The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

PP2A-3 interacts with ACR4 and regulates formative cell division in the Arabidopsis root.

In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

Arabidopsis research requires a critical re-evaluation of genetic tools.

HIGHLIGHT: An increasing number of reports question conclusions based on loss-of-function lines that have unexpected genetic backgrounds. In this opinion paper, we urge researchers to meticulously (re)investigate phenotypes retrieved from various genetic backgrounds and be critical regarding some previously drawn conclusions. As an example, we provide new evidence that acr4-2 mutant phenotypes with respect to columella stem cells are due to the lack of ACR4 and not - at least not as a major contributor - to a mutation in QRT1. In addition, we take the opportunity to alert the scientific community about the qrt1-2 background of a large number of Syngenta Arabidopsis Insertion Library (SAIL) T-DNA lines, a feature that is not commonly recognized by Arabidopsis researchers. This qrt1-2 background might have an important impact on the interpretation of the results obtained using these research tools, now and in the past. In conclusion, as a community, we should continuously assess and - if necessary - correct our conclusions based on the large number of (genetic) tools our work is built on. In addition, the positive or negative results of this self-criticism should be made available to the scientific community.

Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome reveal growth regulators.

Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth.

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions.

Peptides fulfill a plethora of functions in plant growth, development, and stress responses. They act as key components of cell-to-cell communication, interfere with signaling and response pathways, or display antimicrobial activity. Strikingly, both the diversity and amount of plant peptides have been largely underestimated. Most characterized plant peptides to date acting as small signaling peptides or antimicrobial peptides are derived from nonfunctional precursor proteins. However, evidence is emerging on peptides derived from a functional protein, directly translated from small open reading frames (without the involvement of a precursor) or even encoded by primary transcripts of microRNAs. These novel types of peptides further add to the complexity of the plant peptidome, even though their number is still limited and functional characterization as well as translational evidence are often controversial. Here, we provide a comprehensive overview of the reported types of plant peptides, including their described functional and structural properties. We propose a novel, unifying peptide classification system to emphasize the enormous diversity in peptide synthesis and consequent complexity of the still expanding knowledge on the plant peptidome.

The growing story of (ARABIDOPSIS) CRINKLY 4.

Receptor kinases play important roles in plant growth and development, but only few of them have been functionally characterized in depth. Over the past decade CRINKLY 4 (CR4)-related research has peaked as a result of a newly discovered role of ARABIDOPSIS CR4 (ACR4) in the root. Here, we comprehensively review the available (A)CR4 literature and describe its role in embryo, seed, shoot, and root development, but we also flag an unexpected role in plant defence. In addition, we discuss ACR4 domains and protein structure, describe known ACR4-interacting proteins and substrates, and elaborate on the transcriptional regulation of ACR4 Finally, we address the missing knowledge in our understanding of ACR4 signalling.

The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators.

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

Proteome Analysis of Arabidopsis Roots.

Plant health and development are directly depending on a plant's ability to react to a constantly changing environment. Sensing of water and nutrition levels and of the biotic environment is vital for a plant, making the root one of the key plant organs. Proteins are the key molecules that play numerous roles in a cell's everyday life. Quantitative proteome profiling of roots can provide a global overview on the molecular regulatory mechanisms and networks involved in plant growth and development and abiotic and biotic stress responses. Here, we provide a detailed proteomics workflow on Arabidopsis thaliana roots from plant growth up to proteomics data analysis.

The Auxin-Regulated CrRLK1L Kinase ERULUS Controls Cell Wall Composition during Root Hair Tip Growth.

Root hairs facilitate a plant's ability to acquire soil anchorage and nutrients. Root hair growth is regulated by the plant hormone auxin and dependent on localized synthesis, secretion, and modification of the root hair tip cell wall. However, the exact cell wall regulators in root hairs controlled by auxin have yet to be determined. In this study, we describe the characterization of ERULUS (ERU), an auxin-induced Arabidopsis receptor-like kinase, whose expression is directly regulated by ARF7 and ARF19 transcription factors. ERU belongs to the Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) subfamily of putative cell wall sensor proteins. Imaging of a fluorescent fusion protein revealed that ERU is localized to the apical root hair plasma membrane. ERU regulates cell wall composition in root hairs and modulates pectin dynamics through negative control of pectin methylesterase (PME) activity. Mutant eru (-/-) root hairs accumulate de-esterified homogalacturonan and exhibit aberrant pectin Ca2+-binding site oscillations and increased PME activity. Up to 80% of the eru root hair phenotype is rescued by pharmacological supplementation with a PME-inhibiting catechin extract. ERU transcription is altered in specific cell wall-related root hair mutants, suggesting that it is a target for feedback regulation. Loss of ERU alters the phosphorylation status of FERONIA and H+-ATPases 1/2, regulators of apoplastic pH. Furthermore, H+-ATPases 1/2 and ERU are differentially phosphorylated in response to auxin. We conclude that ERULUS is a key auxin-controlled regulator of cell wall composition and pectin dynamics during root hair tip growth.

A phylogenetic approach to study the origin and evolution of the CRINKLY4 family.

Cell-cell communication plays a crucial role in plant growth and development and relies to a large extent on peptide ligand-receptor kinase signaling mechanisms. The CRINKLY4 (CR4) family of receptor-like kinases is involved in a wide range of developmental processes in plants, including mediating columella stem cell identity and differentiation in the Arabidopsis thaliana root tip. Members of the CR4 family contain a signal peptide, an extracellular part, a single-pass transmembrane helix and an intracellular cytoplasmic protein kinase domain. The main distinguishing features of the family are the presence of seven "crinkly" repeats and a TUMOR NECROSIS FACTOR RECEPTOR (TNFR)-like domain in the extracellular part. Here, we investigated the evolutionary origin of the CR4 family and explored to what extent members of this family are conserved throughout the green lineage. We identified members of the CR4 family in various dicots and monocots, and also in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. In addition, we attempted to gain insight in the evolutionary origin of different CR4-specific domains, and we could detect "crinkly" repeat containing proteins already in single celled algae. Finally, we related the presence of likely functional CR4 orthologs to its best described signaling module comprising CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 40 (CLE40), WUSCHEL RELATED HOMEOBOX 5 (WOX5), CLAVATA 1 (CLV1), and ARABIDOPSIS CR4 (ACR4), and established that this module likely is already present in bryophytes and lycophytes.