Sexual differentiation in cucumber: The effects of abscisic acid and other growth regulators on various sex genotypes

AbA, ethephon and gibberellin were applied to cucumber plantsof monoecious, gynoecious, andromoneocious and hermaphroditeinbred lines, as well as to F₁ (gynoecious?monoecious) plants.Exogenous AbA enhanced the male tendency in monoecious cucumberplants and the female tendency in gynoecious plants, irrespectiveof light regime. Exogenous ethephon treatments increased thefemale tendency in monoecious plants, and decreased it in gynoeciousones. These effects were influenced by day length. ExogenousAbA counteracted the effect of gibberellin (A₄₊₇) treatmentin gynoecious plants, but had no such effect in monoecious ones. In addition to its differential effect on sexual differentiation,AbA stimulated flower development in gynoecious plants and inhibitedit in monoecious plants. These responses to AbA are discussedin the light of previously reported effects of plant growthregulators on various sex types of cucumber. The present resultsare being integrated into an updated working hypothesis on sexcontrol in cucumbers. (Received August 30, 1976; )     

Isolation of a partial sequence of a putative nucleotide sugar epimerase, which may involve in stamen development in cucumber (Cucumis sativus L.)

Sex determination is the most widely studied subject in cucumber. The sex of cucumber plants can be monoecious, hermaphrodite, gynoecious, androecious, or andromonoecious. Besides environmental factors, three major genes, F/f, M/m, and A/a mainly govern the sex types in cucumber. Regardless of their sex all floral buds are bisexual at the early bud stage. A stage specific arrest of either stamen or carpel leads to unisexual flower development. The possible downstream product of the interaction of the sex determining genes that may directly allow the growth or selectively arrest stamen or pistil is not yet identified. Therefore, in the current study, we performed suppression subtractive hybridization using floral buds from nearly isogenic gynoecious and hermaphrodite cucumber plants and identified for the first time a cDNA homologous to nucleotide sugar epimerase. The expression level of the isolated putative nucleotide sugar epimerase is weak in female floral buds but strong in bisexual and male flowers. The weak level of the putative nucleotide sugar epimerase may be an indication for its improper functioning, which may influence stamen development in cucumber plants.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

黄瓜花性别分化与内源多胺的关系

研究了黄瓜雌、雄花几个主要发育时期和性别逆转过程中内源多胺的变化。结果表明 ,雄花在不同发育时期 ,内源腐胺含量均高于雌花 ,腐胺含量的显著升高伴随着花粉粒的形成 ,高腐胺含量是雄花发育的特征。雌花在大孢子母细胞时期以后直到雌花发育成熟 ,其内源尸胺含量均高于雄花 ,高尸胺含量可能有利于雌花的发育。高水平的内源多胺、精胺和亚精胺可能有利于雌花大孢子母细胞的形成。亚精胺和腐胺含量随着大小孢子四分体形成和大孢子核的连续分裂而分别表现下降和上升。雌性系黄瓜经硝酸银诱导雄花处理后 ,茎尖内源亚精胺含量下降 ,腐胺含量上升 ,从而诱导雄花形成 ;雄性系黄瓜经乙烯利诱导雌花处理后 ,茎尖内源亚精胺含量上升 ,腐胺含量下降 ,从而诱导雌花形成     

Metabolic engineering of cultured tobacco cells

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.     

Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.     

Characterization of the full-length sequences of phospholipase A2 induced during flower development

The suppression subtractive hybridization (SSH) method was used to isolate developmentally regulated genes during carnation flower maturation. Carnation flower maturation-related clones obtained by the SSH were serially assigned as CFMI (carnation flower maturation-induced) clones. Northern blot analysis showed that several CFMI clones were differentially expressed during flower development. One of the clones, CFMI-3, showed similarity to various animal secretory phospholipases A2 (PLA2). Since little is known about PLA2 gene sequence in plant species, the CFMI-3 clone was selected for further characterization by sequence analysis. Full sequence analysis reveals that the CFMI-3 contains a Ca2+ binding domain, a PLA2 active site, and 12 conserved Cys residues, which is a distinct characteristic of PLA2. Amino acid sequence alignment of CFMI-3 to various putative plant PLA2 confirmed that the CFMI-3 cDNA is the full-length putative PLA2 cDNA identified in plant species.     

Cloning and expression of flavonol synthase from Petunia hybrida

Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl , suggesting that Fl is the structural gene for FLS.     

cDNAs induced by ozone from Atriplex canescens (saltbush) and their response to sulfur dioxide and water-deficit

Ozone effects on plant water relations have been reported to be similar to those of water-deficit. The objective was to identify ozone-inducible (OI) clones from Atriplex canescens (saltbush) and determine if they were also responsive to water-deficit as well as SO₂. cDNA clones derived from four different polyA RNAs which accumulate in 8-month-old shrub leaves exposed to ozone (0.2 μl I⁻¹, 6 h day⁻¹, 7 days) were isolated by differential screening, analyzed by northern blots, sequenced, and gene product homologies with other plant genes were determined. Clone OI12A-3 has homology with wound-inducible proteinase inhibitors, whereas clone OI8–3 protein is homologous to thiol proteases. Clones OI2–2 and OI14–3 putatively code for glycine-rich proteins with repeated motifs (Gly-Gly-Gly-Tyr-Gly-His)_n and putative cell-wall-targeting signal peptides. Clone OI2–2 and particularly clone OI14–3 were also induced by both SO₂ and water-deficit. These data indicate that woody plant genes associated with cell wall protein production and whose expression is induced by several stress factors may be responding to common oxidative stress pathways.     

Characterization of ethylene effects on sex determination in cucumber plants

Sex differentiation in cucumber plants (Cucumis sativus L.) appears to be determined by the selective arrest of the stamen or pistil primordia. We investigated the influence of an ethylene-releasing agent (ethephon) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine) on sex differentiation in different developmental stages of flower buds. These treatments influence sex determination only at the stamen primordia differentiation stage in both monoecious and gynoecious cucumbers. To clarify the relationships between the ethylene-producing tissues and the ethylene-perceiving tissues in inducing female flowers in the cucumber, we examined the localization of mRNA accumulation of both the ACC synthase gene (CS-ACS2) and the ethylene-receptor-related genes (CS-ETR1, CS-ETR2, and CS-ERS) in flower buds by in situ hybridization analysis. CS-ACS2 mRNA was detected in the pistil primordia of gynoecious cucumbers, whereas it was located in the tissues just below the pistil primordia and at the adaxial side of the petals in monoecious cucumbers. In flower buds of andromonoecious cucumbers, only CS-ETR1 mRNA was detected, and was located in the pistil primordia. The localization of the mRNAs of the three ethylene-receptor-related genes in the flower buds of monoecious and gynoecious cucumbers overlap but are not identical. We discuss the relationship between the mRNA accumulation patterns and sex expression in cucumber plants.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.