Differential Characteristics and Subcellular Localization of Two Starch-branching Enzyme Isoforms Encoded by a Single Gene inPhaseolus vulgaris L.

Abstract

Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points. We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3). Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development. Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues. Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds. The determined N-terminal sequence, VKSSHDSD, of LFPvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing. Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2). rLF-PvSBE2 had much higher affinity for amylopectin (K m ‫؍‬ 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas the V max of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg). These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.Starch, one of the most important carbon reserves in plants, is composed of two glucan polymers, amylose and amylopectin.

Biosynthesis of Reserve Starch

“…These include reactivity toward various glucans including branched glucans (amylopectin and glycogen), amylose, and malto-oligosaccharides (MOS) (Tetlow 2012); chemicals such as cyclodextrins (Vikso-Nielsen and Blennow 1998), phosphorylated metabolites (Morell et al 1997), and citrate (Hamada et al , 2002; temperature dependence of activities Hamada et al 2002;Ohdan et al 2011;Sawada et al 2013); and association with starch granules (Rahman et al 1995;Mu-Forster et al 1996;Hamada et al 2001Hamada et al , 2002Grimaud et al 2008;Liu et al 2012b;Abe et al 2014). For other properties of BE, see review by Tetlow (2012).…”

“…Several reports showed that multiple forms of some enzymes such as SSI (Baba et al 1993) and BEIIa (Yamanouchi and Nakamura 1992) in rice endosperm, BEI in wheat endosperm (Båga et al 2000), and BEII from kidney bean (Hamada et al 2002) were generated from a single gene by transcriptional or posttranscriptional regulation and/or modification. However, the physiological significances of multiple forms of the same enzymes were unclear.…”

Two Distinct Forms ofM-Locus Protein Kinase Localize to the Plasma Membrane and Interact Directly withS-Locus Receptor Kinase to Transduce Self-Incompatibility Signaling inBrassica rapa

“…The low levels of BP2 expression throughout the plant suggested a potential role in processes other than meiosis, but its physiological significance remains unknown. Another example of AFE transcripts reported in plants is the SBE gene, which encodes a starch-branching enzyme (Hamada et al, 2002;Kitagawa et al, 2005). One shorter SBE transcript was expressed constitutively, while a longer transcript was expressed in limited tissues, including developing seeds.…”

A review of starch‐branching enzymes and their role in amylopectin biosynthesis

“…Some enzymes of starch biosynthesis are found strongly associated with starch granules, including SBEII isoforms from a range of species (49)(50)(51)(52). The precise mechanism underlying the association of specific enzymes of starch synthesis with the starch granule are not known, although it has been suggested that alternative splicing of SBEII in bean (Phaseolus vulgaris L.) leads to partitioning within the starch granule (27). Recent evidence suggests that granule-associated proteins (including the SBEII class) become entrapped in the granule through association in heteromeric protein complexes (8,53).…”