Growth and osmoregulation in Pisum sativum subhooks as affected by gibberellic acid and cotyledon excision

Growth stimulation by gibberellic acid (GA) of the Alaska pea ( Pisum sativum L.) subhook was observed within 6 h after its application; the stimulation being larger in cuttings with cotyledons than in decotylized ones. The osmotic potential in the subhook increased as it grew, the rate of its increase being faster in cuttings without than in cuttings with cotyledons. GA had no effect on the change in the osmotic potential until 8 h after GA application, but afterwards it suppressed the increase in cuttings with cotyledons. This GA effect was not observed in decotylized cuttings. Changes in the osmotic potential were well correlated with changes in the concentration of soluble sugars, but not with changes in amino acids and K⁺, Soluble sugars accumulated in the subhook of cuttings with and without cotyledons in proportion to growth, irrespective of the presence or absence of GA. Cotyledon excision suppressed sugar accumulation, and GA promoted it in cuttings with cotyledons but not in decotylized ones. These results suggest that GA stimulates the translocation of sugars from the cotyledons to the subhook and, thereby, maintains the osmotic potential low, resulting in enhanced growth.     

Effect of gibberellic acid on epicotyl growth and carbohydrate distribution in derooted Pisum sativum cuttings with or without cotyledons

The effect of gibberellic acid (GA) on subhook growth in derooted cuttings of pea ( Pisum sativum L. cv. Alaska) grown in the dark was studied in relation to the distribution of sugar-related compounds in the epicotyl and cotyledons. GA stimulated subhook growth of cuttings with or without cotyledons. In cuttings with cotyledons, the net inflow of sugar-related compounds (soluble sugars, starch, cell wall polysaccharides and sugars consumed by respiration) to the epicoiyl balanced with the net outflow from the cotyledons. GA stimulated the net inflow of sugar-related compounds to the epicotyl and the net outflow from cotyledons. Among these compounds, GA substantially increased the amount of soluble sugars, starch and cell wall polysaccharides in the subhook. In cuttings without cotyledons, on the other hand, the net inflow of sugar-related compounds to the subhook almost balanced with the net outflow from the epicotyl below the subhook. GA stimulated the net inflow of sugar-related compounds to the subhook and the net outflow from the epicotyl below the subhook. Among these compounds, GA substantially increased the amount of soluble sugars and cell wall polysaccharides in the subhook. These results suggest that GA stimulates an increase in the net inflow of sugar-related compounds to the subhook, thereby preventing an increase in osmotic potential and stimulating cell wall polysaccharide synthesis, when pea subhook growth is stimulated.     

Sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings - Stimulation of sugar exudation and invertase activity in epicotyls by gibberellic acid

The epicotyl of 5-day-old derooted cuttings of pea ( Pisum sativum L. cv. Alaska) with and without cotyledons exuded sucrose and glucose in the presence of EDTA. The amount of sugars exuded was greatly affected by the position at which the epicotyl was cut. The largest amount of sugars was exuded when the epicotyl was cut 2 mm below the hook, leaving the growing subhook. Gibberellic acid (GA) substantially increased the amount of sugars exuded from the epicotyl in the presence of cotyledons but only slightly in their absence. GA stimulated sugar exudation from the cotyledonary node as well as from the epicotyl. In cuttings with cotyledons, GA enhanced invertase activity in the apoplast, and in the intraceUular soluble and bound fractions in the growing subhook. In decotylized cuttings, GA enhanced only soluble invertase activity. GA did not affect invertase activity in the epicotyl below the subhook. These results suggest that GA stimulates sugar accumulation in the growing subhook by stimulating not only phloem loading of sucrose in the cotyledons but also unloading in the subhook.     

Gibberellin-enhanced sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings. Effects of gibberellic acid, indoleacetic acid and cycloheximide on invertase activity, sugar accumulation and growth

The possible involvement of invertase in the action of gibberellic acid (GA) on stimulating sugar accumulation in growing subhooks of Alaska pea ( Pisum sativum L. cv. Alaska) was studied. GA and indoleacetic acid (IAA) stimulated elongation growth to a similar extent. GA, in contrast to IAA, increased the amount of soluble sugars in the subhook. GA substantially increased invertase activity whereas IAA did not. These results suggest that the mode of action of GA and IAA differs, although both stimulate pea subhook growth.
Cycloheximide (CH) inhibited the effect of GA on invertase activity, accumulation of soluble sugars, and elongation growth. Good correlations were found between invertase activity, the amount of soluble sugars and growth. The results suggest that GA-induced enhancement of sugar accumulation in the subhook cells is dependent on increased invertase activity. The sugar accumulated in the subhook may be involved in growth promotion by GA.     

Inhibition of Pisum sativum epicotyl elongation by white light – Different effects of light on the mechanical properties of cell walls in the epidermal and inner tissues

White fluorescent light (5 W m⁻²) inhibited subhook growth in derooted Alaska pea cuttings. In the inner tissue of the subhook, it inhibited the increase in osmotic potential during 18 h incubation. In the epidermis, on the other hand, light did not affect the osmotic potential. Light increased the minimum-stress relaxation time (T₀) of the inner tissue cell walls, but did not change T₀ of the epidermal cell wall. Light decreased tissue stress determined by the split test and the ability of the inner tissue to extend by water absorption. The short-term light effect on subhook growth. T₀, and the tissue stress almost disappeared when pea cuttings were transferred to darkness. These facts suggest that light changes the mechanical properties of the cell wall in the inner tissue of shoots, and decreases tissue stress, which is considered to be the driving force of shoot growth.     

Growth Inhibition, Turgor Maintenance, and Changes in Yield Threshold after Cessation of Solute Import in Pea Epicotyls

The dependence of stem elongation on solute import was investigated in etiolated pea seedlings (Pisum sativum L. var Alaska) by excising the cotyledons. Stem elongation was inhibited by 60% within 5 hours of excision. Dry weight accumulation into the growing region stopped and osmotic pressure of the cell sap declined by 0.14 megapascal over 5 hours. Attempts to assay phloem transport via ethylenediaminetetraacetate-enhanced exudation from cut stems revealed no effect of cotyledon excision, indicating that the technique measured artifactual leakage from cells. Despite the drop in cell osmotic pressure, turgor pressure (measured directly via a pressure probe) did not decline. Turgor maintenance is postulated to occur via uptake of solutes from the free space, thereby maintaining the osmotic pressure difference across the cell membrane. Cell wall properties were measured by the pressure-block stress relaxation technique. Results indicate that growth inhibition after cotyledon excision was mediated primarily via an increase in the wall yield threshold.     

Effects of ethylene and gibberellic Acid on cellular growth and development in apical and subapical regions of etiolated pea seedling

Subhook swelling of 4-day-old etiolated pea seedlings (var. Alaska), caused by 0.5 microliter per liter ethylene, was prevented by preincubation and continued growth in 0.1 mm gibberellic acid (GA). The subhook region exhibited normal elongation and cell size and volume. However, inhibition of elongation and cessation of cell division caused by 0.5 microliter per liter ethylene in the apical hook region of the etiolated pea stem were not overcome by GA. Most of the arrested cells were in G(2). These data suggest a possible interaction of GA and ethylene in cell enlargement in the subhook region of the etiolated pea seedlings. They also suggest a different mode of action by ethylene in the apical hook region where the ethylene effect was not counteracted by GA.     

The role of auxin in growth of the plumular hook section of etiolated pea seedling

Externally applied GA greatly promoted elongation of the plumularhook section of the etiolated Alaska pea seedling, but IAA hadno such effect when given either alone or with GA. PCIB inhibitedelongation of the plumular hook section both in the presenceand absence of applied GA. The PCIB effect in the absence ofGA was partially overcome by IAA, but not completely. On theother hand, the PCIB effect in the presence of GA was completelyovercome by IAA. No antagonic response was, however, obtainedbetween GA and PCIB. CCC also retarded elongation of the sectionand this inhibition was completely overcome by GA, but not byIAA. There was little difference in the amount of endogenous auxindetectable in GA treated and untreated sections. These resultssuggest that auxin is necessary for the growth of both GA treatedand untreated plumular hook sections and that auxin and gibberellinact differently on the growth of the section. (Received April 24, 1968; )     

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.     

Effects of gibberellic acid and indole-3-acetic acid on stress-relaxation properties of pea hook cell wall

The effects of GA, IAA and PCIB on the cell wall propertiesof Alaska pea hooks were examined using stress-relaxation analysis.The results were:

1. GA caused a decrease in the stress-relaxation parameter To ofplumular hook sections after the first 30 min of incubation,long before it induced elongation.
2. PCIB increased To, andIAA tended to negate the PCIB effecton To in GA-treated sectionsafter 90 min of incubation, whenthe effect of PCIB and IAAon the elongation was not yet found.In this case, IAA couldnot be substituted by an extra amountof GA.
3. GA decreasedTo in the middle part of the sections after 24hr of incubation,and then stimulated elongation.
4. In any case, the effect ofGA, IAA or PCIB on To was recognizedin both epidermis and innertissue of plumular hook sections.
5. The stress-relaxation parameterTo appears to represent thecapacity of the cell wall to extend;we thus concluded thatboth gibberellin and auxin increase theextensibility of thecell wall, when they stimulate the elongationof plumular hooksections.

(Received October 4, 1974; )     

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 M and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.     

The Roles of the Cotyledons and Auxin in the Adventitious Root Formation of Hypocotyl Cuttings of Light-Grown Cucumber Seedlings

Adventitious root formation in encumber hypocotyl cuttings was studied. Root formation was quantitatively related to the amount of the cotyledons attached to the hypocotyl. Complete removal of the cotyledons diminished root formation entirely. Hut the removal of the apical bud had no effect. Treatment of the hypocotyl with triiodobenzoic acid resulted in the inhibition of root formation. On t he other hand, IAA promoted root formation. Promoting concentration of IAA was 1 mg/1 for the cuttings with intact cotyledons and 10 mg/1 for those with l/8th of the cotyledons. The first two or three days of treatment was most effective. The presence of auxin (IAA-like. substance) in cucumber seedlings was demonstrated by paper chromatography and the pea straight test. It is concluded that the cotyledon is necessary for root formation in cucumber hypocotyl cuttings and that auxin is at least one of the factors supplied from the cotyledons.     

Auxin Physiology of Dwarfism in Pisum sativum

Similar levels of diffusible auxin are measured for the apices of both Little Marvel (dwarf) and Alaska (normal) cultivars of the pea when grown in sunlight and darkness. In sunlight, however, diffusible auxin disappears in the subtending internode of the Little Marvel plant but remains at 50 per cent of the level of the apex in the subtending internode of the Alaska plant. The enzyme preparation from the apex of the dwarf plant converts tryptophan and tryptamine to IAA more readily than that from the normal plant. Indoleacetyl aspartate synthetase activity is also higher in the dwarf plant than in the normal plant and the dwarf plant contains four times as much conjugate as the normal plant with or without treatment with gibberellic acid. Gibberellic acid (GA) does not affect the induction of the synthetase enzyme nor the enzymatic formation of indoleacetyl aspartate. The growth induced by GA is the result of an increased synthesis of auxin.     

Effect of changing the endogenous concentration of auxins and cytokinins and the production of ethylene in pea stem cuttings on adventitious root formation

Decapitation or treatment with naphthylphthalamic acid (NPA) ndash; an inhibitor of IAA transport ndash; or a synthetic cytokinin N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) caused a decrease in rooting of pea cuttings. However the mode of action of the above treatments was different. Decapitation resulted in a decrease of indole-3-acetic acid (IAA) and a simultaneous increase in cytokinin content at the base of the cuttings. NPA decreased IAA even more, but did not influence cytokinins. CPPU alone or in combination with amino-ethoxy-vinylglycine (AVG) ndash; an ethylene biosynthesis inhibitor ndash; increased IAA at the rooting zone, but CPPU was transported from apex to the base of the cuttings where it inhibited rooting. NAA, applied alone after decapitation, stimulated rooting, probably partly by being an auxin, and partly by inhibiting the accumulation of cytokinins at the base of the cuttings. Treatment with AVG neither influenced rooting nor auxin or cytokinin content. Therefore, ethylene production does not seem to be one of the main factors involved in the reduced rooting after the various treatments.     

Polar transport and accumulation of indole-3-acetic acid during root regeneration by Pinus lambertiana embryos

Summary The relation of indoleacetic acid (IAA) transport to accumulation of auxin at the base of cuttings and to polar root formation was investigated with small cuttings from germinating embryos of Pinus lambertiana.The transport of endogenous auxin participates in regeneration of roots. This is shown by the facts that (1) more than 40% of the cuttings rooted without addition of exogenous indoleacetic acid; (2) the first regeneration always occurred at the basal tip of a slanting cut; and (3) 2,3,5-triiodobenzoic acid (TIBA), a specific inhibitor of auxin transport, totally inhibited rooting. Addition of IAA to the medium increased the number of roots formed per rooting hypocotyl.Sections of hypocotyls excised from dormant embryos and tested immediately after 2 h hydration were capable of polar transport of IAA. This polarity increased during the first 3 days of culture because of a marked increase in basipetal transport. Culturing the cuttings in 1 M IAA for 3–5 days doubled both the basipetal transport of 1-¹⁴C-IAA by hypocotyl segments and the accumulation of radioactivity at the base of cuttings.The extent of the accumulation at the base of cuttings was similar at early (2 days, first mitoses) and late stages (5 days, organized meristem) of regeneration and was not affected by removal of the regenerating region immediately prior to uptake and transport of ¹⁴C-IAA. The accumulation was inhibited by TIBA. In terms of increase in wet and dry weight and mitotic activity, the cotyledons rather than the regenerating root meristems were the most actively growing region of the cuttings. The upper part of the hypocotyl elongated more than the region of the slanting cut where regeneration was occurring.These results provide no support for the idea that the regenerating root controls the direction of polar transport by acting as a sink. The results are consistent with the view that polar auxin transport delivers auxin to the base of the cutting and raises the local concentration to levels sufficient to promote root formation.     

Osmoregulation in hypocotyls of etiolated mung bean seedlings with or without cotyledons in response to water-deficient stress

Effects of water-deficient stress and cotyledon excision on osmoregulation in hypocotyls of dark-grown mung bean seedlings were studied, and following results were obtained. Water-deficient stress inhibited hypocotyl elongation either in intact or decotylized seedlings. The inhibition was more conspicuous in decotylized seedlings than in intact ones. Water-deficient stress decreased osmotic potential in hypocotyls, while cotyledon excision increased it. The concentrations of soluble sugars, free amino acids and potassium ions in hypocotyls of intact or decotylized seedlings increased in response to water-deficient stress. Cotyledon excision reduced the concentration of soluble sugars and free amino acids, but it did not change the concentration of potassium ions, suggesting that a part of soluble sugars and free amino acids is transported from cotyledons. Unlike cotyledon excision, excision of the apex or roots had no influence on osmoregulation in response to water-deficient stress. Segments excised from hypocotyls had the ability to osmoregulate in response to water-deficient stress. Based on these results, the role of cotyledons in osmoregulation in response to water-deficient stress and quantitative relationships between osmotic potential and hypocotyl elongation in etiolated mung bean seedlings are discussed.     

GIBBERELLIN RELATIONSHIPS IN THE ‘ALASKA’ PEA (PISUM SATIVUM)

'Alaska’ peas (Pisum sativum L.) grown under a 16-hr photoperiod at 20 ± 1 C and an 8-hr dark period at 16 ± 1 C in their ontogeny exhibit two periods of sensitivity to applied gibberellin (GA), namely, prior to and subsequent to but not during the linear phase of stem elongation. This paper describes experiments conducted primarily with seedlings. Growth-saturating doses of GA, applied to dry seeds before planting (10⁻³ m) and to the shoot tips of 3-day-old seedlings (10 μg), evoked growth rates equal to the growth rate of etiolated seedlings. Sensitivity of seedlings to applied GA decreased with age through the first 2 to 3 weeks of development; by the time seedlings were about 14 days of age and had four elongating internodes they no longer responded to applied GA. As endogenous growth rate diminished late in ontogeny, the plants again became sensitive to applied GA. Growth response was used as a criterion for determining apparent translocation of applied GA. ‘Alaska’ pea seedlings appeared to transport GA, both acropetally from the cotyledons and basipetally from the shoot tip, to all internodes with remaining extension potential. Excision of both cotyledons at any time during the first 9 days of development caused a significant reduction of growth rate, and applied GA did not restore normal growth rate. No evidence was found that the cotyledons supply endogenous GA to the shoot axis in normal seedling development. It is suggested that the normal growth rate of light-grown ‘Alaska’ peas is correlated with the rate of synthesis of GA and that GA is rate-limiting for stem elongation during early seedling development and during the period of decreasing growth rate and onset of apex senescence.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.