Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence

Of the total ¹⁴C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the ¹⁴C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid-August, 40% of the label in protein occurs in soluble protein. The specific radio-activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of ¹⁴C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits ¹⁴C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of ¹⁴C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll.     

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development

Partitioning and translocation of ¹⁴C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of ¹⁴CO₂ for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total ¹⁴C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered ¹⁴C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the ¹⁴C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The ¹⁴C-organic acids comprised from 3% to 14% of the soluble ¹⁴C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. ¹⁴C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while ¹⁴C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of ¹⁴C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that ¹⁴C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.     

小分子RNA在植物叶发育中的调控作用

叶发育是叶原基细胞有序的分裂、生长和分化的过程,受到植物激素和多个转录因子的严格调控.近年的研究表明,在叶片发育的过程中,小分子RNA是基因调控网络的重要组分.小分子RNA通过对其中一些转录因子的抑制作用,影响其表达水平和空间分布,维持叶的正常发育.本综述介绍了小分子RNA及其靶基因调控模块在叶片发生、 叶片形状、叶子极性发育和叶子衰老等过程中的调控作用,并展望了未来研究中新方向.     

Growth Regulators in Populus tremula IV. Apical Dominance and Suckering in Young Plants

Experiments with small plants of Populus tremula L. growing in solution culture indicate that polarly transported auxin is an important factor in the control of axillary bud growth. If the auxin supply from the growing apex is eliminated, the number of buds released is influenced by factors translocated in the transpiration stream from the roots. Suckers may be induced to develop from aspen roots, the age of which is six weeks or more. Removal of the growing apex and the axillary buds or stoppage of shoot growth by short day treatment were effective in inducing abundant suckering in small aspen plants. Some mature leaves had to be maintained, indicating the dependence of sucker formation on carbohydrate supply. These treatments are known to decrease auxin production in the shoots. Extraction and biological assay showed a decrease in the content of auxin in the roots as a consequence of removal of growing shoot parts. The results indicate that suckering in roots of intact aspen plants is prevented by auxin transported into the roots from growing shoot parts.     

Enhanced Incorporation of Tritium into Glycolate during Photosynthesis by Tobacco Leaf Tissue in the Presence of Tritiated Water

Tobacco (Nicotiana tabacum var. Havana Seed) leaf discs were allowed to photosynthesize for 3 to 20 minutes in the presence of ¹⁴CO₂ and ³H₂O. Several metabolites of the Calvin cycle and photorespiratory pathway were isolated and purified and the ³H:¹⁴C values measured. Glycolate had a 5- to 10-fold higher ³H:¹⁴C than the Calvin cycle intermediate 3-phosphoglyceric acid, or its end product sucrose. The glycolate oxidase inhibitor α-hydroxy-2-pyridinemethanesulfonic acid caused glycolate to accumulate in the tissue and lowered the ³H:¹⁴C in glycolate to a value similar to that in 3-phosphoglyceric acid. Phosphoglycolate, a possible precursor of glycolate arising from the Calvin cycle, exhibited a ³H:¹⁴C value similar to 3-phosphoglyceric acid under all conditions. The finding of a ³H enrichment in glycolate suggests that another source of glycolate, possibly the reduction of glyoxylate, exists in leaf tissue. Analyses of incorporation of ³H into the pro-2R and pro-2S hydrogens of glycolate, in the presence and absence of α-hydroxy-2-pyridinemethanesulfonic acid, suggest an alternative source of glycolate. Biochemical mechanisms to account for ³H enrichment into glycolate are evaluated.     

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F₁-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.     

Distribution of Glycinebetaine in Old and Young Leaf Blades of Salt-Stressed Barley Plants

In barley plants exposed to stepwise salt-stress (up to 200mM NaCl), sodium and chloride ions accumulated preferentiallyin old rather than in young leaf blades. Furthermore, the levelof glycinebetaine in young leaf blades was approximately threetimes that in old leaf blades. ³Present address: Department of Biochemistry, Faculty of Science,Kasesart University, Bangkok, 10900 Thailand     

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.     

Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.     

Changes in Leaf Proteins of Peas, Pisum sativum L., during Development on Deflorated Plants

The soluble (sap) proteins of leaves of pea, Pisum sativum L. cvs. Alaska and Greenfeast, allowed to develop normally or deflowered, to prevent senescence, were separated by isoelectric focusing.     

The Incorporation of d-Glucosamine-14C into Root Tissues of Higher Plants

d-Glucosamine-1-(14)C was rapidly taken up from aqueous solution by both excised bean (Phaseolus vulgaris) and corn (Zea mays) root tips. The labeled glucosamine did not accumulate in the tissues, however, but was metabolized to N-acetyl-d-glucosamine, N-acetyl-d-glucosamine phosphates, and uridine diphosphate N-acetyl-d-glucosamine. Little or no label was detected in respiratory CO(2), glycolytic intermediates, or d-glucosamine 6-phosphate. Between 5 and 10% of the (14)C was recovered in high molecular weight ethanol-insoluble materials which could be solubilized readily with alkali or by treatment with proteases, and which yielded labeled glucosamine upon complete hydrolysis with HCl. Milder hydrolytic conditions released quantities of N-acetylglucosamine-(14)C plus labeled fragments of higher molecular weight. It is concluded that d-glucosamine-(14)C may be used to label specifically the amino sugar residues of plant as well as animal macromolecules. N-Acetyl-d-glucosamine acts similarly as a precursor, except that it is taken up at only about 1/10 the rate of glucosamine and hence is utilized less efficiently.     

Long-Term Chilling of Young Tomato Plants under Low Light. III. Leaf Development as Reflected by Photosynthesis Parameters

Young tomato plants were exposed to two weeks of chilling undernon-photoinhibiting or mild photoinhibiting conditions. Thedevelopment of the leaves was studied under chilling and controlconditions by measuring several physiological parameters. Agradual decrease of the efficiency of the photosynthetic apparatuswith maturation and ageing occurred in unchilled plants. Thiswas reflected by gradual changes in CO₂-saturated photosynthesisand protein and rubisco contents. Except for senescing leaves,a correlation close to 1 : 1 was observed between maximum rubiscoactivity and CO₂-saturated photosynthesis. Chlorophyll (Chl)contents and photochemical chlorophyll fluorescence quenchingshowed strong decreases only in the last phase of senescencein the oldest leaves. In plants chilled under non-photoinhibitingconditions (10C, 100–150 µE m^–2 s^–1or 6C, 30–50 µE m^-2 s^–1), a similar patternof ageing was observed, and no indications were found for aninduction of protein or rubisco degradation by chilling. Sincethese plants stopped growing in the cold, they revealed lowertotal photosynthetic capacities than unchilled plants of thesame size. When the chilling conditions were mildly photoinhibitory(6C, 100–150 µE m^–2 s^–1), a much strongerdepression of rubisco activity and photosynthetic capacity wasfound in all leaves, which was partly reversible in the youngones. This decrease in CO₂fixation capacity, in turn, led toa higher susceptibility of the chilled plants to photoinhibitionat 20C. It is concluded that the decrease of both photosyntheticcapacity and growth after long-term chilling in tomato is aconsequence of the preceeding ageing and senescing of the leavesduring chilling, in contrast to chilling-tolerant species withthe ability for acclimation to low temperatures. (Received April 26, 1993; Accepted September 7, 1993)     

Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking

1. Download : Download high-res image (237KB)
2. Download : Download full-size image

     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition. This revised version was published online in June 2006 with corrections to the Cover Date.