EVIDENCE for AUXIN REGULATION of BORDERED-PIT POSITIONING DURING TRACHEID DIFFERENTIATION in LARIX LARICINA By
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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]. Downloaded from Brill.com09/30/2021 07:43:40PM via free access 290 IAWA Journal, Vol. 16 (3),1995 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. Downloaded from Brill.com09/30/2021 07:43:40PM via free access 292 IAWA Journal, Vol. 16 (3), 1995 14 12 ~ 10 03 u 4 2 o 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 Downloaded from Brill.com09/30/2021 07:43:40PM via free access Leitch & Savidge - Auxin regulation of pit positioning 293 70 60 ~ '"E E 50 .... a.0) ....or. 40 0) .0 E c:::l '5.. 30 -0 ~ 0) "0.... 0 20 .0 10 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). Downloaded from Brill.com09/30/2021 07:43:40PM via free access 294 IAWA Journal, Vol. 16 (3), 1995 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).