Evolution of root apical meristem structures in vascular plants: plasmodesmatal networks.

PREMISE OF THE STUDY: The apical meristem generates indeterminate apical growth of the stem and root of vascular plants. Our previous examination showed that shoot apical meristems (SAMs) can be classified into two types based on plasmodesmatal networks (PNs), which are important elements in symplasmic signaling pathways within the apical meristem. Here, we examined the PNs of root apical meristems (RAMs) in comparison with those of SAMs. METHODS: Root apical meristems of 18 families and 22 species of lycophytes and euphyllophytes were analyzed. Plasmodesmata (PD) in cell walls in median longitudinal sections of RAMs were enumerated using transmission electron micrographs, and the PD density per 1 µm2 of each cell wall was calculated. KEY RESULTS: Root apical meristems with prominent apical cells of monilophytes (euphyllophytes) and Selaginellaceae (lycophytes) had high PD densities, while RAMs with plural initial cells of gymnosperms and angiosperms (euphyllophytes), and of Lycopodiaceae and Isoetaceae (lycophytes) had low PD densities. This correlation between structures of apical meristems and PD densities is identical to that in SAMs already described. CONCLUSIONS: Irrespective of their diversified structures, the RAMs of vascular plants can be classified into two types with respect to PNs: the fern (monilophyte) type, which has a lineage-specific PN with only primary PD, and the seed-plant type, which has an interspecific PN with secondary PD in addition to primary PD. PNs may have played a key role in the evolution of apical meristems in vascular plants.

Extremely thick cell walls and low mesophyll conductance: welcome to the world of ancient living!

Mesophyll conductance is thought to be an important photosynthetic limitation in gymnosperms, but they currently constitute the most understudied plant group in regard to the extent to which photosynthesis and intrinsic water use efficiency are limited by mesophyll conductance. A comprehensive analysis of leaf gas exchange, photosynthetic limitations, mesophyll conductance (calculated by three methods previously used for across-species comparisons), and the underlying ultra-anatomical, morphological and chemical traits in 11 gymnosperm species varying in evolutionary history was performed to gain insight into the evolution of structural and physiological controls on photosynthesis at the lower return end of the leaf economics spectrum. Two primitive herbaceous species were included in order to provide greater evolutionary context. Low mesophyll conductance was the main limiting factor of photosynthesis in the majority of species. The strongest sources of limitation were extremely thick mesophyll cell walls, high chloroplast thickness and variation in chloroplast shape and size, and the low exposed surface area of chloroplasts per unit leaf area. In gymnosperms, the negative relationship between net assimilation per mass and leaf mass per area reflected an increased mesophyll cell wall thickness, whereas the easy-to-measure integrative trait of leaf mass per area failed to predict the underlying ultrastructural traits limiting mesophyll conductance.

Unique Cellular Organization in the Oldest Root Meristem.

Roots and shoots of plant bodies develop from meristems-cell populations that self-renew and produce cells that undergo differentiation-located at the apices of axes [1].The oldest preserved root apices in which cellular anatomy can be imaged are found in nodules of permineralized fossil soils called coal balls [2], which formed in the Carboniferous coal swamp forests over 300 million years ago [3-9]. However, no fossil root apices described to date were actively growing at the time of preservation [3-10]. Because the cellular organization of meristems changes when root growth stops, it has been impossible to compare cellular dynamics as stem cells transition to differentiated cells in extinct and extant taxa [11]. We predicted that meristems of actively growing roots would be preserved in coal balls. Here we report the discovery of the first fossilized remains of an actively growing root meristem from permineralized Carboniferous soil with detail of the stem cells and differentiating cells preserved. The cellular organization of the meristem is unique. The position of the Körper-Kappe boundary, discrete root cap, and presence of many anticlinal cell divisions within a broad promeristem distinguish it from all other known root meristems. This discovery is important because it demonstrates that the same general cellular dynamics are conserved between the oldest extinct and extant root meristems. However, its unique cellular organization demonstrates that extant root meristem organization and development represents only a subset of the diversity that has existed since roots first evolved.

Distribution of gelatinous fibers in seedling roots of living cycads.

UNLABELLED: • PREMISE OF THE STUDY: The presence of gelatinous (tension) fibers (GFs) in the roots of two extant cycadales (Cycas and Zamia) in a recent publication raises interesting issues of GF distribution in seed plants. An immediate question that arises from this discovery is whether GFs occur consistently in the radicle of all extant cycad genera and therefore might have a similar role in root contraction. We present results of a survey of nursery-grown material of all 10 genera.• METHODS: We sequentially sectioned seedling root material and used simple staining and histochemical methods to follow anatomical changes along the radicle of all 10 genera.• KEY RESULTS: We found GFs in nine genera; Stangeria appears to be the only genus without them. In all genera, there is a wide variation in the number of GFs and also variation in the development of thickened, fleshy roots. "Tertiary expansion" is a useful term to describe late cell division and enlargement of both primary and secondary parenchyma, the latter produced by the vascular cambium. Certain other histological features can be diagnostically useful at the generic level.• CONCLUSIONS: The functional interpretation of GFs as being wholly responsible for apparent tissue contraction is now somewhat compromised, especially as distortion of tracheary elements by changes in dimensions of parenchyma cells can falsely suggest root contraction when it may not occur. These preliminary results point the way to a more precise investigation of study material grown in more uniform environments using advanced technological methods.

Slower phloem transport in gymnosperm trees can be attributed to higher sieve element resistance.

In trees, carbohydrates produced in photosynthesizing leaves are transported to roots and other sink organs over distances of up to 100 m inside a specialized transport tissue, the phloem. Angiosperm and gymnosperm trees have a fundamentally different phloem anatomy with respect to cell size, shape and connectivity. Whether these differences have an effect on the physiology of carbohydrate transport, however, is not clear. A meta-analysis of the experimental data on phloem transport speed in trees yielded average speeds of 56 cm h(-1) for angiosperm trees and 22 cm h(-1) for gymnosperm trees. Similar values resulted from theoretical modeling using a simple transport resistance model. Analysis of the model parameters clearly identified sieve element (SE) anatomy as the main factor for the significantly slower carbohydrate transport speed inside the phloem in gymnosperm compared with angiosperm trees. In order to investigate the influence of SE anatomy on the hydraulic resistance, anatomical data on SEs and sieve pores were collected by transmission electron microscopy analysis and from the literature for 18 tree species. Calculations showed that the hydraulic resistance is significantly higher in the gymnosperm than in angiosperm trees. The higher resistance is only partially offset by the considerably longer SEs of gymnosperms.

Structures of xyloglucans in primary cell walls of gymnosperms, monilophytes (ferns sensu lato) and lycophytes.

Little is known about the structures of the xyloglucans in the primary cell walls of vascular plants (tracheophytes) other than angiosperms. Xyloglucan structures were examined in 13 species of gymnosperms, 13 species of monilophytes (ferns sensu lato), and two species of lycophytes. Wall preparations were obtained, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-(1→4)-ß-glucanase preparation. The oligosaccharides released were analysed by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and by high-performance anion-exchange chromatography. The xyloglucan oligosaccharide profiles from the gymnosperm walls were similar to those from the walls of most eudicotyledons and non-commelinid monocotyledons, indicating that the xyloglucans were fucogalactoxyloglucans, containing the fucosylated units XXFG and XLFG. The xyloglucan oligosaccharide profiles for six of the monilophyte species were similar to those of the gymnosperms, indicating they were also fucogalactoxyloglucans. Phylogenetically, these monilophyte species were from both basal and more derived orders. However, the profiles for the other monilophyte species showed various significant differences, including additional oligosaccharides. In three of the species, these additional oligosaccharides contained arabinosyl residues which were most abundant in the profile of Equisetum hyemale. The two species of lycophytes examined, Selaginella kraussiana and Lycopodium cernuum, had quite different xyloglucan oligosaccharide profiles, but neither were fucogalactoxyloglucans. The S. kraussiana profile had abundant oligosaccharides containing arabinosyl residues. The L. cernuum profile indicated the xyloglucan had a very complex structure.

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms.

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

Symplasmic transport and phloem loading in gymnosperm leaves.

Despite more than 130 years of research, phloem loading is far from being understood in gymnosperms. In part this is due to the special architecture of their leaves. They differ from angiosperm leaves among others by having a transfusion tissue between bundle sheath and the axial vascular elements. This article reviews the somewhat inaccessible and/or neglected literature and identifies the key points for pre-phloem transport and loading of photoassimilates. The pre-phloem pathway of assimilates is structurally characterized by a high number of plasmodesmata between all cell types starting in the mesophyll and continuing via bundle sheath, transfusion parenchyma, Strasburger cells up to the sieve elements. Occurrence of median cavities and branching indicates that primary plasmodesmata get secondarily modified and multiplied during expansion growth. Only functional tests can elucidate whether this symplasmic pathway is indeed continuous for assimilates, and if phloem loading in gymnosperms is comparable with the symplasmic loading mode in many angiosperm trees. In contrast to angiosperms, the bundle sheath has properties of an endodermis and is equipped with Casparian strips or other wall modifications that form a domain border for any apoplasmic transport. It constitutes a key point of control for nutrient transport, where the opposing flow of mineral nutrients and photoassimilates has to be accommodated in each single cell, bringing to mind the principle of a revolving door. The review lists a number of experiments needed to elucidate the mode of phloem loading in gymnosperms.

Analysis of the soluble cell wall proteome of gymnosperms.

We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

Fossil woods from Janggi Group (Early Miocene) in Pohang Basin, Korea.

Sixty-four silicified fossil woods were collected from the Early Miocene Upper Coal-bearing Formation of Janggi Group in Pohang City, the Korean Peninsula. Out of them 23 specimens were identified as gymnosperms and 27 specimens as dicotyledons. The taxa identified include Picea palaeomaximowiczii Watari, Taxodioxylon cunninghamioides (Watari) Watari, T. sequoianum (Merckl.) Gothan, Fagus hondoensis (Watari) Watari, Cercidiphyllum sp., Camellia japonoxyla Suzuki et Terada, Distylium chiharu-hirayae Suzuki et Terada, Aesculus sp., Wataria miocenica (Watari) Terada et Suzuki and W. parvipora Terada et Suzuki. All of these species are reported for the first time from the Tertiary basins of Korea. Compared with those of the Miocene Formations in Japan, most taxa we found are common between the paleo-floras in Korea and Japan during the Early to Middle Miocene.

Abietane diterpenoids from suspension cultured cells of Torreya nucifera var. radicans.

Three abietane diterpenoids were isolated from the suspension cultured cells of Torreya nucifera var. radicans along with four known abietane diterpenoids. Based on spectroscopic evidence, the structures of the three were elucidated as (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11-diol, (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11,14-triol and (5R,10S)-3-oxo-7R,12-dimethoxyabieta-8,11,13-trien-11-ol, respectively.

Paleobotanical evidence for near present-day levels of atmospheric Co2 during part of the tertiary.

Understanding the link between the greenhouse gas carbon dioxide (CO(2)) and Earth's temperature underpins much of paleoclimatology and our predictions of future global warming. Here, we use the inverse relationship between leaf stomatal indices and the partial pressure of CO(2) in modern Ginkgo biloba and Metasequoia glyptostroboides to develop a CO(2) reconstruction based on fossil Ginkgo and Metasequoia cuticles for the middle Paleocene to early Eocene and middle Miocene. Our reconstruction indicates that CO(2) remained between 300 and 450 parts per million by volume for these intervals with the exception of a single high estimate near the Paleocene/Eocene boundary. These results suggest that factors in addition to CO(2) are required to explain these past intervals of global warmth.

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) masters.

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22-26 degrees C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees.

Transgenic manipulation of the metabolism of polyamines in poplar cells.

The metabolism of polyamines (putrescine, spermidine, and spermine) has become the target of genetic manipulation because of their significance in plant development and possibly stress tolerance. We studied the polyamine metabolism in non-transgenic (NT) and transgenic cells of poplar (Populus nigra x maximowiczii) expressing a mouse Orn decarboxylase (odc) cDNA. The transgenic cells showed elevated levels of mouse ODC enzyme activity, severalfold higher amounts of putrescine, a small increase in spermidine, and a small reduction in spermine as compared with NT cells. The conversion of labeled ornithine (Orn) into putrescine was significantly higher in the transgenic than the NT cells. Whereas exogenously supplied Orn caused an increase in cellular putrescine in both cell lines, arginine at high concentrations was inhibitory to putrescine accumulation. The addition of urea and glutamine had no effect on polyamines in either of the cell lines. Inhibition of glutamine synthetase by methionine sulfoximine led to a substantial reduction in putrescine and spermidine in both cell lines. The results show that: (a) Transgenic expression of a heterologous odc gene can be used to modulate putrescine metabolism in plant cells, (b) accumulation of putrescine in high amounts does not affect the native arginine decarboxylase activity, (c) Orn biosynthesis occurs primarily from glutamine/glutamate and not from catabolic breakdown of arginine, (d) Orn biosynthesis may become a limiting factor for putrescine production in the odc transgenic cells, and (e) assimilation of nitrogen into glutamine keeps pace with an increased demand for its use for putrescine production.

Phenylpropanoid derivatives and biflavones at different stages of differentiation and development of Araucaria angustifolia.

Chemical investigations carried out with tissues at different developmental stages of Araucaria angustifolia established the presence of E and Z isomers of octadecyl p-coumarate and octadecyl ferulate in undifferentiated callus; in the seedling stems, the source of explants, three biflavones of the amentoflavone-type were isolated, whereas the diterpene, trans-communic acid, was obtained from the seedling roots. Adult stems accumulated the benzaldehydes, vanillin, p-hydroxybenzaldehyde and coniferaldehyde; the lignans, pinoresinol, eudesmin and lariciresinol; and the isoflavones, cabreuvine and irisolidone.

A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues.

Leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalanchoë and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.

[A source of high frequency variations in the tracheid size in annual tree rings of conifers]. / Istochnik vysokochastotnykh kolebanii razmerov trakheid v godichnykh kol'tsakh khvoinykh.

Statistical analysis of high-frequency variations in the radial dimensions of tracheids was carried out using precise measurements of five radial rows in each annual tree ring for several coniferous species: Larix gmelinii, L. leptolepis, L. sibirica, Picea abies, Pinus sylvestris, P. rigida, and P. densiflora. More than 25 tree rings with a varying number of cells and width were measured for each species. High-frequency variations in radial dimensions were found to have a regular (cyclic) characteristic and accumulated 5 to 11% of the total variability. The cyclic pattern was identified using Fourier analysis of time-related or successive series statistical procedure. The first order autocorrelations (-0.45 to -0.74) and the mean cycle of high-frequency variations (2.1-2.5) did not depend on the number of cells in the annual ring, i.e., they are determined by internal causes. The cyclic pattern of high-frequency variation in tracheid dimensions is used to interpret the seasonal mechanism of xylem formation. Specifically, (1) high-frequency variations in the tracheid radial size take place during the last asymmetrical division of the xylem mother cell before transition to the elongation zone, (2) the final tracheid radial size is mostly determined within the zone of division due to the last asymmetrical division, and (3) acceleration of cell cycle from the initial to the periphery of the cambial zone. The results obtained are also discussed with respect to the mechanisms of xylem differentiation.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Temperature variation and distribution of living cells within tree stems: implications for stem respiration modeling and scale-up.

Few studies have examined variation in respiration rates within trees, and even fewer studies have focused on variation caused by within-stem temperature differences. In this study, stem temperatures at 40 positions in the stem of one 30-year-old Norway spruce (Picea abies (L.) Karst.) were measured during 40 days between July 1994 and June 1995. The temperature data were used to simulate variations in respiration rate within the stem. The simulations assumed that the temperature-respiration relationship was constant (Q10 = 2) for all days and all stem positions. Total respiration for the whole stem was calculated by interpolating the temperature between the thermocouples and integrating the respiration rates in three dimensions. Total respiration rate of the stem was then compared to respiration rate scaled up from horizontal planes at the thermocouple heights (40, 140, 240 and 340 cm) on a surface area and on a sapwood volume basis. Simulations were made for three distributions of living cells in the stems: one with a constant 5% fraction of living cells, disregarding depth into the stem; one with a living cell fraction decreasing linearly with depth into the stem; and one with an exponentially decreasing fraction of living cells. Mean temperature variation within the stem was 3.7 degrees C, and was more than 10 degrees C for 8% of the time. The maximum measured temperature difference was 21.5 degrees C. The corresponding mean variation in respiration was 35% and was more than 50% for 24% of the time. Scaling up respiration rates from different heights between 40 and 240 cm to the whole stem produced an error of 2 to 58% for the whole year. For a single sunny day, the error was between 2 and 72%. Thus, within-stem variations in temperature may significantly affect the accuracy of scaling respiration data obtained from small samples to whole trees. A careful choice of chamber position and basis for scaling is necessary to minimize errors from variation in temperature.