Comparative Studies on Differentiation of Regenerated Tissue in Broussonetia papyrifera

This paper describes the differentiation process of regenerated tissue after ordinary girdling or after removal of a section of xylem from the stem, and the disparity in differentiation of the regenerated tissues after being differently treateds in Broussonetia papyrifera. After ordinary girdling for 3–4 weeks, new bark regenerated in the xylem. During the process of rind' formation, many specks of meristematic tissue were formed in the callus, from which vascular tissue clusters were developed. In addition, the new periderm appeared almost at the same time as the new vascular cambium was seen. When a section of xylem was removed from the stem, numerous calli developed rapidly on the inner surface of the bark. Meanwhile, the vascular cambium appeared in the immature phloem. Soon after, discontinued meristematic tissue bands also occurred in the callus. These meristematic tissues then connected with each other to form a concave oblate cambial ring which developed xylem inward and phloem outward. About 2–3 weeks later, the concave oblate trunk grew lengthwisely connecting with the upper anct lower portions of the normal stem. By then, the tree continued to grow. The inner surface tissue of the bark, after the xylem was removed, differentiated about one week earlier than the tissue on the surface of the xylem after girdling.     

龙眼剥皮再生的解剖学研究

龙眼(Dimocarpus tongan Lour.)茎干经过大面积环剥,都能再生出新皮。环剥初期,愈伤组织都由近暴露面的射线细胞产生,稍后,其他未成熟木质部细胞也参加愈伤组织的形成,这些愈伤组织一般在靠近表面都可发生木栓形成层,以后迅速形成正常的周皮。在愈伤组织与木质部交界处的未成熟木质部细胞发生维管形成层。新发生的形成层正常地向外分化出次生韧皮部,向内分化出次生木质部。初期有些原来的射线将新形成层带分割成许多小区,二个月后,由于新的形成层不断平周活动,逐渐将形成层连成一圈,以后基本上与正常树皮维管组织的发育一样。     

Phloem transdifferentiation from immature xylem cells during bark regeneration after girdling in Eucommia ulmoides Oliv

Eucommia ulmoides Oliv. (Eucommiaceae), a traditional Chinesemedicinal plant, was used to study phloem cell differentiationduring bark regeneration after girdling on a large scale. Hereit is shown that new sieve elements (SEs) appeared in the regeneratedtissues before the formation of wound cambium during bark regenerationafter girdling, and they could originate from the transdifferentiationof immature/differentiating axial xylem cells left on the trunk.Assays of water-cultured twigs revealed that girdling blockedsucrose transport until the formation of new SEs, and the regenerationof the functional SEs was not dependent on the substance providedby the axis system outside the girdled areas, while exogenousindole acetic acid (IAA) applied on the wound surface acceleratedSE differentiation. The experiments suggest that the immaturexylem cells can transdifferentiate into phloem cells under certainconditions, which means xylem and phloem cells might share someidentical features at the beginning of their differentiationpathway. This study also showed that the bark regeneration systemcould provide a novel method for studying xylem and phloem celldifferentiation. Key words: Bark regeneration, Eucommia ulmoides Oliv., immature xylem cells, sieve elements, transdifferentiation Received 19 November 2007; Revised 23 January 2008 Accepted 24 January 2008     

MRI links stem water content to stem diameter variations in transpiring trees

In trees, stem diameter variations are related to changes in stem water content, because internally stored water is depleted and replenished over a day. To confirm this relationship, non-invasive magnetic resonance imaging (MRI) was combined with point dendrometer measurements in three actively transpiring oak (Quercus robur L.) trees. Two of these oak trees were girdled to study the stem increment above the girdling zone. MRI images and micrographs of stem cross-sections revealed a close link between the water distribution and the anatomical features of the stem. Stem tissues with the highest amount of water were physiologically the most active ones, being the youngest differentiating xylem cells, the cambium and the youngest differentiating and conductive phloem cells. Daily changes in stem diameter corresponded well with the simultaneously MRI-measured amount of water, confirming their strong interdependence. MRI images also revealed that the amount of water in the elastic bark tissues, excluding cambium and the youngest phloem, contributed most to the daily stem diameter changes. After bark removal, an additional increase in stem diameter was measured above the girdle. This increase was attributed not only to the cambial production of new cells, but also to swelling of existing bark cells. In conclusion, the comparison of MRI and dendrometer measurements confirmed previous interpretations and applications of dendrometers and illustrates the additional and complementary information MRI can reveal regarding water relations in plants.     

Patterned cell determination in a plant tissue: the secondary phloem of trees

The secondary vascular tissues (xylem and phloem) of woody plants originate from a vascular cambium and develop as radially oriented files of cells. The secondary phloem is composed of three or four cell types, which are organised into characteristic recurrent cellular sequences within the radial cell files of this tissue. There is a gradient of auxin (indole acetic acid) across both the cambium and the immediately postmitotic cells within the xylem and phloem domains, and it is believed that this morphogen, probably in concert with other morphogenic factors, is closely associated with the determination and differentiation of the different cells types in each tissue. A hypothesis is developed that, in conjunction with the positional values conferred by the graded radial distribution of morphogen, cell divisions at particular positions within the cambium are sufficient to determine not only each of the phloem cell types but also their recurrent pattern of differentiation within each radial cell file.     

Histogenesis of Xylem of Eucommia ulmoides Cultured in Vitro

Following a complete ringing of the main stem of Eucommia ulmoides Oliv. regeneration of normal new bark has been observed as a subsequence of repeated partial differentiation of the immature xylem into cambium. The immature xylem, when cultured in vitro, showed production of callus tissue from the ray cells. After 15–30 days of continuous culture, meristematic tissue appeared as a discontinuous and more or less regularly arranged.zone was visible within the callus tissue. However, the meristematic cells, unlike the elongated fusif0rm initial cells, are isometric in shape. This meristem differentiated into tracheids centripetally. Nevertheless, no centrifugal differentiation into phloem was evidenced. The vascular tissues were ultimately degenerated after 6 months cultured in vitro.     

EFFECT OF ISOLATION OF BARK ON CAMBIAL ACTIVITY AND DEVELOPMENT OF XYLEM AND PHLOEM IN TREMBLING ASPEN

Circular patches of bark were surgically isolated on the sides of trembling aspen (Populus tremuloides Michx.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated and control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control trees cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) did not prevent initiation of cambial activity and of phloem differentiation in spring but continued normal cambial activity and phloem developmented were prevent. Xylem differentiation was essentially prevented by isolation of tissues during the dormant season. The ultimate effect of isolation of the bark on the cambium, either during the dormant season or during the growing season, was subdivision of all fusiform cambial cells into strands of parenchymatous elements; the ultimate effect on the newly formed phloem was early death of the sieve elements. The most conspicuous effect of isolation of the bark after xylem differentiation had begun was the curtailment of secondary wall formation. Shortening of cells of the cambial region was reflected in the length of the vessel members which differentiated from such cells. These results indicate that normal cambial activity and xylem and phloem development require a supply of currently translocated regulatory substances from the shoots.     

Alcohol dehydrogenase and ethanol in the stems of trees : evidence for anaerobic metabolism in the vascular cambium

Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out.     

Stem anatomy and development of inter- and intraxylary phloem in Leptadenia pyrotechnica (Forssk.) Decne. (Asclepiadaceae)

Modification of external morphology and internal structure of plants is a key feature of their successful survival in extreme habitats. They adapt to arid habitats not only by modifying their leaves, but also show several modifications in their conducting system. Therefore, the present study is aimed to investigate the pattern of secondary growth in Leptadenia pyrotechnica (Forssk.) Decne., (Asclepiadaceae), one such species growing in Kachchh district, an arid region of Gujarat State. A single ring of vascular cambium, responsible for radial growth, divided bidirectionally and formed the secondary xylem centripetally and the phloem centrifugally. After a short period of secondary xylem differentiation, small arcs of cambium began to form secondary phloem centripetally instead of secondary xylem. After a short duration of such secondary phloem formation, these segments of cambium resumed their normal function to produce secondary xylem internally. Thus, the phloem strands became embedded within the secondary xylem and formed interxylary phloem islands. Such a recurrent behavior of the vascular cambium resulted in the formation of several patches of interxylary phloem islands. In thick stems the earlier formed non-conducting interxylary phloem showed heavy accumulation of callose on the sieve plates followed by their crushing in response to the addition of new sieve elements. Development of intraxylary phloem is also observed from the cells situated on the pith margin. As secondary growth progresses further, small arcs of internal cambium get initiated between the protoxylem and intraxylary phloem. In the secondary xylem, some of the vessels are exceptionally thick-walled, which may be associated with dry habitats in order to protect the vessel from collapsing during the dryer part of the year. The inter- and intraxylary phloem may also be an adaptive feature to prevent the sieve elements to become non-conducting during summer when the temperature is much higher.     

THE TIME COURSE OF SIEVE TUBE AND VESSEL REGENERATION AND THEIR RELATION TO PHLOEM ANASTOMOSES IN MATURE INTERNODES OF COLEUS

Quantitative counts of regenerative sieve tubes and vessels were made in a large number of samples of mature internode #5 of C. blumei, with concomitant study of the fine details of vascular regeneration and the occurrence of the normally developing phloem anastomoses. Such anastomoses were found in many of the plants, but their average number in the small regenerating area was low (viz., 0.9 ± 0.2). With the phloem anastomoses excluded from the counts, the time course of regeneration was clear cut—no strands completed their regeneration around the wound until three days after wounding. More regenerative sieve tubes completed their differentiation under all conditions than did regenerative vessels. The number of sieve tubes and vessels regenerated by four days was closely related to the number of preexisting bundles of that type of vascular cell that had been severed by the transverse wound. The ratio of bundles severed by the wound in the phloem to those in the xylem was 2.14, and the ratio of the regenerative sieve tubes to the regenerative vessels was 2.24. For both tracheary and sieve tube cells the initial regeneration was strongly polar (mostly above the wound), as expected from earlier IAA transport data. The path of tracheary regeneration was obviously related to that of the sieve tubes on the other side of the cambium.     

SITES OF CALLUS PRODUCTION IN PRIMARY EXPLANTS OF SPRUCE TISSUE

When explants for tissue cultures taken from mature spruce trees, consisting only of xylem-cambium-phloem without pith or cortex, are placed on nutrient agar, callus forms first from the cambium, then from phloem parenchyma, and finally from the lining of xylem resin ducts. The proliferating cells of the phloem are the tannin cells which in situ give rise to sclereids in the bark.     

Tissue Differentiation of the Regeneration Rind in Jerusalem artichoke Stem

In trees, after removal of the bark, the vascular tissues of the newly-formed bark usually developed as a continuous layer. However, the stem of the herbaceous Jerusalem artichoke, after girdled, gives rise to regeneration of many irregularly arranged vascular bundles. Early July is the best time for girdling as the vascular bundles are well-developed, One week after girdling, some small groups of vascular tissues appeared in callus. Later on the vascular bundles eventually grew close together sooner or later, yet there were some wide pith rays which separated the various sized vascular bundles and exhibited irregularly contours. From these experiments, it is further evidenced that tile stem of herbaceous plants can also be girdled and regenerates a new rind. Furthermore, the girdled portion of this plant regenerates the vascular tissues which in a rather different way from all the plants that previously studied.     

Anomalous secondary vascular tissue inHelminthostachys zeylanica (Ophioglossaceae)

The vascular anatomy ofHelminthostachys zeylanica was examined with special reference to anomalous secondary tissue. Primary xylem development gradually takes place centrifugally. In branched rhizomes with destroyed apices, the vascular cylinder apical to the insertion of branch traces is generally composed of primary xylem, accessory xylem, inner parenchyma of radially arranged cells, outer parenchyma of irregularly arranged cells, and partly crushed phloem, listed in order going outwards. The accessory xylem as well as the inner parenchyma ofHelminthostachys zeylanica is probably secondarily produced, partly to contribute to the branch traces, in a position corresponding to that of secondary vascular tissue developed from a normal cambium inBotrychium sensu lato. It is suggested that although a cambium is lacking inHelminthostachys zeylanica, the secondary vascular tissues are comparable between the genera. The phylogenetic implication of this tissue is discussed.     

Distribution of an Indoleacetic Acid-oxidase-inhibitor in the Storage Root of Daucus carota

Indoleacetic acid (IAA)-oxidase from both secondary phloem and xylem was dependent on 2,4-dichlorophenol for activity, and was enhanced by addition of Mn²⁺. The pH optimum was 6.0 from both tissues. IAA-oxidase and its inhibitors were distributed differently in the secondary phloem and secondary xylem of carrot root. In the phloem a high IAA-oxidase activity was distributed uniformly along the radius but in the xylem a somewhat lower concentration decreased from the cambium. IAA-oxidase inhibitor in the phloem increased exponentially from a very low concentration near the cambium, whereas in the xylem an appreciable concentration was present near the cambium, decreasing linearly with distance from the cambium. Longitudinal gradients in the xylem parallel studies by other workers with the greatest IAA-destroying capacity present in older tissues. In the xylem inhibitor decreased and IAA-oxidase increased from the root apex. In the phloem IAA-oxidase was uniform, whereas the inhibitor increased in older tissue.

The IAA-oxidase inhibitors in phloem and xylem may be different. In the xylem the IAA-oxidase inhibitor may be a lignin precursor present in young cells which disappears as lignification proceeds. In the phloem IAA-oxidase reacting with endogenous IAA appears to form a physiologically active product.

     

Studies on Regeneration after Girdling of Broussonetia papyrifera (L.) Vent

The trunk of Broussonetia payrifera (L.) Vent. following an extensive length of completegirdling could regenerate new bark in the entire growing season after bud-sprouting. Severaldays after girdling, most of ray cells near the surface dilated and proliferated outward to formcallus. Then, other immature xylem cells rehabilitated the ability of cell division and tookpart in the formation of callus Later, a cork cambium developed near the surface of thecallus and a cambium near the middle part of the callus. The newly formed cambium cannormally produce phloem outward and xylem inward. However, when the girdled trunk waswrapped up with a transparent plastic shee during the growing period from late May to earlyAugust causing high temperature and humidity inside the wrapping sheet, the surface of peridem often produced loose sloughy cell layers that could regain it’s normal structure after unwrapping the sheet.     

Regenerative Xylem in Inflorescence Stems of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

By inserting entomological needles into the lower parts of young inflorescence stems of three-month-old Arabidopsis thaliana (L.) Heynh var. Colombia plants, we studied the process of regenerative xylem production. Regenerative xylem was formed only in one- to two-day-old inflorescence stems but not in older ones. The regenerative vessels originated from re-differentiation of cortical parenchyma. To characterize the process of regenerative xylem formation, we conducted a histological study from the time of wounding to day 30 after wounding. In the first day after wounding the tissues showed no structural responses except for the wounding itself. After six days, regenerative vessel members were already differentiating in a basipetal pattern, forming a vascular bypass around the wound. Regenerative vessel member formation reached a maximal level on the twelfth day after wounding. Sixteen days after wounding the pith parenchyma started to become loose as if indicating tissue senescence. Altogether, vascular regeneration following wounding in inflorescence stems of Arabidopsis thaliana is similar to that in other dicotyledon plants. These findings provide the basis for the use of Arabidopsis thaliana as a model system to study the genetics, physiology and cell biology of wound healing and regenerative vascular tissue formation.     

Visualization of lateral water transport pathways in soybean by a time of flight-secondary ion mass spectrometry cryo-system

Water movement between cells in a plant body is the basic phenomenon of plant solute transport; however, it has not been well documented due to limitations in observational techniques. This paper reports a visualization technique to observe water movement among plant cells in different tissues using a time of flight-secondary ion mass spectrometry (Tof-SIMS) cryo-system. The specific purpose of this study is to examine the route of water supply from xylem to stem tissues. The maximum resolution of Tof-SIMS imaging was 1.8 μm (defined as the three pixel step length), which allowed detection of water movement at the cellular level. Deuterium-labelled water was found in xylem vessels in the stem 2.5 min after the uptake of labelled water by soybean plants. The water moved from the xylem to the phloem, cambium, and cortex tissues within 30-60 min after water absorption. Deuterium ion counts in the phloem complex were slightly higher than those in the cortex and cambium tissue seen in enlarged images of stem cell tissue during high transpiration. However, deuterium ion counts in the phloem were lower than those in the cambium at night with no evaporative demand. These results indicate that the stem tissues do not receive water directly from the xylem, but rather from the phloem, during high evaporative demand. In contrast, xylem water would be directly supplied to the growing sink during the night without evaporative demand.     

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.     

Characterization of the plasma membrane proteins and receptor-like kinases associated with secondary vascular differentiation in poplar

The constituents of plasma membrane proteins, particularly the integral membrane proteins, are closely associated with the differentiation of plant cells. Secondary vascular differentiation, which gives rise to the increase in plant stem diameter, is the key process by which the volume of the plant body grows. However, little is known about the plasma membrane proteins that specifically function in the vascular differentiation process. Proteomic analysis of the membrane proteins in poplar differentiating secondary vascular tissues led to the identification 226 integral proteins in differentiating xylem and phloem tissues. A majority of the integral proteins identified were receptors (55 proteins), transporters (34 proteins), cell wall formation related (27 proteins) or intracellular trafficking (17 proteins) proteins. Gene expression analysis in developing vascular cells further demonstrated that cambium differentiation involves the expression of a group of receptor kinases which mediate an array of signaling pathways during secondary vascular differentiation. This paper provides an outline of the protein composition of the plasma membrane in differentiating secondary vascular tissues and sheds light on the role of receptor kinases during secondary vascular development.