From structure to function – a family portrait of plant subtilases

Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant-specific functions, resulting in the present-day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.Instituto de Fisiología Vegeta

The tomato subtilase family includes several cell death-related proteinases with caspase specificity

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

The tomato subtilase family includes several cell death-related proteinases with caspase specificity

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

Post-translational maturation of IDA, a peptide signal controlling floral organ abscission in Arabidopsis

The abscission of sepals, petals and stamens in Arabidopsis flowers is controlled by a peptide signal called IDA (Inflorescence Deficient in Abscission). IDA belongs to the large group of small post-translationally modified signaling peptides that are synthesized as larger precursors and require proteolytic processing and specific side chain modifications for signal biogenesis. Using tissue-specific expression of proteinase inhibitors as a novel approach for loss-of-function analysis, we recently identified the peptidases responsible for IDA maturation within the large family of subtilisin-like proteinases (subtilases; SBTs). Further biochemical and physiological assays identified three SBTs (AtSBT5.2, AtSBT4.12, AtSBT4.13) that cleave the IDA precursor to generate the N-terminus of the mature peptide. The C-terminal processing enzyme(s) remain(s) to be identified. While proline hydroxylation was suggested as additional post-translational modification required for IDA maturation, hydroxylated and non-hydroxylated IDA peptides were found to be equally active in bioassays for abscission

Analysis of Peptide Hormone Maturation and Processing Specificity Using Isotope-Labeled Peptides

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Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation

International audienceMost peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana . Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation

Structural basis of substrate specificity of plant 12-oxophytodienoate reductases

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation

International audienceSignificance Pollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells, which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here, we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development