小麦旗叶Rubisco周转与籽粒含氮量的关系

随着旗叶的衰老,Rubisco含量逐渐减少.延缓小麦旗叶的衰老进程(抽穗期施氮肥),可增加旗叶Rubisco的含量,提高籽粒的全氮含量.在小麦旗叶全展后28d内,Rubisco的15N丰度处于较高水平,表明仍有Rubisco的重新合成;而在28d以后,Rubisco的15N丰度处于低水平,表明无Rubisco的重新合成.但这时籽粒的15N丰度却上升.旗叶全展后14d内Rubisco的15N丰度高于旗叶中全氮的丰度,说明此时期Rubisco重新合成的速率高于其它蛋白质;旗叶衰老过程中Rubisco的15N丰度的净转移高于全氮,Rubi-sco净N转移也高于全氮,表明Rubisco向籽粒中转移的氮素多于其它蛋白质,对籽粒含氮量的影响最大.     

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.     

Reduced atmospheric CO2 inhibits nitrogen mobilization in Festuca rubra

In defoliated grasses, where photosynthesis is reduced due to removal of leaf material, it is well established that remobilization of nitrogen occurs from both older remaining leaves and roots towards the younger growing leaves. In contrast, little is known about the movement of nitrogen within intact grass plants experiencing prolonged inhibition of photosynthesis. We tested the following hypotheses in Festuca rubra L. ssp. rubra cv. Boreal: that both reduction of the atmospheric CO2 concentration and defoliation (1) induce mobilization of nitrogen from roots and older leaves towards growing leaves and (2) elicit similar directional change in the abundance of proteins in roots and older leaves relevant to the process of nitrogen mobilization including, glutamine synthetase (GS), EC 6.3.1.2; papain, EC 3.4.22.2; chymopapain, EC 3.4.22.6; ribulose bisphosphate carboxylase/oxygenase (Rubisco), EC 4.1.1.39; and the light harvesting complex of photosystem II (LHCPII). After growth at ambient atmospheric CO2 concentration, plants of F. rubra were subject to atmospheres containing either ambient (350 micro l l-1) or deplete (< 20 micro l l-1) CO2. Concurrently, plants were either left intact or defoliated on one occasion. Steady state 15N labelling coupled with a series of destructive harvests over a 7-day period enabled changes in the nitrogen dynamics of the plants to be established. Proteins pertinent to the process of nitrogen mobilization were quantified by immunoblotting. Irrespective of defoliation, plants in ambient CO2 mobilized nitrogen from older to growing leaves. This mobilization was inhibited by deplete CO2. Greater concentration of Rubisco and reduced chymopapain abundance in older remaining leaves of intact plants, in deplete compared with ambient CO2, suggested the inhibition of mobilization was due to inhibition of protein degradation, rather than to the export of degradation products. Both deplete CO2 and defoliation induced nitrogen mobilization from roots to growing leaves. In plants at ambient CO2, defoliation did not affect nitrogen uptake or its allocation. Therefore in F. rubra nitrogen mobilization can occur independently of any downregulation of nitrogen uptake. This suggests either different signal compounds may act to downregulate uptake and upregulate mobilization, or if one particular signalling compound is used its concentration threshold differs for induction of mobilization and downregulation of uptake. The abundance of the cysteine proteases papain and chymopapain was low in roots suggesting that they were not involved in protein degradation in this tissue.     

Low stomatal and internal conductance to CO2 versus Rubisco deactivation as determinants of the photosynthetic decline of ageing evergreen leaves

A novel A-Ci curve (net CO2 assimilation rate of a leaf -An- as a function of its intercellular CO2 concentration -Ci) analysis method (Plant, Cell & Environment 27, 137-153, 2004) was used to estimate the CO2 transfer conductance (gi) and the maximal carboxylation (Vcmax) and electron transport (Jmax) potentials of ageing, non-senescing Pseudotsuga menziesii leaves in relation to their nitrogen (N) content and protein and pigment composition. Both gi and the stomatal conductance (gsc) of leaves were closely coupled to Vcmax, Jmax and An with all variables decreasing with increasing leaf age. Consequently, both Ci and Cc (chloroplastic CO2 concentration) remained largely conserved through successive growing seasons. The N content of leaves, as well as the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other sodium dodecyl sulfate-soluble proteins, increased during the first three growing seasons, then stabilized or decreased only slightly afterwards. Thus, the age-related photosynthetic nitrogen use efficiency (PNUE) decline of leaves was not a consequence of decreased allocation of N towards Rubisco and other proteins involved in bioenergetics and light harvesting. Rather, loss of photosynthetic capacity was the result of the decreased activation state of Rubisco and proportional down-regulation of electron transport towards the photosynthetic carbon reduction (PCR) and photorespiratory (PCO) cycles in response to a reduction of CO2 supply to the chloroplasts' stroma. This study emphasizes the regulatory potential and homeostaticity of Cc- rather than photosynthetic metabolites or Ci- in relation to the commonly observed correlation between photosynthesis and gsc.     

Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines

To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI‐TOF‐MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3–10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein‐associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.     

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.     

Variation in the holm oak leaf proteome at different plant developmental stages, between provenances and in response to drought stress

Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions.     

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi)

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

Quantitative proteomics to study the response of wheat to contrasting fertilisation regimes

Negative environmental impacts from mineral fertilisers and pesticides used in conventional cropping have raised concern over the sustainability of arable crop production. Organic cropping uses alternatives that avoid many of these negative environmental effects; however, crop yields can be significantly reduced, possibly due to a lower proportion of plant-available nutrients. To gain insights into the molecular effects of organic compared to conventional cropping systems on plant utilisation of nutrients, we used proteomics to analyse winter wheat (Triticum aestivum). Our aim was to investigate the effects of contrasting fertility management and crop protection regimes in organic and conventional cropping systems on the wheat flag leaf proteome and the association between the proteome and physiological traits. Wheat flag leaves were flash-frozen, lyophilised and milled prior to protein extraction (TCA/acetone) and analysed using 2D gel electrophoresis and MALDI-TOF MS. The abundance of 111 protein spots varied significantly between fertilisation regimes. Flag leaf N and P composition were significant drivers of differences in protein spot abundance, including major proteins involved in nitrogen remobilisation, photosynthesis, metabolism and stress response. These results indicate that molecular-based mechanisms are involved in the effect of contrasting cropping systems on nutrient utilisation and wheat grain yield. Using a functional genomics approach, we were able to identify proteins that are linked to causal genes, enabling the potential development of functional molecular markers for crop improvement in nutrient use efficiency.     

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).     

Seasonal changes in photosynthesis and nitrogen allocation in leaves of different ages in evergreen understory shrub <Emphasis Type="Italic">Daphniphyllum humile</Emphasis>

Seasonal changes in photosynthetic capacity, leaf nitrogen (N) content, leaf chlorophyll (Chl) content and leaf N allocation patterns in leaves of different ages in the evergreen understory shrub, Daphniphyllum humile Maxim, growing at a forest border and an understory site were studied. In current-year leaves at the understory site, the N and Rubisco contents increased from spring to autumn although their light-saturated photosynthetic rate at 22°C (P _max²²) remained stable, indicating that their mesophyll conductance rates declined as they completed their development and/or that they invested increasing amounts of their resources in photosynthetic enzymes during this period. In contrast, seasonal changes in P _max²² in current-year leaves at the forest border site were correlated with changes in Rubisco content. In 1-year old leaves at the understory site, P _max²² and contents of Chl, leaf N, and Rubisco remained stable from spring to autumn, while these parameters decreased in 1-year-old forest border leaves, indicating that N may have been remobilized from shaded 1-year-old leaves to sunlit current-year leaves. When leaves senesced at the forest border site the Rubisco content decreased more rapidly than that of light-harvesting proteins such as LHCII, suggesting that N remobilization from Rubisco may be more efficient, possibly because Rubisco has greater N costs and is soluble, whereas the light-harvesting proteins are membrane components.     

Are contents of Rubisco, soluble protein and nitrogen in flag leaves of rice controlled by the same genetics?

Genetic relations among the contents of Rubisco, soluble protein and total leaf nitrogen (N) in leaves of rice (Oryza sativa L.) were studied by quantitative trait loci (QTL) analysis with a population of backcross inbred lines (BILs) of japonica Nipponbarexindica Kasalath. The ratio of Rubisco to total leaf N in leaves is the main target in improving photosynthetic N-use efficiency in plants. QTLs controlling Rubisco content were not detected near QTLs for total leaf N content. These results indicate that contents of Rubisco and total leaf N are controlled by different genetics. QTLs that controlled the ratio of Rubisco to total leaf N (CORNs) were detected. These results suggest that some mechanism(s) may be involved in determining this ratio, while the contents of Rubisco and total leaf N are controlled in other ways. In elite BILs, the ratios of Rubisco to total leaf N were higher than those of both parents. These results suggest a good possibility of improving N-use efficiency by CORNs in cultivated rice. A QTL controlling Rubisco content was mapped near a QTL for soluble protein content on chromosome 8 at 5 d after heading and on chromosome 9 at 25 d. In each chromosome region, the peaks of both QTLs overlapped accurately, giving a high possibility of pleiotropic effects by the same genes. Different QTLs controlling soluble protein or Rubisco were detected from those detected at 5 d or 25 d after heading. This suggests that these traits are genetically controlled depending on the growth stages of leaves.     

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

SO42- Deprivation Has an Early Effect on the Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Photosynthesis in Young Leaves of Wheat

Wheat (Triticum aestivum cv Chinese Spring) supplied with 0.45 mM SO42- for 14 d with relative growth rates (RGR) of 0.22 to 0.24 d-1 was deprived of S for 7 to 8 d. There was no significant effect on RGR or leaf development (leaf 2 length was constant; leaf 3 expanded for 2-4 d; leaf 4 emerged and elongated throughout the experiment) during the S deprivation. In controls the net assimilation rate (A) closely reflected leaf ontogeny. S deprivation affected A in all leaves, particularly leaf 4, in which A remained at 8 to 10 [mu]mol CO2 m-2 s-1, whereas in controls A rose steadily to >20 [mu]mol CO2 m-2 s-1. In leaf 2, with a fully assembled photosynthetic system, A decreased in S-deprived plants relative to controls only at the end of the experiment. Effects on A were not due to altered stomatal conductance or leaf internal [CO2] ([C]i); decreases in the initial slope of A/[C]i curves indicated an effect of S deprivation on the carboxylase efficiency. Measurement of Rubisco activity and large subunit protein abundance paralleled effects on A and A/[C]i in S-deprived leaves. Negative effects on photosynthesis in S-deprived plants are discussed in relation to mobilization of S reserves, including Rubisco, emphasizing the need for continuous S supply during vegetative growth.