Sequential and coordinated action of phytochromes A and B during Arabidopsis stem growth revealed by kinetic analysis

Photoreceptor proteins of the phytochrome family mediate light-induced inhibition of stem (hypocotyl) elongation during the development of photoautotrophy in seedlings. Analyses of overt mutant phenotypes have established the importance of phytochromes A and B (phyA and phyB) in this developmental process, but kinetic information that would augment emerging molecular models of phytochrome signal transduction is absent. We have addressed this deficiency by genetically dissecting phytochrome-response kinetics, after having solved the technical issues that previously limited growth studies of small Arabidopsis seedlings. We show here, with resolution on the order of minutes, that phyA initiated hypocotyl growth inhibition upon the onset of continuous red light. This primary contribution of phyA began to decrease after 3 hr of irradiation, the same time at which immunochemically detectable phyA disappeared and an exclusively phyB-dependent phase of inhibition began. The sequential and coordinated actions of phyA and phyB in red light were not observed in far-red light, which inhibited growth persistently through an exclusively phyA-mediated pathway.

Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle

The glyoxylate cycle is regarded as essential for postgerminative growth and seedling establishment in oilseed plants. We have identified two allelic Arabidopsis mutants, icl-1 and icl-2, which lack the glyoxylate cycle because of the absence of the key enzyme isocitrate lyase. These mutants demonstrate that the glyoxylate cycle is not essential for germination. Furthermore, photosynthesis can compensate for the absence of the glyoxylate cycle during postgerminative growth, and only when light intensity or day length is decreased does seedling establishment become compromised. The provision of exogenous sugars can overcome this growth deficiency. The icl mutants also demonstrate that the glyoxylate cycle is important for seedling survival and recovery after prolonged dark conditions that approximate growth in nature. Surprisingly, despite their inability to catalyze the net conversion of acetate to carbohydrate, mutant seedlings are able to break down storage lipids. Results suggest that lipids can be used as a source of carbon for respiration in germinating oilseeds and that products of fatty acid catabolism can pass from the peroxisome to the mitochondrion independently of the glyoxylate cycle. However, an additional anaplerotic source of carbon is required for lipid breakdown and seedling establishment. This source can be provided by the glyoxylate cycle or, in its absence, by exogenous sucrose or photosynthesis.

Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, Cytochrome c Oxidase, and Adenine Nucleotide Translocase Activities1

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.

Two Genetically Separable Phases of Growth Inhibition Induced by Blue Light in Arabidopsis Seedlings1

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Rapid Regulation by Acid pH of Cell Wall Adjustment and Leaf Growth in Maize Plants Responding to Reversal of Water Stress1

The role of acid secretion in regulating short-term changes in growth rate and wall extensibility was investigated in emerging first leaves of intact, water-stressed maize (Zea mays L.) seedlings. A novel approach was used to measure leaf responses to injection of water or solutions containing potential regulators of growth. Both leaf elongation and wall extensibility, as measured with a whole-plant creep extensiometer, increased dramatically within minutes of injecting water, 0.5 mm phosphate, or strong (50 mm) buffer solutions with pH ≤ 5.0 into the cell-elongation zone of water-stressed leaves. In contrast, injecting buffer solutions at pH ≥ 5.5 inhibited these fast responses. Solutions containing 0.5 mm orthovanadate or erythrosin B to inhibit wall acidification by plasma membrane H+-ATPases were also inhibitory. Thus, cell wall extensibility and leaf growth in water-stressed plants remained inhibited, despite the increased availability of (injected) water when accompanying increases in acid-induced wall loosening were prevented. However, growth was stimulated when pH 4.5 buffers were included with the vanadate injections. These findings suggest that increasing the availability of water to expanding cells in water-stressed leaves signals rapid increases in outward proton pumping by plasma membrane H+-ATPases. Resultant increases in cell wall extensibility participate in the regulation of water uptake, cell expansion, and leaf growth.

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions1

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψw) in solution culture, decreases O2 movement by increasing solution viscosity. We investigated whether this property causes O2 deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O2 partial pressure (pO2) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO2 (alanine and proline accumulation are responses to hypoxia and low ψw, respectively). Measurements of root pO2 were made using an O2 microsensor to ensure that increased solution pO2 did not increase root pO2 above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO2 was similar to or less than (depending on depth in the tissue) pO2 of roots growing in vermiculite at the same ψw. Even without PEG, high solution pO2 was necessary to raise root pO2 to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O2 to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O2 demand of the root elongation zone may be greater at low relative to high ψw, compounding the effect of PEG on O2 supply. Under O2-sufficient conditions root elongation was substantially less sensitive to the low ψw imposed by PEG than that imposed by dry vermiculite.

Effectiveness of Low-Cost Planting Techniques for Improving Water Availability to Olea europaea Seedlings in Degraded Drylands

Reforestation projects in semiarid lands often yield poor results. Water scarcity, poor soil fertility, and structure strongly limit the survival and growth of planted seedlings in these areas. At two experimental semiarid sites, we evaluated a variety of low-cost planting techniques in order to increase water availability to plants. Treatments included various combinations of traditional planting holes; water-harvesting microcatchments; stone or plastic mulches; small waterproof sheets to increase water harvesting; dry wells; buried clay pots; and deep irrigation. Some of these treatments were also combined with addition of composted biosolids. Waterproof sheets significantly enhanced water harvesting (43%) and soil moisture in the planting hole (40%), especially for low-intensity rainfall events. Treatment effects on the survival and growth of Olea europaea seedlings varied between experimental sites. At the most water-limited site, clay pots, and dry wells improved seedling survival, while no treatment enhanced seedling growth. At the least water-stressed site, the application of composted sludge significantly improved seedling growth. We conclude that nutrient-mediated stress is subordinate to water stress in arid and semiarid environments, and we suggest modifications on the microsite scale to address these limiting conditions in Mediterranean drylands.

Some isolates of the nematophagous fungus Pochonia chlamydosporia promote root growth and reduce flowering time of tomato

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole-3-acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.

Physiological responses to water stress in Eucalyptus cloeziana and E-argophloia seedlings

Effects of water stress duration and intensity on gas exchange and leaf water potential were investigated in 7-month-old seedlings of a humid coastal provenance (Gympie) and a dry inland (Hungry Hills) provenance of E. cloeziana F. Muell. and in a dry inland (Chinchilla) provenance of E. argophloia Blakely supplied with 100% (T-100), 70% (T-70), 50% (T-50) of their water requirements, or were watered only after they were wilted at dawn (T-0). Seedlings of E. argophloia had the highest midday net photosynthetic rate (A), stomata] conductance (g(s)), stomatal density and predawn leaf water potential (Psi(pd)) in all treatments. The E. cloeziana provenances did not differ in these attributes. The T-70 and T-50 treatments caused reductions in A of 30% in E. argophloia, and 55% in the E. cloeziana provenances. Under the T-0 treatment, E. argophloia maintained higher rates of gas exchange at all levels of water stress than E. cloeziana provenances. The estimates of Psi(pd) and midday water potential (Psi(md)) at which plants remained wilted overnight were respectively: -2.7 and -4.1 MPa for E. cloeziana (humid), -2.8 and -4.0 MPa for E. cloeziana (dry) and, -3.7 and -4.9 MPa for E. argophloia. Following stress relief, both A and g(s) recovered more quickly in E. argophloia and in the dry provenance of E. cloeziana than in the humid provenance. We conclude that E. argophloia is more drought tolerant and has a potential for cultivation in the humid and semi humid climates, whilst E. cloeziana has greater potential in the humid subtropical climates.

Auxin dynamics after decapitation are not correlated with the initial growth of axillary buds

One of the first and most enduring roles identified for the plant hormone auxin is the mediation of apical dominance. Many reports have claimed that reduced stem indole-3-acetic acid (IAA) levels and/ or reduced basipetal IAA transport directly or indirectly initiate bud growth in decapitated plants. We have tested whether auxin inhibits the initial stage of bud release, or subsequent stages, in garden pea (Pisum sativum) by providing a rigorous examination of the dynamics of auxin level, auxin transport, and axillary bud growth. We demonstrate that after decapitation, initial bud growth occurs prior to changes in IAA level or transport in surrounding stem tissue and is not prevented by an acropetal supply of exogenous auxin. We also show that auxin transport inhibitors cause a similar auxin depletion as decapitation, but do not stimulate bud growth within our experimental time- frame. These results indicate that decapitation may trigger initial bud growth via an auxin-independent mechanism. We propose that auxin operates after this initial stage, mediating apical dominance via autoregulation of buds that are already in transition toward sustained growth.

Water and nutrient availabilities do not affect lignotuber growth and sprouting ability of three eucalypt species of south-eastern Queensland

Shoot biomass and lignotuber size of seedlings of three eucalypt species, Eucalyptus acmenoides Schauer, E. siderophloia Benth. and Corymbia variegata [syn. E. maculata (F. Muell.)K. D. Hill and L. A. S. Johnson], were measured for glasshouse-grown seedlings established under two water and nutrient regimes. Seedlings were subjected to shoot removal (clipping) at ages from 9 to 19 weeks, and transferred to the high water treatment for a further 8 weeks to assess shoot emergence from lignotubers. Seedling shoot biomass was greater in both the high than the low nutrient and water treatments, but lignotuber diameter was not affected significantly. C. variegata seedlings had the largest lignotuber diameters, followed by E. siderophloia and E. acmenoides, respectively. Although growth of shoots was influenced by nutrient availability, results suggest that species differences in the growth of lignotubers was less affected. It is suggested that lignotuber growth was strongly influenced by genotype. More than 70% of C. variegata seedlings clipped at 9 weeks sprouted, compared with only 5 and 10% of seedlings of E. siderophloia and E. acmenoides, respectively. All C. variegata seedlings sprouted after being clipped at 19 weeks, but < 80% of E. siderophloia and < 60% of E. acmenoides sprouted when clipped at the same age. It was concluded that seedlings forming part of the regeneration stratum in dry sclerophyll forests need to be protected from damage for at least 4 months (for C. variegata) or at least 6 months (for E. siderophloia and E. acmenoides) if they are to survive by sprouting from lignotubers.

Effects of nitrogen source and ectomycorrhizal association on growth and delta N-15 of two subtropical Eucalyptus species from contrasting ecosystems

Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W. Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant N-15 natural abundance (delta N-15). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, delta N-15 of almost all seedlings was lower than source N. The delta N-15 of all studied organisms was higher than the N source when grown on glutathione. This unexpected N-15 enrichment was perhaps due to preferential uptake of an N moiety more N-15-enriched than the bulk molecular average. Grown with ammonium-N, the delta N-15 of non-EM seedlings was mostly higher than that of source N. In contrast, the delta N-15 of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against N-15 was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar delta N-15 of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, delta N-15 of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly N-15-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar delta N-15 may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.

Effect of NaCl on emergence and growth of a range of provenances of Eucalyptus citriodora, Eucalyptus populnea, Eucalyptus camaldulensis and Acacia salicina

High salt levels in mine spoils have been identified as one of the major chemical limitations to plant establishment after coal mining in central Queensland. Soil solution extracts from spoils indicated that EC levels of up to 26 dS/m could be encountered. Glasshouse trials examined the emergence and growth of Eucalyptus citriodora, Eucalyptus camaldulensis and Eucalyptus populnea provenances and Acacia salicina subjected to such EC levels. Relatively low levels of salt (100 mM NaCl, or 11 dS/m) with respect to the levels encountered on mine spoils, were enough to substantially reduce the rate and percentage emergence of all eucalypt provenances. A. salicina was found to be superior to the eucalypts in its ability to emerge and survive under saline conditions. It was the only species to have seedlings emerge and survive at 200 mM NaCl (20 dS/m), and the effect of salt on decreasing seedling dry weight was less pronounced for A. salicina than for any of the eucalypts. Established plants survived the range of salt treatments far better than emerging seedlings, with survival of established plants being reduced only at 300 and 400 mM NaCl (28 and 36 dS/m, respectively). A. salicina performed significantly better at 300 and 400 mM NaCl than most of the eucalypts studied. (c) 2006 Elsevier B.V. All rights reserved.

Effect of physical, chemical and light treatments on germination and growth of tissue-cultured coconuts

The acclimatization and ex vitro establishment of tissue cultured coconut plantlets regenerated either from zygotic or somatic embryos could result to serious losses. Although high germination rates can be achieved in vitro, the survival of zygotic embryo derived plantlets in soil is very low (0-30%). Hence, treatments that could promote development of good quality seedlings having well-developed shoot and root is needed to increase seedling survival ex vitro. The effect of physical, chemical and light quality treatments on germination and growth of coconut embryos and tissue-cultured seedlings respectively, was investigated. The germination of coconut embryos was promoted when placed in a liquid Euwens (Y3) medium and incubated using a roller drum. Gibberellic acid (GA3) significantly affected growth of seedlings as it promoted shoot elongation, shoot and root expansion, and fresh and dry weight increase. However, GA3 did not significantly affect germination. In addition, the blue, red and yellow light significantly affected growth of seedlings as it promoted leaf and shoot elongation, fresh and dry weight increase, and root and leaf production. These conditions could be used to improve the growth and survival ex vitro of tissue cultured coconuts.

Effects of irradiance and spectral quality on leaf structure and function in seedlings of two Southeast Asian Hopea species

We studied the development of leaf characters in two Southeast Asian dipterocarp forest trees under different photosynthetic photon flux densities (PFD) and spectral qualities (red to far-red, R:FR). The two species, Hopea helferi and H. odorata, are taxonomically closely related but differ in their ecological requirements; H. helferi is more drought tolerant and H. odorata more shade tolerant. Seedlings were grown in replicated shadehouse treatments of differing PFD and R:FR. We measured or calculated (1) leaf and tissue thicknesses; (2) mesophyll parenchyma, air space, and lignified tissue volumes; (3) mesophyll air volumes (Vmes/Asurf) and surfaces (Ames/Asurf); (4) palisade cell length and width; (5) chlorophyll/cm2 and a/ b; (6) leaf absorption; and (7) attenuance/absorbance at 652 and 550 nm. These characters varied in response to light conditions in both taxa. Characters were predominantly affected by PFD, and R:FR slightly influenced many characters. Leaf characters of H. odorata were more plastic in response to treatment conditions. Characters were correlated with each other in a complex fashion. Variation in leaf anatomy is most likely a consequence of increasing leaf thickness in both taxa, which may increase mechanical strength and defense against herbivory in more exposed environments. Variation in leaf optical properties was most likely affected by pigment photo-bleaching in treatments of more intense PFD and was not correlated with Amax. The greater plasticity of leaf responses in H. odorata helps explain the acclimation over the range of light conditions encountered by this shade-tolerant taxon. The dense layer of scales on the leaf undersurface and other anatomical characters in H. helferi reduced gas exchange and growth in this drought-tolerant tree.