Tomasz Andrzej Pawłowski

Proteome analysis of Norway maple (Acer platanoides L.) seeds dormancy breaking and germination: influence of abscisic and gibberellic acids

BackgroundSeed dormancy is controlled by the physiological or structural properties of a seed and the external conditions. It is induced as part of the genetic program of seed development and maturation. Seeds with deep physiological embryo dormancy can be stimulated to germinate by a variety of treatments including cold stratification. Hormonal imbalance between germination inhibitors (e.g. abscisic acid) and growth promoters (e.g. gibberellins) is the main cause of seed dormancy breaking. Differences in the status of hormones would affect expression of genes required for germination. Proteomics offers the opportunity to examine simultaneous changes and to classify temporal patterns of protein accumulation occurring during seed dormancy breaking and germination. Analysis of the functions of the identified proteins and the related metabolic pathways, in conjunction with the plant hormones implicated in seed dormancy breaking, would expand our knowledge about this process.ResultsA proteomic approach was used to analyse the mechanism of dormancy breaking in Norway maple seeds caused by cold stratification, and the participation of the abscisic (ABA) and gibberellic (GA) acids. Forty-four proteins showing significant changes were identified by mass spectrometry. Of these, eight spots were identified as water-responsive, 18 spots were ABA- and nine GA-responsive and nine spots were regulated by both hormones. The classification of proteins showed that most of the proteins associated with dormancy breaking in water were involved in protein destination. Most of the ABA- and GA-responsive proteins were involved in protein destination and energy metabolism.ConclusionIn this study, ABA was found to mostly down-regulate proteins whereas GA up-regulated proteins abundance. Most of the changes were observed at the end of stratification in the germinated seeds. This is the most active period of dormancy breaking when seeds pass from the quiescent state to germination. Seed dormancy breaking involves proteins of various processes but the proteasome proteins, S-adenosylmethionine synthetase, glycine-rich RNA binding protein, ABI3-interacting protein 1, EF-2 and adenosylhomocysteinase are of particular importance. The effect of exogenously applied hormones was not a determining factor for total inhibition (ABA) or stimulation (GA) of Norway maple seed dormancy breaking and germination but proteomic data has proven these hormones play a role.

Proteomic approach to analyze dormancy breaking of tree seeds

In forest broadleaves from the temperate zone, a large number of species exhibit seed dormancy phenomena. Tree seeds show some of the most pronounced and complicated forms of dormancy in the plant kingdom. Many seeds are deeply physiologically dormant whatever their moisture level and age. However, dormancy can usually be overcome by a cold or warm stratification for several months. The transition from seed dormancy to germination is a multi-step process. In combination with the availability of genome sequence data, proteomics has opened up enormous possibilities for identifying the total set of expressed proteins as well as expression changes during dormancy breaking. The proteomic approach used for analysis of dormancy breaking of tree seeds offers new data allowing better understanding of the mechanism of deep physiological dormancy. The results of proteomic studies on dormancy breaking and the presence of abscisic and gibberellic acids in tree seeds (beech Fagus sylvatica L., Norway maple Acer platanoides L. and sycamore Acer pseudoplatanus L.), help to explain this process better. Most of the changes in protein expression were observed at the end of stratification and in the germinated seeds. This is the most active period of dormancy breaking when seeds pass from the quiescent state to germination. The analysis of the proteins’ function showed that the mechanism of seed dormancy breaking involves many processes. Energy metabolism, proteasome, transcription, protein synthesis, signal transduction and methionine metabolism proteins have a special importance.

Ferroelasticity of M 3 H(XO 4 ) 2 Crystals: FT NIR Raman, Ferroelastic Domain and Pretransitional Effects Studies

Temperature studies of Raman spectra and ferroelastic domain structure of Rb 3 H(SeO 4 ) 2, Rb 3 H(SO 4 ) 2 and (NH 4 ) 3 H(SO 4 ) 2 crystals are presented. Anomalous behaviour of frequencies and line width of bands related to the internal vibrations of SO 4 and SeO 4 in the vicinity of superprotonic phase transitions T S was found. Significant changes in the ferroelastic domain patterns just below T S are also presented. It was found that Rb 3 H(SO 4 ) 2 is not a ferroelastic in low conductivity phase. The role of ferroelasticity in the superprotonic phase transition is discussed.

Multiple microtubule nucleation sites in higher plants

In most eucaryotes, microtubule (MT) nucleation requires the activity of protein complexes containing -tubulin, Spc98p and Spc97p. These proteins form -tubulin ring complexes ( -TuRCs), which are recruited at structured MT organizing centers (MTOCs) (Moritz and Agard, 2001). In higher plants, the cytoskeleton organization in multiple MT arrays, which succeed one another during the cell cycle and developmental processes, may be linked to the dispersion of MTOC sites. The question remains open to know as to how and where plant MTs are nucleated.

Proteomic Analysis of Embryogenesis and the Acquisition of Seed Dormancy in Norway Maple (Acer platanoides L.)

The proteome of zygotic embryos of Acer platanoides L. was analyzed via high-resolution 2D-SDS-PAGE and MS/MS in order to: (1) identify significant physiological processes associated with embryo development; and (2) identify changes in the proteome of the embryo associated with the acquisition of seed dormancy. Seventeen spots were identified as associated with morphogenesis at 10 to 13 weeks after flowering (WAF). Thirty-three spots were associated with maturation of the embryo at 14 to 22 WAF. The greatest changes in protein abundance occurred at 22 WAF, when seeds become fully mature. Overall, the stage of morphogenesis was characterized by changes in the abundance of proteins (tubulins and actin) associated with the growth and development of the embryo. Enzymes related to energy supply were especially elevated, most likely due to the energy demand associated with rapid growth and cell division. The stage of maturation is crucial to the establishment of seed dormancy and is associated with a higher abundance of proteins involved in genetic information processing, energy and carbon metabolism and cellular and antioxidant processes. Results indicated that a glycine-rich RNA-binding protein and proteasome proteins may be directly involved in dormancy acquisition control, and future studies are warranted to verify this association.

Proteins and polyamines during dormancy breaking of European beech (Fagus sylvatica L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.

Qualitative changes in protein content during cold and warm stratification of Norway maple ( Acer platanoides L.) seeds

Protein synthesis in embryonic axes of Norway maple ( Acer platanoides ) was studied in seeds stratified at 3 or 15°C. At 3°C stratification, germination of seeds started between 6 and 7 weeks; at 15°C stratification germination did not occur. During imbibition of embryonic axes undergoing cold and warm stratification many new proteins became visible. Two of these proteins may be connected with the release from dormancy at 3°C. In seeds stratified at 15°C, these proteins occur in low amounts or are completely absent.

Analysis of the embryo proteome of sycamore (Acer pseudoplatanus L.) seeds reveals a distinct class of proteins regulating dormancy release

Acer pseudoplatanus seeds are characterized by a deep physiological embryo dormancy that requires a few weeks of cold stratification in order to promote germination. Understanding the function of proteins and their related metabolic pathways, in conjunction with the plant hormones implicated in the breaking of seed dormancy, would expand our knowledge pertaining to this process. In this study, a proteomic approach was used to analyze the changes occurring in seeds in response to cold stratification, which leads to dormancy release. In addition, the involvement of abscisic (ABA) and gibberellic acids (GA) was also examined. Fifty-three proteins showing significant changes were identified by mass spectrometry. An effect of ABA on protein variation was observed at the beginning of stratification, while the influence of GA on protein abundance was observed during the middle phase of stratification. The majority of proteins associated with dormancy breaking in the presence of only water, and also ABA or GA, were classified as being involved in metabolism and genetic information processing. For metabolic-related proteins, the effect of ABA on protein abundance was stimulatory for half of the proteins and inhibitory for half of the proteins. On the other hand, the effect on genetic information processing related proteins was stimulatory. GA was found to upregulate both metabolic-related and genetic information processing-related proteins. While seed dormancy breaking depends on proteins involved in a variety of processes, proteins associated with methionine metabolism (adenosine kinase, methionine synthase) and glycine-rich RNA binding proteins appear to be of particular importance.

Dielectric behaviour and conformational disorder in polymer relaxors

Abstract Radiation-induced conformational disorder in ferroelectric P(VDF/TrFE)(75/25) irradiated in the paraelectric phase with fast electrons was studied using IR and NIR Raman spectroscopy and related to changes in dielectric relaxation. The observed radiation-induced changes in the intensity of bands characteristic of all-trans and trans-gauche conformations confirm the scission of long-range all-trans chain conformation. Electron irradiation modifies also the surface topography of the copolymer film: AFM studies of irradiated samples show a considerable increase in the surface roughness.

Preliminary analysis of the cauliflower mitochondrial proteome

Purified cauliflower (Brassica oleracea var. botrytis) mitochondrial proteins fractionated into soluble, membrane, integral membrane and peripheral membrane samples were analyzed by 2D- PAGE (isoelectric focusing/ SDS polyacrylamide gel electrophoresis). 2D gels patterns were compared using the Imager Master 2D Elite software. 561 silver stained protein spots were resolved after electrophoresis under standard conditions of a whole protein extract. In the soluble fraction a prevalent number of more intense protein spots was observed. The cauliflower protein 2D patterns resembled Arabidopsis thaliana 2D patterns. The two protein spots selected which occupied a similar isoelectric point positions on both gels represented the same proteins as revealed by ESI-MS analysis of cauliflower proteins. The third selected spot belongs to unidentified proteins. The comparative analysis of mitochondrial suborganellar fractions proved the usefulness of this approach.