Control of longitudinal and cambial growth by gibberellins and indole-3-acetic acid in current-year shoots of Pinus sylvestris.

We investigated the involvement of gibberellins (GAs) and indole-3-acetic acid (IAA) in the control of longitudinal and cambial growth in current-year shoots of Pinus sylvestris L. Elongating terminal shoots, located at the apex of previous-year (1-year-old) branches in the uppermost whorl on the main stem, were variously decapitated (apical 5 to 10 mm removed), defoliated (all developing needle fascicles removed) and treated with endogenous GA(4/7) or IAA, or both. Shoot length and the radial widths of xylem and phloem were measured, and the concentrations of GA(1), GA(3), GA(4), GA(9) and IAA in the stem were determined by combined gas chromatography-mass spectrometry with deuterated GAs and [(13)C(6)]-IAA as internal standards. Decapitation decreased the production of xylem and phloem and the IAA concentration, but did not alter either longitudinal growth or the concentrations of GAs. Defoliation markedly inhibited shoot elongation, as well as cambial growth, and reduced the concentrations of GA(1), GA(3), GA(4), GA(9) and IAA. Application of GA(4/7) to defoliated shoots promoted longitudinal growth and phloem production, without affecting xylem production or IAA concentration. Application of GA(4/7) and IAA together to decapitated + defoliated shoots increased shoot elongation, xylem and phloem production and IAA concentration, whereas applying either substance alone had a smaller effect or none at all. We conclude that, for elongating current-year shoots of Pinus sylvestris, (1) both the shoot apex and the developing needle fascicles are major sources of the IAA present in the stem, whereas stem GAs originate primarily in the needle fascicles, (2) GAs and IAA are required for both shoot elongation and cambial growth, and (3) GAs act directly in the control of shoot growth, rather than indirectly through affecting the IAA concentration.

Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces.

We have developed a chemical treatment for the mica surface which allows biopolymers to be held in place for atomic force microscopy, even under water, using conventional, untreated force sensing tips. We illustrate the procedure with images of lambda DNA and fd phage. The phage adheres well enough to permit in situ imaging of the adsorption process in water. These experiments yield a mean length for the phage of 883 +/- 72 nm. This compares with a measured length of 883 +/- 33 nm when the phage are imaged after drying following adsorption from water, showing that the effect of dehydration is quite small. Adhesion forces between the force sensing tip and the substrate and the sensing tip and the biomolecules are very different in the three media (air, water and propanol). The apparent height of the phage and the width and height of the DNA depends upon these adhesion forces quite strongly. In contrast, changing the Hookean spring force exerted by the scanning tip makes little difference. These results suggest that the chemical factors involved in adhesion can dominate atomic force images and that the composition of the scanning tip is at least as important a factor as its geometry.

Atomic force microscopy of long DNA: imaging in air and under water.

We have obtained striking atomic force microscopy images of the intact lambda bacteriophage genome and of several lambda restriction fragments both in air and under water. The DNA is unstained and the images are stable under continuous scanning for up to 30 min. Measured contour lengths of fully imaged restriction fragments and intact lambda DNA are accurate to within a few percent. The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces. This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water. This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality. Both the lateral resolution and the contrast increase by a factor of approximately 3 under water.

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.

Electrochemical deposition of molecular adsorbates for in situ scanning probe microscopy.

We have studied gold and graphite electrodes in an electrochemistry cell under various solutions using the scanning tunneling microscope (STM). The gold (111) surface yields quite reproducible images and cyclic voltammograms. In situ voltammograms show that, under certain conditions, nanomolar quantities of DNA fragments can suppress the adsorption of a buffer salt of millimolar concentration. When the DNA concentration is reduced below that required for a monolayer coverage, the salt adsorption is restored. We show images of bare gold, gold covered with an adsorbate produced by the buffer salt, and gold prepared with a concentration of DNA fragments close to that required for monolayer coverage added to the buffer. Under these conditions, the surface is found to be uniformly covered with a characteristic structure.

Quantitation of Gibberellins A(1), A(3), A(4), A(9) and a Putative A(9)-Conjugate in Grafts of Sitka Spruce (Picea sitchensis) during the Period of Shoot Elongation.

The levels of endogenous gibberellin A(1) (GA(1)), GA(3), GA(4), GA(9), and a cellulase hydrolyzable GA(9) conjugate in needles and shoot stems of mature grafts of Sitka spruce (Picea sitchensis [Bong.] Carr.) grown under environmental conditions that were either inductive, hot, and dry, or noninductive, cool, and wet, for flowering, were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated [(2)H(2)]GA(1), GA(3), GA(4), and GA(9) as internal standards. The samples were taken when the shoots had elongated about 30, 70, and 95% of the final shoot length and 17 days after elongation had terminated. The concentration of putative GA(9)-conjugate, estimated by GCSIM of GA(9) after cellulase hydrolysis of the highly water soluble fraction, was 33 nanograms per gram fresh weight in the needles of both heat and drought- and cool and wet-treated plants sampled just after bud burst. The concentration gradually decreased to a final value of 13 nanograms per gram fresh weight in the heat and drought-treated grafts and 6 nanograms per gram fresh weight in the cool and wet-treated grafts. The stems contained no detectable putative GA(9) conjugate. Free GA(9) was highest in heat and drought-treated material. For plants subjected to this treatment, GA(9) increased from 22 to 32 nanograms per gram fresh weight in needles and from 1 to 22 nanograms per gram fresh weight in stems during the rapid stem elongation phase. By day 17, after cessation of shoot elongation, GA(9) had decreased to 12 nanograms per gram fresh weight in needles and 9 nanograms per gram fresh weight in the shoot stems. The cool and wet-treated material also showed an increase in GA(9) concentration during shoot elongation. However, the concentration was not as high and was also delayed compared with heat and drought-treated material. By day 17, after cessation of shoot elongation, GA(9) concentration was 9 nanograms per gram fresh weight in needles and 5 nanograms per gram fresh weight in stems for cool and wet treatment plants. The concentration of GA(4) was very low in tissue from both treatments. Fluctuation in concentration of the more polar gibberellins, GA(1) and GA(3), showed the same pattern as fluctuations in the content of GA(9). However, the heat and drought-treated material had lower amounts of GA(1) and GA(3) during the later phases of shoot elongation, than the cool and wet-treated material. These results imply differential metabolism between clones treated with conditions inductive and noninductive for flowering. Higher concentrations of putative GA(9) conjugate and free GA(9) in the hot and dry treatment indicate a higher capacity of synthesizing, for flowering, the physiologically important GA(4) in the heat and drought-treated material. This synthesis does not, however, result in a buildup of the GA(4) pool, probably because of a high turnover rate of GA(4). The cool and wet-treated material had higher amounts of GA(1) and GA(3), indicating that the differentiation was preferentially directed toward vegetative growth.

Quantitation of gibberellins A1, A 3, A 4, A 9 and an A 9-conjugate in good- and poor-flowering clones of Sitka spruce (Picea sitchensis) during the period of flower-bud differentiation.

The levels of endogenous gibberellin A1 (GA1), GA3, GA4, GA9 and a cellulase-hydrolysable GA9-conjugate in needles and shoot stems of Sitka spruce [Picea sitchensis (Bong.) Carr.] grafts with different coning or flowering histories were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated GA3, GA4 and GA9 as internal standards. The samples were taken at the approximate time of the start of flower-bud differentiation, i.e. when the shoots had elongated approx. 95% of the final length. The needles of the good-flowering clones contained 11-12 ng per g fresh weight (FW) and 15-28 ng· (g FW) (-1) of GA9-conjugate and GA9, respectively. The shoot stems of the same material contained no detectable amounts of GA9-conjugate and 11-15 ng-(g FW)(-1) of GA9. The amounts of GA9-conjugate and GA9 were apparently lower in the poor-flowering clones, the needles containing 4-9 ng-(g FW)(-1) and 7-17 ng·(g FW)(-1), respectively. Also in this material the shoot stems contained no detectable amounts of GA9-conjugate. The amounts of GA4 were very small in both materials, ranging from 1-1.6 ng-(g FW)(-1). The good-flowering clones contained no detectable amounts of the more polar gibberellins, GA1 and GA3. The poor-flowering clones, on the other hand, contained high levels of GA15 17-19ng·(gFW)(-1) in the needles and 10-13 ng·(g FW) (-1) in the shoot stems, and also smaller amounts of GA3, 2-3 ng·(g FW)(-1) in the needles and approx. 1 ng·(g FW)(-1) in the shoot stems. The results demonstrate differences in GA-metabolism between the poor- and the good-flowering clones. The higher amounts of GA9-conjugate and GA9 might indicate a higher capacity for synthesizing GA4 in the good-flowering material. This synthesis does not, however, result in a build-up of the GA4-pool, maybe because of a high rate of turnover. Gibberellin A4 was apparently neither hydroxylated to GA1 nor converted to GA3 in the goodflowering material, as was the case in the poor-flowering material. This might indicate that gibberellin metabolism in the poor-flowering material is directed towards GA1 and GA3, GAs preferentially used in vegetative growth.

Sequence, packing and nanometer scale structure in STM images of nucleic acids under water.

Scanning tunneling microscope (STM) images of random-sequence nucleic acid polymers under water show internal structure which depends strongly on the packing density of the polymer. Images of dense aggregates have a semicrystalline order with the individual polymers adopting simple periodic structures. Loose aggregates (or isolated molecules) show structural variability with considerable local bending and curving on a nanometer scale. It is not clear to what extent this structure is induced by the operation of the microscope. In order to investigate the possibility that the structure is sequence directed, we have imaged various DNA and RNA polymers at low packing densities. We present results here for random sequence DNA, poly(dAT).poly(dAT), poly(dA).poly(dT), poly(dCG).poly(dCG) and for random sequence RNA and poly(U). In contrast to loose aggregates of the random sequence material, the homopolymers show few sharp bends. Furthermore, the homopolymers appear to yield characteristic backbone patterns, usually at resolutions in excess of that obtained with random sequence polymers. The random sequence polymers show much more evidence of image distortion due to tip-molecule interactions, suggesting that they are, on average, mechanically less stable in the STM tunnel-gap than the homopolymers. Thus, while some of the structure observed in STM images is a consequence of tip-molecule interactions, it is related to sequence-directed properties of the polymer.

Identification of Gibberellins in Norway Spruce (Picea abies [L.] Karst.) by Combined Gas Chromatography-Mass Spectrometry.

Gibberellins A(1) (GA(1)), A(3) and A(9) were identified from extracts of shoots of 6-month old Norway spruce (Picea abies) seedlings by the use of sequential reverse and normal phase high performance liquid chromatography (HPLC), bioassay, radioimmunoassay (RIA) and combined gas chromatography-mass spectrometry (GC-MS). The bioassay and RIA were used after fractionation by HPLC to detect the GA-containing fractions, which were then examined by GC-MS. The GAs identified are considered to be endogenous.

Comparison of gas chromatography-mass spectrometry, radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.).

Quantitative estimates of gibberellin A9 in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A9, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.

Abscisic acid in shoots and roots of Scots pine (Pinus sylvestris L.) seedlings grown in controlled long-day and short-day environments.

Scots pine (Pinus sylvestris L.) seedlings grown in nutrient solution in controlled-environment chambers were used. The effects of a shortday (SD, early autumn) treatment on growth and the content of "free" and alkaline hydrolysable abscisic acid (ABA) in shoots and roots were investigated. The weekly relative growth rates of seedlings grown continuously under long-day (LD, summer) conditions were stable at approx. 0.08 g g(-1) d(-1) between weeks four and eight from germination. Weekly relative growth rates of seedlings transferred to SD conditions decreased rapidly to a then stable level of approx. 0.04 g g(-1) d(01). Shoot elongation ceased within two weeks of SD treatment. The content of both "free" and alkaline hydrolysable ABA was approx. 40-50% higher in shoots of seedlings grown for five weeks in LD plus one week in SD than in shoots of seedlings grown for five or six weeks in LD. Two additional weeks of SD did not change the "free" ABA content. Three weeks in simulated late autumn (SD but decreased temperatures) and three weeks in simulated winter (lower light intensity and temperature) further increased the content of "free" ABA in the shoots. A transfer back to LD conditions reduced the ABA content to a level equal to the level found during the first LD period. The recovery of radioactive ABA at certain times after application ofr[(3)H] ABA was the same in shoots and roots of LD-grown and SD-treated seedlings.