Root Browning in Pinus banksiana Lamb. and Eucalyptus pilularis Sm. 1. Anatomy and Permeability of the White and Tannin Zones

Growing tree roots are characteristically brown with white tips. The browning process, which occurs as the white region matures, has often been attributed to the deposition of suberin in various tissues. However, in pouch-grown tree seedlings of jack pine (Pinus banksiana Lamb.) and eucalyptus (Eucalyptus pilularis Sm.), browning was not linked to suberization but was caused by the deposition of condensed tannins in the walls of all cells external to the stele. Therefore, we propose using the term “tannin zone” to refer to this region of the root. Vitality tests indicated that the cells of the epidermis and cortex were alive in white regions but were dead in brown regions. Following sequential treatment with berberine hemisulfate and potassium thiocyanate, the cortical walls external to the endodermal Casparian band were full of berberine thiocyanate crystals, indicating that they were permeable to berberine. These walls should also be permeable to water and ions, which have smaller molecular dimensions than the tracer dye. Based on the anatomy and permeability of the tannin zone, we predict that its capacity for ion uptake would be reduced compared to the white zone because of a reduced absorptive plasmalemma surface area. In jack pine, some uptake could be effected by the passage cells of the endodermis. The tannin zone should be even less absorptive in eucalyptus because the exodermis remains an apoplastic barrier and the endodermis lacks passage cells. It is difficult to predict the difference between the tannin and white zones with respect to water uptake. Death of the cells external to the endodermis would reduce the resistance of the root to water movement, but deposition of tannins would increase it. The deposition of suberin lamellae in increasing numbers of endodermal cells may also retard water flow. The anatomy and physiological properties of the tannin zone are unique from those of the distal, white zone and the proximal, cork-clad zone.     

Root Browning in Pinus banksiana Lamb. and Eucalyptus pilularis Sm. 2. Anatomy and Permeability of the Cork Zone

The periderm in roots of Pinus banksiana Lamb. and the polyderm in roots of Eucalyptus pilularis Sm. originate from the pericycle. This occurs after the roots have turned brown due to deposition of tannins in the walls of cells external to the endodermis. In both species, cork cells form a continuous sheath around the vascular tissues. The cork cell walls are modified by the presence of suberin, lignin and tannin and it is the latter which imparts a brown colour to the tissue. The first layer of cork cells in both species constitutes an apoplastic barrier which prevents the fluorescent dye, berberine, from entering the vascular tissues, despite the absence of an identifiable Casparian band in the cells. Because the roots are still covered with the cortex and epidermis during early stages of periderm and polyderm formation, it is not possible to tell from the external aspect of the root when it makes a transition from the tannin zone to the cork zone.     

Root anatomy of <Emphasis Type="Italic">Pinus taeda</Emphasis> L.: seasonal and environmental effects on development in seedlings

The environmental and seasonal effects on anatomical traits of Pinus taeda L. seedling roots were studied in the laboratory in three contrasting root growth media and also in typical outdoor nursery culture. Growth media with lower water regimen and high penetration resistance caused a reduction in lengths of the white and condensed tannin (CT) zones and acceleration of development of suberin lamellae in the endodermis. As a possible counter to this reduction in zone lengths, second-order laterals were produced closer to the tips of first-order laterals. This suggested there may be an advantage to producing more shorter roots under stressful conditions. Under outdoor nursery conditions (June to mid-December) the white zone was always a rather small part of the root system surface area (4.5% in December), but it dominated as a provider of cortical plasmalemma surface area (CPSA) in contact with modified soil solution (65% in December) because of its live cortex and capacity to increase nearly three fold the amount of CPSA per unit root length. The CT zone always provided most of the total root surface area (80% in December). Although it had no live cortex, a few cells of the CT zone endodermis remained non-suberized passage cells, perhaps giving this major part of the root system some capacity for ion and water absorption. A late summer increase in CPSA was due largely to the rapid production of mycorrhizae. Root systems were capable of very rapid replacement of roots lost due to undercutting and lateral root pruning. The great variation in CPSA per unit root length contained in the white, mycorrhizal and CT zones suggested a capacity to adapt rapidly to changing conditions.     

Pine root structure and its potential significance for root function

Actively growing roots of pouch-grown Pinus banksiana Lamb. are known to have three anatomically distinct zones, i.e., white, condensed tannin, and cork (in order of increasing distance from the root tip). Roots of pouch and pot-grown Pinus taeda L., and field-grown P. banksiana also develop these three zones. The terminal region of a dormant root resembles the condensed tannin zone, with the addition of a suberized metacutis partially surrounding the apical meristem. White roots are anatomically suited for efficient ion uptake due to the presence of a living cortex. The condensed tannin zones of both growing and dormant roots have a dead cortex but retain passage cells in their endodermal layers, through which some ion uptake could occur. The effect of the maturation from white to condensed tannin zone on water uptake is difficult to predict, but some uptake would occur through the endodermal passage cells. In the young cork zone, no ion and little water absorption should occur. The discrepancies between results of separate anatomical and physiological investigations of tree roots need to be resolved by correlative studies incorporating both approaches in individual experiments. This revised version was published online in June 2006 with corrections to the Cover Date.     

白皮松针叶内皮层在盐胁迫中的屏障作用研究

应用荧光显微技术、傅里叶变换显微红外光谱分析(FTIR)、扫描电镜及X-射线能谱微区分析等手段,对白皮松(Pinus bungeana)子叶、初生叶及2a生针叶内皮层细胞径向壁的显微结构特征、化学成分,以及在叶子横切面上Na和Cl的微区分布进行分析。通过荧光显微观察发现,白皮松子叶内皮层不具凯氏带,而初生叶及2a生针叶均存在凯氏带加厚现象。根据FTIR的检测结果显示:子叶内皮层细胞径向壁不含木栓质或极少,2a生针叶内皮层细胞径向壁木栓质含量高于初生叶。对相应区域的X射线微区分析表明,子叶内皮层对Na和Cl在质外体运输中不起障碍作用,而初生叶与2a生针叶内皮层阻碍Na和Cl以质外体途径进入维管组织。研究结果表明:具凯氏带加厚的内皮层细胞壁中木栓质含量决定其在质外体运输过程中的生理功能。     

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na⁺ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na⁺ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na⁺. The average Na⁺ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na⁺ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na⁺ flux varied significantly in different positions along developing rice roots, and a net Na⁺ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na⁺ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.     

Effects of high nitrogen load on growth, photosynthesis and nutrient status of Cryptomeria japonica and Pinus densiflora seedlings

To evaluate the sensitivity of Japanese cedar (Cryptomeria japonica D. Don) and Japanese red pine (Pinus densiflora Sieb. et Zucc.) to high N deposition, 1-year-old seedlings were grown in brown forest soil treated with N as NH₄NO₃ at 0, 25, 50, 100 and 300 mg l^-1 fresh soil volume, equivalent to 0, 28, 57, 113 and 340 kg N ha^-1. Net photosynthetic rate and whole-plant dry mass of C. japonica seedlings were increased by the N treatment, whilst those of P. densiflora seedlings were significantly reduced by the highest N treatment. The reduction in the net photosynthesis of P. densiflora seedlings was mainly due to a depression of carboxylation efficiency accompanied by a decrease in concentration and activity of Rubisco in the needles. In P. densiflora seedlings, needle concentrations of P and Mg were decreased, and the concentrations of N and Mn were increased by the highest N treatment. The reductions in needle protein concentration and Rubisco activity were negatively correlated with the ratios of N/P and Mn/Mg in the needles. These results suggest that nutrient imbalances of these elements may be induced in P. densiflora seedlings grown under high N deposition. We conclude that P. densiflora is more sensitive to high N deposition than C. japonica, and that the relatively high atmospheric N deposition to Japanese forest ecosystems may adversely affect the health of N-sensitive tree species such as P. densiflora.     

Paternal chloroplast inheritance patterns in pine hybrids detected with trnL-trnF intergenic region polymorphism

The inheritance patterns of the chloroplast genomes of shortleaf pine (Pinus echinata Mill.), loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were investigated through the trnL-trnF intergenic spacer polymorphism analysis. The DNA sequences of this spacer differ among these three closely related Pinus species. A modified 'cold' PCR-SSCP (single-strand conformation polymorphism) analysis of this spacer shows that the artificial hybrids (F1) from the shortleaf pine (seed parent) 2 loblolly pine (pollen parent) cross, exhibit the loblolly pine profile. Additionally, nine putative hybrids between shortleaf pine and loblolly pine, previously identified by the IDH (Isocitrate dehydrogenase) allozyme marker, presented the shortleaf pine profile indicating that shortleaf pine, not loblolly pine, sired all of the putative hybrids. Nondenatured polyacrylamide-gel electrophoresis of the trnL-trnF intergenic spacer demonstrated that the artificial hybrids (F1) from the cross, slash pine (seed parent) 2 shortleaf pine (pollen parent), present the shortleaf pine profile. Those results confirmed that the chloroplast genome is paternally inherited in these three species of the genus Pinus. The significance of the trnL-trnF intergenic region polymorphism and our modified 'cold' SSCP protocol for population genetic studies is discussed.     

Transpecific microsatellites for hard pines

Microsatellites are difficult to recover from large plant genomes so cross-specific utilisation is an important source of markers. Fifty microsatellites were tested for cross-specific amplification and polymorphism to two New World hard pine species, slash pine (Pinus elliottii var. elliottii) and Caribbean pine (P. caribaea var. hondurensis). Twenty-nine (58%) markers amplified in both hard pine species, and 23 of these 29 were polymorphic. Soft pine (subgenus Strobus) microsatellite markers did amplify, but none were polymorphic. Pinus elliottii var. elliottii and P. caribaea var. hondurensis showed mutational changes in the flanking regions and the repeat motif that were informative for Pinus spp. phylogenetic relationships. Most allele length variation could be attributed to variability in repeat unit number. There was no evidence for ascertainment bias.     

The use of lanthanum to study the functional development of the Casparian strip in corn roots

Summary Lanthanum in combination with electron microscopy was used to study the functional development of the Casparian strip in the primary root of corn (Zea mays L. WF9 X M14). The Casparian strip was first visible between 13 and 16 mm from the root tip, and was first an effective barrier to lanthanum movement through the apoplast from cortex to stele between 14 and 18 mm from the apex. Until the Casparian strip was formed, lanthanum ion freely penetrated the stelar apoplast. By 18 mm from the apex lanthanum was completely restricted to the extracellular spaces of the cortex. Treatment with 0.02 or 0.03% (v/v) Triton X-100 increased the permeability of the plasma membrane, and allowed lanthanum ion to penetrate the cortical symplast. After the detergent treatment, the endodermis no longer functioned as a barrier to La³⁺ movement from cortex to stele. This confirms that an intact plasma membrane is necessary for the primary endodermis to control the passage of solutes into the vascular tissues of the stele.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.     

Casparian strips in needles of Pinus bungeana: isolation and chemical characterization

By using cell wall degrading enzymes, Casparian strips were for the first time isolated from Pinus bungeana needle endodermis. They appeared as a fine network, similar to those isolated from roots. Fourier transform infrared spectroscopic analysis provided evidence that the Casparian strips were impregnated with lignin, suberin, cellulose and cell wall proteins.     

Functions of passage cells in the endodermis and exodermis of roots

Passage cells frequently occur in the endodermis and exodermis but are not ubiquitous in either layer. Passage cells occur in the form of short cells in the dimorphic type of exodermis. In both layers, Casparian bands are formed in all cells, but the subsequent development of suberin lamellae and thick, cellulosic walls are delayed or absent in the passage cells. Available evidence suggests that passage cells of the endodermis are important for the transfer of calcium and magnesium into the stele and thus into the transpiration stream. They become the only cells which present a plasmalemma surface to the soil solution (and are thus capable of ion uptake) when the epidermis and central cortex die. This occurs naturally in some herbaceous and woody species and is known to be promoted by drought. Most evidence indicates that the development of suberin lamellae in both the endodermis and exodermis increases the resistance of the root to the radial flow of water. Passage cells thus provide areas of low resistance for the movement of water, and the position of these cells in the endodermis (i.e., in close proximity to the xylem) is explained in terms of function. Exodermal passage cells have a cytoplasmic structure suggesting an active role in ion uptake. This may be related to the tendency of the epidermis to die, leaving the passage cells as the only ones with their membranes exposed to the soil solution. Passage cells in the exodermis attract endomycorrhizal fungi while those in the endodermis do not. It is clear that passage cells of the endodermis and exodermis play a variety of roles in the plant root system.     

Mycorrhizal associations in Pinus massoniana Lamb. and Pinus elliottii Engel. inoculated with Pisolithus tinctorius

Dichotomous mycorrhizas were induced in Pinus massoniana and Pinus elliottii seedlings inoculated with Pisolithus tinctorius growing under non-axenic conditions. Six months after inoculation, Pinus massoniana seedlings exhibited a higher degree of infection, bore more mycorrhizas and had developed more abundant extramatrical mycelium than seedlings of Pinus elliottii. Nevertheless, seedlings of Pinus massoniana were stunted and exhibited chorosis of the needles, indicating a possible nutrient deficiency. Histological examination of these pine mycorrhizas showed an ectomycorrhizal association typical of gymnosperms with an intercellular Harting net penetrating between several layers of cortical cells close to the endodermis. However, strong polyphenolic reactions, intracellular hyphae and wall modifications were occasionally observed, indicating that both host-tissue incompatibility and ectendomycorrhizal association can occur in pine species under stressed conditions.     

Method for detecting tree ring boundary in conifers and broadleaf trees using Mask R-CNN and linear interpolation

As tree rings can reveal various information regarding climate and environmental factors, increasing research is being conducted on them. Although tree ring analysis software such as Windendro has been applied, research on the development of analysis software using image preprocessing algorithms and deep learning is recently being attempted as computer vision technology. In this study, Mask R-CNN and linear interpolation were applied from images collected using a smartphone (SM-G973, Samsung, Suwon) and a scanner (CanoScan 9000F, Canon, Otaku) to propose an effective method for detecting tree ring boundaries. Pitch pine (Pinus rigida), Korean pine (Pinus koraiensis), white birch (Betula platyphylla), and cork oak (Quercus variabilis) were selected as tree species. Of the 300 images, 240 were classified as training data and 60 as validation data. As a result of learning, smartphones detected 86.0 % (381 ring boundaries) of the rings in pitch pine, 82.1 % (367 ring boundaries) in Korean pine, 84.7 % (309 ring boundaries) in white birch, and 78.7 % (318 ring boundaries) in cork oak. The scanner detected 93.2 % (413 ring boundaries) of the rings in pitch pine, 90.8 % (405 ring boundaries) in Korean pine, 88.2 % (322 ring boundaries) in white birch, and 89.4 % (361 ring boundaries) in cork oak. In particular, the smartphone showed satisfactory results of 84.7 % and 78.7 % for detecting tree ring boundaries of white birch and cork oak, where the boundaries of the rings were unclear. In the annual growth analysis results, both smartphones and scanners were statistically insignificant, and there was no difference compared with those of Windendro. Therefore, Mask R-CNN might be an effective approach for tree ring boundary detection as it showed satisfactory results, even with smartphones. In addition, although there was distortion in cases where images were acquired with a circular lens, there was no statistically significant difference from Windendro results. Thus, Mask R-CNN and linear interpolation can be used for tree ring boundary detection and growth measurement.     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

Effects of exposure to humid air on epidermal viability and suberin deposition in maize (Zea mays L.) roots

When the basal zones of 4-d-old hydroponically grown maize ( Zea mays L. cv. Seneca Horizon) roots were exposed to moist air for 2 d, the development of both endodermis and exodermis was affected. In the endodermis, Casparian bands enlarged and more cells developed suberin lamellae. The most striking effect was seen in the exodermis. In submerged controls, only 4% of the cells had Casparian bands, whereas in root regions exposed to air, 93% developed these structures. Similarly, in submerged roots 11% of the exodermal cells had either developing or mature suberin lamellae compared with 92% in the air-treated region. The majority of epidermal cells remained alive in the zone exposed to air. Some cell death had occurred earlier in the experiment when the seedlings were transferred from vermiculite to hydroponic culture. The precise stimulus(i) associated with the air treatment which led to accelerated development in both endodermis and exodermis is as yet unknown.     

Casparian strips in needles are more solute permeable than endodermal transport barriers in roots of Pinus bungeana

The structure and development of endodermal Casparian strips in Pinus bungeana needles and roots were studied by scanning electron microscopy and fluorescence microscopy. Primary pit fields (PFs) frequently occurred in radial walls of Casparian strips isolated from needles, whereas PFs were never detected in Casparian strips from roots. Formation of Casparian strips in needles as well as roots started at the outer parts of the radial walls and they finally occupied the entire radial walls of the endodermis. Fourier transform infrared (FTIR) spectroscopy of Casparian strips isolated from roots revealed significant absorption bands characteristic for suberin. However, in Casparian strips of needles, evidence for suberin was rarely detected by FTIR spectroscopy. The apoplastic permeability of Casparian strips in needles and roots was probed by the apoplastic tracers calcofluor and berberine. Casparian strips in roots efficiently blocked the apoplastic transport (AT) of calcofluor and berberine. Casparian strips in needles blocked the AT of calcofluor, but diffusion of berberine was not inhibited and berberine thiocyanate crystals were detectable in the vascular tissue of the needles. From the data presented, it must be concluded that Casparian strips in needles, which are characterized by the absence of suberin, are more solute permeable compared with Casparian strips in roots.