Analysis of Peptide Hormone Maturation and Processing Specificity Using Isotope-Labeled Peptides.

Many peptide hormones and growth factors in plants, particularly the small posttranslationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional posttranslational modifications may contribute to bioactivity. To what extent these posttranslational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not posttranslational modifications contribute to cleavage site recognition. Here, we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of posttranslational modifications on processing and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation.

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis.

Germination sensu stricto in Arabidopsis involves seed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [1, 2]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [3-5]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [6]. Although krs mutants have no defects in germination sensu stricto, they show delayed cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination and thus promotes seed-coat shedding and rapid seedling establishment.

Peptide signalling during angiosperm seed development.

Cell-cell communication is pivotal for the coordination of various features of plant development. Recent studies in plants have revealed that, as in animals, secreted signal peptides play critical roles during reproduction. However, the precise signalling mechanisms in plants are not well understood. In this review, we discuss the known and putative roles of secreted peptides present in the seeds of angiosperms as key signalling factors involved in coordinating different aspects of seed development.