GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate.

Molecular cloning is a crucial technique in genetic engineering that enables the precise design of synthetic transcriptional units (TUs) and the manipulation of genomes. GreenGate and several other modular molecular cloning systems were developed about ten years ago and are widely used in plant research. All these systems define grammars for assembling transcriptional units from building blocks, cloned as Level 0 modules flanked by four-base pair overhangs and recognition sites for a particular Type IIs endonuclease. Modules are efficiently assembled into Level 1 TUs in a hierarchical assembly process, and Level 2 multigene constructs are assembled by stacking Level 1 TUs. GreenGate is highly popular but has three main limitations. First, using ad-hoc overhangs added by PCR and classical restriction/ligation prevents the efficient use of a one-pot, one-step reaction to generate entry clones and domesticate internal sites; second, a Level 1 TU is assembled from a maximum of six modules, which may be limiting for applications such as multiplex genome editing; third, the generation of Level 2 assemblies is sequential and inefficient. GreenGate 2.0 (GG2.0) expands GreenGate features. It introduces additional overhangs, allowing for the combination of up to 12 Level 0 modules in a Level 1 TU. It includes a Universal Entry Generator plasmid (pUEG) to streamline the generation of Level 0 modules. GG2.0 introduces GreenBraid, a convenient method for stacking transcriptional units iteratively for multigene assemblies. Importantly, GG2.0 is backwards compatible with most existing GreenGate modules. Additionally, GG2.0 includes Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing and pre-assembled Level 1 vectors for dexamethasone-inducible gene expression and ubiquitous expression of plasma membrane and nuclear fluorescent markers. GG2.0 streamlines and increases the versatility of assembling complex transcriptional units and their combination.

SNF1-RELATED KINASE 1 and TARGET OF RAPAMYCIN control light-responsive splicing events and developmental characteristics in etiolated Arabidopsis seedlings.

The kinases SNF1-RELATED KINASE 1 (SnRK1) and TARGET OF RAPAMYCIN (TOR) are central sensors of the energy status, linking this information via diverse regulatory mechanisms to plant development and stress responses. Despite the well-studied functions of SnRK1 and TOR under conditions of limited or ample energy availability, respectively, little is known about the extent to which the 2 sensor systems function and how they are integrated in the same molecular process or physiological context. Here, we demonstrate that both SnRK1 and TOR are required for proper skotomorphogenesis in etiolated Arabidopsis (Arabidopsis thaliana) seedlings, light-induced cotyledon opening, and regular development in light. Furthermore, we identify SnRK1 and TOR as signaling components acting upstream of light- and sugar-regulated alternative splicing events, expanding the known action spectra for these 2 key players in energy signaling. Our findings imply that concurring SnRK1 and TOR activities are required throughout various phases of plant development. Based on the current knowledge and our findings, we hypothesize that turning points in the activities of these sensor kinases, as expected to occur upon illumination of etiolated seedlings, instead of signaling thresholds reflecting the nutritional status may modulate developmental programs in response to altered energy availability.