Effect of laterally applied gibberellin A4/7 on cambial growth and the level of indole-3-acetic acid in Pinus sylvestris shoots

Gibberellin A_4/7 (GA_4/7) was applied in lanolin or ethanol around the circumference at the midpoint of the previous-year terminal of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for up to 10 weeks, cambial growth was measured as the radial widths of xylem and phloem, and the level of indole-3-acetic acid (IAA) was determined by combined gas chromatography-mass spectrometry using [¹³₆](IAA) as the internal standard. In intact seedlings, both 1 mg GA_4/7 g^?1 lanolin and 50 mg GA_4/7 I^?1 ethanol increased phloem production and the cambial region IAA level in the current-year terminal, without significantly altering its longitudinal growth. In the previous-year terminal, 1 mg GA_4/7 g^?1 lanolin promoted phloem production at the application point and increased the cambial region IAA level above this point, whereas 50 mg GA_4/7 I^?1 ethanol stimulated the production of both xylem and phloem at the treatment site and elevated the cambial region IAA level beneath it. Laterally applied GA_4/7 at 50 mg I^?1 ethanol stimulated xylem and phloem production in debudded previous-year terminals treated at the apical cut surface with 1 mg IAA g^?1 lanolin, but not in those treated with plain lanolin. However, the promotion of cambial growth in debudded terminals treated apically with 1 mg IAA g^?1 lanolin and laterally with 50 mg GA_4/7 I^?1 ethanol was not associated with an elevated IAA content in the cambial region. The results indicate that exogenous GA_4/7 can promote xylem and phloem production provided an IAA source is present, and that it or a metabolic product acts directly, rather than indirectly by stimulating longitudinal growth and/or raising the cambial region IAA level.     

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.     

Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [¹³C₆]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [¹³C₆]IAA, while [¹²C]IAA of endogenous origin formed a minor pool. However, the pool of [¹³C₆]IAA decreased from 90 to 80% of the total free IAA pool [¹³C₆]IAA+[¹²C]IAA) while being transported down the shoot. This dilution with [¹²C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.     

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     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.     

Identification of endogenous gibberellins, and metabolism of tritiated and deuterated GA4, GA9 and GA20, in Scots pine (Pinus sylvestris) shoots

The application of gibberellin A_4/7 (GA_4/7) to the stem of previous-year (1-year-old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current-year terminal shoot, but the distribution and metabolic fate of the applied GA_4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current-year terminal shoot of 2-year-old seedlings were purified by column chromatography and high-performance liquid chromatography and analysed by combined gas chromatography-mass spectrometry (GC-MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full-scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected-ion monitoring (GA₂₀) and Kovats retention index. Tritiated and deuterated GA₄, GA₉ or GA₂₀ were applied around the circumference at the midpoint of the previous-year terminal shoot, and metabolites were extracted from the elongating current-year terminal shoot, the application point, and the 1-year-old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC-MS, it was evident that, for each applied GA, unmetabolised [²H₂]GA and [³H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [³H]GA₉ and [³H]GA₂₀ were applied, whereas the largest percentage of radioactivity derived from [³H]GA₄ was recovered in the current-year terminal shoot. It was also found that [²H₂]GA₉ was converted to [²H₂]GA₂₀ and to both [²H₂]GA₄ and [²H₂]GA₁, [²H₂]GA₄ was metabolised to [²H₂]GA₁, and [²H₂]GA₂₀ was converted to [²H₂]GA₂₉. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous-year shoots not only are absorbed and translocated extensively throughout the previous-year and current-year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA₉ metabolism follows two distinctly different routes: in one, GA₉ is converted to GA₁ through GA₄, and in the other it is converted to GA₂₀, which is then metabolised to GA₂₉. The results suggest that the late 13-hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA_4/7 may be due to its conversion to GA₁, rather than to it being active per se.     

Cross-colonization of Scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Periodicity of cambial activity in Abies balsamea. I. Effects of temperature and photoperiod on cambial dormancy and frost hardiness

The relationship between from hardiness and growth potential, and their dependence on temperature and photoperiod, was investigated in the one-year-old cambium of balsam fir [Abies balsamea (L.) Mill.]. Six-year-old trees were exposed for 9 weeks to either the natural environment or one of 4 controlled environments in the fall (18 September-18 November), spring (12 April–14 June) and summer (19 July – 19 September). The 4 controlled environments were (1) WS, warm temperature (24/20°C in day/night) + short day (8 h). (2) WL. warm temperature (24/20°C) + long day (8 h + 1 h night break), (3) CS. cold temperature (9/5°C) + short day (8 h) and (4) CL, cold temperature (9/5°C) + long day (8 h + 1 h night break). At the beginning and end of each exposure, cambial activity was measured by recording the number of xylem, cambium and phloem cells, frost hardiness was estimated from the cambium's ability to survive freezing to –40°C, and cambial growth potential was deduced from the duration of the cell cycle and the production of xylem, cambium and phloem cells in cuttings cultured for 4 weeks with exogenous indole-3-acetic acid (IAA) under environmental conditions favourable for cambial activity. In the natural environment, frost hardening began in September and was completed in November, while dehardening occurred when the cambium reactivated. CL, CS, and to a lesser extent WS, promoted hardening in the summer and fall, but did not prevent dehardening in the spring. The cambial growth potential in the natural environment declined from a maximum in April to a low level in June, reached a minimum in September, then increased to a high level in November. This potential was promoted by CL and CS on all dates by WL in the summer and fall. The ratio of xylem to phloem induced by IAA treatment was greatest in June and least in September in cuttings from trees exposed to the natural environment, and was increased by CL and CS in the fall. The cambium in intact branches of trees protected from chilling during the fall and winter resumed cell cycling after less than 9 weeks of dormancy, but produced mostly or only phloem in the subsequent growing period. It is concluded that the frost hardiness of the cambium, the IAA-induced cycling of cambial cells, and IAA-induced xylem to phloem ratio vary independently with season, temperature and photoperiod, and that the periodicity of these processes is regulated endogenously.     

Regulation by light of chlorophyll synthesis in the cotyledons of Scots pine (Pinus sylvestris) seedlings

Seedlings of gymnosperms, unlike angiosperms, synthesize chlorophyll(ide) (Chl) in darkness (D). In Scots pine cotyledons ( Pinus sylvestris L.) Chl accumulation ceases in D at a low level but Chl accumulation is strongly increased by light, red light (R) being more effective than blue light (B), whereas in Pinus maritima Chi synthesis is almost light-independent. In Scots pine the capacity to form Chl can be increased by R pulses, fully reversible by far-red light, demonstrating the involvement of phytochrome. However, when B- or R–grown seedlings were transferred to D, Chl accumulation stopped immediately irrespective of the level of P_fr (far-red light absorbing form of phytochrome), indicating that the conversion of protochlorophyllide (PChl) is light-dependent. Dose response curves in R and B and simultaneous irradiation with R and B show that R and B are perceived by separate photoreceptors. The immunodetected NADPH-dependent protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), assumed to regulate light-dependent Chl synthesis in angiosperms, is not correlated with the capacity of gymnosperm Chi accumulation in darkness. While two FOR bands could be separated in extracts from dark grown material (38 and 36 kDa) of Pinus sylvestris and P. maritima , only the 38 kDa band disappeared consistently in the light. However. the significance of the more light resistant 36 kDa band for chlorophyll synthesis remains unclear as well.     

Changes of morphogenic competence in mature Pinus sylvestris L. buds in vitro

The effects of season and cold storage on morphogenic competence in mature Pinus sylvestris buds were investigated. Peroxidase and polyphenol oxidase activity were measured as markers of oxidative metabolism. No growth in vitro was observed on explants detached from the end of January until the beginning of March. Brachioblasts, each with a couple of needles, formed on 11% of the buds without macrostrobili that were detached in early April and introduced immediately into culture. Of the explants detached in late July, 15% formed shoots with brachioblasts and needles. The lowest activity of peroxidase and polyphenol oxidase in pine buds was observed from the end of April until the beginning of June when morphogenic competence of tissues started to increase. Development of bud explants detached in January was achieved by cold storage for 5 months. Low polyphenol oxidase and peroxidase activity coincided with increased morphogenic potential. Results suggest that reduced or stable activity of peroxidase and polyphenol oxidase is associated with an increased ability of tissues to start growth in vitro.