Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Autumnal changes in total nitrogen, salt-extractable proteins and amino acids in leaves and adjacent bark of black alder, eastern cottonwood and white basswood

Autumnal changes in total nitrogen, salt-extractable protein and amino acid concentrations in leaves and adjacent bark of black alder [ Ainus glutinosa (L.) Gaertn.], eastern cottonwood ( Populus deltoides Bartr. ex Marsh.) and white basswood ( Tilia heterophylla Vent.) were determined for trees growing on minespoils and a prairiederived loamy soil in central Illinois. The composition of free amino acids in foliage was also determined at peak concentration for each tree species during late senescence. Total nitrogen concentration in the leaves decreased slowly throughout most of the fall for all species. In the final stages of senescence, total leaf nitrogen concentrations were about halved in eastern cottonwood and white basswood but continued to decrease slowly in black alder. The concentration of salt-extractable proteins in leaves of all species peaked early in the fall and then declined prior to leaf abscission. This decline coincided with an increase in the concentration of free amino acids in the leaves. The increase stabilized in both eastern poplar and white basswood but continued in black alder. Glutamine in black alder and eastern cottonwood, and asparagine in white basswood were the most abundant free amino acids at the time of peak concentration of total free amino acids in senescent leaves. Bark of trees of all species had higher nitrogen concentrations and higher proportions of salt-extractable proteins to estimated total proteins after leaf senescence than during the preceding summer. Results indicate that autumnal fluxes in leaf and bark nitrogen fractions of alder can differ substantially from fluxes in other broadleaved winter-deciduous trees in a way which suggests that alder does not effectively conserve leaf nitrogen through retranslocation to bark tissue.     

Changes in Urease Activity in Apple Trees as Related to Urea Applications

Changes in urease (E.C.3.5.1.5.) were followed during the growth of 1-year-old MM 106 and 9-year-old Golden Delicious apple trees (Malus pumila Rehd.). Urease was found in leaves, roots, and bark with actively growing tissues containing more activity than senescing tissues. The urease activity in the leaves declined steadily during leaf senescence but abscised leaves still contained about half of their initial urease activity. In the bark the urease activity changed only slightly. Urease activities in the leaves and bark of apple trees were always greater in those trees which had received an application of urea. In senescing apple leaves, urea induced a rapid increase in urease activity. The changes in total activity and specific activity of urease were parallel and suggests that urease was synthesized de novo. After urease activity reached a maximum, a rapid decline occurred. Urease was inhibited by low concentrations of ammonia and this decline may be due to product inhibition.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Influence of nitrogen supply and sink strength on changes in leaf nitrogen compounds during senescence in two wheat cultivars

Changes in various nitrogen compounds during senescence of the fourth leaf were studied in two cultivars of spring wheat (Triticum aestivum L.). One of the cultivars (Yecora) was supplied with two N levels; the other (Tauro) was grown with the high N level and pruned above the fourth leaf, whereas the control was left intact. In both cultivars grown with high N supply, net nitrogen export from the fourth leaf did not occur until 35 days after sowing (DAS). Loss of leaf soluble proteins started earlier than that of chlorophylis, and coincided initially with an increase in insoluble protein. In N deficient plants the level of total N, soluble protein, and the activity of nitrate reductase (NRA. EC 1.6.6.1) started to decrease about 5 days earlier, and along with chlorophyll, continued to decrease at a faster rate, than in high N plants. Also, with low N supply, the large subunit (LSU, 58 kDa) of ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) decreased in greater proportion than other soluble proteins, while with high N supply the decrease in Rubisco LSU was similar to that of other soluble proteins. Nitrogen deficiency caused a greater decrease in soluble proteins than in insoluble proteins, and NRA relative to soluble proteins. The faster senescing Tauro cultivar had lower levels of most parameters, especially NRA, soluble protein and, after 35 DAS. Rubisco LSU as a proportion of soluble protein. The decrease in sink strength due to shoot pruning did generally not affect the level of the various nitrogenous compounds until 35 DAS; thereafter the levels of most parameters, especially soluble protein, Rubisco LSU and, at late stages of senescence, insoluble protein, were higher in pruned than in control shoots. Thus, shoot pruning slows down senescence. The 56- and 78-kDa polypeptides increased, rather than decreased, with leaf age; the level of these two polypeptides showed a negative relationship with Rubisco LSU (r = -0.933 and r = -0.758, respectively).     

Studies on Protein Synthesis by Senescing and Kinetin-treated Barley Leaves

Using sterile conditions, changes in total protein synthesis were followed. over an 8 day incubation period, in detached first seedling leaves of barley from 8 day old plants during senescence and after kinetin treatment. In senescing leaves, total ¹⁴C-alanine incorporation was enhanced by nearly 20% within 6 h of leaf detachment and by about 30 % after 24 h. Kinetin treatment stimulated protein synthesis even more, for total incorporation was promoted ca. 50 % after 6 h and by ca. 60 % after 24 h incubation. The leaf supernatant (30,000 ×g for 30 min) proteins were separated on DEAE-Sephadex (A-50) columns into approximately 14 fractions and changes in ¹⁴C labelling of these fractions were studied following leaf detachment and on incubation on water or kinetin for 6 days. In senescing leaves, ¹⁴C-incorporation into supernatant proteins was sustained, even as protein levels declined rapidly The varied stabilities of the different leaf proteins was suggested by the characteristically changing specific activities of the different protein fractions. Although kinetin greatly promoted incorporation into all protein fractions, no evidence was surmised of specific effects on individual leaf proteins. Studies of changes in total protein synthesis in attached senescing first seedling leaves taken from plants aged 7 to 27 days revealed a relatively small increase in ¹⁴C-incorporation. However, incorporation could be greatly increased in leaves up to 15 days old by detaching and preincubating such leaves for up to 2 days on water, prior to measurement. The promotion of ¹⁴C-incorporation into protcins follwing leaf excision could result from early changes in permeability and precursor pool size.     

Proteins as a potential nitrogen storage compound in bark and leaves of several softwoods

Summary The occurrence of vegetative storage proteins in the leaf and bark tissues of several softwood species during overwintering was investigated by sodium dodecyl sulphate polyacrylamide electrophoresis. Monthly protein profiles from leaves and bark of six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and the bark of one deciduous softwood species (Larix decidua) suggest that storage proteins are present in bark tissues of L. decidua, Pinus sylvestris, and P. strobus. The remaining species did not show similar specific proteins. However, the total soluble protein content which was determined during active growth and during overwintering in the same tissues indicated that protein levels were higher in the winter compared to the summer in the bark of all species and in the leaves of Pinus spp. and T. occidentalis. While vegetative storage proteins do not appear prevalent in all softwood species, proteins may constitute a major form of overwintering nitrogen storage for many species.     

毛竹叶片衰老过程的叶重量、叶面积及元素内吸收率的动态

对福建永春毛竹(Phyllostachys pubescens Mazel ex H.de Lehaie)叶片衰老过程的叶重量、叶面积及元素内吸收率的动态进行了研究,并对元素内吸收率RE_1(以元素的干重含量为计算单位,mg/g)、RE_2(以单位叶片的元素含量为计算单位,mg/leaf)以及RE_3(以单位叶面积的元素含量为计算单位,mg/cm~2)进行了比较。叶片衰老过程中,平均叶重量、叶面积及比叶重分别下降了19.55％、15.16％和5.07％。叶重量与叶面积下降百分率的季节变化趋势一致,说明毛竹叶片存在一定的重量与面积比率。在不同的元素内吸收率比较中,N和K的元素内吸收率均为正,Ca均为负,表明叶片衰老过程中N和K的元素含量从衰老叶片中转移至植株的其他部位,而Ca在老叶中累积。N、P、K、Ca和Mg 5种元素平均的元素内吸收率高低顺序均为RE_2>RE_3>RE_1,反映出以元素的干重含量为计算单位和以单位叶面积的元素含量为计算单位的元素内吸收率偏低。     

Seasonally fluctuating bark proteins are a potential form of nitrogen storage in three temperate hardwoods

The inner bark tissues of three temperate hardwoods contain specific proteins which undergo seasonal fluctuations. Increases in particular proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, occur within the bark of several Acer, Populus and Salix spp. during late summer and early autumn. These proteins are abundant in the bark throughout the winter and their levels decline the following spring. Light and electron microscopy showed that the parenchyma cells of the inner bark are packed with spherical organelles throughout the overwintering period. These organelles are rich in protein and analogous to protein bodies found in cells of mature seeds. The protein bodies of the parenchyma cells are replaced by large central vacuoles during spring and summer, presumably as a result of the mobilization of the storage protein and fusion of the protein bodies. The high levels of specific proteins in inner bark tissues and the presence of protein bodies within the parenchyma cells indicate that the living cells of the bark act as a nitrogen reserve in overwintering temperate hardwoods.Abbreviations FW fresh weight - kDa kilodalton - M _r relative molecular mass     

Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves

Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.     

毛竹叶片衰老过程的叶重量、叶面积及元素内吸收率的动态

对福建永春毛竹(Phyllostachyspubescens Mazel ex H.de Lehaie)叶片衰老过程的叶重量、叶面积及元素内吸收率的动态进行了研究,并对元素内吸收率RE1(以元素的干重含量为计算单位,mg/g)、RE2(以单位叶片的元素含量为计算单位,mg/leaf)以及RE3(以单位叶面积的元素含量为计算单位,mg/cm2)进行了比较.叶片衰老过程中,平均叶重量、叶面积及比叶重分别下降了19.55%、15.16%和5.07%.叶重量与叶面积下降百分率的季节变化趋势一致,说明毛竹叶片存在一定的重量与面积比率.在不同的元素内吸收率比较中,N和K的元素内吸收率均为正,Ca均为负,表明叶片衰老过程中N和K的元素含量从衰老叶片中转移至植株的其他部位,而Ca在老叶中累积.N、P、K、Ca和Mg5种元素平均的元素内吸收率高低顺序均为RE2＞RE3＞RE1,反映出以元素的干重含量为计算单位和以单位叶面积的元素含量为计算单位的元素内吸收率偏低.     

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non‐senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.     

The proteomics of senescence in leaves of white clover,Trifolium repens (L.)

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two-dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one-third of the protein spots present in mature green leaves were also visible by two-dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis

Soluble and thylakoid membrane polypeptides from senescing leaf tissue of Rossa, a normal yellowing Festuca pratensis genotype, were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and compared with those of the non-yellowing mutant Bf 993. Subunits of ribulose-1,5-bisphosphate carboxylase were the major soluble polypeptides and declined to low levels in senescing leaves of both genotypes. The major thylakoid polypeptides were those associated with the chlorophyllprotein complexes CPI and CPII. The levels of all thylakoid polypeptide species fell during senescence of Rossa leaf tissue but Bf993 lamellae retained CPI, CPII and a number of other hydrophobic low molecular weight polypeptides. The increasing hydrophobicity and decreasing protein complement of Bf 993 thylakoids were reflected in a fall in membrane density from 1.16 to 1.13 g cm^-3 over 8 d of senescence and a decline in the extractability of chlorophyll-containing membranes in the same period. In Bf993 the molar ratio of chlorophyll to hydrophobic membrane protein increased from 92 at day 0 to 296 at day 8. In the same time the ratio for Rossa increased from 88 to 722 and 8 d-senesced Rossa tissue yielded less than 2% of the solvent-soluble protein it contained at day 0 as compared with 24% for the protein of Bf993. These results are discussed in relation to the nature of the non-yellowing lesion.Abbreviations RuBPC ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - EDTA ethylenediaminetetraacetate - SDS sodium dodecyl sulphate - CP chlorophyll-protein complex     

Seasonal changes in element contents in mangrove element retranslocation during leaf senescene

The concentrations of nitrogen (N), phosphorus (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg) and chlorine (Cl) were followed monthly in pre-senescence leaves and post-abscission leaves of Kandelia candel (L.) Druce at the Jiulongjiang estuary, and Fujian, China. The element retranslocation efficiency (RE) was studied during leaf senescence. The element RE's evaluated using different methods were compared and a new method was put forward to evaluate element RE during leaf senescence in evergreen trees without concentrated leaf fall. The results showed that during leaf senescence, 77.22% N, 57.53% P, and 44.51% K were translocated out of senescing leaves. Translocation of nutrients out of senescing leaves back into shoots was an important nutnent-conservation mechanism for N and P, was less important for K, and did not occur for Ca, Mg, Na, or Cl. One of the reasons for the high primary productivity of mangroves in nutrient poor sites (especially with low N) is the high nutrient use efficiency.     

Ribulose Bisphosphate Carboxylase Protein and Enzymes of CO2 Assimilation in Barley Infected by Powdery Mildew (Erysiphe graminis hordei)

Effects were examined of barley powdery mildew (Erysiphe graminis f. sp. hordei) on the major leaf protein, ribulose-l,5-bisphosphate carboxylase (RuBPCase), and other enzymes of CO₂ assimilation, phosphoenolpyruvate carboxylase (PEPcase) and malic enzymes, and enzymes associated with RuBPCase in the reductive pentose phosphate pathway. Activity of RuBPCase per unit fresh weight of leaf was inhibited by infection from the first sample, 3 days after inoculation, to the last sample, 24 days after inoculation, when healthy control leaves were visibly senescing. The inhibition occurred because the amount of RuBPCase protein (measured specifically by an immunological technique) was reduced from 6 days after inoculation and because activity per unit protein declined from 3 days until 21 days after inoculation, at which time there was little protein remaining. Activity of PEPcase per unit fresh weight of leaf was initially stimulated by infection but, thereafter, it was inhibited. Inhibition, like that also affecting malic enzymes (NAD) and (NADP), 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenases (NAD) and (NADP), was associated with a decline in amounts of total soluble minus RuBP Case protein per unit fresh weight of leaf. Reduced amounts of leaf protein may be associated with reduced nitrate uptake by roots and fungal demand for nitrogen in mildewed plants.     

Photosynthesis and leaf-nitrogen dynamics during leaf senescence of tropical maize cultivars in hydroponics in relation to N efficiency in the field

The selection process of nitrogen (N)-efficient cultivars during plant breeding could be simplified by a specification of secondary plant traits that are decisive for N efficiency. It was shown that leaf senescence under N deprivation of sixteen tropical maize cultivars in a short-term nutrient solution experiment was related to leaf senescence and grain yield under N deficiency (N efficiency) in field experiments. In this study we investigated if a quantification of leaf- and plant-N flows by ¹⁵N labelling can improve the evaluation of genotypic differences in leaf senescence in short-term experiments. Cultivars differed in leaf-N content prior to senescence; however, this appeared to have no significant impact on the development of leaf senescence. N import into senescing leaves was not related to total plant N uptake, but seems to have been regulated by leaf-inherent factors. Leaf N remaining in the leaf seems to have comprised inefficiently remobilized leaf N, at least during early senescence stages. Photosynthetic rate and chlorophyll contents at early senescence stages depended on additional factors to leaf-N content. Nevertheless, all parameters used to characterize leaf senescence were related to leaf senescence at anthesis in field experiments. However, only photosynthetic rate during late leaf senescence reflected cultivar differences in leaf senescence during reproductive growth and N efficiency in field experiments.