1. Introduction
Leaf is in general the main site of photosynthesis and acts as a carbohydrate source for nutrients to support the growth in sink organs of plants. Therefore, its longevity and senescent level may affect the photosynthesis efficiency and thus crop yield. There are endogenous and exogenous factors affecting leaf senescence, including plant growth regulators, sucrose starvation, dark, cold, drought, salt, wound, pathogen infection and insect attack (Yoshida, 2003; Lim et al., 2007). Leaf senescence is the final developmental stage of leaves and has been considered as a type of programmed cell death. During leaf senescence, it is not only a degradative process but also a recycling one. Therefore, macromolecules and organelles can be degraded into small molecules, salvaged and mobilized from the senescent cells to other sinks, such as young leaves, developing seeds, or storage tissues (Buchanan-Wollaston, 1997; Quirino et al., 2000).
In sweet potato, several morphological, biochemical and physiological changes have also be observed during leaf senescence, including leaf yellowing, decrease of chlorophyll contents, reduction of photochemical Fv/Fm, elevation of H2O2 amount, increase of plastoglobuli number in chloroplast, activation of senescence-associated gene expression, and finally cell death (Chen et al., 2000; Chen et al., 2003; Chen et al. 2010a). Several full-length cDNAs encoding putative isocitrate lyase, papain-like cysteine proteases and asparaginyl endopeptidase, have been cloned from senescent leaves (Chen et al., 2000, 2004, 2006, 2008, 2009, 2010b), which likely play roles in association with lipid degradation and gluconeogenesis, and protein degradation and re-mobilization. These data support the occurrence of macromolecule and organelle degradation into small molecules for recycling and re-mobilization during sweet potato leaf senescence.
During senescence, breakdown of leaf proteins by proteases provides a large pool of cellular nitrogen for recycling (Makino & Osmond, 1991). In plants, different degradation pathways have been described and the vacuolar degradation pathway is assumed to be involved in bulk protein degradation by virtue of the resident proteases in the vacuole (Vierstra, 1996). There are two types of vacuoles described in plants: the storage vacuole and the lytic vacuole (Marty, 1999). Protein storage vacuoles are found in seed tissues and accumulate proteins that are re-mobilized and used as the main nutrient resource for germination (Senyuk et al., 1998; Schlereth et al., 2001). Most cells in vegetative tissues have lytic vacuoles, containing a wide range of proteases in an acidic environment. Substrate proteins must be transported and sequestered into these lytic vacuoles before degradation. Therefore, senescence-associated vacuoles are lytic vacuoles and involved in the degradation of imported chloroplast proteins in tobacco leaves (Martı´nez et al., 2008).
The molecular mechanisms for vacuolar protein degradation and nutrient recycling pathway in senescent leaves are generally not clear. Phylogenetic tree analysis indicated that sweet potato asparaginyl endopeptidase (SPAE) exhibited high amino acid sequence identities and closely-related association with plant vacuolar processing enzymes (VPEs) or legumains, including legumain-like protease LLP of kidney bean (
2. Association of vacuolar processing enzyme and papain-like cysteine protease with seed storage globulin protein degradation
Vacuolar processing enzyme is a novel group of cysteine endopeptidase and has recently been found in seeds. The enzyme exhibits strict cleavage specificity for the peptide bonds of seed globulin storage proteins with asparagines at the P1 position, and is called asparaginyl endopeptidase (Ishii, 1994). The substrate specificity was observed with purified asparaginyl endopeptidases from developing seeds of castor bean (Hara-Nishimura et al., 1991) and soybean (Scott et al., 1992; Hara-Nishimura et al., 1995), from mature seeds of jack bean (Abe et al., 1993), and from germinating seeds of vetch (Becker et al., 1995). Many seeds accumulate protein reserves in the storage vacuoles, and a number of these proteins undergo proteolytic cleavage, including the 7S and 11S seed storage globulins (Müntz & Shutov, 2002). The 11S seed globulin storage proteins are synthesized as precursors and are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase during seed development (Ishii, 1994). In castor bean and soybean seeds, vacuolar processing enzymes were found in the protein bodies and likely associated with the conversion of proproteins into their corresponding mature forms in vacuoles (Hara-Nishimura et al., 1991; Shimada et al., 1994).
Asparaginyl endopeptidases also play a role with bulk degradation and mobilization of storage proteins during seed germination and seedling growth. For example, the asparaginyl endopeptidase, which was also called “legumain-like proteinase” (LLP), was isolated from cotyledons of kidney bean (
For papain-like cysteine protease, the vacuolar SH-EP is synthesized in cotyledons of germinated
Many vacuolar enzymes are synthesized as pro-proteins and become active after proteolytically processed. In seed storage tissues, specific endoplasmic reticulum (ER)-derived compartments containing precursors of cysteine proteases have been described (Chrispeels & Herman, 2000; Toyooka et al., 2000; Hayashi et al., 2001; Schmid et al., 2001). Germination of the seeds induces the expression and processing of those proteases into the mature active forms, which in turn participate in the degradation of cellular materials in storage tissues and provide nutrients to the growing embryo. The mechanism of asparaginyl endopeptidases (VmPE-1) and papain-like cysteine protease (SH-EP) associated with bulk seed storage globulin protein degradation has been studied in
3. Characterization of sweet potato asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2
Recently, similar compartments have also been described in vegetative tissues of
3.1. SPAE
Asparaginyl endopeptidase
In sweet potato,
3.2. SPCP2
4. Ectopic expression of asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2 in transgenic Arabidopsis
Sweet potato full-length cDNAs of asparaginyl endopeptidase
4.1. Expression of sweet potato asparaginyl endopeptidase SPAE altered seed and silique development in transgenic Arabidopsis
Three transgenic
Transgenic
Expression of sweet potato
4.2. Expression of sweet potato papain-like cysteine protease SPCP2 altered seed and silique development and enhanced stress tolerance in transgenic Arabidopsis
Transgenic
Expression of sweet potato SPCP2 in transgenic
Expression of sweet potato
5. Correlation of cysteine protease expression with storage protein degradation in sweet potato storage root during sprouting
In sweet potato storage root, trypsin protease inhibitors (TIs) are the main storage proteins and composed of a multiple gene family. It has been implicated that cysteine proteases are likely associated with the degradation of storage root trypsin inhibitors during sprouting (Huang et al., 2005). Therefore, expression and correlation of sweet potato asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2 with the degradation and mobilization of the two major storage root trypsin inhibitor bands during sprouting were studied. The sprouts appeared and were visible within the first week of incubation of storage root at room temperature, whereas, degradation of trypsin inhibitors became significant in the later incubation. RT-PCR analysis of
Sweet potato asparaginyl endopeptidase
6. Conclusion
Sweet potato asparaginyl endopeptidase
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