Effect of horizontal clinorotation on the root system development and on lipid breakdown in rapeseed (Brassica napus) seedlings

Seedlings of Brassica napus were cultivated on a slowly rotating clinostat (1 rpm) or in the vertical control for 5 d. The root growth, the cotyledonary reserves and the transport of 14C-labeled sucrose from cotyledons to root system were studied in both cultural conditions. The biomass (fresh weight) of the root system was 35% higher in the horizontally clinorotated seedlings than in the controls. This increase was correlated with a greater degradation of reserve lipids and faster accumulation of sucrose in the cotyledons. The activity of isocitrate lyase, one of the two enzymes necessary to conversion of lipids into glucids, was also greater in the cotyledons of clinorotated seedlings. The labeling distribution of 14C in the cotyledons, the hypocotyl and the root system after 30, 60 and 120 min of application of 14C-labeled sucrose on the cotyledons showed higher translocation of the cotyledonary sucrose to the root system of clinorotated seedlings. In addition, we studied the effects of clinorotation on the biomass of the excised root system (of 10 d old seedlings) cultivated in a medium containing 1% sucrose. The horizontally clinorotated root system grew more than that of the controls. These results showed that the horizontal clinorotation acted on the root system growth and provoked a higher sucrose translocation from source to sink, i.e. from cotyledons to root system.     

Automorphosis of etiolated pea seedlings in space is simulated by a three-dimensional clinostat and the application of inhibitors of auxin polar transport

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under microgravity conditions in space show automorphosis: bending of epicotyls, inhibition of hook formation and changes in root growth direction. In order to determine the mechanisms of microgravity conditions that induce automorphosis, we used a three-dimensional clinostat and obtained the successful induction of automorphosis-like growth of etiolated pea seedlings. Kinetic studies revealed that epicotyls bent at their basal region towards the clockwise direction far from the cotyledons from the vertical line (0 degrees) at approximately 40 degrees in seedlings grown both at 1 g and in the clinostat within 48 h after watering. Thereafter, epicotyls retained this orientation during growth in the clinostat, whereas those at 1 g changed their growth direction against the gravity vector and exhibited a negative gravitropic response. On the other hand, the plumular hook that had already formed in the embryo axis tended to open continuously by growth at the inner basal portion of the elbow; thus, the plumular hook angle initially increased; this was followed by equal growth on the convex and concave sides at 1 g, resulting in normal hook formation; in contrast, hook formation was inhibited on the clinostat. The automorphosis-like growth and development of etiolated pea seedlings was induced by auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid), but not by anti-auxin (p-chlorophenoxyisobutyric acid) at 1 g. An ethylene biosynthesis inhibitor, 1-aminooxyacetic acid, inhibited hook formation at 1 g, and ethylene production of etiolated seedlings was suppressed on the clinostat. Clinorotation on the clinostat strongly reduced the activity of auxin polar transport of epicotyls in etiolated pea seedlings, similar to that observed in space experiments (Ueda J, Miyamoto K, Yuda T, Hoshino T, Fujii S, Mukai C, Kamigaichi S, Aizawa S, Yoshizaki I, Shimazu T, Fukui K (1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J Plant Res 112: 487492). These results suggest that clinorotation on a three-dimensional clinostat is a valuable tool for simulating microgravity conditions, and that automorphosis of etiolated pea seedlings is induced by the inhibition of auxin polar transport and ethylene biosynthesis.     

Changes in hormonal balance and meristematic activity in primary root tips on the slowly rotating clinostat and their effect on the development of the rapeseed root system

The morphometry of the root system, the meristematic activity and the level of indole-3-acetic acid (IAA), abscisic acid (ABA) and zeatin in the primary root tips of rapeseed seedlings were analyzed as functions of time on a slowly rotating clinostat (1 rpm) or in the vertical controls (1 rpm). The fresh weight of the root system was 30% higher throughout the growth period (25 days) in clinorotated seedlings. Morphometric analysis showed that the increase in biomass on the clinostat was due to greater primary root growth, earlier initiation and greater elongation of the secondary roots, which could be observed even in 5-day-old seedlings. However, after 15 days, the growth of the primary root slowed on the clinostat, whereas secondary roots still grew faster in clinorotated plants than in the controls. At this time, the secondary roots began to be initiated closer to the root tip on the clinostat than in the control. Analysis of the meristematic activity and determination of the levels in IAA, ABA and zeatin in the primary root tips demonstrated that after 5 days on the clinostat, the increased length of the primary root could be the consequence of higher meristematic activity and coincided with an increase in both IAA and ABA concentrations. After 15 days on the clinostat, a marked increase in IAA, ABA and zeatin, which probably reached supraoptimal levels, seems to cause a progressive disturbance of the meristematic cells, inducing a decrease of primary root growth between 15 and 25 days. These modifications in the hormonal balance and the perturbation of the meristematic activity on the clinostat were followed by a loss of apical dominance, which was responsible for the early initiation of secondary roots, the greater elongation of the root system and the emergence of the lateral roots near the tip of the primary root.     

Early seedling growth and morphogenesis in the <Emphasis Type="Italic">xantha1</Emphasis> mutant of sunflower with alteration of chloroplast biogenesis

In the xantha1 (xan1) mutant of sunflower (Helianthus annuus L.), the effects on organ anatomy and seedling growth did correlate to the alteration of chloroplast biogenesis. The xan1 seedlings grown under 165 μmol(photon) m⁻² s⁻¹ revealed a severely altered chloroplast ultrastructure in cotyledons and leaves. Cross-sections or clarified tissues of the xan1 cotyledons did not show evident alterations with respect to normal cotyledons suggesting that the impairment of chloroplast biogenesis has negligible consequences on embryonic leaves. By contrast, the analysis of xan1 leaves showed that the defects in chloroplast biogenesis were correlated to a drastic reduction of organ size and to a clear enhancement of the trichome growth. The differentiation of palisade and spongy parenchyma in cotyledons and leaves of the xan1 mutant was normal but both organs displayed a drastic reduction in the plastid number with respect to wild type. In addition, xan1 hypocotyls showed a reduced development of the main vascular bundles in comparison with normal seedlings and an undersized central cylinder of the primary root. The exogenous supply of sucrose was not sufficient to revert in vitro the deficit of xan1 growth and the constraints in morphogenetic processes.     

Indole acetic acid mimics the effect of salt stress in relation to enzymes of carbohydrate metabolism in chickpea seedlings

The effect of addition of indole acetic acid (3 M) andNaCl (75 mM) on growth and enzymes of carbohydrate metabolism inchickpea seedlings was compared. In comparison with control seedlings, theseedlings growing in the presence of indole acetic acid (IAA) had reducedamylase activity in cotyledons and enhanced sucrose synthase (SS) and sucrosephosphate synthase (SPS) activities in cotyledons and shoots at all days ofseedling growth. Compared with control seedlings, sucrose content was higher incotyledons, shoots and roots and reducing sugar content was lower in shoots ofIAA treated seedlings. A low invertase (acid and alkaline) activity in shoots ofIAA treated seedlings could lead to reduced sink strength and hence decreasedgrowth of seedlings. Effects of NaCl stress on growth and activities of amylase,SS and SPS in cotyledons and invertase, SS and SPS in shoots were similar tothose observed with addition of IAA.     

The role of the apex in normal and tropic growth of sunflower hypocotyls

Regional growth in vertical and horizontal etiolated sunflower hypocotyls from which the apical hook tissue had been either partly or wholly excised, was measured 24 h later, the regions having been demarcated with resin beads. Removal of the cotyledons (an excision which included the distal end of the shoot apex) had little effect on growth during this period but excision of the apical hook significantly reduced growth. In vertically orientated seedlings, removal of half of the hook severely reduced growth in all other growing regions and removal of the entire hook totally inhibited growth. This inhibition of growth was not a consequence of the removal of the region of growth but a consequence of the removal of a region on which growth was dependent. In horizontal seedlings, the situation was more complex inasmuch as a horizontal orientation itself induced growth in previously non-growing regions. This new growth was localised in its extent and was not as severely affected by progressive excision of the hook as was growth in vertical seedlings. The results are discussed in terms of overall growth co-ordination in the hypocotyl.     

Pea seedling growth and development regulated by cotyledons and modified by irradiance

Growth and development of hydroponically grown pea seedlings ( Pisum sativum L. cv. Alaska) were measured using stem and root length as well as number of leaves and lateral roots. The growth was dependent on the presence of cotyledons and was modulated by the irradiance. All plants were grown in a full nutrient solution. If grown at low irradiance (73 μmol m^-2s^-1) they depended more and for a longer time on the cotyledons than plants grown at high irradiance (220 μmol m^-2s^-1). Low irradiance caused stem elongation but decreased root length and number of lateral roots as compared to plants grown at high irradiance. The dark respiration of the leaves was measured as oxygen uptake. In plants grown at the low irradiance, excision of the cotyledons caused the rate of oxygen uptake to increase by a factor of three, and the increase was sensitive to cyanide. Decotyledonized plants showed a high respiration rate and a diminished leaf growth for their entire life cycle. CO₂ fixation also increased in decotyledonized pea seedlings grown at either irradiance. The mobilization of food reserves from the seeds was positively correlated to seed dry weight, but only if the plants were grown at 73 μmol m^-2s^-1. Increasing dry weight of the seed enhanced top growth, whereas root growth was depressed, so that top and root responds differently with regard to that part of growth which depends on mobilization of reserves from the seed.     

Consumption of wheat seed reserves during germination and early growth as affected by soil water potential

Seed size and weight are important criteria for determining seedling vigour and stand establishment. Evolution of seed dry weight of wheat (Triticum aestivum L.) during germination and early growth was examined because poor stands are often associated with the depletion and exhaustion of seed reserves. Two laboratory experiments were conducted on filter paper and in soil at three water potentials using wheat seeds. Seed, root, and shoot dry weights were recorded at approximately one-day intervals. Coleoptile and first leaf lengths were also measured at all sampling periods. Wheat seedlings grown on filter paper in the dark grew to a length of 90 to 100 mm with 50% of the initial seed weight remaining after eight days when the experiment was terminated. In soil, wheat seedlings grew 15 mm with 25% of the initial seed weight remaining. Seed reserves were depleted more quickly when the soil was wet because seedlings grew more quickly. There were significant and similar negative relationships between seed weight and coleoptile length of wheat seedlings grown on filter paper and in soil. There was no effect of soil water potential on the relationship between seed weight and shoot length. Therefore, it was concluded that poor wheat stands are not likely to occur due to depletion of seed reserves under field conditions without mechanical obstacles.     

Regulation by sucrose of storage compounds breakdown in germinating seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus albus L.) and Andean lupine (Lupinus mutabilis Sweet): I. Mobilization of storage protein

Research of the regulatory function of sucrose in storage protein breakdown was conducted on isolated embryo axes, excised cotyledons and whole seedlings of three lupine species grown in vitro on medium with 60 mM sucrose or without the sugar. Sucrose stimulated growth of yellow, white and Andean lupine isolated embryo axes and cotyledons but growth of seedlings was inhibited. Dry matter content was higher in sucrose-fed isolated organs and in seedling organs. Ultrastructure research revealed that lack of sucrose in the medium caused enhancement in storage protein breakdown. Protein deposits in cotyledons were smaller as well as soluble portion content in all studied organs was lower when there was no sucrose in the medium. In the same conditions, the activity of glutamate dehydrogenase was significantly higher. Increase in vacuolization of cells of white lupine root meristematic zone cells was observed and induction of autophagy in young carbohydrate-starved embryo axes is discussed.     

Correlative Studies on Plant Growth and Metabolism II. Effect of Light and of Gibberellic Acid on the Changes in Protein and Soluble Nitrogen in Lettuce Seedlings

Protein and soluble nitrogen distribution in different parts of lettuce seedling was studied in light and darkness and in presence and absence of gibberellic acid. In dark, applied gibberellic acid failed to show any marked effect on the nitrogen changes in lettuce. Light inhibits translocation of nitrogen reserves from the cotyledons. Gibberellic acid reverses the light inhibition of longitudinal growth but has no effect on the inhibition of translocation from the cotyledons. Light grown, gibberellic acid treated seedlings exhibit a pattern of protein and soluble-N which is characteristic of the dark grown seedlings. Thus gibberellic acid not only causes morphological reversal of light inhibition but also shifts the nitrogen metabolism of light grown plants, close to that of plants grown in darkness.     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Stem length, number of secondary lateral roots, shoot dry weight and reducing sugar concentrations of root were significantly reduced when translocation of reserves from cotyledons to the roots of Afzelia africana seedlings was interrupted by complete or partial cotyledon excision. The sucrose but not the glucose concentration of lateral roots also decreased significantly after complete cotyledon excision. Hartig net development rather than fungal sheath formation was affected after inoculation with the early fungal isolate E1 and by both late-stage fungal isolates L1 and L2 after partial or complete cotyledon excision. However, mycorrhizal colonization by the early fungal isolate E2 was not affected by cotyledonary reserves, suggesting that this fungal isolate has a lower carbohydrate requirement than fungal isolates E1, L1 and L2. The late-stage fungal isolates L1 and L2 induced a hypersensitivity reaction by epidermal cell walls of the host plant after complete cotyledon excision, suggesting they are more dependent than the early fungal isolate E1 on available root carbohydrate substrates for ectomycorrhizal colonization. These results are discussed in the light of the hypothesis that early and late-stage fungi were different carbohydrate requirements, and that the time sequence of colonization was related to the root carbohydrate status, which increased with time.     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Gravity-controlled asymmetrical transport of auxin regulates a gravitropic response in the early growth stage of etiolated pea (Pisum sativum) epicotyls: studies using simulated microgravity conditions on a three-dimensional clinostat and using an agravitropic mutant, ageotropum

Increased expression of the auxin-inducible gene PsIAA4/5 was observed in the elongated side of epicotyls in early growth stages of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown in a horizontal or an inclined position under 1 g conditions. Under simulated microgravity conditions on a 3D clinostat, accumulation of PsIAA4/5 mRNA was found throughout epicotyls showing automorphosis. Polar auxin transport in the proximal side of epicotyls changed when the seedlings were grown in a horizontal or an inclined position under 1 g conditions, but that under clinorotation did not, regardless of the direction of seed setting. Accumulation of PsPIN1 and PsPIN2 mRNAs in epicotyls was affected by gravistimulation, but not by clinorotation. Under 1 g conditions, auxin-transport inhibitors made epicotyls of seedlings grown in a horizontal or inclined position grow toward the proximal direction to cotyledons. These inhibitors led to epicotyl bending toward the cotyledons in seedlings grown in an inclined position under clinorotation. Polar auxin transport, as well as growth direction, of epicotyls of the agravitropic mutant ageotropum did not respond to various gravistimulation. These results suggest that alteration of polar auxin transport in the proximal side of epicotyls regulates the graviresponse of pea epicotyls.     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Effect of Kinetin on Starch and Sucrose Metabolising Enzymes in Salt Stressed Chickpea Seedlings

Higher amylase activity in cotyledons of kinetin treated salt stressed (75 mM NaCl) chickpea (Cicer arietinum L. cv. PBG-1) seedlings, as compared to salt stressed seedlings was observed during a growth period of 7 d. The activities of acid and alkaline invertases were maximum in shoots and minimum in cotyledons under all conditions. The reduced shoot invertase activities under salt stress were enhanced by kinetin with a simultaneous increase in reducing sugar content. Kinetin increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in both the cotyledons and shoots of stressed seedlings. Kinetin appears to increase the turnover of sucrose in the shoots of stressed seedlings.