Occurrence of the Transition of Apical Architecture and Expression Patterns of Related Genes during Conversion of Apical Meristem Identity in G2 Pea

G2 pea exhibits an apical senescence delaying phenotype under short-day (SD) conditions; however, the structural basis for its apical development is still largely unknown. In the present study, the apical meristem of SD-grown G2 pea plants underwent a transition from vegetative to indeterminate inflorescence meristem, but the apical meristem of long-day (LD)-grown G2 pea plants would be further converted to determinate floral meristem. Both SD signal and GA3 treatment enhanced expression of the putative calcium transporter PPF1, and pea homologs of TFL1 (LF and DET), whereas LD signal suppressed their expression at 60 d post-flowering compared with those at 40 d post-flowering. Both PPF1 and LF expressed at the vegetative and reproductive phases in SD-grown apical buds, but floral initiation obviously increased the expression level of PPF1 compared with the unchanged expression level of LF from 40 to 60 d post-flowering. In addition, although the floral initiation significantly enhanced the expression levels of PPF1 and DET, DET was mainly expressed after floral initiation in SD-grown apical buds. Therefore, the main structural difference between LD- and SD-grown apical meristem in G2 pea lies in whether their apical indeterminate inflorescence medstem could be converted to the determinate structure.     

Gibberellin Is Involved in the Regulation of Cell Death-mediated Apical Senescence in G2 Pea

Senescence is the process of programmed degradation. The G2 line of pea exhibits apical senescence-delaying phenotype under short-day （SD） conditions, but the mechanism regulating the apical senescence is still largely unknown. Gibberellin （GA） was proved to be able to delay this apical senescence phenotype in G2 pea grown under long-day （LD） conditions. Here we show that the initiation of cell death signals in the terminal floral meristem was involved in the regulation of apical senescence in pea plants. SD signals prevented the formation of the cell death region in the apical mersitem. Moreover, GA3 treatment could effectively inhibit the occurrence of cell death-mediated apical senescence in LD-grown apical buds. Therefore, our data suggest that the prevention of apical senescence in SD-grown G2 pea through GA3 treatment may be largely responsible for the regulation of occurrence of the DNA fragmentation in apical meristem.     

The role of gibberellins A1 and A3 in fruit growth of Pisum sativum L. and the identification of gibberellins A4 and A7 in young seeds

Gibberellins A₁ and A₃ are the major physiologically active gibberellins (GAs) present in young fruit of pea (Pisum sativum L.). The relative importance of these GAs in controlling fruit growth and their biosynthetic origins were investigated in cv. Alaska. In addition, the non-13-hydroxylated active GAs, GA₄ and GA₇, were identified for the first time in young seeds harvested 4 d after anthesis, although they are minor components and are not expected to play major physiological roles. The GA₁ content is maximal in seeds and pods at 6 d after anthesis, the time of highest growth-rate of the pod (Garcia-Martinez et al. 1991, Planta 184: 53–60), whereas gibberellic acid (GA₃), which is present at high levels in seeds 4–8 d after anthesis, has very low abundance in pods. Gibberellins A₁₉, A₂₀ and A₂₉ are most concentrated in seeds at, or shortly after, anthesis and their abundance declines rapidly with development, concomitant with the sharp increase in GA₁ and GA₃ content. Application of GA₁ or GA₃ to the leaf subtending an emasculated flower stimulated parthenocarpic fruit development. Measurement of the GA content of the pods at 4 d after anthesis indicated that only 0.002–0.5% of the applied GA was transported to the fruit, depending on dose. There was a linear relationship between GA₁ content and pod weight up to about 2 ng · (g FW)⁻¹, whereas no such correlation existed for GA₃ content. The concentration of endogenous GA₁ in pods from pollinated ovaries is just sufficient to give the maximum growth response. It is concluded that GA₁, but not GA₃, controls pod growth in pea; GA₃ may be involved in early seed development. The distribution of GAs within the seeds at 4 d post anthesis was also investigated. Most of the GA₁, GA₈, GA₁₉, GA₂₀ and GA₂₉ was present in the testa, whereas GA₃ was distributed equally between testa and endosperm and GA₄ was localised mainly in the endosperm. Of the GAs analysed, only GA₃ and GA₂₀ were detected in the embryo. Metabolism experiments with intact tissues and cell-free fractions indicated compartmentation of GA biosynthesis within the seed. Using ¹⁴C-labelled GA₁₂, GA₉, 2,3-didehydroGA₉ and GA₂₀ as substrates, the testa was shown to contain 13-hydroxylase and 20-oxidase activities, the endosperm, 3β-hydroxylase and 20-oxidase activities. Both tissues also produced 16,17-dihydrodiols. However, GA₁ and GA₃ were not obtained as products and it is unlikely that they are formed via the early 13-hydroxylation pathway. [¹⁴C]gibberellin A₁₂, applied to the inside surface of pods in situ, was metabolised to GA₁₉, GA₂₀, GA₂₉, GA₂₉-catabolite, GA₈₁ and GA₉₇, but GA₁ was not detected. Gibberellin A₂₀ was metabolised by this tissue to GA₂₉ and GA₂₉-catabolite. Received: 23 July 1996 / Accepted: 2 September 1996     

Photoperiod-induced changes in gibberellin metabolism in relation to apical growth and senescence in genetic lines of peas (Pisum sativum L.)

In an early-flowering line of pea (G2) apical senescence occurs only in long days (LD), while growth in short days (SD) is indeterminate. In SD, G2 plants are known to produce a graft-transmissible substance which delays apical senescence in related lines that are photoperiod-insensitive with regard to apical senescence. Gibberellic acid (GA₃) applied to the apical bud of G2 plants in LD delayed apical senescence indefinitely, while N⁶-benzyladenine and -naphthaleneacetic acid were ineffective. Of the gibberellins native to pea, GA₉ had no effect whereas GA₂₀ had a moderate senescence-delaying effect. [³H]GA₉ metabolism in intact leaves of G2 plants was inhibited by LD and was restored by placing the plants back in SD. Leaves of photoperiod-insensitive lines (I-types) metabolized GA₉ readily regardless of photoperiod, but the metabolites differed qualitatively from those in G2 leaves. A polar GA₉ metabolite, GA_E, was found only in G2 plants in SD. The level of GA-like substances in methanol extracts from G2 plants dropped about 10-fold after the plants were moved from SD to LD; it was restored by transferring the plants back to SD. A polar zone of these GA-like materials co-chromatographed with GA_E. It is suggested that a polar gibberellin is synthesized by G2 plants in SD; this gibberellin promotes shoot growth and meristematic activity in the shoot apex, preventing senescence.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short days - LD long days     

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian “Cerrado”

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah “Cerrado”, is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A₁, A₃, and A₅, and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA₁ and GA₃ significantly stimulated shoot elongation, while GA₅ had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 μg per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA₃ more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.     

Specific proteins in stem meristems during transition to flowering

Immunodiffusion tests were used for studying protein composition of apical buds ofRudbeckia bicolor andPerilla nankinensis during their transition from vegetative to reproductive state under inductive photoperiodic conditions or GA₃ treatment. In both species the induced buds differ from the vegetative ones in the presence of specific proteins (P): P1, P2, P3 appear inRudbeckia apical buds 2, 8, 16 d after the start of inductive treatment; P4 appears inPerilla apical buds 6 d after inductive treatment. P1, P2, P4 are revealed in induced buds in the early period of apex development when morphogenetic changes are not yet present. The similarity between antigenic spectra of induced buds and of those treated by GA₃ appears only inRudbeckia. These observations support the hypothesis of a change in gene expression at floral evocation. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Photoperiodic and genetic control of carbon partitioning in peas and its relationship to apical senescence

Apical senescence but not flower initiation is delayed by short days (SD) compared to long days (LD) in pea plants (Pisum sativum L.) of genotype E Sn Hr. We recently reported that delay of senescence correlated with slower reproductive development, suggesting that fruits are weaker sinks for assimilates under delayed senescence conditions. Thus, we have examined assimilate partitioning in peas to determine if genotype and photoperiod regulate relative sink strength. Assimilate diversion by developing fruit has been implicated in senescence induction. A greater percentage of leaf-exported ¹⁴C was transported to fruits and a smaller percentage to the apical bud of G2 peas (genotype E Sn Hr) in LD than in SD. Relatively more of the ¹⁴C delivered to the apical bud of G2 peas was transported to flower buds than to young leaves in LD as compared to SD. There was no striking photoperiodic difference in carbon partitioning in genetic lines without the Sn Hr allele combination. The Sn Hr allele combination and photoperiod may regulate the relative strength of reproductive and vegetative sinks. Photoperiodic differences in sink strength early in reproduction suggest that these genes regulate sink strength by affecting the physiology of the whole plant. High vegetative sink strength in SD may maintain assimilate supply to the apical bud, delaying senescence.     

Sucrose and Malic Acid as the Compounds Exported to the Apical Bud of Pea following CO(2) Labeling of the Fruit : No Evidence for a Senescence Factor

The G2 line of peas (Pisum sativum L.) displays senescence and death of the apical bud only in long days and in the presence of fruit. As the removal of fruit prevents senescence, one possible mechanism by which fruits induce senescence is that the fruits produce some `senescence factor' under long day conditions, which is then transported to the apical bud. Allowing developing fruits to photosynthesize in the presence of ¹⁴CO₂ results in the recovery of label in the apical bud. In order to determine the chemical nature of this radiolabeled material, fruits of G2 peas, growing under long days, were exposed to ¹⁴CO₂ at the time when the first senescence symptoms start to appear. The radiolabeled material from apical buds was then extracted, purified, and identified. Using HPLC and GC-MS the major labeled compound found in the apical bud following exposure of pea fruits to ¹⁴CO₂ was identified as sucrose, while malic acid was identified as the major ethyl acetate-soluble compound. These compounds accounted for about 73 and 16%, respectively, of the radioactivity in the apical bud. No other compounds were present in significant amounts. As neither of these chemicals is likely to have any kind of senescence effect, we report no evidence for a senescence factor.     

Strigolactone may interact with gibberellin to control apical dominance in pea (Pisum sativum)

The role of strigolactones as plant growth regulators has been demonstrated through research on biosynthesis and signaling mutant plants and through the use of GR24, a synthetic analog of this class of molecules. Strigolactone mutants show a bushy phenotype and GR24 application inhibits the growth of axillary buds in these mutants, thus restoring the phenotype of a wild plant, which is characterized by a stronger apical dominance. In this work, we tested the effectiveness of this chemical on pea (Pisum sativum) plants following apex removal, which disrupts apical dominance and leads to axillary bud outgrowth. Moreover, we searched for relationships between the response to the strigolactone and gibberellin metabolism by applying GR24 to both climbing and dwarf peas, the latters being mutants for gibberellin biosynthesis. The results suggest that the endogenous level of the bioactive gibberellin GA₁ might modulate the response of decapitated pea plants to GR24, by changing bud sensitivity to the applied strigolactone.     

Qualitative and Quantitative Analysis of Endogenous Gibberellins in Onion Plants and Their Effects on Bulb Development

Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁ and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.     

Influence of photoperiod on seed development in the genetic line of peas G2 and its relation to changes in endogenous gibberellins measured by combined gas chromatography — Mass spectrometry

When apical senescence in the genetic line of peas G2 was prevented by short days fruit development was also found to be retarded. The levels of GA₂₀ and GA₂₉ in cotyledons and pods grown under long or short days were measured by gas chromatography — mass spectrometry multiple ion monitoring using extracts derivatised with deuterated trimethylsilyl groups as internal standards. The levels of GA₂₀ but not GA₂₉, were increased by short days. Conventional gas chromatography — mass spectrometry showed that relative to GA₂₉ the levels of GA₁₉, the other GA identified in G2 cotyledons, were also increased in short days. The levels of GA₂₀ in the pods were highest during the main phase of pod growth early in fruit development.Abbreviations GA_n gibberellin A_n - GC/MS gas chromatography — mass spectrometry - MIM multiple ion monitoring - Me methyl ester - SIM single ion monitoring - TIC total ion current - TMS trimethylsilyl ether - TLC thin layer chromatography - TTLC instant thin layer chromatography     

Effect of Gibberellic Acid and Monophenols on Flowering of lmpatiens balsamina in Relation to the Number of Inductive and Non-inductive Photoperiodic Cycles

A single treatment of plants with GA₃ (gibberellic acid) is not adequate to cause induction under LD (long day: 24-h photo-period) condition, but its effect is added to the sub-threshold induction caused by one SD (short day: 8-h photoperiod) cycle. Floral bud initiation is hastened, and the number of floral buds and flowers per flowering plant increases in plants receiving a single treatment with the combination GA₃+ SA (salicylic acid) accompanying a single SD cycle. However, the increase on 10 replicate basis is more marked in plants receiving three treatments with the combination GA₃+β-N (β-naphthol) and five treatments with the combination GA₃+ SA accompanying six and 10 SD cycles, respectively. The number of floral buds and flowers decreases with an increase hi the number of SD cycles, but it is higher in plants treated with GA₃, SA or GA₃+β-N than in the water-treated controls. — Under long days, treatment of plants with the combinations GA₃+ SA or GA₃+β-N accelerates the initiation as well as increases the number of floral buds. While a minimum of five treatments with GA₃ or of 25 with SA or β-N alone is needed for floral bud initiation under a 24-h photoperiod, three treatments are adequate to induce floral buds with the combination GA₃+ SA or GA₃+β-N under continuous illumination. Ten or more treatments with these combinations under a 24-h photoperiod produce more flowers than the same treatments under an 8-h photoperiod.     

Effect of Gibberellic Acid and Phosfon on the Critical Dark Period Reqnirement for Flowering of Impatiens balsamina cv. Rose

The critical dark period requirement for flowering of Impatiens balsamina L. cv. Rose, an obligate short day plant, is about 8.5 hours. While GA₃ completely substituted for the dark period requirement, Phosfon prolonged it to 9.5 hours. GA₃ hastened and Phosfon delayed the initiation of floral buds under all photoperiods. Floral buds opened into flowers only during 8 and 14 hour photoperiods in control and Phosfon-treated plants but during all photoperiods in GA₃-treated ones. The delay in floral bud initiation and flowering was correlated with shifting up of the node bearing the first floral bud and flower respectively. While GA₃ increased the numher of floral buds and flowers in all photoperiods except 8-hour, Phosfon increased their number in the 14-hour photoperiod only. The number of flowering plants decreased with increasing photoperiod regardless of GA₃ and Phosfon application. The effect of Phosfon was completely or partially overcome, depending upon the photoperiod, by simultaneous application of GA₃.     

Effect of photoperiod on polyamine metabolism in apical buds of g2 peas in relation to the induction of apical senescence

Polyamine content and arginine decarboxylase activity of apical buds were measured to determine whether polyamines are required to prevent apical senescence in pea. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. High polyamine concentrations were found in the vigorous apices of plants grown under a short day photoperiod during which senescence is delayed. As the apex senesced in long days, the amounts of polyamines per organ declined in parallel with decreases in the size of the apical bud. However, a decrease in polyamine concentration, due mainly to a change in spermidine, occurred at the time of marked reduction in bud size and growth rate, but not before the onset of the early symptoms of senescence. No correlation was found with arginine decarboxylase. The results suggest polyamines may be required to support bud growth, but the photoperiodic mechanism which governs apical senescence of G2 peas does not exert control through polyamine metabolism.     

The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation

The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA₂₀ and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA₁-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2β-hydroxylated GAs (GA₈ and GA₂₉) in either the mature vegetative tissues or the roots of pea plants.     

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.     

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     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998     

Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.