Effects of short light-dark regimes on in vitro shoot rooting of some fruit tree rootstocks

Experiments were carried out to evaluate the effects of 4/2 light-dark cycles (4 h of light followed by 2 h of dark) on the rooting responses of shoots cultivated in vitro of the fruit tree rootstocks GF 677 (peach × almond hybrid), Mr.S. 2/5 (Prunus cerasifera), MM 106 (apple Nothern Spy × Paradise M1) and BA 29 (Cydonia oblonga). Under this light regime rooting percentage of GF 677, Mr.S. 2/5 and MM 106 shoots reached 100 % as in the control treatment (16/8), while in BA 29 shoots, short light-dark cycles increased rooting response by about 65 %. Moreover, the shoots of all rootstocks submitted to the 4/2 cycle showed an appreciable increase in root number and length, and an earlier root emergence of about 4 – 5 d compared to the 16/8 cycle. Finally, rooting percentage of BA 29 shoots submitted to the 4/2 light regime and treated with 0.2 mg dm⁻³ indolebutyric acid (IBA), was equal to that reported with 0.4 mg dm⁻³ IBA under the 16/8 regime, indicating that the former light regime also amplified the rhizogenic effect of auxin.     

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

Cadaverine turnover in soybean seedlings using 15N-labelled lysine and cadaverine

The synthesis and translocation of the diamine cadaverine during soybean (Glycine max L. Meer cv. Sakai) germination were studied using ¹⁵N-labelled lysine (the cadaverine precursor) and ¹⁵N-labelled cadaverine, both under light/dark (12 h/12 h) and total dark germinating conditions. ¹⁵N-cadaverine and non-labelled polyamines were simultaneously detected using ionspray ionization–mass spectrometry. Both ¹⁵N-cadaverine and ¹⁵N-lysine were taken up by soybean. ¹⁵N-lysine was transported to the shoot and root and converted into ¹⁵N-cadaverine, whereas relatively little ¹⁵N-cadaverine was formed from ¹⁵N-lysine in the cotyledon. The acropetal translocation of ¹⁵N-cadaverine from the cotyledon to the shoot seemed to predominate over basipetal transport to the root. Although no other ¹⁵N-derivatised polyamines were found, supplying exogenous ¹⁵N-lysine seemed to indirectly affect the metabolism of ¹⁴N putrescine, spermidine and spermine, while no significant effect was detected after supplying ¹⁵N-cadaverine.     

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the ¹⁵N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH ₊ ⁴ was replaced by NO _- ² . N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of ¹⁵N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized ¹⁵NO _- ³ and accumulated it as reduced ¹⁵N, predominantly in the shoots. Accumulation of reduced ¹⁵N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced ¹⁵N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.Symbols Anl accumulation of reduced ¹⁵N from ¹⁵NO _- ³ in ¹⁴NO _- ³ -fed roots of divided root system - Ar accumulation in root of reduced ¹⁵N from ¹⁵NO _- ³ - As accumulation in shoot of reduced ¹⁵N from ¹⁵NO _- ³ - Rr ¹⁵NO _- ³ reduction in root - Rs ¹⁵NO _- ³ reduction in shoot - Tp translocation to root of shoot-reduced ¹⁵N from ¹⁵NO _- ³ in phloem - Tx translocation to shoot of root-reduced ¹⁵N from ¹⁵NO _- ³ in xylem     

Uptake and effects of N-benzyladenine in excised watermelon cotyledons: influence of cotyledon age

Some characteristics of uptake of [8-¹⁴C]N⁶-benzyladenine (BA) by watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.

The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower in day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.

In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision.

     

Photoperiodic control of hibernation in Helix pomatia L. (Gastropoda: Pulmonata)

Different groups of Helix pomatia were exposed to short light pulses (1 or 2 hours) during a long dark period (16 or 14 hours) of a 24-hour cycle of light and dark. The effect of the light pulses on the hibernation of the snails was shown to depend on the circadian time the pulses were introduced. Some of these light pulses reduced the hibernation. In other experiments groups of snails were exposed to 12-hour cycles or 24-hour cycles of equal periods of light and dark. Hibernation was reduced by the former as compared to the latter. These results show that Helix pomatia exhibits photoperiodic control of hibernation by a discontinuous or cyclic mechanism of time measurement.     

Dependency of Nitrate Reduction on Soluble Carbohydrates in Primary Leaves of Barley under Aerobic Conditions

Nitrate reduction was studied as a function of carbohydrate concentration in detached primary leaves of barley (Hordeum vulgare L. cv Numar) seedlings under aerobic conditions in light and darkness. Seedlings were grown either in continuous light for 8 days or under a regimen of 16-hour light and 8-hour dark for 8 to 15 days. Leaves of 8-day-old seedlings grown in continuous light accumulated 4 times more carbohydrates than leaves of plants grown under a light and dark regimen. When detached leaves from these seedlings were supplied with NO₃⁻ in darkness, those with the higher levels of carbohydrates reduced a greater proportion of the NO₃⁻ that was taken up. In darkness, added glucose increased the percentage of NO₃⁻ reduced up to 2.6-fold depending on the endogenous carbohydrate status of the leaves. Both NO₃⁻ reduction and carbohydrate content of the leaves increased with age. Fructose and sucrose also increased NO₃⁻ reduction in darkness to the same extent as glucose. Krebs cycle intermediates, citrate and succinate, did not increase NO₃⁻ reduction, whereas malate slightly stimulated it in darkness.

In light, 73 to 90% of the NO₃⁻ taken up was reduced by the detached leaves; therefore, an exogenous supply of glucose had little additional effect on NO₃⁻ reduction. The results indicate that in darkness the rate of NO₃⁻ reduction in primary leaves of barley depends upon the availability of carbohydrates.

     

Dark preincubation improves shoot organogenesis from Rhodiola crenulata leaf explants

An efficient in vitro plant regeneration system has been developed using dark preincubated leaf explants of Rhodiola crenulata, a traditional Tibetan medicinal plant. The leaf explants, preincubated in the dark for 5 d, developed an average of 9.1 shoots per explant on a medium containing 15 μM N ⁶-benzyladenine (BA) and 2.5 μM gibberellic acid (GA₃). The biochemical mechanism underlying dark-induced shoot organogenesis was investigated by measuring polyphenol oxidase (PPO) activity. Dark preincubation significantly reduced PPO activity in leaf explants during the initial period of shoot organogenesis and reduced browning compared to explants cultured in the light. Up to 88.4 % of the regenerated shoots formed roots and developed into complete plantlets on a medium containing 5 μM indoleacetic acid (IAA) within 25 d. Approximately 82 % of the regenerated plantlets survived transplantation and grew vigorously in the greenhouse.     

Diurnal Pattern of Translocation and Carbohydrate Metabolism in Source Leaves of Beta vulgaris L

Transitions in carbohydrate metabolism and translocation rate were studied for evidence of control of export by the sugar beet (Beta vulgaris L. Klein E.) source leaf. Steady-state labeling was carried out for two consecutive 14-hour light periods and various quantities related to translocation were measured throughout two 24-hour periods. Starch accumulation following illumination was delayed. Near the end of the light period, starch stopped accumulating, whereas photosynthesis rate and sucrose level remained unchanged. At the beginning of the dark period there was a 75-minute delay before starch was mobilized. The rate of import to the developing sink leaves at night was similar to that during the day, whereas export decreased considerably at night.

Starch accumulation and degradation seemed to be initiated in response to the level of illumination. Cessation of starch accumulation before the end of the light period was initiated endogenously. Exogenous control appeared to be mediated by the level of sucrose in the source leaf while endogenous control seemed to be keyed to photoperiod or photosynthetic duration.

     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Aspects of 8-[C]Benzylaminopurine Metabolism in Phaseolus vulgaris

Phaseolus vulgaris L. plants were supplied through the root with [8-¹⁴C]benzylaminopurine ([¹⁴C]BA). Collections of root, apex, and leaves were made 8 and 48 hours after labeling; ethanolic extracts of tissues were purified and subjected to thin layer chromatography on silica gel and/or cellulose powder.     

The Effect of Light on the Synthesis of Mitochondrial Enzymes in Division-synchronized Euglena Cultures

The development of the mitochondrial enzymes fumarase and succinate dehydrogenase has been followed in Euglena cultures division-synchronized by 14-hour light periods alternating with 12-hour dark periods. The activity of both enzymes was unaltered over the light phase, doubled in early dark phase, and thereafter remained constant over the rest of the cycle. The increase in enzyme activity in early dark phase probably represented de novo enzyme synthesis because it was prevented by the addition of cycloheximide at a concentration known to inhibit protein synthesis on Euglena cytoplasmic ribosomes.     

The uptake,translocation and release of phosphorus by Elodea densa

Short-term (16 h) laboratory studies of ³²P uptake by Elodea densa rooted in sediment demonstrated both foliar and root uptake, and that translocation occurred acropetally and basipetally. Root absorption is projected to provide 83–85% of total phosphorus uptake during 12–16 h photoperiod days. Measured foliar uptake and excretion rates suggest that there would be no net leakage of phosphorus into the water by undamaged actively-growing E. densa. Foliar uptake decreased and root uptake increased in the dark relative to rates under light.     

Light-enhanced protein synthesis in gravitropically stimulated root caps of corn

Light stimulates gravitropic bending (downward growth) in roots of many cultivars of corn (Zea mays). In this work, using the cultivar Merit, we show that light stimulates protein synthesis in the root cap, with protein levels increasing 1.3 to 1.6 times that recorded for tissues maintained in continuous dark. Light enhances protein levels both in intact caps (attached to the root) and in caps in culture. Protein synthesis is optimal in cultured caps when 1 nanomolar indole-3-acetic acid is included in the culture medium. If cap tissue is illuminated and subsequently returned to the dark, in the 2-hour period following illumination protein levels decline to that observed in dark controls. It is proposed that light-stimulated protein synthesis mediates in part downward bending in roots of these cultivars of corn.     

High responsiveness in <Emphasis Type="Italic">de novo</Emphasis> shoot organogenesis induction of <Emphasis Type="Italic">Passiflora cristalina</Emphasis> (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^?1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^?1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^?1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.     

Water balance of plants during root application of high concentrations of growth substances

Using hydroponic cultures, the effect of high concentrations (10^?3 m) of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in the root medium on the water balance of 8–9 week old plants ofPisum sativum L. and of 9–10 week-old plants ofSinapis alba L. was studied. The water balance was determined in the light and in the dark gravimetrically by measuring the intensity of water uptake and transpiration in plants cultivated by the method of root bridges according to Werner. MCPA present in the root medium in illuminated plants decreased rapidly the intensity of both the uptake and the loss components of the water balance. In permanent darkness, MCPA brought about an increase in the intensity of uptake of water and of transpiration. Simultaneous determination of water uptake and transpiration showed that the intensity of transpiration remained higher than the intensity of water uptake. This indicates that in the presence of MCPA in the root medium the relationship between the uptake and the loss components of water balance is not quantitatively equal, enboth in the light and in the dark. The existing disproportion results in the formation of a passive water balance of plants.     

The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu¹³², the homolog of Asp⁹⁶ of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp⁸⁵ and Asp⁹⁶ (Asp¹²¹ and Glu¹³²). We assigned a pair of FTIR bands (positive at 1749 cm⁻¹ and negative at 1734 cm⁻¹) to the protonation and deprotonation, respectively, of these carboxylic acids.     

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca²⁺. At a constant benzyladenine (BA) level (4.4×10^?5 M) 10^?4 to 10^?2 M Ca²⁺ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10^?6 M) concentration of external Ca²⁺, Chl synthesis was relatively more Ca²⁺-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca²⁺ from external sources to cytosol, and/or (b) interacted with Ca²⁺ directly in the pathway of Chl biosynthesis. The need of external Ca²⁺ was evidenced in experiments with modulators of Ca²⁺-transport systems. The reduction of the inward current of Ca²⁺ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N′N′-tetraacetic acid, EGTA) and Ca²⁺-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca²⁺. Inhibitory action of Ca²⁺-channel blockers depended on their kind and concentration: at the 10^?5 M concentration La³⁺>verapamil>nifedipine inhibited Chl formation. In the presence of Ca²⁺, the Ca²⁺-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.     

A system in which anthocyanin synthesis is induced in regenerated torenia shoots

A system in which anthocyanin synthesis can be induced under defined conditions was established in regenerated torenia shoots. Leaf discs prepared from torenia plantlets grown under sterile conditions were placed on solidified half-strength MS medium containing 3% sucrose and 4.4×10^–6 M benzyladenine (BA) and cultured under 16 h light/8 h dark (standard light) conditions for 10 days, then in the dark for a further 10 days. The discs were transferred to medium containing 7% sucrose without BA and cultured under standard light conditions. Six days after transfer, anthocyanin synthesis started in the regenerated shoots, and thereafter, anthocyanin accumulation increased while chlorophyll content decreased. Experiments in which either the timing of illumination was altered or shoots were retransferred to medium containing 1.5% sucrose or other sugars as well as sucrose indicated that both osmotic stress and light are required to induce anthocyanin synthesis. Once anthocyanin synthesis was induced in the torenia shoots 6 days after transfer, the shoots were fated to the synthesis of anthocyanins and the degradation of chlorophylls, and could not revert to the developmental pathway of shoot regeneration. This system may provide a good model for the investigation of the mechanisms underlying the induction of anthocyanin synthesis.     

Effect of Low-intensity Red and Far-red Light and High-intensity White Light on the Flowering Response of the Long-day Plant Lemna gibba G3

The long-day plant Lemna gibba L., strain G3 exhibits a relatively low sensitivity to short, white-light interruptions given during the dark period of a short-day cycle. However, the plants are fairly sensitive to low-intensity red light treatments given during a 15-hour dark period on the third day of a 2LD-(9L:15D)-2LD-7SD schedule. Far-red light is almost as effective as red light, and attempts to reverse the red light response with subsequent far-red light treatments have not been successful. Blue light proved to be without effect. When plants were grown on a 48-hour cycle with 15 minutes of red light every 4 hours during the dark period, the critical daylength was reduced from about 32 hours to slightly less than 12 hours.

Continuous red light induced a fairly good flowering response. However, as little as 1 hour of white light each day gave a significant improvement in the flowering response over that of the continuous red light control. White light of 600 to 700 ft-c was more effective than white light of 60 to 70 ft-c. The white light was much more effective when divided into 2 equal exposures given 8 to 12 hours apart. These results suggest an increase in light sensitivity with regard to flower induction about 8 to 10 hours after the start of the light period.