Tomato TILLING mutants in Expansin1 gene show alteration in softening and cell wall metabolism during ripening

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Tomato TILLING mutants in Expansin1 gene show alteration in softening and cell wall metabolism during

ripening

S. Minoia, Jacqueline Vigouroux, Bernard Quemener, G. Mosca, G. Sozio, A.

Petrozza, Marie Francoise Devaux, Florence Piron, I. F. Cellin, Abdelhafid Bendahmane, et al.

To cite this version:

S. Minoia, Jacqueline Vigouroux, Bernard Quemener, G. Mosca, G. Sozio, et al.. Tomato TILLING mutants in Expansin1 gene show alteration in softening and cell wall metabolism during ripening. 6.

Solanaceae Genome Workshop (SOL), Nov 2009, New Dehli, India. �hal-02821447�

Tomato TILLING mutants in Expansin1 gene Tomato TILLING mutants in Expansin1 gene

show alteration in softening and cell wall show alteration in softening and cell wall

metabolism during ripening metabolism during ripening

S. Minoia

, J. Vigouroux

, B. Quéméner

, G. Mosca

, G. Sozio

, A. Petrozza

, M.F. Devaux

, F. Piron

, F. Cellini

, A.

Bendahmane

, F. Carriero

, M. Lahaye

(1)

Metapontum Agrobios, S.S. Jonica 106, km 448.2, 75010 Metaponto (MT) Italy;

BIA/INRA, Rue de la Géraudière, F-44300 Nantes, France;

URGV/INRA, 2 rue Gaston Crémieux, 91057 Evry cedex France;

Present adress: ENEA, Casaccia Research Center, PO Box 2400 Roma 00100AD, Italy.

1) Brummell D.A., Harpster M.H., Civello P.M., Palys J.M., Bennett A.B. and Dunsmuir P. (1999), Plant Cell 11:2203-2216

2) McQueen-Mason S.J., Cosgrove D.J. (1995), Plant Physiol. 107: 87-100.

3) Menda N., Semel Y., Peled D., Eshed Y. and Zamir D. (2004), Plant J. 38: 861-872

4) Carriero F., Petrozza A., Sozio G., Cellini F. (2004), XLVIII Annual Congress SIGA Lecce 15/18 September 5) Minoia S., Petrozza A., D’Onofrio O., Piron F., Mosca G., Sozio G., Cellini F., Bendahmane A. and Carriero F.

(2009) Genome Biol (submitted)

6) Moise A., Quéméner B., Bouchet B., Marty I., Guillon F., Lahaye M. (2009) J. Exp. Bot. (submitted) 7) Fry S.C., York W..S, Albersheim P., Darvill A., Hayashi T., Joseleau J..P, Kato Y., Perez Lorences E., MacLachlan G.A., McNeil M., Mort A.J., Reid J.S.G., Seitz H..U, Selvendran R..R, Voragen A..G.J, White A.R.

(1993), Physiol. Plant. 89: 1-3

8) Triques K., Sturbois B., Gallais S., Dalmais M., Chauvin S., Clepet C., Auburg S., Rameau C., Caboche M. and Bendahmane A. (2007), Plant J. 51: 1116-1125

9) Devaux M.F., Bouchet B.,Legland D.,Guillon F., Lahaye M. (2008) Postharvest Biol. Technol. 47:199-209 10) Quéméner B., Bertrand D., Marty I., Causse M. and Lahaye M. (2007), J. Chromatog. A 1141: 41-49 11) Levigne S., Thomas M., Ralet M.C., Quéméner B. and Thibault J.F. (2002), Food Hydrocol. 16: 547-550

The 6

The 6

Solanaceae Solanaceae Genome Genome Workshop Workshop – – 08-13 08-13 November November, 2009, New Delhi , 2009, New Delhi

MATERIAL and METHODS

MATERIAL and METHODS ^{ReferencesReferences}

Plant material: Tomato M3 plants, including wild types (M82 and Red Setter) and genotypes homozygous for both the presence (+/+) and the absence (-/-) of the discovered mutations, were grown in greenhouse conditions according to standard tomato agronomic practice.

TILLING molecular screening: PCR amplification was carried out using labelled primers (IRDye 700 and IRDye 800 dye). Mutation detection was performed with the mismatch specific endonuclease ENDO1 (8) and the LiCor 4300 DNA analyzer. After discovery, mutations were validated by sequence analysis.

Fruit firmness: Penetrometric firmness was measured on whole tomato without peel by using a pressure tester (Forge Gauge, Lutron FG-5000-A) fitted with a cylindrical plunger of 6 mm in diameter. Tomato samples were collected at the breaker stage and analyzed in post-harvest conditions at fixed ripening stages.

Pericarp tissue histology: Cell size distribution in pericarp sections of the different genotypes was established according to (9).

Compression analysis: Compression was measured on pericarp tissue cylinders of 0.96 cm diameter taken at the equator. Compression test was realized at 20 mm/min using an Adamel Lhomargi DY34B (MTS) mechanical testing machine equipped with a 1 kN sensor.

Cell wall polysaccharides chemical composition: Cell walls from pericarp tissue of 5 to 6 fruits per genotype were prepared as alcohol insoluble material. Neutral sugars were quantified by gas-liquid chromatography and uronic acid by colorimetry after sulfuric acid degradation (10). Neutral sugar composition was corrected for residual starch determined by amylolysis and HPLC. The degree of pectin-esterification by methanol (DM) and weight % of acetic acid ester (AA) was measured by HPLC after alkaline hydrolysis (11). Glucanase degradation of cell wall preparations was done as described (10). Oligosaccharides were identified by MALDI-TOF MS on M@ldi LR (Waters) spectrometer using DHB/6ATT matrix.

Data treatment and statistic (ANOVA, pairwise t-test) were performed using R software or MATLAB (histology). AcknowledgementsAcknowledgements

We identified five new SlExp1 allelic variants by TILLING screening of two mutant collections generated in the M82 (3) and Red Setter (4,5) genetic background (Figure 1). Two of the five mutant lines bear mutations within the gene region coding for the active site of the protein: Q213* in M82 and W211S in Red Setter. The nucleotide substitutions led to a nonsense and a missense mutation that affected more strongly the protein function than the three other point mutations mutants (Figure 2).

Mechanical, histological and biochemical analyses were performed on the pericarp tissue of the two nonsense and missense Exp1 mutant lines at different ripening stages. No significant difference was observed in the histology (cell size distributions) between mutant and wild type M82 or Red Setter. Significant differences were observed in the pericarp tissue module of deformation during compression in Q213* mutant lines with respect to the control line (Table 1).

RESULTS and DISCUSSION RESULTS and DISCUSSION

Tomato fruit ripening involves the expression of several types of enzymes and proteins targeted at the cell wall disassembly. Expansin1 gene (SlExp1) codes for the major expansin protein involved in fruit softening (1). This protein controls noncovalent interactions at the hemicellulose- cellulose interface and regulates cell wall polysaccharides metabolism (2). To date, no non-GMO tomato genotypes modulated on expansin are available to realize controlled texture hybrids.

INTRODUCTION INTRODUCTION

Figure 2.

Figure 2. Firmness analysis on two TILLINGFirmness analysis on two TILLING mutants.

mutants. Firmness was calculated from homozygous +/+ (striped bars), homozygous -/- (white bars) and from respective wild type (M82 or Red Setter) (black bars). Tomato samples were collected at the breaker (B0) stage and analyzed in post-harvest conditions at fixed ripening stages: breaker and zero (B0), five (B+5), ten (B+10), fifteen (B+15) and twenty-five (B+25) days after breaker. Bars represents standard errors, and asterisks indicate that homozygous +/+ are significantly different (P<0.05) from homozygous and wild type. (A) Q213*; (B) W211S.

This study is financially supported by EU-SOL fundingFOOD-CT-2006-016214 Figure 1.

Figure 1. SlExp1SlExp1 gene. gene. Schematic structure of SlExp1 gene. The genomic SlExp1 is 1412 bp long and contains 2 exons (filled boxes) and a introns (black line). Numbers indicate the size of introns and exons in bp. Distribution of identified TILLING mutation is reported: in black and red are indicated missense and non-sense mutation, respectively.

U A N S Total A A D M n

M W G21.4±1. 7 55.8±8. 9 77.2±8. 6 1.8±0. 1 44.8±5. 9 6 M 1 G 18.1±2. 9 56.5±8. 2 74.6±6. 2 1.6±0. 1 41.8±10. 7 5 M W B 22.3±2. 1 48.7±3. 3 71.0±4. 9 1.8±0. 1 47.3±8. 1 5 M 1 B 23.0±1. 8 57.4±5. 6 80.4±5. 4 1.7±0. 2 50.7±9. 6 5 MWP 23.3±0. 9 45.3±6. 1 68.6±6. 8 1.7±0. 1 48.0±5. 3 5 M1P 22.7±1. 4 43.4±3. 3 66.0±3. 6 1.7±0. 1 50.6±4. 4 5 M W R 24.5±0. 9 48.8±10. 2 73.3±11. 11.6±0. 2 46.9±5. 1 5 M 1 R 24.2±1. 4 50.8±3. 5 75.0±3. 1 1.6±0. 1 49.7±8. 6 5 MWS 24.6±1. 0 43.3±3. 9 67.8±4. 7 1.5±0. 1 46.4±2. 0 5 M1S 24.9±1. 1 45.9±3. 3 70.8±3. 3 1.5±0. 1 40.0±2. 4 5 R 0 G 20.0±1. 5 45.9±8. 9 65.9±8. 5 1.7±0. 1 50.9±11. 7 6 R 1 G 18.3±0. 9 54.1±7. 7 72.3±6. 9 1.6±0. 1 40.1±12. 7 6 R 0 B 23.2±1. 3 53.6±5. 7 76.7±6. 6 1.7±0. 1 41.5±5. 9 5 R 1 B 23.5±1. 2 55.2±6. 1 78.7±6. 4 1.7±0. 1 47.2±3. 2 6 R 0 R 24.9±1. 7 45.0±4. 0 69.9±5. 0 1.6±0. 1 40.0±2. 6 5 R 1 R 23.6±1. 5 46.4±3. 1 70.1±4. 5 1.7±0. 1 49.2±3. 6 5 Table 2.

Table 2. Cell wall polysaccharide composition.Cell wall polysaccharide composition. Uronic acids (UA), total neutral sugars (NS) and total sugars (Total), acetic acid content (AA) (± standard deviation) on the weight basis of dry alcohol insoluble material and degree of methyl esterification of pectin (DM) in cell walls recovered from pericarp tissue of M82 (M) and Red Setter (R) wild type (W), control (0, -/-) and mutant (1, +/+) at the green (G), breaker (B), pink (P), red (R) and overripe (S) stages. P. value:

results from a pairwise-t-test between data from sild type (M) or control (0) and mutant (1); in bold significantly different results (p<0.05); n=numbers of fruits.

0 2 4 6 8 10 12

B0 B+5 B+10 B+15 B+25

*

* B 0

2 4 6 8 10 12

B0 B+5 B+10 B+15 B+25

*

*

* *

*

A

*

B0 B+5 B+10 B+15 B+25

Firmness (kg/cm2)

Ripening stage +/+

-/- wt

0 2 4 6 8 10 12

B0 B+5 B+10 B+15 B+25

*

* B 0

2 4 6 8 10 12

B0 B+5 B+10 B+15 B+25

*

*

* *

*

A

*

B0 B+5 B+10 B+15 B+25

Firmness (kg/cm2)

Ripening stage +/+

-/- wt +/+

-/- 309 wt

477 27

ATG

342 257

L145F P154L V197IW211S S238FTAA

Q213*

A

309 477

27 ATG

342 257

L145F P154L V197IW211S S238FTAA

Q213*

309 477

27 ATG

342 257

L145F P154L V197IW211S S238FTAA

Q213*

A

Cell wall polysaccharide composition on the dry weight of cell wall preparations is given in Table 2. M82 and Red Setter mutants at the green stage are poorer in uronic acids than the wild type content. On M82 mutant at the breaker stage neutral sugars and total sugar content of cell wall preparations are affected.

Expansin being targeted to hemicellulose/cellulose interactions, the fine structure of hemicelluloses was followed during fruit ripening by β-glucanase degradation and MALDI-TOF MS analysis of the oligosaccharides produced.

Two series of oligosaccharides ion were observed and corresponded to the two major xyloglucan and glucomannan hemicelluloses in tomato. The relative intensity of oligosaccharides attributed to glucomannan oligosaccharides (Hex) markedly decreased with the ripening stage. The fine structure of xyloglucan is also affected during ripening (6). Down-regulation of expansin affects significantly the proportion of xyloglucan fine structures particularly at the breaker stage (Figure 3, Table 3).

Genotype Ripening stage E (kPa) p value n

Mutant green 1.9 ±0.6 0.6794 2 4 Wild type green 2.0 ±0.8 3 6 Mutant breaker

Wild type breaker

3 6 Mutant pin k

Wild type pin k

3 4 Mutant r e d

Wild type r e d

2 6 Mutant overrip e

Wild type overrip e

3 1

Table 1.

Table 1. Compression analysis on M82 and Q213* mutant.Compression analysis on M82 and Q213* mutant.

Modulus of deformation (E) at 20% strain ± standard deviation, analysis of variance between wild type and mutant (p value) and number of measures (n). In bold, statistically significant differences.

Table 3.

Table 3. MALDI-TOF analysis.MALDI-TOF analysis. Analysis of variance (p.values) and interaction (SxM) between ripening stages (S) and genotype (wild-type and mutants for M; 0 and mutant for Red Setter) for M82 and Red Setter genetic background. Oligosaccharide nomenclature:

Hex: hexose based oligosaccharides (glucomannan) of degree of polymerization 4 (Hex4) to 9 (Hex9) bearing 1 to 3 acetic acid ester group (A1-A3); the nomenclature of xyloglucan type oligomers follows that of (7) and was extended by A1 or A2 following the number of acetyl esterification. Data in bold emphasize significant effects.

Figure 3.

Figure 3. Evolution in the proportion of representative Evolution in the proportion of representative glucomannanglucomannan (Hex) (Hex) and xyloglucan fine structure (XXGA1, XXGGA1) in M82 and mutant and xyloglucan fine structure (XXGA1, XXGGA1) in M82 and mutant (Q213*) during ripening.

(Q213*) during ripening. See legend of Table 2 for sample nomenclature.

The two expansin mutant lines show altered softening on ripening. The nonsense Q213* line is more affected as seen from compression test and most likely results from cell wall modifications. This is the first evidence that expansin down-regulation alters the remodelling kinetics of specific xyloglucan fine structures during ripening. The latter may be of particularly importance with regard to xyloglucan-cellulose interactions and thus on fruit texture.

CONCLUSION CONCLUSION

Oligosaccharide M8 2 Red Setter

Stage Mutant S x M Stage Mutant S x M Hex4A1 0.00001 0.88636 0.83015 0.15676 0.17038 0.43510 Hex4A2 0.00239 0.95828 0.30989 0.05734 0.08944 0.55427 Hex5 0.00000 0.08542 0.41524 0.00000 0.39059 0.47101 Hex5A1 0.00000 0.51552 0.88024 0.45702 0.36755 0.31826 Hex5A2 0.02722 0.76714 0.68494 0.08468 0.07602 0.74728 Hex6 0.00000 0.26443 0.28591 0.00000 0.25444 0.69756 Hex6A1 0.00002 0.07173 0.68489 0.47106 0.71269 0.44078 Hex7 0.00000 0.41507 0.18733 0.00000 0.55564 0.84963 Hex8 0.00000 0.63525 0.18743 0.03245 0.86241 0.65086 Hex8A1 0.05810 0.50337 0.50303 0.13584 0.18144 0.73760 Hex8A2 0.09926 0.48581 0.78013 0.18374 0.19239 0.79047 Hex8A3 0.75389 0.32329 0.29484 0.52925 0.25767 0.30015 Hex9 0.22065 0.90017 0.18829 0.04498 0.72834 0.93998 Hex9A2 0.41722 0.48583 0.53466 0.90097 0.12448 0.20580 XLGGA1 0.06787 0.03058 0.72864 0.16083 0.02028 0.68579 XLXGA1/XMGGA1/LSGGA1 0.01071 0.00605 0.48999 0.08959 0.03791 0.30074 XSGGA1 0.06455 0.13093 0.84562 0.04518 0.42256 0.80022 XTGG/Hex7A1 0.00026 0.14281 0.36441 0.00926 0.37359 0.93361 XTGGA1/Hex7A2 0.10384 0.37491 0.72640 0.03372 0.12839 0.84211 XTGGA2/Hex7A3 0.70676 0.52925 0.33718 0.20395 0.84570 0.78440 X X G 0.45467 0.00984 0.23751 0.86762 0.25010 0.66516 XXGA1 0.00059 0.03244 0.73759 0.00000 0.17243 0.43852 XXGG 0.06008 0.83093 0.86841 0.83963 0.13498 0.72858 XXGGA1 0.00000 0.00972 0.02039 0.00000 0.20973 0.60916