 鉱床カリウム : 約 25 億t 岩塩層厚 : 約 100 m 〜 150 m 岩塩層深度 : 地下約 150 m 〜 300 m

(参考) 地盤工学会「土と基礎」,2007.3 PIPATPONGSA THIRAPONG (東京工業大学助教授) 飯 塚 敦 (神戸大学教授) 河 井 克 之 (神戸大学助手)

Area with high-salinity stress-affected damage in Thailand

North-east of Thailand

Nakhon Ratchasima

Sakon nakhon

Bangkok

46

(http://www.ne.jp/asahi/vietnam/agriculture/environment/region.htm#31)

World Bank Report (Dasgupta et al. 2007):Vietnam is one of the countries that are affected most seriously by climate changes、海面が 1 m上昇すると、

人口のおよそ 10%が影響を 受けるとされている。ベトナムの天然資源環境省は、2009 年、「ベトナムにおける気候変動と海面上昇に関するシナリオ」(Bo Tai Nguyen va Moi Truong 2009)を作成し、各省がこのシナリオにもと

づき、堤防の建設など、具体的な対策を講じはじめている。2012 年に最新のシナリオが公表されたが、これによると、気候変動の影響を 3 段階に分け、影響力が「中」 であった場合、It is predicted that if the sea level will rise 1 m, Mekong Delta area will lose 39% of the Area and if the sea level will rise 2 m,more than 92% of the area will be lost (Nguoi Lao Dong 2012).

Drought stress problem occurs.

Areas with drought- and/or high-salinity stress- affected damage (might occur in the future) in Vietnam

Similar situation occurs in India.

Plant CellSignal Perception andSignal TransductionABA

Stress ResponseSalt

ABAindependent

Regulatory ProteinFunctional Protein

Non-coding RNAs RNA regulation

Epigenetic regulation

and Tolerance

Drought ColdHeat

Drought ColdHighHeatsalt

Small peptides

+

+

- - Repress

Alteration of histone modifications

Active-mark

Repressive-mark

HAT

HDAC

HMT

H3K27m3

H3K9ac H3K4m3

HAT: Histone acetyl transferase HDAC: Histone deacetylase HMT: Histone methyl transferase

Transcription

Relaxing

Packing

HDM: Histone demethylase

HDM

HMT Histone modification enzymes

Alteration of histone modifications is deeply and universally correlated with the gene regulations in eukaryotes.

Acetylation

HDAC deacetylation

Ac

Ac Ac

Ac Ac

Ac

Ac Ac Ac Ac

Ac

Ac Ac Ac

Ac

Activation of the genes involved in environmental stress tolerance

遺伝子発現

OFF

Gene Expression

ON

HDAC deacetylation

HDAC inhibitor

Histone Acetylation is involved in activation of gene expression.

Ky-2

Ac Ac

HDAC

Ky-2, an HDAC inhibitor, enhances high-salinity stress tolerance in Arabidopsis.

× SOS1

Transcription ON

SOS1

Na+ Na+

SOS1

Na+

SOS1

SOS1 Na+

Na+

Na+

Na+

Sako et al. (2016) Plant Cell Physiol.

4℃ 22℃ (16h-L/8h-D)9 day after

germination (DAG)0 4 5

5 HDAC inhibitors enhancing high-salinity stress tolerance

0

10

20

30

40

50

60

70

80

90

100

DMSO JNJ MC1568 MS275 SAHA TSA

Survival rate (%)

(n=15, 3 biological replicates)LBH5891 2 3 4 5

Ueda et al. (in preparation)

HDAC inhibitor 1-treated plants + NaCl HDAC inhibitor 1-

treated plants + NaCl

HDAC inhibitor 1-treated plants

Control

HDAC inhibitor 1-treated plants

Control

Shoot (mg) Shoot (mg)

Root

(mg)

Root

(mg)

HDAC inhibitor 1 enhances high-salinity stress tolerance in cassava.

Transcriptome analysis identified many candidate genes of HDAC inhibitor-mediated high-salinity stress tolerance

Genes with higher expression in roots of HDAC Inhibitor 1- treated plants (not high-salinity stress-inducible)

Genes with lower expression in roots of HDAC Inhibitor 1- treated plants (high-salinity stress-inducible)

No

Inhi

bito

r(Co

ntro

l)

No

Inhi

bito

r + 2

h N

aCl

N

o In

hibi

tor +

24

h N

aCl

24 h

Inhi

bito

r

24 h

Inhi

bito

r + 2

h N

aCl

24

h In

hibi

tor +

24

h N

aCl

ＲR RIKEN CSRS has established integrated Omic Analysis Platform. Molecular Analysis to elucidate the mechanism is in progress.

Metabolome

Hormonome

Transcriptome and Epigenome

Agilent Microarray Scanner

Ion Proton

SOLiD5500

Japan Kyushu Univ. (PI: Dr. Takasu) RIKEN Tokyo Univ. Agr. Univ. Tokyo Nagoya Univ.

Vietnam AGI

Main Res. Inst. Sub Res. Inst. Tested Cassava Field

Rayong Field Crop Res. Cent. Thailand

Hung Loc Agr. Cent.

Univ. of Battambang (UBB), Cambodia

55

JST-JICA SATREPS project (2016-2021) : Development and dissemination of sustainable production system based on invasive pest management of cassava in Vietnam, Cambodia and Thailand

56 56 Ajinomoto Co.

Useful biomass, such as amino acids

Fertilizers, Nutrient

materials

Wastes （Rich in nutrients, such as nitrogen）

・Identification of useful genes. ・Development of useful cassava by transformation, chemicals and heavy ion beam irradiation..

Develop to field

Use of stems for propagation

Cassava starch

Cassava pulp

Cellulose Film

Spray on the crops

Harvest of tuber roots

Foods Sweetener Industrial raw materials Materials for food processing

Fermentation

Decompose into glucose

Environmentally friendly reutilization

Green Innovation using Cassava

・Cooperation with Japanese Companies ・Sustainable Human Life through Stable

Supply of Sugars with Low Price for Foods and Biomaterials

Collaborators RIKEN CSRS Yoshinori Utsumi (SP06-22) Chikako Utsumi Yoshio Takei (S09-05) Vu The Ha Onsaya Patanun Minoru Ueda Kaori Sako Minoru Yoshida Hitoshi Sakakibara Kazuki Saito Atsushi Fukushima Miyako Kusano Akihiro Matsui Maho Tanaka Junko Ishida Jong-Myong Kim Tetsuya Sakurai Satoshi Iuchi Masatomo Kobayashi Ken Shirasu Kazuo Shinozaki

Thailand, Mahidol University Jarunya Narangajavana Kanokporn Triwitayakorn Punchapat Sojikul Supajit Sraphet Sukhuman Whankew DOA Opas Boonseng Amonrat Kitjaideaw King Mongkut University of Technology at Thonburi Treenut Saithong Saowalak Kalapanulak

Research Institute for Biological Sciences, Okayama (RIBS) Ken'ichi Ogawa Yoshihiro Narusaka Mari Narusaka

CIAT Manabu Ishitani Joe Tohme Yokohama City University Hiroyuki Tsuji Nara Institute of Science and Technology (NAIST) Ko Shimamoto Masaaki Umeda RIKEN Nishina Center Tomoko Abe Tomonari Hirano

Vietnam, AGI Ham Huy Le Dong Van Nguyen Vu Anh Nguyen

Thank you for your attention

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