Native Aminoacyl-tRNA Synthetase/tRNA Pair Drives Highly Efficient Noncanonical Amino Acid Incorporation in Escherichia coli.

Site-specific noncanonical amino acid (ncAA) mutagenesis in living cells has traditionally relied on heterologous, nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that do not cross-react with their endogenous counterparts. Such heterologous pairs often perform suboptimally in a foreign host cell since they were not evolutionarily optimized to function in the foreign environment. This suboptimal performance restricts the number of ncAAs that can be simultaneously incorporated into a protein. Here, we show that the use of an endogenous aaRS/tRNA pair to drive ncAA incorporation can offer a potential solution to this limitation. To this end, we developed an engineered Escherichia coli strain (ATMY-C321), wherein the endogenous tyrosyl-tRNA synthetase (TyrRS)/tRNA pair has been functionally replaced with an archaeal counterpart, and the release factor 1 has been removed to eliminate competing termination at the UAG nonsense codons. The endogenous TyrRS/tRNACUATyr pair exhibits remarkably efficient nonsense suppression in the resulting cell, relative to established orthogonal ncAA-incorporation systems in E. coli, allowing the incorporation of an ncAA at up to 10 contiguous sites in a reporter protein. Our work highlights the limitations of orthogonal translation systems using heterologous aaRS/tRNA pairs and offers a potential alternative involving the use of endogenous pairs.

Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously.

Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro-electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage-dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.

Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.

High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca(2+) level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K(+) uptake channels with Cs(+), strongly suppressed the cytosolic volume changes. Cs(+) not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca(2+) concentration. A complete loss of voltage-induced Ca(2+) signals occurred when Ca(2+)-permeable plasma membrane channels were simultaneously blocked with La(3+). This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca(2+)-concentration: (i) activation of voltage-dependent Ca(2+)-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca(2+) from intracellular stores.