Efficiency of physical (light) or chemical (ABA, tetracycline, CuSO4 or 2-CBSU)-stimulus-dependent gus gene expression in tobacco cell suspensions.

In this study, the efficiency of inducible promoters to switch on gene expression in the presence of inducer or to switch it off in its absence was evaluated in tobacco cell suspensions transformed with the gus gene coding sequence. Either plant (pats1A, pSalT, pIn2-2) or microbial (pMre, pTet) inducible promoters were used to drive gus expression. The inducers were light, abscisic acid, 2-CBSU, CuSO4, tetracycline, respectively. For each construct (inducible promoter-gus coding sequence), the optimal induction conditions were determined (inducer concentration, induction time, and age of cells in culture cycle before induction). The efficiency of the inducible promoter was then evaluated under optimal induction conditions. GUS-expression levels obtained under non-inducing and inducing conditions were systematically compared. Thirty or forty percent of the clones transformed with the pSalT-gus or pTet-gus construct, respectively, showed high induction rates (>1000) and GUS activities of the same order as those obtained with a constitutive system. However, basal GUS levels were always high for the pTet-gus cell lines. Seventy or eighty-five percent of the cell lines transformed with the pMre-gus or pln2-2-gus construct, respectively, had induction rates of 1.5 to 1000. The pats1A-gus construct gave very low induction rates-55% of cell lines had induction rates less than 1.5. Only the pSalt-gus construct gave both the highest induction rates and basal GUS-levels equivalent to the endogenous GUS background.

The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER.

Proteins are co-translationally transferred into the endoplasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (delta pro) forms of sporamin. The resulting SpoHDEL or delta proHDEL, as well as Spo and delta pro, were expressed at high levels in transgenic tobacco cells (Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or delta pro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or delta proHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.