Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

Isolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.     

Cellular Modelling of Secondary Radial Growth in Conifer Trees: Application to Pinus Radiata (D. Don)

The radial growth of conifer trees proceeds from the dynamics of a merismatic tissue called vascular cambium or cambium. Cambium is a thin layer of active proliferating cells. The purpose of this paper was to model the main characteristics of cambial activity and its consecutive radial growth. Cell growth is under the control of the auxin hormone indole-3-acetic. The model is composed of a discrete part, which accounts for cellular proliferation, and a continuous part involving the transport of auxin. Cambium is modeled in a two-dimensional cross-section by a cellular automaton that describes the set of all its constitutive cells. Proliferation is defined as growth and division of cambial cells under neighbouring constraints, which can eliminate some cells from the cambium. The cell-growth rate is determined from auxin concentration, calculated with the continuous model. We studied the integration of each elementary cambial cell activity into the global coherent movement of macroscopic morphogenesis. Cases of normal and abnormal growth of Pinus radiata (D. Don) are modelled. Abnormal growth includes deformed trees where gravity influences auxin transport, producing heterogeneous radial growth. Cross-sectional microscopic views are also provided to validate the model's hypothesis and results.     

Hormonal control of cambial activity and vessel differentiation in Quercus robur

Cambial activity and vessel differentiation of the Quercus robur stem were investigated in relation to concentration of growth regulators and sucrose, seasonal changes in the sensitivity of cambial cells, and axial polarity of the stem. Basipetal efflux of natural auxin was measured in the oak stem cambial region. IAA, GA₃, kinetin and sucrose affected cambial activity and/or initiation of vessel differentiation differently, depending upon concentration. Depending upon the season, kinetin increased or reduced the stimulation of cambial activity caused by IAA and GA₃, but it did not affect the differentiation of vessels. Supply of sucrose in higher concentrations reduced the number of differentiated vessels but did not decrease the stimulation of cambial divisions.Unlike stimulation of cambial activity by GA₃, auxin stimulation of cambial divisions and differentiation of vessels were highly dependent upon stem polarity, 2,3,5-triiodobenzoic acid (TIBA) inhibited formation of vessels, but not cambial activity. The oscillations in basipetal efflux of natural auxin from the cambial stem region of successive 6 mm long sections substantiate the hypothesis that the histogenesis of xylem tissue in ring-porous species is under control of the vectoriat field that is associated with oscillatory phenomena in polar auxin transport.     

A Radial Concentration Gradient of Indole-3-Acetic Acid Is Related to Secondary Xylem Development in Hybrid Aspen

The radial distribution pattern of indole-3-acetic acid (IAA) was determined across the developing tissues of the cambial region in the stem of hybrid aspen (Populus tremula L. x Populus tremuloides Michx). IAA content was measured in consecutive tangential cryo-sections using a microscale mass spectrometry technique. Analysis was performed with wild-type and transgenic trees with an ectopic expression of Agrobacterium tumefaciens IAA-biosynthetic genes. In all tested trees IAA was distributed as a steep concentration gradient across the developing tissues of the cambial region. The peak level of IAA was within the cambial zone, where cell division takes place. Low levels were reached in the region where secondary wall formation was initiated. The transgenic trees displayed a lower peak level and a wider radial gradient of IAA compared with the wild type. This alteration was related to a lower rate of cambial cell division and a longer duration of xylem cell expansion in the transgenic trees, resulting in a decreased xylem production and a larger fiber lumen area. The results indicate that IAA has a role in regulating not only the rate of physiological processes such as cell division, but also the duration of developmental processes such as xylem fiber expansion, suggesting that IAA functions as a morphogen, conveying positional information during xylem development.     

Indole-3-acetic acid level in wood,bark and cambial sap of apple rootstocks differing in growth vigour

Free and conjugated IAA levels were determined in wood, bark and cambial sap of M.9, M.26 and MM.106 apple rootstock genotypes differing in growth vigour. The measurements were done on May 15^th, June 15^th and July 15^th. The level of free IAA in bark and wood of the tested trees varied from 27.0 to 52.7 ng·g⁻¹ f.w. while the conjugated hormone content averaged 3–5 times higher. In the bark and wood samples, the differences in auxin content between rootstock genotypes and the time of stem harvesting were insignificant. The level of free IAA in cambial sap was on average 10 to 20 times higher than in both bark and wood tissues, while the conjugated hormone level varied from none (below detection limit) to 37 ng·g⁻¹ f.w. Content of free IAA level in cambial sap from dwarf M.9 rootstock was significantly lower than that in either of the more vigorous genotypes. In both vigorous rootstocks IAA level in cambial sap remained at a similar level at all sampling dates but M.9 cambial sap showed a trend towards decreasing auxin content later in the growing season.     

Hormonal Modulation of the Oscillatory System Involved in Polar Transport of Auxin

The amount of natural auxin collected in agar as a result of basipetal efflux from the cambial region of successive short sections of pine stem varies so that a wave-like pattern is formed. The wave-length is several times longer than the cell length in the cambial region, suggesting the existence of a supracellular oscillatory system, which forms a morphogenic field in the stem tissues, The amplitude of the auxin wave is amplified by apical application of IAA to the longer stem sections, particularly at she time of spring initiation of cambial activity. The wave of auxin disappears after simultaneous apical application of IAA and ABA. The modulatory effects of IAA and ABA are translocated along the investigated stem sections faster than known transport velocities of IAA molecules. This fact is considered as evidence of apical control of the morphogenic field by way of influence upon a supracellular system of conjugated oscillators in the tissue.     

Developmental changes in the secondary xylem ofAcer rubrum induced by gibberellic acid,various auxins and 2,3,5-tri-iodobenzoic acid

Summary The effect of GA on the development of the secondary xylem ofAcer rubrum was studied. GA was applied to seedlings (1) separately, (2) simultaneously with TIBA, and (3) simultaneously with TIBA and an auxin. GA alone is without effect on the development of the secondary xylem. In seedlings treated with TIBA-GA or TIBA-GA-auxin, the differentiation of the xylem elements is in general similar to that of seedlings treated with TIBA or TIBA-auxin respectively. GA however, does stimulate cambial activity in a localized region of the stem if the endogenous rate of cambial division is low.The following abbreviations will be used TIBA (2,3,5-tri-iodobenzoic acid) - IAA (indole-3-acetic acid) - GA (gibberellic acid) - NAA (naphthaleneacetic acid) - 2,4-D (2,4-dichlorophenoxyacetic acid) This material was included in a doctoral thesis submitted by P. R.Morey to the graduate school of Yale University, New Haven.     

Effect of indole-3-acetic acid (IAA) and sucrose on vessel size and density in isolated stem segments of oak (Quercus robur)

The effects of several concentrations of indole-3-acetic acid (IAA) and sucrose on xylogenic cambial activity and secondary xylem differentiation were investigated in isolated stem segments of Quercus robur L. supplied with liquid medium in aseptic conditions. After 5 weeks of culture auxin controlled cambial cell division and the number and size of vessel elements even without sugar in the medium. Sucrose modified these IAA effects, although little cambial activity occurred without auxin. The xylem increment correlated with changes of auxin concentration with the optimum at 28.5 μ M IAA. The formation of wide vessels was correlated with the optimal concentration of auxin. The frequency of vessel differentiation increased with auxin concentration. High concentrations of sucrose (0.24 M and 0.96 M ) reduced both the number of vessels and their diameter. The frequency of vessel formation was inhibited more than the vessel size by changes of sugar concentration. The vessels formed under low concentrations of IAA were circular in transverse section. With increase in IAA concentration the shape of the vessel cross-section changed to oval with the largest dimension in the radial direction.     

Seasonal Changes in the Effects of Auxin on Rooting in Stem Cuttings of Ficus infectoria

Ficus infectoria stem cuttings were treated with 10 and 100 μg/ml each of IAA, IBA, 2,4, -D and NAA at monthly intervals and planted to study their rooting response after recording morphophysiological status and cambial activty of the parent branches. Attempts were also made to surgically expose the cambium before auxin treatment to determine the relationship of seasonal variation in auxin effectivity to cambial activity. The results show that: (1) there are two distinct phases in the sensitivity of Ficus infectoria stem cuttings to auxin-induced rooting; (2) the high rooting phase coincides with renovation of growth and high cambial activity starting in March and lasting through August and the low rooting phase coincides with winter dormancy and low cambial activity; (3) roots emerge in longitudinal rows in slitted auxin-treated cuttings; (4) slitted auxin-treated cuttings root profusely in June when cambial activity is high but not in October when cambial activity is low suggesting a close correspondence of seasonal variation between the rooting activity of auxin and cambial activity.     

Distribution of Indole-3-Acetic Acid and the Occurrence of Its Alkali-Labile Conjugates in the Extraxylary Region of Pinus sylvestris Stems

Free and conjugated indole-3-acetic acid (IAA) were measured by quantitative gas chromatography-selected ion monitoringmass spectrometry in the extraxylary region of the stem of large Pinus sylvestris (L.) trees during the annual cycle of cambial activity and dormancy. The extraxylary region at the stem top and bottom was divided into 3 and 4 fractions, respectively, for the free IAA measurements, while the entire extraxylary region was extracted when the IAA-conjugates were analyzed. The effect on the distribution pattern of expressing IAA level as a concentration (per gram fresh weight or dry weight) and as total amount (per square centimeter) was examined. The IAA level was much higher in the cambial region than in the fractions that contained the nonfunctional phloem and the periderm. The largest IAA concentration occurred in the fraction that included the cambium, whereas the total amount of IAA was greatest in the phloemcontaining fraction. The significance of the nonuniform radial distribution of IAA for estimating the IAA concentration in the cambial region is discussed in relation to how the cambial region is sampled. A slight Iongitudinal gradient in IAA concentration, decreasing from the top to the bottom of the stem, was observed in the cambial region when the cambium was in the grand period of activity, but not at the end of the cambial growing period. In all fractions, the total amount of IAA was highest when the cambium was active. However, the IAA concentration in the cambial region did not follow the same pattern, actually being lowest during the tracheid production period at the stem bottom. IAA conjugates were detected on all sampling dates except June 23, but their concentrations were always less than 14% of that of free IAA, and their occurrence did not obviously vary during the year. In general, there was a higher concentration of ester conjugates than of amide conjugates, and the ester conjugates were more abundant at the top of the stem than at the bottom.     

The Role of Auxins in Root-Knot Nematode-Induced Growth on Excised Tobacco Stem Segments

Root-knot nematodes, Meloidogyne incognita, induced lumps of callus tissue on the cambial surfaces of peeled tobacco stem segments cultured in vitro. Except for a layer 1 to 3 cells thick, callus was limited to the basal ends of control segments. Indole-3-acetic acid (IAA) applied in agar blocks to the centers of stem segments, when it had any effect on the cambial surface, induced streaks of callus extending from the blocks toward the basal ends of the segments. IAA in agar blocks also increased callus growth at the basal ends of the segments, increased the growth of pith on the undersides of the segments, promoted root initiation, but inhibited bud initiation. Nematodes produced none of these effects, nor did they change the type of organs induced by various concentrations of IAA in the medium. Callus tissue did grow on the cambial surface of stem segments surrounding agar blocks containing 2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport. Paraffin sections showed that the nematodes were confined to the callus tissue on the cambial surfaces of the segments. Except for occasional syncytia and areas of cell division, nematode-induced callus was composed of thin-walled, irregularly shaped cells arising from the cambium. Differences between the responses of tobacco stem segments to root-knot nematodes and IAA-agar blocks indicate that auxins were not freed from the plant tissue nor secreted by the nematodes. Instead, it is suggested that nematodes enabled the tissue to retain and use endogenous auxins that otherwise would have been transported to the basal ends of the segments.     

The role of auxin stored in scots pine trunk during spring activation of cambial activity

In a 9-year-old pine girdled during the winter cambial activity was observed below the girdle in the next spring. This indicates that cambial activity was initiated without auxin produced in the spring by buds. The auxin produced in apical shoots successively flows down the stem, where as a result of periodic restriction in transport it remains over the winter till the next year. This auxin of apical origin but locally stored over the winter in the stem is responsible for the activation of cambium before the new flow of auxin produced in the apical meristems arrives. Calculations based on seasonal changes in auxin levels can explain both, earlier spring activation of cambium in the crown and the temporary cambial divisions below the girdle, without assumption of direct auxin synthesis in the lateral meristems.     

Participation of auxin and abscisic acid in the regulation of seasonal variations in cambial activity and xylogenesis

Summary The current notion that hormonal level and cell response are clearly correlated has often been challenged recently. During the period of cambial activity, auxin content seems to control the intensity of mitosis and some features of the resulting wood, but not the duration of the active period itself. During cambial rest, the indole-3-acetic acid (IAA) level often remains high in the cambium, but the cell sensitivity to auxin is low. The decrease of auxin transport in autumn is sometimes interpreted as a major qualitative change affecting the pattern of transport, and sometimes as a secondary change occurring later than rest onset. The causes of the seasonal variation of cambial response remain unknown. A hypothesis is proposed that accounts for the structural-functional changes occurring in cambial cells during the onset of dormancy. Abscisic acid (ABA) may reduce wood production and xylem cell enlargement in late summer. An important amount of ABA may be present in the cambial zone in autumn after drought stress and in spring in the young growing shoot. Changes in ABA level do not appear to be clearly correlated with the different steps of cambial rest and activity. Beyond the role of ABA as a stress mediator, its participation in the annual regulation of cambial activity remains unclear. Its distribution in the most alkaline compartments may account for the particularities of its seasonal activity. The involvement of IAA and ABA in cambial growth is discussed within the scope of a possible annual alternation of two different metabolisms in the cambial cell.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA gibberellic acid - GC-MS gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PA phaseic acid - RNA ribonucleic acid - SICM single ion current monitoring - SIM selected ion monitoring     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Fluctuations in ribosomal RNA gene content and nucleolar activity in the cambial region of Abies balsamea (Pinaceae) shoots during reactivation

Tissue was collected from the vascular cambial region of 1-year-old balsam fir shoots over an 11-week period during which cambial reactivation occurred. The amount of rDNA (ribosomal RNA genes) relative to total genomic DNA was determined by quantitative slot blots for three trees, one of which showed a 3-week delay in reactivation. In addition, nucleolar activity was estimated by measuring nucleolar volume, number, and staining intensity. Relative rRNA gene content increased transiently prior to the onset of cambial cell periclinal division. Nucleolar volume also increased transiently, but 1–2 weeks prior to the maximal relative rDNA value. The increases in relative rDNA and nucleolar activity were delayed in the tree in which reactivation was late. We interpret these changes as reflecting the amplification and loss of genes encoding rRNA to facilitate cambial cell reactivation.     

Level of endogenous indole-3-acetic acid in the stem of Pinus sylvestris in relation to the seasonal variation of cambial activity

Abstract. Gas chromatography – selected ion monitoring – mass spectrometry was used to measure the level of indole-3-acetic acid (IAA) in the cambial region at the top and bottom of the branchless portion of the main stem of three large Scots pine trees, at weekly intervals from 28 April to 13 July. During this period, the cambium reactivated from the dormant state and entered its 'grand' period of xylem and phloem production, which was monitored by microscopy. The total amount of IAA (ng cm⁻²) increased steadily from 28 April until late June, and thereafter remained constant. In contrast, the concentration of IAA (ng g⁻¹ fresh weight) was high at the start of cambial reactivation, declined when the number of differentiating tracheids began to increase, and then rose as the number of cells decreased. The timing and magnitude of the changes in xylem and phloem production and in IAA level were similar at the two sampling positions. It is concluded that the seasonal changes in cambial activity in the conifer stem cannot be ascribed simply to a fluctuation in the level of endogenous IAA in the cambial region.     

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22–26 °C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees. Received: 25 May 2000 / Accepted: 12 July 2000     

INFLUENCE OF PHLOEM BLOCKAGE ON CAMBIAL GROWTH OF SUGAR MAPLE

Circular patches of bark were surgically isolated on the sides of sugar maple (Acer saccharum Marsh.) 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 sites cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) prevented initiation of cambial activity and xylem and phloem development in isolated areas of half of the trees. Varying degrees of cambial activity (periclinal divisions) occurred in the remaining isolated areas, but normal cambial activity and xylem and phloem development were prevented. Isolation of bark after initiation of cambial activity and phloem differentiation, but prior to initiation of xylem differentiation, resulted in the formation of very narrow xylem and phloem increments with atypically short vessel members and sieve-tube members, respectively. The xylem increments consisted primarily of parenchyma cells. Isolation of bark after initiation of xylem differentiation resulted in curtailment of secondary wall formation in the last-formed part of many increments. The last-formed vessel members of all these xylem increments were atypically short. Similarly, the last formed sieve-tube members of corresponding phloem increments were atypically short. The atypically short cells in the xylem and phloem of isolated areas reflected the effect of isolation on the cambial region, viz., the subdivision of all fusiform cells into strands of cells. Ultimately, the strands of short fusiform cells lapsed into maturity, leaving only strands of parenchymatous elements between xylem and phloem.     

The regulation of cambial division and secondary xylem differentiation in xanthium by auxins and gibberellin

Cambial division continued in decapitated Xanthium plants without concomitant xylem fiber differentiation. The application of indoleacetic acid to these plants did not affect the production of cambial derivatives or induce xylem fiber differentiation. When naphthaleneacetic acid was applied either to the second internode or to the stump of a lateral shoot, xylem fiber differentiation was induced in the newly formed cambial derivatives on the xylem side of the cambium in the stem. When naphthaleneacetic acid was applied unilaterally, xylem fiber differentiation was restricted to that side of the stem in the first internode and hypocotyl. Naphthaleneacetic acid also enhanced the production of cambial derivatives. Gibberellic acid enhanced cambial derivative production but did not affect the differentiation of xylem fibers. Similar numbers of cambial derivatives were produced in some naphthaleneacetic acid-treated plants in which xylem fiber differentiation was induced and in gibberellic acid-treated plants which did not differentiate xylem. When naphthaleneacetic acid was applied 72 hours after decapitation, the oldest of the cambial derivatives on the xylem side failed to develop into fibers although younger cells did. These results suggest that auxin has its direct effect on the induction of xylem differentiation rather than the induction of divisions prerequisite to differentiation.     

Induction of cambial reactivation by localized heating in a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata)

BACKGROUND AND AIMS: The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata) was investigated. METHODS: Electric heating tape (20-22 degrees C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy. KEY RESULTS: Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems. CONCLUSIONS: The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.