Soybean leghemoglobin targeted to potato chloroplasts influences growth and development of transgenic plants

 Potato tubers were transformed with a chimeric gene made by the fusion of the soybean leghemoglobin encoding gene (lba) with the chloroplastic targeting sequence from Rubisco. This construct was placed under the control of the strong constitutive 35S promoter and the 3′ nontranslated region of Rubisco from pea. Leghemoglobin expression on kanamycin-resistant plants was monitored by RT-PCR. Furthermore, immunodetection of subcellular fractions of transgenic plants revealed that leghemoglobin was imported and correctively processed inside the organelle. In addition, analysis of transgenic plants revealed reduced growth and decreased tuber production compared with the untransformed plants. It is suggested that leghemoglobin expression in potato chloroplasts interferes with aerobic metabolism, leading to physiological and morphological changes. Received: 20 December 1999 / Revision received: 28 May 2000 / Accepted: 16 June 2000     

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the “Class II” hemoglobins. Received: 28 June 1999 / Accepted: 25 November 1999     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.     

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C₁₉-GAs, including the bioactive GAs GA₁ and GA₄, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA₁ and GA₄. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds. C. Dijkstra, E. Adams, A. Bhattacharya and A. F. Page contributed equally to this paper.     

Pleiotropic effect of flavonoid biosynthesis manipulation in transgenic potato plants

Three approaches were successfully used to manipulate content of flavonoids in transgenic plants. Overexpressing either the adaptor 14-3-3 protein or genes coding the key enzymes of the flavonoid biosynthesis pathway resulted in a significant increase in the compound content in potato tuber epidermis. The opposite effect was observed in transgenic plants in which these proteins were repressed; this strongly supports the view that the gene construct determines transgenic plant features. The most effective construct was, however, the one containing single dihydroflavonol reductase (DFR) gene in sense orientation. In all cases the increase in flavonoid content resulted in the expected enhancement of the antioxidant capacity of tuber extract. At the biochemical level a decrease in the starch content in transgenic plant overexpressing proteins regulating flavonoid biosynthesis was detected. In the case of glucosyl transferase (GT) gene overexpression, the content of phenolic compounds remained at the control level, however, the antioxidant capacity of tuber extracts significantly decreased. The GT plants grew faster and were more resistant to pathogen attacks, the tuber yield was significantly higher than that of nontransformants. Thus it is speculated that it is the chemical structure and degree of glucosylation of flavonoids rather than their quantity which determines transgenic plant features.     

The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDA kilodalton - Lb leghemoglobin - TBST Tris-buffered saline plus Tween 20     

Tailored biosynthesis of gibberellin plant hormones in yeast

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.We then built a GA₄-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA₄. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA₇ (trace amounts) and GA₃ (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA₃-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes.ClassificationBiological Sciences, Applied Biological Sciences.     

The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants

Summary The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[¹⁴C]lysine when fed with [¹⁴C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterised by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.     

Reduction of the chloroplastic fructose-1,6-bisphosphatase in transgenic potato plants impairs photosynthesis and plant growth

Transgenic potato plants expressing reduced levels of the chloroplastic isoform of fructose-1,6-bisphosphatase (cp-FBPase) were created via the antisense RNA technique. Transformants with different levels of FBPase activity were selected and analysed with respect to photosynthesis, carbon metabolism, and growth. FBPase activity of less than 15% of wild-type levels led to reduced growth rates, probably due to the reduction of photosynthetic activity. A significant decrease in tuber yield is observed in plants with a FBPase activity below 15% of wild-type levels, whereas plants with 36% of wild-type enzyme activity still give normal tuber yields, even though they demonstrate a lowered photosynthetic capacity. Decreased photosynthesis also results in a reduction of total carbohydrate contents in leaves. Interestingly, increased carbohydrate partitioning towards soluble sugars is observed in plants displaying less than 15% of the wild-type FBPase activity. When excised leaf discs are placed on sucrose-containing media in darkness, discs derived from plants with a reduced FBPase activity accumulate higher amounts of starch. Possible implications are discussed.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.     

Ectopic expression of Hox-2.3 induces craniofacial and skeletal malformations in transgenic mice.

To better understand the role of the Hox-2.3 murine homeobox gene during development, a dominant gain-of-function mutation was generated. The developmental malformations that resulted when the chicken beta-actin promoter was used to direct widespread expression of the Hox-2.3 gene in transgenic mice included early postnatal death as well as craniofacial abnormalities, including open eyes and cleft palate. Ventricular septal defects were also observed in the hearts of three transgenic mice. Skeletal malformations were seen in the bones of the craniocervical transition, with the occipital, basisphenoid, and atlas bones deficient or misshapen. Interestingly, one mutant exhibited an extra pair of ribs as well as alterations in cervical vertebrae identities. Some of the malformations observed in Hox-2.3 gain-of-function mutants overlap with those seen in Hox-1.1 and Hox-2.2 misexpression mutants which suggests functional similarities between paralogous homeobox genes. The results of these experiments are consistent with a role for Hox-2.3 in specifying positional information during development.     

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.     

Ectopic expression of KNOTTED1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice

Some phytohormones such as gibberellins (GAs) and cytokinins (CKs) are potential targets of the KNOTTED1-like homeobox (KNOX) protein. To enhance our understanding of KNOX protein function in plant development, we identified rice (Oryza sativa) genes for adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CK biosynthesis. Molecular and biochemical studies revealed that there are eight IPT genes, OsIPT1 to OsIPT8, in the rice genome, including a pseudogene, OsIPT6. Overexpression of OsIPTs in transgenic rice inhibited root development and promoted axillary bud growth, indicating that OsIPTs are functional in vivo. Phenotypes of OsIPT overexpressers resembled those of KNOX-overproducing transgenic rice, although OsIPT overexpressers did not form roots or ectopic meristems, both of which are observed in KNOX overproducers. Expression of two OsIPT genes, OsIPT2 and OsIPT3, was up-regulated in response to the induction of KNOX protein function with similar kinetics to those of down-regulation of GA 20-oxidase genes, target genes of KNOX proteins in dicots. However, expression of these two OsIPT genes was not regulated in a feedback manner. These results suggest that OsIPT2 and OsIPT3 have unique roles in the developmental process, which is controlled by KNOX proteins, rather than in the maintenance of bioactive CK levels in rice. On the basis of these findings, we concluded that KNOX protein simultaneously decreases GA biosynthesis and increases de novo CK biosynthesis through the induction of OsIPT2 and OsIPT3 expression, and the resulting high-CK and low-GA condition is required for formation and maintenance of the meristem.