Altered Maize Endosperm Adp-Glucose Pyrophosphorylases from Revertants of a SHRUNKEN-2-DISSOCIATION ALLELE.

Four phenotypically wild-type seeds were obtained from separate Activator-induced events in the Dissociation-inhibited allele sh2-ml (shrunken-2, mutable-1). Endosperm adenosine diphosphoglucose pyrophosphorylase, the enzyme controlled by sh2, was extracted and partially purified from the four revertants and was compared to enzyme produced by the progenitor Sh2 allele and the sh2-m allele.The revertants contained 50 to 140% of the activity conditioned by the progenitor allele. Each of the revertants appears to be unique as judged by differences in Km(glucose-1-PO(4)), 3-phosphoglycerate(3-PGA) activation, and phosphate-inhibition. In one case the reversion event apparently increased the sensitivity of ADP-glucose pyrophosphorylate to 3-PGA activation.

Multiple forms of maize endosperm adp-glucose pyrophosphorylase and their control by shrunken-2 and brittle-2.

Heat-labile and heat stable forms of ADP-glucose pyrophosphorylase were identified in the maize endosperm. The heat-labile form is destroyed by normal electrophoretic conditions. The heat-stable form corresponds to pyrophosphorylase B. In wild type, 96% of the total activity is heat labile. Both forms are reduced in 11 brittle-2 (bt2) and 12 shrunken-2 (sh2) mutants. The heat-labile form is reduced to a greater extent than is the heat-stable form in each of the 23 mutants. Deletion of sh2 abolishes both forms. The original ratio of the two forms is restored after sh2 function is expressed via transposition of Dissociation from sh2. The possible roles of these genes in the control of ADP-glucose pyrophosphorylase are discussed.

The mutation bronze-mutable 4 derivative 6856 in maize is caused by the insertion of a novel 6.7-kilobase pair transposon in the untranslated leader region of the bronze-1 gene.

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.

Coordinated Transcriptional Regulation of Storage Product Genes in the Maize Endosperm.

We have demonstrated that expression of genes involved in starch and storage protein synthesis of the maize (Zea mays L.) endosperm are coordinated. Genetic lesions altering synthetic events in one biosynthetic pathway affect expression of genes in both pathways. Initial studies focused on shrunken2 (sh2) and brittle2 (bt2) mutants because these genes encode subunits of the same enzyme, ADP-glucose pyrophosphorylase. Analysis of various sh2- and bt2- mutant alleles showed that the most severe mutations also conditioned the largest increase in transcripts. The analysis was extended by monitoring the transcripts of the genes, shrunken1 (sh1, structural gene for Suc synthase), sh2, bt2, waxy1 (wx1, structural gene for starch synthase), and those of the large and small zeins in isogenic maize lines at 14, 22, and 30 d postpollination. Endosperms were wild type for all of these genes or contained sh1-, sh2-, bt1-, bt2-, opaque2 (o2-), or amylose-extender1 (ae1-) dull1 (du1-) wx1- mutations. Transcripts increased continually throughout kernel development in the mutants relative to the standard W64A used. Variation in the amount of Suc entering the developing seed also altered transcript amounts. The results indicate that starch and protein biosynthetic genes act in a concerted manner, and both are sensitive to mutationally induced differences.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions.

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 degreesC. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

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.

Sugar utilization by developing wild type and shrunken-2 maize kernels.

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.

Development of wild type, shrunken-1 and shrunken-2 maize kernels grown in vitro.

Kernels of wild type maize (Zea mays L.) and the mutants shrunken-1 and shrunken-2 developed as much as in vivo when excised at five days post-pollination and grown in culture using existing methods. Mature kernels from culture exhibited their expected phenotypes. Starch, sugar and enzyme levels of kernels grown in culture were similar to those known to occur in kernels of the same genotypes grown in vivo. Differences in percentage germination of kernels grown in vitro were similar to those of kernels grown in vivo.

Maize transposable element Ds is differentially spliced from primary transcripts in endosperm and suspension cells.

The process by which transposable elements are spliced from the host gene transcripts remains poorly understood. We previously reported that a maize transposable element Ds (dissociation) and a copy of its host site duplication are perfectly spliced from the shrunken-2 transcript in the endosperm. Here, we have monitored splicing of the Ds element and its flanking Sh2 sequence following transient expression in maize suspension cells. The pattern of Ds splicing in suspension cells differs dramatically from that in the endosperm. In contrast to splicing in the endosperm, Ds in suspension cells was completely spliced from the transcripts using multiple donor and acceptor splice sites outside the element. In addition, noncanonical splice sites were utilized in suspension cells. Our results indicate that this difference in splicing is due to the context of Ds placement in the construct and/or to tissue specific differences in splicing.

Characterization of adenosine diphosphate glucose pyrophosphorylases from developing maize seeds.

Electrophoretic examination of 22-day-old, normal maize (Zea mays L.) endosperm extracts revealed two zones of adenosine diphosphate glucose pyrophosphorylase activity. The enzymes are identical in terms of Km for glucose 1-phosphate and the effect of 3-phosphoglyceric acid on apparent Km for glucose 1-phosphate. Both enzymatic activities increase with increasing doses of the functional alleles at the shrunken-2 and brittle-2 loci. Molecular weight differences between the two electrophoretic species were inferred from sucrose gradient centrifugation. It is suggested that the two bands of activity represent different aggregation states of the same enzyme because under different extraction conditions, only one enzyme is found. Molecular weight estimates of 237,000 and 253,000 were obtained for the smaller enzyme. It is suggested that this enzyme is an aggregate of several subunits. Comparison of the embryo and endosperm pyrophosphorylases showed the embryo activity to be more heat stable and probably independent of direct shrunken-2 or brittle-2 control.

ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize.

The Shrunken-2 (Sh2) and Brittle-2 (Bt2) genes of maize encode subunits of the tetrameric maize endosperm ADPglucose pyrophosphorylase. However, in all sh2 and bt2 mutants so far examined, measurable ADPglucose pyrophosphorylase activity remains. We have investigated the origin of the residual activity found in various sh2 and bt2 mutants as well as tissue specific expression and post-translational modification of the Sh2 and Bt2 proteins. Sh2 and Bt2 cDNAs were expressed in Escherichia coli and antibodies were prepared against the resulting proteins SH2 and BT2 specific antibodies were used to demonstrate that SH2 and BT2 are endosperm specific, are altered or missing in various sh2 or bt2 mutants, and have a mol. wt. of 54 and 51 kDa respectively in the wild type. The Sh2 and Bt2 transcripts are also endosperm specific. Ten sh2 and eight bt2 mutants show varying severity of phenotypes expressed at transcript, protein subunit and kernel level. Synthesis of multiple transcripts and proteins commonly occurs as a result of sh2 or bt2 mutation. While all mutants produce detectable enzymic activity, not all produce detectable transcripts and proteins. To examine the origin of the apparent non-SH2/BT2 endosperm enzymic activity, homologs of Sh2 and Bt2, designated Agp1 and Agp2 respectively, were isolated from an embryo cDNA library and found to hybridize to endosperm transcripts distinct from those of Sh2 and Bt2. Thus Agp1 and Agp2 or closely related genes may be responsible for the residual activity in some sh2 and bt2 mutants. Surprisingly, no evidence of post-translational modification of the SH2 and BT2 protein subunits was detected.

Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize.

Electrophoretic characterization of adenosine diphosphate glucose pyrophosphorylase from the developing endosperms of nine shrunken-2 and four brittle-2 mutants revealed that (1) all mutants had low but detectable levels of activity, (2) mutation at either locus decreased activity of pyrophosphorylases A and B, and (3) differences in mobility were not found. However, pyrophosphorylase B extracted from several shrunken-2 and brittle-2 mutants differed from normal in extent of urea denaturation, Km (glucose-1-phosphate) or type of glucose-1-phosphaociation with the sh2 locus) appears to differ from normal in Km (glucose-1-phosphate).

The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize.

Two differentially expressed genes encode isoenzymes of sucrose synthase in Zea mays. A clone of the shrunken 1 (Sh1) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (Css), the structural gene for the isoenzyme expressed in embryo and other tissues. The Css clone was positively identified by RNA blot analysis of RNA from wild type and a sh1 deletion stock and by analysis of the in vitro translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an r-x1 stock, coupled with restriction fragment length polymorphism mapping, placed the Css gene 32 map units from Sh1 on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both Sh1 and Css in root tissue is enhanced by anaerobic conditions, although Css is induced to a lesser extent than is Sh1. Thus, Css appears to be expressed constitutively, whereas Sh1 is expressed at high levels only in response to specific developmental and environmental stimuli.

Origin of the main class of repetitive DNA within selected Pennisetum species.

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 bp and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 bp and 160 bp KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 bp KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 bp KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 bp KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 bp KpnI repeat probably evolved from the 160 bp KpnI repeat since the missing 18 bp segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Increased gene expression by the first intron of maize shrunken-1 locus in grass species.

The first intron of the shrunken-1 (Sh1) locus of maize was incorporated into constructs containing the chloramphenicol acetyltransferase gene (CAT) coupled with the nopaline synthase 3' polyadenylation signal. Transcription was driven with the 35S promoter of the cauliflower mosaic virus (CaMV) or the Sh1 promoter of maize. Transient gene expression was monitored following electroporation into protoplasts of Panicum maximum (guineagrass), Pennisetum purpureum (napiergrass), or Zea mays (maize). The 1028 base pair intron increased gene expression in cells of each species when transcription was driven with the 35S promoter. Eleven to 91-fold increases were observed. Expression levels observed in maize were two and eight times those observed in napiergrass and guineagrass, respectively. The 35S promoter gave CAT activity 10 to 100 times that observed with the Sh1 promoter. Whereas expression driven by the 35S promoter was reproducible, that observed with the Sh1 promoter proved quite variable. In similar constructs the first intron of the alcohol dehydrogenase-1 (Adh1) gene of maize led to increased gene expression of only 7 to 10% of that observed with the Sh1 first intron. The increased level of gene expression caused by the Sh1 first intron is approximately 10 times higher than that caused by any other plant introns that have been used. Thus, the Sh1 first intron may prove quite useful in increasing expression of foreign genes in monocots and possibly other plants.

One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose.

The key regulatory step in starch biosynthesis is catalyzed by the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase). In leaf and storage tissue, the enzyme catalyzes the synthesis of ADP-glucose from glucose-1-phosphate and ATP. Using heterologous probes from maize, two sets (B and S) of cDNA clones encoding potato AGPase were isolated from a tuberspecific cDNA library. Sequence analysis revealed homology to other plant and bacterial sequences. Transcript sizes are 1.9 kb (AGPase B) and 2.1 kb (AGPase S). Northern blot experiments show that the two genes differ in their expression patterns in different organs. Furthermore, one of the genes (AGPase S) is strongly inducible by metabolizable carbohydrates (e.g. sucrose) at the RNA level. The accumulation of AGPase S mRNA was always found to be accompanied by an increase in starch content. This suggests a link between AGPase S expression and the status of a tissue as either a sink for or a source of carbohydrates. By contrast, expression of AGPase B is much less variable under various experimental conditions.

A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation.

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5' terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5' to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5' to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030-2031) led to the identification of 3' intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing.

Comparison of chemical and microbiological methods in the estimation of methionine in cowpea (Vigna unguiculata) seeds.

1. Meals were prepared from the seeds of fifteen varieties of cowpea (Vigna unguiculata), one of lima bean (Phaseolus lunatus) and one of yam bean (Sphenostylis stenocarpa), and their methionine content was determined by six different methods. 2. Total methionine content was determined by two chemical methods (ion-exchange chromatography and a colorimetric procedure) and by two microbiological methods. The 'available' methionine content was determined by microbiological assay with Streptococcus zymogenes. 3. All the different methods for total methionine determination gave similar results, with much the same high extent of precision. 4. The value for 'available' methionine content were similar to or marginally higher than the corresponding microbiological assay value for total methionine content. There was no indication that the methionine in any of the test samples was not completely available.