Jim Giovannoni, USDA-ARS Robert W. Holley Center, Ithaca, NY james.giovannoni@ars.usda.gov

Insights into the genetic control of tomato fruit ripening….......and how they might be leveraged for grape improvement???

Dry Fruit

Fleshy Fruit

UN Dept. of Social and Economic Affairs

Food security is a function ofproduction, quality and decay

Ripening is the coordination of numerous biochemical processes and their underlying pathways

Starch

Sugar

Pigment accumulation

Chlorophyll degradation

AromaVolatiles

Texture

PathogenSusceptibility

Protectivecompounds

Ripening is the coordination of numerous biochemical processes and their underlying pathways

Starch

Sugar

Pigment accumulation

Chlorophyll degradation

AromaVolatiles

Texture

PathogenSusceptibility

Protectivecompounds

Tomato as a Model System

• genetically well characterized• self-pollinating and easily out-crossed• easily propagated and maintained• large selection of compatible germplasm• easily transformed• numerous ripening mutants• qualitatively and quantitatively dramatic ripening phenotype• high marker density genetic map• proliferation of genomics tools• genome sequence• Methylome dynamics

Tomato and its relatives……

…..as a reference genome for the Solanaceae

System 1 C2H4 System 2 C2H4

Transition

C=CH

H

H

H

(-) (+)

Ethylene

IM LIM MG BR RR

Seed development is not required for maturation/ripening

TGRC, UC Davis

Parthenocarpic fruit ripen but……

• tomato pat mutants ripen more slowly• seeds deploy abscisic acid (ABA) as a regulator of dormancy• ABA increases in tomato and other fruit prior to ethylene

- exogenous ABA promotes ripening- tomato ABA mutants ripen more slowly

• there may be an evolutionary advantage for individual fruit to ripen even if pollination was not successful

ripening-inhibitor (rin)

Prof. Henry Munger1916 - 2010

First ripening transcription factor cloned was RIN-MADS

Randy Gardner, NC State

rin/rin Rin/rin

Hybrid Rin/rin tomatoes are the basis of much extended shelf-life fresh market tomato production, are seeing some use in processing varieties and are the basis of many concerns regarding fresh tomato quality and flavor.

- they simply are not fully ripe.

The rin locus encodes a SEPELATA clade MADS-box proteinRin/Rinrin/rin

RIN-MADS MC-MADS(SEP) (AP1)

Vrebalov et al., 2002 Science

(transcription factor)

Tomato MADS-BOX genes provide insight into inflorescence, flower and fleshy fruit development

MADS1

FYFL

J1

RIN

J?

TAGL1FF MC

TAG1

TM29

SEPFUL1/2

Monocots

Dicots

RIN-like MADS-box genesare widely conserved.

Constructs for MaMADS1 and MaMADS2 repression

A.

Control AS-MaMADS2 RNAi-MaMADS2

B.

C.

D.

Control RNAi-MaMADS1

Repression of MaMADS1 or MaMADS2 inhibits banana ripening

Plant Physiology, 2016

RINCNR

CNR is necessary for RINpromoter binding

FUL1/2 and TAGL1 interact with RIN

SBP/SPL

FUL1 FUL2

TAGL1

Martel et al., Plant Phys. 2011

Manning et al. (2006) Nat Genetics

CNR is an epi-allele

TAGL1-1

TAGL1-11

TAGL1-12

Ac +/+

28DPA BB+7d BB+20d cut fruit at BB+7d

The tomato TAGL1 (SHP) MADS-box gene is necessary for fleshy fruit expansion and ripening

Vrebalov et al., 2009, Plant Cell

NOL3-1

NOL3-2

NOL3-3

WT (AC)

NOR (AC)

At least 3 NACs are expressed in kiwi fruit ripeningNOL3 complementation line in NOR background

nor/nor rin/rin

NOR RIN

CNR

TAGL1

C2H4

Multiple ripening regulators upstream of ethylene

FUL1 FUL2NAC3

Ripening

ARF2

U/U u/u

Tomato production is highly dependent upon the uniform mutation

U/U

U is a Golden-like 2 (GLK) transcription factor35S::U/GLK2 in u/u

35S::UNIFORM in u/u

Control u/u

35S::UNIFORM in u/u

Control u/u

Powell and Nyugen et al., 2012, ScienceNguyen et al., 2014, Plant CellCuong Nguyen

ripening inhibitor(rin)

Green-ripe(Gr)

non-ripening (nor)

Never-ripe(Nr)

Never-ripe 2(Nr-2)

Colorless non-ripening(Cnr)

high-pigment 1 (hp-1)

Transcription Factors

Signal Transduction Components

lutescent 2 ( l2)

Plastid Development

green-flesh ( gf)

Non-ripeningethylene

insensitive(nei)

Uniform ripening(u)

RTE1 ERS1

EIN2

DDB1

Much ripening molecular biology focuses on the pericarp, excluding seeds and locule.

NOR::GUS Reporter expression reveals early locule expression

NOR GUS TER

System 1 C2H4 System 2 C2H4

Transition

C=CH

H

H

H

(-) (+)

Ethylene

IM LIM MG BR RR

RNA-seq transcriptome profiling of locule and pericarp tissue

• Most prior studies on fruit development focus on the pericarp tissue.

• The locular tissue differentiates from the placenta and gradually changes to jelly tissue during fruit development and ripening.

• Locular gelling is an early sign of ripening preceding climacteric respiration and ethylene induction.

Ripening control gene expressionin pericarp versus locule

Mat

urat

ion

(rip

enin

g)

T

M

B

IM MG

BR

(B)(A)

Top

Middle

Bottom

seed matures,locule liquefies

System 1 (-)

System 2 (+)

ethylene increase,additional TFs,

ripening phenotypes

RIPENINGCASCADE

DML2CNRRINNOR

Zhangjum FeiCuong Nguyen

5-azacytidine treatment of immature (17 dpa) tomato fruit

Zhong & Fei et al., 2013

Ripening can be achieved through genome demethylation

RIN-MADS bind near DMRs

nor locus

leafIM

MGBr

Ripencnr/cnr

rin/rin

+/+

ripening mutants

RIN ChIP-Seq

http://ted.bti.cornell.edu/epigenome/

Ripening of transgenic DNA demethylase (DML) RNAi fruits is delayed

Liu et al., PNAS, 2015Philippe Gallusci, INRA

Zhangjum Fei

Lukas Mueller

Joss Rose

Carmen Catalá

tea.solgenomics.net

Coffee staging for seed and fruit transcriptome analysis

Dynamic TAGL1 expression in maturing pericarp tissues

TAGL1 repression

Tissue expression of early ripening regulator NOR

NOR

RIN

NOR RIN GRAS

GRASScarecrow-like transcription factor

rin 42DPA/WT 42DPA=0.099Cnr 42DPA/WT 42DPA=0.007

• Ripening induced• RIN regulated• Ethylene responsive• Accumulates first in locule

Ethylene production

0

2

4

6

8

10

12

BB B3 B7 B15

ACGRAS13GRAS20

nl.g

-1h-

1

GRAS mRNA reduced ripening delay is maintained in T1 generation

0

1

2

3

1 10 12 2 3 4 9 Wt

GRAS #20

32.3±

0.829.2

±1.030.2

±0.730.8

±0.631.5

±0.830.3

±1.030.2

±0.626.5

±0.4

7 5 12 4 2 4 14 21

Days from

1cm to breaker

fruits

T2 B+60D 58d after post harvest3/16 phto

AC GRAS 20

AC GRAS 13

T2 B+35D 33d after post harvest3/16 photo

Longer self life

Considerable genetic diversity among wild tomato species………

……but less than 5% is represented in cultivated tomatoes

Eshed and Zamir, 1995

Genotyping of USDA-ARS grape collection (Davis, CA and Geneva, NY)

The range of carotenoid levels within the S. habrochaites IL population and loci influencingthem provides opportunities for discovery of numerous carotenoid

synthesis and regulatory loci.

eQTL mapping using a tomato introgression population

• The ripening transition is regulated by a cascade of genetic events initiating with transcription factors and epigenome changes.

• The MADS-box family is a rich source of fruit development and maturation regulators and evolution has tailored them for specific fruit development and morphology contexts.

• Ripening events do not occur uniformly throughout fruit tissues and more careful examination of tissue-specific responses is needed to fully understand ripening phenomena.

• Genetic diversity provides opportunities to explore the molecular basisofripening control and fruit quality and presents natural allelic variants that can be tested and deployed in practical breeding.

• Primary genetic regulation influencing maturation, quality and self-life characteristics in tomato is likely conserved across diverse species….including grape.

• Not only tomato genes, but tomato infrastructure (e.g. TEA) can be leveraged for additional fruit species.....including grape.

SUMMARY

The people who do the work…………..

Funding …… NSF, ERA-CAPS, USDA-NRI/NIFA, BARD, USDA-ARS, AgroFresh

Catherine Martel Ryan Mcquinn Julia Vrebalov Jemin Lee

Zhangjum Fei

Cuong Nyugen

Rob Alba

Silin Zhomg

Miyoung Chung

Yimin Xu

Graham Seymour, U of Nottingham

Kunsong Chen, Zhejiang U.., ChinaXueren Yin, Zhejiang U., China

Philippe Gallusci, INRA Bordeaux

Cathie Martin, John Innes Center, UK

Mondher Bouzayen, INRA Toulouse

Betsy Ampofo

Steven Tanksley, Cornell

Alisdair Fernie, Max Plank, Golm

Haya Friedman, ARO, IsraelNurit Katzir, ARO IsraelKobi Tadmore, ARO Israel

Tomato Genome Consortium

Lukas Mueller

Ari Feder Itay Gonda Yanna Shi Anquan WangNigel Gapper

Joss Rose Harry Klee