The formation of gametes is a prerequisite for any sexually reproducing organism in order to complete its life cycle. In plants, female gametes are formed in a multicellular tissue, the female gametophyte or embryo sac. Although the events leading to the formation of the female gametophyte have been morphologically characterized, the molecular control of embryo sac development remains elusive. We used single and double mutants as well as cell-specific marker lines to characterize a novel class of gene regulators in Arabidopsis thaliana, the RWP-RK domain-containing (RKD) transcription factors. Morphological and histological analyses were conducted using confocal laser scanning and differential interference contrast microscopy. Gene expression and transcriptome analyses were performed using quantitative reverse transcription-PCR and RNA sequencing, respectively. Our results showed that RKD genes are expressed during distinct stages of embryo sac development. Morphological analysis of the mutants revealed severe distortions in gametophyte polarity and cell differentiation. Transcriptome analysis revealed changes in the expression of several gametophyte-specific gene families (RKD2 and RKD3) and ovule development-specific genes (RKD3), and identified pleiotropic effects on phytohormone pathways (RKD5). Our data provide novel insight into the regulatory control of female gametophyte development. RKDs are involved in the control of cell differentiation and are required for normal gametophytic development.

Summary Plant‐specific EFFECTORS OF TRANSCRIPTION ( ET ) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ET s share a GIY ‐ YIG domain, involved in DNA nicking activity. It was hypothesised that ET s might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana . Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET ‐specific differentially expressed genes and mostly independently characteristic, ET ‐specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

Polystyrene (PS) is a plastic polymer extensively used for food packaging. PS is difficult to decompose and has low recycling rates, resulting in its accumulation in the environment, in the form of microplastic particles causing pollution and harming oceans and wildlife. Degradation of PS by mealworms (Tenebrio molitor) has been suggested as a possible biological strategy for plastic contamination; however, the biodegradation mechanism of PS by mealworms is poorly understood. It is hypothesized that the gut microbiome plays an important role in the degradation of PS by mealworms. This study carried out a comparative analysis of the gut microbiome of Tenebrio molitor larvae under different feeding strategies, and of the formation of degradation compounds (monomers, oligomers). A diet of bran:PS at 4:1 and 20:1 ratios was tested. The diet with the low ratio of bran:PS led to the presence of higher amounts of these compounds, compared to that with the high ratio. In addition, it was demonstrated that the addition of H2O significantly improved the biodegradation of PS monomer and oligomer residues, which could be identified only in the frass. The protein and nitrogen contents in insects’ biomass and frass varied amongst treatments. The diets resulted in differences in the gut microbiota, and three potential bacterial strains were identified as candidates involved in the biodegradation of PS.

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical-chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7-89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.