In Vivo Chemical Genomics with Random Cyclized Peptides.

We have developed and applied a novel strategy that can best be described as in vivo chemical genomics, a concept where populations of any transformable organism may be screened for consequences of novel RNAs or peptides. We created a library of ~800,000 random DNA sequences biased only by third-position nucleotide substitutions that suppress the frequency of termination codons. The sequences may be shuttled to any plant, microbial, or animal expression vector with recombination cloning. We then generated large populations of Arabidopsis thaliana plants, each expressing a randomized DNA sequence, presumably giving rise to synthetic RNA species and/or the peptides they encode. These novel molecules are produced within the context of the cell and have been shown to affect plant biology with a relatively high frequency, as evidenced by diverse phenotypes. This chapter provides the protocols necessary to construct the libraries and isolate plants expressing randomized DNA sequences.

Corrigendum: Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

[This corrects the article on p. 889 in vol. 8, PMID: 28611805.].

Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.