Changes in the water status and rheological characteristics of pea seedling axillary buds during their transitions growth-dormancy-growth in apical dominance

The technique of isopiestic thermocouple psychrometry was used for the analysis of bud transition from dormancy to growth and back in 8-18-day-old pea (Pisum sativum L.) seedlings. We monitored changes in the water (ψ_w) and osmotic (ψ_{s + m}) potentials and also turgor pressure (ψ_p) in dormant buds and threshold turgor (Y) in growing buds, the latter being one of the cell-wall rheological characteristics. Seedling decapitation resulted in a decrease of Y in the bud, which coincided with the start of its outgrowth. The replacement of terminal shoot with exogenous auxin (IAA or NAA) retarded bud outgrowth and maintained the high level of Y, which argues for the auxin control of this parameter. When growth of the first axillary bud was inhibited by the second one, positioned higher and remained on the plant, the beginning of Y increase preceded visible correlative growth suppression; this makes this rheological index an early marker of bud transition from growth to dormancy. The effects of the terminal shoot part and auxin application on the bud osmotic status differed substantially. In fact, bud transition to dormancy in the presence of the terminal shoot, the main or developing from the second axillary bud, was accompanied by the rise in ψ_{s + m}, whereas, in the case of the replacement of the second bud with exogenous auxin, the first bud growth suppression occurred with the decrease in ψ_{s + m}. The low value of the bud ψ_{s + m} is a factor for creating a considerable gradient of the water potential between the stem and bud supporting water transport to the bud, which was much more active than in plants with a terminal shoot. It seems likely that this is the reason for the absence of complete growth suppression observed by us and other researchers even after application of high auxin concentrations. Immediately after seedling decapitation, ψ_{s + m} in the buds reduced; however, this was not the result of trophic metabolite redistribution due to the loss of their active sink because ψ_{s + m} reduced also in experiments with complete isolation of the bud releasing from dormancy in the chamber at 100% humidity. Auxin application to the cut surface of decapitated seedlings did not affect the ψ_{s + m} decrease. Like decapitation, cotyledon removal resulted in the increase in the bud turgor pressure. However, in this case, water stress did not change the bud osmotic status. Thus, the induction of osmotica accumulation in the bud after the removal of the terminal shoot is evidently related to neither trophic, nor auxin, nor hydraulic signal. The data obtained allowed us to conclude that both components of the bud water potential—ψ_{s + m} and Y—play an important role in the control of bud growth at apical dominance. Auxin produced in the shoot apex is involved in the control of Y, whereas the nature of the signal controlling the ψ_{s + m} level is unclear.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Stimulatory effect of cytokinins and interaction with IAA on the release of lateral buds of pea plants from apical dominance

Lateral buds of pea plants can be released from apical dominance and even be transformed into dominant shoots when repeatedly treated with synthetic exogenous cytokinins (CKs). The mechanism of the effect of CKs, however, is not clear. The results in this work showed that the stimulatory effects of CKs on the growth of lateral buds and the increase in their fresh weights in pea plants depended on the structure and concentration of the CKs used. The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) was stronger than that of 6-benzylaminopurine (6-BA). Indoleacetic acid (IAA) concentration in shoot, IAA export out of the treated apex and basipetal transport in stems were markedly increased after the application of CPPU or 6-BA to the apex or the second node of pea plant. This increase was positively correlated with the increased concentration of the applied CKs. These results suggest that the increased IAA synthesis and export induced by CKs application might be responsible for the growth of lateral shoots in intact pea plants.     

Expression of a ribosomal protein gene in axillary buds of pea seedlings

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations ≥10 μm, bud growth and expression of pGB8 in the buds are inhibited.     

Dormancy-associated gene expression in pea axillary buds.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots root apices; fully-elongated stems >elongating stems), and thus is an excellent “dormancy” marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs. Received: 10 December 1997 / Accepted: 23 January 1998     

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [³²P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.     

Two novel transcripts expressed in pea dormant axillary buds

To elucidate the molecular mechanism of apical dominance, the expression patterns of genes that are preferentially expressed in dormant axillary buds of pea (Pisum sativum L. cv. Alaska) seedlings were investigated. We isolated two cDNA clones, cPsAD1 and cPsAD2 whose corresponding genes were named PsAD1 and PsAD2, from a cDNA library of dormant axillary buds using the differential display method. The deduced amino acid sequence of PsAD1 contains 87 residues and is rich in glycine residues in the amino terminal region. A search of the protein databases failed to find any sequences similar to PsAD1 protein except for the glycine-rich region. Northern blot analyses showed that PsAD1 mRNA mainly accumulated in dormant axillary buds and that its amount rapidly decreased after decapitation of the terminal bud. In situ hybridization analyses indicated that PsAD1 mRNA was localized in the apical meristem, procambia, and leaf primordia in dormant axillary buds that were competent to grow out but whose growth was temporarily suspended. That is, the expression of the PsAD1 gene is closely associated with the dormancy of axillary buds. The deduced amino acid sequence of PsAD2 contains 98 amino acid residues and is not similar to those of previously characterized proteins. PsAD2 mRNA accumulated in dormant axillary buds, roots, mature leaflets and elongated stems, suggesting that PsAD2 is involved in not only the dormancy of axillary buds but also the non-growing state in various tissues.     

Changes in abscisic acid and indole-3-acetic acid in axillary buds of Elytrigia repens released from apical dominance

Growth of axillary buds on the rhizomes of Elytrigia repens (L) Nevski is strongly dominated by the rhizome apex, by mechanisms which may involve endogenous hormones. We determined the distribution of indole-3-acetic acid (IAA) and abscisic acid (ABA) in rhizomes and measured (by gas-chromatography-mass spectrometry) their content in axillary buds after rhizomes were decapitated. The same measurements were also made in buds induced to sprout by removing their subtending scale leaves. The ABA content tended to be higher in the apical bud and in the axillary buds than in the adjacent internodes, and tended to decline basipetally in the internodes and scale leaves. IAA was similary distributed, except that there was less difference between the buds and other rhizome parts. After rhizomes were decapitated, the ABA content of the first axillary bud declined to 20% of that of control values within 24 h, while the IAA content showed no marked tendency to change. The ABA content also declined within 12 h in the first axillary bud after rhizomes were denuded, while the content of IAA tended to increase after 6 h. These changes occurred before the length of the first axillary bud increased 24–48 h after rhizomes were decapitated or denuded. We conclude that the release of axillary buds from apical dominance in E. repens does not require IAA content to be reduced, but is associated with reduced ABA content.     

Changes in endogenous level of auxins and cytokinins in axillary buds ofpisutn sativum L. in relation to apical dominance

Decapitation of the stem in one-week-old pea seedlings below the first node causes a rapid outgrowth of the two cotyledonary buds. One of them soon becomes dominant, while the other one is inhibited, but can be released from inhibition by cutting off the dominant bud. The level of endogenous auxins and cytokinins was determined in dominant and inhibited buds, as well as in released buds at different time intervals after deinhibition. It was found that the inhibited buds contained very little acidic, ether soluble auxins, a high level of tryptophan and also a high level of cytokinins, in comparison with dominant buds. When the inhibited buda were released from inhibition, their auxin content rose, while that of tryptophan and cytokinins decreased, reaching the level found in dominant buds within six days. Specific changes in content of two undetermined auxin-like substances were found in released buds during de-inhibition. These results are discussed in relation to the current views on the regulation of apical dominance.     

Expression of beta-tubulin during dormancy induction and release in apical and axillary buds of five woody species

Cell cycle activity was studied in apical and axillary buds of Norway maple ( Acer platanoides L.), apple ( Malus ' M9 ') , pedunculate oak ( Quercus robur L.), Scots pine ( Pinus sylvestris L.) and rose ( Rosa corymbifera 'Laxa') during dormancy induction and release. Flow cytometric analyses revealed that in dormant buds, cells mainly were quiescent at the G₀/G₁ phase, while in non-dormant buds, a significantly higher frequency of G₂ cells was found in all species. In western blots accumulation of 55 kDa beta -tubulin was found in active growing plant material, whereas in dormant buds the accumulation was much lower or below detection level. It was observed for all species that during dormancy induction the amount of beta -tubulin decreased, while during dormancy release a fast accumulation of beta -tubulin occurred. The dynamics of the beta -tubulin accumulation reflected the dormancy status of tree buds of the five species studied suggesting that the beta -tubulin level might be useful as a marker for the dormancy status in buds of temperate woody species.     

Cell cycle regulation during growth-dormancy cycles in pea axillary buds

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G₁ and G₂ nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G₁, at the G₁/S boundary and near the S/G₂ boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G₁/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G₁ is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G₂ and mitosis, cells arrested at G₁/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G₂ boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.     

The effect of exogenous gibberellin and auxin on the dominance between the axillary buds of pea (Pisutn sativum L.) cotyledons

The interaction of GA and IAA in apical dominance was investigated in an experiment in which first of all an IAA paste was applied to the cut areas formed by the decapitation of epicotyl apices of pea seedlings, followed after one week by the application of a 0.25 % GA paste. The latter treatment was able to overcome the growth inhibition of cotylary buds induced by a 0.03 % IAA paste, but not that caused by 0.06 and 0.12 % IAA pastes. The correlative function of a root in the renewal of the apical dominance can, to some extent, be directly simulated by exogenous gibberellin, as has been demonstrated in the experiment with decapitated pea seedlings deprived of one cotyledon, on which the growing axillary of the amputated cotyledon was decapitated. In this case the axillary of the remaining cotyledon grows in the plants where the root has been left, but in those deprived of the root, there appears a serial of the amputated cotyledon (Dostál, Biol. Plant. 9 : 330, 1967). When GA was supplied to the plants treated in this way, the coty lary of the remaining cotyledon grew even in the plants deprived of the root.     

Acropetal transport of kinetin in pea stem sections

Pomocí stimulace r?stu postranních pupen? etiolovaných ?ízk? hrachu kinetinem bylo zji?těno, ?e biologický ú?inek kinetinu není omezen pouze na místo jeho aplikace, ale ?e se m??e projevit i v jiných ?ástech ?ízk?. Tento jev si autor vysvětluje transportem kinetinu.     

Role of water relations and photosynthesis in the release of buds from apical dominance and the early reinvigoration of decapitated poplars

A decrease in xylem pressure potential starting 1 h after decapitation of young hybrid poplars ( Populus deltoides Bartr. × Populus nigra L. cv. DN22) reduced stomatal conductance and transpiration rates for the first 3 days after decapitation. This early moisture stress was alleviated 4 to 5 days after decapitation, resulting in substantial increases in stomatal aperture, transpiration and net photosynthetic rates which continued for the remainder of the one week measurement period. The results suggest the following sequence of events in the decapitated plant: After a brief moisture stress, decapitation increases moisture availability by increasing the root/shoot ratio and by reducing shoot competition for moisture. Improvement in hydration releases buds from apical dominance and increases stomatal conductance and rates of net photosynthesis. This, in turn, leads to the acceleration of growth observed when plants are reinvigorated by decapitation.     

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance. Received: 12 December 1996 / Accepted: 7 January 1997     

On the infrastructural localization of basic proteins in the nucleus and nucleolus of cells in pea axillary meristems submitted to or released from apical dominance

Summary In pea axillary meristems submitted to or released from apical dominance, basic nuclear proteins and their fractions (lysine or arginine-rich) were localized at the infrastructural level using convergent methods. In the inhibited nuclei, the condensed chromatin and the nucleoli are the most reactive regions to alcoholic solution of phosphotungstic acid and to ammoniacal silver nitrate. It is the same in the reactivated nuclei after the release from dominance, but the increase in diameter of the nucleoli is accompanied by the creation of a granular component which is observed around the nucleoli during the G₁ S or G₂ phases. This structure is built up essentially by a lysin-rich ribonucleoprotein complex characteristic of active nuclei.     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

Effects of the auxin-inhibiting substances raphanusanin and benzoxazolinone on apical dominance of pea seedlings

The effects of the auxin-inhibiting substances raphanusanin ((3R*,6S*)-3-[methoxy (methylthio) methyl]-2-pyrrolidinethione, raphanusanin B)and benzoxazolinone (6-methoxy-2-bezoxazolinone, MBOA) on apical dominance of pea(Pisum sativum L. cv. Alaska) seedlings were studied.Application of raphanusanin B or MBOA to the apical bud, internode, or lateralbud of pea seedlings released apical dominance in either intact orindole-3-acetic acid (IAA )-treated, decapitated plants. These results suggestthat the auxin-inhibiting substances raphanusanin B and MBOA have activity inreleasing apical dominance. Conversely, the auxin transport inhibitors2,3,4-triiodobenzoic acid (TIBA) and 1-naphthylphthalamic acid (NPA) did notstimulate lateral bud growth when they were applied directly to the lateralbud,although application to the apical bud or internode released apical dominance.Therefore, the mode of action of raphanusanin B and MBOA in apical dominance isclearly different from that of auxin transport inhibitors. Raphanusanin B andMBOA may suppress the synthesis of growth-inhibiting factor(s) of the lateralbud induced by endogenous auxin transported from the apical bud or exogenouslyapplied auxin, and/or the action of the factor(s).