Influence of ozone on ribonuclease activity in wheat (Triticum aestivum) leaves

Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (P(i)) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O(3)) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O(3). Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O(3) mol(-1)) (control) or non-filtered air plus O(3) (90 nmol O(3) mol(-1)) (O(3)) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O(3)-sensitive cultivar in the O(3) treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [P(i)]. Soluble [P(i)] in leaf tissue extracts from the O(3) and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O(3) treatment. Isozymes stimulated in the O(3) treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [P(i)] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O(3) were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [P(i)] in the leaf tissue samples suggested that O(3)-induced injury and accelerated senescence was atypical of normal senescence processes in that P(i) export was not observed in O(3)-treated plants.     

An assessment of the ability of the stay-green phenotype in lolium species to provide an improved protein supply for ruminants

The stay-green phenotype results from a naturally occurring mutation in which senescent leaves retain their chlorophyll and the associated apoprotein, LHCPII. Protection of this protein pool could deliver grass with enhanced protein content and could decrease the extent of protein degradation by plant proteases in the rumen. This would enhance the efficiency of protein utilization in livestock to the benefit of the environment. Field plots of stay-green and wild-type Lolium perenne were defoliated at intervals to simulate grazing. There were variations in foliar protein content and proteolysis throughout the year, but no significant differences between genotypes when material was analysed fresh or after it was cut and dried to simulate hay-making, which possibly induced senescence. In a subsequent experiment with stay-green and wild-type L temulentum, increased protein retention and decreased protein degradability were observed in stay-green leaves that were allowed to senescence naturally and extensively on the plant. That there is no difference between the two L. perenne genotypes suggests that as a field crop in grazed pastures the stay-green genotype would not confer a nutritional advantage in terms of protein degradability. It is possible that grazing promotes a high proportion of non-senescent to senescent leaf material within the sward and thus any advantage conferred by the stay-green phenotype would be effectively masked by an abundance of mature foliage. It is suggested that the stay-green trait would be of benefit in areas where agricultural practice permits extensive natural senescence to occur.     

Influence of urban tributaries on freshwater mussel populations in a biologically diverse piedmont (USA) stream

We investigated the distribution patterns of senescent and green leaf litter patches on a streambed to evaluate the hypothesis that the different immersion times of senescent leaves in long-term benthic storage and newly retained green leaves affect streambed distribution patterns in summer (June, July, and August). We counted all the leaf litter patches in the streambed of the study reach, comparing the physical condition of patches and classifying the trapping obstacles associated with each patch type. The distribution patterns of senescent and green leaf litter patches differed. Green leaf litter patches were more numerous at every sampling date, with most trapped by cobbles, whereas senescent leaf litter patches were retained by twig obstacles, backwaters, and cobbles. In June and July, senescent leaf litter patches were located in significantly deeper stream areas than were green leaf litter patches. The distribution of senescent leaves would be primarily determined during spring snowmelt-driven floods. We speculate that senescent leaves were originally located at the edges of pools in the main flow pathway of the channel, which overflowed in the floods. We conducted flume experiments to clarify the transport characteristics of senescent and green painted maple and manchurian alder leaves in the water column. Our hypothesis was that the transport characteristics of each leaf type differ when they first enter the water, because of differing leaf properties. The flume experiments showed that duration of surface flotation differed for senescent and green leaves and for the two tree species. These differences in the duration of leaves on the streambed and in the floating time of green leaves of different trees ensure varied food resources for macroinvertebrates in various physical conditions. Handling editor: B. Oertli     

Changes in activity of energy dissipating mechanisms in wheat flag leaves during senescence

Abstract: Excitation energy dissipation, including the xanthophyll cycle, during senescence in wheat flag leaves grown in the field was investigated at midday and in the morning. With progress of senescence, photosynthesis (Pn) and actual PSII photochemical efficiency (ΦPSII) decreased markedly at midday. The decrease in extent of Pn was greater than that of ΦPSII. However, there was no significant decline in Pn and ΦPSII observed in the morning, except in leaves 60 days after anthesis. The kinetics of xanthophyll cycle activity, thermal dissipation (NPQ), and q_f observed at midday during senescence exhibited two distinct phases. The first phase was characterized by an increase of xanthophyll cycle activity, NPQ, and q_f during the first 45 days after anthesis. The second phase took place 45 days after anthesis, characterized by a dramatic decline in the above parameters. However, the qI, observed both at midday and in the morning, always increased along with senescence. A larger proportion of NPQ insensitive to DTT (an inhibitor of the de-epoxidation of V to Z) was also observed in severely senescent leaves. In the morning, only severely senescent leaves showed higher xanthophyll cycle activity, NPQ, q_f, and qI. It was demonstrated that, at the beginning of senescence or under low light, wheat leaves were able to dissipate excess light energy via NPQ, depending on the xanthophyll cycle. However, the xanthophyll cycle was insufficient to protect leaves against photodamage under high light, when leaves became severely senescent. The ratio of (Fj - Fo)/(Fp - Fo) increased gradually during the first 45 days after anthesis, but dramatically increased 45 days after anthesis. We propose that another photoprotection mechanism might exist around reaction centres, activated in severely senescent leaves to protect leaves from photodamage.     

Natural Senescence of Pea Leaves (An Activated Oxygen-Mediated Function for Peroxisomes)

We studied the activated oxygen metabolism of peroxisomes in naturally and dark-induced senescent leaves of pea (Pisum sativum L.). Peroxisomes were purified from three different types of senescent leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. The activities of the O2-- and H2O2-producing enzymes were enhanced by natural senescence. Senescence also produced an increase in the generation of active oxygen species (O2- and H2O2) in leaf peroxisomes and in the activities of two glyoxylate-cycle marker enzymes. A new fraction of peroxisomes was detected at an advanced stage of dark-induced senescence. Electron microscopy revealed that this new peroxisomal fraction varied in size and electron density. During senescence, the constitutive Mn-superoxide dismutase (SOD) activity of peroxisomes increased and two new CuZn-SODs were induced, one of which cross-reacted with an antibody against glyoxysomal CuZn- SOD. This fact and the presence of glyoxylate-cycle enzymes support the idea that foliar senescence is associated with the transition of peroxisomes into glyoxysomes. Our results indicate that natural senescence causes the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence, and reinforce the hypothesis of an effective role of peroxisomes and their activated oxygen metabolism in this stage of the life cycle.     

Induction of leaf senescence by low nitrogen nutrition in sunflower (Helianthus annuus) plants

Different parameters which vary during the leaf development in sunflower plants grown with nitrate (2 or 20 mM) for a 42‐day period have been determined. The plants grown with 20 mM nitrate (N+) showed greater leaf area and specific leaf mass than the plants grown with 2 mM nitrate (N?). The total chlorophyll content decreased with leaf senescence, like the photosynthetic rate. This decline of photosynthetic activity was greater in plants grown with low nitrogen level (N?), showing more pronounced senescence symptoms than with high nitrogen (N+). In both treatments, soluble sugars increased with aging, while starch content decreased. A significant increase of hexose to sucrose ratio was observed at the beginning of senescence, and this raise was higher in N? plants than in N+ plants. These results show that sugar senescence regulation is dependent on nitrogen, supporting the hypothesis that leaf senescence is regulated by the C/N balance. In N+ and N? plants, ammonium and free amino acid concentrations were high in young leaves and decreased progressively in the senescent leaves. In both treatments, asparagine, and in a lower extent glutamine, increased after senescence start. The drop in the (Glu+Asp)/(Gln+Asn) ratio associated with the leaf development level suggests a greater nitrogen mobilization. Besides, the decline in this ratio occurred earlier and more rapidly in N? plants than in N+ plants, suggesting that the N? remobilization rate correlates with leaf senescence severity. In both N+ and N? plants, an important oxidative stress was generated in vivo during sunflower leaf senescence, as revealed by lipid peroxidation and hydrogen peroxide accumulation. In senescent leaves, the increase in hydrogen peroxide levels occurred in parallel with a decline in the activity of antioxidant enzymes. In N+ plants, the activities of catalase and ascorbate peroxidase (APX) increased to reach their highest values at 28 days, and later decreased during senescence, whereas in N? plants these activities started to decrease earlier, APX after 16 days and catalase after 22 days, suggesting that senescence is accelerated in N‐leaves. It is probable that systemic signals, such as a deficit in amino acids or other metabolites associated with the nitrogen metabolism produced in plants grown with low nitrogen, lead to an early senescence and a higher oxidation state of the cells of these plant leaves.     

Increases in Cytosolic Ca2+ in Parsley Mesophyll Cells Correlate with Leaf Senescence

The ability to maintain the cytoplasmic Ca2+ concentration ([Ca2+]cyt) at homeostatic levels has been examined during leaf senescence in detached parsley (Petroselinum crispum) leaves. Fluorescence ratiometric imaging of mesophyll cells isolated from parsley leaves at various senescence stages and loaded with the Ca2+ indicator fura-2 has revealed a distinct elevation of [Ca2+]cyt, which was positively correlated with the progress of leaf senescence. This initial increase of [Ca2+]cyt, which was first observed in cells isolated from 3-d-senescent leaves, occurred 1 d before or in parallel with changes in two established senescence parameters, chlorophyll loss and lipid peroxidation. However, the [Ca2+]cyt elevation followed by 2 d the initial increase in the senescence-associated proteolysis. Whereas the [Ca2+]cyt of nonsenescent cells remained at the basal level, the elevated [Ca2+]cyt of the senescent cells was a long-lasting effect. Experimental retardation of senescence processes, achieved by pretreatment of detached leaves with the cytokinin benzyladenine, resulted in maintenance of homeostatic levels of [Ca2+]cyt in cells isolated from 3-d-senescent leaves. These observations demonstrate for the first time to our knowledge a correlation between elevated [Ca2+]cyt and the process of senescence in parsley leaves. Such senescence-associated elevation of [Ca2+]cyt, which presumably results from a loss of the cell's capability to extrude Ca2+, may serve as a signal inducing subsequent deteriorative processes.     

Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence

Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species-specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

Agonistic Tactics and Size Asymmetries between Opponents in Blattella germanica (L.) (Dictyoptera: Blattellidae)

In this paper we investigated the influence of size differences between opponents on the occurrence of different agonistic attack and response tactics displayed during encounters between Blattella germanica (L.) cockroaches of all developmental stages, competing for a limited food source. The two main results were: 1) attack and response tactics varied during development and 2) size asymmetries between contestants influenced agonistic tactics. Developmental stage of initiator influenced the frequency distribution of the three types of attack patterns (Bite, Kick and Jump) and of the four types of response patterns (Retaliation, Escape, Resettle and No Response). The proportion of kicks increased gradually with developmental stage of the initiator whereas the proportion of bites declined during development. In addition, cockroaches adapted their attack tactics to the developmental stage of their opponent. Similarly the response displayed varied in relation to the developmental stage of the attacked individual, the developmental stage of the initiator and the type of attack displayed. During an interaction, animals appeared to be able to evaluate the relative size of their opponent and to evaluate the consequences of the behavioural pattern they displayed. Larger animals tended to minimize the intensity of the agonistic act they initiated. Escalation in interactions was rare and smaller individuals tended to escape when attacked by larger ones. Wrong estimations of the relative size of opponent, when a smaller animal retaliated after being attacked by a larger animal or when a larger animal fled after being attacked by a smaller one, represented only 6% of the records.     

Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam) senescent leaves

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxyl side of asparagine residues, and is possibly involved in the post-translational processing of proproteins. In this report one full-length cDNA, SPAE, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPAE contained 1479 nucleotides (492 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (c. 61-68%) with asparaginyl endopeptidases of Vicia sativa, Phaseolus vulgaris, Canavalia ensiformis, and Vigna mungo. SPAE probably encoded a putative precursor protein. Via cleavage of the N- and C-termini, it produced a mature protein containing 325 amino acids (from the 51st to the 375th amino acid residues), the conserved catalytic residues (the 173rd His and 215th Cys amino acid residues), and the putative N-glycosylation site (the 332nd Asn amino acid residue). Semi-quantitative RT-PCR and western blot hybridization showed that SPAE gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was much less in mature green leaves, stems, and roots. Phylogenic analysis showed that SPAE displayed close association with vacuolar processing enzymes (legumains/asparaginyl endopeptidases), which function via cleavage for proprotein maturation in the protein bodies during seed maturation and germination. In conclusion, sweet potato SPAE is probably a functional, senescence-associated gene and its mRNA and protein levels were significantly enhanced in natural and induced senescent leaves. The possible role and function of SPAE associated with bulk protein degradation and mobilization during leaf senescence were also discussed.     

Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Effects of light and regulators on senescence-related changes in soluble proteins in detached oat (Avena sativa L.) leaves

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG two-dimensional polyacrylamide gel electrophoresis - ABA abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - IEF isoelectric focusing - Rubisco ribulosebisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - Tris tris (hydroxymethyl) aminomethane     

Factors affecting aggressive behaviour of spawning migratory males towards mature male parr in masu salmon Oncorhynchus masou

This study examined whether dominant migratory males (adopting fighter tactics) of the masu salmon Oncorhynchus masou would more aggressively attack large mature male parr (adopting sneaker tactics) as large mature male parr are expected to have the potential to cause a greater decrease in fertilization success. The frequency of aggressive behaviour was not related to the body size of males, and it increased with the frequency of interactions with mature male parr. The fertilization success of mature male parr was much lower than migratory males, and no relationship was observed between fertilization success and aggressive behaviour. The low fertilization success of mature male parr, despite infrequent aggressive behaviour by migratory males, indicates that there might be little benefit for migratory males to attack mature male parr more aggressively according to their body size.     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.