Jan C. Vendrig

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

SummarySelf-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the Sb-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S1) to 1000 (S2) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S2-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 μg per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S1-, S2- and S3-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.

Are acetylcholine-like cotyledon-factors involved in the growth of the cucumber hypocotyl?

Summary Acetylcholine is highly stimulatory to the growth of cucumber hypocotyl sections. The cotyledons seem to be substituted to a large extent by an application of this compound. Blood pressure assays in rats reveal the presence of acetylcholine-like substances in extracts from cotyledons. From these results we suggest that the cotyledons influence the growth of the seedlings by supplying acetylcholine-like factors to the hypocotyl.

Sterols and polyamines in **ipt**-transformed tobacco plants

Abstract Free sterol and free polyamine contents were determined in the apex and the leaves of control and Pssu-ipt transformed tobacco ( Nicotiana tabacum L. cv. Petit Havana SR1). The older leaves of ipt -transformed plants contained a much higher putrescine (put) content than those of control SR1 plants, whereas no significant differences for spermidine (spd) or spermine (spm) were found between control and ipt plants. Putrescine content corresponded well with endogenous cytokinin (free-bases) content and with ornithine- and ornithine-decarboxylose (ODC and ADC) activities. Plants transformed with ipt were characterized by a higher sterol content in the leaves and by a delay in the increase in the stigmasterol/sitosterol ratio that occurs from the upper to the lower leaves.

The Influence of Plant Growth Regulators on Crown-Gall Initiation on Cotyledonary Leaves of Helianthus giganteus L.in vitro

Summary Tumor initiation in cotyledons of Helianthus inoculated with Agrobacterium tumefaciens B 6 has been studied in vitro .The susceptibility of the cotyledons is dependent on the physiological age and can be changed by exogenously applied growth regulators.About the same results could be obtained with NAA, BAP, GA 3 , and ABA, and also with NETC. The growth of the tumors was unaffected by these additions. It is concluded that tumor initiation in cotyledons in vitro , is not controlled by one of the regulators specifically.Application of plant growth regulators may result in a hormonal imbalance rendering the cells of the cotyledons in an optimal condition for transformation in tumor cells.

Effect of «Penetrant-carriers» in Relation to the Photocontrol of Anthocyanin Synthesis in Mung beans (Phaseolus aureus ROXB.)

Summary Anthocyanin synthesis in the cotyledons of etiolated mung bean seedlings can be enhanced by DMF or DMSO. In seedlings, growing in a solution of those compounds, the anthocyanin synthesis induced by a short red-exposure is also stimulated. The hypocotyl can only synthesize anthocyanins after red-irradiation, but this synthesis can also be increased by DMF or DMSO. It is not possible to reverse the effect of DMSO by FR, which indicates that DMSO and Pfr do not aet at the same level, as may be the case in cabbage seedlings (PECKET and HATHOUT BASSIM, 1974). Treatment of isolated cotyledons with DMF or DMSO leads to the same conclusion.

Metabolism of the synthetic growth regulator α-chloro-β-(3-chloro-o-tolyl) propionitrile by mung bean seedlings

Summary Uptake and metabolism of the synthetic auxin α -chloro- β -(3-chloro-o-tolyl)propionitrile (Cl-ClTPN) were studied in intact Mung bean seedlings and in hypocotyl segments. Cl-ClTPN was taken up much faster by hypocotyl segments than by roots of intact plants. In extracts of plants treated with Cl-ClTPN several metabolites were detected: α -chloro- β -(3-chloro-o-tolyl)propionic acid, α -hydroxy- β -(3-chloro-o-tolyl)propionitrile and α -(3-chloro-o-tolyl)acetic acid (ClTAA). Besides, a lot of unidentified metabolites, most of them being apolar, could be revealed. From the identified metabolites and from the growth promoting activity of several homologues of them in the Avena cylinder test, it is suggested that Cl-ClTPN is converted to ClTAA, which is supposed to be the physiological active form of Cl-ClTPN.

Enhancement of Crown-Gall Tumor Initiation by Extract Fractions from Tumor Forming Cotyledons of Helianthus giganteus L

Summary Tumor initiation in cotyledons of Helianthus giganteus L. inoculated with Agrobacterium tumefaciens B 6 has been studied in vitro . The susceptibility of the cotyledons can be enhanced by exogenously applied active extract fractions derived from 6-days-old cotyledons. About the same results could be obtained with infections on the epi- and hypocotyl of Helianthus plants in vivo . The growth of the tumors was unaffected by these treatments. It is found that the susceptibility of the cotyledons for tumor initiation is clearly correlated with the endogenous content of the active compound(s). This active compound(s), present in the extract fractions, does not require the immediate presence of the bacteria to be physiologically active. No influence of the active compound(s) on the virulence of the bacteria has been observed.

On the Requirement of a Hydroxynitrile Lyase in the Conversion of Orthonil to Chloro-Tolylacetic Acid

Summary The conversion of the synthetic growth regulator α-chloro-β(3-chloro-o-tolyl)propionitrile (CI-CITPN) into α-(3-chloro-o-tolyl)acetic acid (CITAA) was studied at an enzymic level. Cell-free extracts of mung bean seedlings convert very well the metabolite α-hydroxy-β-(3-chloro-o-tolyl)-propionitrile into CITAA. When the enzyme synthesis is blocked by a pre-incubation in p-fluorophenylalanine, CITAA seems to be the sole chloro-tolyl derivative that can induce a growth stimulation. Growth tests, immediately after the application of auxins, gives further evidence that CITAA is the active chloro-tolyl auxin. From the results mentioned it further follows that no enzyme induction is needed. The interaction of vaniline-cyanohydrine in different growth tests indicates the interaction of a hydroxynitrile lyase (already endogeneous present), that catalizes the formation of the effective auxin CITAA from the exogeneously applied CI-CITPN.

Effects of High Temperature on Roots of Etiolated Maize Seedlings: Photosynthetic Activity and Chloroplast Ultra-Structure

It has been often showed that changes in the root environment modify the hormone balance in the root and the hormone export to the shoot. When roots of green bean seedlings are exposed to high temperatures for a short time by dipping cytokinin levels in the xylem exudate are decreased very severely (1). Since cytokinins are believed to play an important role in the chloroplast differentiation process (2), we thought it would be interesting to investigate the effect of heat stress on photosynthetic activity and on chloroplast ultrastructure. The effect of exogeneously applied BA on these phenomena was also studied. Fig.1 summarizes the experimental pattern.