The role of gibberellins and cytokinins in the growth correlative effect of cotyledons in flax and pea

It is wellknown that following the amputation, or darkening of one cotyledon in decapitated flax seedlings, the opposite remaining, or illuminated, cotyledon exerts a stimulatory effect on the growth of its axillary bud. For the induction of this stimulating effect a 21–72 h continuous darkening of the cotyledon is sufficient. Endogenous gibberellins take part in the stimulation effect of the illuminated cotyledon, since their level in the illuminated cotyledon increases as early as 12–48 h following the darkening of the opposite cotyledon. The apical part of the cotyledon has a higher growth stimulatory effect on the growth of the cotyledonary axillary bud than the basal half. This again is associated with endogenous gibberellins the level of which is higher in the apical half of the cotyledon than in the basal one. Upon removal of the root and hypocotyl base in decapitated flax seedlings deprived of one cotyledon, the remaining cotyledon loses its stimulatory influence, so that the bud of the amputated cotyledon grows more vigorously (Dostál 1955). In this growth correlative phenomenon the root may be substituted by cytokinin BA applied in the form of a 0.1–1.0 per cent paste onto the remaining cotyledon, for again in this case the bud of the preserved cotyledon grows more vigorously. Following the decapitation of the axillary of the amputated cotyledon in decapitated pea seedlings with an intact root and deprived of one cotyledon, the axillary of the remaining cotyledon grows more intensively than the serial of the removed one. If the plants operated on in the same way are deprived of the root, the serial of the removed cotyledon gains a correlative growth predominance. If the plants deprived of root are cultivated at the same time in a solution of BA (10–20 mg 1⁻¹), the correlative predominance is acquired by the axillary of the remaining cotyledon. In growth correlations between cotyledons and their axillary buds in pea seedlings the root may thus be substituted by exogenous cytokinin, as well.     

Effect of the amputation of the cotyledon and of the application of growth regulators on the transport of32P in decapitated pea seedlings

When the epicotyl and one cotyledon is cut off from pea seedlings, only the axillary of the amputated cotyledon is known to grow. When³²P is applied to the roots of such plants, then a higher radioactivity appears in the axillary of the amputated cotyledon already 24 hrs. after amputation of one cotyledon, although this axillary is of the same size at this time as that of the remaining cotyledon. This fact indicates a more extensive material transport to the axillary bud of the amputated cotyledon already during the first day after amputation The effect of individual regulators on the³²P transport was investigated in an experiment where pea seedlings cultivated in the dark were decapitated and a 0.5% paste, containing the regulatory compounds was placed either on the cutting surface in the apical part of the epicotyl stump or in its central part. After a week the plant roots were supplied with³²P and its transport to the upper part of the epicotyl stump was followed. This transport increased about 10-fold in the case of a paste, containing indolyl acetic acid, when the paste was spread on the apical cutting surface of the stump. However, the transport was inhibited when the paste was applied in the central part of the stump. These results indicate that only the transport of³²P towards the paste with indolyl acetic acid is accelerated, whereas it is decelerated above this paste. A paste, containing triodobenzoic acid inhibited³²P transport only when applied to the apical cutting surface of the epicotyl stump and not when spread over the middle part. In this case³²P transport was more rapid above the paste than towards the paste. The situation was similar in the case of gibberellin and kinetin.     

Gibberellin-cytokinin interaction in the correlation between the cotyledon and its axillary bud in pea (Pisum sativum L.)

If one cotyledon is removed from decapitated pea seedlings and the remaining one is treated with the cytokinin BA, then a complete correlation reversal occurs in numerous cases: instead of the bud belonging to the removed cotyledon, the bud belonging to the remaining cotyledon starts to grow. However, if GA is applied to the remaining cotyledon together with BA, then the number of these correlation reversals sharply drops. This in respect to cytokinin morphogenetically (correlatively) contradictory effect could play a significant role in apical dominance in plants.     

Interaction of indoleacetic acid with synthetic and native growth regulators during transfer of32P into epicotyls of etiolated pea seedlings

If both cotyledons are amputated from non-decapitated pea seedlings, the intensity of transport of³²P into intact epicotyls is raised more than twofold. This is apparently connected with the regulatory-inhibitory effect of the cotyledons that can be simulated by a 0·1% paste of indoleacetic acid (IAA). If both amputated cotyledons are replaced by this paste, the intensity of³²P transport is again decreased to the level found in plants with intact cotyledons. When etiolated pea seedlings grown in the dark in 100% relative humidity were decapitated, the³²P transport to the top of the epicotyl stumps covered with 0·5% IAA paste was strikingly decreased if the stumps were covered below with a 10% paste containing chlorocholine chloride (CCC) or with a 0·5% paste containing triiodobenzoic acid (TIBA). On the other hand, the intensity of³²P transport was significantly increased if the IAA paste was put above a 0·5% paste with kinetin which alone does not raise the transport intensity. No such synergism toward³²P transport could be established between IAA and gibberellin.     

The antagonism of (2-Chlorethyl) trimethylammonium chloride and indole 3-acetic acid in epicotyl and axillary buds growth in the pea seedlings

The 0·03–0·06% indole-3-acetic acid (IAA) in lanoline paste smeared on the cut surface of decapitated epicotyl of the pea seedling inhibited the growth of the axillary buds of the cotyledons. 0·03–0·06% solution of (2-Chlorethyl) trimethylammonium chloride (CCC) considerably weakened the inhibitory effect of IAA when applied simultaneously to the roots. In a similar way CCC diminishes even the growth inhibition of intact pea epicotyls caused by 0·06% IAA lanoline paste. 0·03% solution of CCC administered to the roots of the decapitated pea seedlings significantly increased growth of the axillary buds. This effect may play a role in the demonstrated antagonism between the low concentration of CCC and IAA.     

The interaction of endogenous gibberellins in correlation with cotyledons and axillary buds in the pea (Pisum sativum L.)

After the decapitation and amputation of one cotyledon in germinating pea seedlings, the axillary bud of the amputated cotyledon always grows and the growth of the axillary bud of the remaining cotyledon is inhibited. Before morphological differences appear between the axillary bud of the amputated and preserved cotyledon, a higher endogenous gibberellin content can be demonstrated chromatographically in the axillary bud of the amputated cotyledon. This indicates that the increased growth of the axillary bud of the amputated cotyledon is in connection with an earlier increase in the activation of endogenous gibberellins.     

Changes in Phytohormone Status in Stems and Roots after Decapitation of Pea Seedlings

Experiments were performed on the first and second internodes and 4-cm-long apical segments of main roots of pea (Pisum sativum L.) seedlings, grown in the light and decapitated above the second node on the seventh day after seed germination. Endogenous phytohormones were measured by the enzyme-linked immunosorbent assay during three days after decapitation of seedlings. The IAA level in the internodes decreased 2–3 times on the second day after decapitation of seedlings while the cytokinin level increased 5–6 times for zeatin and zeatin riboside (Z and ZR) and 1.5–2 times for isopentenyl adenine and isopentenyl adenosine (IP and IPA). In contrast to internodes, the IP and IPA contents in the roots of decapitated seedlings did not change, but the levels of Z and ZR increased 1.5–2 times compared to intact plant roots. The IAA level in the apical region of root remained almost unchanged after the removal of shoot apex. It was concluded that the apical meristem of the main root is not the site of the cytokinin response to the auxin signal coming from the stem apex and that a slight accumulation of Z and ZR after decapitation is due to upper zones of the root. There was no difference in the content of gibberellin-like substances between the internodes of intact and decapitated seedlings. However, the content of gibberellins (GA) in the root tip decreased after decapitation of seedling, which suggests an essential role of apical bud in supplying the root with GA and/or intermediates for their biosynthesis.     

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.     

On the interaction of growth retardants with IAA and kinetin

In the pea test a highly positive response to the treatment with IAA reversed to a negative one or became 5 to 6 times weaker when CCC was applied together with IAA. In cultivating pea seedlings, following their decapitation, for two days in a 0.25 per cent CCC solution and then in water, growth of their cotyledonous axillaries (cotylaries) were inhibited. This inhibitive action of CCC could be made ineffective when the seedlings, following two-days’ cultivation in the CCC solution, were grown further in kinetin solutions (0.37–3 mg per 1). Cotylaries of decapitated pea seedlings, when grown in kinetin solutions were inhibited. With kinetin solutions of 6–12 mg/l a strong inhibition also occured in the growth of roots at the apical parts of which spherical swellings were developing. The CCC supplied to the roots of intact etiolated pea seedlings is translocated acropetally into the stem at a rate of about 5 cm per hour. Decapitation of the plant causes retardation of this transport, yet a coat of 0.00001–1% IAA or kinetin paste produces acceleration of the stream. Existence of an antagonism between CCC and IAA, demonstrated earlier, was found holding true also for B-9 (N, N-dimethyl-aminesuccinamic acid) and IAA, as the inhibitive action of B-9, 0.06% solution on the growth of lettuce hypocotyls was reduced to a highly significant degree when the plants were supplied with B-9 together with IAA at a concentration of 10 mg/l.     

Einfluß von Pasten mit verschiedenen Indolylessigsäure-Konzentrationen auf den Transport von 32P in dekapitierten Erbsenepikotylen

Summary Either a 0.25% or a 0.007% IAA paste was smeared on the apical cut surface of decapitated pea epicotyls and simultaneously ³²P was added to the roots. When subsequently the radioactivity of the apical segment of the epicotyls was measured at different time intervals following the addition of ³²P, no significant differences between the effect of 0.25 and 0.007% IAA paste on ³²P transport were found. Because the 0.25% IAA paste completely retards the growth of cotylary buds whereas the 0.007% paste supports this growth, it might appear that the effect of 0.25% IAA paste on the transport of nutrient substances plays a decisive role in the apical dominance. More probably 0.25% IAA paste imitates the apical dominance only by means of its toxic effect, which, however, requires a detailed investigation.     

The effect of amputation of the epicotyl on the level of endogenous gibberellins in the roots of pea seedlings

The content of endogenous gibberellins was determined chromatographically in the roots 14–17 days old pea seedlings cultivated in water cultures in the dark. When the epicotyls are amputated from these plants, the content of endogenous gibberellins increases significantly within 6–12 hours after amputation as compared with the intact controls, then it falls off again considerably up to 24 hours after amputation. The initial increase of the gibberellin level in the roots can be explained by transport inhibition of the endogenous gibberellins from the root to the epicotyl, the later decrease of this level to be interpreted as inhibition of auxin transport from the epicotyl into the root. This is supported by the observation that spreading of a 0.5% paste with IAA over the epicotyl stump immediately after amputation prevents the mentioned decrease of the gibberellin level in the roots, whereas this decrease is intensified by using a paste with TIBA which inhibits the auxin transport. The results of this work support the possibility of direct gibberellin synthesis in the roots.     

The role of endogenous cytokinins in correlation between cotyledon and its axillary bud and in hypocotyl regeneration of flax

When flax seedlings are decapitated above cotyledons and three days later one of the two cotyledons is removed then the remaining cotyledon stimulates in four to five days growth of its axillary bud. It has been found that content of endogenous cytokinins was higher in the stimulated bud as compared with the other one already 12 h after the cotyledon removal. Flax seedlings decapitated under cotyledons regenerate adventitious buds on thy hypocotyl stump during 5–6 days. The endogenous fytohormonal preparation of this regeneration was investigated in the 20 mm apical part of the hypocotyl stump. Decrease in auxin and increase in gibberellins was already found during the first day after decapitation while the level of cytokinins increased as late as three days after the apex removal.     

Transport of benzyl-8-14C-adenine in pea seedlings in relation to stem apical dominance

In intact, decapitated and decapitated indole-3-acetic acid (IAA) treated pea seedlings the translocation of benzyl-8-^l4C-adenin (¹⁴C-BA) from the roots was studied with regard to the release of lateral buds from apex-induced inhibition. In intact plants (controls) a substantial part of the activity was found in the apical part of the epicotyl. Decapitation resulted in the initiation of growth of lateral buds. As early as 24 h after decapitation and application of¹⁴C-BA a significantly higher activity was found in growing lateral buds (cotylars) of decapitated plants than in inhibited ones of intact or IAA-treated decapitated plants. The accumulation of¹⁴C-activity in stump tops of decapitated plants treated with IAA was associated with the thickening growth.     

Translocation of14C-abscisic acid from roots into the aboveground part of pea (pisum sativum L.) seedlings

The translocation of¹⁴C-ABA from roots into other parts of the plant was followed in intact and decapitated pea seedlings. In intact plants ABA from roots was translocated above all into the apical part of epicotyl. In decapitated plants the regulative ability of intact apex can be partly simulated by exogenous IAA. The growth of lateral buds occurring after decapitation was associated with an intensive flow of¹⁴C-ABA from roots into released lateral buds as late as 72 h after decapitation,i.e. in the stage of intensive elongation growth of buds.     

The decrease in auxin polar transport down the lupin hypocotyl could produce the indole-3-acetic Acid distribution responsible for the elongation growth pattern

The variation of indole-3-acetic acid (IAA) transport along Lupinus albus L. hypocotyls was studied using decapitated seedlings and excised sections. To confirm that the mobile species was IAA and not IAA metabolites, dual isotope-labeled IAAs, [5-³H]IAA + [1-¹⁴C]IAA, were used. After apical application to decapitated seedlings, the longitudinal distribution of both isotopes at different transport periods showed that the velocity of IAA transport was higher in the apical, elongating region than in the basal, non-growing region. This variation in velocity was not a traumatic consequence of decapitation because after application of IAA to the basal region of decapitated seedlings, both the velocity and intensity of IAA transport were lower than in the apical treatment. The variation in IAA transport down the hypocotyl was confirmed when it was measured in excised sections located at different positions along the hypocotyl. The velocity and, to a greater extent, the intensity of IAA transport decreased from the apical to the basal sections. Consequently, if the amount of IAA reaching the apical zones of lupin hypocotyl were higher than the IAA transport capacity in the basal zones, accumulation of mobile IAA might be expected in zones located above the basal region. In fact, an IAA accumulation occurred in the elongating region during the first 4-h period of transport after apical treatment with IAA. It is proposed that the fall in IAA transport along the hypocotyl might be responsible for the IAA distribution and, consequently, for the growth distribution reported in this organ. An indirect proof of this was obtained from experiments that showed that the excision of the slowly transporting basal zones strongly reduced the growth in the remaining part of the organ, whereas excision of the root caused no significant modification in growth during a 20-h period.     

The Role of Endogenous Abscisic Acid in the Correlation Between the Cotyledon and Its Axillary Bud in PeaPisum sativum L

On cutting off one cotyledon from decapitated pea seedlings cultivated in the dark, the apical dominance is restored, as is well-known, by the growth of the bud of the removed cotyledon. As early as 12 h following cotyledon amputation(i.e. at the time when buds of both cotyledons-remaining and removed-are not yet differentiated in size), a decrease in the level of endogenous abscisic acid can be demonstrated in the bud of the removed cotyledon.     

Changes in the content of endogenous IAA and cytokinins in cotyledons of etiolated pea seedlings during their ontogenesis

The inhibitive growth-correlative effect of cotyledons of pea seedlings decreases during their ontogenesis till the age of 14 days. This decrease is associated with an increase in the level of endogenous cytokinins on the one hand and a decrease of endogenous IAA on the other. This is in harmony with the fact that the correlative-inhibiting effect of pea cotyledon upon the growth of its axillary bud can be weakened by exogenous cytokinin and amplified by exogenous IAA.     

Isolation and identification of lateral bud growth inhibitor,indole-3-aldehyde,involved in apical dominance of pea seedlings

A lateral bud growth inhibitor was isolated from etiolated pea seedlings and identified as indole-3-aldehyde. The indole-3-aldehyde content was significantly higher in the diffusates from explants with apical bud and indole-3-acetic acid treated decapitated explants, in which apical dominance is maintained, than in those from decapitated ones releasing apical dominance. When the indole-3-aldehyde was applied to the cut surface of etiolated decapitated plants or directly to the lateral buds, it inhibited outgrowth of the latter. These results suggest that indole-3-aldehyde plays an important role as a lateral bud growth inhibitor in apical dominance of pea seedlings.     

Effect of Abscisic Acid and Other Plant Hormones on Growth of Apical and Lateral Buds of Seedlings

The effect of abscisic acid (AbA) on the growth of lateral and apical buds was studied in seedlings of Pisum sativum and some other species. The hormone was applied in three different ways: 1) directly to the lateral bud on the second node of decapitated pea seedlings as 5 μI droplets in an ethanolic solution; 2) to the cut surface of decapitated seedlings: 3) to the apical bud of intact plants. AbA directly applied in amounts of 5 to 0.1 μg to the lateral bud of the second node of decapitated seedlings had a strong inhibitory effect on the bud. Application to the cut surface of seedlings decapitated about 5 mm above the second node resulted in slight inhibition of the lateral bud on the second node and in growth promotion of the bud on the first node. When AbA at 10 to 0.1 μg was applied to the apical bud of intact seedlings, the growth of this bud was inhibited but the lateral buds grew out. It is concluded that the release of the lateral buds from apícal dominance is the result of the inhibitory effect of AbA on growth of the apical bud and of low transport of AbA. This conclusion is supported by application of GA₃ and IAA, individually and each combined with AbA.