Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv

The seasonal change of free abscisic acid (ABA) and indole-3-acetic acid (IAA) and their relationship with the cambial activity in Eucommia ulmoides trees were investigated by ABA and IAA immunolocalization using primary polyclonal and rhodamine-red fluorescing secondary antibodies, ABA and IAA quantification using high performance liquid chromatography (HPLC), and systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual distribution patterns. In the active period (AP), both immunolocalization and HPLC detected an abrupt decrease of ABA, reaching its lowest level in the summer. During dormancy, ABA started increasing in the first quiescence (Q1) (autumn), peaked in the rest (winter), and gradually decreased from the onset of the second quiescence (Q2) (the end of winter). IAA showed a reverse pattern to that of ABA: it sharply increased in AP, but noticeably decreased from the commencement of Q1. Longitudinally, the ABA distribution increased apico-basally, contrasting with IAA. Laterally, most of the ABA was located in mature vascular tissues, whereas the IAA essentially occurred in the cambial region. The concomitant IAA-ABA distribution and seasonal changes in vascular tissues greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation. Interestingly, the application of exogenous ABA to quiescent E. ulmoides branches, in a water-culture system, inhibited external IAA action on cambial activity reactivation. These results suggest that, in E. ulmoides, ABA and IAA might probably interact in the cambial region. The annual cambial activity could be influenced by an IAA:ABA ratio; and ABA might play a key role in vascular cambium dormancy in higher plants. The relationship between hormonal changes and the (particular) annual life cycle of E. ulmoides is also discussed.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

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     

Patterns of protein synthesis in the cambial region of Scots pine shoots during reactivation

Changes in protein synthesis in cambial region cells were monitored in 1-year-old cuttings of Scots pine ( Pinus sylvestris L.) collected in November, when the cambium was dormant, and subjected to environmental conditions that promoted or inhibited cambial growth. The proteins were labelled in vivo with L-[³⁵S]-methionine and separated using 2-dimensional polyacrylamide gel electrophoresis. In budded cuttings cultured under environmental conditions favoring cambial reactivation, there was a reproducible quantitative change in 55 proteins (33 induced and 22 repressed), a less certain increase or decrease in 40 proteins, and no apparent change in about 150 proteins. Under the same conditions, 8 proteins were induced and 6 others were repressed in debudded cuttings treated apically with 1 mg indole-3-acetic acid (IAA) in 1 g lanolin, in which cambial reactivation occurred, compared with debudded cuttings treated with plain lanolin in which the cambium did not reactivate. Three of the proteins induced in the IAA-reated cuttings only appeared after cambial cell division and derivative differentiation actually began, and the same proteins were found in budded cuttings after their cambium had become reactivated. In contrast, protein expression in cuttings exposed to environmental conditions that prevented cambial reactivation was similar at the beginning and end of the experimental period. These results indicate that the cambium was in the quiescence stage of dormancy at the start of the experiment, that quiescent cambial region cells can synthesize proteins as soon as exposed to environmental conditions favoring reactivation, and that only 3 of the approximately 250 proteins detected were specifically involved in cambial growth     

7. The role of plant growth regulators in forest tree cambial growth

The regulation of cell-division activity in the vascular cambium and of secondary xylem and phloem development is reviewed for temperate-zone tree species in relation to auxins, gibberellins, abscisic acid, cytokinins, and ethylene. Representatives of the first four of these PGR classes (IAA, GA₁, GA₄, GA₇, GA₉, GA₂₀, ABA, Z, ZR, DCA) have been identified conclusively by mass spectrometry in the cambial region in some Pinaceae, but not in any hardwood species. Endogenous ethylene has yet to be definitively characterized in this region in any species. Evidence concerning the source and metabolism of cambial PGRs is scanty and inconclusive for both conifers and hardwoods.Most cambial PGR research has focused on IAA. Much evidence indicates that this PGR is transported primarily in the cambial region at a rate of about 1 cm h^–1, and that the transport is basipetally polar. GC-MS measurements have established that endogenous IAA levels in the cambial region of Pinaceae are highest during earlywood development, and that cambial IAA levels may be considerably lower in hardwoods than in conifers. IAA appears to be involved in the control of cambial growth in conifers and hardwoods in at least three specific ways, viz. maintenance of the elongated form of fusiform cambial cells, promotion of radial expansion in primary walls of cambial derivatives, and regulation of reaction wood formation. In addition, it is well established that exogenous IAA promotes vessel development in hardwoods. In both conifers and hardwoods, exogenous IAA stimulates cambial growth in 1-year-old shoots treated late in the dormant period or after the start of the cambial growing period. However, exogenous IAA has little effect on cambia that are older or are in what is hypothesized to be the resting stage of dormancy. Thus it is uncertain whether IAA is directly involved in the control of cambial growth, or acts indirectly through a process such as hormone-directed transport.It is not yet clear if gibberellins play a role in the control of cambial growth in conifers. However, in hardwoods, there is evidence that they inhibit vessel development and act synergistically with IAA in promoting cambial activity and fiber elongation. In both conifers and hardwoods, foliar sprays of gibberellins increase the accumulation of biomass above-ground, particularly in the main axis, while decreasing it in the roots.There are as yet no definite conclusions to be drawn concerning the involvement of ABA, cytokinins, and ethylene in the regulation of cambial growth in conifers or hardwoods. In conifers, ABA may antagonize the promotory effect of IAA on cambial cell division and tracheid radial expansion under conditions of water stress, but high endogenous ABA levels do not appear to be associated with the formation of latewood or the onset of cambial dormancy. Some evidence suggests that exogenous cytokinins enhance the promotory effect of IAA on cambial growth, particularly ray formation, in both hardwoods and conifers. However, exogenous cytokinins, by themselves, appear to be ineffective. In hardwoods, ethylene-generating compounds satisfy the chilling requirement of the dormant cambium and promote the formation of wood having an apparently greater content of lignin and extractives. Ethylene-generators also affect wood development in conifers and accelerate cambial growth at the application site in both hardwoods and conifers.     

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.     

Distribution and Expression of Auxin Binding Protein 1 (ABP1) in the Thin Cell Layers of Tobacco

Tobacco （ Nicotiana tabacum L.） pedicel in the blooming period was used to study the distribution of auxin binding protein 1 (ABP1) in the tobacco tissue and cell at different time in the culture of thin cell layers (TCL). Using the immunofluorescence marker it was indicated that the main ABP1 was distributed in the epidermis and the 1st and 2nd layers of the subepidermis cells. A little ABP1 was distributed in the cortex. Tobacco ABP1 was induced to express in the protoplast of tobacco pedicel TCL cultured in MS culture medium containing IAA and BA. Expression of ABP1 in the protoplast was stronger in the active period of vegetative bud differentiation. ABP1 expression became weaker in later period of differentiation. The result of SDS-PAGE and Western blotting showed the molecular weight of tobacco ABP1 in TCL was 26 kD.     

烟草薄层培养生长素结合蛋白ABP1的定位和ABP1在细胞分化中的变化

以烟草（Nicotiana tabacum L.)盛花期花梗薄层为材料,研究营养芽分化的不同时期生长素结合蛋白（ABP1)在组织与细胞中的分布变化,免疫荧光标记结果表明,烟草花梗中ABP1主要分布于表皮及亚表皮1－2层细胞内。不同分化期ABP1在烟草花梗薄层原生质体中的表达不同,细胞分化旺盛期ABP1的表达最强,分化后期ABP1的表达有所减弱;Western blotting结果表明,ABP1多克隆抗血清与烟草花梗薄层细胞及分化过程中26kD蛋白有免疫交叉反应。     

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.     

Epicormic Bud Development in Quercus robur L. Studies of Endogenous IAA, ABA, IAA Polar Transport and Water Potential in Cambial Tissues10

Seasonal measurements of IAA,³ made using GC-MS, ⁴ indicatedthat in Q. robur the spring initiation of cambial activity andonset of visible bud outgrowth in the canopy is preceded byan increase in cambial region IAA. The effects of notch-girdlescut into the bole indicated that IAA in the cambial region laterwas present in separate physiological pools, with only the polar-transportedfraction affecting epicormic bud outgrowth. The stage in thespring when the epicormic buds grew out coincided with an increaseboth in cambial region IAA and in the capacity of cambial explantsfor IAA polar transport. Thus the stimulus needed by the epicormicbuds to overcome inhibition by polar-transported IAA appearedto be self-generated. The observed effects of exogenous hormoneson epicormic bud outgrowth from stem explants indicated thatthis stimulus might be cytokinin. The seasonal changes detectedin cambial region ABA³ were consistent with a role for stress-inducedABA in the induction of epicormic bud dormancy after canopydevelopment during the summer. No consistent effects of standthinning on cambial region IAA, ABA, water potentials or watercontents were detected, although polar transport of exogenousIAA by cambial region explants removed in the spring was reducedby thinning. Key words: Epicormic buds, cambium, hormones     

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.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

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 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.     

Modulation of the oscillatory system involved in polar transport of auxin by other phytohormones

A wave-like pattern of the basipetal efflux of natural auxin from the cambial region of a series of consecutive short sections of stems of Larix decidua Mill., Acer pseudoplatanus L. and Picea abies (L.) Karst. has been demonstrated as it was earlier reported for Pinus silvestris L. Apical application of ABA suppressed the IAA-stimulated increase of the auxin-wave amplitude, and zeatin or GA₃ prevented this repression in stem segments of Pinus silvestris . All the exogenously applied substances were highly effective in physiological concentrations. Already 20-min of exposure to IAA or ABA at the apical end produced modulations of the auxin-wave along the whole 6.6 cm long stem segment. Application of 2, 3, 5-triiodobenzoic acid (TIBA) caused suppression of the wave-like pattern of auxin efflux similarly as ABA, supporting the association of the modulatory effects of ABA with the phenomena involved in polar transport of auxin. Abscisic acid applied to the basal end of the stem segment also reduced the auxin-wave amplification caused by simultaneous supply of IAA to the apical end. This finding additionally confirms the hypothesis that: 1) the supracellular auxin-wave generation is associated with the functioning of a system of oscillators coupled at the cellular level and 2) the auxin-wave modulations can be propagated acropetally, that is against the main direction of the auxin molecular transport.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.