| Abstract: | Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.Semicrystalline starch enables photosynthetic eukaryotes to store large quantities of Glc over extended time periods compared with other species, in which the soluble polymer glycogen functions to store carbohydrate reserves (Ball and Morell, 2003). Eukaryotes gained the capacity to photosynthesize after the capture of a cyanobacterial endosymbiont by a glycogen-metabolizing host cell. In the lineage that evolved subsequently, known as the Archaeplastida, select glucan-storage enzymes encoded within the host nucleus, the endosymbiont, and potentially a prokaryotic parasite located within the host cell developed so as to generate the branched glucan polymer amylopectin (Ball et al., 2011, 2013). Such molecules are highly similar to glycogen in terms of chemical structure, but the molecular architecture of amylopectin enables the formation of semicrystalline structures (Buléon et al., 1998). These latter then assemble into higher order structures leading to starch granule formation. The advent of starch granules is likely to have been critical for the evolution of chloroplast-containing organisms, including the spread of land plants on the Earth’s surface, because they enable the storage of photosynthetically generated Glc for many hours in tissues such as leaves during diurnal cycles or for months to years in seeds.An important aspect of the evolutionary change from glycogen to starch is the use of particular α(1→6)-glucosidases, referred to as isoamylase-type starch debranching enzymes (ISA), in the production of amylopectin (Ball et al., 1996; Myers et al., 2000; Hennen-Bierwagen et al., 2012). A suite of genes encoding the enzymes that accomplish starch biosynthesis was established early in the evolution of chloroplast-containing organisms (i.e. the Chloroplastida) prior to the divergence of distantly related groups including green algae and land plants. Included in this gene set are three paralogs that encode the proteins ISA1, ISA2, and ISA3, each of which is highly conserved in chloroplast-containing species. ISA1 of vascular plants and bryophytes, for example, are approximately 70% identical over more than 600 residues, and between land plants and prasinophyte algae this value is about 60%. ISA1 or ISA2 deficiencies in potato (Solanum tuberosum) tuber, Arabidopsis (Arabidopsis thaliana) leaf, Chlamydomonas reinhardtii cells, and cereal endosperms result in reduced starch content, altered amylopectin structure, and the appearance of soluble, branched glucans similar to native glycogen (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Bustos et al., 2004; Delatte et al., 2005; Wattebled et al., 2005). Such soluble polymers, referred to as phytoglycogen, have not been observed in wild-type plants. Thus, ISA1 and ISA2 functions are important determinants of whether storage glucans are semicrystalline or soluble. ISA3, in contrast, functions primarily in starch catabolism (Wattebled et al., 2005; Delatte et al., 2006).ISA1 and ISA2 appear to function together in Arabidopsis leaf as a single entity, because essentially identical phenotypes are observed in single mutants lacking either protein or double mutants lacking both of them (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Biochemical analysis of native and recombinant proteins has shown directly that ISA1 and ISA2 function together in a complex. ISA activity was first purified from potato tuber and found to contain two distinct polypeptides identified as ISA1 and ISA2 (Ishizaki et al., 1983; Hussain et al., 2003). Heteromultimers containing these two proteins were also purified from rice (Oryza sativa) and maize (Zea mays) endosperm (Utsumi and Nakamura, 2006; Kubo et al., 2010). Finally, a mixture of native and recombinant rice proteins demonstrated directly that specific enzymatic activities are provided by ISA1 and ISA2 functioning together in a heteromultimeric complex (Utsumi and Nakamura, 2006). ISA1 is the catalytic subunit within this complex, whereas ISA2 is noncatalytic, owing to amino acid substitutions at residues that are essentially invariant in the GH13 family of glycoside hydrolases (i.e. the α-amylase superfamily), several of which participate in the catalytic mechanism (Hussain et al., 2003; Utsumi and Nakamura, 2006). Despite lacking catalytic activity, ISA2 proteins are conserved in all chloroplast-containing species that have been examined, which rules out recently evolved mutations and, to the contrary, suggests a functional selective advantage.The necessity for the ISA1/ISA2 heteromultimer is not obvious in light of the fact that, in some instances, ISA1 by itself can condition normal levels of starch and the suppression of phytoglycogen accumulation. Cyanidioschyzon merolae, a species within the Rhodophyta lineage of the Archaeplastida family, contains semicrystalline starch and amylopectin with physical characteristics similar to that of Chloroplastida species (Hirabaru et al., 2010). The C. merolae genome contains elements that encode ISA1 and ISA3 yet lacks a homolog encoding ISA2 (Coppin et al., 2005). Thus, in some instances, starch can be generated, and phytoglycogen accumulation suppressed, without an ISA2 protein. Cereal endosperms provide additional evidence that ISA2 is not strictly required for normal starch levels and the suppression of phytoglycogen accumulation. Mutants or transgenic lines lacking ISA2 are known in rice (Utsumi et al., 2011) and maize (Kubo et al., 2010). Endosperm from these plants exhibits normal starch levels, with amylopectin structure essentially the same as the wild type, and lacks phytoglycogen. ISA activity presumably is provided in the endosperm of these mutants by a homomultimeric enzyme containing only ISA1.The reason why ISA2 is strictly conserved in the Chloroplastida is not understood yet. Two explanations can be considered. One possibility is that the inherent structure of ISA1 in cereals, resulting from mutations accumulated specifically in this evolutionary lineage, allows it to act without ISA2. Another possibility is that metabolic differences in specific tissues (e.g. leaf versus endosperm) require specialized enzymatic properties of the ISA1/ISA2 heteromer that ISA1 by itself does not provide. To test these hypotheses, this study combined maize and Arabidopsis ISA1 and ISA2 isoforms both in vitro and in vivo. Maize ISA1 was found to be active without any ISA2 protein, either in vitro or in Arabidopsis leaves, whereas Arabidopsis ISA1 required an ISA2 partner in all instances. Thus, ISA1 appears to have evolved in the cereal lineage so that it no longer requires ISA2 for enzymatic activity or metabolic function in the generation of starch and the suppression of phytoglycogen accumulation. |
