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Sucrose is synthesized from UDP-Glc and Fru-6-phosphate via the activity of sucrose-phosphate synthase (SPS) enzymes, which produce Suc-6-phosphate. Suc-6-phosphate is rapidly dephosphorylated by phosphatases to produce Suc and inorganic phosphate. Arabidopsis has four sps genes encoding SPS enzymes. Of these enzymes, AtSPS1F and AtSPS2F have been grouped with other dicotyledonous SPS enzymes, while AtSPS3F and AtSPS4F are included in groups with both dicotyledonous and monocotyledonous SPS enzymes. In this work, we generated Arabidopsis thaliana transformants containing the promoter region of each sps gene fused to gfp::uidA reporter genes. A detailed characterization of expression conferred by the sps promoters in organs and tissues was performed. We observed expression of AtSPS1F, AtSPS2F and AtSPS3F in the columella roots of the plants that support sucrose synthesis. Hence, these findings support the idea that sucrose synthesis occurs in the columella cells, and suggests that sucrose has a role in this tissue. In addition, the expression of AtSPS4F was identified in embryos and suggests its participation in this developmental stage. Quantitative transcriptional analysis of A. thaliana plants grown in media with different osmotic potential showed that AtSPS2F and AtSPS4F respond to osmotic stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proteins of the Split ends (Spen) family are characterized by an N-terminal domain, with one or more RNA recognition motifs and a SPOC domain. In Arabidopsis thaliana, the Spen protein FPA is involved in the control of flowering time as a component of an autonomous pathway independent of photoperiod. The A. thaliana genome encodes another gene for a putative Spen protein at the locus At4g12640, herein named AtSpen2. Bioinformatics analysis of the AtSPEN2 SPOC domain revealed low sequence similarity with the FPA SPOC domain, which was markedly lower than that found in other Spen proteins from unrelated plant species. To provide experimental information about the function of AtSpen2, A. thaliana plants were transformed with gene constructs of its promoter region with uidA::gfp reporter genes; the expression was observed in vascular tissues of leaves and roots, as well as in ovules and developing embryos. There was absence of a notable phenotype in knockout and overexpressing lines, suggesting that its function in plants might be specific to certain endogenous or environmental conditions. Our results suggest that the function of Atspen2 diverged from that of fpa due in part to their different transcription expression pattern and divergence of the regulatory SPOC domain.

Light-regulation of photosynthesis-associated nuclear genes is mediated by multipartite cis-regulatory units located in their promoter regions. The combination, spacing, and relative orientation of transcription factor binding sites in these units influences the assembly of specific multiprotein complexes that control gene expression. In this work, the functional architecture of the conserved modular array 5 (CMA5), a previously characterized minimal light-regulatory unit of rbcS gene promoters, has been analysed. With the aim of defining the sequences of CMA5 that, besides the I- and G-box elements, are essential for CMA5 responsiveness to light and chloroplast-derived signals, a series of mutations affecting the sequences flanking these elements was performed. It was found that an I-box associated module, named IbAM5, is essential for CMA5 functionality and is able to bind nuclear proteins in vitro. The spacing requirements of the I- and G-box elements in achieving adequate combinatorial interaction were also studied as well as the effect of interchanging the relative position of these elements in the native rbcS promoter arrangement. The results show that helical phasing and distance between the I- and G-box motifs are critical to determine the functionality and transcriptional strength of CMA5. Furthermore, it is shown that the relative position of the IbAM5/I-box composite element and the G-box element is not critical for the enhancer activity of CMA5, as long as the proper distance between them is maintained. Taken together, these results suggest that the light-responsive, plastid-dependent activity of CMA5 requires the synergistic interaction of several DNA-binding transcription factors assembled in a higher-order nucleoprotein complex.

Light-inducible promoters are able to respond to a wide spectrum of light through multiple photoreceptor systems. Several cis-acting elements have been identified as components of light-responsive promoter elements; however, none of these regulatory elements by itself appears to be sufficient to confer light responsiveness; rather, the combination of at least two elements seems to be required. Using phylogenetic structural analysis, we have identified conserved DNA modular arrays (CMAs) associated with light-responsive promoter regions that have been conserved throughout the evolutionary radiation of angiosperms. Here, we report the functional characterization of CMA5, a native 52-bp fragment of the Nicotiana plumbaginifolia rbcS 8B promoter, which contains an I- and a G-box cis-element. CMA5 behaves as a light-responsive minimal unit capable of activating a heterologous minimal promoter in a phytochrome-, cryptochrome-, and plastid-dependent manner. We also show that CMA5 light induction requires HY5 and that downstream negative regulators COP (constitutive photomorphogenic)/DET (de-etiolated) regulate its activity. Our results show that the simplest light-responsive promoter element from photosynthesis-associated genes described to date is the common target for different signals involved in light regulation. The possible mechanism involved in light-transcriptional regulation and tissue specificity of combinatorial elements units is discussed.

The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation., (Copyright 1999 Academic Press.)

In transgenic tobacco plants the regulatory region of the Agrobacterium genes involved in mannopine synthesis (pmas) directs expression in vascular tissues and is induced upon wounding. To identify cis-acting sequences required for the cell-type-specific and wound-inducible expression of this regulatory region, the expression pattern of chimeric genes in which 5' upstream deletions of the pmas 1' promoter direct the expression of the beta-glucuronidase coding sequence, was analyzed in transgenic tobacco plants. It was found that the pmas1' promoter is specifically expressed in phloem cells and that this tissue-specific pattern of expression is the result of the interaction of at least two negative regulatory elements with a positive element that directs expression in many cell types. It was also found that wound induction is mediated by two different mechanisms, one of them involving a dramatic change in tissue-specific expression.