The peptide SCOOP12 acts on reactive oxygen species homeostasis to modulate cell division and elongation in Arabidopsis primary root.

Small secreted peptides have been described as key contributors to complex signalling networks that control plant development and stress responses. The Brassicaceae-specific PROSCOOP family encodes precursors of Serine riCh endOgenOus Peptides (SCOOPs). In Arabidopsis SCOOP12 has been shown to promote the defence response against pathogens and to be involved in root development. Here, we explore its role as a moderator of Arabidopsis primary root development. We show that the PROSCOOP12 null mutation leads to longer primary roots through the development of longer differentiated cells while PROSCOOP12 overexpression induces dramatic plant growth impairments. In comparison, the exogenous application of synthetic SCOOP12 peptide shortens roots through meristem size and cell length reductions. Moreover, superoxide anion (O2·-) and hydrogen peroxide (H2O2) production in root tips vary according to SCOOP12 abundance. By using reactive oxygen species scavengers that suppress the proscoop12 phenotype, we showed that root growth regulation by SCOOP12 is associated with reactive oxygen species metabolism. Furthermore, our results suggest that peroxidases act as potential SCOOP12 downstream targets to regulate H2O2 production, which in turn triggers cell wall modifications in root. Finally, a massive transcriptional reprogramming, including the induction of genes from numerous other pathways, including ethylene, salicylic acid, and glucosinolates biosynthesis, was observed, emphasizing its dual role in defence and development.

The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins.

The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.

Imaging and analysis of mitochondrial dynamics in living cells.

One of the most striking features of plant mitochondria when visualized in living tissue is their dynamism. The beauty of cytoplasmic streaming, driving, and being driven by the motility of mitochondria and other small organelles belies the complexity of the process. Equally, capturing that dynamism and investigating the genes, proteins, and mechanisms underpinning the processes using molecular cell biology and bioimaging is a complex process. It requires the generation of fluorescent protein constructs, stable transgenic plants sometimes expressing multiple fusions, and generation of mutants, even before one is ready for analytical experimentation. Here, we describe some of the key tools and methods necessary to investigate plant mitochondrial dynamics.

A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis.

Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Pausing of Golgi bodies on microtubules regulates secretion of cellulose synthase complexes in Arabidopsis.

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane-bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325'615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.