Xylem Ray Parenchyma Cells Research Articles

Cambial Age Influences PCD Gene Expression during Xylem Development and Heartwood Formation

Heartwood formation is an important ontogenetic stage in Scots pine (Pinus sylvestris L.). The amount of heartwood determines the proportion of functionally active sapwood in the total trunk biomass as well as the quality of wood. The key criterion for heartwood formation is the death of xylem ray parenchyma cells. Previously, models that described the patterns of heartwood formation, depending on the cambial age, were derived from Scots pine trees of different ages. The cambial age is the number of annual xylem layers at the core sampling site at a certain trunk height. We studied the features of the occurrence of programmed cell death (PCD) processes during the xylem differentiation and heartwood formation of 80-year-old Scots pine trees, depending on the cambial age, under the lingonberry pine forest conditions in the transition area of the northern taiga subzone and tundra. We have shown that the distance from the cambial zone to the heartwood boundary does not change significantly with stem height. As the cambial age increases, the lifespan of the formed xylem ray parenchyma cells increases and the activity of PCD genes decreases during the formation of both (1) xylem (in the outer layers of sapwood) and (2) heartwood (in the inner layers of sapwood and transition zone). We hypothesized that the decisive factor in the PCD initiation during heartwood formation is the distance of the xylem ray parenchyma cells from the cambial zone. The younger cambium forms wider annual increments, and therefore the xylem ray parenchyma cells in these parts of the trunk reach the distance from the cambial zone earlier, which is necessary for PCD initiation.

Anatomy and cell wall ultrastructure of sunflower stalk rind

The anatomy and ultrastructure of sunflower stalk rind are closely related to its conversion and utilization. We studied systematically the anatomy and ultrastructure of the stalk rind using light, scanning electron, transmission electron and fluorescence microscopy. The results showed that the stalk rind consisted of phloem fibers (PF), xylem fibers (XF), vessel elements (V), ground parenchyma cells (GPC), axial parenchyma cells (APC), xylem ray parenchyma cells (XRPC), and pith ray parenchyma cells (PRPC). These cell walls were divided into the middle lamella, primary wall, and secondary wall (S). It was found that the S of PF, XF and V was further divided into three layers (S1–S3), while the S of APC, GPC, XRPC and PRPC showed a non-layered cell wall organization or differentiated two (S1, S2) to seven layers (S1–S7). Our research revealed the plasmodesmata characteristics in the pit membranes (PMs) between parenchyma cells (inter-GPCs, inter-XRPCs, and inter-PRPCs). The morphology of the plasmodesmata varied with the types of parenchyma cells. The thickness and diameter of PMs between the cells (inter-Vs, V–XF, V–APC, and V–XRPC) were greater than that of PMs between parenchyma cells. The cell corners among parenchyma cells were intercellular space. The lignification degree of vessels was higher than that of parenchyma cells and fibers. The results will provide useful insights into the biological structure, conversion and utilization of sunflower stalk rind.

In Planta Analysis of the Radial Movement of Minerals from Inside to Outside in the Trunks of Standing Japanese Cedar (Cryptomeria japonica D. Don) Trees at the Cellular Level

Although the radial movement of minerals in tree trunks is a widely accepted phenomenon, experimental evidence of their movement in standing trees and underlying mechanisms is very limited. Previously, we clarified that cesium (Cs) artificially injected into the outer part of the sapwood of standing Japanese cedar (Cryptomeria japonica D. Don) trunks moved to the inner part of the sapwood, including the intermediate wood, via active transport by xylem parenchyma cells and diffusion through cell walls and then moved into the heartwood by diffusion. To understand the mechanism underlying the radial movement of minerals in the standing tree trunk, it is necessary to clarify their movement in the opposite direction. Therefore, the present study aimed to determine the radial movement of minerals from inside to outside in the trunks of standing trees at the cellular level. For this, a long hole across the center part of the trunk, which reached the heartwood, intermediate wood, and sapwood, was made in standing Japanese cedar trunks, and a solution of stable isotope Cs was continuously injected into the hole for several days as a tracer. The injected part of the trunk was collected after being freeze-fixed with liquid nitrogen, and the frozen sample was subjected to analysis of Cs distribution at the cellular level using cryo-scanning electron microscopy/energy-dispersive X-ray spectroscopy. The Cs injected into the inner sapwood or intermediate wood rapidly moved toward the outer sapwood via xylem ray parenchyma cells together with diffusion through the cell walls. In contrast, the Cs injected into the heartwood barely moved to the sapwood, although it reached a part of the inner intermediate wood. These results suggest that minerals in xylem ray parenchyma cells in the sapwood are bidirectionally supplied to each other; however, the minerals accumulated in the heartwood may not be supplied to living cells.

Xylella fastidiosa causes transcriptional shifts that precede tylose formation and starch depletion in xylem.

Pierce's disease (PD) in grapevine (Vitis vinifera) is caused by the bacterial pathogen Xylella fastidiosa. X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant‐derived xylem blockages, primarily in the form of tyloses. Tylose‐mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera. We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine‐scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X‐ray computed microtomography and machine‐learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up‐regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down‐regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

6. 低温馴化によるシラカンバ木部放射柔細胞の細胞壁の変化(平成11年度第45回低温生物工学会研究報告)

6. 低温馴化によるシラカンバ木部放射柔細胞の細胞壁の変化(平成11年度第45回低温生物工学会研究報告)

3. 樹木木部放射柔細胞の凍結適応機構の変遷(平成9年度第43回低温生物工学会研究報告)

3. 樹木木部放射柔細胞の凍結適応機構の変遷(平成9年度第43回低温生物工学会研究報告)

21. 樹木木部柔細胞の深過冷却をもたらすと考えられている細胞壁マイクロキャピラリーサイズを解析する試み(第49回低温生物工学会研究報告)

21. 樹木木部柔細胞の深過冷却をもたらすと考えられている細胞壁マイクロキャピラリーサイズを解析する試み(第49回低温生物工学会研究報告)

Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Histochemical and Anatomical Studies of Phloem and Xylem Cells of Jackfruit (<i>Artocarpus heterophyllus</i> Lam.) Tree

The localization of starch, lipid and protein, nuclei in the phloem and xylem cells of stem of Jackfruit trees (Artocarpus heterophyllus) has been studied. The optical digital images of anatomical features and localization of nuclei and reserve materials were obtained by light microscopy and fluorescence microscopy. Starch granules in the ray parenchyma cells were more abundant in the outer xylem close to the cambium than in the inner xylem and phloem. Radial localization of starch granules provided more clear data than transverse and tangential observations. Lipid and protein appeared as droplets and were uniformly distributed in the outer xylem. The parenchyma cells of phloem have large amount of lipid bodies but those were almost absent in cambium and xylem ray parenchyma cells. The results on the localization of storage starch, lipid droplets and proteins in phloem, cambium and xylem cells indicating that reserve materials might be important for wood formation in jackfruit trees. This data would be helpful for further study in tree breeding program and clarification of the mechanism of utilization of such reserve materials and their distribution pattern within the cells. DOI: http://dx.doi.org/10.3329/ijns.v2i1.10876 International Journal of Natural Sciences (2012), 2(1): 01- 07

Temporally and spatially controlled death of parenchyma cells is involved in heartwood formation in pith regions of branches of Robinia pseudoacacia var. inermis

Focussing on four types of parenchyma cell around pith regions of branches of Robinia pseudoacacia L. var. inermis, we examined the timing and role of cell death during heartwood formation. Large parenchyma cells that were located in the inner part of the pith died within a year. By contrast, other parenchyma cells died within 4 years, with the timing of cell death depending on the type of cell. Axial parenchyma cells of the xylem close to the pith died first. Then, small parenchyma cells died in the perimedullary zone in the outer part of the pith. Finally, ray parenchyma cells in the xylem close to the pith died. Variations in the autofluorescence of cell walls, which might have been due to deposition of heartwood substances, were observed first in xylem ray parenchyma cells and small parenchyma cells in the perimedullary zone. Our results indicate that the initiation of heartwood formation occurs within 4 years in pith regions of branches in Robinia pseudoacacia L. var. inermis. Moreover, it appears that not only xylem ray parenchyma cells but also small parenchyma cells in the perimedullary zone might be involved in the synthesis of heartwood substances.

Cold stability of microtubules in wood-forming tissues of conifers during seasons of active and dormant cambium

The cold stability of microtubules during seasons of active and dormant cambium was analyzed in the conifers Abies firma, Abies sachalinensis and Larix leptolepis by immunofluorescence microscopy. Samples were fixed at room temperature and at a low temperature of 2-3°C to examine the effects of low temperature on the stability of microtubules. Microtubules were visible in cambium, xylem cells and phloem cells after fixation at room temperature during seasons of active and dormant cambium. By contrast, fixation at low temperature depolymerized microtubules in cambial cells, differentiating tracheids, differentiating xylem ray parenchyma and phloem ray parenchyma cells during the active season. However, similar fixation did not depolymerize microtubules during cambial dormancy in winter. Our results indicate that the stability of microtubules in cambial cells and cambial derivatives at low temperature differs between seasons of active and dormant cambium. Moreover, the change in the stability of microtubules that we observed at low temperature might be closely related to seasonal changes in the cold tolerance of conifers. In addition, low-temperature fixation depolymerized microtubules in cambial cells and differentiating cells that had thin primary cell walls, while such low-temperature fixation did not depolymerize microtubules in differentiating secondary xylem ray parenchyma cells and tracheids that had thick secondary cell walls. The stability of microtubules at low temperature appears to depend on the structure of the cell wall, namely, primary or secondary. Therefore, we propose that the secondary cell wall might be responsible for the cold stability of microtubules in differentiating secondary xylem cells of conifers.

Efficacy and Duration of Trunk-Injected Imidacloprid in the Management of Hemlock Woolly Adelgid (Adelges tsugae)

Hemlock woolly adelgid (Adelges tsugae) (HWA) is an introduced piercing, sucking insect that affects hemlocks (Tsuga spp.) by extracting carbohydrates from the xylem ray parenchyma cells. Left untreated, HWA will result in reduced shoot growth, branch dieback, and ultimately tree death. In this study, the insecticide IMA-jet (5% imidacloprid w/w) was applied by trunk microinjection with the Arborjet Tree I.V. using the VIPER method. Sixteen randomly selected HWA-infested hemlocks were treated in 2002 and 2003 and eight trees were selected as untreated controls. Eight infested branch samples per tree were cut each year (2003, 2004, and 2005) and assessed. Four parameters were used to assess the efficacy and duration of treatments. These were percent HWA mortality, total and live HWA per linear centimeter shoot growth, and current-year shoot growth. The eastern hemlock (T. canadensis) in this study had high initial HWA pressure. In the 3 years of the study, winter low temperatures were insufficient to cause an appreciable or sustainable reduction in infestation levels. In the controls, HWA increased and hemlock growth decreased. Treatment with stem-injected imidacloprid did not provide a quick knockdown of the HWA; rather, it required time (i.e., at least 1 year). In the year after a second treatment, we observed sufficiently high HWA mortality for hemlock to resume growth. We have a high degree of confidence that a repeat treatment increased the levels of imidacloprid for the duration of efficacy observed. We recommend a 2× dosage (e.g., for trees in the 30 to 59 cm [12 to 23.6 in] size class, increase from 0.08 g A.I. to 0.16 gm A.I./cm trunk diameter at breast height [dbh]) for an increased level of efficacy to extend the injection interval (to once every 2 years) and to limit the number of wounds a tree receives to centimeters dbh/5 (dbh in/2). The new rate recommendations are reflected on the IMA-jet label amended in 2006. These results demonstrate that hemlock with high HWA pressure can be successfully treated using IMA-jet and the Arborjet Tree I.V. system.

Sirococcus clavigignenti‐juglandacearum

<i>Sirococcus clavigignenti‐juglandacearum</i>

18. 深過冷却で寒冷環境に適応するカラマツ木部柔細胞の遺伝子発現(第49回低温生物工学会研究報告)

18. 深過冷却で寒冷環境に適応するカラマツ木部柔細胞の遺伝子発現(第49回低温生物工学会研究報告)

Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation.

It has been accepted that xylem ray parenchyma cells (XRPCs) in hardwood species respond to subfreezing temperatures either by deep supercooling or by extracellular freezing. Present study by cryo-scanning electron microscopy examined the freezing responses of XRPCs in five boreal hardwoods: Salix sachalinensis Fr. Schmit, Populus sieboldii Miq., Betula platyphylla Sukat. var japonica Hara, Betula pubescens Ehrh., and red osier dogwood (Cornus sericea), in which XRPCs have been reported to respond by extracellular freezing. Cryo-scanning electron microscopy observations revealed that slow cooling of xylem to -80 degrees C resulted in intracellular freezing in the majority of XRPCs in S. sachalinensis, an indication that these XRPCs had been deep supercooled. In contrast, in the majority of XRPCs in P. sieboldii, B. platyphylla, B. pubescens, and red osier dogwood, slow cooling to -80 degrees C produced slight cytorrhysis without clear evidence of intracellular freezing, suggesting that these XRPCs might respond by extracellular freezing. In these XRPCs exhibited putative extracellular freezing; however, deep etching revealed the apparent formation of intracellular ice crystals in restricted local areas. To confirm the occurrence of intracellular freezing, we rewarmed these XRPCs after cooling and observed very large intracellular ice crystals as a result of the recrystallization. Thus, the XRPCs in all the boreal hardwoods that we examined responded by deep supercooling that was accompanied with incomplete desiccation. From these results, it seems possible that limitations to the deep-supercooling ability of XRPCs might be a limiting factor for adaptation of hardwoods to cold climates.

Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar.

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.

Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants.

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula x Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content.

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.

Seasonal Changes in the Freezing Behavior of Xylem Ray Parenchyma Cells in Four Boreal Hardwood Species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely,Betula platyphylla, Populus canadensis, P. sieboldii,andSalix sachalinensis,was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates.