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.

AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response.

An environmentally responsive root system is crucial for plant growth and crop yield, especially in suboptimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density while simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture. Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here, we describe a search for proteins that regulate root system architecture (RSA) by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co-purifying proteins. Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here, we show that, in Arabidopsis, AZG1 is a cytokinin (CK) import protein that co-localizes with and stabilizes PIN1, linking auxin and CK transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1-dependent under salt stress. This report therefore identifies a potential point for auxin:cytokinin crosstalk, which shapes RSA in response to NaCl.

Arabidopsis AZG2 transports cytokinins in vivo and regulates lateral root emergence.

Cytokinin and auxin are key regulators of plant growth and development. During the last decade transport mechanisms have turned out to be the key for the control of local and long-distance hormone distributions. In contrast with auxin, cytokinin transport is poorly understood. Here, we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as cytokinin transporter involved in root system architecture determination. Even though purines are substrates for both AZG1 and AZG2, we found distinct transport mechanisms. The expression of AZG2 is restricted to a small group of cells surrounding the lateral root (LR) primordia and induced by auxins. Compared to the wild-type (WT), mutants carrying loss-of-function alleles of AZG2 have higher LR density, suggesting that AZG2 is part of a regulatory pathway in LR emergence. Moreover, azg2 is partially insensitive to exogenous cytokinin, which is consistent with the observation that the cytokinin reporter TCSnpro :GFP showed lower fluorescence signal in the roots of azg2 compared to the WT. These results indicate a defective cytokinin signalling pathway in the region of LR primordia. The integration of AZG2 subcellular localization and cytokinin transport capacity data allowed us to propose a local cytokinin : auxin signalling model for the regulation of LR emergence.

Ureide metabolism in Arabidopsis thaliana is modulated by C:N balance.

Plants can respond and adapt to changes in the internal content of carbon and nitrogen by using organic compounds that widely differ in their carbon/nitrogen ratio. Among them, the amides asparagine and glutamine are believed to be preferred by most plants, including Arabidopsis. However, increases in the ureides allantoin and/or allantoate concentrations have been observed in different plant species under several environmental conditions. In this work, changes in the ratio between carbon skeletons and reduced nitrogen were investigated by varying the concentrations of nitrogen and sucrose in the growth media. Allantoin accumulation was observed when plants were grown in media with high ammonia concentrations. This increase was reverted by adding sucrose as additional carbon source. Moreover, mutant plants with a decreased capability to degrade allantoin showed a compromised growth compared to WT in ammonia supplemented media. Together, our results indicate that allantoin accumulation is induced by low carbon/nitrogen ratio and suggest that its degradation is critical for proper plant growth and development.

Ureide Permease 5 (AtUPS5) Connects Cell Compartments Involved in Ureide Metabolism.

Allantoin is a purine oxidative product involved in long distance transport of organic nitrogen in nodulating legumes and was recently shown to play a role in stress tolerance in other plants. The subcellular localization of enzymes that catalyze allantoin synthesis and degradation indicates that allantoin is produced in peroxisomes and degraded in the endoplasmic reticulum (ER). Although it has been determined that allantoin is mostly synthesized in roots and transported to shoots either for organic nitrogen translocation in legumes or for plant protection during stress in Arabidopsis (Arabidopsis thaliana), the mechanism and molecular components of allantoin export from root cells are still unknown. AtUPS5 (Arabidopsis UREIDE PERMEASE 5) is a transmembrane protein that transports allantoin with high affinity when expressed in yeast. The subcellular fate of splicing variants AtUPS5L (long) and AtUPS5S (short) was studied by tagging them with fluorescent proteins in their cytosolic loops. The capability of these fusion proteins to complement the function of the native proteins was demonstrated by nutritional and salt stress experiments. Both variants localized to the ER, but the AtUPS5L variant was also detected in the trans-Golgi network/early endosome and at the plasma membrane. AtUPS5L and AtUPS5S localization indicates that they could have different roles in allantoin distribution between subcellular compartments. Our data suggest that under nonstress conditions UPS5L and UPS5S may function in allantoin degradation for nutrient recycling, whereas under stress, both genes may be involved in vesicular export allowing allantoin translocation from roots to shoots.