The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis).

The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of beta-carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as "membranous" chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.

A novel gene mutation that confers abnormal patterns of beta-carotene accumulation in cauliflower (Brassica oleracea var. botrytis).

The Or gene of cauliflower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is beta-carotene, which can reach levels that are several hundred-fold higher than those in comparable wild-type tissues. The beta-carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in flower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild-type tissues. Thus the unpigmented wild-type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidentified mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid-sequestering sheets.

Wood biomechanics and anatomy of PACHYCEREUS PRINGLEI.

We report the longitudinal, biomechanical, and anatomical trends observed for tissue samples drawn from the parallel aligned, prismatic woody vascular bundles running the length of a Pachycereus pringlei plant measuring 5.22 m in height. The main vertical stem of this plant was cut into five segments (labeled A through E in the acropetal direction) measuring ∼1.02 m in length. Four of the 14 vascular bundles in each segment were surgically removed to obtain 20 vascular bundle segments that were tested in bending to determine their stiffness measured in the radial E(R) and tangential E(T) direction. We also determined the lignin content of representative samples of wood.A nonlinear trend in stiffness was observed: E(R) and E(T) were highest in segments B or C (1.67 GN/m and 1.09 GN/m, respectively), lower in segment A (E(R) = 1.18 GN/m and E(T) = 0.35 GN/m), and lowest in segment E (E(R) = 0.03 GN/m and E(T) = 0.20 GN/m). Similar longitudinal trends were seen for axial tissue volume fraction and fiber wall thickness, which achieved their highest values in segment B (69.8% and 6.59 µm, respectively). Wood stiffness also correlated significantly with cell wall lignin content: with respect to segment B (which had the highest lignin content, and was thus used as the standard reference for percent lignin content), lignin content, was 15, 60, 85, and 43% in segments E, D, C, and A, respectively. Fiber cell length, which increased toward the base of the stem and toward the vascular cambium in the most proximal vascular bundle segment, did not correlate with E(R) or E(T).Basic engineering principles were used to calculate stem stresses resulting from self-loading and any wind-induced bending moment (produced by drag forces). Calculations indicated that the less stiff wood produced in segment A eliminates a rapid and potentially dangerous increase in stresses that would otherwise occur in segments B or C. The less stiff wood in segment A also reduces the probability of shear failure at the cellular interface between the wood and surrounding tissues in this portion of the stem.We conclude that P. pringlei wood stiffness is dependent on the volume fraction and lignification of axial tissues, less so on fiber wall thickness, and that wood development in this species is adaptively responsive to self-loading and differentially applied external mechanical forces.

Cucumber mosaic virus is restricted from entering minor veins in transgenic tobacco exhibiting replicase-mediated resistance.

Transgenic tobacco plants expressing an altered form of the 2a replicase gene from cucumber mosaic virus (CMV) strain Fny exhibited a suppression of viral replication and restricted viral movement when inoculated mechanically or by insect vectors. Resistant plants could be infected, however, through a graft-union with an infected nontransformed plant. The infectious entity moved quickly through intergrafts of resistant tissue, indicating that it could move without replicating in the vascular system. Viral replication continued to be suppressed in systemically infected transgenic portions of grafted plants, as demonstrated by the synthesis of lower levels of viral RNA than in systemically infected nontransformed portions of the same grafted plants. Cell-to-cell spread within this tissue also occurred much more slowly than in nontransformed tobacco. Young inoculated levels of transgenic-resistant plants exhibited limited cell-to-cell virus movement, revealed as chlorotic lesions, but no long-distance virus movement occurred. The results of in situ hybridization studies on these lesions indicated that CMV RNA does not traffic from bundle-sheath cells to vascular parenchyma or companion cells in chlorotic lesions on the inoculated leaves of transgenic-resistant tobacco plants. The inhibition of long-distance movement was a consequence of restricted entry of the infectious entity into the vascular system.

Brassica S-Proteins Accumulate in the Intercellular Matrix along the Path of Pollen Tubes in Transgenic Tobacco Pistils.

A tobacco plant transformed with a Brassica oleracea SLG-22 gene was analyzed by immunocytochemical methods to determine the localization of the transgene-encoded protein product. Immunolabeling was observed in the pistil along the path followed by pollen tubes after pollination. S-antigen accumulated in the intercellular matrix of the transmitting tissue of the style and its continuation in the basal portion of the stigma and outside a few special cells of the placental epidermis of the ovary. This pattern of S-antigen distribution closely resembles that described for the S-associated glycoproteins of self-incompatible Nicotiana alata and differs from its distribution in B. oleracea.

The S-locus specific glycoproteins of Brassica accumulate in the cell wall of developing stigma papillae.

Self-incompatibility in Brassica oleracea is now viewed as a cellular interaction between pollen and the papillar cells of the stigma surface. In this species, the inhibition of self-pollen occurs at the stigma surface under the influence of S-locus specific glycoproteins (SLSG). We used antibodies specific for a protein epitope of SLSG to study the subcellular distribution of these molecules in the stigmatic papillae. The antibodies have uncovered an interesting epitope polymorphism in SLSG encoded by subsets of S-alleles, thus providing us with useful genetic controls to directly verify the specificity of the immunolocalization data. Examination of thin sections of Brassica stigmas following indirect immunogold labeling showed that SLSG accumulate in the papillar cell wall, at the site where inhibition of self-pollen tube development has been shown to occur. In addition, the absence of gold particles over the papillar cell walls in the immature stigmas of very young buds, and the intense labeling of these walls in the stigmas of mature buds and open flowers, correlates well with the acquisition of the self-incompatibility response by the developing stigma.

Autoradiographic evidence for the effects of light on RNA and DNA synthesis during chloroplast replication in spores of Polytrichum.

Light stimulates the incorporation of [3H]uridine and [3H]thymidine in addition to plastid replication in germinating Polytrichum spores. Significant amounts of [3H]uridine and [3H]thymidine are incorporated in darkness but not to the same level as in light. Plastids continue to produce nucleic acids when their capacity to multiply is suspended due to the absence of light. However, a higher amount of DNA synthesis in the light is correlated with the result that chloroplast replication begins again in the light after prolonged dark incubation. An imperfect association of plastid replication and nucleic acid synthesis is suggested by the lack of stimulation of DNA synthesis by light during plastid replication in the first 8 h of incubation. A temporal separation can be demonstrated for chloroplast and nuclear DNA synthesis at the beginning of germination in Polytrichum spores, with DNA synthesis in the chloroplasts preceding that in the nucleus. The mitotic S phase is longer than 16 h for at least half of the nuclei.

INTERCALARY MERISTEMATIC ACTIVITY IN THE SPOROPHYTE OF FUNARIA (MUSCI).

Marking procedures were combined with anatomical techniques to establish that in Funaria (1) the apical region does not act as an apical meristem contributing to seta growth, and (2) the subapical region contains an intercalary meristem the derivatives of which account for the elongation of the seta. In sporophytes that are 8 mm long there is a distinctive difference in the pattern of cell division in the apical and subapical regions. Large, undivided endothecial cells exist in the apical region, and a central strand of elongated cells occupies the analogous position in the subapical region. The apical region is earmarked to form the operculum and spore sac and part of the apophysis. There is an ontogenetic continuity between the seta and the lower portion of the apophysis, but the uppermost cells of the subapical region do not contribute to seta formation. Instead, these cells and those at the base of the apical region form a transitional zone between apical and subapical influences, and they account for most of the stomates that develop on the apophysis.

Spermatogenesis in Polytrichum juniperinum : I. The origin of the apical body and the elongation of the nucleus.

Spermatogenesis in Polytrichum juniperrinum includes a series of precise and coordinated morphogenetic movements among the organelles of the androcyte. The basal bodies, the underlying microtubules, and the multilayered structure (MLS) are positioned as an integrated unit at the periphery of the cell, and the nucleus migrates into contact with them. The shape of the nucleus begins to change, with the formation of an anterior projection, or beak. The mitochondrial sheath that has coalesced on the plastid divides to form the apical body, and elongation of the nucleus begins. The posterior basal body and one microtubule undergo lateral displacement from the rear forward, as elongation of the nucleus and the microtubules continues. The mitochondrial mass that is now the apical body grows rearward along the side of the elongated nucleus, and the two groups of microtubules in the MLS rearrange themselves. The lower elements of the MLS also participate in the morphogenetic rearrangement. By the time the nucleus has elongated once around the cell and the apical body has begun it s rearward growth, the lower elements of the MLS are found only beneath the anterior basal body. Subsequently these layers disappear from this location also.

Spermatogenesis in Polytrichum juniperinum : II. The mature sperm.

In the mature sperm of Polytrichum, the nucleus contains only condensed chromatin. The apical body is as long as the posterior basal body, and the anterior basal body remains short. The microtubules that were part of the multilayered structure persist to form the filamentous appendage of the mature sperm. The cytoplasmic remnant contains the plastid and a few mitochondria that did not participate in the formation of the apical body.