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  • The transcriptome analysis of barley seeds in two tissue

    2019-11-15

    The transcriptome analysis of barley seeds in two tissue fractions: starchy endosperm/aleurone and embryo/scutellum, during maturation, desiccation and germination, revealed the large group of GA-responsive genes (Sreenivasulu et al., 2008). These transcripts have been divided into two groups. Nearly all genes from the first group were synthesized and stored during seed maturation and used during germination, whereas the transcripts of the second group were newly synthesized during germination (Sreenivasulu et al., 2008). Among genes of both groups there are cysteine proteases from the papain family. Finnie et al. (2011) in the study of intracellular and secreted proteomes of barley aleurone layer also identified three intracellular and nine extracellular proteases from the papain family. Numerous studies indicate that plant cysteine proteases from the papain family (C1A) are crucial in degradation and mobilization of storage proteins in cereal seeds (Mikkonen et al., 1996, Prabucka and Bielawski, 2004, Shi and Xu, 2009). The increasing activity of these proteases during the ongoing process of germination, their high affinity for natural storage proteins and their broad substrate specificity confirm their role in storage protein degradation (Mikola et al., 2001). The proteases are synthesized as inactive or weakly active precursors and become active autocatalytically or with the aid of processing amprolium (Grudkowska and Zagdańska, 2004). Subsequently, they are transported via the trans-Golgi network and stored in the vacuoles or externally secreted (Cambra et al., 2012, Grudkowska and Zagdańska, 2004). All of the known C1A proteases that have been isolated from many types of seeds are characterized by a low molecular mass (21–40kDa), acidic pH optimum, low pI and high sensitivity to specific inhibitors called cystatins (Bottari et al., 1996, Koehler and Ho, 1990). Due to their similarity to mammalian proteases, they have been grouped as cathepsin L-, H-, B- and F-like proteases (Martínez and Diaz, 2008). Plant cathepsin B-like proteases are characterized by a high sequence similarity to the human cathepsin B, while H- and L-like proteases, despite conserved common motifs in the prosequence, are evolutionarily separated (Martínez and Diaz, 2008). In turn, plant F-like enzymes, like human cathepsin F, have an additional N-terminal segment which shares structural similarities with cystatins (Cambra et al., 2012, Wang et al., 1998, Santamaría et al., 1999, Nagler et al., 1999). Cathepsin L- and H- proteases have been identified in several cereal seeds. Cysteine proteases amprolium EP-A and EP-B, belonging to the cathepsin L-like family, are the main enzymes responsible for degradation of storage proteins in barley seeds (Mikkonen et al., 1996). In wheat, three isoforms of protease EP-A are known. In barley EP-A and EP-B are synthesized in the aleurone layer in response to gibberellins. EP-A begins the process of protein degradation and then is supported by protease EP-B (Kiyosaki et al., 2007, Mikkonen et al., 1996). Gibberellins also control the synthesis of the barley cysteine protease, aleurain, from the cathepsin H-like family (Rogers et al., 1985). Based on an aleurain cDNA library, three cysteine proteases from rice seeds (oryzains α, β and γ) were identified (Watanabe et al., 1991). Oryzain α and β are cathepsin L-like proteases while oryzain γ belongs to the cathepsin H-like family (Watanabe et al., 1991). The cathepsin L-like family includes also REP-1 and WEB-1 which digest the reserve proteins of rice and wheat, respectively (Kato and Minamikawa, 1996, Sutoh et al., 1999). Moreover, cysteine protease activities dominate in the germinating endosperm of triticale seeds. Prabucka and Bielawski (2004) observed the activities of seven triticale cysteine proteases, EP2–EP8. EP3 and EP4, were active during the first two days after imbibition, whereas EP8, a homologue of barley EP-A, was the most active on the third day of germination (Prabucka and Bielawski, 2004). Analysis of its transcript level revealed that EP8 is expressed in both developing and germinating triticale seeds, although the activity of this protease was not detected in any of the extracts of the developing seeds (Prabucka et al., 2013). These results suggest that mRNA of EP8 can be synthesized during development and stored in the dry seed, then is used during germination, or the activity of this enzyme is inhibited by specific inhibitors (Prabucka et al., 2013, Sreenivasulu et al., 2008). The genes encoding cathepsin B-like proteases are present as small multi-gene families in some species such as Nicotiana and wheat (Cejudo et al., 1992). In wheat six independent clones have been identified and classified in three groups in which Al16, Al20 and Al21 are representatives (Cejudo et al., 1992). The genes of cathepsin B-like protease, orthologous to the wheat A121, have been also identified in seeds of barley (HvCathB) and Brachypodium distachyon (BdCathB) (Cejudo et al., 1992, González-Calle et al., 2014, Martínez et al., 2003b). The expression of these genes is highly induced in the aleurone layer of germinating seeds, while barley HvCathB was also detected in developing endosperm and wheat Al21 was expressed in the scutellar parenchyma (Cejudo et al., 1992, Martínez et al., 2003b).