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Control of Stomatal Distribution on the Arabidopsis Leaf Surface
Jeanette A. Nadeau and Fred D. Sack

Supplementary Material

Materials and Methods

Cloning of TMM.TMM was fine-mapped to chromosome 1 between GL2 and the T7 end of BAC clone F5D15 using molecular markers (see for markers). Overlapping TAC clones (S1) K27D14, K6H17, and K11P20 spanning this interval were transformed into tmm-1 plants to test for complementation. Only K27D14 was found to complement, further localizing TMM to a 40 kb region. Sequence from two independent alleles revealed polymorphisms in a single ORF when compared to the Genbank database. A ~3 kb fragment of genomic DNA spanning the predicted coding region of At1g80080 (Genbank accession NC003070) and flanking noncoding sequence was transformed into tmm-1 plants by Agrobacterium transformation to test for complementation (S2). Ten of twelve transformants were completely wild-type in stomatal spacing in both the cotyledon and leaf, while two were partly wild-type.

Construction of transgenic plants harboring reporter constructs. A TMM reporter construct was created by replacing the CaMV 35S promoter in pCAMBIA1303 with 511 bp of 5� upstream sequence in frame with the GUS-GFP coding sequence. This was equivalent to the noncoding region between TMM and an adjacent upstream gene. The omission of the neighboring gene coding sequence resulted in a fragment that was 178 bp shorter in the TMM promoter reporter construct than in the genomic complementation construct. A carboxy-terminal TMM fusion protein (TMMpro::TMM-GFP) was created by replacing the CaMV 35S promotor in pCAMBIA1302 with the 511 bp of 5� TMM sequence described above plus the complete open reading frame of TMM and a spacer coding for eight additional amino acids in frame with GFP. An amino-terminal fusion protein (TMMpro::GFP-TMM) was created by cloning a pCAMBIA-derived GFP fragment containing PCR-introduced KpnI and SalI restriction sites into complementary PCR-introduced restriction sites in the TMM gene in pCAMBIA2300. This placed GFP in frame with TMM between amino acids 6 and 7 of the predicted mature TMM protein, downstream of the putative signal peptide cleavage site and upstream of the conserved sequences of the NNL. All constructs were fully sequenced and introduced into Col gl1-1 (wild type) and Col tmm-1 plants by Agrobacterium transformation. Complete complementation of the mutant phenotype was observed in tmm-1 plants transformed with TMMpro::GFP-TMM. Transformation with TMMpro::TMM-GFP resulted in substantial but not complete complementation (Table S1).

Supporting Online Text

Comparison of the expression pattern of the TMMpro::GUS-GFP reporter protein with the TMMpro::TMM-GFP and TMMpro::GFP-TMM translational fusion proteins.

The pattern of expression derived from analysis of fusion protein constructs is comparable to that from the TMM promoter-reporter, but is restricted to fewer cells. In both COOH- and NH2-terminal GFP fusions to TMM, fluorescence is absent from many older epidermal cells and guard cells. In contrast, the reporter GUS-GFP protein is present in older stomata. While this might reflect developmentally regulated removal of the TMM-GFP fusion protein, it is equally likely that this observation reflects the long half-life of the GUS-GFP reporter protein. Because GUS-GFP may persist in older cells and in the progeny of cells that expressed it, the promoter-reporter construct may not accurately report transcriptional and posttranscriptional TMM regulation. Because the GFP fusion proteins result in complete or mostly complete complementation, the fusion proteins were used for quantitative analysis.

Supplemental Figure 1. Stomatal patterning involves reiterative developmental modules. Meristemoid mother cells (MMC; yellow) divide asymmetrically to produce a meristemoid (M; red). Meristemoids have limited capacity for self-renewal, dividing one or more times to produce a new meristemoid and a sister cell that may ultimately differentiate as a pavement cell. Cells adjacent to a single stoma or precursor can divide asymmetrically to produce a satellite meristemoid (SM; red). The orientation of this division is controlled so that an intervening cell is produced. Each meristemoid eventually differentiates into a guard mother cell (GMC) which divides symmetrically to produce the two guard cells of the stoma (S).

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Supplemental Figure 2. The developmental patterns of expression of amino- and carboxy-terminal GFP fusion proteins are equivalent. Stomata (S) and satellite meristemoids (SM) are indicated. (A) Developing abaxial leaf of a TMMpro::TMM-GFP plant. (B) Developing abaxial leaf of a TMMpro::GFP-TMM plant. Fluorescence from the TMM-GFP fusion protein was significantly brighter than the GFP-TMM fusion protein. This most likely reflects reduced fluorescence when the GFP moiety is extracellular, where it is exposed to low apoplastic pH, as compared to cytoplasmic localization.

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Supplemental Figure 3. Subcellular localization of TMM fusion protein using confocal microscopy. (A and B) Control ER localization in abaxial epidermal cells of a plant transformed with CaMV 35S::mGFP5-ER (S3). (A) Outer focal plane showing reticulate cortical ER. (B) Same cell as in (A) in deeper focal plane. The cortical ER appears as punctate spots (arrow) outside the large central vacuole. The region of the nuclear envelope is also fluorescent (n). (C and D) As in (A) and (B) except for a plant transformed with the COOH-terminal GFP fusion to TMM (TMMpro::TMM-GFP). Fluorescence is localized to membrane compartments. Note the diffuse fluorescence from the plasma membrane region that is present in addition to cortical ER. Both the COOH- and NH2-terminal GFP translational fusion proteins exhibit similar patterns.

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Supplemental Table 1. Complementation of the tmm-1 phenotype with TMM-GFP or GFP-TMM translational fusion proteins expressed from the native TMM promoter. Six T2 individuals from five independent transgenic lines for each transgene were scored for the frequency and size of clustered stomatal units (stomata in contact) on the abaxial cotyledon of soil-grown plants. Clusters containing Greater Than or Equal to Symbol3 stomata were scored as large. Data are expressed as a percent reduction in cluster frequency and size compared to tmm-1. The tmm-1 mutant exhibits 58% overall cluster frequency, and 46% of stomatal units in large clusters. Wild-type Col plants show 1% overall cluster frequency, and were never observed to produce large clusters. The carboxy-terminal placement of GFP produces a fusion protein slightly less effective in correctly patterning stomata than the amino-terminal fusion, which completely complements. Complementation suggests that the fusion proteins are active and the cell-specific pattern of expression is sufficient for TMM function.
TransgeneReduction in Overall Cluster Frequency
Reduction in Large Cluster Frequency
(% ± SD)
TMMpro::TMM-GFP71 ± 590 ± 3
TMMpro::GFP-TMM95 ± 1100 ± 0


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