Distinct Functional Properties of Isoamylase-Type Starch Debranching Enzymes in Monocot and Dicot Leaves

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.

Replacement of the Endogenous Starch Debranching Enzymes ISA1 and ISA2 of Arabidopsis with the Rice Orthologs Reveals a Degree of Functional Conservation during Starch Synthesis

“…However, this raises the possibility that the isoamylase may somehow determines not only the starch structure but also the amount produced. Interestingly, the ZmISA1 expressing plants produced starch with the same amylopectin CLD as the Arabidopsis wild type [28]. This is in contrast to our results, but might be explained by the fact that the amylopectin CLDs of maize and Arabidopsis are more similar to each other than Arabidopsis and rice (Fig.…”

Biosynthesis of Reserve Starch

“…The roles of ISAs, their presence and composition, and their contributions to starch biosynthesis were dramatically different depending on plant species and tissues (Hennen-Bierwagen et al 2012;Facon et al 2013). One example is that the complete loss of ISA1 gene caused most cells to synthesize both starch-type glucans and WSP in many plant sources, while starch was completely replaced by WSP in rice endosperm (Nakamura et al 1997) and Chlamydomonas (Mouille et al 1996), although the exact reason remains to be clarified.…”

Starch

“…CLg1 (Cenci et al 2013) (also see review by Hennen-Bierwagen et al 2012), although in this chapter experimental results with maize and rice endosperm are mainly described. It is interesting that both ISA1 homomer and ISA1-ISA2 heteromer were present in maize endosperm ) and leaf (Lin et al 2013), rice endosperm (Utsumi and Nakamura 2006;, and Chlamydomonas . The essential role of ISA1 was proven by the molecular studies showing that the sugary-1 phenotype reverted to wildtype one when normal ISA1 gene was introduced into the sugary-1 mutant of rice (Kubo et al 2005; (Fig.…”