Targeting of mRNAs to domains of the endoplasmic reticulum.

The targeting of proteins to specific regions of the cell by signal elements within the polypeptide sequence has received much attention, but proteins can also be directed to their appropriate cellular locations by localization of their mRNAs. This mechanism is seen clearly in polar cells like germ and embryonic cells, neurons and epithelia. Recent evidence indicates that mRNAs may also be localized to morphologically and functionally distinct endoplasmic reticulum membranes, thereby facilitating sorting of the proteins they encode to subdomains of the reticulum or to polarized plasma membranes.

Rice prolamine protein body biogenesis: a BiP-mediated process.

Rice prolamines are sequestered within the endoplasmic reticulum (ER) lumen even though they lack a lumenal retention signal. Immunochemical and biochemical data show that BiP, a protein that binds lumenal polypeptides, is localized on the surface of the aggregated prolamine protein bodies (PBs). BiP also forms complexes with nascent chains of prolamines in polyribosomes and with free prolamines with distinct adenosine triphosphate sensitivities. Thus, BiP retains prolamines in the lumen by facilitating their folding and assembly into PBs.

Engineering starch for increased quantity and quality.

The characterization and production of starch variants from mutation studies and transgene technology has been invaluable for our understanding of the synthesis of the starch granule. The knowledge gained has allowed for genetic manipulation of the starch biosynthetic pathway in plants. This in vivo approach can be used to generate novel starches and diminishes the need for post-harvest chemically and enzymatically treated starches. Thus, the modification of the starch biosynthetic pathway is a plausible means by which starches with novel properties and applications can be created.

Role of silicon on diatom metabolism. IX. Differential synthesis of DNA polymerases and DNA-binding proteins during silicate starvation and recovery in Cylindrotheca fusiformis.

During recovery from silicate-starvation, a period of active DNA synthesis, synchronized cells of Cylindrotheca fusiformis incorporated 3 times more L-[U-14C]aspartate than did starved cells. Of the diatoms's four DNA polymerases, A and D are synthesized during silicate recovery, indicating that they are involved in silicate-dependent DNA replication. Polymerase B, and the chloroplast enzyme, polymerase C, are synthesized during silicate-starvation and their levels are unaffected by the addition of silicate. DEAE-Sephadex analysis of the DNA-binding proteins, labeled with [14C]- and [3H]asparate, shows that only three proteins are synthesized in cells recovering from silicate-starvation. Two of these proteins correspond to polymerases A and D, while the function of the third protein is not known. At least 15 other proteins are present in silicate-starved cells and their synthesis is repressed upon the addition of silicate. Models are proposed which describe the modes by which silicate might regulate DNA synthesis in the diatom.

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.

Analyses of alpha/beta-type gliadin genes from diploid and hexaploid wheats.

The alpha/beta-gliadin genes isolated from both hexaploid wheat (cv. Yamhill) and the diploid A genome progenitor Triticum urartu had remarkably similar sequences and differ by only a few point mutations. Primer extension analysis indicated that the transcriptional start points for individual genes in the family cluster within a few nucleotides. Comparison of the promoter region of several alpha/beta-gliadin and B-hordein genes reveals two conserved regions at about -130 and -250 bp. DNA from the hexaploid cultivars, Cheyenne and Chinese Spring, and the diploid progenitors T. urartu and Aegilops squarrosa was analysed by Southern blotting. Restriction fragment lengths of the alpha/beta-gliadin genes varied only slightly between the various wheats, although the overall copy number varied significantly. A region between approx. -1700 and -700 bp upstream from the TATA box was highly repeated in all three wheat genomes. For the hexaploid-derived gene, over 1700 bp of sequence upstream from the TATA box was determined, revealing an additional open reading frame between approx. -1550 and -1250 bp relative to the gliadin TATA box. Northern blot analysis indicated that RNA homologous to this repeated sequence family was present only in developing seed and accumulated to a maximum at late stages of maturation.

Molecular characterization of the gene encoding a rice endosperm-specific ADPglucose pyrophosphorylase subunit and its developmental pattern of transcription.

The gene encoding a rice endosperm-specific ADPglucose pyrophosphorylase (AGPP) subunit was isolated and its structure determined by nucleotide (nt) sequencing. A comparison of the genomic and cDNA nt sequences revealed a complex gene structure with ten exons and nine introns spanning over 6 kb. The exons ranged in size from 293 to 99 nt and the introns were between 1435 and 84 nt in size, with the first intron being the largest. All of the intron splice sites, except intron-2, contained GT/AG borders and were similar to the published splice site consensus sequences. Intron-2 had CA/CC borders at the 5' and 3' ends, but sequences adjacent to the splice site borders shared homology to the splice site consensus sequence, suggesting that the overall splice region, rather than the specific GT/AG sequence, determines the splice site. Several sequence motifs which may play a role in the regulation of plant genes were evident upstream from the transcriptional start point. Analysis of the developmental pattern of expression revealed a maximum level of mRNA transcripts for AGPP at five days after flowering coincident with starch accumulation. This result suggests that starch biosynthesis is controlled at the transcriptional level during seed development.

Nucleotide and primary sequence of a major rice prolamine.

A recombinant cDNA clone encoding a major rice seed storage prolamine was isolated by antibody screening of a cDNA lambda gt 11 library. This clone contained a single open reading frame encoding a putative rice prolamine precursor (Mr 17,300). In contrast to other cereal prolamines, the primary sequence of the rice prolamine was devoid of any major tandem repetitive sequences, a feature prevalent in all cereal prolamines studied to date. No significant homology was detected between the rice prolamine and other cereal prolamines, indicating that the rice gene evolved from unique ancestral DNA segments.

Isolation and characterization of a higher plant ADP-glucose pyrophosphorylase small subunit homotetramer.

ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the LS. TG-15 has a leucine to phenylalanine change at position 48 (L(48)F) that corresponds to a phenylalanine residue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V(59)I). TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.

Increasing rice productivity and yield by manipulation of starch synthesis.

Plant productivity and yield are dependent on source-sink relationships, i.e. the capacity of source leaves to fix CO2 and the capacity of developing sink tissues and organs to assimilate and convert this fixed carbon into dry matter. Studies from our laboratories as well as others have demonstrated that rice productivity and yield are mainly sink-limited during its development because of limited capacity to utilize the initial photosynthetic product (triose phosphate). This limitation in triose phosphate utilization, evident at both the vegetative and reproductive stages of rice development, may be associated with limited capacity for carbohydrate synthesis in rice leaves (which are poor accumulators of starch) or feedback due to limited sink strength of developing seeds. Strategies in improving triose phosphate utilization by enhancing starch production in leaves and developing seeds by the expression of engineered genes for ADP glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, are discussed.

Substrate binding mutants of the higher plant ADP-glucose pyrophosphorylase.

To explore the structure-function relationships of the heterotetrameric higher plant ADP-glucose pyrophosphorylase, composed of a pair of large and small subunits, the small subunit cDNA was subjected to chemical mutagenesis and then co-expressed with the wild-type large subunit cDNA. Mutants were selected for their inability to complement a defective bacterial ADP-glucose pyrophosphorylase gene and, in turn, to accumulate glycogen as viewed by iodine staining of the cells. Based on these initial analyses, we subsequently identified four distinct classes of mutations which were glycogen-deficient but exhibited enzyme activity levels comparable to the normal recombinant enzyme under saturating reaction conditions. Three classes, each a product of single amino acid substitution, showed altered kinetic constants for substrates. Substitution of Asp252 to Asn conferred the enzyme lower affinity for glucose-1-phosphate, replacement of Asp121 to Asn resulted in an enzyme less responsive to both glucose-1-phosphate and ATP, while the Ala106 to Thr substituted enzyme contains altered sensitivity primarily to ATP. The fourth class, a Pro43 to Ser substitution, resulted in an enzyme with decreased sensitivity (8-fold) to the activator 3-PGA. Overall, the results of this study suggests that the two subunit types do not have identical roles in enzyme function and that the small subunit plays a more dominant role in catalysis than the large subunit.

Is there an alternative pathway for starch synthesis?

In leaf tissue, carbon enters starch via the gluconeogenesis pathway where d-glycerate 3-phosphate formed from CO(2) fixation is converted into hexose monophosphates within the chloroplast stroma. In starch-containing sink organs, evidence has been obtained indicating that the flow of carbon into starch follows a different pathway whereby hexose monophosphates formed from sucrose are transported into the amyloplast, a plastid specialized in starch accumulation. In both chloroplasts and amyloplasts, the formation of ADPglucose, the substrate for starch synthase, is controlled by the activity of ADPglucose pyrophosphorylase, a key regulatory enzyme of starch synthesis localized in the plastid. Recently, an alternative pathway of starch synthesis has been proposed in which ADPglucose is synthesized from sucrose and transported directly into the plastid compartment, where it is used for starch synthesis. On the basis of the biochemical phenotypes exhibited by various plant mutants with defined genetic lesions, it is concluded that ADPglucose pyrophosphorylase is essential for starch synthesis, whereas the alternative pathway has only a minor role in this process.

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA.

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat.

Recombinant cDNA plasmids possessing the coding sequences for the γ-type gliadins were isolated from a cDNA library prepared from wheat seed poly (A(+)) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A(+)) RNA molecules 1 400-1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the γ-type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3' untranslated region as well as 245 amino acids from the C-terminus of the γ-type gliadin polypeptide. The 5'-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the α/ß-type and γ-type gliadin cDNA sequences.

Structure, expression, and heterogeneity of the rice seed prolamines.

By screening two rice (Oryza sativa L.) seed cDNA libraries, recombinant cDNA clones encoding the rice prolamine seed storage protein were isolated. Based on cross-hybridization and restriction enzyme map analyses, these clones can be divided into two homology classes. All clones contain a single open reading frame encoding a putative rice prolamine precursor (molecular weight = 17,200) possessing a typical 14-amino acid signal peptide. The deduced primary structures of both types of prolamine polypeptides are devoid of repetitive sequences, a feature prevalent in other cereal prolamines. Clones of these two homology classes diverge mainly by insertions/deletions of short nucleotide stretches and point mutations. An isolated genomic clone about 15.5 kilobases in length displays a highly conserved 2.5-kilobase EcoRI fragment, repeated in tandem four times, each containing the prolamine coding sequence. Close homology is exhibited by the coding segments of the genomic and cDNA sequences, although the 5' ends of the untranslated regions are widely divergent. The sequence heterogeneity displayed by these genomic and cDNA clones and large gene copy number ( approximately 80-100 copies/haploid genome) indicate that the rice prolamines are encoded by a complex multigene family.

Structural Relationship among the Rice Glutelin Polypeptides.

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the alpha and beta subunits are proteolytic products of the 51 kD precursor protein. The poly(A)(+) RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.

Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells.

A simple method is presented for the preparation of nuclear extracts from suspension cultures of rice, wheat and tobacco cells. These extracts are shown to be capable of RNA Polymerase II-dependent transcription from two plant promoters in vitro; a 250 bp fragment of a wheat gliadin promoter containing sequences from -167 bp to +83 relative to the in vivo transcriptional initiation site and two fragments of the CaMV 35S promoter, containing sequences from -419 to +17, and from -90 to +17. Using the rice extract, transcription is shown to be extract-dependent, DNA-dependent, alpha-amanatin-sensitive, promoter-dependent, and accurate with respect to initiation site selection on the gliadin promoter and the -90 to +17 35S promoter, but not accurate on the -419 to +17 35S promoter.

Interactions of the glutelin Gt3 5' flanking regulatory regions with rice nuclear proteins.

Three DNA binding activities, BP-1, BP-2 and BP-3, which interact with the 5' flanking region of the rice glutelin Gt3 gene, were identified by gel retardation assays of rice seed nuclear extracts. The DNA binding activities were seed-specific as identical DNA-protein complexes were not observed when nuclear extracts from leaf tissue or suspension culture cells were analyzed, suggesting that these DNA binding activities are involved in seed-specific expression of the Gt3 gene. The DNA sequences recognized by these DNA binding activities were identified by DNaseI foot-printing and Bal 31 nuclease mapping analyses. BP-1 recognizes DNA sequences located at -272 bp to -259 bp relative to the transcriptional start site. This DNA segment contains a sequence motif that is conserved among several seed protein genes, implicating that the motif may be a common cis-regulatory element determining seed-specific expression of these genes. BP-2 interacts with sequences located between -861 bp to -838 bp while BP-3 interacts with sequences upstream of -788 bp. The temporal levels of BP-2 binding activity parallel the steady state levels of the Gt3 mRNAs during seed development. Overall, these results and those obtained from in vivo promoter analysis in transgenic plants [Zhao et al. (1994) Plant Mol. Biol. 25: 429] indicate that multiple regulatory elements located at two spatially separated regions of the Gt3 promoter are involved in endosperm-specific and temporal regulation.

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.