Thread by @dolfweijers

We are extremely pleased to present something quite new from our team. In these TWO @biorxivpreprint, Mark Roosjen, @AndreKuhn8 @WUR and colleagues report the existence of an ancient, rapid auxin response system, its regulators and function. A Tweetorial… (1/23)

Wonderful work by many colleagues has pieced together a response system for the plant signaling molecule auxin to regulate gene expression, a mechanism that found applications in animal cell biology (auxin degron). Two reasons convinced us that another system must exist…(2/23)

First, many cellular responses are simply too fast to involve gene expression. Second, algae respond to auxin, but lack known response proteins elifesciences.org/articles/33399. We hypothesized that an intrinsically rapid system is conserved between land plants and algae… (3/23)

…and explored the idea that protein phosphorylation may be central to such a mechanism. In the first preprint biorxiv.org/cgi/content/sh…, we describe the existence of a phosphorylation-based auxin response system in Arabidopsis. (4/23)

We were shocked to see that within 2 minutes after treatment with natural auxin (but not synthetic or chemically related molecules), close to 1000 proteins changed phosphorylation…(5/23)

Mark Roosjen pushed the sampling process as much as he could, and managed to record phosphoproteomes after 30 seconds of treatment, as well as several other timepoints. Lo and behold… hundreds of differential phosphosites after 30 seconds, and nearly 2 thousand in total…(6/23)

The pathways and proteins targeted are diverse, and likely rewires many cellular processes. We developed a simple web-tool with #AlphaFold2 integration weijerslab.shinyapps.io/AuxPhos to navigate these (7/23)

Interestingly, the phosphoresponse not only requires extracellular auxin binding by ABP1 and its partner TMK1 (as we reported recently –nature.com/articles/s4158…), but also the cytosolic AFB1 receptor – antagonizing ABP1 and TMK1 (8/23)

The dense temporal sampling allowed to use an inference strategy to extract protein kinase – substrate relationships, which indeed recovered known relationships and predicted key kinases (more later…) (9/23)

We used this new resource to explore the regulation of ion fluxes. Many proton pumps are rapidly regulated by auxin. By using Fusicoccin, we found that pH changes themselves are sufficient to trigger part of the auxin-induced phosphorylation changes… (10/23)

…including rapid Calcium influx. This shows that the complex response can be deconvoluted into branches and waves. We hope that this data and resource will be a starting point for renewed, molecular investigation into a poorly illuminated part of auxin biology (11/23)

Auxin also triggers physiological cellular responses in algae, sister to land plants that lack the well-known response system. In the second preprint biorxiv.org/cgi/content/sh…, we asked whether perhaps the rapid phosphorylation response may be deeply conserved (12/23)

Through phosphoproteomics in a liverwort, a moss and two algal species, we find not only that each species has a rapid phosphorylation response, but that a small set of proteins is targeted in ALL species, spanning half a billion years of evolution…(13/23)

We show that algae and land plants share at least two rapid cellular responses to auxin: plasma membrane depolarization and acceleration of cytoplasmic streaming (14/23)

By mining phosphorylation site motifs across species and through kinase-substrate network inference (see before), we identify a deeply conserved RAF-like kinase (MARK) as a likely mediator. Indeed, mutants in both Arabidopsis and Marchantia show strong growth defects…(15/23)

…some linked to auxin response. However, transcriptional responses to auxin are normal. But…mark kinase mutants are strongly affected in auxin phosphoresponse! (16/23)

Finally…we find that in both Arabidopsis and Marchantia, mutants in this MARK kinase are completely insensitive to the rapid response to auxin in cytoplasmic streaming. Thus, the kinase connects rapid phosphorylation to a rapid cellular response (17/23)

We believe that we have found the elusive, ancient auxin response system that explains BOTH rapid responses AND responses in algae. This has a number of important implications (18/23)

First, auxin response is ancient, and mechanistically shared between algae and land plants. One may wonder what its ancestral function is… (19/23)

Second, a rapid response system predated the emergence of a nuclear response system, but was kept in land plants, suggesting distinct roles of the two systems (20/23)

Third, something for auxin researchers to keep in mind: hundreds to thousands of proteins are differentially phosphorylated in the minutes spent waiting for auxin to change gene expression…(21/23)

We hope that these findings will spark new approaches and directions in auxin biology, perhaps unifying the sometimes eclectic set of auxin responses in algae and land plants (22/23)

This has been a fantastic collaboration both within @WUR @SumanthMutte @PoletCarrillo1 @JorisSprakel @_Jasperrx, Sjef Boeren and with teams of Jiri Friml, @matesakf, Takyuki Kohchi, Ryuichi Nishihama. We are grateful to @NWO_Science and @ERC_Research for €€€ (23/23)