The premature abscission of flowers and fruits limits crop yield under environmental stress. Drought-induced flower drop in tomato plants was found to be regulated by phytosulfokine (PSK), a peptide hormone previously known for its growth-promoting and immune-modulating activities. PSK formation in response to drought stress depends on phytaspase 2, a subtilisin-like protease of the phytaspase subtype that generates the peptide hormone by aspartate-specific processing of the PSK precursor in the tomato flower pedicel. The mature peptide acts in the abscission zone where it induces expression of cell wall hydrolases that execute the abscission process. Our results provide insight into the molecular control of abscission as regulated by proteolytic processing to generate a small plant peptide hormone.

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions.

In plants, low molecular weight terpenes produced by terpene synthases (TPS) contribute to multiple ecologically and economically important traits. The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production. Two carrot mutants, yellow and cola, which are contrasting in the content of low molecular weight terpenes, were crossed to develop an F2 mapping population. The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene, α-thujene, α-terpinene, γ-terpinene, terpinen-4-ol and 4-carene. The genomic region of this locus includes a cluster of five terpene synthase genes (DcTPS04, DcTPS26, DcTPS27, DcTPS54 and DcTPS55). DcTPS04 and DcTPS54 displayed genotype- and tissue-specific variation in gene expression. Based on the QTL mapping results and the gene expression patterns, DcTPS04 and DcTPS54 were selected for functional characterization. In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene, whereas DcTPS04 is a multiple-product terpene synthase producing α-terpineol as a major product and four additional products including sabinene, β-limonene, β-pinene and myrcene. Furthermore, we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.

Transient expression in Nicotiana benthamiana holds great potential for recombinant protein manufacturing due to its advantages in terms of speed and yield compared to stably transformed plants. To continue improving the quantity of recombinant proteins the plant host will need to be modified at both plant and cellular levels. In attempt to increase leaf mass fraction, we transformed N. benthamiana with the At-CycD2 gene, a positive regulator of the cell cycle. Phenotypic characterization of the T1 progeny plants revealed their accelerated above-ground biomass accumulation and enhanced rate of leaf initiation. In comparison to non-transgenic control the best performing line At-CycD2-15 provided 143 and 140% higher leaf and stem biomass fractions, respectively. The leaf area enlargement of the At-CycD2-15 genotype was associated with the increase of epidermal cell number compensated by slightly reduced cell size. The production capacity of the At-CycD2-15 transgenic line was superior to that of the non-transgenic N. benthamiana. The accumulation of transiently expressed GFP and scFv-TM43-E10 proteins per unit biomass was increased by 138.5 and 156.7%, respectively, compared to the wild type. With these results we demonstrate the potential of cell cycle regulator gene At-CycD2 to modulate both plant phenotype and intracellular environment of N. benthamiana for enhanced recombinant protein yield.