Light-Independent and Tissue-Specific Accumulation of Light-Harvesting Chlorophyll a/b Binding Protein and Ribulose Bisphosphate Carboxylase in Dark-Grown Pine Seedlings

The presence of light-harvesting chlorophyll a/b binding proteinsand the small and large subunits of ribulose bisphosphate carboxylasewas revealed in dark-grown pine seedlings. The light-independentaccumulation of the proteins was observed in photosyntheticand/or green tissue, such as cotyledons and hypocotyls, butnot in non-photosynthetic roots. ¹ This work was supported by Grants-in-Aid from the Ministryof Agriculture, Forestry and Fisheries of Japan (IntegratedResearch Program for the Use of Biotechnological Proceduresfor Plant Breeding), and from the Science and Technology Agencyof Japan (Encouragement of Basic Research). (Received May 2, 1991; Accepted August 13, 1991)     

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.     

His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR)⁴ from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1–3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4–6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans→13-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7, 8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family.⁵One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the protein active site and specifically to the Schiff base counterion Asp-97 inferred from the x-ray crystal structure of BR suggests its involvement in spectral tuning of the visible absorption (10) and potentially PR photochemical reactions. Because the pK_a of histidine in solution is close to neutral pH (11), its imidazole group often plays a major role in intramolecular proton transfers in enzymes, including NADPH oxidase (12), alcohol dehydrogenase (13), carbonic anhydrase II (14), and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic charge displacement measurements, and site-directed mutagenesis to study the role of His-75 in GPR. We report evidence that protonated His-75 interacts directly with Asp-97 in the unphotolyzed protein and during the photocycle undergoes a deprotonation in response to the protonation of Asp-97.     

Chlorophyll Formation in the Dark I. Chlorophyll in Pine Seedlings

In black pine (Pinus nigra) chlorophyll was first detected two days after planting of the soaked seeds. There was a slight change in the chlorophyll content between two and five days, then a faster chlorophyll synthesis started. The maximum chlorophyll content was found after 15 days of germination. Thereafter the content showed a gradual decrease. The chlorophyll a/b ratio decreased from 10 at two days to 3 after five days. Protochlorophyllide did not accumulate after 6 days of germination.     

Kinetics of Freezing Damage in Apple Bark and Pine Needles

The time course of freezing damage in pine needles and in bark of apple trees was followed at different subzero temperatures. From these data the killing rate by freezing was determined for trees which differed in degree of cold hardiness. The activation energy of the killing reaction was also calculated. The killing rate was lowest in cold-acclimated trees, but the activation energy of the killing reaction was very high indicating a high degree of structured water in the cells. Non-acclimated trees showed uniform low values of the activation energy of the killing reaction at all subzero temperatures studied. It is suggested that intracellular supercooling could be a part of the mechanism of frost protection in cold-acclimated apple trees within the – 30 to – 20°C range, but not in the –20 to –10°C range.     

Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.     

Removing Phosphorus from Aqueous Solutions Using Lanthanum Modified Pine Needles

The renewable pine needles was used as an adsorbent to remove phosphorus from aqueous solutions. Using batch experiments, pine needles pretreated with alkali-isopropanol (AI) failed to effectively remove phosphorus, while pine needles modified with lanthanum hydroxide (LH) showed relatively high removal efficiency. LH pine needles were effective at a wide pH ranges, with the highest removal efficiency reaching approximately 85% at a pH of 3. The removal efficiency was kept above 65% using 10 mg/L phosphorus solutions at desired pH values. There was no apparent significant competitive behavior between co-existing anions of sulfate, nitrate, and chloride (SO₄^2-, NO₃^- and Cl^-); however, CO₃^2- exhibited increased interfering behavior as concentrations increased. An intraparticle diffusion model showed that the adsorption process occurred in three phases, suggesting that a boundary layer adsorption phenomena slightly affected the adsorption process, and that intraparticle diffusion was dominant. The adsorption process was thermodynamically unfavorable and non-spontaneous; temperature increases improved phosphorus removal. Total organic carbon (TOC) assays indicated that chemical modification reduced the release of soluble organic compounds from 135.6 mg/L to 7.76 mg/L. This new information about adsorption performances provides valuable information, and can inform future technological applications designed to remove phosphorus from aqueous solutions.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Chlorophyll Formation in the Dark II. Chlorophyll in Wheat Leaves Transplanted to Pine Megagametophytes

Chlorophyll production in wheat (Triticum vulgare) leaves takes place in the dark when wheat embryos are transplanted to the megagametophytes of black pine (Pinus nigra). The chlorophyll content in the wheat leaves is about half that in black pine cotyledons of the same age.     

Protoplasmic Changes in Cambial Cells Induced by a Tracheid-Differentiation Factor from Pine Needles

Direct differentiation of ‘dormant’ fusiform cambialcells (fees) into tracheids in stem cuttings of Pinus contortaDougl. was investigated by light and transmission electron microscopy.In cuttings from which all but one pair of needles had beenremoved, differentiation occurred close to the needle tracewithout prior cell division or expansion. The highly osmiophillic,granular cytoplasm of differentiating fees exhibited numerousmitochondria, dictyosomes and endoplasmic reticulum (ER) cisternaeand was enriched with vesicles and polyribosomes. The protoplasmdid not become highly vacuolated, as it normally does, untildifferentiating tracheids were approaching maturation. During differentiation, secondary walls were deposited in feesas ribs of annular and spiralled thickenings and also as borderedpit-likestructures devoid of margo and torus and lacking the typicallamellar structure of secondary-xylem tracheids. The fee plasmamembrane at sites of secondary-wall growth was in dynamic flux,dilated vesicles derived from both ER and dictyosomes apparentlyfusing with it at these locations. In addition, electron-denseblebs were invariably plasma membrane-associated and appearedto be exocytotic; these blebs became rare during autolysis andthey were never seen in non-differentiating cells. Depositionof oriented nascent cellulose microfibrils appeared to occurin the absence of associated cortical microtubules. As secondary-walldeposition neared completion, vacuoles derived from rough ERgrew and fused; concomitantly, the protoplasm disappeared resultingin fully autolysed tracheids. Auxin (indol-3-ylacetic acid, IAA) promoted the gel-like protoplasmof dormant fees to became highly vacuolated, and cell divisionand expansion followed; however, tracheid differentiation didnot occur. Polyribosomes, rough ER, and dictysomes were lessabundant than in differentiating fees and the cytoplasm of auxin-treatedfees was fine grained and less densely stained. The electron-denseexocytotic blebs found at the plasmalemma in differentiatingfees were not induced by IAA treatment. Fees of control cuttingsenlarged somewhat and became more vacuolated but otherwise remaineddormant. Key words: Cambium, exocytosis, Gymnospermae, ultrastructure, xylogenesis     

Growth and Nutrient Retranslocation in Needles of Radiata Pine in Relation to Nitrogen Supply

The effects of N application on tree growth and the retranslocationof N, P, and K from young needles to new growth were examinedin young radiata pine (Pinus radiata D. Don) trees. Nitrogen fertilization increased the number and size of needles,rates of shoot production, stem volume growth and tree biomass.Foliar N and P contents (µg per needle) fluctuated ina cyclic fashion with prominent phases of accumulation, retranslocationand replenishment. The patterns of these fluctuations in controland N-fertilized trees were similar, although the fluxes ofN, P and K in and out of needles were increased by N fertilization.Greater translocation (g per tree) of N and K from needles ofN fertilized trees occurred because fertilization increasedthe needle weight and the proportion of N and K retranslocatedfrom individual needles. Nitrogen fertilization increased theretranslocation of P largely as a result of higher needle mass.Trees supplied with more than adequate amounts of P in the soilretranslocated up to 58 per cent of the initial pool of P fromyoung needles. The periods of high retranslocation coincidedwith periods of high concentrations of soil mineral N and withshoot production. Conversely, the periods of rapid replenishmentof N and P into the needles coincided with the time of slowshoot growth and low concentration of soil mineral N. The growthrate of trees, rather than the availability of nutrients inthe soil was the main factor controlling retranslocation. For radiata pine, retranslocation from needles is not a mechanismspecific for coping with low soil fertility. It seems to bea mechanism which enhances the nutrient supply to apical growingpoints, especially during periods of flushing. Pinus radiata, nitrogen supply, shoot growth, nutrient fluctuations and retranslocation, nutrient use and adaptation     

Movement of Nutrients in Radiata Pine Needles in Relation to the Growth of Shoots

The pattern of needle growth and the movement of mineral nutrients(nitrogen, phosphorus, potassium and calcium) in needles ofradiata pine (Pinus radiata D. Don) were studied from needleinitiation to 2 years of age. During this period, very prominentcyclic patterns of nutrient accumulation, retranslocation andreplenishment were observed for nitrogen, phosphorus and potassium,which highlighted the potential role of needles as a nutrientreserve for growth. Significant retranslocation occurred from very young needlesabout 3 months after initiation. The phases of retranslocationcoincided with new flushes of shoot growth, and the growth ofnew shoots on a branch resulted in nutrient withdrawal frompre-existing needles, regardless of needle age and season. Suchwithdrawal occurred even in fertilized and irrigated trees onhigh quality sites and under environmental conditions conducivefor nutrient uptake. At all times, except for the short periodafter initiation when needles were actively growing, the nutrientsin the needle were readily available for retranslocation. Contraryto the general view, retranslocation of nutrients was not necessarilyrelated to senescence and ageing of needles. Because new shoots are the primary ‘sinks’ for retranslocatednutrients, an ongoing competition between different parts ofa branch for internal nutrients can be envisaged, preferencebeing for the youngest shoot in the hierarchy. The relevanceof these results to our understanding of ‘optimum nutrition’of pine trees is discussed. Pinus radiata D. Don, radiata pine, mineral nutrition, retranslocation, phosphorus, nitrogen, shoot growth     

Needles in the Haystack: Identifying Individuals Present in Pooled Genomic Data

Recent publications have described and applied a novel metric that quantifies the genetic distance of an individual with respect to two population samples, and have suggested that the metric makes it possible to infer the presence of an individual of known genotype in a sample for which only the marginal allele frequencies are known. However, the assumptions, limitations, and utility of this metric remained incompletely characterized. Here we present empirical tests of the method using publicly accessible genotypes, as well as analytical investigations of the method''s strengths and limitations. The results reveal that the null distribution is sensitive to the underlying assumptions, making it difficult to accurately calibrate thresholds for classifying an individual as a member of the population samples. As a result, the false-positive rates obtained in practice are considerably higher than previously believed. However, despite the metric''s inadequacies for identifying the presence of an individual in a sample, our results suggest potential avenues for future research on tuning this method to problems of ancestry inference or disease prediction. By revealing both the strengths and limitations of the proposed method, we hope to elucidate situations in which this distance metric may be used in an appropriate manner. We also discuss the implications of our findings in forensics applications and in the protection of GWAS participant privacy.     

Influence of Plant-Parasitic Nematodes on Longleaf Pine Seedlings

Seedlings of longleaf pine (Pinus palustris) were grown in 20-cm pots for 5 to 7 months in the greenhouse following inoculation with a high or low level of one of seven species of plant-parasitic nematodes. Belonolaimus longicaudatus and Helicotylenchus dihystera had no effect on seedling growth. High inoculum densities of Hoplolaimus galeatus and Tylenchorhynchus claytoni caused a significant reduction of fresh weight of seedling roots. Root and top weights of seedlings grown in soil infested with Meloidodera floridensis or Pratylenchus brachyurus were significantly less than those of seedlings in noninfested soil. Root growth of seedlings was stimulated by the higher inoculum density of Scutellonema brachyurum.