Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

A Comparison of Two Sucrose Synthetase Isozymes from Normal and shrunken-1 Maize

The maize sucrose synthetase isozyme (SS2) present in sh1 endosperm, sh1 seedlings, and in suspension culture cells was purified to homogeneity from each of these tissues by sequential ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, gel filtration chromatography, and affinity elution with UTP from a carboxymethyl-cellulose column. Cyanogen bromide digests were used to demonstrate that the SS2 isozymes in these different tissues are structurally identical and are therefore the product of the same gene. The sucrose synthetase produced by the Sh1 gene (SS1) was purified by modification of the SS2 procedure and was used in comparative analyses of the two isozymes. Ouchterlony assays demonstrated that SS1 and SS2 have partial antigenic identity. The two isozymes have similar enzyme kinetics in the sucrose cleavage reaction but differ in their relative activities with ADP and TDP. The amino acid compositions of SS1 and SS2 are similar, and proteolytic digests revealed that they share limited structural homologies.     

The enzymatic deficiency conditioned by the shrunken-1 mutations in maize

Evidence is presented to show that the Sh locus specifies sucrose synthetase in the developing endosperm of maize. The sh/sh/sh endosperm possesses less than 10% sucrose synthetase activity as compared to the normal Sh/sh/sh endosperm. The residual enzyme activity in five independently derived mutant genotypes is attributable to a protein molecule of different electrophoretic and immunochemical specificities that is presumably independent of the sh locus. Sucrose synthetase activity in the embryo in both the genotypes is electrophoretically indistinguishable from the one present in the mutant endosperm. Mutant endosperm has a reduced starch content as compared to the normal. This observation constitutes genetic evidence supporting a critical role for sucrose synthetase in starch biosynthesis.     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Identification and characterization of intraspecific variability of the sucrose synthase gene <Emphasis Type="Italic">Sus4</Emphasis> of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Nucleotide and amino acid variability of fragments of the Sus4 gene encoding the sucrose synthase enzyme was studied in 24 potato cultivars bred in Russia and other countries and differing in starch content in tubers. Both SNPs and indels were detected in a chosen Sus4 gene fragment including the sequence from exon 3 to exon 6 and corresponding to the main part of the sucrose synthase domain. Four types of Sus4 sequences were revealed depending on the presence of an insertion in introns 4 and 5 and of the mononucleotide octamer (T)₈ in intron 5. Differentiation of these sequences was confirmed by statistical methods. Sixteen amino acid substitutions were identified in the translated sequence, of which eleven were nonsynonymous. Specific cultivar-specific nucleotide and amino acid substitutions were also revealed, which can be used in future for identifying potato cultivars/genotypes. No direct associations between the mutational changes and the starch content were found in the potato cultivars studied.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998     

Genetic evidence that invertase-mediated release of hexoses is critical for appropriate carbon partitioning and normal seed development in maize

 Cell wall-bound invertase (CWI) is spatially and temporally the first enzyme which metabolizes the incoming sucrose in developing seed of maize (Zea mays). Our previous studies have shown that the cell wall-bound invertase-2 (INCW2) isozyme encoded by the wild-type gene of the Miniature1 (Mn1) seed locus plays a critical role in seed development. Null mutations of the gene, such as the mn1 seed mutant which lacks invertase activity, are associated with a loss of ∼70–80% of the normal seed weight. We show here that under in vitro kernel culture conditions the hexose-based medium was similar to the sucrose-based medium in promoting the normal development of kernels of the Mn1, but not of the mutant mn1, genotype. Anatomical, biochemical, and immunohistological data showed that the mn1 kernels retain their mutant phenotype regardless of the presence of sucrose or hexoses in the culture media. The most drastic changes in the mn1 seed mutant were associated with a significant reduction in the size of the endosperm, but not in the pattern or the level of starch localization. Because Mn1 expression was temporally coincident with the endosperm cell divisions, INCW2 must play a critical role in providing hexose sugars for mitotic division, and only a minor role in generating carbon skeletal substrates for starch biosynthesis in the early stages of endosperm development. Furthermore, a lack of the wild-type seed phenotype of the mn1 mutant in hexose media suggests that a metabolic release of hexoses catalyzed by INCW2, rather than an exogenous source, is critical for both generating appropriate sugar-sensing signals for gene expression and for normal endosperm development. Received: 8 April 1998 / Accepted: 14 August 1998     

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.     

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.     

The wheat (T. aestivum) sucrose synthase 2 gene (TaSus2) active in endosperm development is associated with yield traits

Sucrose synthase catalyzes the reaction sucrose + UDP → UDP-glucose + fructose, the first step in the conversion of sucrose to starch in endosperm. Previous studies identified two tissue-specific, yet functionally redundant, sucrose synthase (SUS) genes, Sus1 and Sus2. In the present study, the wheat Sus2 orthologous gene (TaSus2) series was isolated and mapped on chromosomes 2A, 2B, and 2D. Based on sequencing in 61 wheat accessions, three single-nucleotide polymorphisms (SNPs) were detected in TaSus2-2B. These formed two haplotypes (Hap-H and Hap-L), but no diversity was found in either TaSus2-2A or TaSus2-2D. Based on the sequences of the two haplotypes, we developed a co-dominant marker, TaSus2-2B _tgw , which amplified 423 or 381-bp fragments in different wheat accessions. TaSus2-2B _tgw was located between markers Xbarc102.2 and Xbarc91 on chromosome 2BS in a RIL population from Xiaoyan 54 × Jing 411. Association analysis suggested that the two haplotypes were significantly associated with 1,000 grain weight (TGW) in 89 modern wheat varieties in the Chinese mini-core collection. Mean TGW difference between the two haplotypes over three cropping seasons was 4.26 g (varying from 3.71 to 4.94 g). Comparative genomics analysis detected major kernel weight QTLs not only in the chromosome region containing TaSus2-2B _tgw, but also in the collinear regions of TaSus2 on rice chromosome 7 and maize chromosome 9. The preferred Hap-H haplotype for high TGW underwent very strong positive selection in Chinese wheat breeding, but not in Europe. The geographic distribution of Hap-H was perhaps determined by both latitude and the intensity of selection in wheat breeding.     

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-¹⁴C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-¹³C]-, [2-¹³C]- or [U-¹³C]-glucose followed by the quantification by ¹H-NMR and ¹³C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.