Eva Hrabětová

Developmental changes in gene expression during pollen differentiation and maturation inNicotiana tabacum L

Total and polysome-bound ribosomes and the uptake and incorporation of3H-uridine and14C-leucine were examined in dividing microspores and in pollen grains isolated from anthers of 6 different developmental stages. Direct evidence was obtained that the formation of cytoplasm of the vegetative cell following microspore division is related to a rapid activation of RNA and protein synthesis and of ribosomes in differentiating pollen. Total ribosomes associated with gametophytic programme rose about 10times and the process of differentiation was accompanied by a rapid increase in uptake capacity of pollen grains for both uridine and leucine. Pollen development after cytoplasm synthesis and starch deposition continued by pollen maturation, which was characterized by a decline in RNA synthesis, dissociation of polysomes and by a further rise of transport activity of pollen grain wall for exogenous substrates, indicating probable pollen adaptation for utilization of metabolites from the degenerating tapetal cytoplasm.

Protein changes during pollen development inNicotiana tabacum L.

Developmental variations in the amount and SDS-PAGE pattern of soluble proteins and of those. bound to heavy cell structures were examined during transition of the dividing microspore into mature pollen in tobacco cultivars Samsun and White Burley. Both protein fractions exhibited an initial rapid rise associated with young pollen grain filling with cytoplasm and a slight decrease during pollen maturation after starch deposition. These changes were more marked in the soluble fraction and the total protein content at its maximum was more than three times higher than at the stage of microspore mitosis. Most of about 70 distinct polypeptides detected were found in all stages. Stage specific variations concerned a progressive increase and changes in the proportion of existing proteins and in appearance of some minor peaks. Small differences between the cultivars were observed at the time of differentiation and in proportions of some polypeptides.

Protein synthesis in pollen tubes: preferential formation of new species independent of transcription

SummaryPollen tubes grown in vitro were pulselabelled for 5–30 min with [14C]amino acids, and the proteins analysed by SDS-PAGE and fluorography. In Nicotiana tabacum, the qualitative pattern of the synthesized proteins did not change significantly when the pollen was cultured for 1–24 h in different media, but total protein synthesis declined as pollen tube growth slowed down and was very low in non-growing tubes. The patterns showed preferential synthesis of proteins with apparent molecular masses of 63 and 65 kDa. The 65 kDa species, which is not synthesized during pollen maturation and is absent in ungerminated pollen grains, appears as the most intensely labelled band in both soluble and insoluble protein fractions, but accumulates as a protein non-covalently bound to pollen structures. It is also preferentially synthesized in N. sylvestris, N. alata, and in apple pollen tubes, and probably plays an important role in tube wall formation. In Nicotiana tabacum, the labelling of the 65 kDa, protein is not specifically reduced upon strong inhibition of transcription by actinomycin D, which indicates a post-transcriptional level of induction of its synthesis in germinating pollen.

The technique of obtaining germinating pollen without microbial contamination

Sterilizují se dospělé prašníky roztoky Famoseptu nebo Chloraminu B. Po opláchnutí vodou se rychle vysuší ve vakuu za nízké vlhkosti vzduchu. Z otevřených prašníků se pyl vyklepává v jednoduchém zařízení (obr. 1).AbstractЗрелые пыльники стерплизуют растворами фамосента или Хлорамина Б. После ополаскивания водой их быстро высушивают в вакууме в условиях пониженной влажности. Из открытых пыльников вытряхивают пыльыу в простом приборчике (рис. 1.).

Protein Changes in Tobacco Pollen Culture; a Newly Synthesized Protein Related to Pollen Tube Growth

Summary Pollen of Nicotiana tabacum L. was grown for various intervals of up to 48 h. At pH optimum of culture medium enabling linear tube growth, the rate of protein synthesis was relatively constant and the uptake of external leucine declined only slightly. In addition to some changes in proportions of individual proteins separated by SDS-PAGE, the growth of pollen tubes was associated with the appearance of a novel non-covalently bound protein with apparent molecular mass of 65 kD. Its de novo synthesis was already detected during the first hours of germination but not in non-germinating immature pollen. At optimal pH, the amount of this protein increased proportionally to the length of pollen tubes throughout 48 h, while in non-buffered medium its increase ceased after 8 h, before growth came to a stop due to external pH decrease. In comparison with other pollen proteins, the synthesis of the new protein is much greater and seems to be involved in tube wall formation.

A simple rapid method of determining pollen tube growth in mass culture

Abstract Pollen tubes cultivated in liquid media are separated from the solution by filtration on a simple device under constant conditions and immediately weighed. The weight corresponds to the volume of the tubes and is a measure of growth activity of the culture. The results on growth of apple pollen in a shaken suspension as estimated by tube length measurement and by the “weight” method, are presented and an equation is derived for the calculation of the tube length from the weight of the culture. The paper also describes an improved procedure for obtaining large quantities of pollen free of microbial contamination.

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.

Amino acids and bivalent cations in the growth of tobacco pollen in mass culture

Abstract Glutamic acid, aspartic acid, their amides and particularly casein hydrolysate (CH) enhance the growth of Nicotiana tabacum pollen tubes in shake suspension culture. At optimal CH concentration (1 mg ml−1) growth of the pollen tubes usually proceeds for about 24h and the final mass of tubes is enhanced 3-fold. This enhancement of growth is dependent on the presence of calcium and magnesium, a mixture of the two being more effective than either cation alone. In the medium with CH and the cations tube growth is greatest with a pollen density of 4 × 104 grains (0.4 mg) per ml. It is suggested that the observed effect of amino acids is related to a progressive dependence of pollen tubes on exogenous source of organic nitrogen.

Amino Acid Uptake and Protein Synthesis in Cultured Tobacco Pollen

Summary The uptake of amino acids and their incorporation into proteins by tobacco pollen tubes in suspension culture was studied as a function of pollen tube growth. Leucine transport over a 0.01–0.5 mM concentration range occurs against the concentration gradient via an energy-dependent saturable system with an apparent K m × 10 -4 M and maximum velocity 1.25 fmol min -1 per pollen tube in a 4 h culture. A part of the absorbed leucine is released into the medium, the efflux being about 6 % of influx in 10 min preloaded pollen tubes. The rate of uptake per pollen tube increases proportionally to the duration of growth up to at least 8 h and decreases after 12 h of culture. In the period between 6 and 12 h, the first callose plug is formed nearly in 90 % of pollen tubes, separating the about 0.9 mm apical part. The uptake is linear for 30 min and in 2 h an equilibrium between the endogenous and exogenous amino acid pools is achieved in 1 mg · ml -1 of casein hydrolysate. The rate of protein synthesis, as estimated from the proportion of incorporated to free amino acids absorbed, does not decrease and the amount of incorporated exogenous amino acids increases continuously for at least 18 h of culture, provided that pollen tube growth is not depressed. A comparison of the results from different periods of labelling with 14 C-leucine suggest that the free leucine pool is compartmentalized. The protein precursor poll appears to be small and rapidly fed from exogenous source.

Effect of some mineral ions on pollen tube growth and release of proteins in culture

In the absence of cations, the release of proteins from pollen tubes ofNicotiana tabacum in culture is greatly dependent on boron concentration and inversely related to growth stimulation. The minimum of proteins in the medium occurs at 100 mg 1− of boric acid, which is the optimum concentration for growth. The shift of boron level to this optimum further increases the proportion of proteins bound to the insoluble pollen tube fraction; on the other hand the amount of soluble proteins is not affected inside pollen tubes, but greatly decreased in the medium.The loss of proteins into the medium is considerably reduced by oalcium and also at the optimal boron concentration and in the presence of K+ and Mg2+ ions. The rate of tube elongation, however, is slightly decreased and the duration of pollen growth activity is not extended. The release of proteins is not affected by potassium and is slightly reduced by magnesium.The possible mechanism of calcium and boron action on protein release is discussed in relation to the exocytotic function of secretory vesicles and it is suggested that boron supports the incorporation of proteins transported to the growing tip into the pollen tube wall structures.