Seasonal changes in leaf lipids of Diapensia lapponica, with special reference to storage lipid bodies

Seasonal fluctuations in storage lipids in the cushion plant Diapensia lapponica , growing in Northern Finland (70°N 27°E), were studied by microscopy and chemical analysis. Lipid bodies in the mesophyll cells were stained with Sudan Black for quantitative observation by light microscope. Electron microscope observations were made using aldehyde prefixed and osmium tetroxide postfixed sections of leaf blades. Thin layer and gas capillary chromatographic techniques were used to analyse total lipids and total fatty acids in green shoots of Diapensia . Free sugars and starch were extracted separately and determined by the anthrone method.
A mesophyll cell was characterized by a large lipid body (storage lipid) in summer but by several small spherules in winter. Total surface area of the cross-sectioned lipid globules was at its lowest from April to September; the maximal value was in March. The amount of total lipids in the leafy tops of D. lapponica was 91–200 mg g^-1 dry weight. Values were lowest at the end of June, when the total carbohydrate level was at its highest. Accordingly, the decrease in the total lipid level in the early growing season, when new leaves were developing, can be attributed primarily to the increase in the level of carbohydrates, particularly starch. The amount of total fatty acids varied from 21 to 30 mg g^-1 dry weight. The level increased in the early growing season and remained elevated throughout the summer. Like the total lipids, the total fatty acids are derived from structurally different parts of the sclerophyllous leaves, including the well-developed cuticle and epicuticular wax layer. The discrepancies in the results obtained from microscopic and chemical analyses are discussed.     

木姜子油细胞的发育解剖学研究

利用薄切片法对木姜子茎叶油细胞的发育以及油细胞分布的研究结果表明：油细胞最早发生于第一叶原基以及茎端皮层和髓的基本分生组织中,在未出现油细胞以痛,上述器官的基本分生组织和原分生组织,难以区分油细胞的原始细胞与周围细胞。当油细胞原始细胞呈现出体积较大,液泡化程度较低,细胞核大而明显的特征才明显可辩,以后经过液泡融合,油细胞成熟和油细胞细胞质解体阶段而成为一贮油的囊,油细胞中未出现杯形构造。叶和茎中,     

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.     

Effects of shoot inversion on stem structure in Pharbitis nil

The effects of shoot inversion on stem structure over 72 hr were investigated in Pharbitis nil by analyzing cell number, cell length, and the cross sectional areas of cells, tissues, and regions. An increase in stem diameter can be attributed to an increase in both cell number and cross sectional area of pith (primarily) and vascular tissue (secondarily). Qualitative observations of cell wall thickness in the light microscope did not reveal any significant effects of shoot inversion on this parameter. The inhibition of shoot elongation was accompanied by a significant decrease in cell length in the pith. The results are generally consistent with an ethylene effect on cell dimensions, especially in the pith.     

Seasonal variation of organogenetic activity and reserves allocation in the shoot apex of Pinus pinaster Ait

BACKGROUND AND AIMS: To understand better the basic growth characteristics of pines and the fundamental properties of the shoot apical meristem (SAM), variations within the shoot apex of buds were studied. METHODS: A detailed structural comparison of meristem dimensions, organogenetic activity, and the presence of lipids, starch grains and tannins was performed on shoot apices of juvenile, and male and female adult Pinus pinaster at five different times in the annual growth cycle. KEY RESULTS: There were significant correlations among traits and differences in the pattern for juvenile and adult shoots. In juvenile shoots, peaks of organogenesis were present in spring and autumn, but not in summer. In adult shoots, one peak, characterized by an increase in meristem dimensions, was present in summer. The accumulation of starch grains beneath the SAM and of tannin in sub-apical pith parenchyma were at their maximum when organogenetic activity was high in spring and autumn in juvenile plants, and in summer in adult plants. In juvenile and adult plants, lipids were stored within the SAM in autumn, filling a large part of the bud in winter, and were depleted in the cortical parenchyma and then in the pith during shoot elongation. CONCLUSIONS: Depending on the sites of accumulation within the SAM and on the stage of the annual growth cycle, lipids, starch and tannins may be involved in different processes. In spring, energy and structural materials released by lipid hydrolysis may contribute to stem elongation and/or cell-to-cell communication. During organogenesis, energy and structural materials released by starch hydrolysis may influence developmental programmes in the SAM and adjacent cells. Tannins may be involved in cellular detoxification. At the end of the growing season, accumulation of lipid and starch is positively correlated with the onset of dormancy.     

Chlorophyll distribution pattern in inner stem tissues: evidence from epifluorescence microscopy and reflectance measurements in 20 woody species

The occurrence of functional chloroplasts in internal stem tissues and their distribution profiles in 20 woody species have been investigated. Chloroplasts were identified from the red chlorophyll auto-fluorescence using epi-fluorescence microscopy. Chloroplasts were detected in the cortex of all species examined, in the xylem rays of 19 and in the perimedullar and the pith cells of 16 out of the 20 investigated species. Chloroplast containing cell clusters in the pith were identified in some species. In addition, we report on the semi-quantitative distribution of chlorophylls in various internal stem tissues. Chlorophyll level was estimated by reflectance measurements at specific wave bands. Although decreasing chlorophyll gradients from cortex to pith were observed in half of the species, chlorophyll distribution in the remaining species was irregular with occasionally high levels in the pith. According to our data, chlorophyll occurrence in stem internal tissues is quite widespread, even in the light remote, deeply shaded central compartments like pith, provided that corresponding cells are viable. The species-specific tissue distribution of chlorophyll levels may be used to select suitable plants to investigate further this neglected area of photosynthesis research.     

WOUND RECOVERY BY PITH CELL REDIFFERENTIATION: STRUCTURAL CHANGES

The redifferentiation of tobacco pith cells was examined in two experimental systems: wounds recovering from an incision that severed vascular tissue of the stem, and induced differentiation of excised pith responding to indoleacetic acid supplied locally via pipets inserted into the tissue. In both systems there was an initial period during which cell division was resumed and the pith cells were cleaved into numerous small cells. This was followed by redifferentiation of some of the divided cells as tracheary elements and, especially in the stem, by the formation of a cambial meristem that produced further xylem and phloem. In the stem the size of the wound meristem decreased as the wound was made further from the shoot apex, and in the cultured pith tissue it was demonstrated that the size of the dividing zone increased with the concentration of auxin supplied. Auxin was, therefore, demonstrated to be limiting in the division phase of redifferentiation. The sequence of redifferentiation in the two experimental systems resembled the normal ontogeny of vascular tissues in the intact plant sufficiently that these systems could be used to investigate the relationship between cell differentiation and auxin transport.     

A localised decrease of GA1 in shoot tips of Salix pentandra seedings precedes cessation of shoot elongation under short photoperiod

By application of a recently developed method allowing analysis of gibberellins (GAs) in mg amounts of tissue, the effect of photoperiod on levels of GAs in shoot tips of individual seedlings of the woody species Salix pentandra was studied. In elongating long day-grown seedlings, maximum levels of GA₁ were found 5–20 mm below the apex, approximately twice the levels in other segments. After exposure of plants to 5 or 15 short days, the levels of GA₁ were about 50% lower within this specific region of the stem, as compared with seedlings grown under long days. Short day-induced cessation of shoot elongation also correlated with overall declines in the levels of GA₅₃, GA₁₉, GA₂₀ and GA₈, Within each photoperiodic treatment the levels of these GAs were generally relatively similar throughout the upper 35 mm of stems. No differences in internode lengths or in lengths of pith or epidermal cells were found in plants grown under long days compared with those exposed to 5 short days. In both cases, cells in mitosis were observed in the subapical stem tissues of shoot tips. After 15 short days, stem elongation was completed, and dividing cells were generally not found in the subapical part of the stem. However, short day exposure did not prevent elongation of internodes and cells differentiated before the treatment was started. Thus, the localised decrease in level of GA₁ in shoot tips under short days precedes the morphological and anatomical changes connected with the short day-induced cessation of elongation growth. This supports the hypothesised role for GA₁ in photoperiodic control of shoot elongation in S. pentandra .     

Sodium fluxes in sweet pepper exposed to varying sodium concentrations

The sodium transport and distribution of sweet pepper (Capsicum annuum L.) under saline conditions were studied after transferring the plants to a sodium-free nutrient solution. Sodium stress up to 60 mM did not affect the growth of sweet pepper, as it appears able to counteract the unfavourable physiological effects of sodium efficiently. Sodium was particularly accumulated in the basal pith cells of the stem and in the root cells, while almost no sodium was directed to the leaves or the fruits. The sodium concentration in the pith cells and xylem sap gradually decreased towards the shoot tip. Removal of sodium from the medium resulted in a 50% release of sodium from the plant after 1 week without affecting the gradient in the pith cells. In contrast, the concentration profile in the xylem sap was completely changed: the sodium concentration in the xylem sap at the stem base was similar to that at the top.Phloem transport was studied in a split root experiment, in which both portions of the roots were exposed to 15 mM NaCl and one part was fed with additional ²²NaCl. During continuous exposure to 15 mM NaCl no label was detected in unlabelled root parts. However, after transferring the plants to a sodium-free solution, ²²Na was rapidly released from the unlabelled roots, indicating a downward phloem transport.It was concluded that pith cells, the intermediates between the xylem and phloem, play a decisive role in the recirculation of sodium throughout the plant. Release of sodium from the plants following transfer to a sodium-free solution may be explained by changes in the diffusion resistance for passive sodium efflux from the cells.Key words: Xylem, phloem, sodium, fluxes, sweet pepper      

ONTOGENY AND DIFFERENTIATION OF SCLEREIDS IN RAUWOLFIA

An interesting anatomic feature of Rauwolfia is the occurrence of a remarkable type of sclereid in the stem and root. The initials of the sclereids in the stem arise in the ground tissue element of the pith in a region between 50 and 70μ below the surface of the shoot apex. This region of the shoot remains surrounded by a whorl of either 3 or 4 leaves. Sclereids initiate in succession in association with each whorl of leaves. Thus, the sclereids are restricted to the nodes. The sclereids in the stem arise as a primary element of the shoot from the ground tissue of the pith. In the root, they differentiate from the cells of the phelloderm and are secondary in origin. Morphologically, the sclereids in these 2 organs are basically the same, except that the sclereids in the stem are larger in size than those in the root. A solitary cell, or 2 to several cells in a longitudinal cell file (originated from a single mother cell), may differentiate into sclereid initials. The growth of the sclereids through relatively compact ground tissue of the pith is possibly accomplished by a combination of gliding growth and apical intrusive process. The sclereid initials grow rapidly and force their way between the parenchymatous cells. As a result, the neighboring cells lose their original surface contacts. Sclereids increase in size rapidly, and, therefore, very enlarged sclereids with thin primary walls may be observed in the second node. They mature progressively in basipetal direction in the subjacent nodes. In the fifth or sixth node, mature sclereids with massive secondary walls are most common. The secondary walls of sclereids contain much lignin as determined by the phloroglucinol-HCl test. The walls of sclereids at maturity show a variable number of lamellae ranging from 10 to 15 in the lateral walls. A remarkable feature of the sclereids is their canal-like pits in the secondary walls. Two adjacent pits may coalesce uniquely to form a Y-like configuration directed centrifugally from the lumen of the sclereids. The sclereids are ventrically symmetrical, joined end-to-end by their transverse walls like 2 superimposed young fibers.     

Distribution of leaf assimilates in the stem of Picea abies L.

Summary Autoradiographic and microautoradiographic studies of 2-year-old Picea abies plants show that in summer leaf assimilates from the second-year shoot are translocated basipetally. Leaf assimilates are first transported to the stem via leaf trace phloem, then to the base of the stem in the sieve cells of the latest increment of secondary phloem. On the way down leaf assimilates move radially from sieve cells into cells of the phloem parenchyma, the vascular cambium, the rays, the inner periderm and certain cells of pith and cortex, including the epithelial cells surrounding the resin ducts. Other cells of pith and cortex remain nearly free of label, despite the long translocation time (20 h). With the exception of the vascular cambial cells, the stem cells that gain leaf assimilates by radial distribution coincide with those that contain chlorophyll and starch.     

Competency of Nicotiana tabacum L. stem tissues to dedifferentiate is associated with differential levels of cell cycle gene expression and endogenous cytokinins

The expression of the mitotic cyclin Arath; CYCB1;1 and of the cyclin-dependent kinase Arath; CDC2a was located by beta-glucuronidase histochemical detection and in situ hybridization, and was quantified by 4-methylumbelliferyl beta- D-glucuronide assay in tobacco stem tissues during both in vivo differentiation and in vitro dedifferentiation. The changes in localization of endogenous cytokinins were also determined during both processes using quantitative analysis and in situ immunocytochemistry. The CDC2a promoter was expressed continuously during stem development, with particularly high expression in the shoot apical meristem and in the internal and external primary phloem. CYCB1 expression was not restricted to the dividing cells; its expression in the shoot apical meristem was particularly high in the leaf-forming peripheral cells but the gene was also expressed throughout development in the internal and external phloem in which the rate of cell division was reduced or zero. Following in vitro culture, the internal phloem cells appeared to be particularly competent to re-enter the cell cycle within a short lag phase while the pith tissue reactivated later. In culture, cells that resumed division were found to accumulate cytokinins. The high competency of primary phloem to dedifferentiate was associated with its capacity to express CDC2a and CYCB genes and the presence of high cytokinin levels, providing some insights into the determinants of competency for resuming cell division.     

MEDULLARY BUNDLES AND THE EVOLUTION OF CACTI

Medullary bundles are absent from the pith of the leafy, relictual cacti (genus Pereskia) but are present in most members of subfamily Cactoideae. They are absent only from tribes Hylocereeae, Rhipsalideae, and some members of Cacteae and Notocacteae. Presence of medullary bundles tends to be correlated with presence of a broad pith, but exceptions occur. Most medullary bundles are collateral, and in all genera phloem is produced and accumulates throughout the lifetime of the bundle. Xylem definitely accumulates as medullary bundles age in some groups, but it definitely does not accumulate in others, being produced only while the bundle is young. Pith can be broad (up to 75 mm in diameter), can constitute half the shoot volume, and is long-lived, remaining alive as long as the shoot is alive. Medullary bundles appear to be adaptive in allowing this large pith to be used for storage of water and starch. Medullary bundles have fewer, narrower tracheary elements than does the stele xylem in the same region; medullary bundles probably could not carry out significant longdistance transport if a major part of the stele becomes damaged.     

Anatomy of shoots and tumors of in vitro habituated Rhododendron 'Montego' (Ericaceae) cultures with tissue proliferation

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons that were initially propagated in vitro. In the cultivar 'Montego', TP-like symptoms are first observed in vitro as shoot clusters with small leaves and nodal tumors. In addition, unlike the normal in vitro non-TP (TP-) shoots, in vitro TP (TP+) shoots proliferate rapidly without the presence of the plant growth regulator cytokinin in the tissue culture medium. Comparisons of the anatomy of TP+ and TP- shoot tips showed that TP+ shoots had a less developed vascular system, longer cells in the pith and cortex, and altered internodal elongation at the shoot apex. In addition, TP+ axillary buds were abnormal in that they were displaced onto the stem above the leaf axil, and a small group of proliferating cells replaced the shell zone at the base of the bud. Initiation of tumor formation began with the expansion of this region of cell proliferation (RCP) and shoot growth from the abnormal axillary bud (tumor bud). Organization of the tumor bud and extension of the RCP characterized the further development of two types of tumors. In polar shoot tumors, shoot growth continued from the persistent tumor bud and the tumor at the base of the shoot remained small in size. The RCP extends downward to the vascular junction of the subtending leaf and the stem of the TP+ shoot. In nonpolar tumors, continuous de novo meristem formation led to the development of large tumors with or without shoots. The RCP is present throughout the tumor and is associated with de novo meristem formation. Comparisons to the anatomy of other tumor-like structures showed that TP tumors of Rhododendron 'Montego' are most similar to tobacco genetic tumors.     

Environmental factors affecting basic nitrogen metabolism and seasonal levels of various nitrogen fractions in tissues of bilberry, Vaccinium myrtillus

Bilberry tissues accumulated nitrogen for the winter in the form of reduced low-molecular weight amino compounds. The storage organ was principally the underground stem and the oldest parts of the aerial shoot. Most of the nitrogen was stored in arginine and ammonium compounds, and less in glutamine and other amino acids. Proteins did not accumulate during the winter. The soluble nitrogenous compounds were discharged from storage in May, when nitrogen was translocated from the lower parts of the stem to the growing leaves and buds. Amino acid compositions and concentrations in winter were almost identical under the snow and in snowless areas, only the concentration of glutamine being lower and that of glutathione higher in the snowless area. The level of total protein, particularly in the leaves and buds was much higher in a nitrogen-polluted industrial area than in unpolluted urban forests. The same difference was observed in total amino compounds, but among individual substances it only appeared in ammonium compounds. Certain species differences in the amino acid pool were recorded between V. myrtillus and V. vitis-idaea.     

Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith

Pith tissue from Nicotiana tabacum L. cv `Maryland Mammoth' or `Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.     

Structural aspects of the regulation of starch accumulation in stem pith explants of kale

Anatomical aspects were studied of starch deposition in primary explants of stem pith of marrow-stem kale. During cultivation on semisolid media containing glucose and growth substances starch is accumulated in the inner part of the explant but absent on its periphery. The starch localization pattern formed within originally quite homogeneous tissue seems to be caused both by gradients of substances absorbed from culture medium and by physiological gradients manifested in the explanted tissue. Strange enlarged grains were abundant in cells with high starch content.     

SHOOT GROWTH AND HISTOGENESIS OF TREES POSSESSING DIVERSE PATTERNS OF SHOOT DEVELOPMENT

Shoot growth and histogenesis were followed in five unrelated tree taxa possessing inherently diverse patterns of shoot development. Following the resumption of growth in spring, each species differs quantitatively in the number of internodes elongating contemporaneously, in rates and duration of internodal elongation and seasonal periodicity of shoot growth. The basic pattern of internode elongation and histogenesis is qualitatively similar in each of the dicotyledonous species observed irrespective of growth habit or final form of the shoot produced. During the intial phase of internode development, growth is essentially uniform throughout young internodes, corresponding to an active period of cell division during which time pith cells increase in size to about one-third their final length. Subsequently, the pattern of cell division shifts progressively upward concomitant with increased elongation and maturation of pith cells in the basal portion of developing internodes. Thereafter, a wave of cell division accompanied by cell elongation continues to proceed acropetally until growth finally ceases in the distal portion of each internode. As long as internode elongation continues, frequently at distances 15–20 cm below the shoot apex, cell divisions still occur in the distal growing portion. As successive portions of each internode mature acropetally, final length of pith cells becomes relatively uniform throughout the internode. During the process of internode growth and development, cell lengths increase only two- to threefold, whereas cell numbers increase ten- to 30-fold, indicating the dominant role of cell division and increases in cell number to final internode length. Morphological patterns of shoot expression associated with differences in internode lengths along the axis of either preformed or neoformed shoots, as well as sylleptic branches, are due to differences in cell number rather than final cell length. Significant variations in final internode lengths along the axis of episodic shoots, caused by either endogenous or exogenous factors, are also attributed to differences in cell number.     

Anatomical features, monomer lignin composition and accumulation of phenolics in 1-year-old branches of the Mediterranean Cistus ladanifer L.

Mediterranean shrub species are described as having phenology, habitus , reproductive biology and anatomical alterations in certain tissues, allowing their survival during the dry season and protecting them from herbivory. Anatomical and chemical analyses were conducted in 1-year-old branches of Cistus ladanifer L. in order to investigate the role played by shoot structure in the adaptive strategies of this species in the Mediterranean environment. Results showed that both xylem and pith underwent lignification. Pith parenchyma cells had thickened walls, higher lignin content than xylem and different monomer composition. Xylem presented features aiding safe water transport. A large accumulation of phenolic substances was found in xylem, pith and cortical parenchyma. Observations reported in this paper suggest the occurrence of adaptive strategies in 1-year-old branches of C. ladanifer whose structural features: (1) allow mechanical reinforcement of tissues to withstand drought without suffering permanent damage; (2) favour safety rather than efficiency in water transport; (3) defend the plants from animal predation and pathogens by accumulating phenolics in various tissues, and (4) protect inner tissues against UV-B radiation through deposition of phenolic compounds in cortical layers.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155 , 361–371.     

Histochemical Studies of Sclereid Induction in the Shoot of Rauwolfia Species: I. CYTOCHROME OXIDASE

Results of histochemical tests for cytochrome oxidase activityin four species of Rauwolfia have been reported. The cells beneaththe terminal shoot meristem constitute the pith. Histochemicaltests showed intensified enzymatic activity in those cells ofthe pith which would differentiate as sclereid initials. Similarcytochrome oxidase activity also occurred in the sclereid initialsand the developing sclereids. The cytochrome oxidase activitywas associated with two types of particulate formation, thegranular and rod-shaped. The ground parenchymatous cells ofthe pith and the leaf-base showed very little enzymatic activityof cytochrome oxidase. The characteristic indophenol blue colorationdue to cytochrome oxidase activity did not appear in controlsections. Physiological changes correlated with morphogeneticexpression of some pith cells demonstrate that the physiologicalchanges occur before the initiation of sclereids in the morphologicallyhomogeneous parenchymatous cells of the pith. Intensified cytochromeoxidase activity was also recorded in the meristematic tissuesof shoot apex, procambium, axillary buds and the laticiferouscells of Rauwolfia.