Generation of an integrated Hieracium genomic and transcriptomic resource enables exploration of small RNA pathways during apomixis initiation.

BACKGROUND: Application of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts. RESULTS: A genomic and transcriptomic resource was developed for Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified. CONCLUSIONS: The established Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium.

Cell-free protein synthesis of membrane (1,3)-ß-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-ß-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.

Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi.

PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na+ or K+ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4-6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.

Characterization of the wheat endosperm transfer cell-specific protein TaPR60.

The TaPR60 gene from bread wheat encodes a small cysteine-rich protein with a hydrophobic signal peptide, predicted to direct the TaPR60 protein to a secretory pathway. It was demonstrated by heterologous expression of recombinant TaPR60 protein that the signal peptide is recognized and cleaved in yeast cells. The full-length gene including promoter sequence of a TaPR60 orthologue was cloned from a BAC library of Triticum durum. A transcriptional promoter-GUS fusion was stably transformed into wheat, barley and rice. The strongest GUS expression in wheat and barley was found in the endosperm transfer cells, while in rice the promoter was active inside the starchy endosperm during the early stages of grain filling. The TaPR60 gene was also used as bait in a yeast two-hybrid screen. Five proteins were identified in the screen, and for some of these prey proteins, the interaction was confirmed by co-immunoprecipitation. The signal peptide binding proteins, TaUbiL1 and TaUbiL2, are homologues of animal proteins, which belong to proteolytic complexes, and therefore may be responsible for TaPR60 processing or degradation of the signal peptide. Other proteins that interact with TaPR60 may have a function in TaPR60 secretion or regulation of this process. Examination of a three dimensional model of TaPR60 suggested that this protein could be involved in binding of lipidic molecules.