SERKs regulate embryonic cuticle integrity through the TWS1-GSO1/2 signaling pathway in Arabidopsis.

The embryonic cuticle integrity is critical for the embryo to separate from the neighboring endosperm. The sulfated TWISTED SEED1 (TWS1) peptide precursor generated in the embryo diffuses through gaps of the nascent cuticle to the surrounding endosperm, where it is cleaved by ABNORMAL LEAF SHAPE1 (ALE1) and becomes an active mature form. The active TWS1 is perceived by receptor-like protein kinases GASSHO1 (GSO1) and GSO2 in the embryonic epidermal cells to start the downstream signaling and guide the formation of an intact embryonic cuticle. However, the early signaling events after TWS1 is perceived by GSO1/2 are still unknown. Here, we report that serk1/2/3 embryos show cuticle defects similar to ale1, tws1, and gso1/2. Genetic and biochemical analyses were performed to dissect the signaling pathway mediated by SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs) during cuticle development. SERKs function with GSO1/2 in a common pathway to monitor the integrity of the embryonic cuticle. SERKs interact with GSO1/2, which can be enhanced dramatically by TWS1. The phosphorylation levels of SERKs and GSO1/2 rely on each other and can respond to and be elevated by TWS1. Our results demonstrate that SERKs may function as coreceptors of GSO1/2 to transduce the TWS1 signal and ultimately regulate embryonic cuticle integrity.

CASPARIAN STRIP INTEGRITY FACTOR (CIF) family peptides - regulator of plant extracellular barriers.

In multicellular organisms, water and most of the small molecules, such as nutrients, toxic substances, and signaling compounds, move freely through extracellular spaces, depending on their biochemical nature. To restrict the simple diffusion of small molecules, multicellular organisms have evolved extracellular barriers across specific tissue layers, such as tight junctions in the animal epithelium. Similar extracellular barriers are also generated in plants through the accumulation of hydrophobic chemicals, such as lignin or cutin, although the detailed molecular mechanisms underlying this process remain elusive. Here, I summarize recent advances in extracellular barrier formation in plants by focusing mainly on CASPARIAN STRIP INTEGRITY FACTOR (CIF) family peptides, which trigger the spatially precise deposition of designated cell wall components, enabling plants to establish transcellular barrier networks correctly. The genome of Arabidopsis thaliana, a model plant species, harbors five CIF genes, which encode propeptides which are processed into small secreted peptides of 21-24 amino acids. Sulfation of tyrosine residues in CIF peptides ensures their full bioactivity and high-affinity binding to their receptors SCHENGEN3/GASSHO1 (SGN3/GSO1) and GSO2 in vitro. Additionally, in vivo analysis shows that physical restriction of CIF peptide diffusion and the subcellular localization of a signaling module and expression patterns of a peptide processing enzyme specify the location of signal activation. Thus, the CIF peptide family provides fascinating models for understanding mature peptide biogenesis and spatially limited signal activation with small diffusive molecules.

Pronymphs, hatching, and proboscis assembly in leafhoppers and froghoppers (Hemiptera, Cicadellidae and Aphrophoridae).

Pharate 1st instar nymphs enclosed in the embryonic cuticle, referred to as pronymphs, were studied in a froghopper Aphrophora pectoralis Mats. (Aphrophoridae) and the leafhoppers Oncopsis flavicollis (L.), Populicerus populi (L.), Alebra wahlbergi (Boh.), Igutettix oculatus (Lindb.), and Scenergates viridis (Vilb.) (Cicadellidae). The species vary in the relative length of the pronymphal antennae and details of sculpturing of the cephalic region. No egg bursting structures were observed, except small denticles on the crown region of S. viridis pronymphs. Rudimentary mandibular and maxillary stylets of a pronymph are external, short, tubular appendages containing tips of the corresponding nymphal stylets, whose more basal parts develop inside of the head. Casting off of the embryonic cuticle results in the nymphal stylets being passively pulled out and assuming a close-set parallel orientation. Once the sheaths of unsclerotized cuticle secreted by the peripodial epithelium and enveloping each developing stylet have been cast off with the exuviae, the bare stylets become squeezed and interlocked into a functional bundle. The roles of the maxillary plates, clypeus, labrum, and labium in the stylet bundle assembly are discussed. The process repeats after each molt.

Molecular characterization of Rhodnius prolixus' embryonic cuticle.

The embryonic cuticle (EC) of Rhodnius prolixus envelopes the entire body of the embryo during hatching and provides physical protection, allowing the embryo to pass through a narrow chorionic border. Most of the knowledge about the EC of insects is derived from studies on ultrastructure and secretion processes during embryonic development, and little is known about the molecular composition of this structure. We performed a comprehensive molecular characterization of the major components extracted from the EC of R. prolixus, and we discuss the role of the different molecules that were identified during the eclosion process. The results showed that, similar to the post-embryonic cuticles of insects, the EC of R. prolixus is primarily composed of carbohydrates (57%), lipids (19%), and proteins (8%). Considering only the carbohydrates, chitin is by far the major component (approximately 70%), and it is found primarily along the body of the EC. It is scarce or absent in its prolongations, which are composed of glycosaminoglycans. In addition to chitin, we also identified amino (15%), neutral (12%) and acidic (3%) carbohydrates in the EC of R. prolixus. In addition carbohydrates, we also identified neutral lipids (64.12%) and phospholipids (35.88%). Proteomic analysis detected 68 proteins (55 were identified and 13 are hypothetical proteins) using the sequences in the R. prolixus genome (http://www.vectorbase.org). Among these proteins, 8 out of 15 are associated with cuticle metabolism. These proteins are unequivocally cuticle proteins, and they have been described in other insects. Approximately 35% of the total proteins identified were classified as having a structural function. Chitin-binding protein, amino peptidase, amino acid oxidase, oxidoreductase, catalase and peroxidase are all proteins associated with cuticle metabolism. Proteins known to be cuticle constituents may be related to the function of the EC in assisting the insect during eclosion. To our knowledge, this is the first study to describe the global molecular composition of an EC in insects.

Chitin deposition on the embryonic cuticle of Rhodnius prolixus: the reduction of CHS transcripts by CHS-dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph.

In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R. prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R. prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R. prolixus females lasted up to 3 egg-laying cycles (∼100 days), pointing to R. prolixus as a useful model for developmental studies.