hannele tuominen, laurence puech, siegfried fink, and ... · hannele tuominen, laurence puech,...

Plant Physiol. ( 1 997) 1 1 5 : 577-585

A Radial Concentration Gradient of Indole-3-Acetic Acid 1s Related to Secondary Xylem Development in Hybrid Aspen'

Hannele Tuominen, Laurence Puech, Siegfried Fink, and Bjorn Sundberg* Department of Forest Genetics and Plant Physiology, Swedish University of Agricultura1 Sciences, 901 83 Ume3,

Sweden (H.T., B.S.); and lnstitut für Forstbotanik und Baumphysiologie, Albert-Ludwigs-Universitat, Bertoldstrasse 17, 79085 Freiburg, Germany (L.P., S.F.)

l h e 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 mas spectrometry technique. Analysis was performed with wild-type and transgenic trees with an ectopic expression of Agrobacterium fumefaciens IAA-biosynthetic genes. In all tested trees IAA was distributed as a steep concentration gradient across the developing tissues of the cambial region. lhe 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. l h e 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.

The vascular cambium is responsible for xylem formation, which constitutes the bulk of secondary growth. De- velopment of xylem elements involves severa1 consecutive phases, including division in the cambial zone, cell expansion, secondary wall formation, and autolysis of xylem elements destined for water transport and mechanical sup- port (Larson, 1994). This development is coordinated in space, resulting in a radial pattern of discrete zones where the different developmental phases take place. The rate of xylem cell production, i.e. the number of divisions in the cambial zone per unit of time, is determined by two com- ponents: (a) the rate of cell cycling of the individual cambial zone cells and @) the number of dividing cells, which is determined by the duration of meristematic capacity for each xylem mother cell. Similarly, the rate and duration of expansion and secondary wall formation can modulate final cell size and wall thickness of the xylem elements.

This work was supported by grants from the Swedish Council for Forestry and Agricultura1 Research, the Swedish Natural Sci- ences Research Council, the Academy of Finland, and the Kempe Foundation.

* Corresponding author; e-mail bjorn.sundberg8genfys.slu.se; fax 46-90-165901.

These aspects are important in forest trees, where they determine critica1 wood properties such as annual ring width and the seasonal pattem of earlywoodllatewood formation (Wilson and Howard, 1968; Gregory, 1971; Wodzicki and Zajaczkowski, 1974; Denne and Wilson, 1977; Dodd and Fox, 1990; Ridoutt and Sands, 1994).

The presence and concentration of IAA is critical for basic cellular processes such as division and expansion (Evans, 1984; Jacobs, 1995). Consequently, its role in controlling different aspects of cambial growth has been in- vestigated in many studies (Roberts et al., 1988; Little and Pharis, 1995; Savidge, 1996). An important observation is that polarly transported IAA maintains cambial activity. This is shown by the finding that remova1 of the IAA source by various treatments decreases the endogenous concentration of IAA in the tissues of the cambial region. This decrease results in an inhibition of cambial activity, which can be restored by exogenous application of auxin (Digby and Wareing, 1966; Little and Wareing, 1981; Sav- idge, 1983; Sundberg and Little, 1990; Rinne et al., 1993; Sundberg et al., 1994). In addition, numerous experiments with exogenous auxin in both hardwoods and conifers have demonstrated the potential of IAA to affect most aspects of cambial growth in a dose-dependent manner, including xylem and phloem production and size and secondary wall thickness of the xylem elements (for review, see Little and Savidge, 1987; Little and Pharis, 1995).

In spite of the well-documented role of IAA in cambial growth, the exact function of endogenous IAA in the regulation of xylem production and structure is still uncertain. IAA is generally considered to control the rate of different processes such as cell division and expansion. IAA may also act as a morphogen, controlling the amount of time each cambial derivative stays in the different developmental phases of cell division, cell expansion, or secondary wall formation. These times are a function of the rate of cell production in the meristem and the radial width of each developmental zone (Wilson and Howard, 1968). By anal- ogy to the positional information theory in animal development (Wolpert, 1996), it is conceivable that the coordi- nation of xylem development, and hence the radial width of the developmental zones, is determined by concentration gradients of morphogens. Recently, a steep radial concentration gradient of IAA was demonstrated across the developing vascular tissues of Pinus sylvestris (Uggla et al., 1996). This observation suggested a role for IAA in posi-

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578 Tuominen et al. Plant Physiol. Vol. 11 5, 1997

tional signaling in cambial growth. This concept does not exclude a further role for IAA in controlling rates of developmental processes through changes in absolute concentration of IAA in specific cell types.

To investigate the role of IAA in cambial growth we studied the effects of altered patterns of endogenous levels of IAA on cambial growth and xylem cell I1-iorphology. Apart from application of auxin, internal IAA balance can be manipulated by transforming plants with bacterial IAA- biosynthetic genes (Klee and Romano, 1994). We previ- ously described transgenic hybrid aspen (Populus tremula L. X Populus tremuloides Michx.) lines expressing the IAA- biosynthetic genes from Agrobacterium tumefaciens T-DNA (Tuominen et al., 1995). These trees had a decreased rate of xylem production and an altered xylem anatomy. Despite these differences, an increased IAA concentration was found only in roots and mature leaves, whereas the IAA concentration in the bulk tissues of the cambial region was similar to that of wild-type plants. In this study we show that hybrid aspen, a hardwood species, displays an IAA concentration gradient across the cambial region similar to that found in P. sylvestris and that the transgenic trees exhibit an aberrant radial IAA gradient, which can be related to altered xylem cell morphology and xylem production.

MATERIALS AND METHODS

Plant Material and Cultivation Conditions

gansformation and regeneration of the transgenic hybrid aspen (Populus tremula L. X Populus tremuloides Michx.) line G1'2'D used in this study were described earlier (Tuominen et al., 1995). This line expresses the bacterial IAA-biosynthetic genes iaaM and iaaH from the mannopine synthase (mas) 2' and 1' promoters (Velten et al., 1984), respectively.

Wild-type and transgenic G1'2'D plants were micro- propagated in sterile culture, potted in fertilized peat, and acclimatized to greenhouse conditions under plastic bags for 2 weeks. The plants were cultivated in a greenhouse under natural light conditions and temperatures above 15"C, during six months from March until the end of August. During this period the plants were fertilized once a week with a 1% dilution of SuperbaS (Supra Hydro, Landskrona, Sweden).

Plant material was collected at the end of August, when the trees had reached a height of 2.5 to 3.5 m and were still actively growing. Stem samples were collected from three positions: 30 cm above the ground (denoted base), at the midpoint of the stem (denoted middle), and 50 to 70 cm from the apical shoot tip (denoted top). For RNA and IAA analysis, the harvested tissues were immediately frozen in liquid N, and stored at -70°C. For microscopy, sub- samples were isolated and stored in a fixative.

Measurement of IAA Content

Frozen samples from the base of the stem were trimmed into blocks of approximately 2 mm (tangentially) X 10 mm

(radially) x 10 mm (vertically), consisting of part of the mature xylem and a11 extraxylary tissues. Tissue-specific samples for IAA quantification were obtained from the trimmed blocks by tangential cryosectioning with a microtome (HM 505 E, Microm Laborgerate, Walldorf, Ger- many) at -20"C, according to the method of Uggla et al. (1996). Briefly, the barkside of the block was placed facing the knife and tangential 30-pm-thick sections were cut through the specimen. To align the block, hand-cut, transverse sections from both ends of the block were obtained with a razor blade and inspected under a microscope. After the block was oriented to give tangential sections parallel to the cambial zone, samples were obtained throughout the tissues of the cambial region of the stem, from the mature phloem to the mature xylem. To determine the tissue type(s) in each tangential section, transverse, hand-cut sections were cut at both ends with a razor blade after every third tangential section.

IAA content in each tangential section was quantified using a recently developed micromethod (Edlund et al., 1995). Briefly, the samples were extracted in 0.05 M phosphate buffer (pH 7.0) for 1 h with 1 ng of [I3C,]IAA (Cam- bridge Isotope Laboratories, Woburn, MA) as an internal standard. Semipurification was done by ion exchange with Amberlite XAD-7 resin (Serva, Heidelberg, Germany) and then extraction was done with dichloromethane. Quantifi- cation was performed by an isotope-dilution technique and GC-selected reaction monitoring-MS using a JMS-SXI SX102A instrument (Jeol).

RNA Gel-Blot Analysis

Total RNA was isolated from different parts of transgenic G1'2'D trees. RNA from root tips (about 1 cm), a developing leaf, a fully expanded young leaf, and an old leaf was extracted with hot phenol buffer and then selec- tively precipitated by 2 M LiCl (Verwoerd et al., 1989). Stem tissues were separated in different fractions. The bark of each stem sample was peeled, and the tissues on the ex- posed xylem and bark sides were carefully scraped with a scalpel. The fraction on the xylem side (denoted xylem) consisted of radially expanding, primary-walled xylem elements in the late stage of expansion and some xylem elements forming a secondary wall. The fraction on the bark side (denoted phloem) consisted of xylem elements in the early stage of expansion, cambial zone cells, and differentiating and mature phloem elements. For the cortex fraction, the very thin cork layer was removed from the surface of the stem, and the outermost tissues, consisting of periderm and cortex, were collected by scraping until the appearance of primary phloem fibers. Total RNA from the xylem and phloem fractions was isolated using an RNeasy plant total RNA kit (Qiagen, Hilden, Germany). Total RNA from the cortex fractions was isolated by cetyltrimethylam- monium bromide extraction buffer and then precipitated by LiCl according to the method of Chang et al. (1993).

Thirty micrograms of the total RNA from each sample was separated in a formaldehyde agarose gel (Sambrook et al., 1989) and blotted onto a Hybond-N membrane (Amer- sham). Equal loading of the different RNA samples on the

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Radial IAA Gradient in Aspen Stem 579

gel was controlled both by ethidium bromide-staining of the gel and by methylene blue-staining of the membrane (Herrin and Schmidt, 1988). The membrane-bound RNA was hybridized according to the method of Church and Gilbert (1984). An interna1 1697-bp BglII fragment of the iaaM gene or a 2093-bp BamHI fragment of the iaaH gene spanning the whole coding sequence was labeled with [CI-~~PI~CTP in a randomly primed reaction to a high specific activity and used as a probe. The probe was removed between the two hybridizations by treatment of the membrane with boiling 0.1% SDS.

Anatomical Characterization

Specimens from the base of the stems, consisting of mature xylem and extraxylary tissues, were fixed in cold 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for at least 1 d. The samples were then dehydrated in an ascend- ing series of acetone and embedded in a methacrylate resin (S. Fink, unpublished data). Transverse and longitudinal sections, 3 and 5 pm thick, were cut with a no. 2065 microtome (Leica, Nussloch, Germany) using a diamond knife. The sections were stained polychromatically with successive incubations in 0.1% acriflavin/ 3% safranin O, 1% auramin O, and 2% methylene blue. Parallel sections were also stained with periodic acid-Schiff reagent, as described by Gerlach (1977). The sections were mounted in Eukitt (Thoma, Freiburg, Germany) and observed under a light microscope (Axiophot, Zeiss).

The widths of different developmental zones and the number and size of fusiform cells in the cambial zone were measured for 10 radial files on one section per tree under the microscope using an ocular measuring scale. The developmental zones measured on transverse sections fn- cluded (a) the cambial zone, consisting of dividing cells; (b) the expansion zone, defined by the presence of primary- walled xylem fibers; and (c) the maturation zone, defined by the presence of fibers depositing secondary walls. The distinction between the different phases was made according to radial dimensions of the cells, thickness of tangential walls, presence of birefringence, and presence or absence of cytoplasm (Skene, 1969; Wodzicki, 1971). Widths of the phloem and the remaining bark (cortex and periderm) were measured on radial sections, where the transition from phloem to cortex was recognized by the disappear- ance of sieve tubes.

The densities of vessels and rays were determined on photomicrographs of transverse sections by measuring the number of vessels and rays that were crossing a circular line drawn in the recently differentiated xylem. The length of the line was approximately 2 mm and the distance from the cambial zone was 400 pm.

The cross-sectional lumen area of fibers and the radial and tangential diameter of vessels and ray cells were measured on transverse sections using a computer-assisted image-analyzing system. These sections were stained with periodic acid-Schiff reagent to intensify the contrast of the cell walls and to reduce staining of the cell contents. Before analysis, fibers and vessels were distinguished by the use of UV fluorescence in combination with periodic acid-

Schiff reagent staining (Puech and Mehne-Jakobs, 1997). Three images of 400 X 400 pm in the recently differentiated xylem on each section (see Fig. 4A) were captured with a 3CCD color video camera (DXC-930P, Sony). The images were improved by removing cell content, restoring broken cell walls, and enhancing contrast and sharpness using Adobe-Photoshop D1-2.5.1 (Adobe Systems, Mountain View, CA). Image analysis was done with Image-Pro Plus 1.1 for Windows (Media Cybernetics, Silver Spring, MD). In the digitalized images the radial and tangential diameter of vessels and ray cells was determined manually, and the lumen area of fibers was measured automatically. For the automatic measurements of the fibers, threshold values were always set to allow the whole cell lumen to be counted as an object. Border objects were excluded automatically and ray cells were removed manually. An upper size limit was set to exclude vessels, and a lower limit was set at 50 pm2 to exclude fiber endings.

To measure fiber length, a 1-cm-long sample was collected from a position in the stem having a xylem diameter of 1 cm. The xylem was chopped into thin sticks and macerated in acidified sodium chlorite solution (Spearin and Isenberg, 1947). The length of 600 fibers in each sample was manually measured with an ocular measuring scale under a microscope (Axioplan, Zeiss) using Nomarski optics.

RESULTS

Concentration and Distribution of IAA across the Tissues of the Cambial Region

The radial distribution of IAA across the cambial region was visualized in three wild-type and three transgenic hybrid aspen trees by measuring the IAA content in a series of tangential30-pm sections (Fig. 1). The series covered the tissues between the recently differentiated phloem fibers and mature xylem. Endogenous IAA content, calculated per square centimeter section, displayed a steep radial gradient across the cambial region in both wild-type and transgenic trees. The peak level was in the cambial zone or at the border between the cambial zone and the expansion zone toward the xylem. In a11 cases, the level of IAA decreased from the peak level more rapidly toward the phloem than toward the xylem. The average reduction in IAA level from the peak value was 85-fold on the phloem side and 32-fold on the xylem side over a 300-pm radial distance. Because the average fresh weight per section area did not vary significantly and the water content varied by less than a factor of 2 across the various tissues of the cambial region (data not shown), it can be concluded that the gradient in IAA content reflects a true concentration gradient.

The shape and the peak level of the radial IAA gradient differed between the wild-type and transgenic lines (Fig. 1). The peak level in the wild type ranged from 1.7 to 3.2 ng per square centimeter section. Using the average fresh weight per section these amounts correspond to an approximate concentration of 592 to 1115 ng 8.' fresh weight. In the transgenic trees the peak level ranged from 1.1 to 1.9 ng

67



580 Tuominen et al. Plant Physiol. Vol. 115, 1997

STEM LEAF Ro

0.6 0.9Radial width (mm)

Figure 1. Radial distribution pattern of IAA across the cambial regionat the base of three wild-type and three transgenic hybrid aspenstems. Each column represents a 30-/im tangential section and itsrelative composition of cell types from different tissues. EndogenousIAA content for each section is indicated with a black dot. The totalamount of IAA in the cambial region per square centimeter of stemarea (i.e. the integrated area under the gradient) is indicated at theupper right for each tree. Ph, Phloem; CZ, cambial zone; EZ, expan-sion zone of the xylem; and MZ, maturation zone and mature xylem.

per square centimeter section, corresponding to a concen-tration of 383 to 662 ng g~ ] fresh weight. In spite of thedifferent peak levels of IAA in the wild-type and transgenictrees, the total amount of IAA per square centimeter ofstem area (i.e. the integrated area under the gradient) wassimilar in both lines (Fig. 1). This was because the radialIAA gradient in all cases was wider in the transgenic trees.

TOP MIDDLE BASEXy Ph Co Xy Ph Co Xy Ph Co Y YF O

Figure 2. RNA gel-blot analysis of the iaaM and iaaH genes at threedifferent positions of the stem, three different leaf types, and root tipsof the transgenic line G1 '2'D. For control of loading of the differentRNA samples, an ethidium bromide-staining of the total RNA on theformaldehyde gel is shown. The approximate sizes of the iaaM andiaaH transcripts are 2.5 and 1.7 kb, respectively. Xy, Radially ex-panding xylem elements in the late stage of expansion and somexylem elements forming secondary wall; Ph, xylem elements in theearly stage of expansion, cambial zone cells, and differentiating andmature phloem elements; Co, cortex and periderm; Y, young, devel-oping leaf; YF, fully expanded young leaf; O, old leaf; and Ro, roottips.

The increase in width was most obvious on the xylem sideof the gradient peak but was also apparent on the phloemside. The similar amounts of total IAA across the cambialregion explains why no differences in IAA concentrationswere found between the lines in our previous study(Tuominen et al., 1995), in which the stem samples con-sisted of all tissues of the cambial region.

2.5

2.0

1.5•a'•£"c3

1.0

0.5

0

Cortex andperiderm

Phloem *

Cambial zone*Expansionzone **

Maturationzone ***

WILD-TYPE G1'2'D

Figure 3. Radial width of different stem tissues measured on trans-verse sections sampled from the base of five wild-type and fivetransgenic G1'2'D trees. The bars indicate SE. The asterisks indicatestatistical significance of the difference between the means of thewild-type and the transgenic trees (Student's f test): *,P s 0.05; **,P £ 0.01; and ***, P s 0.001.https://plantphysiol.orgDownloaded on December 23, 2020. - Published by

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WILD TYPE TRANSGENIC Gl'2'D

Figure 4. Anatomical appearance of wild-type (A, C, E, and C) andtransgenic (B, D, F, and H) hybrid aspen trees in representativetransverse sections sampled from the base of the stem. The position

Whereas the shape of the radial IAA gradient differedbetween the wild-type and transgenic trees, the distribu-tion pattern in relation to different developmental zoneswas similar (Fig. 1). In both lines the gradient covereddividing and expanding tissues, reaching low or undetect-able levels in cells in which secondary wall formation hadbegun.

Expression Pattern of the lAA-Biosynthetic Genes

In the two-step bacterial biosynthesis pathway of IAA,the expression of the first gene, iaaM, is crucial for the levelof IAA production (Klee et al., 1987; Sitbon et al., 1992). Weanalyzed the expression level of both the iaaM and iaaHgenes, which were under the control of the mas 2' and Vpromoters, respectively. These two promoters are knownto be expressed in a coordinate manner, the 2' generallydirecting a higher level of expression than the 1' promoter(Harpster et al., 1988; Langridge et al., 1989). The expres-sion has also been shown to increase in a basipetal mannerand reside mainly in the vascular tissues (Leung et al.,1991; Saito et al., 1991; Ni et al., 1995).

The RNA-blot analysis revealed a much higher expres-sion level of the iaaM gene compared with the iaaH gene,but the expression pattern seemed to be similar for bothgenes (Fig. 2). Both genes showed the highest level ofexpression in the basal parts of the tree, i.e. the old leavesand the roots. This basipetally increasing expression pat-tern was not as obvious in the stem samples, where thehighest gene expression was present in the sample from themiddle of the stem. In each stem sample the gene expres-sion was always strongest in the phloem fraction, lower inthe xylem fraction, and barely detectable in the cortexfraction.

Anatomical Characterization of the Stem Tissues

The secondary tissues of the stem are composed of cellsin various phases of division, expansion, and maturation,as well as mature xylem and phloem. These tissues areembedded in the primary body of the stem, which includesthe cortex, primary phloem and xylem, and pith. Radialwidths of most of these tissues (for definition, see "Mate-rials and Methods") were characterized at the base of thewild-type and transgenic Gl'2'D stems. Compared withthe wild-type trees, the transgenic trees had significantlywider zones of phloem, cambial zone, and expanding xy-lem, but they had a narrower zone of maturing xylem (Figs.3 and 4, A-D). The radial width of the mature xylem was13.4 ± 0.6 mm (average ± SD, n = 5) in the wild type and9.3 ± 2.0 mm (average ± so, n = 5) in the transgenic lines.

of the 0.16-mm2 square that was used for measurement of the fiber,vessel, and ray properties is shown in A. A and B, Different devel-opmental zones of the stem; bar = 200 /xm. C and D, Cambial zoneand cambial derivatives on the xylem side; bar = 50 jum. E to H,Recently differentiated xylem; bar = 250 jum (E and F); bar = 100 /j,m(G and H). Cx, Cortex; Ph, phloem; CZ, cambial zone; EZ expansionzone; MZ, maturation zone and mature xylem; F, fiber; V, vessel; andR, ray. https://plantphysiol.orgDownloaded on December 23, 2020. - Published by

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The morphology of cells in the cambial zone and the recently formed xylem was characterized in more detail in the wild-type and transgenic lines (Table I). The radial diameter of the fusiform cells in the cambial zone was larger in the transgenic line compared with the wild type, but since the width of the cambial zone was also larger, the number of the cambial zone cells remained unchanged. Morphology of the xylem elements differed in severa1 aspects between the transgenic and the wild-type trees (Table I; Fig. 4). The fibers were larger in the cross-sectional lumen area but not longer in the transgenic trees. The ray cells of the transgenic plants were larger in tangential diameter, and the rays were present in a significantly lower density. The vessels showed a tendency toward a smaller tangential diameter and higher density. Additional differences between the two lines were recognized in the pattern of vessel grouping and the structure of rays (Fig. 4, G and H).

DISCUSSION

A steep radial concentration gradient of endogenous IAA across the cambial region of hybrid aspen was visualized by using cryosectioning combined with a microscale GC-MS technique (Fig. 1). The leve1 of IAA was generally highest within the cambial zone. It decreased rapidly both centrifugally and centripetally and approached low or un- detectable levels in the cells where secondary wall formation was initiated. The radial distribution pattern of IAA in hybrid aspen, an angiosperm tree, is thus similar to the one recently demonstrated in a gymnosperm, P. sylvestvis (Ug- gla et al., 1996). This indicates a general occurrence of a

radial IAA gradient across the developing secondary vascular tissues in plants.

Concentration gradients of morphogens have been demonstrated to convey positional information in various animal systems (Gurdon et al., 1995; Cohn and Tickle, 1996; Wolpert, 1996). This means that cell fate is determined by its position in a morphogenetic field and that gradients of different factors coordinate complex developmental processes. Analogous to this, it has been proposed that developmental patterns in primary and secondary meristems of plants are controlled by concentration gradients of regula- tory factors conveying positional information (Warren Wil- son and Warren Wilson, 1984; Callos and Medford, 1994; Hake et al., 1995). To our knowledge, the radial concentration gradient of IAA across the cambial region is the first evidence for the existence of morphogenetic fields in plants, suggesting a role for IAA in positional signaling. In line with this idea, the radial IAA gradient was related to two developmental phases of cambial growth in hybrid aspen. First, the outer end of the IAA gradient was always associated with the transition from the expansion to the maturation zone. IAA stimulates cell expansion in many experimental systems and tissues used for studies of plant growth (Little and Savidge, 1987; Kutshera, 1994); there- fore, it can be suggested that significant IAA concentrations maintain cell expansion and thus determine the width of the xylem expansion zone. Second, the center of the IAA gradient seemed to define the cell division zone, which supports the role of IAA in maintaining the meristematic state.of the cambial zone cells. However, the position of the cell division zone was not associated with a particular IAA

Table 1. Morphological properties of developing and mature xylem elements in wild-type and transgenic G 1’2‘ D hybrid aspen

All measurements except fiber length were done on transverse sections sampled from the base of the tree. The radial and tangential diameter of vessels and ray cells and the lumen area of fibers were measured in three images (0.16 mm’, Fig. 4A) per section, captured from the recently differentiated xylem. The density of vessels and rays was determined by measuring the number of vessels and rays crossing a circular line in the recently differentiated xylem at a distance of 400 p m from the cambial zone. The fusiform cells in the cambial zone were characterized in 10 randomly selected rows per section. The length of the fibers was measured manually from a macerated xylem sample from a position in a tree having a xylem diameter of 1 cm. For each property, a mean value was calculated for each tree. The results represent the means from five wild-type (WT) and five transgenic trees. P values were determined bv Student’s t test.

Cell Type WT C1’2’D P

Fibers Lumen area (pm‘) Length (pm)

Diameter, radial (pm) Diameter, tangential (pm) Density (no./mm)

Cell diameter, radial (pm) Cell diameter, tangential (pm) Density (no./“)

Cambial zone cells No. per radial row Diameter, radial (wm)

Vessels

Rays

133 460

44.6 34.9

7.2

77.1 5.5

14

9.9 5.4

178 454

44.6 30.2

9.7

60.2 7.0

11

10.6 6.6

0.043 0.601

0.988 0.228 0.1 38

0.145 0.081 0.008

0.337 0.002




concentration along the gradient, and thus the spatial control of the cell division zone cannot be determined by IAA alone.

The radial IAA gradient is most likely created by lateral transport / diffusion of polarly transported IAA (Uggla et al., 1996). The transgenic hybrid aspen trees expressing the IAA-biosynthetic genes displayed a wider radial gradient of IAA but a lower peak level in the cambial zone compared with the wild type (Fig. 1). We suggest that the wider IAA gradient of the transgenic trees was related to the expression of the IAA-biosynthetic genes, even though the pattern of gene expression did not perfectly correlate with the alterations in the IAA gradient. The expression of the genes was always highest in the fraction of the stem consisting of the phloem and tissues of the cambial zone, but the width of the IAA gradient seemed to be mostly affected on the xylem side. This discrepancy, however, may have resulted from minor differences in posttranscriptional gene regulation, Trp availability, IAA metabolism, or IAA transport between different tissue types. In addition, interpre- tation of the results is confounded by the fact that it is impossible to differentiate between the IAA originating from the native genes and the IAA originating from the inserted genes. The lower peak level of IAA in the transgenic trees could have various causes. Although ectopic expression of the IAA-biosynthetic genes did not significantly affect the pool of conjugated IAA in cambial region tissues (Tuominen et al., 1995), it may affect IAA biosynthesis from the native genes (Ribnicky et al., 1996). Also, the reduced growth vigor of the transgenic trees will result in a decreased supply of native IAA through the polar transport system.

The altered concentration and distribution pattern of IAA in the transgenic trees was associated with differences in xylem formation (Table I; Fig. 4). First, the transgenic trees had a wider zone of cambial cell division but an equal number of cambial zone cells and a lower rate of xylem production. This indicates a lower rate of cell division in each cambial zone cell. Second, the transgenic plants ex- hibited a larger cross-sectional lumen area of the xylem fibers and a wider zone of xylem fiber expansion, indicat- ing an increased duration of fiber expansion. These alterations in the transgenic trees, i.e. the lower rate of cambial cell division and the increased duration of fiber expansion, were related to a lower peak level of IAA in the cambial zone and a wider radial IAA gradient, respectively. The results suggest that IAA can affect both the rate of developmental processes through changes in concentration in specific cells and the duration of the developmental phases through changes in the gradient width, thus acting as a positional signal. Other investigators (Wodzicki and Zajac- zkowski, 1974; Denne and Wilson, 1977; Porandowski et al., 1982) have demonstrated that applied auxin can affect both the rate and duration of xylem cell development, although the results are somewhat contradictory.

It is interesting that the larger cross-sectional xylem fiber area was not accompanied by an increase in the diameter of xylem vessels but rather a reduction in vessel diameter (Table I). IAA has been demonstrated to increase the lumen area of both vessels and fibers when applied to stem seg-

ments of Fraxinus excelsior (Doley and Leyton, 1968). Our observation that developing vessels and fibers had a different response to the altered IAA balance strongly suggests that the physiological regulation of expansion of the two types of xylem elements, fibers and vessels, is uncou- pled. This is also supported by the fact that secondary wall formation of vessels is initiated before that of fibers. (Doley and Leyton, 1968; Ridoutt and Sands, 1994). Experiments with exogenous IAA in conifers have also implicated a role for IAA in the control of secondary wall thickening of xylem elements (Jenkins, 1974; Wodzicki and Zajacz- kowski, 1974; Denne and Wilson, 1977; Sheriff, 1983). From the radial distribution pattern of IAA in hybrid aspen and P. sylvestris (Uggla et al., 1996), it is evident that the IAA concentration in this developmental phase is low. This observation suggests the importance of factors other than IAA in the control of secondary wall formation (Savidge, 1994). Other alterations in the transgenic plants, such as altered ray density, vessel patterning, and xylem-to- phloem ratio, cannot easily be explained by the altered concentration and distribution pattern of endogenous IAA, as visualized in this study.

Concentration gradients of IAA along the stem have been suggested to explain the axial variation in cambial growth and xylem structure (Larson, 1962; Aloni and Zim- mermann, 1983). However, measurements of endogenous IAA in the cambial region tissues have not convincingly supported this idea. In fact, no consistent correlation has been found between endogenous IAA concentration and any aspect of cambial growth (Little and Pharis, 1995). From our demonstration that endogenous IAA is distributed as a steep radial concentration gradient across cambial region tissues, it is evident that IAA measurements of bulk cambial region tissues are of limited value for evaluating the role of IAA in the control of specific phases of xylem development. It is also clear that models explaining the role of IAA in cambial growth must consider not only putative longitudinal gradients but also radial concentration gradients, together with the possible involvement of IAA in controlling both the rate and duration of the different developmental phases. Such thinking is important for un- derstanding IAA perception and signal transduction path- ways.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Karin Waldmann and Mr. Kjell Olofsson for technical assistance and Drs. Sharon Regan and Patrick von Aderkas for valuable comments and critica1 reading of the manuscript.

Received May 19, 1997; accepted June 10, 1997. Copyright Clearance Center: 0032-0889/ 97/ 115/0577/09.

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