Gene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

SummaryGene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

Summary Glycine betaine (GB) is known to accumulate in plants exposed to cold, but the underlying molecular mechanisms and associated regulatory network remain unclear. Here, we demonstrated that PtrMYC2 of Poncirus trifoliata integrates the jasmonic acid (JA) signal to modulate cold‐induced GB accumulation by directly regulating PtrBADH‐l, a betaine aldehyde dehydrogenase (BADH)‐like gene. PtrBADH‐l was identified based on transcriptome and expression analysis in P. trifoliata. Overexpression and VIGS (virus‐induced gene silencing)‐mediated knockdown showed that PtrBADH‐l plays a positive role in cold tolerance and GB synthesis. Yeast one‐hybrid library screening using PtrBADH‐l promoter as baits unraveled PtrMYC2 as an interacting candidate. PtrMYC2 was confirmed to directly bind to two G‐box cis‐acting elements within PtrBADH‐l promoter and acts as a transcriptional activator. In addition, PtrMYC2 functions positively in cold tolerance through modulation of GB synthesis by regulating PtrBADH‐l expression. Interestingly, we found that GB accumulation under cold stress was JA‐dependent and that PtrMYC2 orchestrates JA‐mediated PtrBADH‐l upregulation and GB accumulation. This study sheds new light on the roles of MYC2 homolog in modulating GB synthesis. In particular, we propose a transcriptional regulatory module PtrMYC2‐PtrBADH‐l to advance the understanding of molecular mechanisms underlying the GB accumulation under cold stress.

Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in cold stress response, and raffinose is known to accumulate in plants exposed to cold. However, it remains elusive whether ERFs function in cold tolerance by modulating raffinose synthesis. Here, we identified a cold-responsive PtrERF108 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-tolerant plant closely related to citrus. PtrERF108 is localized in the nucleus and has transcriptional activation activity. Overexpression of PtrERF108 conferred enhanced cold tolerance of transgenic lemon, whereas virus-induced gene silencing (VIGS)mediated knockdown of PtrERF108 in trifoliate orange greatly elevated cold sensitivity. Transcriptome profiling showed that PtrERF108 overexpression caused extensive reprogramming of genes associated with signaling transduction, physiological processes and metabolic pathways. Among them, a raffinose synthase (RafS)-encoding gene, PtrRafS, was confirmed as a direct target of PtrERF108. RafS activity and raffinose content were significantly increased in PtrERF108-overexpressing transgenic plants, but prominently decreased in the VIGS plants under cold conditions. Meanwhile, exogenous replenishment of raffinose could recover the cold tolerance of PtrERF108-silenced plants, whereas VIGS-mediated knockdown of PtrRafS resulted in cold-sensitive phenotype. Taken together, the current results demonstrate that PtrERF108 plays a positive role in cold tolerance by modulation of raffinose synthesis via regulating PtrRafS. Our findings reveal a new transcriptional module composed of ERF108-RafS underlying cold-induced raffinose accumulation in plants.