Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains

Abstract

An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1. In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS-PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce

“…Next, in‐solution protein digestion was performed with commercially available trypsin, as described previously (León et al ., 2013). The tryptic peptides were desalted and fractionated with the use of a custom reversed‐phase (C18) microcolumn (Franc et al ., 2012) and subsequently analyzed by LC‐MS (Simerský et al ., 2017). The acquired MS data were searched against P. abies ‐specific protein database (Grebe et al ., 2019) employing MaxQuant software v.1.6.10.43 (Beck et al ., 2015; Tyanova et al ., 2016) with Andromeda search engine (Cox et al ., 2011).…”

Chemical Driving the Subtype Selectivity of Phytohormone Receptors Is Beneficial for Crop Productivity

“…Leveraging CBP1 as a prototypical protein model in wheat, a total of 21 proteins displaying a marked affinity for 6-BA were successfully isolated. These proteins were found to be intricately involved in diverse aspects related to plant cell functions, such as responses to abiotic stress, carbohydrate metabolism, senescence, embryonic development, cell death, and other life processes . While the current evidence supporting CBP1 as a CTK receptor remains preliminary, there exists substantial room for further exploration of other CBPs.…”

“…These proteins were found to be intricately involved in diverse aspects related to plant cell functions, such as responses to abiotic stress, carbohydrate metabolism, senescence, embryonic development, cell death, and other life processes. 63 While the current evidence supporting CBP1 as a CTK receptor remains preliminary, there exists substantial room for further exploration of other CBPs. This exploration encompasses investigating the binding capabilities of these affinity proteins with natural hormones, elucidating their roles in plant signal transduction pathways and potentially classifying them as novel hormone receptors.…”

The origin and early evolution of cytokinin signaling

“…In fact, completely different signaling pathways for cytokinin might exist, or cytokinins might work as allosteric regulators of key enzymes in specific metabolic pathways. A proteomic approach identified numerous cytokinin-binding proteins that could be involved in such pathways ( Simerský et al., 2017 ).…”