Fig. 7.

Glc signalling assays. (A) Transient expression assays using leaf protoplasts from WT Col or hkl1-1. Protoplasts were co-transfected with pRBCS-LUC and an internal control, pUBQ10-GUS, plus or minus effectors HXK1-HA and/or HKL1-HA. Protoplast treatments include without glc or effectors (Control), with 2 mM glc (Glc), with 2 mM glc+HXK1-HA (Glc+K1), with 2 mM glc+HKL1-HA (Glc+L1), and with 2 mM glc+HXK1-HA+HKL1-HA (Glc+K1+L1). Values are means ±SD of the relative LUC units to GUS activities for replicated assays normalized to the control. GUS activity was not affected by the presence of glc or either effector. (B) Expression of glc regulated genes in HKL1 transgenic lines and mutants. Semi-quantitative RT-PCR was used to determine the transcript levels of asparagine synthase (ASN), glycerate kinase (GLYK), trehalose 6-phosphate synthase (T6P), and ubiquitin (UBQ). Seedlings grown in liquid medium were challenged without (–) or with (+) 2% glc for 8 h (see Materials and methods for further details). The number of PCR cycles was varied in each case, but for the presented data are as follows: ASN, 32 cycles, GLYK, 32 cycles, T6P, 33 cycles, UBQ, 31 cycles.