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BackgroundPlant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood.ResultsHere, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes’ expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees.ConclusionsThese findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.

Radial growth has long been a subject of interest in tree biology research. Recent studies have brought a significant change in the understanding of some basic processes characteristic to the vascular cambium, a meristem that produces secondary vascular tissues (phloem and xylem) in woody plants. A new hypothesis regarding the mechanism of intrusive growth of the cambial initials, which has been ratified by studies of the arrangement of cambial cells, negates the influence of this apical cell growth on the expansion of the cambial circumference. Instead, it suggests that the tip of the elongating cambial initial intrudes between the tangential (periclinal) walls, rather than the radial (anticlinal) walls, of the initial(s) and its(their) derivative(s) lying ahead of the elongating cell tip. The new concept also explains the hitherto obscure mechanism of the cell event called ‘elimination of initials’. This article evaluates these new concepts of the cambial cell dynamics and offers a new interpretation for some curious events occurring in the cambial meristem in relation to the radial growth in woody plants.

High-throughput sequencing and Degradome sequencing revealed that miRNAs play crucial roles during the juvenile-to-mature stages in Chinese fir. In woody plants, the vascular cambium is a continuous cylinder of meristematic cells in the stem that produces secondary xylem (wood) and secondary phloem via cell division, expansion, secondary wall formation, lignification, and programmed cell death. MicroRNAs (miRNAs) are endogenous single-stranded non-coding RNAs that play important roles in plant growth and development. However, little is known about the roles of miRNAs in age-related changes in the vascular cambium, especially in conifers. Here, we compared age-related miRNAs in the vascular cambium of 3-(juvenile), 13-(transition), and 35-year-old (mature) Chinese fir (Cunninghamia lanceolata) trees. Transmission electron microscopy revealed changes in the morphology and cell wall thickness of the vascular cambium during the juvenile-to-mature phase transition. In addition, high-throughput sequencing and subsequent analyses identified 700 miRNAs, including 651 known and 49 novel miRNAs. We identified 105, 120, and 94 differentially expressed miRNAs at the three stages of growth. These miRNAs included various differentially expressed development-related and phytohormone-related miRNAs involved in cambium activity, cell division, and cell wall modification. Degradome sequencing revealed 38 target genes that were cleaved by 88 known miRNAs and 16 target genes that were cleaved by seven novel miRNAs. Additionally, miR159, miR164, and miR166 were highly expressed in the 13-transition stage; these miRNAs are involved in the auxin-signaling pathway. Moreover, miR156 levels were abundant in VC13 along with miR172 levels decreased during the juvenile-to-mature stages. These results provide insight into the roles of age-specific miRNAs in the growth of C. lanceolata and shed light on vascular cambium development in conifers.

In Hancornia speciosa, the latex is synthesized in the cytosol of the ground meristem cells and in plastids of the fusiform derivatives of secondary phloem. Laticifer development is related to climatic seasonality. Hancornia speciosa is a latescent species that grows in the Brazilian Cerrado (neotropical savanna) and shows significant potential for rubber production and the extraction of bioactive compounds from its latex. We examined the development of laticifers in its stem apex and secondary phloem in relation to climate seasonality. Morphometric evaluations of elongating branches and micromorphometric evaluations of the cambial zone were carried out monthly for 1 year, with structural and ultrastructural analyses of the stem apex and secondary phloem. Laticifer development in both the stem apex and secondary phloem is related to increasing day length and maximum temperature and humidity. Laticifers are formed by anastomosis of the transverse and longitudinal walls of the ground meristem cells of the stem apex, and the fusiform derivatives of secondary phloem in the cambial zone. The process of latex secretion involves the synthesis of terpenic droplets in the cytoplasm of the ground meristem cells of the stem apex and in the plastids of the fusiform derivatives of secondary phloem. Latex is an emulsion formed by the cytosol terpenic droplets engulfed by vacuoles contaning mucilages and proteins secreted by dictyosomes and rough endoplasmic reticulum, and alkaloids secreted by the phloem cells. This work expands our knowledge concerning the development of laticifers in Apocynaceae and contributes to our better understanding of the influence of environmental factors on latex secretion.

During secondary growth, the thickening of plant organs, wood (xylem) and bast (phloem) is continuously produced by the vascular cambium. In Arabidopsis hypocotyl and root, we can distinguish two phases of secondary growth based on cell morphology and production rate. The first phase, in which xylem and phloem are equally produced, precedes the xylem expansion phase in which xylem formation is enhanced and xylem fibers differentiate. It is known that gibberellins (GA) trigger this developmental transition via degradation of DELLA proteins and that the cambium master regulator BREVIPEDICELLUS/KNAT1 (BP/KNAT1) and receptor like kinases ERECTA and ERL1 regulate this process downstream of GA. However, our understanding of the regulatory network underlying GA-mediated secondary growth is still limited. Here, we demonstrate that DELLA-mediated xylem expansion in Arabidopsis hypocotyl is mainly achieved through DELLA family members RGA and GAI, which promote cambium senescence. We further show that AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, which physically interact with DELLAs, specifically repress phloem proliferation and induce cambium senescence during the xylem expansion phase. Moreover, the inactivation of BP in arf6 arf8 background revealed an essential role for ARF6 and ARF8 in cambium establishment and maintenance. Overall, our results shed light on a pivotal hormone cross-talk between GA and auxin in the context of plant secondary growth.

Aims Exposure of Eucalyptus tree stems to the radiant heat of forest fires can kill cambial cells and their embedded regenerative meristems, thus preventing epicormic resprouting and recovery of the tree. Currently, there is no tissue-level method to quantify the viability of cambial cells in Eucalyptus following heat exposure. The first aim of this study was to adapt and validate the tetrazolium reduction method of testing for cell viability in Eucalyptus. The second aim was to apply the method to establish a threshold level of cambium cell viability in Eucalyptus obliqua to enable the identification of a critical temperature. Methods The study used the tetrazolium reduction method to quantitatively determine phloem–cambium cell viability in Eucalyptus. Circular sections of bark with underlying phloem and cambium were cut from mature E. obliqua and samples ranging in mass from 1 to 30 mg were exposed for 1 min to temperature treatments ranging from 20 to 85 °C and kept for 20–22 h at room temperature in 0.8% 2,3,5-triphenyl tetrazolium chloride (TTC) to test for cell viability. The 1,3,5-triphenyl tetrazolium formazan (TPF) formed was cold extracted with ethanol and quantified as absorbance at 485 nm. Important Findings The TTC reduction method reliably quantified a decline in cell viability with rising temperature in tissue sections that included vascular cambium, and identified 60 °C as the critical temperature for cambium–phloem cells of Eucalyptus species. Cell viability, calculated as [TPF Treatment °C]/[TPF 20 °C], declined by 90% between 20 and 85 °C. The cell viability results confirmed that significant tissue necrosis occurred in Eucalyptus at temperatures between 50 and 70 °C, after 1 min of in vitro tissue heating. The decline in cell viability with increasing temperature shown by the TTC method was consistent with an independently derived count of live cells following temperature treatment and neutral red staining.

Chinquapin (Castanea henryi) is a dual-purpose tree species in China valued for as a source of timber and starch. We investigated the effect of four cutting mediums (pure vermiculite; peat:river sand at 3:1 v/v; peat:krasnozem at 1:1 v/v; and pure krasnozem) and three stem cutting periods (March, May, and July) on rooting performance of C. henryi cuttings. Different cutting periods and cutting mediums greatly influenced the rooting rate of C. henryi, ranging from 3.35 to 77.31%. Principal component analysis indicated that the best combination of cutting period and cutting medium was semi-hardwood cuttings (May cuttings) + krasnozem. Histological evidence indicated that adventitious root initials were present by week 5–6, and that the site of root primordia initiation was observed in the vascular cambium. Stem anatomical structures observed at different periods indicated that a xylem/radius ratio of 29.90–37.42% and a fractured phloem fiber ring are indicative of rooting success. The relational model between rooting index and medium properties indicated that nutrient content and porosity significantly influenced callus production. However, pH strongly affected C. henryi root formation, with the Pearson correlation coefficients for May and July cuttings of − 0.856 and − 0.947, respectively. Our protocol is helpful to achieve mass clone propagation of improved C. henryi genotypes, thus overcoming a common hurdle in chinquapin breeding programs.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum , an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo ). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA , which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness—one of the key biological innovations associated with the earliest phases of angiosperm evolution.

New observations of radial sieve tubes in the secondary xylem of two genera and four species of agarwood — Aquilaria sinensis, A. crasna, A. malaccensis and Gyrinops versteeghii (Thymelaeaceae) — are presented in this study. The earliest radial sieve tubes in Gyrinops are formed in the secondary xylem adjacent to the pith. The radial sieve tubes originate from the vascular cambium and develop in both uniseriate and multiseriate ray tissue. In addition to sieve plates in lateral and end walls, scattered or clustered minute sieve pores are localized in the lateral wall of radial sieve tubes. There is a direct connection between radial sieve tubes in ray tissue and axial sieve tubes in interxylary phloem strands (IP), such as (i) connection by bending of radial sieve tube strands, (ii) connection of two IP strands by an oblique bridge, and (iii) connection of two IP strands at a right angle. The average number of radial sieve tubes and interxylary phloem was found to be 1.7 per mm3 and 9.1 per mm2 in the secondary xylem. Considering the higher frequency of radial sieve tubes with the increasing thickness of the secondary xylem, the direct connections between radial and axial sieve tubes could play a significant role in assisting the translocation of metabolites in Aquilaria and Gyrinops.

The periderm acts as the first line of defence for a plant, protecting wood and phloem from abiotic and biotic stresses. During secondary growth, through the increase in girth of plant organs, the periderm replaces the epidermis as the outermost tissue. The phellogen, a bifacial post-embryonic meristem, forms the phelloderm inwards (toward the vasculature) and the suberized phellem outwards (toward the environment). These three tissues are collectively referred to as the periderm. Here, we summarize recent findings on the molecular mechanisms of periderm development by describing periderm formation in connection to the fate of the surrounding tissues, by discussing common regulatory hubs between the vascular cambium and the phellogen, and by highlighting transcription factors (TFs) controlling phellem differentiation.

Aesculus hippocastanum trees are commonly infested by the leaf miner Cameraria ohridella, whose larval activity causes the destruction of the leaf parenchyma and induces defoliation. Pest attacks result in, e.g., production of smaller fruits and tree re-flowering in autumn. Concerning pest influence on stem structure only scarce information of narrower annual growth rings of wood has been published. Therefore, we determined the effect of the presence of the leaf miner infestation on intra-annual cambial activity and on differentiation of conductive tissues. These data were compared with phenological phases and pest activity. Pest feeding resulted in changes in onset, cessation and duration of cambial divisions, and differentiation of secondary xylem. The duration of cambial activity was about a month shorter in heavily infested trees and was connected with premature tree defoliation. Affected trees were characterised by a reduction in cambial divisions and earlier cessation of wood differentiation resulting in narrower wood rings. Furthermore, the infested trees exhibited altered wood structure, with more vessels of smaller diameters, however these changes did not affect its theoretical hydraulic conductivity. Interestingly, pest attack did not influence secondary phloem differentiation. The probable influence of long-term infestation on tree growth and condition was discussed. info:eu-repo/semantics/openAccess

Wild ginseng (Panax ginseng) can survive in their natural habitat for hundreds of years, reflecting a remarkable plasticity. Plant stem cells (SCs) play a key role in the regenerative capacity and lifelong activity of these plants. WUSCHEL-RELATED HOMEOBOX (WOX) genes are master regulators of plant SC pluripotency, but their functions in medicinal plants have not been previously reported. To investigate whether these genes define different SC niches in ginseng, we cloned and analysed five WOX genes in ginseng (PgWOXs) and found that they might regulate root reconstruction. Then, the whole-mount RNA in situ hybridization was used to characterize the 3D gene expression pattern of PgWOXs in ginseng seedlings and cultured adventitious roots. PgWOX4 was expressed in vascular cambium SCs; PgWOX5 and PgWOX11 were mainly expressed in the tips of seedling and adventitious roots, which are the energetic centre of the meristem; and PgWOX13a and PgWOX13b were detected in the parenchyma cells of the main root of seedlings and cultured adventitious roots, suggesting that they are important for maintaining the balance between SC differentiation and self-renewal in the phloem and xylem. This is the first report of SC regulation in medical herbs; we expect that P. ginseng can serve as a model herb for investigating the relationship between SCs and their herbal morphological features, which would be a new research direction to improve the yield and quality of the medicinal materials by regulating the herbal SCs.

The early life history of Rafflesia R.Br. is hidden by its cryptic nature. Being a holoparasitic plant, early growth and development of species in this genus have been suggested to occur inside the host plant. In this study, using microscopic analyses, we examined how Rafflesia patma Blume grows from a simple clump of cells or protocorm inside the cambium tissue of Tetrastigma leucostaphylum (Dennst.) Alston, its host plant. We further show how the protocorm expands in about 2 months into an early form of a flower knob or bud, just before the flower begins to emerge from the host plant vine. Our study suggests that the endophyte, R. patma, spreads within T. leucostaphylum vascular cambium tissue in a linear manner, but not as a continuous strand. We suspect that the parasitic endophyte spreads inside the host vascular cambium and is pushed farther away from its point of origin to another part of the host as the host vine cambium fusiform initial cells divide and enlarge over months. Moreover, the protocorm matures in the cambium region of the T. leucostaphylum vine which lies close to the xylem, and grow outwards, bursting through the host phloem, then the host cortex, and finally towards the host periderm as it begins to emerge. Although we believe this might be the closest prediction on how Rafflesia develops at an early stage and is distributed within its host body, a future analysis of endophytic tissue growth in a controlled environment, such as the use of tissue culture, is required to strengthen our findings.

Stem cell populations in meristematic tissues at distinct locations in the plant body provide the potency of continuous plant growth. Primary meristems, at the apices of the plant body, contribute mainly to the elongation of the main plant axes, whereas secondary meristems in lateral positions are responsible for the thickening of these axes. The stem cells of the vascular cambium—a secondary lateral meristem—produce the secondary phloem (bast) and secondary xylem (wood). The sites of primary and secondary growth are spatially separated, and mobile signals are expected to coordinate growth rates between apical and lateral stem cell populations. Although the underlying mechanisms have not yet been uncovered, it seems likely that hormones, peptides, and mechanical cues orchestrate primary and secondary growth. In this review, we highlight the current knowledge and recent discoveries of how cambial stem cell activity is regulated, with a focus on mobile signals and the response of cambial activity to environmental and stress factors.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications. Published version

Understanding the changing patterns of vascular cambium during seasonal cycles is crucial to reveal the mechanisms that control cambium activity and wood formation, but this area has been underexplored, especially in conifers. Here, we quantified the changing cellular morphology patterns of cambial zones during the active, transition and dormant stages. With the help of toluidine blue and periodic acid–Schiff staining to visualize cell walls and identify their constituents, we observed decreasing cambial cell layers, thickening of newly formed xylem cell walls and increased polysaccharide granules in phloem from June to the following March over the course of our collecting period. Pectin immunofluorescence showed that dormant-stage cambium can produce highly abundant de-esterified homogalacturonan and (1–4)-β-d-galactan epitopes, whereas active cambium can strong accumulate high methylesterified homogalacturonan. Calcofluor white staining and confocal Raman spectroscopy analysis revealed regular changes in the chemical composition of cell walls, such as relative lower cellulose deposition in transition stage in vascular cambium, and higher lignin accumulation was found in dormant stage in secondary xylem. Moreover, real-time quantitative polymerase chain reaction analysis suggested that various IAA (Aux/IAA protein), CesA, CslA and HDZ genes, as well as NAC, PME3 and PME4, may be involved in cambium activities and secondary xylem formation. Taken together, these findings provide new information about cambium activity and cell differentiation in the formation, structure and chemistry in conifers during the active–dormant transition.

During secondary growth, the thickening of plant organs, wood (xylem) and bast (phloem) are continuously produced by the vascular cambium. In Arabidopsis hypocotyl and root, we can distinguish two phases of secondary growth based on cell morphology and production rate. The first phase, in which xylem and phloem are equally produced, precedes the xylem expansion phase in which xylem formation is enhanced and xylem fibers differentiate. It is known that Gibberellins (GA) trigger this developmental transition via the degradation of DELLA proteins and that the cambium master regulator BREVIPEDICELLUS/KNAT1 (BP/KNAT1) and the receptor like kinases ERECTA and ERL1 regulate this process downstream of GA. However, our understandings on the regulatory network underlying GA-mediated secondary growth, are still limited.Here, we demonstrate that DELLA-mediated xylem expansion is mainly achieved through RGA and GAI and that RGA and GAI promote cambium senescence. We further show that AUXIN RESPONSE FACTOR (ARF6) and ARF8, which physically interact with DELLAs, specifically repress phloem proliferation and induce cambium senescence during the xylem expansion phase. Moreover, the inactivation ofBPinarf6 arf8background revealed an essential role for ARF6 and ARF8 in cambium establishment and maintenance. Overall, our results shed light on a pivotal hormone cross-talk between GA and auxin in the context of plant secondary growth.

Mg deficiency affected only the differentiation zone in Citrus sinensis roots. Phloem impairment in Mg-deficient leaves resulted in cell wall lignifications of both vascular cambium and spongy parenchyma cells. Decrease of Mg content in the roots hampered secondary lignification of endodermal and protoxylem cell walls. Magnesium (Mg) is a vital nutrient for plant photosynthesis and biochemical reactions. Mg deficiency is a widespread problem in citrus production, directly affecting yield and quality. With the objective of investigating the relationship among root and leaf anatomy, photosynthetic efficiency and mineral nutrient uptake and transport in Mg-deficient citrus, the effects of Mg deficiency in ‘Xuegan’ (Citrus sinensis) seedlings on plant biomass, leaf gas exchange and chlorophyll, nutrient contents, root and leaf anatomy and lignin biosynthesis were studied. Mg deficiency decreased root and shoot dry weight, leaf CO2 assimilation, chlorophyll and Mg, phosphorus (P), boron (B), cupper (Cu) and iron (Fe) concentration in roots, stems and leaves, and increased potassium (K) and calcium (Ca) concentration. Mg deficiency in the leaves resulted in phloem disorder that blocked phloem transport, leading to carbohydrate accumulation in chloroplasts and secondary lignifications of both vascular cambium and spongy parenchyma cell walls. In contrast, decrease of Mg content in the roots hampered lignin deposition on the protoxylem and endodermal cell walls at the root differentiation zone. Poorly lignified Casparian strip and xylem cell walls in Mg-deficient root tips might decrease xylem loading efficiency of nutrients, lowering their contents in the above ground parts, which ultimately inhibited whole plant growth.

For more than 225 million years, all seed plants were woody trees, shrubs, or vines (1–4). Shortly after the origin of angiosperms ~135 million years ago (MYA) (5), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium (6), the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem (7–9), which are produced by apical meristems and retained in nearly all seed plants. Here we sequence and assemble a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compare it to genomes from other cambium-bearing and cambium-less lineages (like monocots and Nelumbo). This reveals lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also find the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent vascular cambium loss reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent trait loss events. Our results shed light on the evolution of herbaceousness – one of the key biological innovations associated with the earliest phases of angiosperm evolution.Significance StatementFor ~225 million years, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of flowering plants ~135 million years ago, Nymphaeales (water lilies) became one of the first seed plant lineages to become herbaceous through loss of the meristematic cell population known as the vascular cambium. We sequence and assemble the draft genome of the water lily Nymphaea thermarum, and compare it to genomes of other plants that have retained or lost the vascular cambium. By using both genome-wide and candidate-gene analysis, we find lineage-specific patterns of gene loss and divergence associated with cambium loss. Our reveal divergent genomic signatures of convergent trait loss in a system characterized by complex gene-trait relationships.

We used sector analysis to study cambium development and dynamics and to test whether fundamental developmental and functional differences exist between cambial initials as true 'stem cells' and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet's forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uidA reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees, we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not pre-determined.

Phloem and xylem conduit structure vary greatly throughout the body of Populus trichocarpa trees, particularly between roots and shoots. This has implications for understanding organ and whole plant vascular function. Woody plant vascular transport occurs predominantly within secondary xylem and phloem, which are both produced by the vascular cambium during secondary growth. We examined how vessel and sieve tube structure varied throughout the plant body of P. trichocarpa trees and whether xylem and phloem conduit structure was correlated across different positions within the plant. We excavated entire juvenile P. trichocarpa trees and measured vessel and sieve tube structural traits of current-year growth in 1 m increments along the main root:shoot axis. Trees were > 4 m tall and had roots that extended 4–5 m at their longest length. We found that both sieve tube and vessel diameters greatly varied throughout the plant body and with organ diameter. Roots had wider diameter conduits than shoots. Sieve tube diameter was strongly correlated with vessel diameter, which may be related to their common developmental origin. Other structural traits, such as pit membrane area and pit density for xylem, and sieve plate area and number of sieve areas per plate for phloem, also varied and were correlated with changes in conduit diameter. The median air-seeding pressure of vessels (Pm) and vessel length did not differ between roots and shoots. Understanding plant vascular function will likely require increased knowledge of whole plant structure and function, since plant performance may be limited by any point along the transport pathway. Considering intra-organismal variation may be a way to evaluate structure–function hypotheses while controlling for confounding sources of variation that may impact inter-specific comparisons.

The structure and development of interxylary phloem (IP) and external phloem in Aquilaria sinensis were investigated using light and scanning electron microscopy. Complete IP strands were isolated, measuring 14 ± 4 mm in length and 417 ± 124 μm in width. The outer margin of IP was composed of two to three layers of fusiform parenchyma cells. The development of IP can be divided into five stages: 1) Locally IP starts its differentiation within a small segment of a broad cambial zone, at the cost of xylem differentiation. 2) Inward growth of IP advances, and fibres and sieve tubes differentiate. 3) IP is constricted by the encroachment of immature xylem cells between cambium and immature IP. 4) IP is isolated from the cambium and surrounded by immature, non-lignified xylem tissue. 5) IP is surrounded by lignified xylem tissue, and the fibres within IP become lignified.In all the phloem islands in a ten-year-old stem, sieve elements showed positive staining of callose with aniline blue. However, no staining of callose was observed in the external secondary phloem of agarwood trees collected from two different sites. No sieve tubes or sieve pores were detected by SEM observation of numerous serial cross and radial sections of the external phloem. We therefore conclude that sieve tubes are absent from the external phloem or extremely rare and that the transport of photosynthetic products in the stem of A. sinensis takes place in the interxylary phloem.

Bignoniaceae is known for the presence of different growth forms like trees, shrubs, lianas and herbs that show a wide range of variations in their wood structure. Trees, shrubs and lianas are characterised by the presence of normal secondary growth except the lianoid forms of the tribe Bignonieae that have a unique type of the cambial variant (that is, wedged/furrowed xylem) whereas other lineages lack it. Campsis radicans belongs to tribe Tecomeae and thus is expected to lack the cambial variant but it develops a quantifiable amount of intraxylary secondary xylem and phloem in the pith. Therefore, the main aim of the present investigation is to elucidate the ontogeny of intraxylary phloem and the secondary xylem formed by internal vascular cambium. Development of intraxylary phloem is delayed significantly as compared to other families of eudicots that show the presence of intraxylary phloem. Subsequently, a functionally bidirectional complete ring of internal vascular cambium initiates along the pith margin that produces secondary xylem centrifugally and secondary phloem centripetally. In thick stems, an internal vascular cambium forms quantifiable amounts of inversely oriented secondary xylem and phloem that completely replace the pith. Structurally, this secondary xylem remains similar to the xylem formed by the regular (external) cambium. It is composed of vessels, fibres, axial and ray parenchyma cells while the secondary phloem consists of sieve elements, companion cells, axial and ray parenchyma cells. The development of internal vascular cambium completely replaced the parenchyma of the pith by producing intraxylary secondary xylem and phloem. The reason for the occurence of intraxylary phloem in C. radicans remains unknown. However, to the lesser extent it may be playing a secondary role in conductive safety of the conduit.

Aragoa is a shrubby genus endemic to páramo in the northern Andes representing the sister group to Plantago and Limosella. Stem and leaf structure of Aragoa corrugatifolia were studied to clarify the evolutionary pathways and ecological significance of their anatomical traits. Aragoa and Plantago share a non-fascicular primary vascular system, rayless wood and secondary phloem, and anomocytic stomata. Aragoa is distinctive from most Plantaginaceae in the presence of cortical aerenchyma and of helical thickenings in vessels. Its procambium emerges in the primary meristem ring as a continuous cylinder. The view on the ring meristem and procambial strands as developmental stages in the formation of a primary vascular system is not relevant for Aragoa, and probably for other Plantaginaceae. The raylessness is synapomorphic for the crown clade of Plantaginaceae comprising Aragoa, Littorella, Plantago, Veronica, Picrorhiza, Wulfenia, and Veronicastrum. The loss of rays is thought to be predetermined by procambium rather than by the vascular cambium. The extremely narrow vessels with helical thickenings are presumably adaptive to hydric and thermic conditions of páramo. Cortical aerenchyma is thought to be a response to the local hypoxia caused by the water retained by ericoid leaves. Trichomes on juvenile leaves are expected to be the traits of considerable taxonomic importance.

Basic knowledge of the intra-annual timings of leaf development and radial growth (including the phloem part) in different tree parts is generally missing although such rudimentary data are crucial to link the structure and function of vascular tissues at the whole tree level. To understand better the time course of leaf development and radial growth patterns in different tree parts, we studied leaf phenology and intra-annual xylem and phloem formation and structure in the stem and at two locations in branches of sub-Mediterranean Quercus pubescens in 2015. Onset and end dates of cambial cell production were synchronized at the two locations in branches, but were different at the stem base. The period of cell production was thus a month longer in the stem, resulting in 82.8 and 45.1% wider xylem and phloem increments, respectively. In addition, the xylem ring was wider than the phloem ring in all three parts. Thus, phloem ring widths in stem represented 24.8% and in branches 79.4% of the xylem ring width. Earlywood occupied 52.9% (stem) and 74.9% (branches) of the xylem ring, and early phloem 53.7% (stem) and 43.3% (branches) of the phloem ring. Most of the annual radial increment (i.e. xylem and phloem increments) in stem and branches was formed prior to full leaf development. Latewood and late phloem were formed in the period of full leaf unfolding. Our study confirmed that the temporal sequence of leaf development and radial growth are not contemporary in Q. pubescens. Different intra-annual patterns of radial growth in different tree parts result in different structures of xylem and phloem, which is in line with different roles of stem and branches in terms of tree functioning.

Bark is an important functional structure in woody plants. However, fossil tree axes are commonly decorticated. The development of bark during the evolutionary history of fossil plants thus remains poorly understood. Here, we describe exceptionally well-preserved extraxylary tissues of the Lopingian (Late Permian) conifer Ningxiaites specialis Feng, including vascular cambium and bark, from the Sunjiagou Formation (Changhsingian) of northern China. The vascular cambium bears one or two layers of cambial cells. The bark comprises secondary phloem and periderm. The secondary phloem consists of rays, axial parenchyma and sieve cells. The rays of the secondary phloem are uniseriate and continuous from the rays of the xylem. They are more frequently present in the inner zone of the secondary phloem. Axial parenchyma cells are vertically aligned and appear more regularly distributed in the outer zone of the secondary phloem. Elliptical or subcircular sieve areas are placed on the radial walls of the sieve cells. The periderm located outside the secondary phloem is composed of imbricate flattened cork cells. The cork cells show suberised cell walls and are generally filled with dark contents. Remains of the secondary phloem present between layers of periderm indicate the formation of rhytidome-type bark. This is the first detailed report of the bark anatomy of a conifer from the upper Palaeozoic of Cathaysia, and shed light on the early diversity of bark structure during the evolutionary history of conifers.

Stem ontogeny and structure of two neotropical twining vines of the genus Callaeum are described. Secondary growth in Callaeum begins with a typical regular cambium that gradually becomes lobed as a result of variation in xylem and phloem production rates in certain portions of the stem aligned with stem orthostichies. As development progresses, lignified ray cells of the initially formed secondary xylem detach on one side from the adjacent tissues, forming a natural fracture that induces the proliferation of both ray and axial nonlignified parenchyma. At the same time, parenchyma proliferation takes place around the pith margin and generates a ring of radially arranged parenchyma cells. The parenchyma generated in this process (here termed disruptive parenchyma) keeps dividing throughout stem development. As growth continues, the parenchyma finally cleaves the lignified axial parts of the vascular system into several isolated fragments of different sizes. Each fragment consists of xylem, phloem and vascular cambium and is immersed in a ground matrix of disruptive parenchyma. The cambium present in each fragment divides anticlinally to almost encircle each entire fragment and maintains its regular activity by producing xylem to the centre of the fragment and phloem to the periphery. Additionally, new cambia arise within the disruptive parenchyma and produce xylem and phloem in various polarities, such as xylem to the inside and phloem to the outside of the stem, or perpendicularly to the original cambium. Unlike the very distinctive stem anatomical architecture resulting from this cambial variant in Callaeum, its secondary xylem and phloem exhibit features typical of lianas. These features include very wide conducting cells, abundant axial parenchyma, high and heterocellular rays and gelatinous fibres.

Strands of phloem that are present at the periphery of pith are known as intraxylary phloem. Its presence remains restricted to a small portion of eudicots and considered as a characteristic feature for certain families. In the present study, Ipomoea turbinata Lag. showed development of intraxylary protophloem on adaxial tips of protoxylem from the procambial derivatives. Subsequently, additional intraxylary sieve elements were added from the adjacent parenchyma cells that were morphologically different from the pith cells. In thick stems, thin walled cells located between protoxylem and intraxylary protophloem acquired meristematic characters and formed several small segments of internal cambium. Initially, these cambial segments produced only phloem derivatives. Soon after, these segments became bidirectional and began to produce secondary xylem centrifugally and secondary phloem centripetally. Interestingly, in some of the samples development of secondary phloem and xylem was observed in the same direction. The secondary xylem formed by the internal cambium was composed of wide and fibriform vessels, fibres and axial parenchyma. Phloem possessed sieve tube elements, companion cells and parenchyma cells while rays in both xylem and phloem were mostly uniseriate but multiseriate rays were also observed.

Sector analysis in poplar tree stems was used to shed light on cell identity, cell fate, and patterns of development within the vascular cambium., We used sector analysis to study cambium development and dynamics and to test whether fundamental developmental and functional differences exist between cambial initials as true ‘stem cells’ and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet’s forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uidA reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees, we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not pre-determined.

During plant growth and development mineral elements are preferentially delivered to different organs and tissues to meet the differential demand. It has been shown that the preferential distribution of mineral nutrients in gramineous plants is mediated by node-based transporters, but the mechanisms of preferential distribution in dicots are poorly understood. Here, we report a distinct mechanism for the preferential distribution of phosphorus (P) in Arabidopsis plants, revealed by detailed functional analysis of AtSPDT/AtSULTR3;4 (SULTR-like P Distribution Transporter), a homolog of rice OsSPDT. Like OsSPDT, AtSPDT is localized at the plasma membrane and showed proton-dependent transport activity for P. Interestingly, we found that AtSPDT is mainly expressed in the rosette basal region and leaf petiole, and its expression is up-regulated by P deficiency. Tissue-specific analysis showed that AtSPDT is mainly located in the vascular cambium of different organs, as well as in the parenchyma tissues of both xylem and phloem regions. Knockout of AtSPDT inhibited the growth of new leaves under low P due to decreased P distribution to those organs. The seed yields of the wild-type and atspdt mutant plants are similar, but the seeds of mutant plants contain – less P. These results indicate that AtSPDT localized in the vascular cambium is involved in preferential distribution of P to the developing tissues, through xylem-to-phloem transfer mainly at the rosette basal region and leaf petiole.

Stem cell populations in meristematic tissues at distinct locations in the plant body provide the potency of continuous plant growth. Primary meristems, at the apices of the plant body, contribute mainly to the elongation of the main plant axes, whereas secondary meristems in lateral positions are responsible for the thickening of these axes. The stem cells of the vascular cambium-a secondary lateral meristem-produce the secondary phloem (bast) and secondary xylem (wood). The sites of primary and secondary growth are spatially separated, and mobile signals are expected to coordinate growth rates between apical and lateral stem cell populations. Although the underlying mechanisms have not yet been uncovered, it seems likely that hormones, peptides, and mechanical cues orchestrate primary and secondary growth. In this review, we highlight the current knowledge and recent discoveries of how cambial stem cell activity is regulated, with a focus on mobile signals and the response of cambial activity to environmental and stress factors.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium(1). Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown(2). Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium(3). Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

Modification of external morphology and internal structure of plants is a key feature of their successful survival in extreme habitats. They adapt to arid habitats not only by modifying their leaves, but also show several modifications in their conducting system. Therefore, the present study is aimed to investigate the pattern of secondary growth in Leptadenia pyrotechnica (Forssk.) Decne., (Asclepiadaceae), one such species growing in Kachchh district, an arid region of Gujarat State. A single ring of vascular cambium, responsible for radial growth, divided bidirectionally and formed the secondary xylem centripetally and the phloem centrifugally. After a short period of secondary xylem differentiation, small arcs of cambium began to form secondary phloem centripetally instead of secondary xylem. After a short duration of such secondary phloem formation, these segments of cambium resumed their normal function to produce secondary xylem internally. Thus, the phloem strands became embedded within th...

Despite the crucial roles of phytohormones in plant development, comparison of the exact distribution profiles of different hormones within plant meristems has thus far remained scarce. Vascular cambium, a wide lateral meristem with an extensive developmental zonation, provides an optimal system for hormonal and genetic profiling. By taking advantage of this spatial resolution, we show here that two major phytohormones, cytokinin and auxin, display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which has its highest concentration in the actively dividing cambial cells, cytokinins peak in the developing phloem tissue of a Populus trichocarpa stem. Gene expression patterns of cytokinin biosynthetic and signaling genes coincided with this hormonal gradient. To explore the functional significance of cytokinin signaling for cambial development, we engineered transgenic Populus tremula × tremuloides trees with an elevated cytokinin biosynthesis level. Confirming that cytokinins function as major regulators of cambial activity, these trees displayed stimulated cambial cell division activity resulting in dramatically increased (up to 80% in dry weight) production of the lignocellulosic trunk biomass. To connect the increased growth to hormonal status, we analyzed the hormone distribution and genome-wide gene expression profiles in unprecedentedly high resolution across the cambial zone. Interestingly, in addition to showing an elevated cambial cytokinin content and signaling level, the cambial auxin concentration and auxin-responsive gene expression were also increased in the transgenic trees. Our results indicate that cytokinin signaling specifies meristematic activity through a graded distribution that influences the amplitude of the cambial auxin gradient.

Rubber tree (Hevea brasiliensis) is a pivotal source for natural rubber production. Polyisoprene is synthesized in laticifer vessels, which are developed from vascular cambium in the phloem area of the inner bark tissue. The transcription factors and phytohormones were reported to be involved in network regulation of plant vascular tissue differentiation. Methyl jasmonate (MeJA) was reported to act as a stimulator for laticifer vessel formation in rubber tree, but the regulatory mechanism remains largely unknown. In this study, the correlation of laticifer vessel proliferation in phloem tissue upon MeJA treatment and the MeJA-responsive gene expression was investigated. Histochemical study of the laticifer revealed the circle and diffused structure around the secondary phloem of the stem. The number of laticifer vessels was increased from the top shoot to the lower stem part. The higher average number of laticifer vessels in stems of high latex-producing clones than in low latex/high wood yield clones suggested the correlation of laticifer vessel number and latex yield potential. This study demonstrated the temporal differential expression of MeJA-responsive genes upon MeJA treatment and adjusted back to a normal level after 3 months in two high latex-producing clones. The increasing number of laticifer vessels in MeJA-treated plants confirmed the consequent effect of MeJA treatment, and the possible roles of these genes in relation with laticifer vessel proliferation are discussed. A better understanding of gene function in laticifer development would be beneficial in rubber tree improvement and exploitation.

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four lateral organ boundaries (LBD) transcription factors have important roles in the regulation of secondary (woody) growth with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. We studied gene copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across species, and gene copy number was strongly and significantly correlated with tangential ray width. The climbing vines, cucumber and grape, with wide multiseriate rays (>10 cells wide) showed the highest gene copy number. Because the growth habit of woody lianas like grape requires significant twisting and bending, it was suggested that the unlignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in LBD18 protein sequences specific to woody taxa downstream of the LBD domain. Neutrality tests showed evidence for strong purifying selection on these regions across various orders, indicating important functional roles in woody taxa. Additionally, structural modeling demonstrates that these regions have a significant impact on tertiary protein structure and thus are likely of significant functional importance.

Piercing-sucking insects cause mechanical and physiological injury to plants. Ceresa nigripectus Remes Lenicov is a pest of alfalfa in subtropical regions of South America and a carrier of the ArAWB phytoplasma. The aim of this study was to determine the feeding habits of this treehopper and to describe the effects of the feeding injuries on stem vascular tissues in alfalfa. Adults and nymphs of C. nigripectus inserted their stylets repeatedly girdling the stem. One week after feeding, alfalfa stems exhibited numerous feeding canals with salivary deposits, most of which reached the phloem. Two weeks after feeding, cortex and phloem cells next to the salivary sheath collapsed, mature tracheal elements became sparse and appeared with an increased cross-section area, and phenolic compounds increased in cells and cell walls compared to undamaged plants. Three weeks after feeding, an annular callus, formed by abnormal cell division and hypertrophy of preexisting cortex and vascular cambium cells, appeared immediately above the stem girdle. Parenchyma cells from the outer layers of the callus differentiated to form secondary anomalous amphicribal bundles in the wound. The aerial parts above the stem girdle eventually withered and died. Fil: Perez Grosso, Tomas. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mercado, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Cs.naturales E Instituto Miguel Lillo. Departamento de Morfología Fisiología y Taxonomia Vegetal. Laboratorio de Anatomia Vegetal; Argentina Fil: Ponesa, G. I.. Universidad Nacional de Tucuman. Facultad de Cs.naturales E Instituto Miguel Lillo. Departamento de Morfología Fisiología y Taxonomia Vegetal. Laboratorio de Anatomia Vegetal; Argentina. Fundación Miguel Lillo; Argentina Fil: Conci, Luis Rogelio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina Fil: Virla, Eduardo Gabriel. Fundación Miguel Lillo. Dirección de Zoología. Instituto de Entomología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

474 I. 474 II. 475 III. 475 IV. 477 V. 477 VI. 477 VII. 479 VIII. 481 482 References 482 SUMMARY: A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue. Xylem is required for water transport, and is present in the vascular tissue with a second conductive tissue, phloem, required primarily for nutrient transport. Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, including cell division in the vascular meristem, vascular organization and differentiation of vascular cell types, is a signalling module characterized by a peptide ligand called TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and its cognate receptor, PHLOEM INTERCALATED WITH XYLEM (PXY). In this review, we explore the literature that describes signalling components, phytohormones and transcription factors that interact with these two central factors, to control the varying outputs required in vascular tissues for normal organization and elaboration of plant vascular tissue.

Propagation by cuttings is an effective method for ex-situ conservation of plants. In the present study, propagation of Ginkgo biloba L. was conducted using hardwood and semi hard wood cuttings. The cuttings from basal part of the tree were selected for the experiment and planted during rainy (i.e. July) and summer season (i.e. April) at Central Department of Botany, T.U. Cuttings beds were prepared using 3:1 sand and soil. Different concentration of auxins such as IAA, NAA and IBA were found effective in inducing rooting. During July, the stem cuttings treated with 1000 ppm IAA for 30 minutes induced highest percentage (90 %) and statistically signifi cant (P< 0.05) rooting while those treated with same concentration and time induced only 40% rooting during April. Like wise other auxins NAA and IBA induced more rooting during July and less rooting during April. Cuttings planted as control, during July showed 10 % rooting while during April did not show any rooting. Among all the auxins studied, IAA hormone and rainy season was found to be the most effective for rooting of G. biloba cuttings. From the anatomical study it was revealed that vascular cambium, secondary phloem, cortical cells and pericycle cells took part on rooting. Successfully rooted cuttings were then transferred to the natural soil condition. J. Nat. Hist. Mus. Vol. 26, 2012: 175-180

Successive cambia are often associated with the climbing or shrub habit, and is less common in trees. We studied formation of successive cambia and structure of secondary xylem in young stems of Cocculus laurifolius DC., a tree species of Menispermaceae. Cell division in the vascular cambium ceased in pencil-thick stems. Subsequently, parenchyma cells located outside the perivascular fibre cap re-differentiated and gave rise to several small segments of meristematic cells, of which the central cells divided repeatedly to initiate the first successive cambium which produces secondary xylem centripetally and phloem centrifugally. Cells located on the inner side of the newly initiated cambium differentiated into conjunctive tissue while cells on the outer side of it divided further and differentiated into sclereids. Xylem was diffuse porous and composed of vessels, fibre tracheids and ray parenchyma cells, and only differed in vessel diameter from wide-vessel climbing relatives.

Bark beetles and their fungal associates are integral parts of forest ecosystems, the European spruce bark beetle (Ips typographus Linnaeus, 1758) and the associated pathogenic blue stain fungus Ceratocystis polonica (SIEM.) C. MOREAU, are the most devastating pests regarding Norway spruce [Picea abies (L.) H. KARST.]. Bark beetles commonly inhabit weakened and felled trees as well as vital trees. They cause physiological disorders in trees by destroying a phloem and cambium or interrupt the transpiration -ow in the xylem. Conifers have a wide range of effective defence mechanisms that are based on the inner bark anatomy and physiological state of the tree. The basic function of bark defences is to protect the nutrient-and energy-rich phloem, the vital meristematic region of the vascular cambium, and the transpiration -ow in the sapwood. The main area of defence mechanisms is secondary phloem, which is physically and chemically protected by polyphenolic parenchyma (PP) cells, sclerenchyma, calcium oxalate crystals and resin ducts. Conifer trunk pest resistance includes constitutive, inducible defences and acquired resistance. Both constitutive and inducible defences may deter beetle invasion, impede fungal growth and close entrance wounds. During a successful attack, systemic acquired resistance (SAR) becomes effective and represents a third defence strategy. It gradually develops throughout the plant and provides a systemic change within the whole tree’s metabolism, which is maintained over a longer period of time. The broad range of defence mechanisms that contribute to the activation and utilisation of SAR, includes antioxidants and antioxidant enzymes, which are generally linked to the actions of reactive oxygen species (ROS). The presented review discusses the current knowledge on the antioxidant defence strategies of spruce inner bark against the bark beetle (Ips typographus) and associated blue stain fungus (Ceratocystis polonica).

The structure and development of successive cambia and its products were studied in the stems of the halophyte Sesuvium verrucosum Raf. The young stem has several collateral vascular bundles arranged in a circle and separated by interfascicular zones. The secondary thickening begins with the differentiation of fascicular and interfascicular cambia which give rise to secondary xylem, phloem and lignified cells. This normal cambium ceases to divide after a limited period of activity. A new segment of anomalous cambium was developed from the phloem parenchyma cells outside the normal previous cambium. These cells are served as a site for the origin of the anomalous cambium and subjected to repeated periclinal divisions. This cambium has fusiform cells with semi-storied appearance. The activity of this anomalous cambium produces secondary xylem, phloem, fibers and soft parenchyma as conjunctive tissues. The formation of subsequent cambia followed a similar pattern of development and causes vascular increments in the old stem. So, the old stem of S. verrucosum is constructed of concentric fibrovascular bands separated from each other by cylinders of conjunctive parenchyma tissue. This internal structure has a great adaptive potential to the halophyte S. verrucosum . This can be detected by the following points: (1) Production of large number of vessels and sieve tubes elements increases the conductive activity; (2) Occurrence of fibers alongside the vessels increases the mechanical strength that helps and protects water columns from embolism and ensures this water to store and transport in the succulent leaves; and (3) Thin walled parenchyma conjunctive tissue offers flexibility to the plant stem which forms a mat like and able to bend toward the ground without harm. Perhaps these features matched well with the S. verrucosum plant habitat.