Restriction of cytosolic sucrose hydrolysis profoundly alters development, metabolism, and gene expression in Arabidopsis roots

A reduction in cytosolic invertase in Arabidopsis roots changes the sucrose to hexose ratio, resulting in profound alterations of metabolism, growth, development, and patterns of gene expression.

Table S1.Primers used in this study.

AGI Number
P r i m e r s q-PCR primers: Genotyping primers: Log2 fold difference  S5) show that transcript levels for SUS2, SUS4, A/N-InvB and A/N-InvD, a vacuolar INV and two mitochondrial neutral INV were slightly reduced and transcript levels of cwINV1 and of the phloem-specific protein SUS6 were higher in mutant than in wild-type roots; (B) Photographs of four-day-old mutant and wild-type seedlings grown without glucose.All images are at the same magnification; (C) Multi-dimensional scaling plot visualizing the distance between the libraries in the RNA-seq experiment, based on the 500 tags with the largest variation between the four treatments.The distance between each pair of libraries is equivalent to the square root of the common dispersion between these two libraries.
Libraries were from roots of four-day-old seedlings.Green symbols, wild-type grown without glucose; blue symbols, wild-type grown with 55 mM glucose; black symbols, cinv1 cinv2 grown without glucose; red symbols, cinv1 cinv2 grown with 55 mM glucose.For each genotype/treatment, the three points represent the three independent biological replicates (see Materials and Methods); (D) Validation of RNA-seq analysis.For 38 selected transcripts representing a wide range of different cellular functions (loci given in Supplementary Table S1 below), the graphs show log2 fold differences in transcript abundance in cinv1 cinv2 relative to wild-type from qPCR measurements (blue diamonds) and RNA-seq (red squares: see Supplementary Table S5).For qPCR, values are means of measurements on three independent biological replicates.In all cases SD was less than 10% of the mean.

Wild-type cinv1 cinv2
Wild-type +Glc cinv1 cinv2 +Glc Figure S1 Fig. S1.Transcript levels for sucrose metabolizing enzymes in cinv1 cinv2 roots, and validation of RNA-seq data.(A) Levels of transcripts of cytosolic INV and SUS in 14day-old seedlings, measured by q-PCR.Values are expressed as log2 fold difference in transcript abundance in cinv1 cinv2 relative to wild-type.Values are means of measurements on three independent biological replicates ± SD.There is essentially no difference between 14-day-old mutant and wild-type roots with respect to transcript levels for SUS1, SUS2, SUS5 and SUS6, and only a modest elevation (≤1.6-fold) of transcripts for SUS1, SUS4, A/N-InvB and A/N-InvD.For four-day-old plants RNAseq data (Supplementary TableS5) show that transcript levels for SUS2, SUS4, A/N-

Fig
Fig. S2.Original, uncropped micrographs from which the composite Fig. 1D was assembled.

Table S2 .
Transcript levels for cytosolic invertases in roots a a Data are from www.bar.utoronto.ca,extractedfromDinnenyetal. (2008).Seedlings were grown for five days on 1x Murashige and Skoog salt mixture, 1% agar, 1% sucrose.Cell type or section-specific data were generated by fluorescence-activated cell sorting or sectioning of roots, followed by RNA extraction and microarray analysis.bLongitudinal zone 1 (~150 µm) is from the root tip to the point at which shape changes from

Table S3 .
Leaf area and stomatal density in wild-type and mutant shoots.For leaf area, values are from measurements on eight plants ± SD.Wild-type and mutant values are statistically significantly different (Student's T-test, P<0.0001).
b c For stomatal density, values are means ± SD of measurements on fully-expanded leaf 6 of eight plants per genotype.For each leaf, stomata were counted on ten images, each of 0.138 mm 2 of leaf surface.Wild-type and mutant values are statistically significantly different for both adaxial and abaxial surfaces (Student's T-test, P<0.01).

Table S4 .
Metabolite contents of wild-type and mutant shoots.