Expression of C4-like photosynthesis in several species of Flaveria

Abstract Photosynthetic metabolism was investigated in leaves of five species of Flaveria (Asteraceac), all previously considered to be C₄ plants. Leaves were exposed to ¹⁴CO₂ for different intervals up to 16s. Extrapolation of ¹⁴C-product curves to zero time indicated that only F. trinervia and F.bidentis assimilated atmospheric CO₂ exclusively through phosphoenolpyruvate carboxylase. The proportion of direct fixation of ¹⁴CO₂ by ribulose-I, 5-bisphosphate carboxylase/oxygenase (Rubisco) ranged from 5 to 10% in leaves of F. australasica. F. palmeri and F. vaginata. Protoplasts of leaf mesophyll and bundle sheath cells were utilized to examine the intercellular compartmentation of principal photosynthetic enzymes. Leaves of F. australasica, F. palmeri and F. vaginata contained 5 to 7% of the leaf's Rubisco activity in the mesophyll cells, while leaves of F. trinervia and F. bidentis contained at most 0.2 to 0.8% of such activity in their mesophyll cells. Thus, F. trinervia and F. bidentis have the complete C₄ syndrome, while F. australasica, F. palmeri and F. vaginata are less advanced, C₄-like species.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Effect of maltose on the response of potato anthers in culture

Anthers of the Solanum tuberosum genotype H3703 were cultured on medium containing equimolar concentrations of sucrose or maltose. It was found that significantly more pollen embryos became plants after culture on maltose and hence the yield of plants per 100 anthers cultured increased significantly. Mechanisms by which carbohydrate source may influence response to anther culture are discussed.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Formation of zeatin allylic phosphate by the microsomal fractions of bulb disks of Iris × hollandica Tub. and tubers of Helianthus tuberosus L.

It is shown that in bulbous Iris zeatin originates from a nucleotide. This nucleotide is probably zeatin-allylic-phosphate, in which a phosphate group is attached to the isoprenoid side-chain of zeatin. The formation of zeatin-allylic-phosphate from t-zeatin and 8-[¹⁴C]-zeatin by the microsomal fractions of Iris bulb disks and Helianthus tubers was demonstrated. The responsible enzyme was partially purified. 5-AMP was found to be a phosphate group delivering substrate. Adenosine and adenine inhibited the enzyme reaction. The significance of the results is discussed in relation to cytokinin biosynthesis and the occurrence of bud blast in Iris.     

Gibberellin involvement in dormancy-break and germination of seeds of celery (Apium graveolens L.)

The temperature-dependent, primary dormancy of cv. Florida 683 celery seeds in darkness was partially broken by a 30 min light exposure on the third day of incubation at 20–22°C, resulting in c 50 percent germination after 20 days. This light stimulation was negated by including different inhibitors of gibberellin biosynthesis in the incubation medium. Subsequent addition of a solution of the gibberellins A₄ and A₇ or of the gibberellin-active compound (1-3-chlorophthalimido)-cyclohexane carboxamide (AC94,377) overcame the inhibitory effects on germination of these GA-biosynthesis inhibitors. It is suggested that light stimulates the biosynthesis of gibberellins which are essential for dormancy-break in celery seeds and that this biosynthesis is either directly or indirectly controlled through phytochrome.Abbreviations AC94,377 1-(3-chlorophthalimido)-cyclohexane carboxamide; ancymidol, -cyclopropyl--(4-methoxyphenyl)-5-pyrimidinemethanol - AMO1618 N,N,N-2-tetramethyl-5-(1-methyl ethyl)-4-(1-piperidinylcarbonyl)oxy-benzenaminium chloride - BTS44584 S-2,5-dimethyl-4-pentamethylenecarbamoyloxyphenyl-SS-dimethyl sulphonium - P toluenesulphonate; chlormequat chloride, 2-chloroethyltrimethylammonium chloride; daminozide - N dimethylaminoscuccinamic acid; paclobutrazol, (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl pentan-3-ol)