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The diversity of rice phytocystatins

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Abstract

Phytocystatins encompass a family of plant competitive cysteine proteinase inhibitors. They are encoded by part of a conserved monophyletic group of genes that are found in all eukaryotes. The primary targets of phytocystatins are papain-like cysteine proteinases. However, a group of larger phytocystatins is also able to inhibit proteinases such as legumains. Phytocystatins have been implicated in several physiological processes and act within an intricate proteolytic regulatory network. The present work characterizes the gene family of rice phytocystatins, which contains twelve genes with different features. Phylogenetic analyses cluster rice phytocystatins into three main groups. Group 1 is composed of OcI, OcIII and OcXII and is nearly ubiquitous and highly expressed in plants under normal and stressed conditions including salt, wounding, ABA or a fungal elicitor such as chitosan. Rice phytocystatins can contribute to plant senescence and may exhibit an inverse correlation between their gene expression and the activities of their target proteinases. This work contributes to clarifying the roles of individual phytocystatin genes in plant processes such as germination and response to environmental stresses.

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Acknowledgments

This work was supported by CNPq Grant 478417/2012-8. R. Margis has a research fellowship 307868/2011-7 from CNPq and A. P. Christoff a CAPES Ph.D. fellowship.

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Correspondence to Rogerio Margis.

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Communicated by A. K. Tyagi.

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Supplementary material 1 (DOC 241 kb)

438_2014_892_MOESM2_ESM.tif

Supplementary material 2 (TIFF 5592 kb). Fig. S1. Phylogenetic tree of phytocystatins. The protein sequences of 26 plant and algae genomes were analyzed to reconstruct a phylogenetic tree with the Bayesian method. Only the common N-terminal gene sequences were used in the analysis. The branch supports represent the posterior probability values, and the three main groups are identified with circled numbers. Rice phytocystatins are highlighted in red

438_2014_892_MOESM3_ESM.tif

Supplementary material 3 (TIFF 4141 kb). Fig. S2. Rice phytocystatin gene expression patterns. a RNAseq libraries from root and leaf (Arenhart et al. 2013) were analyzed. The RPKM method was used to normalize the read numbers. b Microarray datasets from the Rice Oligonucleotide array were evaluated to identify the phytocystatin gene expression pattern through several plant development stages. Gene expression levels are measured according to the right-hand scale bar, on which yellow represents the more expressed genes. c RNAseq libraries from different rice tissues SRP008821 were mapped against the rice phytocystatins transcripts and the number of reads normalized with RPKM method. d RNAseq experiments from rice infected with compatible and incompatible blast fungus (Magnaporthe grisea), DRP000568. Reads were normalized using RPKM and letters above the error bars indicate statistical Kruskal–Wallis differences for each experimental triplicate

438_2014_892_MOESM4_ESM.tif

Supplementary material 4 (TIFF 739 kb). Fig. S3. Relative cysteine protease activity during rice germination. Papain and legumain protease activities were measured in total protein extracts (per mg of protein) from stages of dry seed (0), 0.5, 1, 2, 4, and 8 days after the beginning of plant germination. Rice samples from 0, 0.5, 1, 2, 4 and 8 dag were collected in triplicate, frozen and macerated in liquid nitrogen. The ground material was thawed on ice and mixed with 1 mL of extraction buffer per 100 mg of sample. Papain-like activities were measured at 410 nm, with 2 mM Bz-DL-Arg-βNA-HCl colorimetric substrate (BACHEM). The extraction buffer was a phosphate buffer pH 6.0, containing 1 mM DTT and 2 mM EDTA. Legumain proteinase activity was measured with 1 mM Z-Ala-Ala-Asn-AMC fluorigenic substrate (BACHEM) in an extraction buffer of citrate–phosphate pH 5.4, containing 1 mM DTT and 2 mM EDTA. Free AMC release was measured at excitation and emission wavelengths 360 and 460 nm, respectively. The protein concentration of the samples was determined in triplicate with a serial Bradford dilution (Bradford 1976). These data represent the results of three independent experiments. The replicate averages were compared using an ANOVA one-way statistical test and Duncan’s test; significance set at p < 0.05

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Christoff, A.P., Margis, R. The diversity of rice phytocystatins. Mol Genet Genomics 289, 1321–1330 (2014). https://doi.org/10.1007/s00438-014-0892-7

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