figure s1 l nma-1 col-0 nma-3 - plant physiology · scale bar = 5 cm ler-0 nma-1 col-0 nma-3 figure...
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Figure S1: Adult phenotype of nma-1 (Ler-0 back-ground) and nma-3 (Col-0 background) in comparison to corresponding wild-type plants. Scale bar = 5 cm
Ler-0 nma-3Col-0nma-1
Figure S1
[number of cells]30
20
10
25
15
5
nma-1n=15
Ler-0n=15
ssp-2n=15
Col-0n=15
* *
Figure S2
Figure S2: Number of cells in a central optical sectionof the proembryo 3 days after pollination.Significant differences as indicated by Mann Whitney U-test (p<0.001) are highlighted by an asterisk. Boxes represent lower and upper quartiles around median, whiskers indicate maximum and minimum values.The number of examined embryos is given below each genotype.
pollen tube length [µm]
nma-1n=189
Ler-0n=146
350
300
250
200
150
100
50
Figure S3:
Figure S3: Pollen tube length of in vitro germinated pollen 6h after inocculation.Boxes represent lower and upper quartiles around median, mean values are shown as triangles, whiskers indicate maximum and minimum values.The total number of analyzed pollen tubes is givenbelow the genotype.
suspensor length [µm]
100
WT x WT WT x nma nma x WT nma x nma
20
40
60
80
(n=107) (n=92) (n=94) (n=38)
Figure S4:
Figure S4: Measurements of suspensor length after reciprocal crosses of wild-type (Ler-0) and nma-1 mutant plants. Number of measured suspensors is given in brackets. Significant differences to wild-type cross (WT x WT) as indicated by Mann Whitney U-test (p<0.001) are highlighted by an asterisk. Boxes represent lower and upper quartiles around median. Mean values are shown as triangles.
* *
0
20
10
[recombinants]
13.7 13.8 14.414.314.214.114.013.9position [Mb]
Figure S5:
Figure S5: Fine mapping of nma-1. Number of recombinants in a 563 kb genomic interval around nma-1 on chromosome II are shown. Dashed lines indicate an intervall without further recombinations.
1kb nma-3(SALK_015991)
nma-2(SALK_126968)
nma-1(G>A)
A
signal peptidenma-1
(G279>D)
cleavage site
glycoside hydrolase, family 28
N C
100 aa
B
Figure S6:
Figure S6: Graphic representation of the NMA gene and protein.A, Gene model of the genomic NMA locus. Exons are depicted as white boxes, 3’UTR as black box, intergenic regions as line. The position of nma alleles are shown below the model. B, protein model of NMA. The predicted signal peptide is shown in black, the predicted polygalacturonase domain in grey. The position of the nma-1 substitution is shown on top of the model.
A CB
Figure S7:
Figure S7: Embryonic phenotype of T-DNA insertion alleles of nma.A, nma-2; B, nma-3; C, Col-0 wild-type. Scale bar = 20µm
20
120
100
80
60
40
[µm]Le
r-0
nma-
1
pNM
A::N
MA
pNM
A::Y
FP-N
MA
pSU
C5:
:YFP
-NM
A
pJ10
::YFP
-NM
A
pOB
P1:
:YFP
-NM
A
suspensor length
140
Figure S8:
Figure S8: Complementation assay based on suspensor length.The genotype of representative transgenic lines is given below chart. Boxes represent lower and upper quartiles around median. Whiskers depict maximum and minimum values.
normalized expression level
Figure S9:
RL SIPOINCL
Figure S9: quantitative RT-PCR data of NMA expression in various tissue types. RL, rosette leaves; CL, cauline leaves; IN, inflorescences; PO, pollen; SI, siliques; SH, seedling shoot including cotyledons and first leaves; RT, seedling root.Values are normalized to average expression levels in pollen. Mean values of three biological replicates with standard deviations are shown.
RTSH0
12
10
8
6
4
2
14
16
Figure S10:
Figure S10: Graphic representation of NMA expression data in publicly available microarray data.Screen shot of the Arabidopsis eFP browser (Winter et al. 2007).
Figure S11:
Figure S11: Expression of promoter::reporter gene fusions. A, pOBP1::n3xGFP; B, pJ10::n3xGFP. Scale bar = 10µm.
A B
Figure S12:
Figure S12: Average developmental stage of embryos 4 dap in pJ10::YFP-NMA, nma-1, and Ler-0. Embryos were classified in 6 developmental stages. Numerical values were asigned to each stage. Averages and standard deviations of at least three biological replicates are shown as bar graphs. Asterisks indicate significant differences in pair-wise comparison (t-test; p<0.05).
pJ10::YFP-NMA Ler-0nma-1
Developmental stage
n=500n=374 n=1102
mg
lg
tr
eh
lh
et
1
6
5
4
3
2
*
Table S1: Primer sequences
Primer name Primer sequence
160-3, R ctctcttcttcttcctcgcc
At2g33160P-FC cggatctgttgttttttctcag
At2g33160P-RC tctctcttctttggtttggt
At2g33160-RC aaggtagaggacgatgtgattc
LBb1.3 attttgccgatttcggaac
NMA dC-F aacaaactttgaatgagctgtaat
NMA dC-R tgcgatacggacttagcccatgtcttgatatttaaa
NMAi1 AP-F accaaaccaaagaagagagaggcgcgccaaaaattaattaaagcgtctagggttcttctgat
NMAi1 AP-R atcagaagaaccctagacgctttaattaatttttggcgcgcctctctcttctttggtttggt
NMAi1 ASP-F ccacgaaggaatatcccggcgcgccagtcgacttaattaaaatctttgacgtcagaaa
NMAi1 ASP-R tttctgacgtcaaagattttaattaagtcgactggcgcgccgggatattccttcgtgg
NMAi1-F ttggatatctctgatgtcagatgcggaccagggcatggta
NMAi1-R taccatgccctggtccgcatctgacatcagagatatccaa
qRT-F gcaaatctacaacgtcact
qRT-R gacactaataccatgccct
NRT-F3 gtcgtacactagctatgaa
NRT-R4 aatgacgatagggtttccaa
p35Sas aaaaaaggtaacccccgtgttctctccaaatg
p35Ss ttttttctcgagtcgacctgcaggcggccgca
pJ10as aaaaaaggttaccctttacaccgtctcttgctctc
pJ10s ttttttctcgaggtgccatgttgttctatattct
pOBP1as aaaaaaggttaccaatcggagaaaggttgaagctt
pOBP1s ttttttctcgagcatagatttggtgttgatgtgg
pSUC5as aaaaaaggttacctatgaaaagaaaaacgagcag
pSUC5s ttttttctcgagtgagtaagaacaggtagtcc
RT 160.2 F tttgtgtgagaacgttggtg
RT 160.2 R ggttttagtagcctcagta
RT-5F ttttgtgtgtgccttctctc
RT-5R aagtaatccaccgccagtaa
SSP1-I ttagagaccacacgagaaggc
SSP1-R taacatggcttggtctgatcc