Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrUs was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.     

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation： Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on tour1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants （Col-0） and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels （-tensile strength） of the hypocotyls of the mutants with pectin alterations （mur1, qua2） were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for tour1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.     

Alteration in Secondary Wall Deposition by Overexpression of the Fragile Fiber1 Kinesin-Like Protein in Arabidopsis

Secondary walls in fibers and vessels are typically deposited in three distinct layers, which are formed by the successive re-orientation of cellulose microfibrils. Although cortical microtubules have been implicated in this process, the underlying mechanisms for the formation of three distinct wall layers are not known. The Fragile Fiber1 （FRA1） kinesin-like protein has been previously shown to be involved in the oriented deposition of cellulose microfibrils and important for cell wall strength in Arabidopsis thaliana. In the present report, we investigated the expression pattern of the FRA 1 gene and studied the effects of FRA1 overexpression on secondary wall deposition. The FRAI gene was found to be expressed not only in cells undergoing secondary wall deposition including developing interfascicular fibers and xylem cells, but also in dividing cells and expanding/elongating parenchyma cells. Overexpression of FRA1 caused a severe reduction in the thickness of secondary walls in interfascicular fibers and deformation of vessels, which are accompanied with a marked decrease in stem strength. Close examination of secondary walls revealed that unlike the wild-type walls having three typical layers with the middle layer being the thickest, the secondary walls in FRA1 overexpressors exhibited an increased number of layers, all of which had a similar width. Together, these results provide further evidence implicating an important role of the FRA1 kinesin-like protein in the ordered deposition of secondary walls, which determines the strength of fibers and vessels.     

PtrCel9A6, an Endo-l,4-β-Glucanase,Is Required for Cell Wall Formation during Xylem Differentiation in Populus

ABSTRACT Endo-l,4-β-glucanases （EGases） are involved in many aspects of plant growth. Our previous study found that an EGase, PtrCel9A6, is specifically expressed in differentiating xylem cells during Populus secondary growth. In this study, the xylem-specific PtrCel9A6 was characterized for its role in xylem differentiation. The EGase is localized on the plasma membrane with catalytic domain toward the outside cell wall, hydrolyzing amorphous cellulose. Suppression of PtrCel9A6 expression caused secondary cell wall defects in xylem cells and significant cellulose reduction in Populus. Heterologous expression of PtrCelgA6 in Arabidopsis enhanced plant growth as well as increased fiber cell length. In addition, introduction of PtrCel9A6 into Arabidopsis resulted in male sterility due to defects in anther dehiscence. Together, these results demonstrate that PtrCel9A6 plays a critical role in remodeling the 1,4-β-glucan chains in the wall matrix and is required for cell wall thickening during Populus xylem differentiation.     

Na~ and Water Uptake in Relation to the Radial Reflection Coefficient of Root in Arrowleaf Saltbush Under Salt Stress

The response of halophyte arrowleaf saltbush(Atriplex triangularis Willd)plants to a gradient of salt stress were investigatedwith hydroponically cultured seedlings.Under salt stress,both the Na~ uptake into root xylem and negative pressures inxylem vessels increased with the elevation of salinity(up to 500 mol/m~3)in the root environment.However,the increment innegative pressures in root xylem far from matches the decrease in the osmotic potential of the root bathing solutions,evenwhen the osmotic potential of xylem sap is taken into consideration.The total water potential of xylem sap in arrowleafsaltbush roots was close to the osmotic potential of root bathing solutions when the salt stress was low,but a progressivelyincreased gap between the water potential of xylem sap and the osmotic potential of root bathing solutions was observedwhen the salinity in the root environment was enhanced.The maximum gap was 1.4 MPa at a salinity level of 500 mol/m~3without apparent dehydration of the tested plants.This discrepancy could not be explained with the current theories inplant physiology.The radial reflection coefficient of root in arrowleaf saltbush decreased with the enhanced salt stress wasand accompanied by an increase in the Na~ uptake into xylem sap.However,the relative Na~ in xylem exudates based onthe corresponding NaCl concentration in the root bathing solutions showed a tendency of decrease.The results showedthat the reduction in the radial reflection coefficient of roots in the arrowleaf saltbush did not lead to a mass influx of NaClinto xylem when the radial reflection coefficient of the root was considerably small;and that arrowleaf saltbush could usesmall xylem pressures to counterbalance the salt stresses,either with the uptake of large amounts of salt,or with thedevelopment of xylem pressures dangerously negative.This strategy could be one of the mechanisms behind the highresistance of arrowleaf saltbush plants to salt stress.     

Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UVRESISTANCE LOCUS 8 （UVR8） gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.     

The Ca2+-dependent DNases are Involved in Secondary Xylem Development in Eucommia ulmoides

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca 2+ -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca 2+ -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca 2+ -dependent DNases are involved in secondary xylem development.     

Genome-wide Expression Profiling in Seedlings of the Arabidopsis Mutant uro that is Defective in the Secondary Cell Wall Formation

Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economic usefulness. Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentially for water and nutrient transport, respectively. On the other hand, products of plant secondary growth are important raw materials and renewable sources of energy. Although advances have been recently made towards describing molecular mechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood. Secondary cell wall formation in plants shares some common mechanisms with other plant secondary growth processes. Thus, studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms for plant secondary growth. We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant, upright rosette （uro）, which is defective in secondary cell wall growth and has an unusually soft stem. Here, we show that lignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem. We also show genome-wide expression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes. Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling, cell division and plant secondary tissue growth. These results provide useful information for further characterizations of the regulatory network in plant secondary cell wall formation.     

On the Function of Wall Sculpturing in Xylem Conduits

The water-conducting network of capillaries in vascular plants has evolved over hundreds of millions of years in order to beable to cope with bubble clogging,a problem which also affects modern microfluidic devices.Decades of anatomical studieshave revealed that plants growing in habitats in which the formation of bubbles,or emboli,is likely to be a frequent occurrenceoften have various forms of geometrical sculpturing on the internal surfaces of the xylem conduits.The possible function of suchwall sculpturing has long been the subject of speculation.We have investigated the hypothesis that wall sculpturing is a functionaladaptation designed to increase the wettability of the walls of xylem conduits,an effect which could be described as theinverse of the well-known lotus-effect.Our results show that wall sculpturing does enhance wettability.Importantly,theoreticalcalculations reveal that the geometric parameters of various types of wall sculpturing are such that the resulting surfaces aresufficiently rough to enhance wettability,but not significantly rougher.The results provide an appealing answer to thelong-standing debate on the function of wall sculpturing in xylem conduits,and may provide biomimetic clues for new approachesto the removal of bubbles in microfluidic channels.     

Microanalysis of Plant Cell Wall Polysaccharides

Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee （Clausena lansium Skeels） axes and maize （Zea mays L.） embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase （SOD）, ascorbate peroxidase （APX） and catalase （CAT） of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum （Bacillariophyta）

Responses of the photosynthetic activity of Phaeodactylum tricornutum （Bacillariophyta） to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem （PS） Ⅰ and PS Ⅱ and the values of F685/F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.