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Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives.

The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. x P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours.

Cell Wall↗

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of microfilaments.

The cortical microfilament (MF) component of the cytoskeleton within axial elements of the secondary vascular system of the angiosperm tree, Aesculus hippocastanum L. (horse-chestnut) was studied using transmission electron microscopy of ultrathin sections and indirect immunofluorescence microscopy of actin in thick sections. As seen by electron microscopy, MF bundles have a net axial orientation within fusiform cambial cells and their secondary vascular derivatives (i.e. in the axial xylem and phloem parenchyma, xylem fibres, vessel and sieve elements, and companion cells). Immunofluorescence studies, however, reveal that this axial orientation can be more accurately described as a helix of extremely high pitch; it is a persistent feature of all axial secondary vascular elements during their development. Helical MF arrays are the only arrangement seen in secondary phloem cells. However, in addition to helices, other MF arrays are seen in secondary xylem cells. For example, fibres possess ellipses of MFs associated with simple-pit formation, and vessel elements possess circular arrays of MFs that associate with the developing inter-vessel bordered pits, ray-vessel contact pits, and with the perforation plate. Linear MF arrays are seen co-oriented with the developing tertiary wall-thickenings in vessel elements. The possible roles of MFs during the cytodifferentiation of secondary vascular cells is discussed, and compared with that of microtubules.

Actin Cytoskeleton↗

4-Coumarate:coenzyme A ligase in black locust (Robinia pseudoacacia) catalyses the conversion of sinapate to sinapoyl-CoA.

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.

Acyl Coenzyme A↗

Strategies for the engineered phytoremediation of toxic element pollution: mercury and arsenic.

Plants have many natural properties that make them ideally suited to clean up polluted soil, water, and air, in a process called phytoremediation. We are in the early stages of testing genetic engineering-based phytoremediation strategies for elemental pollutants like mercury and arsenic using the model plant Arabidopsis. The long-term goal is to develop and test vigorous, field-adapted plant species that can prevent elemental pollutants from entering the food-chain by extracting them to aboveground tissues, where they can be managed. To achieve this goal for arsenic and mercury, and pave the way for the remediation of other challenging elemental pollutants like lead or radionucleides, research and development on native hyperaccumulators and engineered model plants needs to proceed in at least eight focus areas: (1) Plant tolerance to toxic elementals is essential if plant roots are to penetrate and extract pollutants efficiently from heterogeneous contaminated soils. Only the roots of mercury- and arsenic-tolerant plants efficiently contact substrates heavily contaminated with these elements. (2) Plants alter their rhizosphere by secreting various enzymes and small molecules, and by adjusting pH in order to enhance extraction of both essential nutrients and toxic elements. Acidification favors greater mobility and uptake of mercury and arsenic. (3) Short distance transport systems for nutrients in roots and root hairs requires numerous endogenous transporters. It is likely that root plasma membrane transporters for iron, copper, zinc, and phosphate take up ionic mercuric ions and arsenate. (4) The electrochemical state and chemical speciation of elemental pollutants can enhance their mobility from roots up to shoots. Initial data suggest that elemental and ionic mercury and the oxyanion arsenate will be the most mobile species of these two toxic elements. (5) The long-distance transport of nutrients requires efficient xylem loading in roots, movement through the xylem up to leaves, and efficient xylem unloading aboveground. These systems can be enhanced for the movement of arsenic and mercury. (6) Aboveground control over the electrochemical state and chemical speciation of elemental pollutants will maximize their storage in leaves, stems, and vascular tissues. Our research suggests ionic Hg(II) and arsenite will be the best chemical species to trap aboveground. (7) Chemical sinks can increase the storage capacity for essential nutrients like iron, zinc, copper, sulfate, and phosphate. Organic acids and thiol-rich chelators are among the important chemical sinks that could trap maximal levels of mercury and arsenic aboveground. (8) Physical sinks such as subcellular vacuoles, epidermal trichome cells, and dead vascular elements have shown the evolutionary capacity to store large quantities of a few toxic pollutants aboveground in various native hyperaccumulators. Specific plant transporters may already recognize gluthione conjugates of Hg(II) or arsenite and pump them into vacuole.

Arabidopsis↗

Analysis of tree rings using inductively coupled plasma mass spectrometry to record fluctuations in a metal pollution episode.

Analysis of Acer pseudoplatanus L. (sycamore) tree rings using ICP-MS was used to assess the impact of metal deposition on trees growing in the vicinity of a metal refinery at Prescot, north-west England compared to a reference site at Croxteth 6 km distant receiving minimal deposition. No difference in tree growth between sites was recorded. Large reductions in Cu and Cd deposition since the late 1970s was accompanied by a steep decline in Cd concentrations in the outer rings in trees close to the refinery. A similar reduction in Cu concentrations was less apparent due to a tendency for Cu to increase in the outer rings. Zinc and Ni were higher in xylem at Prescot compared to Croxteth. There was no trend in Zn concentrations at either site although Ni concentrations increased in trees close to the refinery after 1982. Manganese concentrations in xylem were much higher at Prescot; Mn levels declined until the late 1970s and then increased slightly in later years interspersed by large concentration peaks within individual years during the 1980s. There was little change in Mn concentrations in trees at Croxteth during this period. Calcium, Mg and Sr concentrations remained steady or declined slightly in rings formed since 1965 in trees at Croxteth. Concentrations of Ca, Mg and Sr were higher in rings formed prior to the mid 1970s in trees at Prescot, but declined steadily after this period, although peaks in concentrations of each element were recorded in individual years during the 1980s. Phosphorous concentrations in rings increased towards the cambium at Croxteth, although P levels decreased in rings formed after 1982 at Prescot. No difference in K concentrations between sites was recorded. Lead concentrations in xylem at both sites declined steadily in rings formed after 1970, although concentrations were higher at the reference site. Analysis of individual sycamore tree rings appears to record short-term changes in pollution episodes, with little lateral movement of elements occurring. It is suggested that changes in element concentrations in trees close to the refinery are a result of reduced metal deposition combined with increased soil acidity due to reduced buffering capacity of metal ions in rainfall.

Journal Article↗

Differential expression patterns of two new primary cell wall-related cellulose synthase cDNAs, PtrCesA6 and PtrCesA7 from aspen trees.

Based on elegant molecular genetic analyses, distinct classes of cellulose synthase (CesA) genes have been associated with either primary or secondary cell wall development in Arabidopsis. Here, we report on cloning of two new CesA cDNAs, PtrCesA6 and PtrCesA7 involved in the primary cell wall development in aspen (Populus tremuloides) trees. Both these distinct cDNAs, isolated from a developing xylem cDNA library, share only 60-67% identities with each other as well as with five other previously known aspen CesA cDNAs. Interestingly, PtrCESA6 from aspen, a dicot species, shares maximum identity of 81-84% with three CESA isoforms from maize and rice, two monocot species. On the other hand, PtrCESA7 shares a maximum identity of 86% with AtCESA2, a primary wall-related CesA member from Arabidopsis, a dicot species. Gene expression analyses by reverse transcriptase-polymerase chain reactions (RT-PCRs) suggested that both these genes are expressed at a low level in all aspen tissues examined but PtrCesA7 is expressed at a higher level than PtrCesA6. While corroborating these results, in situ mRNA hybridization studies using three different aspen organs also suggested that PtrCesA6 and PtrCesA7 genes are expressed in all expanding cells depositing primary cell wall but PtrCesA7 is expressed at a higher level than PtrCesA6. These differential gene expression profiles suggest that each of these CesAs may be playing a specific role during primary cell wall development in aspen trees. Isolation of two primary wall related CesA genes from xylem tissues also suggest their importance during xylem development, which is traditionally considered to be enriched in secondary cell wall forming cells of economical significance.

Blotting, Southern↗

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

Citrus sinensis↗

Functional imaging of plants: a nuclear magnetic resonance study of a cucumber plant.

Functional magnetic resonance imaging was used to study transients of biophysical parameters in a cucumber plant in response to environmental changes. Detailed flow imaging experiments showed the location of xylem and phloem in the stem and the response of the following flow characteristics to the imposed environmental changes: the total amount of water, the amount of stationary and flowing water, the linear velocity of the flowing water, and the volume flow. The total measured volume flow through the plant stem was in good agreement with the independently measured water uptake by the roots. A separate analysis of the flow characteristics for two vascular bundles revealed that changes in volume flow of the xylem sap were accounted for by a change in linear-flow velocities in the xylem vessels. Multiple-spin echo experiments revealed two water fractions for different tissues in the plant stem; the spin-spin relaxation time of the larger fraction of parenchyma tissue in the center of the stem and the vascular tissue was down by 17% in the period after cooling the roots of the plant. This could point to an increased water permeability of the tonoplast membrane of the observed cells in this period of quick recovery from severe water loss.

Biophysics↗

Proteolytic activity in relation to seasonal cambial growth and xylogenesis in Pinus banksiana.

Proteolytic activity in the cambial zone and developing xylem of Pinus banksiana Lamb. was investigated over an annual cycle of growth and dormancy. Highest proteolytic activity was associated with the most active period of primary-wall radial expansion of cambial derivatives, in early spring, before protoplasmic autolysis was initiated in developing earlywood. Three pH maxima of proteolytic activity, near pH 3.0, 6.5 and 9.5, were observed at that time. In general, activities measured at pH values below 7.0 were greater than those determined above pH 7.0 at all stages in the annual cycle, in both cambial zone and developing xylem, although elevated activity at alkaline pH was also observed during springtime growth. Polyvinylpolypyrollidone (PVP) treatment markedly enhanced pH 7.5 but not pH 4.0 proteolytic activity in the cambial zone, but not in developing xylem, indicating the presence of PVP-binding proteinase regulators in the cambium. By fractionation and effector studies total proteolysis was determined to comprise interactions between serine, cystine, aspartate and metallo-proteases having MWs, by gel chromatography, between 10 and 100 kDa. The observations point to a complex regulatory mechanism controlling the presence and catalytic rates of the distinct types of proteases in the cambial region throughout an annual cycle of growth and dormancy.

Cycadopsida↗

Water transport in trees: current perspectives, new insights and some controversies.

This review emphasizes recent developments and controversies related to the uptake, transport and loss of water by trees. Comparisons of the stable isotope composition of soil and xylem water have provided new and sometimes unexpected insights concerning spatial and temporal partitioning of soil water by roots. Passive, hydraulic redistribution of water from moister to drier portions of the soil profile via plant root systems may have a substantial impact on vertical profiles of soil water distribution, partitioning of water within and among species, and on ecosystem water balance. The recent development of a technique for direct measurement of pressure in individual xylem elements of intact, transpiring plants elicited a number of challenges to the century-old cohesion-tension theory. The ongoing debate over mechanisms of long-distance water transport has stimulated an intense interest in the phenomenon and mechanisms of embolism repair. Rather than embolism being essentially irreversible, it now appears that there is a dynamic balance between embolism formation and repair throughout the day and that daily release of water from the xylem via cavitation may serve to stabilize leaf water balance by minimizing the temporal imbalance between water supply and demand. Leaf physiology is closely linked to hydraulic architecture and hydraulic perturbations, but the precise nature of the signals to which stomata respond remains to be elucidated. When water transport in trees is studied at multiple scales from single leaves to the whole organism, considerable functional convergence in regulation of water use among phylogenetically diverse species is revealed.

Journal Article↗

Ammonium, bicarbonate and calcium effects on tomato plants grown under saline conditions.

Tomato plants (70 days old) were grown in hydroponic culture into a greenhouse, where supply of inorganic carbon, ammonium and calcium to saline nutrient solution, was investigated in order to reduce the negative effect of salinity. After 70 days, an ameliorating effect upon the decrease in growth observed under salinity was only observed with the treatments NaCl+Ca(2+) and NaCl+HCO(3)(-)+NH(4)(+)+Ca(2+). A large reduction of hydraulic conductance (L(0)) and stomatal conductance (G(s)) was observed with all treatments, compared with the control. However, the reductions were less when NaCl and Ca(2+) were added together. Organic acids (mainly malic acid) in the xylem were decreased with all treatments except with NaCl+NH(4)(+) and with all single treatments added together (NaCl+HCO(3)(-)+NH(4)(+)+Ca(2+)). Amino acid concentrations in the xylem (mainly asparagine and glutamine) decreased when plants were treated with NaCl and NaCl+Ca(2+), but there was a large increase in the plants treated with NaCl+NH(4)(+) or with all treatments together. As HCO(3)(-) is an important source of carbon for NH(4)(+) assimilation, the increase in the concentration of amino acids and organic acids caused by the treatments that contained NH(4)(+), support the idea that fixation of dissolved inorganic carbon was occurring and that the products were transported via the xylem to the shoot. The ameliorating effect of Ca(2+) on root hydraulic conductivity plus the increase of NH(4)(+) incorporation into the amino acid synthesis pathway possibly due to dissolved inorganic carbon fixation, could reduce the negative effect of salinity on tomato plants.

Journal Article↗

Growth of Prunus tree stems under simulated microgravity conditions.

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees. Grant Numbers: 07456073, 1D 215.

Gravitation↗

Functional magnetic resonance imaging in intact plants--quantitative observation of flow in plant vessels.

Quantitative magnetic resonance (MR) images of flow velocities in intact corn plants were acquired using magnetization-prepared MR microscopy. A phase contrast flow imaging technique was used to quantitate water flow velocities and total volume flow rates in small xylem vessels. The simultaneous measurement of the transpiration of the whole plant was achieved by using a closed climate chamber within the MR magnet. The total volume flow rate and the transpiration values were in close correlation. Functional magnetic resonance imaging in intact plants was performed by light stimulation of the transpiration inside of the magnet. The change in the flow velocities in the xylem vessels of single vascular bundles was in correlation with the changes in the transpiration. Significant differences were observed between the xylem vessels in different vascular bundles. Furthermore, flow velocity measurements were performed on excised plant stems and visualized by the uptake of the MR contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). A comparison between the phase contrast flow imaging and the contrast media uptake showed to be in good agreement with each other.

Hydroponics↗

Water ascent in plants: do ongoing controversies have a sound basis?

The cohesion-tension theory of the ascent of sap in plants is fundamental to the understanding of water movement in plants. According to the theory, water is pulled upwards by high tensions (low negative pressures) created in the xylem vessels and tracheids of higher plants by the evaporation of water vapour from leaves. However, much lower tensions (less negative pressures) have been found from direct measurements using a pressure probe. These do not appear to be compatible with the cohesion-tension theory. As a consequence, the validity of the cohesion-tension theory has been questioned and alternative mechanisms for sap ascent have been proposed. Recent experiments show that the conclusions drawn from the pressure probe work were premature. New direct measurements of xylem pressure support the cohesion-tension theory and the previous indirect measurements of xylem pressure.

Journal Article↗

Uptake by cucurbitaceae of soil-Bome contaminants depends upon plant genotype and pollutant properties.

Three Cucurbitaceae, Cucurbita pepo L. subsp. pepo (cv. Black Beauty, true zucchini), Cucurbita pepo L. intersubspecific cross (cv. Zephyr, summer squash), and Cucumis sativis (cv. Marketmore, cucumber), were grown in rhizotrons containing soil contaminated with three classes of highly weathered, hydrophobic organic contaminants: (1) technical chlordane, (2) dichlorodiphenylethanes (DDT and DDD) and -ethene (DDE), (3) polyaromatic hydrocarbons (PAHs), and heavy metal residues. Movement of the contaminants through the soil/plant system was studied by comparing contaminant concentration in the bulk soil, the rhizosphere soil pore water, the xylem sap, and aerial tissue. This permitted, for the first time, calculation of bioconcentration factors (BCFs) based on concentration in the xylem sap versus that in the rhizosphere soil pore water. The bioconcentration factors so determined for the sum of five chlordane residues (two enantiomers of trans-chlordane, TC; two enantiomers of cis-chlordane, CC; and achiral trans-nonachlor, TN) were 36, 40, and 23 for Black Beauty, Zephyr, and Marketmore, respectively. In addition, the xylem sap of each cultivar had a consistent enantioselective profile for some of the chiral chlordane components. For the sum of dichlorodiphenylethanes and -ethene, comparable BCF values were 19, 4, and 0.8, respectively. In the case of PAHs, different BCF patterns among the cultivars were noted for three- versus four-ring compounds. Similarly, movement of heavy metals was cultivar-dependent, with cadmium BCF values 9.5, 3.5, and 0.6for Black Beauty, Zephyr, and Marketmore, respectively; the analogous BCFs for zinc were 9, 11, and 2. Thus, passage from ex planta to in planta regions of the soil/plant system is dependent not only on properties of the plant, but also on those of the pollutant. Such data will provide insight into transport mechanisms of highly hydrophobic organic contaminants, as well as heavy metal contaminants, in the soil/plant system.

Animals↗

The poplar bark storage protein gene (Bspa) promoter is responsive to photoperiod and nitrogen in transgenic poplar and active in floral tissues, immature seeds and germinating seeds of transgenic tobacco.

In Populus, seasonal nitrogen storage involves the accumulation of a 32 kDa bark storage protein (BSP) in the inner bark parenchyma and xylem rays. Poplar BSPs are encoded by a multigene family and one member, bspA, has been cloned and sequenced. The regulation of bspA was investigated by transforming either hybrid poplar or tobacco with a chimeric gene consisting of the 2.8 kb bspA promoter fused to the coding region of beta-glucuronidase (uidA). In transformed poplar, the bspA 2.8 kb promoter conferred both short-day (SD) and nitrogen (N) inducibility to GUS and activity was localized to the bark (primary and secondary phloem, and cortex) and xylem rays. Night-break treatments inhibited SD induction of GUS. Deletion of the 1.6 kb distal DNA sequences from the bspA promoter eliminated SD induction of GUS while some N induction was retained. These results indicate that although poplar BSP is encoded by a multigene family, transcriptional activation of bspA per se can account for bsp expression in bark and xylem rays in response to either SD or N treatment. These results also show that the elements responsible for SD or N induction are separable. Because of the long generation intervals associated with trees, the developmental regulation of bspA in flowers, developing seeds, and germinating seeds was investigated by transforming the 2.8 kb bspA-promoter::uidA chimeric gene into tobacco. The bspA promoter was active in developing tobacco floral tissues and in seeds during early stages of embryogenesis, decreased progressively during seed maturation and regained activity upon seed germination. Although seed storage proteins of poplar share some similarities to poplar BSP, the observed developmental expression patterns in tobacco are consistent with a role for bspA in vegetative rather than seed storage protein storage.

Gene Expression Regulation, Developmental↗

New applications for an old lignified element staining reagent.

The use is reported of Mirande's reagent in epifluorescence microscopy which permits a clear distinction between cellulosic and lignified tissues. Homogeneous Prespermatophytae and gymnosperm xylem appeared entirely green with Mirande's reagent under ultraviolet excitation, whereas heteroxyled angiosperm wood showed a mixed pink and blue-green colour. This coloration was due to the fluorescence of cellulose, since certain elements in dicotyledonous wood (parenchyma, fibres, xylem rays) are not entirely lignified. Monocotyledonous (Poaceae) lignin showed an intense blue fluorescence due to hydroxycinnamic acids bound to the cell wall. The method showed that lignification occurs first in the middle lamella, and later in the secondary wall of xylem cells. In addition, this staining technique proved useful in the study of lignin and suberin deposition in response to various stress factors.

Cellulose↗

A proteoglycan mediates inductive interaction during plant vascular development.

Inductive cell-cell interactions are essential for controlling cell fate determination in both plants and animals; however, the chemical basis of inductive signals in plants remains little understood. A proteoglycan-like factor named xylogen mediates local and inductive cell-cell interactions required for xylem differentiation in Zinnia cells cultured in vitro. Here we describe the purification of xylogen and cloning of its complementary DNA, and present evidence for its role in planta. The polypeptide backbone of xylogen is a hybrid-type molecule with properties of both arabinogalactan proteins and nonspecific lipid-transfer proteins. Xylogen predominantly accumulates in the meristem, procambium and xylem. In the xylem, xylogen has a polar localization in the cell walls of differentiating tracheary elements. Double knockouts of Arabidopsis lacking both genes that encode xylogen proteins show defects in vascular development: discontinuous veins, improperly interconnected vessel elements and simplified venation. Our results suggest that the polar secretion of xylogen draws neighbouring cells into the pathway of vascular differentiation to direct continuous vascular development, thereby identifying a molecule that mediates an inductive cell-cell interaction involved in plant tissue differentiation.

Amino Acid Sequence↗