Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual 11 aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis 12 thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 13 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was 14 overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were 15 characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown 16 to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide 17 substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases 18 was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse 19 peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may 20 indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the 21 cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in 22 the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the 23 cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses 24 both important similarities with and distinctions from the already known phytaspases, and is likely to be the most 25 divergent member of the phytaspase family. 26Additional keywords: apoplast, aspartate specificity, proteolysis, subtilisin-like protease. 27 N. V. Chichkova et al. 28 Identification and properties of A. thaliana phytaspase 29Although plant proteases of the phytaspase family are important contributors to stress-induced plant cell death, 30phytaspase of a classical model plant Arabidopsis thaliana has escaped identification thus far. We identified the 31Arabidopsis phytaspase-encoding gene and characterised the recombinant enzyme. Substrate specificity and 32
The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.
Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein ( Nt Phyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of Nt Phyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the Nt Phyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein ( At Phyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The At Phyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from At Phyt, though the elevated level of intracellular At Phyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.
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