Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Expression, purification, and characterization of cytokinin signaling intermediates: Arabidopsis histidine phosphotransfer protein 1 (AHP1) and AHP2

Key message

We have expressed, purified, and biophysically characterized recombinant AHP1 and AHP2. Also, using computational homology models for AHP1, ARR7, and AHP1–ARR7 complex, we identified three-dimensional positioning of key amino acids.

Abstract

Cytokinin signaling involves activation of Arabidopsis Response Regulators (ARRs) by Arabidopsis Histidine Phosphotransfer Proteins (AHPs) by phosphorylation. Type-A ARRs are key regulators of several developmental pathways, but the mechanism underlying this phosphorylation and activation is not known in plants. In this study, we report the successful expression and purification of recombinant AHP1 and AHP2. Biophysical characterization shows that these two recombinant proteins were purified to homogeneity and possess well-defined secondary structures. Brief attempts to purify recombinant ARR7 posed problems during size-exclusion chromatography. Nevertheless, we generated computational homology models for AHP1, ARR7, and AHP1–ARR7 complex using crystal structures of homologous proteins from other organisms. The homology models helped to identify the three-dimensional positioning of the key conserved residues of AHP1 and ARR7 involved in phosphorylation. The similarity in positioning of these residues to other homologous proteins suggests that AHPs and type-A ARRs could be structurally conserved across kingdoms. Thus, our homology models can serve as valuable tools to gain structural insights into the phosphorylation and activation of cytokinin response regulators in plants.     

AXR1 promotes the Arabidopsis cytokinin response by facilitating ARR5 proteolysis

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multi‐step phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): type B ARRs (response activators) and type A ARRs (negative‐feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the RUB (related to ubiquitin) modification pathway, lead to decreased cytokinin sensitivity. Here we show that the cytokinin resistance of axr1 seedlings is suppressed by loss of function of the type A ARR family member ARR5. Based on the established role of the RUB pathway in ubiquitin‐dependent proteolysis, these data suggest that AXR1 promotes the cytokinin response by facilitating type A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity.     

Effects of Exogenous Growth Regulators on Fruit Development in Citrus unshiu

The effects of applied growth regulators on fruit developmenthave been determined in the parthenocarpic Satsuma mandarin(Citrus unshiu Marc.). The application of either gibberellicacid or benzyladenine at flower opening, caused a transientincrease in cell division in the ovary wall, but had no significanteffect on final fruit size. Late fruit growth and final fruitsize were increased by the application of the synthetic auxin2,4,5-trichlorophenoxyacetic acid, which had a specific effecton the enlargement of the juice vesicles. The three growth regulators enhanced vascularization in thepedicel, but the growth effects observed were unrelated to theirinfluence on the transport capacity of the phloem but causedby their direct effects on the fruit tissues. The sensitivityof the fruit tissues to the applied growth regulators changedmarkedly during early fruitlet development, and was characterizedculturing the fruit tissues in vitro.Copyright 1993, 1999 AcademicPress Citrus unshiu Marc., fruit growth, hormone treatment, in vitro culture, phloem formation, phloem transport, xylogenesis     

Proteasome-dependent proteolysis has a critical role in fine-tuning the feedback inhibition of cytokinin signaling

The ubiquitin/26S proteasome-dependent proteolysis of response regulators is a critical element of many plant hormone signaling pathways. We have recently shown that cytokinin signaling requires the AXR1 component of the related to ubiquitin (RUB) protein modification pathway to promote the proteasome-dependent degradation of the cytokinin response inhibitor ARR5. Here, we show that ARR5 also accumulates in the 26S proteasome mutant rpn12a-1, and leads to a marked resistance to cytokinins. Collectively, these results suggest that proteasome-dependent proteolysis of feedback inhibitors such as ARR5 is essential for the maintenance of optimal responsivity and plasticity in cytokinin signaling.     

Hormonal Control of Nuclease Level in Excised Avena Leaf Tissues1

In excised Avena leaves, kinetin and benzyladenine decreased,while abscisic acid and benzimidazole increased the over-allnuclease level. Significant effects were observed as early as2 h after treatment. Not all the nucleases of the Avena leafwere affected by the growth regulators. Changes in over-allnuclease activity were accounted for almost entirely by changesin the amount of a relatively purine-specific endo-ribonuclease,which produces 2',3'-cyclic phosphates as breakdown products.Slight changes induced by the growth regulators were also detectedin the amount of a sugar non-specific endo-nuclease which produces5'-nucleotides and has a relative specificity for adenylic acid.The level of an alkaline phosphodiesterase, an exo-nucleasewhich produces 5'-nucleotides, was not affected by any of thegrowth regulators tested. Gibberellic acid and indol-3yl-aceticacid did not influence the level of Avena nucleases.     

Fire Tolerance and the Fire-related Sprouting Characteristics of Two Cool-temperate Broad-leaved Tree Species

This study was conducted in a secondary broad-leaved forestin northern Hokkaido, Japan, which burned on 20–21 April1998. The study plot, set up on 13 July 1998, contained Quercusmongolica var. grosseserrata and Betula platyphylla var. japonicatrees. The latter were more severely damaged by the fire. Size-dependentsurvivorship was observed in both species with larger treesbeing more likely to survive the fire. However, many largeBetulatrees were completely destroyed. Both species developed abundantroot collar sprouts immediately after the fire. The number ofsprouts ranged from 0 to 296 in Betula and from 0 to 34 in Quercus.Trees with no sprouts were not necessarily dead. The frequencydistribution of the sprouting pattern in Betula was bimodal.This was related to damage severity, with the most abundantsprouting manifest in the most severely damaged trees. Thispattern was not observed inQuercus . Stem diameter also appearedto influence the extent of sprouting in Betula, while in Quercusthere was no distinct correlation between basal area and thenumber of sprouts. In Betula, the relationship between the numberof sprouts and the damage severity suggests a trade-off in resourceallocation for sprouting vs. shoot flushing in the crown. Copyright2000 Annals of Botany Company Betula platyphylla var. japonica, fire, fire tolerance, Quercus mongolica var.grosseserrata , resource allocation, root collar sprouting, stem diameter size.     

FIELD OBSERVATIONS ON FEEDING OF THE LAND SNAIL HELIX ASPERSA MULLER

Feeding of the land snail Helix aspersa (Müller) was observedat monthly intervals. Three natural populations in Galicia (NW-Spain)were studied. At two sites only a few plants constituted thebulk of the diet and in spring the snails' diet had the highestdiversity (H'). In the third population feeding and distributionof Helix aspersa (Müller) were observed in a small plotwith permanent patches of Urtica dioica. Nearly one half offeeding snails fed upon Urtica dioica. Most of the other observations wereon Mentha suaveolens, Ranunculus repens and Gramineae. The diversity ofthe snails' diet showed seasonal variation with the maximumin the autumn months. Comparison between the availability ofthe different plant species and their contribution to the snails'diet showed that the snails did not eat at random; Urtica dioicawas eaten much more than expected from its occurrence and grasseswere strongly under-represented in the snails' diet. Temporalchanges of availability were significantly correlated with the amountseaten in the case of Urtica, but not for the other food plants.The distribution of the snails in the plot was significantlycorrelated with that of Urtica. Chemical analyses of the foodplants revealed Urtica as the species with the higher protein,ash and calcium contents. The strong preference of Helix aspersafor Urtica dioica could be explained by the value of Urticaas food or by its suitability as habitat for the snails. The largestproportions of green material in the snails' diet occurred inthe spring and juveniles ate more green material than adultsin the three populations. (Received 16 March 1998; accepted 30 November 1998)     

Interaction Between Cryopreservation, Rewarming Rate and Seed Humidification on the Germination of Two Spanish Endemic Species

The effects of humidification, storage in liquid nitrogen (1or 30 d) and rewarming rate on seed germination were studiedin two Spanish endemics. Humidification resulted in higher germinationpercentages only in the species with hard covers, especiallyin slowly rewarmed seeds. In an experiment lasting 21 weeks,seeds stored in liquid nitrogen were removed for 10 min eachweek to mimic the withdrawal of samples from a seed bank; thishad no effect on germination.Copyright 1998 Annals of BotanyCompany Centaurea hyssopifolia,Limonium dichotomum, cryopreservation, cypsela, endemics, germination, humidification, seeds.     

Reproductive Biology ofDalbergia miscolobiumBenth. (Leguminosae-Papilionoideae) in SE Brazil: The Effects of Pistillate Sorting on Fruit-set

The breeding system ofDalbergia miscolobiumBenth. was investigatedby means of controlled pollinations and studies of post-pollinationevents in pistils. This species does not have homomorphic self-incompatibility,but crossed flowers always have higher fruit set than selfs,despite the fact that embryo development may be initiated inselfed pistils. It is suggested that deleterious recessive alleles,which lead to abortion of most selfed seeds, together with somematernal resource limitation, result in low fruiting success.Copyright1998 Annals of Botany Company Dalbergia miscolobiumBenth., Leguminosae, breeding system, pistillate sorting, fruiting success, seed set.