Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding beta-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

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Peach ERF3b is a potent transcriptional repressor for defense-related genes even in the presence of similar levels of transcriptional activators and can interfere with plant development through pathways independent of the EAR motif. Ethylene response factors (ERFs) are a major group of plant transcription factors with either activation or repression capabilities on gene transcription. Repressor-type ERFs are characterised by an intrinsic motif, namely the ERF-associated amphiphilic repression motif (EAR). Here we report the identification of three genes from peach (Prunus persica), PpERF12, PpERF3a and PpERF3b, encoding for ERF repressors. The transcription kinetics of these genes was investigated by qRT-PCR after inoculation of peach leaves with Xanthomonas campestris pv. pruni. All three genes showed higher induction in the susceptible 'BabyGold 5', than in the resistant 'Venture' peach varieties suggesting a negative role for these genes in disease resistance. The functional potency of PpERF3b has been confirmed in vivo by its ability to repress the expression of GUS-reporter gene. To better understand the functional role of PpERF3b, the full-length and the EAR-truncated (PpERF3b∆EAR) genes were overexpressed in tobacco (Nicotiana tabacum). Both transgenic plants (PpERF3b and PpERF3b∆EAR) uniformly exhibited precocious side branching, which suggests the interference of PpERF3b with auxin-mediated dormancy of lateral shoots. Consistent with that the expression of auxin-response factors (Nt-ARF1, Nt-ARF6 and Nt-ARF8) was significantly downregulated in transgenic plants compared to the wild type (WT). Although side branching was independent of EAR motif, the response of transgenic plants to inoculation by Pseudomonas syringae pv. tabaci was EAR dependent. Transgenic plants overexpressing PpERF3b∆EAR showed less disease symptoms than those overexpressing the full-length gene or WT plants. Resistance of PpERF3b∆EAR plants was associated with enhanced induction of pathogenesis-related (PR) genes. Our results indicate that repressor-type ERFs might act through pathways that are dependent or independent of the EAR motif.

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Plant cell culture provides a unique opportunity to manipulate morphogenesis in a controlled environment, thus providing crop improvement with a powerful, complementary tool. Since the late 1970s, the process of in vitro selection has been applied to several cell culture systems to generate mutants with useful agronomic traits such as disease resistance. However, the promise of genetic engineering technology and some early failures among the in vitro selected plants stifled research in this area. Recent advances in molecular characterization of stress-related responses and the emergence of sensitive molecular analytical tools have reinvigorated research on in vitro selection. This technology is easy to use, and not encumbered by intellectual property issues and social concerns currently inhibiting development of transgenic crops. Thus it is an attractive complement to existing crop improvement strategies. The sub-cellular mechanisms that lead to altered phenotypes after in vitro selection are discussed.