ABP1-TMK auxin perception for global phosphorylation and auxin canalization.

The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.

Phytaspase Is Capable of Detaching the Endoplasmic Reticulum Retrieval Signal from Tobacco Calreticulin-3.

Soluble chaperones residing in the endoplasmic reticulum (ER) play vitally important roles in folding and quality control of newly synthesized proteins that transiently pass through the ER en route to their final destinations. These soluble residents of the ER are themselves endowed with an ER retrieval signal that enables the cell to bring the escaped residents back from the Golgi. Here, by using purified proteins, we showed that Nicotiana tabacum phytaspase, a plant aspartate-specific protease, introduces two breaks at the C-terminus of the N. tabacum ER resident calreticulin-3. These cleavages resulted in removal of either a dipeptide or a hexapeptide from the C-terminus of calreticulin-3 encompassing part or all of the ER retrieval signal. Consistently, expression of the calreticulin-3 derivative mimicking the phytaspase cleavage product in Nicotiana benthamiana cells demonstrated loss of the ER accumulation of the protein. Notably, upon its escape from the ER, calreticulin-3 was further processed by an unknown protease(s) to generate the free N-terminal (N) domain of calreticulin-3, which was ultimately secreted into the apoplast. Our study thus identified a specific proteolytic enzyme capable of precise detachment of the ER retrieval signal from a plant ER resident protein, with implications for the further fate of the escaped resident.

Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach.

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.

Rapid translocation of NGR proteins driving polarization of PIN-activating D6 protein kinase during root gravitropism.

Root gravitropic bending represents a fundamental aspect of terrestrial plant physiology. Gravity is perceived by sedimentation of starch-rich plastids (statoliths) to the bottom of the central root cap cells. Following gravity perception, intercellular auxin transport is redirected downwards leading to an asymmetric auxin accumulation at the lower root side causing inhibition of cell expansion, ultimately resulting in downwards bending. How gravity-induced statoliths repositioning is translated into asymmetric auxin distribution remains unclear despite PIN auxin efflux carriers and the Negative Gravitropic Response of roots (NGR) proteins polarize along statolith sedimentation, thus providing a plausible mechanism for auxin flow redirection. In this study, using a functional NGR1-GFP construct, we visualized the NGR1 localization on the statolith surface and plasma membrane (PM) domains in close proximity to the statoliths, correlating with their movements. We determined that NGR1 binding to these PM domains is indispensable for NGR1 functionality and relies on cysteine acylation and adjacent polybasic regions as well as on lipid and sterol PM composition. Detailed timing of the early events following graviperception suggested that both NGR1 repolarization and initial auxin asymmetry precede the visible PIN3 polarization. This discrepancy motivated us to unveil a rapid, NGR-dependent translocation of PIN-activating AGCVIII kinase D6PK towards lower PMs of gravity-perceiving cells, thus providing an attractive model for rapid redirection of auxin fluxes following gravistimulation.

Clathrin-Mediated Endocytosis Delivers Proteolytically Active Phytaspases Into Plant Cells.

Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein (NtPhyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of NtPhyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the NtPhyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein (AtPhyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The AtPhyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from AtPhyt, though the elevated level of intracellular AtPhyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.