A. F. Croes

Protein synthesis in germinating pollen of Petunia: role of proline.

Pollen of Petunia hybrida was germinated in artificial medium. At the beginning of the incubation, a large amount of proline, which comprises about half of the total free amino acid pool, was released into the medium. Part of this proline is reutilized by the pollen. Uptake of radioactive amino acids and their incorporation into proteins were studied. The highest rate of protein synthesis was found directly after the onset of germination. The endogenous free proline pool was found to be compartmentalized; one of the compartments is the protein precursor pool; its size is probably much less than 50% of the total free proline in the pollen.

The 5′-Untranslated Region of the ntp303 Gene Strongly Enhances Translation during Pollen Tube Growth, But Not during Pollen Maturation

Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the protein only accumulates to detectable levels after pollen germination. In an attempt to explain the divergence in the accumulation profiles of the mRNA and the protein, we investigated the role of the untranslated regions (UTRs) in enhancing ntp303 translation during the transition from developing to germinating pollen. Luciferase reporter gene fusion constructs containing the ntp3035′-UTR gave rise to luciferase activity that was up to 60-fold higher during pollen tube growth than that of constructs containing different 5′-UTRs. No apparent differences in the luciferase activity of these constructs were observed during pollen development. Thentp303 5′-UTR-mediated increase in luciferase activity was not significantly influenced by coding region or 3′-UTR sequences. Furthermore, enhanced luciferase activity directed by thentp303 5′-UTR occurred predominantly at the post-transcriptional level. A series of 5′-UTR deletion constructs was created to identify putative regulatory sequences required for the high level of translation during pollen tube growth. Two predicted stem loop structures (H-I and H-II) caused a complete inhibition of the enhanced translation after their total or partial deletion. A (GAA)8repeat within the H-I stem loop structure was demonstrated to be important for the modulation of translation efficiency. The H-II stem loop structure was found to be essential for the determination of mRNA stability.

Thiophene accumulation in relation to morphology in roots of Tagetes patula : Effects of auxin and transformation by Agrobacterium.

Roots of marigold (Tagetes patula L.) accumulate thiophenes, heterocyclic sulfurous compounds with strong biocidal activity. In detached roots cultured in vitro, the thiophene content was 5 μmol·(g fresh weight)-1 which is 25-times higher than in roots attached to the plant. In roots derived from tissues transformed by Agrobacterium tumefaciens and A. rhizogenes, the morphology and thiophene content varied with the bacterial strain used. Transformation stimulated the elongation of the root tips and the formation of lateral roots but lowered the thiophene level to 20–50% relative to the concentration in untransformed detached roots. A negative correlation was found between the number of laterals in a root system and the thiophene content. Extensive branching and a decrease in thiophene accumulation was evoked in untransformed roots by indole-3-acetic acid (1–10 μmol·l-1) added to the medium. Within the roots, the highest thiophene concentrations were found in the tips. The results indicate that auxin directly or indirectly plays a role in the regulation of the thiophene level in root tips.

Metabolism of 1-naphthaleneacetic acid in explants of tobacco: Evidence for release of free hormone from conjugates

Abstract1-Naphthaleneacetic acid (1-NAA), required for in vitro flower bud formation, was taken up by pedicel explants of tobacco (Nicotiana tabacum L.) in large amounts and rapidly metabolized into various conjugates. These conjugates have been tentatively identified in four thin-layer Chromatographic systems using authentic standards as references. The major metabolite formed during the first hours of culture comigrated with 1-NAA-glucoside (1-NAGlu). From the 6th hour on, most 1-NAA had been converted into a yet unidentified metabolite. 1-NAglu was an intermediate in the formation of this metabolite. After 24 h, 1-NAA-aspartate (1-NAAsp) became the second major metabolite. The increase in 1-NAAsp formation was induced by 1-NAA. The inactive analog 2-naphthaleneacetic acid (2-NAA) was metabolized similar to 1-NAA, but was unable to increase the formation of the aspartate conjugate. When explants were fed labeled 1-NAGlu, 1-NAAsp or the major unidentified metabolite, radioactivity became associated with free 1-NAA and all major conjugates, indicating interconversion of conjugates and breakdown to free 1-NAA. A regulatory role of conjugation in maintaining a particular level of free 1-NAA in the tissue is proposed herein.

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.

Regulation of anthraquinone biosynthesis in cell cultures ofMorinda citrifolia

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

In silico identification of putative regulatory sequence elements in the 5 '-untranslated region of genes that are expressed during male gametogenesis

During pollen development, transcription of a large number of genes results in the appearance of distinct sets of transcripts. Similar mRNA sets are present in pollen of both mono- and dicotyledonous plant species, which indicates an evolutionary conservation of genetic programs that determine pollen gene expression. In pollen, regulation of gene expression occurs at the transcriptional and posttranscriptional level. The 5′-untranslated region (UTR) of several pollen transcripts has been shown to be important for regulation of pollen gene expression. The important regulatory role of 5′-UTR sequences and the evolutionary conservation of genetic programs in pollen led to the hypothesis that the 5′-UTRs of pollen-expressed genes share regulatory sequence elements. In an attempt to identify these pollen 5′-UTR elements, a statistical analysis was performed using 5′-UTR sequences of pollen- and sporophytic-expressed genes. The analysis revealed the presence of several pollen-specific 5′-UTR sequence elements. Assembly of the pollen 5′-UTR elements led to the identification of various consensus sequences, including those that previously have been demonstrated to play a role in the regulation of pollen gene expression. Several pollen 5′-UTR elements were found to be preferentially associated to genes from dicots, wet-type stigma plants, or plants containing bicellular pollen. Moreover, three sequence elements exhibited a preferential association to the 5′-UTR of pollen-expressed genes from Arabidopsis andBrassica napus. Functional implications of these observations are discussed.

Isochorismate synthase isoforms from elicited cell cultures of Rubia tinctorum

Abstract Elicitation of Rubia tinctorum cell cultures with a Pythium aphanidermatum elicitor leads to a doubling of anthraquinone content which is preceded by a large rise in isochorismate synthase (ICS; EC 5.4.99.6) activity. Two ICS isoforms were purified from the elicited cultures. Both isoforms had an absolute requirement for Mg 2+ . Isoform I and II had a K m for chorismate of 365 and 466 μM, respectively. Gel filtration indicated a molecular mass of approximately 67 kD for both isoforms.

Development of dormancy in different lily genotypes regenerated in vitro

Dormancy development in four Lilium genotypes,L. speciosum, Star Gazer, C. King and Snow Queenregenerated in vitro was compared. Major factorsinfluencing dormancy development were the same for different genotypes andespecially L. speciosum and Star Gazer, that are closelyrelated, reacted similarly. Temperature was the main factor in dormancyinduction and breaking. The range of temperatures that induced dormancy and thelevel of dormancy that developed differed per genotype. In Star Gazer, dormancydeveloped gradually but in Snow Queen, dormancy developed very fast. Thereactions to temperature, reflected the climate in the area of origin. Abscisicacid deepened the level of dormancy induced by temperature but had no effectunder non-inductive temperature conditions. When abscisic acid synthesis wasblocked, no dormancy developed. Dormancy in all genotypes was broken by coldincubation for severalweeks. The cold requirement of the genotypes differed in line with the naturalwinter conditions in their habitat. The effect of hormones on dormancy breakingwas also investigated. A gibberellin treatment of 24 h brokedormancy in L. speciosum, Star Gazer and Snow Queen.

Thiophene interconversions in Tagetes patula hairy-root cultures

Abstract On the basis of feeding experiments with 35 S-labelled intermediates, we present a modified biosynthetic pathway of the bithienyls in Tagetes . The monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene, which is present in small amounts in Tagetes hairy roots, is the precursor of all bithienyls that have been described for this species but not of α-terthienyl. The current hypothesis that 5-(3-buten-1-ynyl)-2,2′-bithienyl originates from 5′-methyl-5-(3-buten-1-ynyl)-2,2′-bithienyl after oxidative decarboxylation proved untrue. The latter compound is only converted into (5′-but-3-en-1-ynyl-[2,2′]bithiophenyl-5-yl)-methyl acetate, probably via (5′-but-3-en-1-ynyl-[2,2′]bithiophenyl-5-yl)-methanol. Substitution of the butenynyl side chain of 5-(3-buten-1-ynyl)-2-2′-bithienyl results in the formation of 5-(3,4-dihydroxy-1-butynyl)-2,2′-bithienyl and 5-(4-hydroxy-1-butynyl)-2,2′-bithienyl, which are subsequently converted into respectively 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl and 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl. The end products of this biosynthetic pathway are all bithienyl-acetate esters.