Carb

ohyd

rate

cont

ent (

mg/

g FW

)

Col-0 i1 i1i2

i1AtP6C3

i1AtP1D1

i1AtP1A4

i1ZmP7E5

i1ZmP7E4

i1ZmP2D7

i1i2ZmP4F8

i1i2ZmP4F1

i1i2ZmP4F5

i1i2ZmP4D3

0

2

4

6

8

10

12

14

16

StarchWSP

WT isa1- isa1- isa2-

isa1- host isa1- isa2- host

AtISA1 (P6C3)

AtISA1 (P1D1)

AtISA1 (P1A4)

ZmISA1 (P7E5)

ZmISA1 (P7E4)

ZmISA1 (P2D7)

ZmISA1 (P4F8)

ZmISA1 (P4F1)

ZmISA1 (P4F5)

ZmISA1 (P4D3)

Scale bars = 10 µm

WT isa1- isa2-

P4D3P4F8

P7E4

P4F5

P6C3

P1A4 P7E5

P4F1

P2D7

P1D1

A

B

Supplemental Figure S5:Panel A: starch and WSP contents determined in host and transgenic lines (as indicated in the figure). Contents are expressed in mg of carbohydrate per g of fresh weight (FW) and are the means of three independent biological replicates. Black and grey vertical bars indicate starch and WSP content respectively. Vertical lines on bars stand for standard error. Typically no or very low amount of WSP were detected for WT (Wild Type) and transgenic lines expressing AtISA1 or ZmISA1 protein. Panel B: scanning electron microscopy images of purified starch granules (host and transgenic lines are indicated). The starch samples were prepared from Arabidopsis leaves harvested at the end of the illuminated period (16h light / 8h dark). Scale bars = 10µm.Note that some transformants were not complementing although they were hygromycin resistant: P7E5 (isa1- exp. ZmISA1) and P4F1 (isa1- isa2- exp. ZmISA1). Rearrangement of the transgene may have occurred during integration into the nuclear genome of the plant leading to an alteration of the ZmISA1 open reading frame and consequently of its expression.