Kaurene Synthetase Activity in Helianthus annuus L. : Increases in Enzyme Activity after Storage of Seedlings in Liquid Nitrogen

In previous studies, the conversion of geranylgeranyl pyrophosphate to ent-kaurene (kaurene synthetase AB activity) could not be detected readily in crude extracts of sunflower (Helianthus annuus L.) seedlings (Shen-Miller, West 1982 Plant Physiol 69: 637-641). These investigations also revealed the presence of inhibitors for Marah macrocarpus kaurene synthetase AB activity in crude extracts of sunflower seedlings. It has now been found that crude extracts prepared from intact sunflower seedlings stored in liquid N₂ for several days have greatly enhanced AB activity in comparison with frozen, but not stored, controls. The levels of activity for the conversion of copalyl pyrophosphate to ent-kaurene (kaurene synthetase B activity) are affected only slightly by storage of intact seedlings in liquid N₂. Extracts from intact seedlings that had been stored in liquid N₂ also showed less inhibitory activity for Marah macrocarpus endosperm kaurene synthetase AB activity.     

Effects of Indoleacetic Acid on the Quantity of Mitochondria, Microbodies, and Plastids in the Apical and Expanding Cells of Dark-grown Oat Coleoptiles

We determined the number of mitochondria, microbodies, and plastids in dark-grown oat (Avena sativa) coleoptiles following incubation in indoleacetic acid (IAA) for a period of 60 minutes at 6-minute intervals. In the apical outer epidermis of coleoptiles, the mitochondria increased from 31.4 to 35 per cell section with a 6-minute incubation in IAA, and this trend persisted over the 60-minute incubation. Neither the microbodies, plastids, nor the dicytosomes (Gawlik and Miller 1974 Plant Physiol 54:217-221) responded to the hormone. The apical parenchyma showed no change in quantity of any of the organelles including the dictyosomes during IAA incubation. The quick response of mitochondria in the coleoptile tip could be interpreted as an association of this organelle with hormone transport, growth, or perhaps with gravity perception. In the subapical expansion region, IAA caused significant reductions of mitochondria, microbodies, and dictyosomes in the outer epidermis compared to the control, the timing of which preceded the IAA-induced elongation and of geotropism. The fast response of organelles in the various cells is probably a change in organelle volume rather than number. That microbodies show a response to the plant hormone in the permanently achlorophyllous epidermis indicates that these organelles, in addition to their peroxisomal functions in green leaves, also may have a growth regulation function. IAA treatment was without effect on the quantity of the various types of plastids (including the amyloplasts) in the different oat coleoptile cells.     

Spectral response of corn (Zea mays) in root geotropism

The primary roots of corn (Zea mays, Wisconsin hybrid 64A ?22R) show positive geotropism following exposure to light. Thisconfirms the works of other investigators. The curvature responsebegins at about 1 hr following irradiation and reached a plateauat 5 hr. A study of wavelengths 350–760 nm, using energiesof 2.24 ? 10¹⁴ photon cm^–2 and exposure times 60 sec,shows that the most effective light is at 660 nm with lessereffectiveness at 460 and 560 nm. The responses at 660 and 460nm are reversible by a far-red (730 nm) exposure, indicativeof the possible participation of phytochrome. Analyses of freshtips of corn roots with a dual-wavelength difference photometershow the phytochrome content in the root to be about 0.16 (OD) per gram fresh wieght. The requirement of light for thegeotropic growth response of corn roots might be an adaptivephenomenon. The occurrence of photomorphogenic activity in thegreen light should be of concern to those who use green as the"safe" light in "dark" experiments. ¹Work supported by U. S. NASA and U. S. ERDA. (Received September 26, 1977; )     

Quantitative Assessment of Ultrastructural Changes in Primary Roots of Corn (Zea mays L.) after Geotropic Stimulation: II. Curving and Noncurving Zones of the Root Proper

In outer cortical cells of corn (Zea mays L.) roots we made ultrastructural comparisons between the geotropically responding (661 nm irradiated) and nonresponding (dark control) roots in both the curving and noncurving regions of the root. In the control treatment, Golgi apparatus (dictyosomes) and mitochondria exhibited centrifugal distribution (taking the stele as the center) in both regions of the roots (the organelles localized in the top of the cells in the upper tissues, and in the bottom of the cells in the lower tissues). In the geotropically responding roots, the distribution patterns were the same as those of the controls. However, in the zone of curvature the dictyosomes (but not the mitochondria) were randomly distributed in the cells of the upper tissues. This change in pattern of dictyosome distribution could be related to the change in cell elongation of upper cells.     

Distribution of ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus

Kaurene synthetases catalyse the biosynthesis of ent-kaurene, a precursor of the gibberellins. In 4-day-old dark- or light-grown Helianthus annuus seedlings, the cotyledons contained over 90% of the synthetase activity. The low enzyme activity in the seedling hypocotyls and roots is not a consequence of inhibitory factors in these tissues. The cotyledons not only have the highest kaurene synthetase activity, but also have the highest inhibitory activity. The differences in kaurene synthetase activities in the different tissues cannot be explained on the basis of the levels of inhibitor(s) in the extracts. The mature perennial root of Marah macrocarpus has very low kaurene synthetase activity, in contrast to the liquid endosperm of immature seeds of the same plant which is a rich source of the enzyme.     

Effects of indoleacetic Acid on dictyosomes of apical and expanding cells of oat coleoptiles

We found that the auxin-induced growth is mediated through the activation of the dictyosomes (collectively, the Golgi apparatus). Incubation of oat (Avena sativa) coleoptile segments in indoleacetic acid-sucrose-phosphate buffer changes significantly the number of dictyosomes in the expanding cells. A further indication of auxin enhancement of dictyosome activity is a decrease in dictyosomal cisternae (flattened membranous sacs) number. This decrease occurred after 6 minutes of incubation in auxin, and then was followed by a reduction in the organelle number per se. These times are in keeping with the rapid action of auxin-induced cell elongaton, and the latent period of geotropism. In the apical cells, the effect of indoleacetic acid is more subtle and complex. The periods of increased dictyosome utilization and of increased dictyosome synthesis in auxin-treated segments alter with those of the control. These observations indicate that dictyosomes not only have a function in cell elongation, but also may participate in processes such as auxin transport and stimuli perception. The expanding cells have five times as many dictyosomes as the cells in the apex. Dictyosome number within a cell appears to be directly proportional to the length of the cell. The fluctuation of dictyosome number and the effect of auxin on the rate of elongation of individual outer epidermis are discussed.     

Exceptional seed longevity and robust growth: ancient Sacred Lotus from China

A 1,288 ± 271-yr-old (1,350 ± 220 yr BP, radiocarbon age) seed of Sacred Lotus (Nelumbo nucifera Gaertn.) from an ancient lake bed at Pulantien, Liaoning Province, China, has been germinated and subsequently radiocarbon dated. This is the oldest demonstrably viable and directly dated seed ever reported, the preserved relict of one of the early crops of lotus cultivated by Buddhists at Pulantien after introduction of the religion into the region prior to 372 A.D. A small portion of the dry pericarp of a second lotus fruit from the same locale has been dated as being 332 ± 135-yr-old (270 ± 60 yr BP, radiocarbon age) by accelerator mass spectroscopy at the Lawrence Livermore National Laboratory. This polycentenarian seed not only germinated but is still growing (since March, 1994). Of six old lotus fruits tested, two-thirds germinated, almost all in fewer than 4 d, as rapidly as fruits harvested from the progeny of Pulantien Sacred Lotus plants (under cultivation by the National Park Service in Washington, DC), and more rapidly than fresh fruits of Yellow Lotus [N. lutea (Willd.) Pers.]. Growth of the old lotus is robust: rhizome formation and leaf emergence at rhizome nodes are more rapid than those of the Pulantien progeny, although the leaf width is smaller. Activity of the protein-repair enzyme L-isoaspartyl methyltransferase in the old lotus seed is persistent during germination and is as robust as that in the progeny, and the degree of aspartyl racemization in proteins of the two groups of plants is minimal and essentially identical. The six dated ancient Sacred Lotus fruits range in age from 95 to 1,288 yr (with a mean age of 595 ± 380 yr), evidently reflecting their production, deposition, and preservation at varying times during the intervening millennium.     

Phototropism and photoinhibition of basipolar transport of auxin in oat coleoptiles

We have proposed that the lateral inequality of auxin associated with the phototropic response is a consequence of a light-induced impairment of basipolar transport of the hormone. If this is so, the dose-response curve for photoinhibition of auxin transport should resemble that for phototropism. Further, the wavelength dependencies of the transport and tropic response should also be similar. Oat coleopilles were irradiated equilaterally with white light and with broad- and narrow-spectral bands of blue light. Indoleacetic acid 2-¹⁴C was then applied apically to the intact coleoptile. Irradiation by the 3 sources inhibited basipolar transport of the auxin. The photoinhibition of transport increases with exposure, reaches a maximum at radiant energies depending on the source used, and then decreases. The dose response for transport inhibition matches that for phototropism. A correspondence in the spectral response of the 2 phenomena is also found for coleoptiles exposed to first positive energies at various wavelengths from 340 to 730 nm. We interpret these correlations as support for the hypothesis that phototropism is mediated by a photoinhibition of basipolar transport of auxin.     

Intracellular distribution of mitochondria after geotropic stimulation of the oat coleoptile

The number of mitochondria is greater in the bottom than in the top of cells in geotropically stimulated oat (Avena sativa) coleoptiles. In the avascular tip and outer epidermis of subapical regions this difference occurs only in the lower tissues. These inequalities are found both in the KMnO₄⁻ and in the glutaraldehyde-fixed tissues; however, they are significant only in the former. Also, the number of mitochondria scored is consistently lower when the tissues were fixed in KMnO₄. These results suggest that mitochondria undergo a small degree of sedimentation after geostimulation, a redistribution reduced by the slower fixation with glutaraldehyde. Differences in mitochondrial number begin later than those in the amyloplast and the Golgi apparatus after geotropic stimulation. The cells in the avascular-tip region (a region having an important role in geotropism) have two to three times more mitochondria than the subapical cells.     

Rhythmicity in the Basipetal Transport of Indoleacetic Acid through Coleoptiles

¹⁴C-Indoleacetic acid was applied to coleoptiles of corn (Zea mays) and oat (Avena sativa). The coleoptiles were detached from the endosperms at 6-minute intervals after indoleacetic acid application, and the radioactivity was determined in successive 2-millimeter regions. The rate (per cent per minute) of basipetal transport of indoleacetic acid is periodic in various regions of the coleoptile, with a period of about 20 minutes. The possible relation of this cyclic phenomenon to other rhythmic processes of similar periodicities is discussed. A distinct acropetal transport (against the concentration gradient) from the subapical region to the apical 2-millimeter region of the coleoptile was detected.