WT pg

lct-1/

mex1

pGlcT

rRNA

(a)

(b)

(c)

TPT

rRNA

pglct

-1/tpt

-2

tpt-2/

mex1

WTW

T

pGlcT

rRNA

pglct

-1/tpt

-2

TPT

rRNA

WT

tpt-2

WT

pGlcT

rRNA

pglct

-2

pglct

-1

Wild-type

Homozygousmutant

TGG

TGA

Fig. S1 Characterization of homozygous plastidic sugar transporter mutants. The null expression of the corresponding genes in the single (a) and double (b) mutants was confirmed by Northern blot analysis. Ethidium bromide-stained rRNAs are shown as the respective controls. (c) The homozygous mex1 allele in the pglct-1/mex1 double mutant was confirmed by sequencing, because mex1 is a single point mutation.

Fig. S2 Digital Northern analysis of plastidic sugar transporters. Microarray data for adult rosette leaf, roots, inflorescence stem and flower of Arabidopsis were obtained from GEO (Gene Expression Omnibus) database at the Genvestigator site (https://www.genvestigator.com) and used for the digital Northern analysis.

Sig

nal i

nten

sity

pGlcT

MEX1

TPT

Fig. S3 Maltose content of mutant and wild-type plants at the end of the day and night. Maltose content was determined in the mature leaves of 4-week-old single (pglct-1, pglct-2 and mex1) and double (pglct-1/mex1) mutants at the end of the day and night. Each point represents the mean ( SD) from four different measurements within each line. Asterisks indicate significant differences (p < 0.05, t-test) between mutant and wild-type plants. FW; fresh weight.

*

*

* *

No sugar + Glucose + Maltose

WT

pglct-1/mex1

tpt-2/mex1

Fig. S4 Recovery of the defective phenotypes of the pglct-1/mex1 and tpt-2/mex1 mutants by supplying external sugars, glucose and maltose. Mutant and wild-type plants were grown on agar plates containing Gamborg B5 media complemented with 2% of each sugar. The pictures shown are of 25-day-old mutant and wild-type plants.

Time of the day (h)

0 4 8 12 16 20 24

Fruc

tose

con

tent

(m

ol/g

FW)

0.0

0.5

1.0

1.5

2.0

2.5

3.0WT pglct-1/tpt-2 pglct-1/mex1tpt-2/mex1

Time of the day (h)

0 4 8 12 16 20 24

Fruc

tose

con

tent

(m

ol/g

FW)

0.0

0.5

1.0

1.5

2.0

2.5

3.0WT pglct-1 pglct-2 tpt-2 mex1

Time of the day (h)

0 4 8 12 16 20 24G

luco

se c

onte

nt (

mol

/gFW

)

0

1

2

3

4

5WT pglct-1/tpt-2 pglct-1/mex1tpt-2/mex1

Time of the day (h)

0 4 8 12 16 20 24

Glu

cose

con

tent

(m

ol/g

FW)

0

1

2

3

4

5WT pglct-1 pglct-2 tpt-2 mex1

(a) (b)

(c) (d)

Fig. S5 Diurnal changes in glucose and fructose levels in the plastidic transporter mutants. Glucose (a and b) and fructose (c and d) contents were determined in the mature leaves of 4-week-old single (a and c) and double (b and d) mutants during the diurnal cycle. Each point represents the mean ( SD) from five different measurements within each line. Asterisks indicate significant differences (p < 0.1, t-test) between mutant and wild-type plants. The black shading in the upper bar indicates the dark period. FW; fresh weight.

*

**

*

*

*

**

**

**

**

** **

*

DPE2

SEX1

TUB

DPE1

BAM3

PHS1

ISA3

pglct

-1

pglct

-2

WT

tpt-2

mex1

pglct

-1/tpt

-2

tpt-2m

ex1

pglct

-1/mex

1

Fig. S6 Effects of plastidic sugar transporter defects on the expression of starch degradation-related enzymes. Expression levels of starch degradation-related genes, isoamylase 3 (ISA3), β-amylase 3 (BAM3), disproportionating enzyme 1 (DPE1), DPE2, glucan-water dikinase (SEX1), and glucan phosphorylase (PHS1) in the middle of day were analyzed by RT-PCR. Total RNA was isolated from the leaves collected at the middle of day and night of Arabidopsis plants grown for 4 weeks. The tubulin 2 gene (TUB) was amplified as a control.