Figure 2.

Phenotypes Associated with NMT1 Knockout in the nmt1-1 Line and Rescue with At NMT1.

(A) Global phenotype of the nmt1-1 line. Left: Phenotypes of various independent nmt1-1 lines at various time points and their comparison with the wild type. A close-up of the nmt1-1 line is shown. The seedling was dissected to uncover the cotyledons, which are normally covered by the seed coat (see left part).

(B) RT-PCR analysis of the NMT1 transcript in the nmt1-1 line. The EF transcript was used as the positive control probe. The reasons for transcript overproduction are given in Supplemental Figure 1 online.

(C) Immunoblot analysis of seedlings 10 DAI, showing the absence of At NMT1 in the nmt1-1 line. We analyzed 80-μg aliquots of total protein. The band migrated at the expected M_r of ∼50 kD.

(D) Complementation of the nmt1-1 line with the N1At1 construct leads to reversion to the wild type. Left: Comparison of the phenotypes of wild-type and nmt-1 plants stably expressing the N1At1 transgene (nmt-1-N1At1). Growth 35 DAI is shown. Right: Immunoblot analysis of the amount of At NMT1 in wild-type, nmt-1, or nmt-1-N1At1 lines. Proteins were extracted 7 DAI. PEPC, phosphoenolpyruvate carboxylase. Antibodies were provided by J. Vidal.

(E) Analysis of the cytological phenotype of the nmt1-1 line and comparison with the wild type as observed 4 DAI. Left panel: Confocal imaging of the root meristem region stained with FM464. Center panel: Thin cross section of the SAM region stained with toluidine blue. The circle indicates the SAM. Right panel: Whole-mount cleared seeds observed with differential interference contrast microscopy at the heart embryo stage. The position of the SAM is indicated with an arrow.