Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens (D.Don.) Endl.)

A rapid and effective system of somatic embryogenesis and organogenesis from the in vitro needles of redwood (Sequoia sempervirens (D.Don.) Endl.) had been established. The influences of plant growth regulators (PGRs) and days of seedlings in vitro on adventitious bud regeneration and somatic embryogenesis were studied. The process of somatic embryo formation was also observed. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l). SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l). The optimal days of seedling in vitro for adventitious bud and somatic embryogenesis were 30 days and 30–40 days, respectively. The developments of somatic embryos were similar to that of zygotic embryogenesis. The result of histocytological studies indicated that proteins were gradually accumulated in the process of somatic embryo formation and there were two peaks of starch grains accumulation that one was in the embryogenic callus and the other was in the globular embryos. These results indicated that starch and protein were closely related with the energy supply and the molecular base of somatic embryogenesis, respectively.     

Hydraulic efficiency and safety of branch xylem increases with height in Sequoia sempervirens (D. Don) crowns

The hydraulic limitation hypothesis of Ryan & Yoder (1997, Bioscience 47, 235-242) suggests that water supply to leaves becomes increasingly difficult with increasing tree height. Within the bounds of this hypothesis, we conjectured that the vertical hydrostatic gradient which gravity generates on the water column in tall trees would cause a progressive increase in xylem 'safety' (increased resistance to embolism and implosion) and a concomitant decrease in xylem 'efficiency' (decreased hydraulic conductivity). We based this idea on the historically recognized concept of a safety-efficiency trade-off in xylem function, and tested it by measuring xylem conductivity and vulnerability to embolism of Sequoia sempervirens branches collected at a range of heights. Measurements of resistance of branch xylem to embolism did indeed show an increase in 'safety' with height. However, the expected decrease in xylem 'efficiency' was not observed. Instead, sapwood-specific hydraulic conductivities (Ks) of branches increased slightly, while leaf-specific hydraulic conductivities increased dramatically, with height. The latter could be largely explained by strong vertical gradients in specific leaf area. The increase in Ks with height corresponded to a decrease in xylem wall fraction (a measure of wall thickness), an increase in percentage of earlywood and slight increases in conduit diameter. These changes are probably adaptive responses to the increased transport requirements of leaves growing in the upper canopy where evaporative demand is greater. The lack of a safety-efficiency tradeoff may be explained by opposing height trends in the pit aperture and conduit diameter of tracheids and the major and semi-independent roles these play in determining xylem safety and efficiency, respectively.     

Fog interception by Sequoia sempervirens (D. Don) crowns decouples physiology from soil water deficit

Although crown wetting events can increase plant water status, leaf wetting is thought to negatively affect plant carbon balance by depressing photosynthesis and growth. We investigated the influence of crown fog interception on the water and carbon relations of juvenile and mature Sequoia sempervirens trees. Field observations of mature trees indicated that fog interception increased leaf water potential above that of leaves sheltered from fog. Furthermore, observed increases in leaf water potential exceeded the maximum water potential predicted if soil water was the only available water source. Because field observations were limited to two mature trees, we conducted a greenhouse experiment to investigate how fog interception influences plant water status and photosynthesis. Pre-dawn and midday branchlet water potential, leaf gas exchange and chlorophyll fluorescence were measured on S. sempervirens saplings exposed to increasing soil water deficit, with and without overnight canopy fog interception. Sapling fog interception increased leaf water potential and photosynthesis above the control and soil water deficit treatments despite similar dark-acclimated leaf chlorophyll fluorescence. The field observations and greenhouse experiment show that fog interception represents an overlooked flux into the soil–plant–atmosphere continuum that temporarily, but significantly, decouples leaf-level water and carbon relations from soil water availability.     

Small RNAs of Sequoia sempervirens during rejuvenation and phase change

In this work, the population of small RNAs (sRNAs) was studied in the gymnosperm Sequoia sempervirens during phase changes, specifically in the juvenile, adult and rejuvenated plants obtained in vitro. The potential target genes of Sequoia sRNAs were predicted through bioinformatics. Rejuvenation is a pivotal process in woody plants that enables them to regain their growth potential, which results in the recovery of physiologic and molecular characteristics that were lost when the juveniles mature into adult plants. The results from the five repeated graftings of juvenile, adult and rejuvenated plants in vitro showed that sRNAs could be classified into structural RNAs (Group I), small interfering RNAs (Group II), annotated microRNAs (Group III, and unannotated sRNAs (Group IV). The results indicate that only 573 among 15,485,415 sRNAs (Groups III and IV) had significantly different expression patterns associated with rejuvenation and phase change. A total of 215 sRNAs exhibited up‐regulated expression patterns in adult shoots, and 358 sRNAs were down‐regulated. Expression profiling and prediction of possible target genes of these unique small RNAs indicate possible functions in the control of photosynthetic efficiency and rooting competence abundance during plant rejuvenation. Moreover, the increase in SsmiR156 and decrease in SsmiR172 during plant rejuvenation suggested that these two microRNAs extensively affect phase transition.     

Rhizonema sequoiae n.gen. n.sp. from Coast Redwood Sequoia sempervirens (D.Don) Endl.

Rhizonema sequoiae n.gen, u. sp. is described from the roots of Coast Redwood, Sequoia sempervirens (D. Don) Endl., growing near Lake Lagunitas, Marin County, California. Rhizonema females are annulated over their entire bodies, are wholly embedded in host tissue, and secrete an abundant amount of gel material. Mature females do not form a cyst. The vulva is located on a large posterior terminal cone, and the anus is on the dorsal vulval lip. Esophageal glands of the second-stage larvae fill more than half of the body cavity. Tails of the vermiform males are blunt, and a cloacal tubus is present.     

Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco,Calocedrus decurrens (Torr.), and Pinus taeda L.

Summary The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis.     

The contribution of fog to the water relations of Sequoia sempervirens (D. Don): foliar uptake and prevention of dehydration

Fog is a defining feature of the coastal California redwood forest and fog inputs via canopy drip in summer can constitute 30% or more of the total water input each year. A great deal of occult precipitation (fog and light rain) is retained in redwood canopies, which have some of the largest leaf area indices known (Westman & Whittaker, Journal of Ecology 63, 493–520, 1975). An investigation was carried out to determine whether some fraction of intercepted fog water might be directly absorbed through leaf surfaces and if so, the importance of this to the water relations physiology of coast redwood, Sequoia sempervirens. An array of complimentary techniques were adopted to demonstrate that fog is absorbed directly by S. sempervirens foliage. Xylem sap transport reversed direction during heavy fog, with instantaneous flow rates in the direction of the soil peaking at approximately 5–7% of maximum transpiration rate. Isotopic analyses showed that up to 6% of a leaf's water content could be traced to a previous night's fog deposition, but this amount varied considerably depending on the age and water status of the leaves. Old leaves, which appear most able to absorb fog water were able to absorb distilled water when fully submersed at an average rate of 0.90 mmol m² s^?1, or about 80% of transpiration rates measured at the leaf level in the field. Sequoia sempervirens has poor stomatal control in response to a drying atmosphere, with rates of water loss on very dry nights up to 40% of midday summer values and rates above 10% being extremely common. Owing to this profligate water use behaviour of S. sempervirens, it appears that fog has a greater role in suppressing water loss from leaves, and thereby ameliorating daily water stress, than in providing supplemental water to foliar tissues per se. Although direct foliar absorption from fog inputs represents only a small fraction of the water used each day, fog's in reducing transpiration and rehydrating leaf tissues during the most active growth periods in summer may allow for greater seasonal carbon fixation and thus contribute to the very fast growth rates and great size of this species.     

Microsatellite markers in coast redwood (Sequoia sempervirens)

Five polymorphic microsatellite markers are described for coast redwood, a hexaploid conifer. Dinucleotide (three), trinucleotide (one) and tetranucleotide (one) repeat loci were isolated from a genomic plasmid library and amplified in a test population of 25 second‐growth redwoods. There were three to 11 alleles per locus and observed heterozygosities ranged from 0.00 to 0.80. The nonheterozygous locus (a CTTA repeat) is probably of chloroplast origin and may have potential as a marker for paternal inheritance.     

Proteomic profiling of proteins associated with lymph node metastasis in colorectal cancer

Lymph node metastasis (LNM) is associated with poor prognosis in colorectal cancer (CRC). The presence or absence of lymph node metastases is a strong independent prognostic factor for CRC survival. Investigation of proteins associated with the process of lymph node metastasis (LNM) is crucial for understanding of the molecular mechanisms underlying the LNM process and for predicting the CRC prognosis. In the present study, proteins from CRC tissues and adjacent normal mucosa (NMC) were examined using two‐dimensional gel electrophoresis coupled with MALDI‐TOF‐MS. The expression levels of Ferritin Heavy Chain (FHC) were decreased in LNM CRC as compared to those in non‐LNM CRC, while the expression of Cathepsin D and Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1) were increased in LNM CRC. The results were confirmed by Western blotting and immunohistochemical staining. Furthermore, in vitro cell invasion assay showed that the overexpression of UCH‐L1 through gene transfection increased the invasive ability of HCT8 cells, suggesting that UCH‐L1 is not only a biomarker for LNM in CRC, but also a functional protein that may play a significant role in cell migration. The proteins identified in the present study should further our understanding of the LNM process of CRC and may become useful markers for diagnosis and targets for therapeutic interventions. J. Cell. Biochem. 110: 1512–1519, 2010. © 2010 Wiley‐Liss, Inc.     

Temporal changes in the leaf monoterpenes of Sequoia sempervirens

Sequoia sempervirens leaf monotorpenes were analysed from bud burst through maturity. Significant changes occurred in both quantitative composition and total yield/dry wt during the first six months of leaf development. Composition then remained stable until initiation of twig abscission. Total yield also remained relatively stable except for a sharp increase at month 15, followed by a return to earlier levels and stability until initiation of abscission. The age of the shoot was important only for individuals less than 1 m high. Simulated herbivory by leaf removal showed only minor compositonal changes when plants were resampled one year later. Results support the concept that variation in individual compounds could provide an effective defensive strategy against herbivores and pathogens, and also establish a suitable time period for minimizing variation in sample collections.     

Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain

It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.     

Anti-tumor abietane diterpenes from the cones of Sequoia sempervirens

A new abietane, namely, 20-hydroxyferruginol (1), together with known ferruginol (2), 18-hydroxyferruginol (3), sugiol (4), and 6alpha-hydroxysugiol (5), were isolated from the cones of Sequoia sempervirens. Their structures were elucidated through spectral data. Compounds 1 and 5 strongly inhibited colon, lung, and breast human tumors and oncogene transformed cells with GI(50) 2-5 microg/mL.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Proteomic profiling of giant skeletal muscle proteins

Introduction: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys.

Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults.

Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.     

A hydraulic-photosynthetic model based on extended HLH and its application to Coast redwood (Sequoia sempervirens)

Although recent investigations [Ryan, M.G., Yoder, B.J., 1997. Hydraulic limits to tree height and tree growth. Bioscience 47, 235-242; Koch, G.W., Sillett, S.C.,Jennings, G.M.,Davis, S.D., 2004. The limits to tree height. Nature 428, 851-854; Niklas, K.J., Spatz, H., 2004. Growth and hydraulic (not mechanical) constraints govern the scaling of tree height and mass. Proc. Natl Acad. Sci. 101, 15661-15663; Ryan, M.G., Phillips, N., Bond, B.J., 2006. Hydraulic limitation hypothesis revisited. Plant Cell Environ. 29, 367-381; Niklas, K.J., 2007. Maximum plant height and the biophysical factors that limit it. Tree Physiol. 27, 433-440; Burgess, S.S.O., Dawson, T.E., 2007. Predicting the limits to tree height using statistical regressions of leaf traits. New Phytol. 174, 626-636] suggested that the hydraulic limitation hypothesis (HLH) is the most plausible theory to explain the biophysical limits to maximum tree height and the decline in tree growth rate with age, the analysis is largely qualitative or based on statistical regression. Here we present an integrated biophysical model based on the principle that trees develop physiological compensations (e.g. the declined leaf water potential and the tapering of conduits with heights [West, G.B., Brown, J.H., Enquist, B.J., 1999. A general model for the structure and allometry of plant vascular systems. Nature 400, 664-667]) to resist the increasing water stress with height, the classical HLH and the biochemical limitations on photosynthesis [von Caemmerer, S., 2000. Biochemical Models of Leaf Photosynthesis. CSIRO Publishing, Australia]. The model has been applied to the tallest trees in the world (viz. Coast redwood (Sequoia sempervirens)). Xylem water potential, leaf carbon isotope composition, leaf mass to area ratio at different heights derived from the model show good agreements with the experimental measurements of Koch et al. [2004. The limits to tree height. Nature 428, 851-854]. The model also well explains the universal trend of declining growth rate with age.     

Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus

The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of 2-DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.