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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.

In the context of an increased risk of extreme drought events across Europe during the next decades, the capacity of trees to recover and survive drought periods awaits further attention. In summer 2005, 4-year-old beech (Fagus sylvatica L.) saplings were watered regularly or were kept for 4 weeks without irrigation in the field and then re-watered again. Changes of plant water status, leaf gas exchange and Chl a fluorescence parameters, as well as alterations in leaf pigment composition were followed. During the drought period, stomatal conductance (g(s)) and net photosynthesis (P(n)) decreased in parallel with increased water deficit. After 14 days without irrigation, stomata remained closed and P(n) was almost completely inhibited. Reversible downregulation of PSII photochemistry [the maximum quantum efficiency of PSII (F(v)/F(m))], enhanced thermal dissipation of excess excitation energy and an increased ratio of xanthophyll cycle pigments to chlorophylls (because of a loss of chlorophylls) contributed to an enhanced photo-protection in severely stressed plants. Leaf water potential was restored immediately after re-watering, while g(s), P(n) and F(v)/F(m) recovered only partially during the initial phase, even when high external CO(2) concentrations were applied during the measurements, indicating lasting non-stomatal limitations. Thereafter, P(n) recovered completely within 4 weeks, meanwhile g(s) remained permanently lower in stressed than in control plants, leading to an increased 'intrinsic water use efficiency' (P(n)/g(s)). In conclusion, although severe drought stress adversely affected photosynthetic performance of F. sylvatica (a rather drought-sensitive species), P(n) was completely restored after re-watering, presumably because of physiological and morphological adjustments (e.g. stomatal occlusions).

The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.

Leaf senescence can be described as the dismantling of cellular components during a specific time interval before cell death. This has the effect of remobilizing N in the form of amino acids that can be relocalized to developing seeds. High levels of carbohydrates have previously been shown to promote the onset of the senescence process. Carbohydrate accumulation in barley (Hordeum vulgare) plants was induced experimentally by steam-girdling at the leaf base, occluding the phloem, and gene regulation under these conditions was investigated using the Affymetrix Barley GeneChip array and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Transcript levels of plastidial (aminopeptidases, cnd41) and vacuolar (thiol and serine) proteases clearly increase in girdled leaves. Of special interest are cnd41, a plastidial aspartyl peptidase that has been implicated in Rubisco degradation in tobacco; and cp-mIII, a highly upregulated carboxypeptidase. SAG12, hexokinases and other senescence-specific genes are also upregulated under these conditions. Applying a genomic approach to the innovative experimental system described here significantly enhances our knowledge of leaf proteolysis and whole-plant N recycling.

The capability to withstand and to recover from severe summer droughts is becoming an important issue for tree species in central Europe, as dry periods are predicted to occur more frequently over the coming decades. Changes in leaf gas exchange, chlorophyll a fluorescence and leaf compounds related to photoprotection were analysed in young Quercus pubescens trees under field conditions during two summers (2004 and 2005) of progressive drought and subsequent rewatering. Photochemistry was reversibly down-regulated and dissipation of excess energy was enhanced during the stress phase, while contents of leaf pigments and antioxidants were almost unaltered. Plant water status was restored immediately after rewatering. Net photosynthesis (P(n)) measured at ambient CO2 recovered from inhibition by drought within 4 wk. P(n) measured at elevated CO2--to overcome stomatal limitations--was restored after a few days. A network of photoprotective mechanisms acted in preserving the potential functionality of the photosynthetic apparatus during severe drought, leading to a rapid recovery of photosynthetic activity after rewatering. Thus, Q. pubescens seems to be capable of withstanding and surviving extreme drought events.

The translocation of manganese (Mn), nickel (Ni), cobalt (Co), zinc (Zn) and cadmium (Cd) in white lupin (Lupinus albus cv. Amiga) was compared considering root-to-shoot transport, and redistribution in the root system and in the shoot, as well as the content at different stages of cluster roots and in other roots. To investigate the redistribution of these heavy metals, lupin plants were labelled via the root for 24 h with radionuclides and subsequently grown hydroponically for several weeks. 54Mn, 63Ni and 65Zn were transported via the xylem to the shoot. 63Ni and 65Zn were redistributed afterwards via the phloem from older to younger leaves, while 54Mn remained in the oldest leaves. A strong retention in the root was observed for 57Co and 109Cd. Cluster roots contained higher concentrations of all heavy metals than noncluster roots. Concentrations were generally higher at the beginning of cluster root development (juvenile and immature stages). Mature cluster roots also contained high levels of 54Mn and 57Co, but only reduced concentrations of 63Ni, 65Zn and 109Cd.

Background and Aims: The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum 'Arina') plants were investigated., Methods: After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides., Key Results: The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co., Conclusions: The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xylem.

As a nontolerant plant to a large number of toxic compounds, Arabidopsis thaliana is a suitable model to study regulation of genes involved in response to heavy metals. Using a cDNA-microarray approach, we identified some ABC transporters that are differentially regulated after cadmium treatments, making them putative candidates for being involved in Cd sequestration and redistribution in plants. Regarding yeast and fission yeast, in which Cd is able to form complexes either with glutathione (GSH) or phytochelatins (PC) subsequently transported into vacuoles via ABC transporters, it is also very likely that some plant ABC transporters are able to transport GS(2)-Cd or PC-Cd complexes into subcellular compartments or outside of the cell. The characterization of such transporters is of great interest for developing molecular biology approaches in phytoremediation.

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Senescence is a highly organized and well-regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C(3)-plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll-binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnected.

A (1-->3,1-->4)-beta-D-glucan endohydrolase [(1-->3,1-->4)-beta-glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1-->3,1-->4)-beta-glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1-->3,1-->4)-beta-glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1-->3,1-->4)-beta-glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1-->3,1-->4)-beta-glucanase accumulation under sugar depletion remains to be elucidated.

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30 degreesC, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40 degreesC. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45 degreesC. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40 degreesC. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40 degreesC for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713-1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

The catabolism of nuclear-encoded stromal proteins was investigated in intact chloroplasts isolated mechanically from pea (Pisum sativum) leaves. Glutamine synthetase, phosphoribulokinase, and nitrite reductase (quantified by immunoblotting) were more rapidly degraded in the light than in the dark. Furthermore, the degradation rates depended on exogenously supplied metabolites. For example, 2-oxoglutarate accelerated the catabolism of all three enzymes in chloroplasts incubated in the light, whereas oxaloacetate stabilized glutamine synthetase and at the same time destabilized the other two enzymes.

Some proteolytic enzymes occurring in the leaves of field-grown corn (Zea mays) (B73) were identified and partially characterized. Changes in activities of several proteolytic enzymes and in concentrations of protein and chlorophyll as a function of intraleaf segments (tip to base), leaf position, and leaf senescence during grain development and maturation were followed in crude leaf extracts.The aminopeptidase (not affected by sulfhydryl or fluoride reagents) was most active at pH 7, while the carboxypeptidase(s) (sensitive to fluoride, but insensitive to sulfhydryl reagents) was most active in the acid range, pH 3 to 6. The presence of two or more endopeptidases is indicated. Endopeptidase (caseolytic) activity at pH 5.4 appeared to be stimulated by sulfhydryl groups or EDTA, while caseolytic activity at pH 7.5 was not.Visually, individual leaf senescence starts at the leaf tip and the necrotic (brown) V-shaped area enlarges progressively toward the leaf base. Canopy senescence occurs in two phases. Foliar symptoms are first observed on the bottom leaf and then in sequential order up the plant. Subsequently, senescence occurs on the top leaf and moves downward. These foliar senescence symptoms are paralleled by decreases in exopeptidase activities, protein, and chlorophyll concentrations and by increases in endopeptidase activities.During development and maturation of the grain, both aminopeptidase and carboxypeptidase activity of the middle half of the ear leaf increased (2- to 3-fold) during the onset of the visual reproductive phase (tassel and car emergence). However, during grain development and plant senescence, both activities decreased rapidly and concurrently with the loss of protein and chlorophyll from this leaf section. In contrast, caseolytic activity at both pH 5.4 and 7.5 increased gradually during the early reproductive phase and rapidly with leaf senescence. The fastest rate of increase in caseolytic activities was concurrent with the most rapid loss of protein from the leaves. The coincidence of these events suggests a major role for the caseolytic enzymes in initiating the rapid hydrolysis of leaf protein.

Chick embryo chorioallantoic membrane, infected with the Blacksburg strain of Newcastle disease virus, was examined with an electron microscope to investigate the sequence of viral-induced host cell alterations. These were evident mostly in the endodermal epithelial cells lining the allantoic sac and were divided arbitrarily into three stages. Stage 1 was characterized by commencement of cell hypertrophy and hyperplasia and presence of fewer cytoplasmic inclusion bodies normally found in the cells; in stage 2, juxtanuclear nucleocapsid-glycogen aggregates appeared, and there were increased numbers of microvilli; stage 3 was characterized by increased cytoplasmic density and evidence of viral assembly and release. The morphological features of viral assembly and the virion are also described.