Sugar-responsible elements in the promoter of a gene for beta-amylase of sweet potato.

Expression of genes coding for sporamin and beta-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately inducible in atypical vegetative tissues by sugars. A sweet potato gene for beta-amylase (beta-Amy) with introns as well as a beta-Amy::GUS fusion gene composed of the beta-Amy promoter and the GUS coding sequence, both showed sugar-inducible expression in leaves of transgenic tobacco which occurred via a hexokinase-independent pathway. Analyses using various 5'-terminal and internal deletions of the beta-Amy promoter indicated that truncated promoters of beta-Amy containing a sequence between -901 and -820, relative to the transcription start site, and the basic promoter region can confer sugar-inducible expression. This 82 bp region contained the TGGACGG sequence that plays an essential role in the sugar-inducible expression of the truncated promoter of the sporamin gene. Deletion or base substitutions of this element in the truncated beta-Amy promoter abolished the sugar-inducible expression, the results suggesting that the TGGACGG element plays an important role in the coordinate induction of expression of genes for beta-amylase and sporamin by sugars.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins.

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.

Expression patterns of two genes for the delta-subunit of mitochondrial F1-ATP synthase from sweet potato in transgenic tobacco plants and cells.

Two nuclear genes, F1 delta-1 and F1 delta-2, coding for the delta-subunit of mitochondrial F1-ATP synthase, which corresponds to oligomycin-sensitivity conferring protein in animal and yeast mitochondria, were isolated from sweet potato. The gene for the delta-subunit was composed of 6 exons and these two genes shared high sequence similarities to each other not only in exons but also in introns and in the 5'-upstream regions. However, the 5'-upstream regions of F1 delta-1 and F1 delta-2 were distinguishable by the presence of novel sequences, designated Ins-1 and Ins-2, respectively. Ins-1 and Ins-2 contained a terminal direct repeat of 10 bp and 12 bp, respectively, and various forms of repeat sequences. The promoter fusion of both F1 delta-1 and F1 delta-2 with the GUS coding sequence gave expression of GUS activity in transformed tobacco BY-2 cells, although the levels of GUS activity and the patterns of expression during the growth of cells were different between the two. In transgenic tobacco plants, the two fusion genes showed similar levels of expression in leaves and stems, while F1 delta-2:GUS gave significantly higher levels of expression in roots than F1 delta-1:GUS. Deletion of Ins-1 from the 5'-upstream region of F1 delta-1:GUS did not affect the expression of the fusion gene in various organs of transgenic plants. However, it caused significant enhancement of expression in transformed tobacco BY-2 cells.

A major jasmonate-inducible protein of sweet potato, ipomoelin, is an ABA-independent wound-inducible protein.

Treatment of sweet potato plants cultured in vitro with a vapor of methyl jasmonate (MeJA) induced an accumulation in leaves of a large amount of protein with an apparent molecular mass of 18 kDa. This protein, designated ipomoelin, was purified, and the amino acid sequences of proteolytic fragments were determined. Screening a cDNA library of MeJA-treated leaves by oligonucleotide probes designed from the peptide sequences identified a clone that could code for a polypeptide with 154 amino acids. The deduced amino acid sequence of ipomoelin showed an overall amino acid identity of 25% with the salt-inducible SalT protein of rice. In addition, the C-terminal 70 amino acid sequence of ipomoelin showed about 50% identity with the C-terminal amino acid sequences of seed lectins from Moraceae. The gene for ipomoelin was present in a few copies in the genome of sweet potato. The mRNA for ipomoelin was detected in leaves and petioles, but not in stems and tuberous roots, of sweet potato plants grown in the field. Mechanical wounding of leaves induced ipomoelin mRNA both locally and systemically, while treatment of leaves with ABA, salt, or a high level of sucrose did not induce ipomoelin mRNA. By contrast, ABA-inducible mRNA for sporamin was not induced by MeJA. These results suggest that ipomoelin is involved in defensive reactions of leaves in response to wounding and that JA-mediated wound-induction of ipomoelin occurs independently of ABA.

Conditional root expansion mutants of Arabidopsis.

Regulation of cell expansion is essential to the formation of plant organs. We have characterized 21 mutations, representing six loci, that cause abnormal cell expansion in the root of Arabidopsis thaliana. The phenotype of these mutants is conditional upon the rate of root growth. Calculation of cell volumes indicated that the mutations resulted in defects in either the orientation or the extent of expansion or in both. Analysis of cortical microtubules in the mutants suggested that a shift in the orientation of cell expansion may not be dependent on a change in the orientation of the microtubules. Double mutant combinations resulted in loss of the conditional phenotype suggesting that the genes may act in a similar pathway or encode partially redundant functions.

Transcript map of oppositely oriented pea mitochondrial genes encoding the alpha-subunit and the subunit 9 of F1F0-ATPase complex.

The atpA gene of pea mitochondria coding for the alpha-subunit of F1F0-ATPase (ATP synthase) is carried on four types of genomic segments generated by recombination, and two of these segments contain the atp9 gene coding for the subunit 9 of F1F0ATPase in the 5'-upstream region of atpA in an opposite orientation. The atpA and atp9 genes were separated by a spacer of 2112 bp, which does not contain any open reading frame. Pea mitochondria contained at least four atpA transcripts of 4.5, 2.7, 2.3, and 1.8 kb and at least three atp9 transcripts of 1.35, 0.6, and 0.4 kb. Mapping of these transcripts by northern blot hybridization, primer extension, and S1-nuclease mapping indicated that multiple transcripts of atpA differ in their length both at their 5'- and 3'-termini. On the other hand, three transcripts of atp9 differed in their length only at their 5'-termini. It was found that the 5'-terminal sequence of the 4.5-kb transcript of atpA is complementary to the 5'-terminal part of the 1.35- and 0.6-kb transcripts of atp9 for 705 and 4 nucleotides, respectively.

Molecular cloning and characterization of cDNAs for the gamma- and epsilon-subunits of mitochondrial F1F0 ATP synthase from the sweet potato.

Mitochondrial F1F0 ATP synthases purified from dicotyledonous plants contain six different subunits named alpha, beta, gamma, delta, delta', and epsilon. Our previous N-terminal amino acid sequence analyses indicated that the gamma- and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma- and epsilon-subunits of animal mitochondrial F1, respectively (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). A cDNA clone for the gamma-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide hybridization selection of a cDNA library, and a cDNA clone for the epsilon-subunit was isolated by reverse polymerase chain reaction and hybridization selection of a cDNA library by the polymerase chain reaction product. The 1.4-kilobase long cDNA for the gamma-subunit contained a 978-base pair open reading frame coding for a precursor for the gamma-subunit. The mature gamma-subunit is composed of 281 amino acids, and its sequence showed significantly higher similarities with the gamma-subunit of animal mitochondrial F1 and bacterial F1 compared with the gamma-subunit of chloroplast CF1 from plants. The precursor for the gamma-subunit contained N-terminal presequence of 45 amino acid residues. By contrast, the 0.46-kilobase long cDNA for the epsilon-subunit contained a coding sequence of 207-base pairs for the mature epsilon-subunit of 69 amino acid residues that is preceded by an ATG codon suggesting that the epsilon-subunit is synthesized without the cleavable presequence for mitochondrial import. The amino acid sequence of the epsilon-subunit of sweet potato mitochondrial F1 showed similarities of 25 and 36% amino acid positional identity with the epsilon-subunits of mitochondrial F1 from yeast and bovine, respectively.

The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.

High-level expression of a sweet potato sporamin gene promoter: beta-glucuronidase (GUS) fusion gene in the stems of transgenic tobacco plants is conferred by multiple cell type-specific regulatory elements.

Genes coding for sporamin, the most abundant protein of the tuberous root of the sweet potato, are expressed at a high levels in the stems of plantlets cultured axenically on sucrose-containing medium. Their expression is also induced in leaf-petiole explants by high concentrations of sucrose. A fusion gene comprising of the 1 kb 5' upstream region of the gSPO-A1 gene coding for the A-type sporamin and the coding sequence of bacterial beta-glucuronidase (GUS) was introduced into the tobacco genome by Agrobacterium-mediated transformation. Transgenic tobacco plants cultured axenically on sucrose-containing medium expressed GUS activity predominantly in their stems. Histochemical examination of GUS activity using a chromogenic substrate showed a distinct spatial pattern of GUS staining in the stem. Strong GUS activity was detected in the internal phloem of the vascular system and at the node, especially at the base of the axillary bud. Relatively weaker GUS activity was also detected in pith parenchyma. A 5' deletion of the promoter to nucleotide -305, relative to the transcription start site, did not alter significantly the level of GUS activity or the spatial pattern of GUS staining in the stem. However, further deletions to -237 and -192 resulted in a decrease in the level of GUS activity in the stem that occurred simultaneously with the loss of GUS staining in both the internal phloem and at the base of the axillary bud. However, plants with these deletion constructs still exhibited the predominant expression pattern of GUS activity in the stem and GUS staining in the pith parenchyma cells. Deletion to -94 completely abolished the expression of GUS activity. These results indicate that a sequence between -305 and -237 contains a cis-regulatory element(s) that is required for expression of the GUS reporter gene in both the internal phloem and at the base of the axillary bud, while a sequence between -192 and -94 contains a cis-acting element(s) that is required for expression in pith parenchyma cells.

Structure and expression of pea mitochondrial F1ATPase alpha-subunit gene and its pseudogene involved in homologous recombination.

We have characterized four pea mitochondrial DNA segments carrying the F1ATPase alpha-subunit coding sequences. These four types share a common 1.7-kb repeat sequence flanked by four combinations of two different left- and right-hand sequences. These results suggest that the alpha-subunit genes locate at the homologous recombination sites in the pea mitochondrial genome and that homologous recombination between two of these loci generates the other two types of structures. The uninterrupted alpha-subunit coding sequence of 1,521 bp is present in two of these loci. A rearrangement of 965 bp 3' to the ATG initiation codon generates two copies of pseudogenes where the C-terminal two-thirds of the alpha-subunit coding sequence is replaced with an unidentified coding frame. The other border of sequence divergence is located 733 bp upstream of the ATG initiation codon. Although multiple forms of alpha-subunit gene transcripts are present in mitochondria, the pseudogenes do not seem to express any alpha-subunit-related polypeptide.

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein.

By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.