Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation.

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.

Cis-elements important for the expression of the ADP-glucose pyrophosphorylase small-subunit are located both upstream and downstream from its structural gene.

ADP-glucose pyrophosphorylase (AGP) is a key regulatory enzyme in the biosynthesis of starch in higher plants. Previous studies have suggested that, unlike other plants that display tissue-specific AGP genes, potato expresses the same AGP small-subunit gene (sAGP) in multiple tissues. This view was confirmed by the spatial patterns of expression of the sAGP gene in transgenic potato plants observed when a promoter-dependent-beta-glucuronidase (beta-GUS) system was used. sAGP-beta-GUS chimeric gene fusions were expressed at high levels in tubers and in many other starch-containing cells throughout the plant. Deletional analysis of the 5'-upstream region of sAGP revealed that the observed spatial patterns of expression were due to different regions of the promoter of sAGP functioning in combination to confer cell- and organ-specific patterns of expression. Depending on the tissue examined, the patterns of reporter-gene expression were enhanced, suppressed, or altered when the 3'-nopaline-synthase terminator was replaced by the 3'-flanking sequence of sAGP. The observed cellular expression patterns of sAGP only partially overlap with the reported expression patterns of the major large-subunit gene (lAGP) in leaves. Since AGP is a heterotetrameric enzyme, composed of two sAGP and two lAGP subunits, this difference in the cellular expression patterns as well as quantitative differences in expression of the two AGP genes may account for the observed post-transcriptional regulation, i.e., relatively high levels of transcript but low levels of sAGP subunit in leaves.

Differential Regulation of ADP-Glucose Pyrophosphorylase in the Sink and Source Tissues of Potato.

Expression of potato (Solanum tuberosum L.) ADP-Glc pyrophosphorylase (AGP) was analyzed to assess whether the expression patterns of the individual subunit genes play a role in effectuating AGP activity and hence starch biosynthesis. Temporal analysis revealed that the coordinate expression of the large (IAGP) and small (sAGP) subunits, which collectively make up the heterotetrameric AGP holoenzyme, is primarily under transcriptional control during tuber development. In contrast, noncoordinate expression of the subunit transcripts was evident in leaves in which the relative level of the sAGP mRNA was present at severalfold excess compared to the level of IAGP mRNA. Immunoblot analysis, however, revealed that the levels of sAGP and IAGP polypeptides were present at near equimolar amounts, indicating that a posttranscriptional event co-ordinates subunit polypeptide levels. This posttranscriptional control of subunit abundance was also evident in leaves subjected to a photoperiod regime and during sucrose-induced starch synthesis. The predominant role of transcriptional and posttranscriptional regulation of AGP in tubers and leaves, respectively, is consistent with the distinct pathways of carbon partitioning and with the type and function of starch synthesis that occurs within each tissue.

Structure and expression of the potato ADP-glucose pyrophosphorylase small subunit.

ADP-glucose pyrophosphorylase (AGP) catalyzes a key regulatory step in starch synthesis. To elucidate the molecular basis for the expression of the potato (Solanum tuberosum L.) AGP during tuber development, the structure of the small subunit AGP (sAGP) gene and its patterns of expression were examined. DNA sequence analysis revealed that the sAGP gene is over 5.5 kilobases long and has a complex structure including eight introns. Unlike the situation in other plants where tissue-specific sAGP are found, our Southern and Northern blot analysis indicated that the same sAGP gene is expressed both in tubers (non-photosynthetic tissue) and leaves (photosynthetic tissue). These data were supported by comparing sequences of isolated sAGP leaf cDNAs to the tuber cDNA sequence, by primer extension analysis of leaf and tuber poly(A)+ RNAs, and by the spatial expression patterns of a gusA (beta-glucuronidase) reporter gene driven by the potato sAGP promoter in transgenic potato plants. Although the sAGP gene appeared to be transcriptionally controlled in both developing tubers and in leaves, the relative level of leaf antigen was significantly lower than its level of transcript, indicating that sAGP expression in leaves is primarily regulated post-transcriptionally. The observed tissue type-dependent regulation of sAGP expression appears to control the extent of starch biosynthesis by regulating the levels of this enzyme and, thus, alleviate the need for tissue-specific forms of the sAGP in potato.

Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.

cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADP-glucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrophosphorylase activity. Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity. Maximal enzyme activity required both subunits. The expressed ADP-glucose pyrophosphorylase was purified and characterized. The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber. The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum. 3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol. In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A0.5 value of 57 microM. The recombinant ADP-glucose pyrophosphorylase was less sensitive to P(i) inhibition and more sensitive to heat denaturation than the potato tuber enzyme. Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.

Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits.

Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to the Escherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.

The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase.

ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.