Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants

Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Antiquity and evolution of the MADS-box gene family controlling flower development in plants

MADS-box genes in plants control various aspects of development and reproductive processes including flower formation. To obtain some insight into the roles of these genes in morphological evolution, we investigated the origin and diversification of floral MADS-box genes by conducting molecular evolutionary genetics analyses. Our results suggest that the most recent common ancestor of today's floral MADS-box genes evolved roughly 650 MYA, much earlier than the Cambrian explosion. They also suggest that the functional classes T (SVP), B (and Bs), C, F (AGL20 or TM3), A, and G (AGL6) of floral MADS-box genes diverged sequentially in this order from the class E gene lineage. The divergence between the class G and E genes apparently occurred around the time of the angiosperm/gymnosperm split. Furthermore, the ancestors of three classes of genes (class T genes, class B/Bs genes, and the common ancestor of the other classes of genes) might have existed at the time of the Cambrian explosion. We also conducted a phylogenetic analysis of MADS-domain sequences from various species of plants and animals and presented a hypothetical scenario of the evolution of MADS-box genes in plants and animals, taking into account paleontological information. Our study supports the idea that there are two main evolutionary lineages (type I and type II) of MADS-box genes in plants and animals.     

Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene

A cDNA encoding chalcone isomerase (CHI) was isolated from the petals of Nicotiana tabacum and the effect of its suppression on flavonoid biosynthesis was analyzed in transgenic tobacco plants. CHI-suppression by RNA interference (RNAi) showed reduced pigmentation and change of flavonoid components in flower petals. The plants also accumulated high levels of chalcone in pollen, showing a yellow coloration. Our results first demonstrated that suppression of CHI by genetic transformation is possible in higher plants. This suggests that CHI plays a major part in the cyclization reaction from chalcone to flavanone, and that spontaneous reactions are few, if any, in tobacco plants.     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.     

Changes in free polyamines and gene expression during peach flower development

Free polyamine contents and expressions of five genes encoding for polyamine biosynthetic enzymes were investigated at four different stages during peach (Prunus persica L. Batsch cv. Akatsuki) flower development. Fresh mass of peach flowers increased, accompanied by reduction in contents of total polyamines and putrescine/spermidine ratio due to decrease in putrescine content. Spermidine, the largest fraction, and spermine, the least part, underwent minor change. Expressions of the five key genes involved in polyamine biosynthesis during flower development did not parallel the changes in free polyamines.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Genome-wide analysis of gene expression during early Arabidopsis flower development

Detailed information about stage-specific changes in gene expression is crucial for the understanding of the gene regulatory networks underlying development. Here, we describe the global gene expression dynamics during early flower development, a key process in the life cycle of a plant, during which floral patterning and the specification of floral organs is established. We used a novel floral induction system in Arabidopsis, which allows the isolation of a large number of synchronized floral buds, in conjunction with whole-genome microarray analysis to identify genes with differential expression at distinct stages of flower development. We found that the onset of flower formation is characterized by a massive downregulation of genes in incipient floral primordia, which is followed by a predominance of gene activation during the differentiation of floral organs. Among the genes we identified as differentially expressed in the experiment, we detected a significant enrichment of closely related members of gene families. The expression profiles of these related genes were often highly correlated, indicating similar temporal expression patterns. Moreover, we found that the majority of these genes is specifically up-regulated during certain developmental stages. Because co-expressed members of gene families in Arabidopsis frequently act in a redundant manner, these results suggest a high degree of functional redundancy during early flower development, but also that its extent may vary in a stage-specific manner.     

Abnormal flower formation of tobacco plants regenerated from callus cultures

The plantlets regenerated from tobacco calluses, subcultured for prolonged periods, were weak and generally could not be cultivated into mature flowering specimens. However, some of them flowered and various kinds of abnormal flowers as well as leaves were observed in all the tobacco plants bearing flowers. They were sterile at had abnormal immature pollen grains. Some of them occasionally germinated in the anther. Abnormalities were also found in the nucleus of immmature pollen grains. Such drastic floral abnormalities were not found in tobacco plants derived from seeds and callus cultures subcultured for a relatively short period under the same conditions of cultivation. Part XVIII in the series “studies on plant tissue cultures”; for Part XVII, see Chem. Pharm. Bull., in press (1972).     

Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants

To study the role of ethylene in plant reproduction, we constructed transgenic tobacco plants in which the expression of a pistil-specific gene coding for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylate oxidase was inhibited. Flowers from transgenic plants showed female sterility due to an arrest in ovule development. Megasporogenesis did not occur, and ovules did not reach maturity. When pollinated, pollen tubes were able to reach the ovary but did not penetrate into the immature ovule in transgenic plants. Flower treatment with an ethylene source resulted in a functional recovery of ovule development and restored guidance of the pollen tube tip into the ovule micropyle that resulted in seed set. The recovery was abolished if inhibitors of ethylene action were present. These results demonstrate that the plant hormone ethylene is required during the very early stages of female sporogenesis and ultimately to enable fertilization.     

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.     

Scopoletin expression in elicitor-treated and tobacco mosaic virus-infected tobacco plants

Localized acquired resistance (LAR) characterizes a narrow zone of living cells expressing strong defense responses and surrounding cells undergoing a hypersensitive response (HR). In Samsun NN tobacco plants, tissues undergoing tobacco mosaic virus-induced or elicitor-induced LAR exhibit a strong blue fluorescence under UV light. We have shown that scopoletin and its glucoside, scopolin, accounted for the fluorescence: (1) both compounds were identified after extraction and purification by thin layer and high performance liquid chromatography; (2) there was a strict correlation between the occurrence of fluorescence and accumulation of high amounts of scopoletin; and (3) infiltration of commercial scopoletin caused a similar fluorescence to that occurring in LAR tissues. There was a 20-fold increase in scopoletin levels in LAR tissues compared to tissues treated with a non-HR dose of elicitor, while PR1 protein accumulated in similar amounts in both types of tissues. Scopoletin was able to suppress the elicitor-induced HR only when co-infiltrated with very low HR-dose of elicitor. These two observations suggested that, although scopoletin alone would not be able to control the development of the HR through its known antioxidant activity, it may nevertheless participate to such function of LAR tissues in combination with other antioxidant molecules.     

Mechanically stimulated TCH3 gene expression in Arabidopsis involves protein phosphorylation and EIN6 downstream of calcium

Mechanical signals are important both as environmental and endogenous developmental cues in plants. Among the quickest measurable responses to mechanical stimulation (MS) in plants is the up-regulation of specific genes, including TCH3, in Arabidopsis. Little is known about the signaling events and components that link perception of mechanical signals to gene expression in plants. Calcium has been identified previously as being potentially involved, and a role for ethylene has also been suggested. Using the protein kinase inhibitor staurosporine, we determined that MS up-regulation of TCH3 expression requires protein kinase activity in young Arabidopsis seedlings. Our data from studies on the Arabidopsis ein6 mutant demonstrate that the EIN6 protein is also required, but that its role in mechanically induced TCH3 expression appears to be independent of ethylene. Challenge of seedlings with protein phosphatase inhibitors calyculin A and okadaic acid stimulated TCH3 expression even in the absence of MS, implying protein phosphatase activity acting to negatively regulate TCH3 gene expression. This phosphatase activity acts either downstream or independently of EIN6. EIN6 and protein kinase activity, on the other hand, operate downstream of calcium to mediate mechanically stimulated TCH3 expression.     

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M _r50000 together with a range of smaller polypeptides.     

Effects of codon modification on human BMP2 gene expression in tobacco plants

Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused β-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants.     

Mosaic expression pattern of the nptII gene in transgenic tobacco plants Nu 21

Mosaic expression pattern of the nptII gene in transgenic tobacco Nu 21 leaf somatic cells was demonstrated. Inheritance of this phenotype (in T1-T4 and F1 backcrosses) was revealed. Three plant groups were distinguished, with low frequency of variegation manifestation (0-21.8%), with the high frequency of mosaic progeny (63.1 to 100%), and the intermediate type, where the frequency of the appearance of mosaic plants varied in a wide range, from 0 to 100%. The data obtained suggested the existence of two metastable states of a transgene in the leaf disk somatic cells (active and silenced), which could be associated with DNA modification, i.e., methylation of cytosine within the nptII gene sequence.