Online Resources
Online Resource 1. Materials and methods
Detection of cell death
For detection of cell death, trypan blue staining was performed. Leaf discs were covered with an alcoholic lactophenol trypan blue mixture (30 mL of ethanol, 10 g of phenol, 10 mL of double sterilized water, 10 mL of glycerol, 10 mL of lactic acid, and 10 mg of trypan blue), placed in a boiling water bath for 4 min, and then transferred to room temperature for overnight in the staining solution. After overnight staining, the samples were destained by replacing the staining solution with chloral hydrate solution (2,5 g mL -1) for several times until the background was removed, and then transferred into fresh 50 % ethanol for observation.
Genetic analysis of sy-2
Phenotypic analysis of wild type and sy-2 plants at 20 C were performed. All the F1 plants obtained from the cross between cold tolerance (wild type) and cold susceptible (mutant) plants were studied at 20C. The F2 seeds were collected from the F1 plants and the segregation analysis of 148 individuals of F2 population was performed according to phenotypic study at 20C.
Online Resource 2.Genetic analysis of the sy-2 mutant
Plant phenotype / Observed value (O) / Expected value (E) / χ2 / P valueNumber of tolerant plant / 118 / 148 × 3/4=111
Number of susceptible plants / 30 / 148 × 1/4=37 / 2.04 / 0.10
Total number of plants / 148 / 148
Online Resource 3. Fatty acid analysis of sy-2 plants
Fatty acid / Plants / Temperature / Fatty acid content
c16:0 / Wt / 20C / 16.25±0.31
26C / 20.07±0.17
sy-2 / 20C / 20.08±0.71**
26C / 22.89±1.28*
c18:0 / Wt / 20C / 1.24±0.18
26C / 1.90±0.23
sy-2 / 20C / 3.18±1.63
26C / 2.45±0.39
c18:1 / Wt / 20C / 1.93±0.81
26C / 4.66±0.16
sy-2 / 20C / 1.63±0.88
26C / 3.25±0.50
c18:2 / Wt / 20C / 18.00±1.04
26C / 19.15±1.05
sy-2 / 20C / 21.84±3.58*
26C / 20.00±1.65
c18:3 / Wt / 20C / 37.34±2.03
26C / 24.25±0.22
sy-2 / 20C / 27.22±4.49*
26C / 23.51±3.23
The values are the mean ± SD obtained from three independent wild type and sy-2 plant grown at 26C or 20C.
Asterisks indicate the significance of difference between wild type and mutant plants at 26C or 20C (*, P<0.1; **, P<0.01).
Online Resource 4- The list of predicted protein in restricted tomato scaffold sequence.Number / Start / End / Length / Direction / Description
1 / 3685404 / 3686228 / 433 aa / chain - / C2_At1g09070 / protein kinase
2 / 3689632 / 3700066 / 534 aa / chain + / zinc finger (C3HC4-type RING finger) family protein
3 / 3706723 / 3709111 / 433 aa / chain + / chloroplast-targeted copper chaperone
4 / 3713318 / 3714601 / 427 aa / chain - / xyloglucan-specific endoglucanase inhibitor protein
5 / 3715374 / 3718678 / 167 aa / chain - / signal peptidase subunit family protein
6 / 3720361 / 3721990 / 487 aa / chain + / heat shock protein-like protein
7 / 3722654 / 3730110 / 680 aa / chain - / Ca2+ antiporter/cation exchanger
8 / 3730407 / 3733510 / 313 aa / chain + / heat shock protein
9 / 3735597 / 3742869 / 821 aa / chain +
10 / 3745280 / 3749978 / 577 aa / chain + / Mevalonate kinase [Lipid metabolism]
11 / 3750552 / 3751998 / 293 aa / chain - / 3-ketoacyl-(acyl-carrier-protein) reductase
12 / 3753849 / 3754519 / 163 aa / chain + / GPI-anchored protein-like protein II
13 / 3755417 / 3771266 / 1656 aa / chain + / seryl-tRNA synthetase / serine--tRNA ligase
14 / 3775347 / 3780134 / 374 aa / chain - / Yip1 domain
15 / 3783084 / 3786849 / 317 aa / chain - / mitochondrial FAD carrier
16 / 3794234 / 3805617 / 828 aa / chain + / Ras-like GTPase superfamily
17 / 3808570 / 3809321 / 226 aa / chain - / Cyclin superfamily
18 / 3810766 / 3815192 / 568 aa / chain - / Plant invertase/pectin methylesterase inhibitor
19 / 3818135 / 3821353 / 357 aa / chain + / glyceraldehyde-3-phosphate dehydrogenase
20 / 3822662 / 3826256 / 138 aa / chain + / succinic semialdehyde dehydrogenase
21 / 3828823 / 3830691 / 513 aa / chain + / PGSIP5 (Plant Glycogenin-Like Starch Initiation Protein 5)
22 / 3831797 / 3835192 / 569 aa / chain + / PGSIP5 (Plant Glycogenin-Like Starch Initiation Protein 5)
23 / 3836159 / 3850929 / 1612 aa / chain - / ATP binding / microtubule motor
24 / 3851979 / 3857366 / 350 aa / chain + / unknown
25 / 3857816 / 3862811 / 1065 aa / chain - / plant_lipase_like_hydrolase superfamily
26 / 3867494 / 3868978 / 380 aa / chain - / plant_lipase_like_hydrolase superfamily
27 / 3879720 / 3883189 / 375 aa / chain - / plant_lipase_like_hydrolase superfamily
28 / 3888809 / 3890394 / 380 aa / chain - / Acylhydrolase family protein
29 / 3892481 / 3895238 / 150 aa / chain - / alpha-L-fucosidase 2
30 / 3896712 / 3898181 / 377 aa / chain - / GDSL-like Lipase/Acylhydrolase family protein
31 / 3904645 / 3906121 / 343 aa / chain - / alpha-L-fucosidase 2
32 / 3909233 / 3918394 / 1189 aa / chain - / endo-beta-mannosidase
33 / 3923081 / 3928273 / 343 aa / chain + / acyl-CoA synthetase 6
34 / 3929330 / 3930833 / 398 aa / chain - / Endonuclease/exonuclease/phosphatase
35 / 3931971 / 3938322 / 495 aa / chain + / acyl-CoA synthetase 6
36 / 3938930 / 3939308 / 59 aa / chain + / acyl-CoA synthetase 6
37 / 3940158 / 3949064 / 756 aa / chain - / xaa-pro aminopeptidase
Number / Start / End / Length / Direction / Description
38 / 3950024 / 3953080 / 478 aa / chain + / AXR4 (Auxin Resistant 4)
39 / 3954173 / 3956701 / 288 aa / chain + / LysM
40 / 3957631 / 3964438 / 447 aa / chain - / transmembrane protein 49
41 / 3971492 / 3973013 / 301 aa / chain + / putative dessication-related protein
42 / 3975019 / 3977042 / 512 aa / chain + / lipoxygenase
43 / 3983497 / 3998131 / 2142 aa / chain + / lipoxygenase
44 / 3998836 / 4002271 / 859 aa / chain - / lipoxygenase
45 / 4010933 / 4014506 / 864 aa / chain + / lipoxygenase
46 / 4016385 / 4020017 / 894 aa / chain + / lipoxygenase
47 / 4021539 / 4040355 / 2445 aa / chain + / putative DNA polymerase zeta catalytic subunit
48 / 4041628 / 4042737 / 369 aa / chain + / zinc finger (Ran-binding) family protein
49 / 4043629 / 4044564 / 311 aa / chain + / 6-phosphogluconate dehydrogenase NAD-binding domain
50 / 4045447 / 4047618 / 665 aa / chain - / serine-threonine protein kinase,
51 / 4046438 / 4052026 / 317 aa / chain + / C2_At4g29210 / heat shock protein binding
Online Resource 5. Light microscopic study of leaf sections of sy-2 plants. A, B Photographs of second leaves of wt and sy-2 mutant at 26℃. C, D Photographs of second leaves of wt and sy-2 mutant at 20℃. E, F Sections of the middle portion of (A) and (B) respectively. G, H Sections of the middle portion of (C) and (D) respectively. The images were taken at 50X magnification.
Online Resource 6. Study of cell death in sy-2 leaves.A, B Trypan blue staining of wild type and sy-2 mutant leaves grown at 26℃. C, D Trypan blue staining of wild type and sy-2 mutant leaves grown at 20℃. E, F and G, H were 50X magnified images of (A, B) and (C, D) respectively.