Group B phytocystatins are also expressed in caryopses Altho
Group B phytocystatins are also expressed in caryopses. Although their expression patterns during germination are similar to those of the group A inhibitors, their expression patterns during seed development vary, indicating that they play different roles in the latter process. The highest levels of expression of the corn CC10 (Massonneau et al., 2005) and the triticale TrcC-6 (Simińska et al., 2015) phytocystatins were observed at the end of development and in mature caryopses. Martínez et al. (2009) also found transcripts encoding HvCPI-5 and HvCPI-9 in the mature embryos of barley caryopses but found only a low level of these transcripts or none in developing endosperms during the first 22days after flowering. The presence of inhibitors at late stages of development may be associated with the regulation of cysteine protease activity while the limited proteolysis of storage proteins is ongoing, as well as with the prevention of extensive proteolysis of the accumulated materials (Fig. 3A). The members of the subgroup C1 phytocystatins include triticale (TrcC-7) (Simińska and Bielawski, 2015), barley (HvCPI-6 and 8) (Martínez et al., 2009), rice (OC-VI and VIII) (Martínez et al., 2005a) and corn (CC6 and CC7) phytocystatins (Massonneau et al., 2005). According to Martínez et al. (2009), two barley phytocystatins, HvCPI-6 and HvCPI-8 (group C1), are preferentially expressed in seeds, particularly in the developing endosperm, and are effective inhibitors of the C1A proteases. Simińska and Bielawski (2015) and Massonneau et al. (2005) showed that the highest levels of triticale TrcC-7 and corn CC6 and CC7 phytocystatin transcripts occur during the middle stages of caryopsis development. These cystatins most likely regulate protein accumulation and maturation in the A 419259 trihydrochloride receptor during seed development. Many genes encoding members of the subgroup C2 phytocystatins (Fig. 2) are expressed at barely detectable levels, and the gene products have a low inhibitory capacity or even fail to inhibit most endogenous cysteine protease activities. However, Corre-Menguy et al. (2002) showed that the expression of the gene encoding WC5 occurs during the early stages of seed development. At 8–10days after pollination (DAP), when the triploid endosperm differentiates into the aleurone layer and the starchy endosperm, WC5 reaches its maximal level in the fraction corresponding to the testa, nucellus and aleurone layer. It is known that the surrounding nucellus undergoes a degenerative process during this period, which implies the expression of proteolytic enzymes as well as plasma-membrane disruption (Domínguez and Cejudo, 1998). Therefore, an additional function of WC5 in the aleurone layer or in the embryo is to protect these tissues from the intensive proteolytic activity occurring in the surrounding tissues. In wheat, the period between 12 and 17 DAP corresponds to the transition stage between endosperm differentiation and maturation, when the storage proteins and starch begin to accumulate (Young and Gallie, 2000). At 13 DAP, WC5 protein is found in the endosperm near the aleurone layer. Therefore, this protein is secreted by the aleurone layer and is involved in endosperm differentiation and might control the accumulation of storage proteins. The triticale phytocystatin TrcC-5, which has a high degree of sequence similarity with WC5 and which is expressed only until 17 DAP, most likely controls proteolysis during seed development and storage-protein accumulation. No mRNA corresponding to WC5 or TrcC-5 has been detected in shoots, roots, germinating seeds or young seedlings. These results indicate that phytocystatins of the C2 subgroup also take part in the regulation of proteolysis during seed development. Additionally, studies of Wang et al. (2015) have revealed that transcripts of all identified rice cystatins, independently of their phylogenetic position, have been found during the whole seed development, however their expression levels were different depending on the stage of this process. Most of these cystatins were also detected in the germinating seeds and their expression level decreased gradually upon germination, which contribute to the increase of the activities of cysteine proteases (Wang et al., 2015). These results indicate that phytocystatins have a stage-specific role during the growth of cereal seeds.