Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement.
Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined.
Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf.
The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.
Isoamylase-type starch debranching enzymes (ISAs) provide functions in starch biosynthesis that are subject to evolutionary conservation, as indicated by their high degree of primary sequence identity in chloroplast-containing organisms (i.e. the Chloroplastida; for recent reviews, see Hennen-Bierwagen et al., 2012; Streb & Zeeman, 2012). ISAs catalyze hydrolysis of α(1→6) glycoside bonds in branched glucose homopolymers, and are thought to be involved in determining a clustered arrangement of branched linkages in precursor glucans. This structure is proposed to favor crystallization of unbranched regions of α(1→4)-linked linear chains and subsequent assembly into higher order structures culminating in insoluble starch granules. In support of this hypothesis, mutations in genes encoding certain ISA proteins typically cause decreased starch content and accumulation of water-soluble polysaccharides (WSPs) that are not present in nonmutant plants. Such changes have been observed in the endosperm of several monocots, in dicot leaves and tubers, and in the unicellular green alga Chlamydomonas reinhardtii. In all instances a gene encoding an ISA protein was mutated, and this distinctive phenotype has not been observed in the absence of altered ISA function.
Three separate genes encoding ISA proteins are conserved in the genomes of all Chloroplastida species characterized to date (Deschamps et al., 2008). ISA3 functions primarily in starch catabolism (Wattebled et al., 2005, 2008; Delatte et al., 2006), whereas ISA1 and ISA2 are involved in starch biosynthesis. ISA1 provides the catalytic activity for glucan debranching, and in all species tested is required for normal contents of crystalline starch and to repress accumulation of the plant WSP termed phytoglycogen, which is similar in structure to animal glycogen. ISA2 is a noncatalytic protein as a result of conserved substitutions in amino acid residues required for activity in members of the well-characterized glycosyl hydrolase family 13 (Henrissat, 1991; Hussain et al., 2003; Hennen-Bierwagen et al., 2012). In dicot tissues, specifically the Arabidopsis thaliana leaf and potato (Solanum tuberosum) tuber, ISA1 and ISA2 are involved at the same step in starch metabolism and both proteins are needed to execute that function (Zeeman et al., 1998; Bustos et al., 2004; Delatte et al., 2005; Wattebled et al., 2005). In the rice (Oryza sativa) or maize (Zea mays) endosperm, by contrast, loss of ISA2 is tolerated without a significant reduction in starch content or the appearance of phytoglycogen (Kubo et al., 2010; Utsumi et al., 2011). Chlamydomonas reinhardtii is intermediate in this regard, with loss of either ISA1 or ISA2 causing a decrease in starch content and the accumulation of phytoglycogen, but with less severe effects when ISA2 is affected (Mouille et al., 1996; Dauvillee et al., 2001). These observations raise the question of why noncatalytic ISA2 would be strictly conserved in all chloroplast-containing species even though in some plant tissues the biosynthetic ISA function(s) can be provided by ISA1 alone.
To address this question, the roles of ISA1 and ISA2 in starch metabolism in maize leaves were characterized in this study. ISA function in the leaf has been characterized genetically only in A. thaliana, where essentially identical metabolic and biochemical phenotypes result from loss of ISA1, ISA2, or both together (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Leaf starch content is reduced in either single or double mutants by c. 80%, the structure of the amylopectin component of starch is characteristically altered, and phytoglycogen accumulates to a level equivalent to c. 40–50% of the starch present in wild type. A single ISA activity is detected by in-gel enzyme assays (i.e. zymograms) of leaf soluble extracts, and this is absent in null mutants lacking ISA1 or ISA2 and in double mutants. From these data, ISA1 and ISA2 appear to function together in the A. thaliana leaf in a heteromultimeric enzyme complex, and both proteins are necessary for activity and physiological function.
Loss of ISA1 in the cereal endosperm causes a phenotype similar to that seen in the A. thaliana leaf, specifically a starch content reduced by 80% or more, accumulation of high concentrations of phytoglycogen, and altered amylopectin structure (James et al., 1995; Nakamura et al., 1996; Dinges et al., 2001; Burton et al., 2002). Genetic alterations that cause loss of ISA2 are known in rice and maize (Kubo et al., 2010; Utsumi et al., 2011). In contrast to the A. thaliana leaf, these did not cause a substantial reduction in endosperm starch or accumulation of phytoglycogen. Endosperm tissues from rice and maize were shown by biochemical purification to possess a heteromultimeric enzyme comprising both ISA1 and ISA2 (Utsumi & Nakamura, 2006; Kubo et al., 2010), consistent with the genetic data from A. thaliana. These tissues also possess a homomultimeric enzyme that contains only ISA1, which was not observed in the A. thaliana leaf.
Two potential explanations can be suggested for the differences between ISA functions, that is, whether or not ISA2 is required for normal starch metabolism, in dicot tissues and the monocot endosperm. One possibile explanation is distinctions in carbohydrate metabolism between tissues. In particular, accumulation of transitory starch in the leaf might require the ISA1/ISA2 heteromultimeric enzyme, whereas ISA1 alone might be sufficient for storage starch in the endosperm, which is not degraded during the diurnal cycle. Thus, mutations that affect ISA2 would affect leaf starch metabolism but not affect the endosperm. Alternatively, evolutionary divergence between dicots and some or all cereal species may have resulted in different ISA1 primary structures. The ISA1 sequence in cereals may possess the intrinsic ability to function as a homomultimer, whereas the protein from dicots may be functional only in heteromultimeric complexes that contain ISA2. According to this hypothesis, mutation of ISA2 should have species-specific rather than tissue-specific effects.
Characterization of the effects of mutations eliminating ISA1 or ISA2 on starch metabolism in leaves of a monocot species will help to distinguish these possibilities. The results presented here show that ISA functions differently in maize leaves than in A. thaliana leaves. In contrast to A. thaliana leaves, the effects of eliminating either ISA1 or ISA2 are distinct from each other in maize. The data are consistent with the hypothesis that evolutionary divergence has resulted in an ISA1 protein in monocots that is enzymatically competent in the absence of ISA2. This enzyme does not entirely supply the physiological role of ISA in leaf starch biosynthesis, however, because ISA2 mutations had discernible effects on starch content and granule size.
Materials and Methods
Maize (Zea mays L.) lines used in this study were essentially congenic in the W64A inbred genetic background as a result of a minimum of five backcrosses to the nonmutant standard. Molecular characterization was described previously for mutations of the gene sugary1 (su1) that encodes ISA1, specifically su1-4582 and su1-Ref (James et al., 1995; Dinges et al., 2001), or the isa2-339 allele of the isa2 locus that encodes ISA2 (Kubo et al., 2010). Seedlings were grown in the glasshouse until emergence of the fourth leaf. Late in the light phase of the diurnal cycle, the outer half of the third leaves was harvested and used fresh for enzyme activity gels or immediately frozen in liquid nitrogen and stored at −80°C until use for carbohydrate analyses. Seedlings for analysis of gene expression in leaves were grown in a growth chamber under a diurnal cycle with alternating periods of 8-h darkness at 25°C and 16-h light at 28°C with c. 280 μmol quanta m−2 s−1 of mixed fluorescent and incandescent light. Tissues were harvested at various times throughout the diurnal cycle, immediately frozen in liquid nitrogen, and stored at −80°C until use.
Leaf starch was isolated and quantified as previously described (Yandeau-Nelson et al., 2011) after lysis of c. 200 mg of tissue in 0.7% ice-cold perchloric acid, with biological replicate analyses performed on individual plants of the same genotype. WSP was collected from the soluble phase of the same extracts, and quantified after neutralization to approximately pH 7 by dropwise addition of 2 M KOH, 0.4 M 2-(N-morpholino) ethanesulfonic acid and 0.4 M KCl. Both starch and WSP were quantified after conversion to free glucose by treatment with thermostable α-amylase and amyloglucosidase using commercial reagents (K-TSTA; Megazyme, Bray, Co. Wicklow, Ireland). The amylopectin linear chain length distribution was determined by fluorophore-assisted carbohydrate electrophoresis (FACE) using a P/ACE capillary electrophoresis instrument (Beckman Coulter Inc., Indianapolis, IN, USA) to separate glucans (O'Shea et al., 1998; Lin et al., 2011). Glucans for amylopectin chain length distribution analysis were extracted from the insoluble leaf fraction by boiling in DMSO before FACE analysis. The frequency of chains of each degree of polymerization (DP) was calculated as the percentage of the total chains within a given DP range. Glucans from a minimum of three biological replicates were analyzed, and the frequency values for each chain length were averaged among individuals of the same genotype. Difference plots were calculated by subtracting the wild-type average value from the mutant average value of the same DP.
The relative abundance of ISA1 and ISA2 mRNAs standardized to the 18S rRNA abundance was measured using quantitative real-time PCR (qPCR) amplification of leaf cDNA. Methods for mRNA isolation, cDNA preparation, and qPCR analysis of these two transcripts were described previously (Kubo et al., 2010).
Immunoblot and zymogram analyses of total soluble extracts
Fresh leaf tissue (c. 1 g) was ground to a powder under liquid nitrogen in a mortar and pestle. The powder was ground further in 4 ml of ice-cold extraction buffer (50 mM Tris-acetate, pH 7.5, 10 mM DTT, and 1X protease inhibitor cocktail (Sigma P-2714)), and then the lysate was transferred to a 15-ml polypropylene tube and centrifuged at 10 000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.45-μm syringe filter (431220; Corning Inc., Tewksbury, MA, USA) and then concentrated by centrifugal filtration to c. 0.3 ml (UFC801024; EMD Millipore, Darmstadt, Germany). The protein concentration in the total soluble lysate was determined by the Bradford method (500-0006; Bio-Rad, Hercules, CA, USA). For immunoblot analysis, 180 μg of extract was fractionated by SDS-PAGE and probed with affinity-purified immunoglobulin G (IgG) fractions specific for maize ISA1 or ISA2, as previously described (Kubo et al., 2010). For zymogram analysis of total leaf extracts, 100–400 μg of soluble extract was fractionated by native-PAGE using precast 7.5% acrylamide gels (Bio-Rad 345-0010). Conditions for electrophoresis and processing for detection of in-gel ISA activity were described previously (Kubo et al., 2010). Endosperm extracts used as controls in the zymograms were prepared as described previously from kernels harvested 20 d after pollination (DAP; Kubo et al., 2010).
Fractionation of ISA activities
Leaf extracts were prepared as described in the previous section, except that 5 g of leaf tissue was extracted in 12 ml of buffer. The lysate was clarified by centrifugation at 17 000 g for 10 min at 4°C, and the supernatant was passed twice through 0.45-μm syringe filters. Eight milliliters of lysate was passed through a 1-ml bed volume HiTrap Q HP anion exchange column (17-1153-01; GE Healthcare Biosciences). Proteins were eluted in a linear gradient of 0–1 M NaCl in 50 mM Tris-acetate, pH 7.5, with 10 mM DTT added immediately before use, and 1-ml fractions were collected. ISA1 and ISA2 were identified by immunoblot analyses, and both proteins were found to elute in the same three fractions (data not shown). These fractions were pooled and concentrated to c. 1 ml by centrifugal filtration and DTT was added to a final concentration of 20 mM. The pooled AEC fractions (500 μl) were applied to a Superdex 200 10/300 GL gel permeation column (17-5175-01; GE Healthcare Life Sciences, Pittsburgh, PA, USA) and eluted in 50 mM Tris-acetate, pH 7.5, 150 mM NaCl and 10 mM DTT, while collecting 1-ml fractions. Samples of each fraction were tested by SDS-PAGE and immunblot analysis for the presence of ISA1 and ISA2. Fractions containing those proteins (25 μl) were applied to native-PAGE gels and subjected to zymogram analysis. Duplicate native-PAGE gels were analyzed for ISA1 and ISA2 proteins by immunoblot analysis. Endosperm extracts were analyzed similarly, beginning with 5 g of tissue from kernels harvested 20 DAP.
Transmission electron microscopy and scanning electron microscopy
The outermost regions of maize leaves from glasshouse-grown seedlings were harvested at the end of the light period of the diurnal cycle. Tissue was cut with a fresh razor blade into small pieces, then immersed in fixative 0.1 M cacodylate, pH 7.2, 2% paraformaldehyde and 2% glutaraldehyde. Vacuum was applied for 30 min. Leaf samples were embedded in Spurr's resin, then postfixed with 1% osmium tetroxide, sectioned to a thickness of 80 nm, affixed to grids and stained with uranyl acetate and lead citrate. Transmission electron microscopy (TEM) observation was performed with a Jeol 2100 microscope operating at 200 kV.
Starch granules to be analyzed by scanning electron microscopy (SEM) were purified from insoluble leaf material as previously described (Yandeau-Nelson et al., 2011). The pellet from the leaf crude lysate was resuspended in sterile deionized water and filtered through two layers of Miracloth (Calbiochem, Darmstadt, Germany). The starch pellet was further purified by isopicnic centrifugation in Percoll (GE Healthcare) at 10 000 g for 1 h at 4°C. The pellet was rinsed twice with sterile deionized water and stored in 20% ethanol at 4°C until use. Droplets of starch granule suspensions were allowed to dry on freshly cleaved mica. After coating with gold/palladium, they were observed in secondary electron mode using a Jeol JSM6300 microscope (Jeol, Peabody, MA, USA) operating at 8 kV. Size-distribution histograms were determined by measuring the apparent diameter of at least 300 particles per sample from the SEM images.
Transcript and protein abundance
The relative steady-state abundances of ISA1 and ISA2 mRNAs in seedling leaves over the course of a 16-h light : 8-h dark diurnal cycle were measured by qPCR. The abundances of these transcripts were reported previously for developing endosperm tissue, where ISA1 was c. 2.5-fold more abundant than ISA2 (Kubo et al., 2010). In seedlings leaves, the relative abundances of ISA1 and ISA2 mRNAs were approximately equal at the end of the night. ISA2 mRNA abundance was elevated within the first 3 h of the light phase to approximately five-fold higher than ISA1 mRNA, and remained more abundant by about the same factor throughout the day (Fig. 1a). The qPCR results are in agreement with publicly available whole-transcriptome sequencing data (RNAseq) showing ISA2 to be two- to three-fold more abundant than ISA1 in leaf tissue harvested during the day (Fig. 1b). Thus, the relative abundances of ISA1 and ISA2 transcripts are reversed in the maize endosperm and leaf, with ISA1 more abundant in the nonphotosynthetic tissue and ISA2 present at higher abundances in leaves throughout the light phase of the diurnal cycle.
ISA1 and ISA2 proteins were both detected by immunoblot analysis in soluble extracts of seedling leaves harvested during the day (Fig. 1c). The specificity of the IgG fractions used for this analysis was demonstrated using leaf extracts from plants homozygous for the null mutations su1-4582, in the gene sugary1 (su1) that codes for ISA1 (James et al., 1995), or isa2-339, in the gene that codes for ISA2 (Kubo et al., 2010). Leaves homozygous for su1-4582 and thus lacking ISA1 also were completely deficient for ISA2 as a secondary effect (Fig. 1c), as has been observed previously in the endosperm (Kubo et al., 2010). This result is consistent with the conclusion that ISA1 is required for the stability of ISA2. By contrast, ISA1 continued to accumulate in isa2-339 mutant leaves lacking ISA2, although at a reduced concentration compared with nonmutant tissue (Fig. 1c). An analogous secondary effect of an isa2- null mutation was seen in A. thaliana leaves (Delatte et al., 2005); however, in the dicot only a trace amount of ISA1 remained, whereas in maize substantial amounts of ISA1 accumulated in the absence of ISA2.
Leaf ISA activity
The ISA enzymes in maize leaves were first characterized by zymogram analyses of total soluble extracts. Proteins were separated by native-PAGE, and then electrophoretically transferred to a gel impregnated with 0.3% solubilized potato starch. The starch gel was stained with iodine-potassium iodide solution (I2/KI) so that starch-modifying enzymes could be detected by changes in color. In such analyses, ISAs are known to generate light-blue bands, whereas starch branching enzymes generate red or pink bands and amylases produce white bands (Colleoni et al., 2003). Endosperm tissue subjected to such analyses revealed three forms of ISA activity, including a faster-migrating form of the ISA1 homomultimer (form I) and two slower-migrating forms of the ISA1/ISA2 heteromultimeric enzyme (forms II and III; Kubo et al., 2010). Leaf extracts generated two bands of ISA activity, which migrated slightly faster in native-PAGE than the two heteromultimeric forms from the endosperm (Fig. 2). Both of these activities were missing in isa2-339 or su1-4582 leaves, consistent with their identification as the ISA1/ISA2 heteromultimeric enzyme. A third activity band corresponding to form I was not obvious in leaf extracts, although a diffuse, faster-migrating light-staining area was present near the position of the endosperm ISA1 homomultimer. This diffuse activity was missing in extracts deficient for ISA1 but was not affected by loss of ISA2 (Fig. 2).
To better resolve ISA activities, enzymes from leaf and endosperm crude extracts were partially purified by anion-exchange chromatography (AEC) on HiTrapQ HP columns, concentrated, and then separated by gel permeation chromatography (GPC) on a Superdex 10/300 GL column. Fractions were then analyzed by zymogram and also by immunoblot of duplicate lanes to determine whether the ISA1 and/or ISA2 protein co-eluted in GPC and co-migrated in native-PAGE with specific ISA activities. Endosperm extracts provided standards for the presence of the ISA1 homomultimer and ISA1/ISA2 heteromultimers in specific GPC fractions (Fig. 3a). As seen previously (Kubo et al., 2010), both ISA1 and ISA2 co-migrated with enzyme forms II and III, whereas ISA1 co-migrated with form I but ISA2 clearly was not present in that activity band (Fig. 3a).
GPC fractions from the leaf also contained three forms of ISA. These activities eluted in the same fractions as ISA forms I, II and III from the endosperm, and co-migrated with those enzymes in native-PAGE (Fig. 3b). ISA form I in the pooled AEC fractions from nonmutant leaves co-migrated in native-PAGE with ISA1 but did not contain any ISA2 (Fig. 3b). SDS-PAGE immunoblots revealed that the ISA2 protein in leaf extracts co-eluted in GPC fractions B6 to B4 with activity forms II and III, but was not detected in fraction B3 which was enriched for enzyme form I (Fig. 3b). These data indicate the presence of the ISA1 homomultimeric enzyme in the maize leaf. ISA form II in the pooled AEC fractions from the leaf co-migrated with both ISA1 and ISA2, indicating the presence of an ISA1/ISA2 heteromultimer in that tissue. ISA form III in the leaf AEC pool co-migrated with ISA2. The fact that ISA1 was not detected in leaf form III probably is attributable to the low sensitivity of the native-PAGE immunoblots and the relatively low abundance of that form of the enzyme. ISA1 is expected to be present in that band because form III is absent from whole-cell extracts of either the endosperm or the leaf from su1-4582 mutants. Taken together, the data indicate that both the ISA1 homomeric enzyme and the ISA1/ISA2 heteromultimer exist in nonmutant maize leaves. The leaf differs from the endosperm, however, in the relative proportions of the different ISA activities, as far as can be judged from these semiquantitative assays. The ISA1 homomultimer appears as the most prevalent activity in endosperm extracts, whereas there is proportionally less of this form of the enzyme in leaf extracts compared with the heteromultimeric form II (Fig. 3a,b).
ISA activity was also partially purified from leaves of an isa2-339 mutant. In this instance, a single ISA activity band was present, indicated by its blue color, that co-migrated in native-PAGE with the homomeric enzyme from the endosperm, that is, form I (Fig. 3c). This is in contrast to the extracts from nonmutant leaves that exhibited three blue bands of ISA activity (Fig. 3b). Immunoblot analysis of duplicate fractions showed that ISA1 co-eluted with the activity band in GPC and co-migrated with that enzyme in native-PAGE. These data confirm that an ISA activity exists in maize leaves that does not utilize ISA2, because this protein is entirely lacking in isa2-339 mutants (Fig. 1c).
Granule morphology in leaves lacking ISA1 or ISA2
The appearance of starch granules in seedling leaf chloroplasts was compared between wild type and mutant lines homozygous for a defect in the gene encoding ISA1 or ISA2. Leaf samples were collected from glasshouse-grown seedlings at the end of the light period of the diurnal cycle and immediately fixed for TEM. Genotypes analyzed were the wild-type inbred W64A, and congenic mutants homozygous for isa2-339, su1-4582, or su1-Ref. The su1-Ref allele is a missense mutation that results in an enzymatically inactive form of ISA1 present at a low level compared with wild type (Kubo et al., 2010), whereas isa2-339 and su1-4582 are null alleles that result in complete losss of ISA2 or ISA1, respectively. Bundle sheath chloroplasts were examined because these are the site of starch granule accumulation in the mature maize leaf as the result of C4 metabolism. As a control, palisade mesophyll cells from wild-type and homozygous isa1-1 A. thaliana leaves were analyzed similarly. The A. thaliana mutant plastids exhibited loss or a reduced number of starch granules, and a distended stromal volume apparently resulting from phytoglycogen accumulation, as previously reported (Fig. 4; Delatte et al., 2005).
Plastids from maize leaves lacking ISA2 exhibited a nearly normal appearance with the exception that starch granules appeared to be slightly smaller than in wild type (Fig. 4). This is in contrast to isa2-1 mutants of A. thaliana, in which mesophyll or bundle sheath chloroplasts typically lack recognizable starch granules and contain amorphous glucan particles with a range of structures (Zeeman et al., 1998; Delatte et al., 2005). Such abnormal glucan particles were never observed in maize isa2-339 mutants. Maize bundle sheath chloroplasts from either su1-4582 or su1-Ref mutants contained starch granules that were noticeably smaller and more irregularly shaped than in wild type, although distended organelles and apparent phytoglycogen accumulation were not obvious (Fig. 4). The granule morphology within bundle sheath plastids was distinctly different between leaves mutated for either ISA1 or ISA2.
The size ranges of seedling leaf starch granules were quantified by SEM. Granules were extracted from W64A, isa2-339, or su1-Ref seedlings at the end of the light phase and imaged by SEM (Fig. 5a), and then individual particle diameters were measured. The distributions of granule diameters differed among all genotypes (Fig. 5b), and the average diameters differed between any two lines with a high degree of statistical significance (P value < 0.0001). The results demonstrated that starch granules from su1-Ref mutant leaves were smaller than those from nonmutants, and that granules generated in the absence of ISA2 were intermediate in diameter between the particles from nonmutant and ISA1-defective leaves. As a biological replication, the experiment was repeated with W64A, isa2-339, and su1-4582 seedlings from a separate planting. The diameter distributions again differed among gentoypes (Fig. 5b) and the average values were significantly different (P value < 0.0001). The replicate analyses were consistent in the sense that starch granules lacking functional ISA1 were smaller than those present in either nonmutant leaves or mutants lacking only ISA2. These data confirm the results of the TEM analysis of granule morphology within chloroplasts, and indicate that in maize, unlike A. thaliana, the effects of ISA2 mutation on starch granule morphology are different from those resulting from loss of ISA1 function.
Storage glucan content and structure in leaves lacking ISA1 or ISA2
Starch, WSP, and free glucose were quantified in glasshouse-grown seedling leaves of wild-type, su1-4582, and isa2-339 maize plants harvested at the end of the light phase. Loss of either ISA1 or ISA2 caused a reduced starch content to c. 36–46% of the wild-type content (Fig. 6). Despite this defect there was no obvious difference in plant growth as judged by the appearance of su1-4582 or isa2-339 mutants compared with the nonmutant standard over the course of the plant life cycle (data not shown). Differences between the mutant and wild-type starch contents were statistically significant (P value < 0.002), whereas the two mutant lines were not significantly different from each other (P value = 0.36). WSP, that is, glucose units generated by exhaustive α-amylase and amyloglucosidase treatment, was not detected above the nonmutant background in either su1-4582 or isa2-339 leaves (Fig. 6). This is in contrast to A. thaliana, where WSP contents increase significantly in the absence of either ISA1 or ISA2 (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Free glucose content was not significantly different between either of the mutants and the nonmutant standard (P value ≥ 0.19), nor between the two mutants (P value = 0.07).
The amylopectin linear chain length distribution was compared between mutants affected in either ISA1 or ISA2 and the nonmutant standard line. Leaf starch was completely debranched with commercial isoamylase, and then the linear chains were derivatized at the reducing end with a fluorescent functional group, separated by capillary electrophoresis, and quantified. Chains of DP 5 or less were not detected. The frequency of chains in the DP range 6–38 was calculated as a percentage of the total, and for each DP the value in nonmutant starch was subtracted from the mutant value to reveal structural differences (Fig. 7). Mutants affected in ISA1 as a result of carrying either the su1-Ref or su1-4582 allele exhibited an increased frequency of short chains in the range of DP 6–12 compared with normal. The effect was stonger for the null allele than the point mutation for the shortest chains of DP 6–8. This structural pattern is similar to amylopectin from other plant sources carrying mutations of ISA1, including the A. thaliana leaf (Delatte et al., 2005; Wattebled et al., 2005) and maize endosperm (Dinges et al., 2001). As previously mentioned, in the A. thaliana leaf the changes observed in isa2- mutants were essentially identical to those of isa1- mutants (Delatte et al., 2005; Wattebled et al., 2005). In maize leaf starch, the effects of isa2- mutations were similar but not identical to those of the isa1- mutants. The changes were quantitatively less severe and the range of chain lengths affected was narrower in isa2- leaf starch compared with the isa1- chain length profiles (Fig. 7).
Distinctions in ISA1 primary structure conserved in monocots and dicots
Multiple amino acid sequence alignments of ISA1 from five monocot species and six dicots were obtained using the program ClustalW (Thompson et al., 1994). In general, the alignment between monocot and dicot ISA1s extends throughout the proteins with a high degree of sequence identity. Amino acids that are identical in all the monocots but vary by the presence of nonconserved residues at those positions in the dicots were noted (Supporting Information Fig. S1). Two extended blocks of such monocot/dicot divergence were identified (Fig. 8). A span of residues that differs entirely between monocots and dicots, beginning at residue 287 of full-length maize ISA1, is intercalated between regions of extremely high sequence conservation in both the N- and C-terminal directions (Fig. 8). All of the dicots have five additional amino acids in this region compared with the monocots. The monocot sequences contain three successive Ser residues, or in one instance a Cys and two Ser residues, none of which are present in the dicots. The dicots possess a different sequence of 10 residues, six of which are identical or essentially conserved in all six species examined.
Prediction of the maize ISA1 three-dimensional structure based on comparison to the known structure of isoamylase from Pseudomonas amyloderamosa (Katsuya et al., 1998) suggests that this region of the protein is accessible on the surface (Fig. S2).
The second structural feature divergent between monocot and dicot ISA1s is located close to the N terminus (Fig. 8) of the mature proteins. All five monocot proteins analyzed contain a contigous sequence close to their mature amino terminus of 8–10 residues that are entirely Asp or Glu acidic groups or contain a single neutral residue within the span of acidic amino acids. None of the dicot ISA1 proteins possess such a sequence. Furthermore, adjacent to the acid-rich sequence is a span of 14 residues in which ten amino acids are identical in all the monocots and divergent in all the dicots. Thus, the amino termini of ISA1 from monocots and dicots diverge greatly in character, whereas the remainder of the proteins are highly conserved (Fig. S1).
ISA1 homomultimer function in the maize leaf
Biochemical characterization and genetic analysis both demonstrated that ISA1 functions as a homomeric enzyme, in the absence of ISA2, in maize leaves. ISA enzymatic activity was detected by zymogram in isa2-339 null mutant leaves. This enzyme eluted as a multimeric complex in GPC analysis, at approximately the same volume as the ISA1 homomultimer from the endosperm. ISA1 protein detected by immunoblot analysis co-eluted in GPC and co-migrated in native-PAGE with this ISA activity. An ISA activity with the same purification properties was present in nonmutant leaves and did not contain any ISA2, as shown by immunoblot. The function of the ISA1 homomultimer was implied by the finding that leaf starch granules generated in the presence of ISA1 alone, that is, in isa2-339 mutants, differed in size and morphology from those of su1- mutants lacking ISA1 activity. Also, the amylopectin chain length distribution was different in starches generated in the presence of ISA1 alone compared with su1- mutants. Taken together, the data provide strong evidence that ISA1 functions enzymatically and physiologically in the maize leaf, in the absence of ISA2.
The ISA1 homomer, however, does not provide the full physiological function of ISA activity in maize leaves. This is in contrast to the endosperm, where loss of ISA2, and thus the ISA1/ISA2 heteromeric enzyme, can be tolerated without major defects in starch content or structure (Kubo et al., 2010; Utsumi et al., 2011). The need for ISA2 in leaves is evident from the fact that starch content is reduced, starch structure is altered, and granules are smaller on average in mutants lacking the protein compared with nonmutants. Thus, the ISA1 homomultimer cannot support starch metabolism that is normal in all of these aspects. Taken together, the data indicate that both homomeric and heteromeric ISA forms are capable of function in maize leaf starch biogenesis, raising the possibility that they each play specific roles necessary for normal metabolism. The ISA1/ISA2 heteromultimer appears to affect granule growth because the starch particles were smaller in the mutant lacking ISA2 than in the nonmutant standard.
The fact that the ISA1 homomultimer functions in the maize leaf but not in A. thaliana is most simply explained by inherent differences in protein structure between the two species. The A. thaliana leaf possesses only a single electrophoretic mobility form of ISA activity, whereas one ISA1 homomultimer and two ISA1/ISA2 heteromeric forms are present in the maize leaf as they are in the endosperm. Specific structural differences between ISA1 proteins clearly exist that are monocot- or dicot-specific, within the context of very close sequence homology throughout the protein in all vascular plants examined. The positions of the localized regions of sequence divergence between monocot and dicot, based on the structure of Pseudomonas amyloderamosa isoamylase (Katsuya et al., 1998), are consistent with this suggestion. The Ser-rich sequence specific to monocots aligns with a loop on the external surface of the isoamylase protein (Fig. S2). This region could thus influence interactions with other subunits in the quaternary structure of the plant enzyme and allow monocot ISA1 to function without ISA2. The acid-rich region at the N terminus of the monocot ISA1 is not represented in the P. amyloderamosa protein, so its position within the three-dimensional structure cannot be predicted. The high degree of conservation specifically among the monocots, however, suggests that this region is functionally significant.
Contrary to this view, however, is the fact that the ISA1 homomultimer was not detected in the leaves of rice, another monocot species (Utsumi et al., 2011). From this observation, the possibility must be considered that particular aspects of leaf metabolism determine whether or not the ISA1 homomultimer forms and/or functions physiologically, and that these aspects differ in maize compared with both rice and A. thaliana. For example, A. thaliana and rice are C3 plants that fix CO2 directly using RUBISCO, whereas maize is a C4 plant that initially fixes CO2 using pyruvate as the acceptor. Transfer of maize ISA1 to A. thaliana plants lacking both endogenous ISA1 and ISA2, however, showed that the C4 monocot enzyme could support starch biosynthesis in the C3 dicot (M. Facon & F. Wattebled, unpublished). This result supports the interpretation that the maize ISA1 structure inherently provides the ability for homomultimer function in the absence of ISA2. The reason that the ISA1 homomultimer was not detected in rice leaves, despite its conserved primary structure compared with maize, is not obvious. The different results could relate to the stability of the enzyme, variation in the relative abundances of ISA1 and ISA2 in the leaves of the two species, and/or regulation of the enzymes by post-translational mechanisms.
Physiological aspects of the mutant phenotypes
Allele-specific distinctions were observed in the effects of su1-Ref and su1-4582 on leaf starch, although qualitatively the two mutations caused similar changes. For example, although both mutations resulted in granules that were smaller than those present in nonmutant or isa2-339 mutant leaves, su1-4582 seemed to have more severe effects than su1-Ref (Fig. 5). Quantitatively different effects of the two alleles on amylopectin chain length distribution were also observed (Fig. 7), and the appearance of granules visualized by TEM also differed slightly (Fig. 4). As noted, su1-Ref mutants contain reduced abundances of an inactive form of ISA1, whereas su1-4582 plants completely lack ISA1. Furthermore, ISA2 accumulates to normal levels in the su1-Ref endosperm, whereas it is completely missing from either the leaf or the endosperm of su1-4582 mutants (Fig. 1; Kubo et al., 2010). Thus, the allele-specific effects on leaf starch potentially could result from the differences in ISA1 or ISA2 protein abundance in the different mutants, even though enzymatic activity is missing in both instances. Alternatively, the differences could result from the growth environments of the two biological replicates, which utilized seedlings grown in the glasshouse at different times of the year. The latter explanation is supported for the granule-size phenotype by the fact that the average diameters of all three genotypes were smaller in the second biological replication of the experiment (Fig. 5).
A clear difference between maize and A. thaliana leaves was the absence of phytoglycogen in the monocot when ISA1 or ISA2 was missing. This result is unusual in comparison to other genetic analyses of ISA function, where in general phytoglycogen accumulation is a hallmark of the mutant phenotype (Hennen-Bierwagen et al., 2012). Tissue specificity is part of the explanation, because ISA1 mutants of maize exhibit high concentrations of phytoglycogen in the endosperm. The requirement for ISA function in the A. thaliana leaf varies by cell type, as shown by the presence of starch granules in some cells and their apparent replacement by phytoglycogen in others (Delatte et al., 2005). Bundle sheath and epidermal cell chloroplasts, for example, contain abnormally shaped starch granules in isa1- or isa2- mutants, whereas adjacent palisade or spongy mesophyll cells lack granules. Thus, ISA activity is not entirely responsible for whether or not glucans assemble into granules, but rather it appears to be a contributing factor that influences the equilibrium between crystallization and solubility. Other enzymes are known to affect the distribution between soluble and insoluble glucans, as shown by multiple mutant combinations where ISA is compromised, for example starch synthase III in the maize endosperm (Lin et al., 2011) and the α-amylase AMY3 in A. thaliana (Streb et al., 2008). Mesophyll chloroplasts in mature maize leaves do not accumulate starch granules during normal vegetative growth because the Calvin cycle is not operating in those cells as a consequence of C4 metabolism (Majeran et al., 2010). Starch in maize leaves accumulates for the most part in bundle sheath cells, where RUBISCO and subsequent photosynthetic carbon metabolism reactions occur. Potentially, the metabolic nature of maize bundle sheath cells allows crystallization of precursor glucans into starch granules in the absence of ISA1, as it does in certain cell types in A. thaliana leaves, and this could explain why phytoglycogen was not observed in su1- or isa2- mutant maize leaves. Despite the absence of phytoglycogen, the formation of starch in the maize leaf in the absence of ISA1 or ISA2 is abnormal. This was shown by reduced contents of total granular material, abnormal granule size and shape, and changes in amylopectin fine structure. Another consideration regarding the lack of measurable WSP in mutant maize leaves is the fact that in A. thaliana phytoglycogen is degraded to some extent during the day, as shown by accumulation of the breakdown product maltose (Delatte et al., 2005; Streb et al., 2008). Whether or not such degradation occurs in maize su1- or isa2- mutant leaves and contributes to the lack of observed WSP and the reduced total glucan contents remains to be determined.
The decrease in total glucan polymer in the maize su1- or isa2- mutants had no obvious effect on plant health or growth rate, consistent with the fact that plants with su1-Ref and other alleles are widely grown as commercial sweetcorn varieties (Tracy et al., 2006). This might be explained by the fact that the amount of starch that accumulates in the mutants, c. 36–46% of normal, is sufficient to meet the physiological demands of the plant during the dark phase of the diurnal cycle. Some amount of leaf starch is required for normal maize growth, as shown by analysis of a near-starchless mutant affected in leaf ADPglucose pyrophosphorylase (Schlosser et al., 2012). In these mutants grown in field conditions, slight effects were observed on plant height, flowering time, and seed yield; however, plant health was not severely compromised. Thus, there appears to be some degree of physiological plasticity regarding how maize plants utilize photosynthate and transitory starch.
In summary, homomeric ISA1 was detected in maize leaves and shown to partially supply the in vivo requirement for ISA activity in starch biosynthesis. This is the first demonstration that homomeric ISA1 functions in leaf tissue. The ISA1/ISA2 heteromultimer was also shown to function in the maize leaf, although whether or not this form by itself could supply full physiological activity remains to be determined. The functional differences between maize and A. thaliana leaves, where ISA1 alone apparently is not active, are probably explained by structural differences that arose by evolutionary divergence in the monocot lineage.
The authors acknowledge T. Pepper and the Iowa State University Microscopy and NanoImaging Facility for performing the TEM analyses and C. Lancelon-Pin (CERMAV) for the SEM observation of purified starch granules.