Fig 1.
Sequence analysis of WRI1 homologues.
Alignments of two domains shown to be functionally relevant to this clade are displayed, with the functionally critical valine residue indicated with a black box.
Fig 2.
WRI1 homologues molecular phylogeny.
Molecular phylogeny of characterized members of the McWRI1 clade. Multiple alignments of amino acid sequences were performed using DNAMAN 7. A phylogenetic tree was then constructed by the neighbor-joining method with MEGA 7.
Fig 3.
McWRI1 expression level in the fruits of Malus asiatica Nakai, Malus prunifolia, Malus asiatica Nakai ‘binzi’, Malus sieversii and Malus crabapple ‘Royalty’.
The error bars represent the mean ± SD of three biological replicates. Differences from the control samples were determined using Duncan’s test: P ≤ 0.05.
Fig 4.
Description of fruits anatomic structures of low temperature and WRI1 silencing group compared with wild type.
A,B,C,D. Phenotypes and light microscopy image of Malus asiatica Nakai after low temperature and WRI1 silencing stimulating compared with wild type and CK. E, F, G, H. Phenotypes and light microscopy image of Malus sieversii after low temperature and WRI1 silencing stimulating compared with wild type and CK.
Fig 5.
Main components of cuticular wax under low temperature stimulated condition by GC-MS.
A. The contents of pentadecane (Pent), heneicosane (Hene), docosane (Doco), tricosane (Tric), heptacosane (Hept), octacosane (Octa),nonacosane (Nona), hentriacontane (Hent), hexadecanoic acid (Hexa-acid), octadecanoic acid (Octa-acid), linoleic acid (Lino-acid), oleic acid (Olei-acid), docosanoic acid (Doco-acid), linoleic acid butyl ester (Labe), erucic acid ethyl ester (Eaee), hexadecanoic acid—octadecane ester (Haoe), hexadecanoic acid—eicosane ester (Haee) in Malus asiatica Nakai. B. The contents of henicosane, docosane, tricosane, heptacosane, octacosane, nonacosane, hentriacontane, hexadecanoic acid, octadecanoic acid, linoleic acid, oleic acid, erucic acid ethyl ester, hexadecanoic acid—octadecane ester, hexadecanoic acid—eicosane ester in Malus sieversii.
Table 1.
Main components content of cuticular wax under low temperature and VIGS stimulated.
Fig 6.
Comparison of relative gene expression level under low temperature stimulated and room temperature conditions in the fruits of two classes.
A. The expression of McWRI, McLACS, McKCS, McWAX, McPKM2 and McKPMHT in Malus asiatica Nakai. B. The expression of McWRI, McLACS, McKCS, McWAX, McPKM2 and McKPMHT in Malus sieversii. The error bars represent the mean ± SD of three biological replicates. Differences from the control samples were determined using Duncan’s test: P ≤ 0.05.
Fig 7.
Main components analysis of cuticular wax between WRI1 silencing group and wild type by GC-MS.
A.The contents of pentadecane (Pent), heneicosane (Hene), docosane (Doco), tricosane (Tric), heptacosane (Hept), octacosane (Octa), nonacosane (Nona), hentriacontane (Hent), hexadecanoic acid (Hexa-acid), octadecanoic acid (Octa-acid), linoleic acid (Lino-acid), oleic acid (Olei-acid), docosanoic acid (Doco-acid), linoleic acid butyl ester (Labe), erucic acid ethyl ester (Eaee), hexadecanoic acid—octadecane ester (Haoe), hexadecanoic acid—eicosane ester (Haee) in Malus asiatica Nakai. B. The contents of henicosane, docosane, tricosane, heptacosane, octacosane, nonacosane, hentriacontane, hexadecanoic acid, octadecanoic acid, linoleic acid, oleic acid, erucic acid ethyl ester, hexadecanoic acid—octadecane ester, hexadecanoic acid—eicosane ester in Malus sieversii.
Fig 8.
Comparison of relative gene expression level between wild type and McWRI1 silencing in the fruits of two classes.
A. The expression of McWRI, McLACS, McKCS, McWAX, McPKM2 and McKPMHT in Malus asiatica Nakai. B. The expression of McWRI, McLACS, McKCS, McWAX, McPKM2 and McKPMHT in Malus sieversii. The error bars represent the mean ± SD of three biological replicates. Differences from the control samples were determined using Duncan’s test: P ≤ 0.05.
Fig 9.
Y1H assay between McWRI1 and the promoter of McKCS, McLAC and McWAX genes.
A.The growth of the yeast strains of McKCS promoter, Y1Hgld [pG-box & GCC-box AbAi]-pGADT7-WRI1,Y1Hgld [pG-box & Mutant-AbAi]-pGADT7-WRI1 and Y1Hgld [pMutant & GCC-box-AbAi]-pGADT7-WRI1on the media supplemented with leucine and Aureobasidin A (AbA) in 800ng. B. The growth of the yeast strains of the McWAX promoter, Y1Hgld [pGCC-box & CAACA-AbAi]-pGADT7-WRI1,Y1Hgld [pMutant & CAACA-AbAi]-pGADT7-WRI1 and Y1Hgld [pGCC-box & Mutant-AbAi]-pGADT7-WRI1 on the media supplemented with leucine and Aureobasidin A (AbA) in 1000ng. C. The growth of the yeast strains of the McWAX promoter, Y1Hgld [pLAC-AbAi]-pGADT7-WRI1 and Y1Hgld [p53-AbAi]-pGAD-Rec-p53on the media supplemented with leucine and Aureobasidin A (AbA) in 800ng.