EVIDENCE for AUXIN REGULATION of BORDERED-PIT POSITIONING DURING TRACHEID DIFFERENTIATION in LARIX LARICINA By

IAWA Journal, Vol. 16 (3),1995: 289-297

EVIDENCE FOR AUXIN REGULATION OF BORDERED-PIT POSITIONING DURING TRACHEID DIFFERENTIATION IN LARIX LARICINA by

Mathew Adam Leitch & Rodney Arthur Savidge 1 Faculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, New Brunswick, E3B 6C2, Canada

SUMMARY

Chips containing cambium intact between xylem and phloem were cut from 8-year-old stem regions of 20-30-year-old dormant Larix laricina in late spring and cultured under controlled conditions for six weeks on a defined medium containing varied concentrations of I-naphthalene acetic acid (NAA, a synthetic auxin). Microscopy revealed that auxin was essential for cambial growth and tracheid differentiation. Low (0.1 mg/l) and high (10.0 mg/l) auxin concentrations were conducive to bordered pits forming in tangential walls, whereas an intermediate concentration (1.0 mg/l) of NAA favoured positioning of pits in radial walls.

INTRODUCTION

Two decades ago, Barnett and Harris (1975) pointed out that the processes involved in bordered-pit formation were incompletely understood, in particular the way in which the pit site is determined and how adjoining cells form a perfectly symmetrical bor• dered-pit pair. Observing that radial walls of enlarging cambial derivatives were vari• ably thick and thin, Barnett and Harris (1975) advanced the hypothesis that bordered• pit development occurs at sites where the primary wall is thinned through the process of centrifugal displacement of microfibrils. Others, however, considered that a thick• ening rather than a thinning of the primary wall was indicative of the site where pit development commenced (FengeI1972; Parham & Baird 1973). It has long been known that the pit border begins to develop before general secondary wall formation occurs (Sachs 1882), but whether primary wall thinning or thickening is associated with bor• dered-pit development remains to be conclusively demonstrated, and the factor(s) con• trolling bordered-pit development still remain unknown (Savidge 1985). In most conifer species, bordered pits are produced exclusively within radial walls of developing earlywood tracheids. On the other hand, experimental induction of earlywood formation in young stem cuttings of Pinus spp., by application of auxin, revealed that positioning of bordered pits in the radial walls was not an absolute

1) Author to whom correspondence should be addressed; E-mail: [email protected].

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Downloaded from Brill.com09/30/2021 07:43:40PM via free access Leitch & Savidge - Auxin regulation of pit positioning 291 requirement (Savidge & Wareing 1981; Savidge 1983). Similarly, when earlywood was induced in 'chip' cultures from stems of Pinus contorta and Larix laricina, bor• dered pits were common in both radial and tangential walls (Savidge 1983, 1993). Auxin was essential for induction of wood formation in the chip cultures; hence, it seemed appropriate to investigate what, if any, effect varied auxin concentration would have on bordered-pit development. As detailed below, the results indicate that auxin is a factor controlling pit placement.

MATERIALS AND METHODS Trees Three dormant tamarack trees [Larix laricina (Du Roi) K. Koch] between 20 and 30 years old and 6 to 8 metres in height and having no visible deformities were felled in the University of New Brunswick Forest (Fredericton, N.B., Canada) on March 30th. Following removal of lateral shoots, the 8-year-old section of the main-stem axis from each tree was removed and transferred to the laboratory for in vitro investiga• tions.

Chip preparation and culturing Surface sterilization, tissue explant procedures, culture conditions (under ordinary white fluorescent illumination, 12 Watts m- 2) and visual assessments were done as previously detailed (Savidge 1993). I-Naphthalene acetic acid (NAA, Sigma Chern. Co.), a synthetic auxin, was incorporated at 0.0,0.1,1.0, and 10.0 mg/l to provide four distinct media. The media were adjusted to pH 5.8 with 0.1 N KOH, and agar (0.8% w/v, Difco-Bacto agar) was added before autoclaving (120°C, 140 kPa, 20 min.). Sterilized media were poured into pre-sterilized petri dishes (Fisher Sci., No. 8-757- 14) to a depth of 4-6 mm (approximately 80 ml of medium/dish) and allowed to solidify. Fifteen chips per treatment (NAA concentration) per tree were investigated.

Microscopy Transverse and radial sections, cut by hand with a razor blade, were mounted in glycerin on glass slides. Unstained sections were examined with brightfield or Nomarski interference contrast illumination using a Reichart Polyvar photomicroscope. To avoid

Fig. 1. Growth response of a chip on 1.0 mg/l NAA as seen in transverse section cut by hand. c: cambial zone; r: ray; curved arrow: latewood-earlywood boundary; arrowheads: border• ed pits in tangential walls; arrow: bordered pits in a transverse end wall. Bar = 10 J..IIll. - Fig. 2. Radial hand section showing radial wall pitting in induced earlywood of a chip grown on 1.0 mg/l NAA. c: cambial zone; a: axial parenchyma as the first cells produced adjacent to the latewood boundary (curved arrow); arrowheads: bordered pits in transverse end walls of shortened fusiform cells. Bar = 10 J..IIll. - Fig. 3. Radial hand section showing biseriate radial wall pitting in induced earlywood of a chip grown on 0.1 mg/l NAA. The curved arrow indi• cates the earlywood-Iatewood boundary. Bar = 10 J..IIll. - Fig. 4. Radial hand section showing tangential wall pitting (arrowhead) in induced earlywood of a chip grown on 0.1 mg/l NAA. a: axial parenchyma adjoining the latewood.

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~ 10

03 u 4

NAA concent rat ion (mg /I )

_ CZceli s IZ22 RE ce ll s. xy logen ic chips D TE·s. xy logen ic chips

Fig. 5. Response of cultured Larix laricina chips to different concentrations ofNAA. CZ: cambial zone; RE: radial enlarged primary walled cambial derivatives; TE: mature tracheary elements produced during the culture period. Bars are standard errors of means. possible effects of wounding near the chip edges, analyses were done of the earlywood produced only at the mid-point of the chips. Radial and tangential wall numbers of bordered pits were counted in radial sections by variable focussing using a x 40 ob• jective. For quantitative data, Student's t-test was used to compare responses at the 95 % confidence level (Zar 1984). Standard errors of the means were calculated for the results. RESULTS

The cambium was dormant when the cultures were initiated. Cambial cell divisions occurred only in chips that produced a compact, striated type of callus as noted previ• ously (Savidge 1993). During chip growth, fusiform cambial cells divided periclinally. maintaining radial file continuity (Fig. 1, 2). Pseudotransverse anticlinal divisions oc• curred occasionally also, resulting in doubling of some radial files (Fig. 1). True trans• verse divisons in the fusiform cells yielded shortened axial parenchyma and tracheids

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~ '"E E 50 .... a.0) ....or. 40 0) .0 E c:::l '5.. 30 -0 ~ 0) "0.... 0 20 .0

0 0.0 0.1 1.0 10.0 NAA concentration (mg/l)

o radial wa ll pits ~ tangential wall pits

Fig. 6. Distribution of bordered pits between radial and tangential walls in reponse to varied NAA concentration. Bars are standard errors of means.

(Fig. 2). The first cells to differentiate adjacent to latewood usually appeared as axial parenchyma (Fig. 1-4). Tracheid production occurred only when NAA was included in the medium (Fig. 5). Controls (0.0 mg/l NAA) showed no cambial growth response, nor did cal• lus form on these chips. NAA at 1.0 mg/l yielded a stronger cambial response than did NAA at 0.1 and 10 mg/l (Fig. 5). Bordered pits were conspicuously present in both radial (Fig. 3) and tangential (Fig. 4) walls. Bordered pits were also commonly seen in the transverse walls of both axial and ray tracheids (Fig. 1, 2); however, these end-wall pits were not counted in this study. NAA at 0.1 mg/l was associated with the highest number per unit area of bordered pits in tangential walls. On the other hand, NAA at 10.0 mg/l yielded the highest ratio of tangential to radial wall pitting (Fig. 6). In contrast to the low and high NAA treatments, NAA at 1.0 mg/l resulted in bordered pits being produced pri• marily in the radial walls (Fig. 6).

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DISCUSSION

NAA in the medium was essential for both cambial and callus growth to occur, In a previous study with Larix laricina, endogenous auxin (indol-3-ylacetic acid, IAA) concentrations in the dormant cambial region were determined by combined gas chro• matography-mass spectrometry to be low throughout the winter months (Savidge 1991). The existence of low endogenous IAA agrees with our present observation that neither cambial nor callus growth occurred in explants on the medium lacking auxin. It fol• lows that the observed in vitro responses required the presence of exogenous auxin in the medium. Nevertheless, the cambial response rarely extended to 20 earlywood tracheids per radial file. Following this initial response, cambial activity and tracheid production ceased. Sub-culturing of chips onto media identical to those on which they had first been explanted did not encourage continuing wood formation. Hence, al• though the results indicate that auxin is limiting for reactivation and growth of the dormant cambium, they also point to there being one or more additional endogenous factors, present in the stem at the time of explanting, which becomes depleted during in vitro growth (Savidge 1993, 1994). This factor(s) evidently is essential for cell divi• sion activity and, conceivably, could also have a role in bordered pit placement during tracheid differentiation. In many conifer species, bordered pits appear almost exclusively in radial walls of earlywood axial tracheids, but they also develop in tangential walls of the last few latewood elements in each growth layer (Howard & Manwiller 1969; Siau 1970; Wilson & White 1986; Core et al. 1979; Panshin & DeZeeuw 1980). This is not invari• ably the case, however. In the southern pines, Howard and Manwiller (1969) reported that bordered pits in the tangential walls were not confined to latewood but were present throughout the growth ring. Our data indicate that the concentration of auxin in the medium influenced whether bordered pits developed in tangential or radial walls. Pre• sumably, the content of auxin in the cambial region of the chip was directly related to the concentration in the medium; however, this remains to be shown. Seasonal studies into endogenous auxin revealed that IAA is high during radial wall pitting (earlywood formation), and low when tangential wall pitting occurs during latewood formation (Savidge et al. 1982; Savidge & Wareing 1984; Savidge 1991). The in vitro responses indicate that low endogenous auxin content in the cambial region is more likely to result in a bordered pit forming in a tangential wall, whereas an intermediate concen• tration is conducive to radial wall pitting. On the other hand, the data also indicate that very high auxin content in the cambial region would be conducive to tangential wall pitting. NAA at 10 mg/l is probably supraphysiological and may not be relevant to in vivo development, but the effect of this high NAA concentration may nevertheless be important in understanding how auxin influences bordered pit placement. All of the NAA concentrations investigated, viz. 0.1, 1.0 and 10.0 mg/l, were rela• tively high considering that 0.01 mg/l NAA promoted growth in callus cultures of Cryptomeria (Yamamoto et al. 1983). On the other hand, Watson and Halperin (1981) found that NAA at 10 mg/l was maximally stimulatory to cell division, while a con• centration of 1 mg/l was the most effective for tracheid differentiation, in Jerusalem

Downloaded from Brill.com09/30/2021 07:43:40PM via free access Leitch & Savidge - Auxin regulation of pit positioning 295 artichoke cultures. The concentrations selected were based on previous work with Larix laricina chip cultures where it was concluded that the range 0.5-1.7 mg/l NAA was best for promoting wood formation (Savidge 1993). The requirement for higher than normal NAA concentrations in our experiments probably is explainable in terms of the xylem surface, only, contacting the medium. Hence, for NAA to reach the cambium it must diffuse or be actively transported (e.g., through the rays) through several milli• metres of wood. NAA is a synthetic auxin. Unlike IAA, NAA is stable when exposed to heat, air, light, or the salts present in culture media (Dunlap & Robacker 1988; Nissen & Sutter 1990). As far as is known, the metabolic machinery for NAA biosynthesis does not exist in higher plants; hence, NAA probably cannot be catabolized either. Neverthe• less, NAA when supplied to conifer tissues can, like IAA, be conjugated through its carboxyl group with glucose, aspartic acid, and possibly related molecules (Zenk 1962; Greenwood et al. 1974; Riov et al. 1979). The aspartate conjugate ofIAA promotes tracheid differentiation in similar manner to IAA (Savidge 1994); however, there is no clear evidence that the conjugate can act directly, before hydrolysis, in this respect. Conjugation presumably tends to lower the physiological action of auxin, but it may also serve to extend the duration of auxin physiological effects over the longer term (Wodzicki 1993). Further research will be necessary to distinguish between aspects of cambial growth and development occurring in response to immediately available auxin and those occurring in response to conjugated forms. It is well established that auxin promotes primary wall loosening and growth, and there is evidence that auxin acts differentially at the plasma membrane (Rossignol et al. 1991), as would be necessary if auxin acts directly to determine where bordered pit development occurs. It seems likely that a factor distinct from auxin may also be involved in bordered pit placement (Savidge 1985, 1993). Ethylene and jasmonic acid are plausible candidates, and the unidentified factor may also be linked to changing intra- and inter-cellular forces within the chip in association with wound healing around the periphery (Kutschera 1989). In vivo, considerable tension exists tangentially be• tween cambial cells when xylem is developing (Hejnowicz 1980). Applied pressure can also influence vascular development (Brown & Sax 1962). During the process of chip excision at time of explanting, any tangential tension in the chip must have been reduced. Once callus had healed the wounded surfaces, tensional stresses may have been restored. If this was the case, the first cambial derivatives to enlarge and differen• tiate in chips probably experienced primarily radial stress whereas subsequent cells may have differentiated under both tangential and radial stresses. Plausibly, the direc• tion of the stress vector influences the placement of the pit. Microtubules are thought to be involved in bordered pit formation and their placement plausibly could be af• fected by physical forces (Stein et al. 1971; Uehara & Hogetsu 1993); however, there also is evidence that microtubules are unnecessary for bordered pit development (Savidge & Barnett 1993). Within the concept that desmotubules traversing the compound middle lamella en• able intercellular communication such as that presumed necessary for formation of symmetrical pit pairs, the hypothesis that bordered pits develop where 'pit fields' of

Downloaded from Brill.com09/30/2021 07:43:40PM via free access 296 IAWA Journal, Vol. 16 (3),1995 plasmodesmata exist has been advocated (Tschernitz & Sachs 1975; Murmanis & Sachs 1969; Kerr & Bailey 1934). However, it is not finally established that plasmodesmata actually occur in cambial fusiform cells. Although some investigators considered they were present (Wardrop 1958; Murmanis & Sachs 1969; Murmanis 1971), Barnett and Harris (1975) noted that plasmodesmata were scarcely, if at all, present in fusiform cambial derivatives either prior to or during bordered pit development. Plasmodesmata certainly are present in the tangential walls of cambial ray cells, a location where bor• dered pit development rarely occurs (Barnett & Harris 1975; RA Savidge, unpub• lished observations). More research is clearly needed into both microtubules and plasmodesmata, to determine if either is obligately associated with or have any role in formation of bordered pits.

ACKNOWLEDGMENTS

This research was funded by grants from the Pulp and Paper Research Institute of Canada, the Canada Forestry Service, and the Natural Sciences and Engineering Research Council of Canada under the Research Partnerships program.

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