Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development

Abstract

The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley.

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…If SP plays a role in granule initiation, as indicated by these studies (and those of Malinova et al [254] in the above Section 5.2.2), it is not yet clear whether this is in concert with the SSIII/SSIV-mediated pathway or if the two pathways operate separately under different circumstances. It has been argued that SP and SBE activities acting in concert produce branched glucans resistant to phosphorolysis in the early stages of endosperm development and potential initiation points for granule synthesis to continue [302,362]. Interestingly, Pho1 from a number of cereal endosperms has been shown to form protein complexes with SBE isoforms [288,291,363] which supports a role for SP in starch biosynthesis.…”

“…By contrast, in maize endosperm, SBEIIb is the predominant form, being expressed at approximately 50 times the level of the SBEIIa [294], and it is the most abundant protein in the amyloplast stroma [299], whereas in barley, both isoforms are expressed to an equivalent degree in the endosperm [300,301]. Studies with barley at very early stages of endosperm development (0-1 DAP) indicate that SBEIIa is the only detectable SBE isoform, and it has been suggested that SBEs and SP (see later) play a role in granule initiation [302]. SBEII isoforms appear to play a major role in determining amylopectin structure, since the loss of SBEII activity produces clear starch phenotypes.…”

“…However, studies with rice endosperm Pho1 have shown that the SP reaction, in fact, favours α-glucan synthesis, even in the presence of a high Pi:Glc1P ratio ( [359] Hwang 2010). Studies with barley and rice Pho1 indicate that the protein is expressed at very early stages of endosperm development (0-3 DAP) [302,360], leading to the suggestion that SP plays a role in granule initiation. Early stages of endosperm development are characterized by relatively low ADP-Glc levels, in part as a result of an active ADP-Glc pyrophosphatase [361], and allowing for SP to elongate short MOS (DP 4-19).…”

“…Early stages of endosperm development are characterized by relatively low ADP-Glc levels, in part as a result of an active ADP-Glc pyrophosphatase [361], and allowing for SP to elongate short MOS (DP 4-19). Moreover, recent studies with the rice and barley Pho1 indicate that the enzyme is capable of producing and extending MOS solely from Glc1P (i.e., in the absence of α-glucan primer [302]. If SP plays a role in granule initiation, as indicated by these studies (and those of Malinova et al [254] in the above Section 5.2.2), it is not yet clear whether this is in concert with the SSIII/SSIV-mediated pathway or if the two pathways operate separately under different circumstances.…”

“…Phosphorylases from a number of plant tissues occur as homotetrameric or homodimeric assemblies [302,[364][365][366]. Elution profiles of maize Pho1 from gel permeation chromatography is consistent with a homotetrameric form of SP, although evidence suggests SP may exist as monomers or lower complexity multimers when associating with other enzymes of starch synthesis [315,363].…”