Nature's dominoes of hormones

When humans are injured, they feel pain
.
There is a series of indirect relay reactions
between these occurrences.
Similar signal transduction systems are present in all living organisms, including plants
.
When a stimulus emerges, such as as a drought factor that pathogens attack, the plant receives this information through the receptor, analyzes it, sometimes amplifies it, and forms a corresponding response
.
Thus, the signal from the receptor activates a variety of elements such as proteins, ions, low-molecular compounds, etc.
, through which it relays this information to the effectors
that form the answer.
The message is passed between elements in a defined way, which requires the activation of a specific "Google Maps"
.

It's like dominoes, says
Dr.
Krzysztof Jaworski, a professor at the Norwegian National University.
The causative factor, in our case, is the phytohormone auxin, the main intracellular regulator of plant growth and development, like pushing the fingers of dominoes
.
The first block is the receptor, whose activation moves other blocks
.
At the end of this chain is a globule, which is the ultimate factor, which alters the expression of a specific set of genes, inhibits or activates the formation
of proteins that affect the final physiological response of an organism.
But it turns out that the structure and function of the first block (auxin receptor) is not fully understood, and the role of auxin is more complex, which we are demonstrating in our research
.
Scientists knew that the second block would fall, but they didn't know the whole mechanism
of how the first block transferred energy to the next.
In order to do this, adenylate cyclase had to take over, something they didn't know before
.
Adenylate cyclase is an enzyme
that produces periodic AMP (cAMP) cells from ATP.
cAMP is a chemical compound, a signal particle that changes the activity of proteins by linking them, which in turn leads to the rearrangement
of cellular processes.

For 20 years, scientists have thought that auxin receptors do not have any intracellular activity, they have a domain with adenylate cyclase activity, although the processes in which cyclases and their products such as cAMP are involved have not been fully discovered
.
To be sure, they do affect the phenotype, that is, the appearance of plants, mainly their roots
.
They are involved in inhibiting root growth as a response to gravity (organ response to changes in position of reference gravity vectors).

For example, put a plant in a horizontal position – its branches will rise, while its roots will move
down.

Although the main initiators of the collaboration are world authorities in the field of auxins, Polish scientists from NCU have extensive experience in the field of phytocyclic nucleotides, labeling them, analyzing cyclases and phosphodiesterases, enzymes that eat enzymes involved in their metabolism
.
Dr.
Adrianna Szmidt-Jaworska has been expanding their knowledge in the field for over 15 years
.

It was tough at first, says Jaworska, and we were pioneers
in finding plant adenylate cyclases.
We started our research and made some small achievements that began to prove that we were right that both cyclic nucleotides and cyclases are integral elements
in plant transduction trajectories.
Appropriate bioinformatics equipment and high-resolution detection techniques, such as liquid chromatography combined with mass spectrometry tandem (LCMSMSMS), facilitate the work, which allows quantitative analysis
at the picomolar level.
Using the experience of researchers from Tolunia and Professor Chris Gehring from the University of Perugia, scientists from Austria identified regions
of adenylate cyclase in auxin receptors.
"We received a genetic structure with a template, on the basis of which we multiplied the recombinase bacteria," explains
Mateusz Kwiatkowski, a doctoral student at Prof.
Jaworski.
"We wash them and run all the necessary biochemical markers, setting the key parameters
of the enzyme reaction.
At the same time, during directed mutagenesis, we change the structure of the protein, which means that we exclude three functional amino acids in one sequence or in one group, which completely takes away the activity
of the studied adenylate cyclase.
”

Professor Jaworski added: "So far, in our research on adenylate cyclases, we have only focused on the cognitive aspects without mentioning the physiological role
of this group of enzymes.
New articleIt connects our achievements so far and integrates them into existing information transmission systems
.
What's more, it changes our perception
of plant hormone signaling.
”

The study of signaling in plant cells began nearly 30 years ago, when biologists began analyzing signaling cells
.
Calcium ions and periodic AMP and GMP are so-called messengers, which appear for a moment
in response to stimuli.
The presence of periodic nucleotides in plant cells was confirmed
as early as the early 70s of the 20th century.
Although many other reports on the same issue have emerged, many have questioned
the results.
Professor Jaworska said: "I think these doubts are due to the very low cyclic density of cyclic nucleotides in plant cells, 1000 times
higher than those recorded in animal cells.
It is also necessary to add that for many years no tool has been able to accurately mark such low densities
.
Thus, some scientists claim that what we are recording is the hum of measuring devices
.
Currently, we know that periodic nucleotides in plant cells act locally, and the low density seems reasonable
.
We don't need more of them
.
They appear, trigger a reaction, and then disappear.

Thus, two groups of enzymes are involved in the efficient regulation of cAMP and cGMP levels: cyclases are responsible for their synthesis, and phosphorus denitrogenases are involved in their inactivation processes
.
There is no doubt that the moment these enzymes were discovered was a breakthrough
in the study of periodic plant nucleotides.
We started this research thanks to Professor Gehring, because it was he who experimentally demonstrated in 2003 the presence of an enzyme in plant cells that converts the cycle of GTP into circular GMP.

The road to success is not easy because, despite known amino acid sequences and appropriate bioinformatics tools, researchers have not been able to find similarities
between well-characterized animal cyclases and potential plant cyclases.
”

These similarities are found by comparing amino acid sequences – explains Mateusz Kwiatkowski
.

Professor Gehring took a different approach
.
He claims that if we do not observe similarity in sequences, we should look for amino acids, so-called functional amino acids, such as those responsible for binding substrates, which are cofactors
.
Hit the jackpot this time! By using various tools and observing how functional amino acids are arranged, Professor Gehring noticed some kind of
dependence between them.
Building on this discovery, he created a search motivation that allows searching for and identifying protein domains that are characteristic of
cyclases.

In 2003, Professor Gehring discovered and described the first plant guanosine cyclase
.
In 2013, they pioneered the characterization of plant adenylate cyclases and, in the process, created their own research motivation
.

Professor Jaworski said: "Once we started comparing the existing motivations of plant and animal cyclases, we found that they had two significant differences, firstly, their domain structure differed
.
Second, plant cyclase does not constitute a single protein, which is common
in the animal world.
They are part of multi-domain functional proteins, which means that there are at least two functions
.
We already know that there may be more regions with different functions in a protein, and the discovery of this enzyme itself has not caused much confusion
in the scientific community.
If there are only a few centers dealing with this problem, it is difficult to pass the information, and in addition to our team, there are four other centers
in the world.
Fortunately, we are all working together, as one of our colleagues described, to form a family
of circulating nucleotides.
Now, a new center has been added
.
While its adventure began with cyclases and periodic nucleotides, together we are disrupting previous knowledge
of auxins and periodic nucleotides.
I remember when I first started, it was just Professor Gehring and our team
at NCU.
There was a lot of competition between us at first, but then we joined forces
.
The cooperation has gone very well for several years and we have achieved a lot of success
.
Year after year, things get better and better
.
”

The scientists have just returned from a conference dedicated to auxin, which was attended by 150 other experts in the field
.
They presented their research there, which aroused the interest
of many.
Professor Jaworski said the region where cyclase was found in ghrelin receptors changed the authoritative view
of hormone signaling.
Now, it is necessary to consider the circulating nucleotide, which now plays a role that is not very superior, but very critical
.
Mateusz Kwiatkowski predicts that the search for similar centers
of activity in plant hormones will soon begin.
Professor Jaworski said the outlook for plant hormone transduction for plant signaling was sure to change
in the coming years.
I believe it will go
the way Mateusz mentioned.
We also have good reason to think that auxin receptors are not an isolated case
.
We can expect that most proteins with receptor properties can use a similar mechanism that takes into account the involvement
of cyclases and cyclic nucleotides.
It is not necessarily adenylate cyclase, it can be guanosine phosphocyclase
.
The principle is the same, only the subtraction is a little different
.

Mateusz Kwaitkowski says (who just defended his doctoral thesis by demonstrating the existence of phosphodiesterase in plants, the second group of enzymes involved in periodic nucleotide metabolism): "In our next study, we want to focus on finding effectors, proteins that interact with cAMP and cGMP, and enzymes
that inactivate that signaling cell.
Once we reach our goal, the signal transduction process in plant cells, involving plant hormones and periodic nucleotides, will be less mysterious
.
”

Until then, no one would question the presence of
cyclases and cyclic nucleotides in plants.

Adenylate cyclase activity of TIR1/AFB auxin receptor in plants