Light-dependent Elongation of Anaerobically Maintained Green Pea Stem Segments and Its Implications

Anaerobic conditions reversibly inhibit the elongation of isolated green pea (Pisum sativum L. var Alaska) stem segments. Illumination of segments maintained under anoxia causes a resumption of growth. Polarographic studies show pea stem segments are photosynthetically competent as determined by O₂ evolution. Although O₂ production is totally inhibited by dichlorophenyldimethylurea (DCMU) and dinitrophenol (DNP) inhibits O₂-dependent growth, neither DCMU nor DNP completely abolishes light-dependent growth, although both reduce the effect markedly. Phenazine methosulfate promotes the growth of anaerobically maintained, illuminated, DCMU-treated segments. The data indicate that the principal effect of light in inducing growth under anaerobic conditions is the photosynthetic provision of O₂ for respiration. There is also some evidence that, at least in the absence of O₂, a small amount of elongation is due to some other light-driven process, perhaps cyclic photophosphorylation.     

Control of Auxin-induced Stem Elongathn by the Epidermis

Segments of the 4th and 5th internodes of light-grown pea seedlings were used for the study of control of stem elongation. With 5th internodes, at low turgor as well as at water saturation auxin primarily appeared to cause a change in cell wall properties of the epidermis but it showed little effect on expansion af the inner tissue. This was confirmed by comparison of expansion between peeled and unpeeled segments, split tests and by measurements of stress-relaxation properties of the epidermal cell wall. Segments with the central part re-moved elongated well in response to auxin, but the isolated epidermis showed neither auxin-induced elongation nor cell wall loosening. A fungal β-1,3-glucanase appeared, at least partly, to have a similar effect as that of auxin on elongation, by changing cell wall properties of the epidermal cell wall. Peeled segments of 4th internodes expanded very little and auxin had little effect on their epidermal cell wall properties.     

The Role of the Epidermis in the Control of Elongation Growth in Stems and Coleoptiles

The peripheral cell wall(s) of stems and coleoptiles are 6 to 20 times thicker than the walls of the inner tissues. In coleoptiles, the outer wall of the outer epidermis shows a multilayered, helicoidal cellulose architecture, whereas the walls of the parenchyma and the outer wall of the inner epidermis are unilayered. In hypocotyls and epicotyls both the epidermal and some subepidermal walls are multilayered, helicoidal structures. The walls of the internal tissues (inner cortex, pith) are unilayered, with cellulose microfibrils oriented primarily transversely. Peeled inner tissues rapidly extend in water, whereas the outer cell layer(s) contract on isolation. This indicates that the peripheral walls limit elongation of the intact organ. Experiments with the pressure microprobe indicate that the entire organ can be viewed as a giant, turgid cell: the extensible inner tissues exert a pressure (turgor) on the peripheral wall(s), which bear the longitudinal wall stress of the epidermal and internal cells. Numerous studies have shown that auxin induces elongation of isolated, intact sections by loosening of the growth-limiting peripheral cell wall(s). Likewise, the effect of light on reduction of stem elongation and cell wall extensibility in etiolated seedlings is restricted to the peripheral cell layers of the organ. The extensible inner tissues provide the driving force (turgor pressure), whereas the rigid peripheral wall(s) limit, and hence control, the rate of organ elongation.     

Control of Stem Elongation and Flowering in Scrophularia marilandica

The influence of temperature, photoperiod. and certain metabolities was determined for stem elongation and flowering in Scrophularia marilandica. Induction for flowering did not occur until several weeks after the beginning of rapid stem elongation. From the experiments reported it is concluded that S. marilandica is a high-temperature quantitative long-day plant. Temperatures above 20°C negate the absolute requirement for long days for flowering. Plants exposed to photoperiods as brief as 4 hours flowered, given high temperatures. Stem elongation was found to be a necessary prerequisite for flowering. The process of stem elongation was somewhat more sensitive to inhibition by low temperatures than flowering and to a great extent more sensitive than leaf formation and leaf growth. Vernalization was found to be unnecessary for stem elongation and flowering. Gibberellic acid promoted stem elongation and branching without flowering under conditions resembling cool short days. Other metabolities were tested but had no observable effects.     

Localization of Stem Elongation Control in Cucumis sativus L

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA₃, GA₄₊₇, and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA₄₊₇ was more effective than GA₃. Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA₄₊₇ or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.     

山茶属的叶表皮形态及其分类学意义

在光学显微镜下观察了山茶属（Camellia)19个组36个代表种的叶表皮形态,发现上述植物的叶上下表皮细胞表面观形状均为不规则形,气孔器仅在下表皮存在,且一致为环列型;对上述植物中18个组30个种的叶下表皮进行扫描电镜观察,其特片为：气孔形状为梭形或长卵形,气孔外拱盖内缘光滑或呈浅波状,波状,气孔器附近的角质膜平坦或皱褶,表面光滑或有多种纹饰,这些特征可作为区分种或变种的依据,但与植物的外部形态等特征没有相关性。     

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.     

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: II. Metabolic Requirements

The 2- to 4-fold rise in particle-bound β-glucan synthetase (uridine diphosphate-glucose: β-1, 4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca²⁺ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by α, α′-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.     

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: I. Kinetic Aspects

Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

A Microautoradiographic Study of Auxin Transport in the Stem of Intact Pea Seedlings (Pisum sativum L.)

Soluble-compound microautoradiography was used to determinethe distribution of radioactivity in transverse sections ofintact dwarf pea stems (Pisum sativum L.) following the applicationof [³H]IAA to the apical bud. Near the transport front labelwas confined to the cambial zone of the axial bundles, includingthe differentiating secondary vascular elements. Fully differentiatedphloem and xylem elements remained unlabelled and no radioactivitywas detected in the leaf or stipule traces. Similar resultswere obtained in experiments with Vicia faba L. plants. Nearerthe labelled apical bud of the pea there was a more generaldistribution of label and evidence was found of free-space transportof radioactive material in the pith. When [³H]IAA was applied to mature foliage leaves the greatestconcentration of label was found in the differentiated phloemelements of the appropriate leaf trace and in the phloem ofthe adjacent axial bundles. Both basipetal and acropetal transportwas detected in this case. These results are consistent with the conclusions drawn fromearlier transport experiments which indicated that in the intactplant the long-distance basipetal transport of auxin from theapical bud takes place in a system which is separated from thephloem transport system and suggests that the vascular cambiumand its immediate derivatives may function as the normal pathwayfor the longdistance movement of auxin in the plant. The physiologicalsignificance of such a transport system for auxin is discussed.     

Magnitude and Kinetics of Stem Elongation Induced by Exogenous Indole-3-Acetic Acid in Intact Light-Grown Pea Seedlings

Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tall (cv Alaska) peas (Pisum sativum L.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3 M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effectively promotes growth as it is transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (IGR) and prolonged growth response (PGR) induced by auxin. The IGR was inducible by at least 1 order of magnitude lower IAA concentrations than the PGR, suggesting that the process underlying the IGR is more sensitive to auxin induction. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tall seedlings. These results suggest that endogenous IAA is more growth limiting in dwarf plants than in tall plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of action as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tall plants, auxin cannot be the only limiting factor for stem growth in peas.     

Auxin Biosynthesis in Pea: Characterization of the Tryptamine Pathway

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.Auxin is a key plant growth hormone, involved in processes as diverse as branching, gravitropism, phototropism, and seed development (Davies, 2004). However, the biosynthetic pathways leading to the main auxin in plants, indole-3-acetic acid (IAA), are not well understood. Although there is good evidence that the amino acid Trp is an early precursor (Gibson et al., 1972; Wright et al., 1991; Tsurusaki et al., 1997), several routes from Trp to IAA have been proposed, and for any given species it is not clear which route or routes occur. The possible Trp-dependent pathways in higher plants are the indole-3-pyruvic acid (IPyA) pathway (Stepanova et al., 2008; Tao et al., 2008), the tryptamine (YUCCA) pathway (Zhao et al., 2001), the indole-3-acetaldoxime (IAOx) pathway (Bartel et al., 2001), and the indoleacetamide pathway (Pollmann et al., 2002), on the basis of the first metabolite of Trp (Fig. 1). In addition, a possible Trp-independent pathway has been proposed (Normanly et al., 1993), bypassing Trp completely, further complicating the process of IAA biosynthesis.Open in a separate windowFigure 1.Left: The putative tryptamine (red), Trp-independent (light blue), IPyA (green), indoleacetamide (yellow), and IAOx (dark blue) biosynthetic pathways to IAA in Arabidopsis. The steps shown in gray appear not to occur in peas. Right: The simplified pathway scheme suggested to occur in pea based on the present results and Sugawara et al. (2009). N-hydroxytryptamine was not detected as a metabolite in this study, suggesting that tryptamine might be converted directly to IAAld in pea roots. The Trp-independent, indoleacetamide, and IPyA pathways were not studied.Since 2001, there has been renewed interest in the tryptamine route to IAA, after the discovery and functional analysis of the Arabidopsis (Arabidopsis thaliana) YUCCA gene, reported to encode the enzyme for converting tryptamine to N-hydroxytryptamine (Zhao et al., 2001, 2002). On the basis of the Zhao et al. (2001, 2002) reports, tryptamine pathways have generally been proposed in the sequence: Trp, tryptamine, N-hydroxytryptamine, IAOx, indole-3-acetaldehyde (IAAld), IAA (Fig. 1; Woodward and Bartel, 2005). Recently, however, Sugawara et al. (2009) suggested that IAOx be removed from the tryptamine pathway, and that IAOx-dependent IAA biosynthesis operates only in the Brassicaceae. In Arabidopsis, this pathway can be important, at least in some circumstances, because when a side branch is impaired, as in the sur1 mutant, IAA levels increase dramatically (Sugawara et al., 2009). On the other hand, the IAOx pathway is not the only pathway operating in Arabidopsis, because genetically blocking the step Trp to IAOx does not always reduce IAA content, compared with wild-type plants (Sugawara et al., 2009). This means that in Arabidopsis, the tryptamine and/or IPyA and/or indoleacetamide pathways compensate for the loss of the IAOx pathway. Interestingly, Sugawara et al. (2009) do not include IAAld in their tryptamine pathway, and their model implies instead that N-hydroxytryptamine is directly converted to IAA.Turning to other species, it has been reported that tryptamine is not present in pea (Pisum sativum; Schneider et al., 1972), despite being present in tomato (Solanum lycopersicum; Cooney and Nonhebel, 1991), rice (Oryza sativa; Ishihara et al., 2008), Arabidopsis (Sugawara et al., 2009), and barley (Hordeum vulgare; Schneider et al., 1972). In tomato, although tryptamine is relatively abundant, and early metabolism studies indicated the conversion of tryptamine to IAA via IAAld (Schneider et al., 1972), Cooney and Nonhebel (1991) cast doubt on the role of tryptamine after studying patterns of labeling after incubation of plants with deuterated water. Again, in tobacco (Nicotiana tabacum), Songstad et al. (1990) showed that while tobacco plants overexpressing a Trp decarboxylase accumulated very high levels of tryptamine, IAA levels were unaffected. Although this result has been interpreted as evidence against the involvement of tryptamine (Bartel et al., 2001), another explanation is that excess tryptamine is converted to compounds via a side branch or side branches, although these are not well studied. Finally, the compound N-hydroxytryptamine is relatively unknown, with no reports of its presence in plants to date.It is clear, therefore, that the tryptamine pathway to IAA remains poorly understood. In this article, we further characterize the pathway, using the garden pea as a model species. We report on the presence/absence and levels of the putative endogenous intermediates, as determined by gas chromatography/tandem mass spectrometry (GC/MS/MS), and investigate their metabolic fates using [¹⁴C] and deuterated versions of the compounds. Our evidence indicates that key elements of the tryptamine pathway are operative in pea roots.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Circadian Control of the Accumulation of mRNAs for Light- and Heat-Inducible Chloroplast Proteins in Pea (Pisum sativum L.)

The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically in pea (Pisum sativum L.) plants either grown in a dark-light cycle or under constant light conditions. This has been observed for the early light-inducible protein, the light-harvesting chlorophyll a/b protein, and the small subunit of the ribulose-1,5-bisphosphate carboxylase. The mRNA levels are high in the morning, exhibit a minimum in the first half of the night, and increase again during the second half of the night. The amplitude of fluctuation is between 5- and 10-fold. A similar change in the mRNA abundance was found for four nuclear encoded heat-shock proteins of 18, 24, 26, and 30 kilodaltons. The ability of plants to transcribe heat-shock genes upon heat-shock for 2 hours varies through the day. The maxima for induction are found in the second half of the night and the morning. The minima are reached during the afternoon. The degree of fluctuation is between 3- and 5-fold. The levels of mRNAs for cytosolic as well as for plastid heat-shock proteins oscillate in parallel.