Lignified Cell Research Articles

Pod shattering resistance associated with domestication is mediated by a NAC gene in soybean

Loss of seed dispersal is a key agronomical trait targeted by ancient human selection and has been regarded as a milestone of crop domestication. In this study, in the legume crop soybean Glycine max (L.) Merr. which provides vegetable oils and proteins for humans, we show that the key cellular feature of the shattering-resistant trait lies in the excessively lignified fibre cap cells (FCC) with the abscission layer unchanged in the pod ventral suture. We demonstrate that a NAC (NAM, ATAF1/2 and CUC2) gene shattering1-5 (SHAT1-5) functionally activates secondary wall biosynthesis and promotes the significant thickening of FCC secondary walls by expression at 15-fold the level of the wild allele, which is attributed to functional disruption of the upstream repressor. We show that strong artificial selection of SHAT1-5 has caused a severe selective sweep across ~ 116 kb on chromosome 16. This locus and regulation mechanism could be applicable to legume crop improvement.

Strawberry resistance to the aphid Chaetosiphon fragaefolii Cockerell (Homoptera: Aphididae)

Cross-sections of leaf blades and petioles of different strawberry genotypes exhibiting different levels of resistance to Chaetosiphon fragaefolii were studied using the paraffin method, and staining with safranin, crystal violet and light green. Besides thicker cell walls of the cortex collenchyma in the more resistant genotypes, and a proportionally wider collenchyma than parenchyma in the cortex, their midribs are also encircled by a ring of more intensely stained lignified cells forming a physical ring. This stain reaction of cells to safranin and crystal violet occurs also in lateral vascular bundles, as well as in leaf palisade tissue. The tissue cross-sections of the sensitive genotypes revealed a predominance of green on the cellulose cell walls and protoplasts due to the reaction to light green SF, while stain reactions to safranin and crystal violet were not evidenced.

Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification, while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against cinnamoyl CoA-reductase1 driven by the promoter from cellulose synthase7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.

Visualization of plant cell wall lignification using fluorescence‐tagged monolignols

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development.

Effect of density on the compression behaviour of cork

The compression properties of cork were studied for samples of different density. The densities were grouped into three classes: low density (0.13–0.15), mid density (0.15–0.19) and high density (0.19–0.25). The porosity of the cork samples increased from the low to the high density class, with porosity coefficients of 5.1%, 6.9% and 9.4%, respectively. The difference in the porosity was associated with structural features, namely the presence of thick walled cork cells and the presence of lignified cells lining the pores.The stress–strain curves were similar for all cases, showing an elastic compression up to a yield point of about 5% strain, followed by a plateau with a small slope. The cork strength was higher in the radial direction than in the other directions. The density influenced the compression such that the corks with high density presented higher stiffness in compression in three directions: Young’s modulus was 17.4, 22.6 and 26.1MPa for low, mid and high density corks respectively. This density effect was more evident in the plateau region of the progressive buckling of the cell walls (σ30 was respectively 1.07, 1.29 and 1.54MPa for the three density classes).The recovery of dimensions after compression in each direction was also studied following compression to 50% strain. The recovery was on average 50% of the initial deformation on the first day, and almost total after 15days. The recovery was higher for corks with low density and in non-radial directions.

Aerial stem and leaf morphoanatomy of some species of Smilax

This study aimed to describe the morphoanatomy of the aerial vegetative organs of seven Smilax species, used in Brazilian folk medicine. Samples of leaves and stems were fixed with FAA 50, embedded in historesin, sectioned on a rotary microtome, stained and mounted in synthetic resin. Cuticle ornamentation was analyzed with standard scanning electron microscopy. In the frontal view, the walls of the adaxial epidermis are straight in S. brasiliensis, S. cissoides, S. goyazana and sinuous in the other species. The walls of the epidermis on the abaxial surface are straight in S. brasiliensis, S. goyazana, S. rufescens, sinuous in S. campestris, S. fluminensis, S. oblongifolia, and wavy in S. cissoides. The stomata are paracytic in S. brasiliensis, S. goyazana, S. oblongifolia, and S. rufescens, anomocytic in S. cissoides, S. campestris; anisocytic and paracytic in S. fluminensis. The midrib has three vascular bundles that are individually wrapped by lignified cells in S. brasiliensis, S. cissoides, and S. fluminensis. In the other, the three vascular bundles are surrounded by a single lignified sheath. In the stems the vascular cylinder is surrounded by a sclerenchymatous ring with the exception of Smilax fluminensis, which has a starch sheath and internal layers of thin-walled cells.

Morpho-anatomy and chemical profi le of native species used as substitutes of quina (Cinchona spp.) in Brazilian traditional medicine. Part I: Polyouratea hexasperma

This research is part of a larger study of the Brazilian species that are commonly referred to as quinas and are usually used as substitute of Cinchona species. The purpose of the present study was the botanical characterization of the whole and powdered stem bark of Polyouratea hexasperma (A. St.-Hil.) Tiegh., Ochnaceae, by morphological and anatomical description, and the analysis of its chemical profile. The external texture of the bark, the whitened pit in the inner bark and the presence of cristarque cells, as well as the shape and arrangement of other lignified cells, are the most important macroscopic and microscopic features for the characterization of the bark. Chlorogenic and cafeic acids were detected in the chemical analysis and can also be used in the identification of the bark.

PHARMACOGNOSTICAL EVALUATION OF FRUITS OF HELICTERES ISORA LINN (STERCULIACEAE)

All information about the herbal drug including all its organoleptic characters, phytoconstituents, pharmacological actions and its standardization in respect to various parameters is required before developing an herbal drug formulation. Helicteres isora Linn, Syn. East Indian Screw Tree belonging to family Sterculiaceae was used during the 16th century for its astringent and blood - purifying properties in diarrhoea and worm infection. We are do ing this work because it was thought worthwhile to explore this most functional plant on the basis of this standardization parameter. Objective of the work is to study pharmacognostic and physicochemical characteristics of fruits of Helicteres isora Linn. Fresh and dried fruits, fruit powder were studied for its morphology, microscopy, physicochemical & phytochemical characteristics, fluorescence analysis and other WHO recommended methods for standardization. The fruit was greenish brown in color, beaked; c onsist of 5 follicles twisted spirally with numerous angular and wrinkled seeds. Microscopy revealed the presence of pericarp showing an epicarp, covering trichomes and mucilaginous cells. The testa showed rectangular thin walled lignified cells. Prelimina ry phytochemical screening revealed the presence of carbohydrates, proteins, flavonoids, steroids, alkaloids, saponins and phenolic compounds. It can be concluded that the pharmacognostic profile of Helicteres isora Linn fruit is useful in standardization for quality, purity and sample identification.

Cytology of infection, development and expression of resistance to Plasmodiophora brassicae in canola

AbstractThe timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi‐nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of non‐inoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.

Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant

Lignin is a complex phenolic heteropolymer deposited in the secondarily thickened walls of specialized plant cells to provide strength for plants to stand upright and hydrophobicity to conducting cells for long-distance water transport. Although essential for plant growth and development, lignin is the major plant cell-wall component responsible for biomass recalcitrance to industrial processing. Peroxidases and laccases are generally thought to be responsible for lignin polymerization, but, given their broad substrate specificities and large gene families, specific isoforms involved in lignification are difficult to identify. This study used a combination of co-expression analysis, tissue/cell-type-specific expression analysis, and genetic complementation to correlate a sugarcane laccase gene, SofLAC, to the lignification process. A co-expression network constructed from 37 cDNA libraries showed that SofLAC was coordinately expressed with several phenylpropanoid biosynthesis genes. Tissue-specific expression analysis by quantitative RT-PCR showed that SofLAC was expressed preferentially in young internodes and that expression levels decrease with stem maturity. Cell-type-specific expression analysis by in situ hybridization demonstrated the localization of SofLAC mRNA in lignifying cell types, mainly in inner and outer portions of sclerenchymatic bundle sheaths. To investigate whether SofLAC is able to oxidize monolignols during lignification, the Arabidopsis lac17 mutant, which has reduced lignin levels, was complemented by expressing SofLAC under the control of the Arabidopsis AtLAC17 promoter. The expression of SofLAC restored the lignin content but not the lignin composition in complemented lac17 mutant lines. Taken together, these results suggest that SofLAC participates in lignification in sugarcane.

What is ‘wood’ – An anatomical re-definition

The technical definition of ‘wood’ is well accepted, but its botanical understanding remains vague. Different degrees and amounts of lignification in plants and their imprecise description, together with a conceptually doubtful life form catalog including trees, shrubs and herbs further complicate our understanding of ‘wood’. Here, we use permanent micro sections to demonstrate that the xylem and bark of terrestrial plants can vary from one tissue with a few lignified cells to an almost fully lignified tissue. This universal principle of plant growth and stabilization, accounting for all taxonomic units within vascular plants, suggests that the classical life form separation into herbs, shrubs and trees is not valid. An anatomical-based differentiation between ‘wood’, ‘woody’ and ‘woodiness’ is also only meaningful if supplemented by insight on the particular plant section and its lignified proportion. We therefore recommend utilizing the botanically more neutral term ‘stem anatomy’ instead of ‘wood anatomy’, which further implies integration of the xylem and bark of all terrestrial plants. Since dendrochronology considers shrubs, dwarf shrubs and perennial herbs in addition to trees, its semantic expansion toward ‘xylemchronology’ might be worthwhile considering.

Histological analysis of spindle and spheroid root galls caused by Plasmodiophora brassicae

Clubroot of crucifers, caused by the obligate parasite Plasmodiophora brassicae, is characterized by the formation of conspicuous root galls. These galls usually have a club- or spindle-shaped morphology, and interfere with water and nutrient uptake by infected plants. Smaller galls, historically regarded as resistance structures and distinct from the typical spindle-shaped galls, have also been identified and termed ‘spheroid galls’ because of their spherical or nearly spherical form. An assessment of various Brassica species and varieties revealed that spheroid galling could be observed in all genotypes investigated, but occurred regularly only in a few particular host/P. brassicae combinations. While spindle gall formation was coincident with the expansion of the stele and infection of secondary tissues by P. brassicae, spheroid galls typically had a region of proliferating tissue that corresponded to the secondary cortex and periderm of the healthy plants, with the outer proliferating tissue less infected than the inner portions. The underlying host tissue showed limited secondary tissue development, was largely uninfected, and, where infection occurred, a continuous stele was maintained. An active host defensive reaction, in the form of cell lignification or the hypersensitive response, was not observed, while pathogen resting spores were visible in one longitudinal section of a spheroid gall. These findings suggest that while the proliferation of P. brassicae is restricted in spheroid galls, these structures are not indicative of complete resistance to clubroot.

Impact of the Absence of Stem-Specific β-Glucosidases on Lignin and Monolignols

Monolignol glucosides are thought to be implicated in the lignin biosynthesis pathway as storage and/or transportation forms of cinnamyl alcohols between the cytosol and the lignifying cell walls. The hydrolysis of these monolignol glucosides would involve β-glucosidase activities. In Arabidopsis (Arabidopsis thaliana), in vitro studies have shown the affinity of β-GLUCOSIDASE45 (BGLU45) and BGLU46 for monolignol glucosides. BGLU45 and BGLU46 genes are expressed in stems. Immunolocalization experiments showed that BGLU45 and BGLU46 proteins are mainly located in the interfascicular fibers and in the protoxylem, respectively. Knockout mutants for BGLU45 or BGLU46 do not have a lignin-deficient phenotype. Coniferin and syringin could be detected by ultra-performance liquid chromatography-mass spectrometry in Arabidopsis stems. Stems from BGLU45 and BGLU46 mutant lines displayed a significant increase in coniferin content without any change in coniferyl alcohol, whereas no change in syringin content was observed. Other glucosylated compounds of the phenylpropanoid pathway were also deregulated in these mutants, but to a lower extent. By contrast, BGLU47, which is closely related to BGLU45 and BGLU46, is not implicated in either the general phenylpropanoid pathway or in the lignification of stems and roots. These results confirm that the major in vivo substrate of BGLU45 and BGLU46 is coniferin and suggest that monolignol glucosides are the storage form of monolignols in Arabidopsis, but not the direct precursors of lignin.

The peach (Prunus persica L. Batsch) genome harbours 10 KNOX genes, which are differentially expressed in stem development, and the class 1 KNOPE1 regulates elongation and lignification during primary growth

The KNOTTED-like (KNOX) genes encode homeodomain transcription factors and regulate several processes of plant organ development. The peach (Prunus persica L. Batsch) genome was found to contain 10 KNOX members (KNOPE genes); six of them were experimentally located on the Prunus reference map and the class 1 KNOPE1 was found to link to a quantitative trait locus (QTL) for the internode length in the peach×Ferganensis population. All the KNOPE genes were differentially transcribed in the internodes of growing shoots; the KNOPE1 mRNA abundance decreased progressively from primary (elongation) to secondary growth (radial expansion). During primary growth, the KNOPE1 mRNA was localized in the cortex and in the procambium/metaphloem zones, whereas it was undetected in incipient phloem and xylem fibres. KNOPE1 overexpression in the Arabidopsis bp4 loss-of-function background (35S:KNOPE1/bp genotype) restored the rachis length, suggesting, together with the QTL association, a role for KNOPE1 in peach shoot elongation. Several lignin biosynthesis genes were up-regulated in the bp4 internodes but repressed in the 35S:KNOPE1/bp lines similarly to the wild type. Moreover, the lignin deposition pattern of the 35S:KNOPE1/bp and the wild-type internodes were the same. The KNOPE1 protein was found to recognize in vitro one of the typical KNOX DNA-binding sites that recurred in peach and Arabidopsis lignin genes. KNOPE1 expression was inversely correlated with that of lignin genes and lignin deposition along the peach shoot stems and was down-regulated in lignifying vascular tissues. These data strongly support that KNOPE1 prevents cell lignification by repressing lignin genes during peach stem primary growth.

Demethylesterification of the Primary Wall by PECTIN METHYLESTERASE35 Provides Mechanical Support to the Arabidopsis Stem

Secondary cell walls, which contain lignin, have traditionally been considered essential for the mechanical strength of the shoot of land plants, whereas pectin, which is a characteristic component of the primary wall, is not considered to be involved in the mechanical support of the plant. Contradicting this conventional knowledge, loss-of-function mutant alleles of Arabidopsis thaliana PECTIN METHYLESTERASE35 (PME35), which encodes a pectin methylesterase, showed a pendant stem phenotype and an increased deformation rate of the stem, indicating that the mechanical strength of the stem was impaired by the mutation. PME35 was expressed specifically in the basal part of the inflorescence stem. Biochemical characterization showed that the activity of pectin methylesterase was significantly reduced in the basal part of the mutant stem. Immunofluorescence microscopy and immunogold electron microscopy analyses using JIM5, JIM7, and LM20 monoclonal antibodies revealed that demethylesterification of methylesterified homogalacturonans in the primary cell wall of the cortex and interfascicular fibers was suppressed in the mutant, but lignified cell walls in the interfascicular and xylary fibers were not affected. These phenotypic analyses indicate that PME35-mediated demethylesterification of the primary cell wall directly regulates the mechanical strength of the supporting tissue.

The Presequence of Arabidopsis Serine Hydroxymethyltransferase SHM2 Selectively Prevents Import into Mesophyll Mitochondria

Serine hydroxymethyltransferases (SHMs) are important enzymes of cellular one-carbon metabolism and are essential for the photorespiratory glycine-into-serine conversion in leaf mesophyll mitochondria. In Arabidopsis (Arabidopsis thaliana), SHM1 has been identified as the photorespiratory isozyme, but little is known about the very similar SHM2. Although the mitochondrial location of SHM2 can be predicted, some data suggest that this particular isozyme could be inactive or not targeted into mitochondria. We report that SHM2 is a functional mitochondrial SHM. In leaves, the presequence of SHM2 selectively hinders targeting of the enzyme into mesophyll mitochondria. For this reason, the enzyme is confined to the vascular tissue of wild-type Arabidopsis, likely the protoxylem and/or adjacent cells, where it occurs together with SHM1. The resulting exclusion of SHM2 from the photorespiratory environment of mesophyll mitochondria explains why this enzyme cannot substitute for SHM1 in photorespiratory metabolism. Unlike the individual shm1 and shm2 null mutants, which require CO(2)-enriched air to inhibit photorespiration (shm1) or do not show any visible impairment (shm2), double-null mutants cannot survive in CO(2)-enriched air. It seems that SHM1 and SHM2 operate in a redundant manner in one-carbon metabolism of nonphotorespiring cells with a high demand of one-carbon units; for example, during lignification of vascular cells. We hypothesize that yet unknown kinetic properties of SHM2 might render this enzyme unsuitable for the high-folate conditions of photorespiring mesophyll mitochondria.

Extraction of polymers from enzyme-treated softwood

In a biorefinery context it is an advantage to fractionate and extract different wood components in a relatively pure form. However, one major obstacle for efficient extraction of wood polymers (lignin, polysaccharides etc.) is the covalent lignin-polysaccharide networks present in lignified cell walls. Enzymatic catalysis might be a useful tool for a controlled degradation of these networks, thereby enhancing the extraction of high molecular weight polymers. In this work, a methanol-alkali mixture was used to extract two different wood samples treated with endoxylanase and gammanase, respectively. Wood chips were pretreated with alkali prior to enzymatic treatment to enhance the cell-wall accessibility to enzymes. Extractions were also carried out on non-enzyme-treated samples to evaluate the enzymatic effects. Results showed that the enzymatic treatment increased the extraction yield, with gammanase as the more efficient of the two enzymes. Furthermore, polymers extracted from xylanase-treated wood had a higher degree of polymerization than the reference.

Standardisation of homoeopathic drug - Syzygium jambos (L.) alston

Syzygium jambos, a small tree belonging to the family Myrtaceae is a potential drug in Homoeopathy. The drug is useful in clinical conditions like acne, pimples, nausea, comedones, headache, heartburn, abdominal colic, diarrhoea and fever. The seeds are up to 2.5 cm long, ellipsoid to oblong, narrowed at middle. Seed coat is rough, thick, sclerotic and papery, made of tangentially elongated lignified cells with dense contents and separating from the endosperm. Cortex is 20 - 40 layered, interspersed with groups of sclerenchyma fibers. The endosperm possess large secretory cavities in the hypodermal areas. The endosperm is filled with starch grains often encapsulated in the plastids. A few tracheary bundles are present in the endosperm. Powder microscopical and organoleptic characters are presented. Physico-chemical parameters of raw drugs viz., extractive values, ash values, formulation, besides wt. per mL, total solids, alcohol content and TLC and UV studies are given for the mother tincture.

XYLEM INTERMIXED WITH PHLOEM1, a leucine-rich repeat receptor-like kinase required for stem growth and vascular development in Arabidopsis thaliana

The regulation of cell specification in plants is particularly important in vascular development. The vascular system is comprised two differentiated tissue types, the xylem and phloem, which form conductive elements for the transport of water, nutrients and signaling molecules. A meristematic layer, the procambium, is located between these two differentiated cell types and divides to initiate vascular growth. We report the identification of a receptor-like kinase (RLK) that is expressed in the vasculature. Histochemical analyses of mutants in this kinase display an aberrant accumulation of highly lignified cells, typical of xylem or fiber cells, within the phloem. In addition, phloem cells are sometimes located adjacent to xylem cells in these mutants. We, therefore, named this RLK XYLEM INTERMIXED WITH PHLOEM 1 (XIP1). Analyses of longitudinal profiles of xip1 mutant stems show malformed cell files, indicating defects in oriented cell divisions or cell morphology. We propose that XIP1 prevents ectopic lignification in phloem cells and is necessary to maintain the organization of cell files or cell morphology in conductive elements.

Caracterização morfo-anatômica do colmo de genótipos de milho

The objective was to evaluate the morpho-anatomical characteristics of ten genotypes of corn to silage, five normal (F2, Lu16, F271, LU5 and LU6) and five bm3 (F2bm3, Lu16bm3, F271bm3, Lu5bm3 and Lu6bm3), grown in Druelle- France, in plots of 150 m² during the period from May 2002 to September 2002, totaling a period of 150 days. All evaluations were performed in internode below the primary ear. The experimental design was completely randomized, with five replications per treatment. The genotypes showed significant differences on the anatomical aspects evaluated, and that all bm3 genotypes showed lower amounts of both lignified cells in the medullar region (1.24) as in the cortex (1.72). The statistic analysis pointed positive correlation (r= 0.70) between the amount of lignified cells in the medullar region and the percentage of medullar region. There was also positive correlation (r= 0.67) between the thickness of the cell wall with the xylem % and negative correlation (r= -0.67) between the thickness of cell wall with the phloem %. The results are consistent to the types of strains studied, because the bm3 lines are characterized by smaller proportions of lignin compared to normal genotypes.