Effect of 6-Benzylaminopurine on the Structure of the Photosynthetic Apparatus of Faba Bean (Vicia faba L.)

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (S _max), was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of S _max) and decreased in leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of the terminal phloem; however, it did (1) stimulate elongation of mesophyll cells; (2) increase the area and thickness of the leaf blade, amount of photosynthetic pigments, and assimilation potential; and (3) delay senescence of the leaves and defoliation, thereby increasing the biomass of the aboveground plant part. It is inferred that BAP has the potential for inducing the development of photosynthetic apparatus and increasing the yield of the green mass of faba bean.     

Cytokinins in Populus×robusta: Qualitative Changes during Development

Qualitative changes of cytokinins in leaves of different ages from Populus x robusta (Schneid.) have been determined, together with seasonal changes in cytokinin activity in mature leaves and xylem sap. Chromatography on Sephadex LH-20 has shown that total cytokinin activity and diversity are at a maximum in expanding leaves. As leaves age, the amount and number of cytokinins decrease, with yellow senescent leaves having only one detectable cytokinin, thought to be a glucoside. Seasonal changes were followed by chromatography of the extracts on paper in butan-2-ol: 25 % NH₄OH (4:1). Maximum cytokinin levels, due to Fraction Z (Rf 0.5–0.8), in leaves and xylem sap were found in mid-summer. Prior and subsequent to cessation of shoot elongation growth, fraction Z decreased and fraction N (Rf 0–0.2) increased to predominate in senescent leaves. Removal of the apex resulted in an increase of fraction N in leaves from decapitated plants when compared to similar leaves from intact plants. It is suggested that, once apical sink activity has ceased, cytokinins in the xylem sap are diverted into leaves and converted to a cytokinin glucoside, possibly a storage form of the hormone.     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Structure of the photosynthetic apparatus of the bean Vicia faba L. when treated with 6-benzylaminopurine

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (Smax) was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of Smax) and decreased in the leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of terminal phloem; however, it stimulated elongation of mesophyll cells; increases in the area and thickness of leaf blade, amount of photosynthetic pigments, and assimilation potential; and delayed senescence of leaves and defoliation, thereby increasing biomass of the aboveground plant part. It was inferred that BAP had a potential for induction of photosynthetic apparatus development and increase in the yield of faba bean green mass.     

Does the photosynthetic light‐acclimation need change in leaf anatomy?

There is a strong correlation between leaf thickness and the light‐saturated rate of photosynthesis per unit leaf area (P_max). However, when leaves are exposed to higher light intensities after maturation, P_max often increases without increasing leaf thickness. To elucidate the mechanism with which mature leaves increase P_max, the change in anatomical and physiological characteristics of mature leaves of Chenopodium album, which was transferred from low to high light condition, were examined. When compared with leaves subjected to low light continuously (LL leaves), the leaves transferred from low to high light (LH leaves) significantly increased P_max. The transfer also increased the area of chloroplasts facing the intercellular space (S_c) and maintained a strong correlation between P_max and S_c. The mesophyll cells of LL leaves had open spaces along cell walls where chloroplasts were absent, which enabled the leaves to increase P_max when they were exposed to high light (LH). However, the LH leaves were not thick enough to allow further increase in P_max to the level in HH leaves. Thus leaf thickness determines an upper limit of P_max of leaves subjected to a change from low to high light conditions. Shade leaves would only increase P_max when they have open space to accommodate chloroplasts which elongate after light conditions improve.     

The physiological significance of the leaf nodules of psychotria

Summary In 6-month growth experiments it was found that leaf-nodulatedPsychotria mucronata seedlings grown in N-poor soil showed a restricted growth and developed severe nitrogen-deficiency symptoms in the leaves. Plants in the same soil supplied with NO₃-N showed healthy growth and dark green leaves. Detached Psychotria leaves bearing leaf nodules exposed to an atmosphere containing N¹⁵-labelled nitrogen gas or acetylene gas gave no evidence of nitrogen fixation, either in the light or in the dark or in both in succession. Therefore nitrogen fixation is probably not associated with the leaf nodules. Chlorophyll retention was observed around the leaf nodules in senescent Psychotria leaves. Psychotria leaf-nodule discs placed on oat leaves cause chlorophyll retention in the oat leaves below the discs. As chlorophyll retention is a common bioassay for cytokinins, these results indicate that a cytokinin-like substance is involved. With the aid of autoradiography and C¹⁴-labelled α-amino-isobutyric acid it was shown that this amino acid accumulates in the leaf nodules. Such directed transport is also a property of cytokinin.     

Role of roots in regulating the growth rate and cytokinin content in leaves

The role of roots in the enhancement of cytokinin content and leaf growth of Phaseolus vulgaris plants after decapitation and partial defoliation was investigated. Partial excision of the roots of plants which were decapitated above the primary leaf node resulted in a reduction of leaf growth and soluble proteins accumulation in the primary leaves. Roots excision was done at time of decapitation and repeated 8 days later. Endogenous cytokinins, known to be involved in enhancing shoot growth, accumulated in the leaves and stems of decapitated and partially defoliated plants. Lower levels of cytokinins were detected in the leaves of decapitated plants with only a partial root system. The level of cytokinins in the roots of decapitated plants was reduced by partial root excision. The growth and accumulation of cytokinins in leaves were, however, not totally suppressed by removing a large proportion of the roots. At the commencement of the experiment the stem had a higher cytokinin content than both the leaves and roots. This suggests that the stem could be an alternative source of cytokinins to the leaves. The cytokinin complement in the leaves of decapitated plants is not identical to that in the roots. It appears that cytokinins supplied by the roots are metabolized in the leaves, or that alternatively certain cytokinins are synthesized in the leaves themselves.     

Functional consequences of stenophylly for leaf productivity: comparison of the anatomy and physiology of a rheophyte, Farfugium japonicum var. luchuence, and a related non-rheophyte, F. japonicum (Asteraceae)

We investigated the anatomical and physiological characteristics of stenophyllous leaves of a rheophyte, Farfugium japonicum var. luchuence, and sun and shade leaves of a non-rheophyte, F. japonicum, comparing three different populations from coastal, forest floor, and riparian habitats. Light adaptation resulted in smaller leaves, and riparian adaptation resulted in narrower leaves (stenophylly). The light-saturated rate of photosynthesis (P _max) per unit leaf area corresponded to the light availability of the habitat. Irrespective of leaf size, the P _max per unit leaf mass was similar for sun and shade leaves. However, the P _max per mass of stenophyllous leaves was significantly lower than that of sun and shade leaves. This was because the number and size of mesophyll cells were greater than that required for intercellular CO₂ diffusion, which resulted in a larger leaf mass per unit leaf area. Higher cell density increases contact between mesophyll cells and enhances leaf toughness. Stenophyllous leaves of the rheophyte are frequently exposed to a strong water flow when the water level rises, suggesting a mechanical constraint caused by physical stress.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

Early stages of leaf development in <Emphasis Type="Italic">has</Emphasis> mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The elucidation of molecular mechanisms underlying the leaf development can be facilitated by the detailed anatomical study of leaf development mutants. We present an analysis of leaf anatomy and morphogenesis during early developmental stages in has mutant of Arabidopsis thaliana. The recessive has mutation affects a number of aspects in plant development, including the shape and size of both cotyledons and leaves. The earliest developmental observations suggest almost synchronous growth of the first two leaf primordia of has mutant. No significant disruption of the cell division pattern in the internal tissue is observed at the earliest stages of development, with the major anatomical difference compared to wild type primordia being the untimely maturation of mesophyll tissue cells in has mutant. At the stage of leaf blade formation, structure disruption becomes clearly evident, by irregular arrangement of the cell layers and the lack of polarity in juvenile has leaves. One distinguishing feature of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. Altered has mutant leaf morphology could be at least partially accounted for by the ectopic STM activity that was found at the base of leaf primordia during early stages of leaf development in has plants.     

The apparent loss of tissue culture competence during leaf differentiation in yams (Dioscorea bulbifera L.)

Explants taken from the leaves of yams (Dioscorea bulbifera L.) at different stages of development were cultured in vitro on a checkerboard using various combinations and/or concentrations of auxin (2,4-d) and cytokinin (6-BAP). An addition of cytokinin to the culture media was not essential for callus induction from explants derived from young leaves in the very early stages of expansion. When the leaves expanded further they required cytokinin and the requirement increased considerably during expansion. Explants taken from fully expanded leaves were no longer able to proliferate, even when extremely high concentrations of cytokinins were applied. Callus grown from highly immature leaves was able to continue proliferating in the absence of cytokinin when subcultured. Callus, that initially required cytokinin in the medium, proliferated in the absence of exogenous cytokinin when subcultured.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine - 1-NAA 1-naphthalenacetic acid     

Palisade mesophyll cell expansion during leaf development in Zinnia elegans (Asteraceae)

Temporal and spatial patterns of palisade mesophyll cell expansion in Zinnia elegans were characterized as a basis for developing a suspension culture model for mesophyll cell expansion. Our objectives were to 1) identify the leaf regions from which cells in various stages of expansion could be selectively isolated for culture, and 2) develop a basis for comparison of rate and extent of mesophyll cell expansion in culture with that in the leaf. Palisade mesophyll cells were isolated from expanding leaves by gentle physical maceration without the use of enzymes. Isolated cells from leaves in different stages of expansion were then measured by computer image analysis. Analysis of size frequency distributions showed that unexpanded cells can be isolated from the entire blade of small leaves or the basal regions of partially expanded leaves. Fully expanded cells can be obtained from the apical and middle regions of partially expanded leaves. Within the leaf, Zinnia mesophyll cells expanded from about 400 μm² to about 2.300 μm² at an estimated rate of 160 μm² d^-1. The percent increase in cell length exceeded the percent increase in cell width. Expansion of mesophyll cells continued for 6–8 d after epidermal expansion ceased. This difference in the timing of cell expansion in epidermal and mesophyll cells indicates that different regulatory factors may be operating in these adjacent tissues and underscores the importance of investigating the regulation of mesophyll cell expansion at the cellular level.     

The interplay between cytokinins and light during senescence in detached Arabidopsis leaves

Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild‐type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis‐zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N⁶‐(Δ²‐isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.     

Hormonal and cell division analyses in Watsonia lepida seedlings

The regeneration ability, cell division activity, auxin and cytokinin content of seedling regions and hypocotyl subsections of Watsonia lepida were studied. A total of 21 different cytokinins or conjugates were found in seedlings, with the highest cytokinin content in meristematic regions (root and shoot apical meristems). The greatest contribution to the cytokinin pool came from the biologically inactive cZRMP, suggesting that significant de novo synthesis was occurring. Five different auxins or conjugates were detected, being concentrated largely in the shoot apical meristem and leaves, IAA being the most abundant. Analysis of hypocotyl subsections (C1–C4) revealed that cell division was highest in subsection C2, although regeneration in vitro was significantly lower than in subsection C1. Anatomically, subsection C1 contains the apical meristem, and hence has meristematic cells that are developmentally plastic. In contrast, subsection C2 has cells that have recently exited the meristem and are differentiating. Despite high rates of cell division, cells in subsection C2 appear no longer able to respond to cues that promote proliferation in vitro. Auxin and cytokinin analyses of these subsections were conducted. Possibly, a lower overall cytokinin content, and in particular the free-base cytokinins, could account for this observed difference.     

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Changes in mesophyll anatomy and sink–source relationships during leaf development in Quercus glauca, an evergreen tree showing delayed leaf greening

Changes in mesophyll anatomy, gas exchange, and the amounts of nitrogen and cell wall constituents including cellulose, hemicellulose and lignin during leaf development were studied in an evergreen broad‐leaved tree, Quercus glauca, and in an annual herb, Phaseolus vulgaris. The number of chloroplasts per whole leaf in P. vulgaris increased and attained the maximal level around 10 d before full leaf area expansion (FLE), whereas it continued to increase even after FLE in Q. glauca. The increase in the number of palisade tissue cells per whole leaf continued until a few days before FLE in Q. glauca, but it had almost ceased by 10 d before FLE in P. vulgaris. The radius and height of palisade tissue cells in Q. glauca, attained their maximal levels at around FLE whereas the thickness of the mesophyll cell wall and concentrations of the cell wall constituents increased markedly after FLE. These results clearly indicated that, in Q. glauca, chloroplast development proceeded in parallel with the cell wall thickening well after completion of the mesophyll cell division and cell enlargement. The sink–source transition, defined to be the time when the increase in daily carbon exchange rate exceeds the daily increase in leaf carbon content, occurred before FLE in P. vulgaris but after FLE in Q. glauca. During leaf area expansion, the maximum daily increase in nitrogen content on a whole leaf basis (the maximum leaf areas were corrected to be identical for these species) in Q. glauca was similar to that in P. vulgaris. In Q. glauca, however, more than 70% of nitrogen in the mature leaf was invested during its sink phase, whereas in P. vulgaris it was 50%. These results suggest that Q. glauca invests nitrogen for cell division for a considerable period and for chloroplast development during the later stages. We conclude that the competition for nitrogen between cell division and chloroplast development in the area of expanding leaves can explain different greening patterns among plant species.     

Leaf anatomy and photosynthetic acclimation in <Emphasis Type="Italic">Valeriana jatamansi</Emphasis> L. grown under high and low irradiance

Relationship of leaf anatomy with photosynthetic acclimation of Valeriana jatamansi was studied under full irradiance [FI, 1 600 mol(PPFD) m^–2 s^–1] and net-shade [NS, 650 mol(PPFD) m^–2 s^–1]. FI plants had thicker leaves with higher respiration rate (R _D), nitrogen content per unit leaf area, chlorophyll a/b ratio, high leaf mass per leaf area unit (LMA), and surface area of mesophyll cell (S _mes) and chloroplasts (S _c) facing intercellular space than NS plants. The difference between leaf thickness of FI and NS leaves was about 28 % but difference in photon-saturated rate of photosynthesis per unit leaf area (P _Nmax) was 50 %. This indicates that P _Nmax can increase to a larger extent than the leaf thickness with increasing irradiance in V. jatamansi. Anatomical studies showed that the mesophyll cells of FI plants had no open spaces along the mesophyll cell walls (higher S _c), but in NS plants wide open spaces along the mesophyll cell wall (lower S _c) were found. Positive correlation between S _c and P _Nmax explained the higher P _Nmax in FI plants. Increase in mesophyll thickness increased the availability of space along the mesophyll cell wall for chloroplasts (increased S _c) and hence P _Nmax was higher in FI plants. Thus this Himalayan species can acclimate to full sunlight by altering leaf anatomy and therefore may be cultivated in open fields.     

Cytokinin-induced changes in the chlorophyll content and fluorescence of in vitro apple leaves

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846–2176 μg/1 g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1 g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (F_v/F_m test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (F_v/F_m) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.