Tissue-specific variation of softwood nanostructure revealed by scanning wide-angle X-ray scattering and clustering analysis

Cytoskeletal involvement in cotton fiber growth and development

Cellulose in plant cell walls are synthesized as crystalline microfibrils with diameters of 3-4 nm and lengths of around 1-10 μm. These microfibrils are known to be the backbone of cell walls, and their multiscale three-dimensional organization plays a critical role in cell wall functions including plant growth and recalcitrance to degradation. The mesoscale organization of microfibrils over a 1-100 nm range in cell walls is challenging to resolve because most characterization techniques investigating this length scale suffer from low spatial resolution, sample preparation artifacts, or inaccessibility of specific cell types. Here, we report a sum frequency generation (SFG) study determining the mesoscale polarity of cellulose microfibrils in intact plant cell walls. SFG is a nonlinear optical spectroscopy technique sensitive to the molecular-to-mesoscale order of noncentrosymmetric domains in amorphous matrices. However, the quantitative theoretical model to unravel the effect of polarity in packing of noncentrosymmetric domains on SFG spectral features has remained unresolved. In this work, we show how the phase synchronization principle of the SFG process is used to predict the relative intensities of vibrational modes with different polar angles from the noncentrosymmetric domain axis. Applying this model calculation for the first time and employing SFG microscopy, we found that cellulose microfibrils in certain xylem cell walls are deposited unidirectionally (or biased in one direction) instead of the bidirectional polarity which was believed to be dominant in plant cell walls from volume-averaged characterizations of macroscopic samples. With this advancement in SFG analysis, one can now determine the relative polarity of noncentrosymmetric domains such as crystalline biopolymers interspersed in amorphous polymer matrices, which will open opportunities to study new questions that have not been conceived in the past.

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging

Wall Organization in Plant Cells

1 We searched for antifeedant activity in predomonantly non‐host woody plants to find new compounds for seedling protection of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) against feeding by pine weevil Hylobius abietis. In total, 38 species from 25 families were compared in choice and no‐choice tests.2 In choice tests with Empetrum, Juniperus, Ledum, Populus, Betula, Evonymus, Sorbus, Salix, Myrica and Pinus, the weevils preferred Pinus to all others. In no‐choice tests with the same species, the insects removed a similar or even greater area of the bark in three of 10 species than Pinus. The results were clearly different between the test modes.3 In experiment 4, the areas of outer and inner bark (phloem) removed were quantified separately.The weevils removed significantly less of both outer and inner bark in Ilex, Evonymus, Populus, Syringa, Taxus, Tilia, Viburnum, Lonicera and Sorbus than Pinus.4 Large areas of outer bark were removed in Juglans, Fraxinus, Sambucus, Aesculus, Quercus, Corylus, Fagus, Salix, Alnus and Acer. However, in the latter cases the insects stopped when reaching the inner bark. Thus, certain plant species have the outer bark removed by the insects but possessed an inner bark with antifeedant qualities.

Dibenzodioxocin, an 8-ring substructure of lignin identified in the mid- 1990's, is known to occur in softwood cell walls especially in the S3-layers of normal wood. In this study the lignin substructure was immunolocalised in juvenile and mature wood as well as in different degrees of compression wood of Norway spruce (Picea abies (L.) H. Karst.) and Scots pine (Pinus sylvestris L.). In juvenile wood of Norway spruce, dibenzodioxocin was hardly present in the tracheid cell wall, while in Scots pine some dibenzodioxocin was found evenly distributed in the S2-layers. In mature normal wood, dibenzodioxocin was localised in the S3-layers in both Scots pine and Norway spruce. In contrast, in compression wood tracheids of Scots pine, where the S3-layer is not present, dibenzodioxocin was found in the S1-layers and in the outer part of the S2-layers, while in Norway spruce the innermost cell wall layer showed a strong signal. These findings support the idea that in mature wood the condensed dibenzodioxocin structure is formed in Norway spruce at the end of lignification, when the supply of monolignols and probably also hydrogen peroxide is diminishing. The reasons for Scots pine juvenile and compression wood showing a different pattern of dibenzodioxocin labelling is discussed.

Цель исследований − изучение сезонного роста деревьев Pinus sylvestris и Picea abies. Задачи – установить зависимость интенсивности и продолжительности формирования хвои, стеблей и стволов от температуры воздуха. Исследования показали, что первыми (в мае) трогаются в рост стебли, через 1–4 недели появляется молодая хвоя и начинается деление клеток камбия стволов. Кульминация роста вегетативных органов происходит в следующем порядке: стебли, стволы и хвоя. Установлено влияние температуры воздуха на динамику формирования изучаемых органов. The authors studied the peculiarities of seasonal growth of Pinus sylvestris and Picea abies during three vegetation periods in the zone of coniferous forests (Northwest Russia). The length of stems of shoots of the second order of branching from the southwestern part of the crown was measured at a height of about 2 m from the moment of swelling of vegetative buds. The length of needles was measured every 5 days from the moment of budding of vegetative buds until the complete cessation of growth (at the base of the same auxiblasts). To study the seasonal radial increment of trunk wood of each accounting tree, wood samples were taken every 5 days after the start of cambium activity. Results. The studies showed that the growth of vegetative organs of the studied species began according to a particular progression. In May, stems start to grow. In 1-4 weeks after that, new needles appear, and division of cells of cambium of trunks starts. Culmination of growth goes in the following order: stems, trunks, needles. At first, growth of stems ceases, than needles stop to grow and, at last, trunks. In comparison with Picea abies, growth of stems, needles and trunks of Pinus sylvestris begins 16, 15, 6 days earlier. Culmination of growth begins 30, 15, 11 days earlier. Growth of stems for both species ceases simultaneously. Growth of needles and trunks for Pinus sylvestris trees ceases 10 and 11 days earlier, in comparison with Picea abies. Growth of stems, needles and trunks of Pinus sylvestris begins and reaches its peak at the air temperature lower by 4-6 °С and the sum of positive temperatures lower by 100-500 °С than for Picea abies. The air temperature regime at the end of the vegetative organ growth period is almost equal. The period of Pinus sylvestris stems, needles and trunks forming is larger for 17, 9 and 24 days in comparison with Picea abies. Conclusion. The study showed that Pinus sylvestris was better adapted to the severe conditions of the taiga in comparison with Picea abies.

Rice false smut, caused by the fungal pathogen Villosiclava virens, is one of the most important rice diseases in the world. Previous studies reported that the pathogen has less number of cell wall-degraded genes and attacks dominantly rice stamen filaments and extends intercellularly. To reveal why the fungus infects plant stamen filaments, inoculation test on barley was carried out with the similar protocol to rice. The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly, usually resulting in severe damage of the stamen filament tissues. It also attacked young barley lodicules and grew intercellularly by chance. The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps. Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent. The cell walls were very soft and flexible, and often folded. However, V. virens extended dominantly in the noncellulose regions and seemed never to degrade microfibrils in barley and rice cell walls. This suggested that the unique structures of rice and barley stamen filaments should be fit for their function of elongation in anthesis, and also endow with the susceptibility to the fungus, V. virens.

Chiral asymmetry is important in a wide variety of disciplines and occurs across length scales. While several natural chiral biomolecules exist only with single handedness, they can produce complex hierarchical structures with opposite chiralities. Understanding how the handedness is transferred from molecular to the macroscopic scales is far from trivial. An intriguing example is the transfer of the handedness of helicoidal organizations of cellulose microfibrils in plant cell walls. These cellulose helicoids produce structural colors if their dimension is comparable to the wavelength of visible light. All previously reported examples of a helicoidal structure in plants are left-handed except, remarkably, in the Pollia condensata fruit; both left- and right-handed helicoidal cell walls are found in neighboring cells of the same tissue. By simultaneously studying optical and mechanical responses of cells with different handednesses, we propose that the chirality of helicoids results from differences in cell wall composition. In detail, here we showed statistical substantiation of three different observations: 1) light reflected from right-handed cells is red shifted compared to light reflected from left-handed cells, 2) right-handed cells occur more rarely than left-handed ones, and 3) right-handed cells are located mainly in regions corresponding to interlocular divisions. Finally, 4) right-handed cells have an average lower elastic modulus compared to left-handed cells of the same color. Our findings, combined with mechanical simulation, suggest that the different chiralities of helicoids in the cell wall may result from different chemical composition, which strengthens previous hypotheses that hemicellulose might mediate the rotations of cellulose microfibrils.

Microdistribution of non-cellulosic polysaccharides in pit membranes of bordered pits (intertracheid pits between adjacent tracheids), cross-field pits (half bordered pits between tracheids and ray parenchyma cells) and ray pits (simple pits in nodular end walls of ray parenchyma cells) was investigated in mature earlywood of juvenile Scots pine and Norway spruce seedlings using immunocytochemistry combined with monoclonal antibodies specific to (1→4)-β-galactan (LM5), (1→5)-α-arabinan (LM6), homogalacturonan (HG, LM19, LM20), xyloglucan (LM15), xylan (LM10, LM11) and mannan (LM21, LM22) epitopes. Using phloroglucinol-HCl and KMnO4 staining, lignin distribution in pit membranes was also examined. Apart from cross-field pit membranes in Scots pine, all pit membranes observed showed a positive reaction for lignin with differences in staining intensity. Ray pit membranes showed strongest reaction with lignin staining in both species. Intensity of lignin staining in bordered pit membranes was stronger in Norway spruce than in Scots pine. With localization of non-cellulosic polysaccharide epitopes, Scots pine showed differences in cross-field pit membranes (rhamnogalacturonan-I (RG-I), HG and xyloglucan epitopes) from bordered and ray pit membranes (RG-I and HG epitopes). In contrast, Norway spruce showed significant differences in ray pit membranes (RG-I, HG, xyloglucan, xylan and mannan epitopes) from bordered and cross-field pit membranes (HG and no/trace amount of RG-I epitopes). Distributional differences in HG epitopes depending on antibody type/ membrane regions were also observed in cross-field pit membranes between the two species. Together, the results suggest that distribution patterns of lignin and non-cellulosic polysaccharides in pit membranes differ significantly between pit types and between Scots pine and Norway spruce. Compared with the same types of pit membranes in hardwoods, the results for Scots pine and Norway spruce (softwoods) differed significantly.

Wood is a natural porous material, obtained from trees, whose cell walls consist of cellulose micrifibrils, lignin and hemicelluloses. Cellulose microfibrils in wood cell walls, whose crystalline regions have high Young’s modulus and strength in the direction parallel to the molecular chain axis, play an important rule in mechanical and physical properties of wood. Chemical treatments that cause the transformation of cellulose crystals have been used to improve the mechanical and physical properties of cellulose fibers for a long time. These treatments also cause the changes in cell wall structures of wood and alter the properties of wood.

The composition of nonvolatile extractives soluble in petroleum ether was investigated separately for inner and outer bark of Norway spruce (Picea abies (L.) Karst) and Scots Pine (Pinus silvestris (L.)). The bark extractives of one spruce and one pine tree were prefractionated by thin-layer chromatography and the detailed composition of free and esterified fatty acids, free and esterified sterols, triterpenoid alcohols and fatty alcohols, resin acids and diterpene aldehydes was determined by gas chromatography. Bark of four other trees was analysed by a routine method based on direct gas chromatography of the extracts. Fatty acids, resin acids and sterols accounted for ca 80% of extractives in inner bark and for 50% in outer bark. Qualitative differences between the extractives in inner and outer bark were noticed. The total amount of fatty and resin acids was about 1.54 of spruce and pine bark dry weight and the amount of sterols was 0.2–0.5%. These levels are low considering possible technical...

Key messageDistribution patterns of pectin and hemicellulose epitopes in the phloem of Pinaceae conifers differ between cell types including sieve cells, axial/ray parenchyma cells and stone cells, which is more prominent in seedlings than mature trees.Although there is considerable information on the gross chemistry of conifer bark, little is known on the chemistry of secondary phloem at the individual cell level. This study investigated distribution of pectins and hemicelluloses in the phloem of two conifer species (Norway spruce and Scots pine) at an individual cell wall level using nine monoclonal antibodies specific for pectin and hemicellulose epitopes combined with immunofluorescence and TEM immunogold labeling. Differences in phloem cell wall chemistry between juvenile (seedlings) and mature conifer trees were also examined. The two conifer species showed qualitatively similar distribution patterns of epitopes in sieve- and (axial/ray) parenchyma cells, irrespective of seedlings and mature trees. Sieve- and parenchyma cell walls showed the presence of rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and xyloglucan epitopes, but revealed the absence of heteroxylan epitopes. Heteromannan epitopes were only detected in sieve cell walls, showing a chemical difference between sieve- and parenchyma cells. In contrast to qualitative similarity, there were several quantitative differences of epitope localization in sieve- and parenchyma cells between the two conifer species, indicating variations in the chemical structure and/or the amount of pectins and hemicelluloses between the two conifer species. These differences were more significant in seedlings than mature trees. Immunogold labeling of Norway spruce seedlings further indicated the possibility of chemical variations between cell wall regions within a single sieve cell wall. Phloem stone cells detected in mature Norway spruce showed the presence of heteromannans/heteroxylans and RG-I/HG/xyloglucans in secondary cell wall and middle lamellae, respectively.

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.

Structural studies of Chondrus crispus: the effect of extraction of carrageenan