3.1. Approximate Growth Parameters of the Species
The climatic conditions varied considerably in 2010, 2011, and 2012, especially in temperature and water supply. These tree species have different crown architectures and a rough estimate of tree heights was 12 m for the pyramid-shaped
Tsuga canadensis and 3 m for the bushy growing
Taxus baccata. The small subspecies
Taxus baccata aurea is 1.5 m tall and the extremely dwarfed nana types of
Taxus and
Tsuga both measure 0.5 m. Histological changes in development during the initial phase of cell cycling are presented in
Figure 1. Stem cells of shoot tips showed no special activity towards any differentiation. In this state there is no synthesis of specialized metabolites, such as phenols or nuclear flavanols. Outside the stem cells, the peripheral flanks of the apex form lateral primordia, which are active in cell division and flavanol synthesis.
Figure 1.
The beginning of sprouting in the normal and the Tsuga canadensis nana types; the dwarf type has smaller stem cells and a major group of the descendant cells, including the nuclei, is reduced in size; both cell expansion and cell division are delayed; in the end, mitotic areas are smaller. Stem cell nuclei have no flavanols.
Figure 1.
The beginning of sprouting in the normal and the Tsuga canadensis nana types; the dwarf type has smaller stem cells and a major group of the descendant cells, including the nuclei, is reduced in size; both cell expansion and cell division are delayed; in the end, mitotic areas are smaller. Stem cell nuclei have no flavanols.
At the onset of needle growth the sizes of both the cells and nuclei of the nana type of
Tsuga were slightly smaller than those usually found in needles of the normally growing
Tsuga canadensis (
Figure 1). Presumably, the small cells suffered from a lack of expansion proteins and also the nuclei (3–5 µm in diameter) were more compacted than those of the vigorous tree (7–8 µm in diameter). Such tightly condensed nuclei had fewer mobile domains because of a lack of interchromosomal spacing. In the end, mitosis was restricted and delayed (
Figure 1).
Based on data from three years (
Figure 2), the mean length of the needles ranged from 35 mm in
Taxus baccata to 15 mm in
Tsuga canadensis. The needles of the longest leader shoots of the top crown are excluded from the calculations.
Taxus baccata aurea is a small bush that develops a yellow color in the distal area of the needles of the upper light-exposed canopy. The yellow pigmentation is mainly due to the thick cell walls of the upper and lower epidermal tissue. Some faint yellow staining was also seen in the cytoplasm. The partially yellow needles of
Taxus baccata aurea reached 15 mm in length whereas the green needles were 19 mm long. Both extremely stunted nana types had much smaller needles, namely 13 mm and 14 mm (SD values were about 1 mm for all needle groups). The overview presented in
Figure 2 shows that blue staining flavanols were dominant in the
Taxus baccata nuclei, which had the longest needles. The yellow flavonols of the nuclei increased in color towards the end of the growing season and this phenomenon was particularly evident in the dwarf genotypes.
Figure 2.
Appearance of yellow flavonols in the blue staining nuclei during the annual growth period of the conifers; as a result, the blue nuclei take on a greenish color; an early change to greenish nuclei is correlated with reduced needle size and tree height.
Figure 2.
Appearance of yellow flavonols in the blue staining nuclei during the annual growth period of the conifers; as a result, the blue nuclei take on a greenish color; an early change to greenish nuclei is correlated with reduced needle size and tree height.
3.2. Seasonal Changes of Phenolic Compounds from Entire Needles of Tsuga Canadensis and the Importance of Cell Structures
The phenolic pattern of the entire needles is presented in
Figure 3 and gives an overview of the proportions of diverse phenol groups in relation to flavanols. The relatively inactive needle group, namely 1-year old needles, was sampled in April before bud break and compared with just sprouting needles from early May and also with needles from mid-July which had down-regulated cell cycling.
Figure 3.
Total content of phenols at different stages of Tsuga canadensis needle development; the different cell sizes and thickness of cell walls together with different amounts of vacuolar flavanols made it impossible to quantify nuclear flavanols; however, it was possible to sample seed wings in developing cones which have no vacuolar flavanols.
Figure 3.
Total content of phenols at different stages of Tsuga canadensis needle development; the different cell sizes and thickness of cell walls together with different amounts of vacuolar flavanols made it impossible to quantify nuclear flavanols; however, it was possible to sample seed wings in developing cones which have no vacuolar flavanols.
The flavanols of all three-needle groups are located in the nuclei and vacuoles of both mesophyll cells and the epidermis. During the winter rest period up to April, the nuclei have no flavanols. However, in May the nuclei of young developing cells contain flavanols in relatively high concentrations; but the high value of 17.4 mg flavanols per mg DW needs to be explained. Some tiny vacuoles with flavanols are repeatedly found in cells outside the meristem clusters. Furthermore, the relatively high amount of flavanols is partly due to the small cell size and therefore a relatively high number of cells and nuclei per mg DW is the result. Also, the relative thin cell walls in May contribute to a higher proportion of cytoplasm per unit DW. Cell wall thickness was greater in April and July.
In April, the hydroxycinnamic acids accounted for only a third or half the amount compared to May and July. The flavone group was lower during rapid cell cycling (May) compared with the two other time periods. The kaempferol-glycoside concentrations were slightly higher in May and July compared with the resting needles from April. The flavanols were mainly located in vacuoles. The results clearly demonstrate that the diverse phenolic compounds changed constantly and that it was not possible to measure only the nuclear flavanols.
Based on the above results, it is clear that only highly specialized tissue is suitable for determining nuclear flavanols. Fortunately, most seed wings of
Tsuga canadensis have no vacuoles (inset in
Figure 3) although it is advisable to check them before analysis. It is advantageous that the seed wings of entire cones of the same branch behave synchronously. This means that at any one sampling date seed wings are either with or without vacuoles. Thus far, the main flavanol compounds of the nuclei, as determined by HPLC, were catechin and epicatechin. In addition, small quantities of B1 and B type proanthocyanidins were detected. The striking result of this study is the verification of the presence of flavanols in the nuclei.
3.3. Micrometric Histology of Nuclear Flavanols from Needles of the Tsuga Genotypes
For this type of study it is very important to compare only small clusters of nuclei, which had developed in the same microenvironment of a small tissue sector because throughout a needle there are substantial changes in physiological conditions. The 3 nuclei of
Tsuga canadensis (
Figure 4a) were located in close spatial proximity and were 6, 7, and 9 µm in diameter. This corresponded to a volume of 31, 38, and 60 µm
3. It is remarkable that the most activated nucleus reaches double the volume of the adjacent one. The largest nucleus is characterized by wide interchromatin spaces allowing a high directional transport of metabolites. In contrast, the two smaller nuclei indicated a more diffuse and less defined blue mosaic expression, a pattern that may be characteristic of lower activity that could be assigned to early G 1 phase. Their blue colored chromatins revealed absorbance values of 40 and 47 compared with 62 for the enlarged S phase nucleus.
The two lineages (
Figure 4b) allowed fine-tuned differences to be observed in the flavanol densities of telophase, metaphase, and interphase nuclei. The lineage on the left displayed a changing density from very dark blue to moderate blue (from AU 44 to 38). The structures of the stretched chromosomes at transit from metaphase to rod-like anaphase suffered from pulling stress. This apparently caused some diffusivity of the flavanols, thereby promoting slight viscoelastic dilation as often observed in stretched molecules and often found in Taxus in our previous investigations (unpublished data).
Figure 4.
Nuclear flavanols as blue and, if combined with flavonols, as greenish clusters of the Tsuga and Taxus genotypes.
Figure 4.
Nuclear flavanols as blue and, if combined with flavonols, as greenish clusters of the Tsuga and Taxus genotypes.
As shown in
Figure 4c, fully developed nuclei of
Tsuga canadensis have a certain elasticity in their structures. Two nuclei are shown which were pressed by the cytoplasm towards the cell walls. Thus, they have a high degree of physical compaction and a high flavanol density (AU 86, SD 4.5,
n 15), compared to three non-pressed ones. The latter were larger in size and located in the center of the cell (AU 69, SD 4.3,
n 15). (For simplicity of presentation, these nuclei were removed from the original photos and combined in
Figure 4c.)
As frequently observed within the same needle of
Tsuga Canadensis, there were cell clusters with nuclei showing either blue or more greenish colors (
Figure 4d). Physiological changes were apparently in play, causing differential activation of the flavonoid branches within a needle. The somewhat greenish nuclei consist of blue staining flavanols combined with yellow flavonols. The blue group of nuclei reached AU values of 65 (SD 2.0,
n 15).
As shown in the dwarfed
Tsuga nana (
Figure 4e), after the spring flush all mesophyll cells of a needle could be covered with a yellow tint in the cytoplasm. This is linked to the appearance of green nuclei. As the growth season progressed, the yellow color of the flavonol component intensified. To sum up, the nuclei of
Tsuga nana give the following message: the more yellow, the less active.
Furthermore, application of cytokinin to
Tsuga nana (
Figure 4f) resulted in a fairly strong promotion of nuclear flavanols, attaining AU values up to 73 (SD 2.2,
n 40) compared with controls (AU 47, SD 3.5,
n 40). One pale control nucleus is shown in the left row (asterisk *). Many more nuclei, up to 300, were stained in this respect (not shown). A central finding of this work is the fact that cytokinin is engaged in delivering more flavanols to the nuclei.
3.4. Micrometric Histology of Nuclear Flavanols from Needles of the Taxus Genotypes
Commonly, meristematic cells of the bushy
Taxus baccata undergo periclinal divisions to form a cell lineage. However, sometimes the plane of division changed from periclinal to anticlinal (
Figure 4g). Strikingly, despite the clonal character of the 4 descendants, each one had its own “blue face”, indicating an individual spatial and temporal epigenetic modification of the flavanol pattern with AU values ranging between 55 and 65 (SD 5). Furthermore, the maximum cell cycling activity was at a distinct stage associated with one or two large whitish nucleoli each being as much as 3–4 µm in diameter (
Figure 4g). Such nucleoli reflect a highly active interphase state, taking place when ribosomal RNAs are produced.
In other cases, mitotic nuclei were in interphase (
Figure 4h), evidently running through a short period of low activity (number 1, 2, and 4, with AU values of 57, 55, and 56). Mainly in early G1 the interphase nuclei generally have a lower active state. In addition, there was one quadrate, fairly blue colored nuclear area in transit from late telophase to G1 (number 3, AU 77) and also a correct telophase showing two sickle-shaped compressed “half-moon” nuclei (number 5 and 6, AU 75 and 83). The telophase conformations are about 20% smaller in area than the interphases. This means that at telophase the flavanols are triggered to accumulate up to a dark blue compaction.
Under rare specific metabolic conditions, the four cells even of a meristematic cell lineage curiously underwent a metabolic shift towards extreme deposition of starch grains (
Figure 4i). Nevertheless, the nuclei retained their ability to modify the flavanols (AU 85–91,
n 4), thereby forming deep blue patches of heterochromatin, which points to a strict down-regulation of sizeable sets of genes. (Between the starch grains there are black shadows, which are somewhat misleading.)
In conifers, to our knowledge, the meristem cells have never been found to be vacuolated. A really unique example ever observed in a 4-celled lineage (
Figure 4j) is shown by medium-sized vacuoles located adjacent to the nuclei. Hypothetically, yellow staining phenols are kept away from the nuclei. The color of the rather diffuse nuclei is extremely dark blue, obviously due to unusually over-expressed flavanol synthesis (AU 90, SD 0.6). Studies of the needles from the dwarf
Taxus aurea (
Figure 4k) revealed that the cytoplasm can attain a strong yellow color and the nuclei turn to a greenish tint.
The nuclear activities of the dwarf
Taxus baccata nana are documented by examples of different needles (
Figure 4l). During spring, the ability to form cell lineages is linked to light blue and clearly mottled blue nuclei (AU 42, SD 2.0). Within a recently formed 4-celled lineage, there is commonly, as expected, no variation in flavanol density of the nuclei and also no yellow surrounding cytoplasm. However, as the growth slowed down, 6 more greenish nuclei appeared and two cells of this lineage underwent an anticlinal cell division (
Figure 4l). A greenish colored lineage is exceptional and apparently related with the early appearance of yellow flavonols in the
Taxus nana genotype. Interestingly, a further situation is also shown in
Figure 4l as a single large cell was subjected to a fairly high developmental activity in flavanol synthesis and deposition of these flavanols into about 10 small vacuoles. The nucleus responsible for such a differentiation obviously displays pale blue staining (53 AU). By contrast, two adjacent large cells with a yellow cytoplasm developed slightly greenish nuclei as a sign of relative inactivity. Indeed, the presence of notable amounts of flavonols in nuclei of
Taxus nana is documented by yellow fluorescence as indicated clearly by DPBA (
Figure 4m).