Yuri V. Gamalei

Structure and function of leaf minor veins in trees and herbs

SummaryThe structure of leaf minor veins in 700 species from 140 families of dicotyledons, monocotyledons and conifers has been studied by light and electron microscopy. The presence of several structural types of minor veins has been shown. The main types are open and closed veins characteristic of trees and herbs, respectively. These vein types differ by the structure of intermediate cells, and by the mechanisms of phloem loading and sugar transport. Most woody plants have intermediate cells with numerous plasmodesmal fields, symplastic transport as the main phloem loading mechanism, as well as oligosaccharides and other complex sugars as the main phloem transport substances. By contrast, the majority of herbs have intermediate cells without plasmodesmal connections, and apoplastic loading of sucrose occurs only by membrane proton cotransport. The closed type is divided into three subtypes, differing in the degree of development of the structures used for sugar uptake from the apoplast. A list of the plants investigated with their vein types is given. The evolution of the minor vein structure and phloem loading mechanism are discussed in relation to the evolution of life forms of higher plants.

Phloem loading and its development related to plant evolution from trees to herbs

SummaryMinor vein structure in various taxonomic groups was described in a previous paper (Gamalei 1989). Here, these results are used to correlate minor vein structure with plant evolutionary, ecological and growth form schemes. The following pattern emerges: reductive evolution from evergreen trees to annual herbs is accompanied by gradually increasing symplastic isolation of the mesophyll and the phloem. This evolutionary tendency is confirmed by the ecological spreading and life-form distribution of modern plants with different types of minor vein structure. The meaning of this phenomenon is discussed. Chilling sensitivity of plasmodesmal translocation is considered to be the main reason. It is suggested that phloem loading for assimilate transport is double-routed. The symplastic route is more ancient and more economical for loading. The apoplastic pathway becomes the main or the only route under unfavorable conditions. The existence of a symplast/apoplast regulatory loading mechanism is suggested. The two loading routes differ in their selectivity for products of photosynthesis which changes their symplast/apoplast ratio which, in turn, determines the composition of the sieve tube exudate. The latter will influence growth and morphogenesis. Correlated changes of structure and function related to photosynthesis, loading, translocation and growth, are analysed with respect to life-form evolution. The influence of the pathway of loading on other processes is discussed.

Effects of temperature on the conformation of the endoplasmic reticulum and on starch accumulation in leaves with the symplasmic minor-vein configuration

The phloem-loading-related effects of temperature on leaf ultrastructure were studied in seven species having numerous plasmodesmatal connections between the mesophyll and phloem (symplasmic minor-vein configuration). The response to temperature (between 5 and 30 °C) was characterized by drastic changes in the endoplasmic-reticulum labyrinth (ER labyrinth) of intermediary cells, in the position of the vacuole in bundle-sheath cells, and in the starch content in the chloroplasts of bundle-sheath cells and mesophyll cells. At temperatures above 20 °C, the ER system in the intermediary cells reached its maximal volume, while the vacuole in bundlesheath cells was positioned centripetally (proximal to the intermediary cell). With decreasing temperature, the ER labyrinth in intermediary cells gradually contracted till the ER was fully collapsed at 10 °C and the vacuole in bundle-sheath cells moved to a more centrifugal position. The apparent elimination of photosynthate transport via the ER and plasmodesmata at temperatures lower than 10 °C led to starch accumulation in the chloroplasts of bundle-sheath cells and mesophyll cells. All of these changes were fully temperature-reversible and probably reflect changes in the balance between photosynthate transport and storage. The ultrastructural shifts appear to be correlated with the passage of photosynthate through the intermediary cells and, as a consequence, with the rate of phloem loading at various temperatures. A contraction of the ER/plasmodesmata system imposed by cytoskeletal reorganisation is discussed as the reason for the blockage of phloem loading at low temperatures in association with the general chilling sensitivity of these species.

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to 14CO2. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.

Different ratios of sucrose/raffinose-induced membrane depolarizations in the mesophyll of species with symplasmic (Catharanthus roseus, Ocimum basilicum) or apoplasmic (Impatiens walleriana, Vicia faba) minor-vein configurations

In mesophyll cells of species with a symplasmic (Ocimum basilicum, Catharanthus roseus, Magnolia denudata) or an apoplasmic (Vicia faba, Impatiens walleriana, Bellis perennis) minor-vein configuration, membrane depolarizations in response to 20 or 200 mol·m−3 raffinose and sucrose were measured. Ageing period and resting potential marginally affected the degree of depolarization. The symplasmic species showed similar depolarization responses to 20 and 200 mol·m−3 sucrose or raffinose. In the apoplasmic species, depolarization increased statistically significantly from 20 to 200 mol·m−3 sucrose, whereas the depolarization response to raffinose was equal at both concentrations. In the apoplasmic species, moreover, the depolarization response to raffinose was significantly weaker than to sucrose at all concentrations. A major difference between symplasmic and apoplasmic species seems to lie in the scantiness of raffinose carriers in the mesophyll plasma membrane of species with the apoplasmic mode of phloem loading.

Cytology of the minor-vein phloem in 320 species from the subclass Asteridae suggests a high diversity of phloem-loading modes.

The discovery of abundant plasmodesmata at the bundle sheath/phloem interface in Oleaceae (Gamalei, 1974) and Cucurbitaceae (Turgeon et al., 1975) raised the questions as to whether these plasmodesmata are functional in phloem loading and how widespread symplasmic loading would be. Analysis of over 800 dicot species allowed the definition of “open” and “closed” types of the minor vein phloem depending on the abundance of plasmodesmata between companion cells and bundle sheath (Gamalei, 1989, 1990). These types corresponded to potential symplasmic and apoplasmic phloem loaders, respectively; however, this definition covered a spectrum of diverse structures of phloem endings. Here, a review of detailed cytological analyses of minor veins in 320 species from the subclass Asteridae is presented, including data on companion cell types and their combinations which have not been reported previously. The percentage of Asteridae species with “open” minor vein cytology which also contain sieve-element-companion cell complexes with “closed” cytology, i.e., that show specialization for both symplasmic and apoplasmic phloem loading, was determined. Along with recent data confirming the dissimilar functional specialization of structurally different parts of minor vein phloem in the stachyose-translocating species Alonsoa meridionalis (Voitsekhovskaja et al., 2009), these findings suggest that apoplasmic loading is indispensable in a large group of species previously classified as putative symplasmic loaders. Altogether, this study provides formal classifications of companion cells and of minor veins, respectively, in 24 families of the Asteridae based on their structural features, opening the way to a close investigation of the relationship between structure and function in phloem loading.

Acquisition of carbon in Elodea canadensis Michx.

Summary Mature shoots of Elodea canadensis Michx., grown in artificial pond water, were exposed to 14 CO 2 orsubmersed in solution containing H 14 CO 3 − for 30 min under PAR. The macroautoradiographs indicated that bicarbonate was used for photosynthesis, while gaseous 14 CO 2 was not incorporated. Accordingly, gas exchange measurements showed no reduction of CO 2 -content in the cuvette. Microautoradiographs indicated that 14 C was taken up by penetrating the epidermis of the stem and that of the lower side of the leaves. In the symplast, label was accumulated in the chloroplasts of both epidermal layers and in the plastid envelope of the chloroplasts of cortex cells. In the stem no label occurred inside the central cylinder. Among sieve tubes, only those of the leaf traces were labeled. Thin layer chromatograms of extracts of labeled plants were autoradiographed. The main spots were identified by co-chromatography as malic acid, glucose, asparagine, proline and hexose phosphates, while sucrose appeared only slightly labeled. By SEM, strands of stretched cells with inflated ends were observed and named ≪canal cells≫. In the light microscope, canal cells were found among cells of the lower leaf epidermis. Upon illumination with PAR they increasingly were filled with gas, but in the dark no gas collected in the canal cells. They probably are used for collecting and transferring photosynthetically produced oxygen. Accordingly, acquisition of carbon and release of oxygen seem to follow different pathways in Elodea canadensis .

Chloroplast Movement as Response to Wounding in Elodea canadensis

Summary Chloroplast movement was studied in intact, detached or cut leaves of Elodea canadensis Michx. The influence of external factors (light, temperature, water supply) and of leaf age on activity and pattern of chloroplast movement was observed. Chloroplast movement does not occur in unsevered leaves; in leaves severed either by detachment or by a lateral incision, chloroplast movement occurs at room temperature in similar patterns. In midrib cells axial chloroplast movement starts within 10 min after setting the wound, almost independently from external conditions. In epidermis cells, lateral chloroplast movement is time-limited depending on the pretreatment of the plant (light, dark, cold). Lateral chloroplast movement is minimal in young leaves and maximal in mature leaves. In the morning hours chloroplast movement is restricted to midrib cells, but in the afternoon additional lateral movement occurs. Predarkening of plants reduced lateral chloroplast movement, but continuous illumination activated the movement. Cooling of plants to the temperature of melting ice (+5 °C) inhibited chloroplast movement in detached and incised leaves. However, after transfer to room temperature, axial chloroplast movement started within 20 min, and lateral chloroplast movement started within 40 min. The velocity of movement was highest after several warm-cold-warm changes following detachment or incision (maximum 36.8 µm s -1 ). The time course of chloroplast movement coincides with rapid changes of the membrane potential with low amplitudes (humming, 4 to 7 mV), which was recorded with microelectrodes impaled into the midrib of the attached leaf. Cutting a leaf of the third, last-formed generation initiated a sudden depolarization (from -200 mV to -140 mV) and a subsequent hyperpolarization to -240 mV, which slowly increased, but after 10 min suddenly changed to the humming period in the range of -200 mV ± 1.5 mV. It is concluded that chloroplast movement is initiated and enhanced by wound reactions transmitted from cell to cell via plasmodesmata.