Auxin visualized for the first time

Auxin plays a central role in plant life. The hormone regulates various processes, from embryonic development to the formation of roots and the directional growth in response to light and gravity. Auxin binds to specific receptors in the nucleus of a cell, leading to an activation of signaling cascades that coordinate the plant's response to external stimuli.

Although the molecular action of auxin is well understood mechanistically, it has not been feasible so far to directly observe auxin in individual cells. Instead it was only possible to determine the overall response to, and the general presence of auxin. With the new biosensor, it is now possible to visualize auxin directly in individual plant cells. This way, the rapid and dynamic redistribution of auxin, e.g. upon changing the directional root growth, could be viewed for the first time, almost 100 years after the physiological effects of auxin in plants were first described.

The development of this biosensor, in short AuxSen, is the result of an interdisciplinary collaboration between two teams that built strong synergies in plant biology and protein biochemistry. The goal was easily phrased: Plants should produce a protein that glows when auxin is present, allowing the team to visualize auxin distribution with an optical method. However, the implementation was a bit more complex.

The researchers chose a protein from the bacterium E. coli that binds specifically to the amino acid tryptophan and rather poorly to the chemically related auxin. This protein was coupled to two other proteins that fluoresce when excited with light of a specific wavelength. When these two fluorescent proteins come close to each other, for example due to binding another molecule, the excitation energy of one is transferred to the other protein, and a so-called fluorescence resonance energy transfer (FRET) occurs.

AuxSen combines sensitivity with high specificity of auxin binding

The goal of the engineering process was that this FRET effect should only occur when AuxSen is bound to auxin. For this purpose, the starting protein had to be substantially altered in order to bind strongly to auxin but no longer to tryptophan. Biochemistry became crucial in this experimental phase. Crystal structures of the protein complex with tryptophan or auxin were generated, and the team was able to derive predictions about the effects of amino acid exchanges on the binding to auxin. "For us, it was amazing to see that tryptophan and auxin, two closely related molecules, are oriented very differently in the binding pocket," said co-first author Andre C. Stiel. "This made it easier to improve binding to auxin at the expense of tryptophan." In total, about 2,000 variants were generated in an iterative process and these were tested for their specific binding to auxin, finally leading to AuxSen.

Transferring the AuxSen biosensor from the test tube to transgenic plants posed another challenge of finding the right conditions for AuxSen expression. There was a dilemma. On the one hand, a protein with a high binding affinity to auxin was needed in order to detect auxin with high sensitivity, and this protein should be present in sufficient quantity in all cells; on the other hand, the researchers worried about a disruption of normal auxin activity and that plants might suffer. After some testing, a compromise was found. AuxSen is ubiquitously and strongly expressed, but only after induction with a chemical agent and then for a relatively short time, so that the plants would not be harmed.

New and surprising findings on the redistribution dynamics of auxin in plants

What are the key findings? One unexpected finding is the rapid uptake of auxin into the cells, reflecting their ability to respond fast to changing conditions -- after addition of auxin, the team observed a maximum response of AuxSen in the cell nucleus within one to two minutes. Auxin export from the cell appears to be slower, taking about ten minutes for the AuxSen signal to disappear. This difference between fast uptake and slower export might facilitate the directional transport of auxin, for example towards the root tip.

Of particular interest was the rapid auxin redistribution after rotating the plant such that the root tip pointed no longer downward but diagonally upward. Just after one minute, auxin accumulated on the new bottom side of the root tip, and after turning back the root, the former distribution of auxin was restored. "This completely amazed us," commented co-first authors Ole Herud-Sikimic and Martina Kolb. This rapid and reversible response had not been expected. Nor had it been possible to measure this.

The publication of AuxSen is first and foremost a technological breakthrough that has enormous application potential. The conclusion of the two senior authors Birte Höcker from the University of Bayreuth and Gerd Jürgens from the Max Planck Institute for Developmental Biology: "We have shown that both increases and decreases of auxin concentration can be visualized in tissue in real time, which was not possible before. In addition, AuxSen reveals auxin in subcellular areas to which other, indirect auxin reporters have no access. The goal now is to improve the possible applications to other biological problems by optimizing expression systems and using fluorescent proteins with different characteristics. We are now providing the necessary material to the scientific community." AuxSen might well be the starting point to elucidate in the near future how rapid redistribution, in time and space, of auxin in diverse biological contexts mediates the multitude of physiological effects attributed to this remarkable small molecule over the past 100 years.