Paraveinal Mesophyll Research Articles

Proteins homologous to leaf glycoproteins are abundant in stems of dark-grown soybean seedlings. Analysis of proteins and cDNAs

We report here the cloning and sequence analysis of cDNAs for a pair of closely related proteins from soybean (Glycine max [L.] Merr. cv. Williams 82) stems. Both proteins are abundant in soluble extracts of seedling stems but not of roots. One of these proteins (M r=28 kDa) is also foundd in the cell wall fraction of stems and actumulates there when seedlings are exposed to mild water deficit for 48 h. The mRNA for these proteins is most abundant in the stem region which contains dividing cells, less abundant in elongating and mature stem cells, and rare in roots. Using antiserum against the 28 kDa protein, we isolated cDNA clones encoding it and an antigenically related 31 kDa protein. The two cDNAs are 80% homologous in nucleotide and amino acid coding sequence. The predicted proteins have similar hydropathy profiles, and contain putative NH2-terminal signal sequences and a single putative N-linked glycosylation site. The two proteins differ significantly in calculated pI (28 kDa=8.6; 31 kDa=5.8), and the charge difference is demonstrated on two-dimensional gels. The proteins described here may function as somatic storage proteins during early seedling development, and are closely related to glycoproteins which accumulate in vacuoles of paraveinal mesophyll cells of fully expanded soybean leaves when plants are depodded.

Sink Removal and Leaf Senescence in Soybean

Three cultivars of soybean (Glycine max [L.] Merr. cvs Harper, McCall, and Maple Amber) were grown in the field and kept continuously deflowered throughout the normal seedfill period. For all cultivars, deflowering led to delayed leaf abscission and a slower rate of chlorophyll loss. Compared to control plants, photosynthesis and ribulose 1,5-bis-phosphate carboxylase/oxygenase (Rubisco) level declined slightly faster for deflowered Harper, but for both McCall and Maple Amber, leaves of deflowered plants maintained approximately 20% of maximum photosynthesis and Rubisco level 1 month after control plants had senesced. Deflowering led to decreased leaf N remobilization and increased starch accumulation for all cultivars, but cultivars differed in that for McCall and Maple Amber, N and starch concentrations slowly but steadily declined over time whereas for Harper, N and starch concentrations remained essentially constant over time. SDS-PAGE of leaf proteins indicated that for all cultivars, deflowering led to accumulation of four polypeptides (80, 54, 29, and 27 kilodaltons). Western analysis using antisera prepared against the 29 and 27 kilodalton polypeptides verified that these polypeptides were the glycoproteins previously reported to accumulate in vacuoles of paraveinal mesophyll cells of depodded soybean plants. The results indicated that depending on the cultivar, sink removal can lead to either slightly faster or markedly slower loss of photosynthesis and Rubisco. This difference, however, was not associated with the ability to synthesize leaf storage proteins. For any particular cultivar, declines in chlorophyll, photosynthesis, and Rubisco were initiated at approximately the same time for control and deflowered plants. Thus, even though cultivars differed in rate of decay of photosynthetic rate and Rubisco level in response to sink removal, the initiation of leaf senescence was not influenced by presence or absence of developing fruits.

Locating active proton extrusion pumps in leaves

Abstract Stabilized microscopic preparations of an apoplastic fluorescent tracer, sulphorhodamine G (SR), have previously shown it confined to leaf cell walls. SR has a pK of 3.2, is dissociated at normal wall pH, and therefore does not enter cells. In transpiring soybean leaves, the SR showed a major internal water pathway in the walls of the paraveinal mesophyll (PVM), which has been implicated in the temporary storage of protein. Also the SR penetrated the PVM and bundle sheath cells, staining organelles and vacuoles, but not other leaf cells. This implies that sufficient SR is undissociated in these walls to allow penetration, and that the pH of the PVM walls is lower than that of most other cells. It is proposed that proton extrusion pumps are revealed by the low wall pH, and that these pumps are probably involved in collecting ammo acids from the transpiration stream.

Allantoinase activity and ureide content of mesophyll and paraveinal mesophyll of soybean leaves

Paraveinal mesophyll (PVM) is a specialized soybean ( Glycine max Merr.) leaf tissue which represents a significant biochemical compartment. Stereological measurements showed that PVM makes up 23% of the mesophyll volume in nodulated soybean. To get an indication of the extent of involvement of PVM in ureide metabolism, physical characteristics, distribution of allantoinase activity and ureide content were determined in isolated PVM protoplasts (PVMP) and mesophyll protoplasts (MP). PVMP were larger and contained less chlorophyll and protein than MP. PVMP had twice as much allantoinase activity per protoplast but only half as much allantoinase activity when expressed on a volume basis as compared to MP. Total leaf ureide concentration was high and nearly equally distributed between MP and PVMP. PVMP had a higher ureide content per protoplast, a higher concentration of allantoic acid and a lower ratio of allantoin to allantoic acid. These results suggest that both tissues have the capacity to assimilate allantoin in vivo. The data are discussed with reference to the relative access of the two mesophyll tissues to incoming ureides.

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): MORPHOLOGY AND ANATOMY

Mature field‐ and growth‐chamber‐grown leaves of Populus deltoides Bartr. ex Marsh. were examined with light and scanning electron microscopes to determine their vasculature and the spatial relationships of the various orders of vascular bundles to the mesophyll. Three leaf traces, one median and two lateral, enter the petiole at the node. Progressing acropetally in the petiole these bundles are rearranged and gradually form as many as 13 tiers of vascular tissue in the petiole at the base of the lamina. (Most leaves contained seven vertically stacked tiers.) During their course through the midrib the tiers “unstack” and portions diverge outward and continue as secondary veins toward the margin on either side of the lamina. As the midvein approaches the leaf tip it is represented by a single vascular bundle which is a continuation of the original median bundle. Tertiary veins arise from the secondary veins or the midvein, and minor veins commonly arise from all orders of veins. All major veins–primaries, secondaries, intersecondaries, and tertiaries–are associated with rib tissue, while minor veins are completely surrounded by a parenchymatous bundle sheath. The bundle sheaths of tertiary, quaternary, and portions of quinternary veins are associated with bundle‐sheath extensions. Minor veins are closely associated spatially with both ad‐ and abaxial palisade parenchyma of the isolateral leaf and also with one or two layers of paraveinal mesophyll that extend horizontally between the veins. The leaves of growth‐chamber‐grown plants had thinner blades, a higher proportion of air space, and greater interveinal distances than those of field‐grown plants.

Isolation of mesophyll and paraveinal mesophyll protoplasts from soybean leaves

Mesophyll (MP) and paraveinal mesophyll protoplasts (PVMP) have been prepared with high yields from primary and trifoliate leaves of soybean, using a Cellulase CEL or CELF and Pectolyase Y-23 enzyme mixture. The PVMP, which are specialized for nitrogen metabolism and storage, are larger than MP and contain very few chloroplasts. This gives rise to considerable difference in the buoyant density of the two protoplast types, which has been exploited to obtain pure preparations of each. The MP and PVMP preparations were free of cellular debri and were stable for several days on ice. The purity of the preparations was further indicated by radial immunodiffusion assay using antibody to a glycoprotein specifically located in the paraveinal mesophyll (PVM) vacuole.

Paraveinal Mesophyll of Soybean Leaves in Relation to Assimilate Transfer and Compartmentation

Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var ;Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling.

The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation

The paraveinal mesophyll (PVM) is a unique and specialized, one-cell-thick tissue spanning the vascular bundles at the level of the phloem in soybean (Glycine max) (L.) Merr.) leaves. Its position within the leaf dictates that all photosynthate produced in the palisade and spongy mesophyll must pass through this specialized layer enroute to the phloem. Symplastic continuity, via plasmodesmata, exists between the PVM and bundle sheath, palisade parenchyma and spongy mesophyll. During leaf ontogeny the PVM is the first tissue to differentiate and at maturity these cells are six to eight times larger than other mesophyll cells, are highly vacuolate, and are interconnected by tubular arms. The PVM undergoes several unique structural and metabolic modifications during leaf development. The PVM cytoplasm, in vegetative plants, is dense, enriched in rough endoplasmic reticulum and dictyosomes, but contains few, small starch-free chloroplasts and few microbodies. Unlike the tonoplast of mesophyll cells, the tonoplast of the PVM is unusually thick and dense-staining. During leaf development the vacuoles of PVM cells accumulate a glycoprotein derived from the dictyosomes which reacts with the protein staining reagents, mercuric bromophenol blue and sulfaflavine, and is degraded by Pronase. Both the vacuolar material and tonoplast are also stained by phosphotungstic acid, which at low pH is relatively selective for glycoprotein. A unique role of the PVM in the transport and compartmentation of nitrogen reserves in soybeans is discussed.

The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation

Nitrogen and carbohydrate assimilates were temporally and spatially compartmented among various cell types in soybean (Glycine max L., Merr.) leaves during seed filling. The paraveinal mesophyll (PVM), a unique cell layer found in soybean, was demonstrated to function in the synthesis, compartmentation and remobilization of nitrogen reserves prior to and during the seed-filling stages. At anthesis, the PVM vacuoles contain substantial protein which completely disappears by two weeks into the seed filling. Distinct changes in the PVM cytoplasm, tonoplast and organelles were correlated with the presence or absence of the vacuolar material. Microautoradiography following the accumulation of several radiolabeled sugars and amino acids demonstrated the glycoprotein nature of the vacuolar material. Incorporation of methionine, leucine, glucose, and glucosamine resulted in heavy labelling of the PVM vacuole, in contrast to galactose, proline, and mannose which resulted in a much reduced labelling pattern. In addition, starch is unequally compartmented and degraded among the various leaf cells during seed filling. At the end of the photoperiod at the flowering stage, the highest starch accumulation was in the second palisade layer followed by the spongy mesophyll and the first (uppermost) palisade layer. Starch in the first palisade layer was completely degraded during the dark whereas the starch in the second palisade and spongy mesophyll was not remobilized to any appreciable extent. By mid-podfilling (approximately five weeks postanthesis) starch was absent in the first palisade layer at the end of the photoperiod while the second palisade and spongy mesophyll layers contained substantial starch. Starch was remobilized from these latter cells during the remainder of seed filling when current photosynthetic production is low. Structural changes associated with cell senescence first appear in the upper palisade layer and then progress (excluding the PVM) to the second palisade and spongy mesophyll layer. The PVM and phloem appear to retain their structural integrity into the leaf yellowing stage. Reducing sink capacity by pod removal resulted in a continued accumulation of vacuolar protein, an increase in cytoplasmic volume, and fragmentation of the vacuole in the PVM. Pod removal also resulted in an increased amount of accumulated starch (which did not turn over) in all mesophyll layers, and an increase in cell size and cell-wall thickness.

Specialized cellular arrangements in legume leaves in relation to assimilate transport and compartmentation: comparison of the paraveinal mesophyll

Leaves of eight species of Leguminosae-Papilionoideae were examined for the presence of a highly specialized cell layer called the paraveinal mesophyli (PVM). Three species, Glycine max (L.) Merr, Psophocarpus tetragonolobus D.C. and Vigna radiata L., contained PVM; five (Medicago sativa L., Phaseolus vulgaris L., Pisum sativum L., Vicia faba L., Vigna unguiculata L.) did not. The PVM of G. max and P. tetragonolobus was anatomically identical and consisted of large, interconnected, multiarmed cells forming a network, one cell thick, spanning the region between vascular bundles and abutting the bundle sheath at the level of the phloem. The PVM of V. radiata differed in that elaborate extensions of individual bundle-sheath cells comprised the entire intervascular network. The PVM cells of all three species were large, contained a dense, thin peripheral layer of cytoplasm, and a large central vacuole. The cytoplasm contained few small chloroplasts and few microbodies, but was enriched in rough endoplasmic reticulum. Plasmodesmata were common in crosswalls between adjacent PVM cells and between PVM cells and other cell types abutting them. Vacuolar material was present in all three species, but was variable in appearance. That of G. max was present in large amounts, semifibrillar and finely dispersed. That of P. tetragonolobus was also present in large amounts but primarily as large aggregates, although some fibrillar material was also present. Vigna radiata had small amounts of vacuolar material evenly distributed between small aggregates and dispersed fibrils. Removal of flowers or young pods resulted in further increase of the vacuolar material in G. max PVM and increase of the fibrillar material in P. tetragonolobus, but had no appreciable affect on the vacuolar material in V. radiata. Histochemical staining indicated the vacuolar material in G. max and P. tetragonolobus was proteinaceous.

Differential Starch Accumulation in the Leaf Mesophyll Layers of Soybean1

This study was undertaken to examine a common assumption that starch is uniformly distributed in soybean [Glycine max( L.) Merr.] leaves. Observations from leaf samples collected during 4 years, including at least 12 cultivars and a shading experiment, showed this assumption to be false. In fact, a consistent hierarchy was found in the distribution of starch. Starch was first deposited in the abaxial palisade layer, followed by the spongy mesophyll, and then each of the remaining palisade layers in sequence. Unloading occurred in the reverse order in a shading experiment. Seldom was starch observed in the paraveinal mesophyll layer.

Leaflet Anatomy of Phaseoleae (Leguminosae:Papilionoideae) and Its Relation to Taxonomy

Leaflet anatomy of the tribe Phaseoleae was studied by light microscopy of cleared material and by scanning electron microscopy of leaflet surfaces. Anatomical results compared favorably with the recently revised subtribal classification of the tribe. The following taxonomically useful characters were found: (1) Bulbous-based and vesicular glands are restricted to the subtribe Cajaninae; (2) hooked hairs are present in three generic groups: (a) Phaseolus, Alepidocalyx, and Minkelersia, (b) Clitoria, Periandra, Clitoriopsis, and Centrosema, and (c) one species each of Canavalia and Camptosema; (3) glandular hairs are particularly well developed in the Diocleinae, Phaseolinae, and Kennediinae; (4) branched (two- to several-armed) hairs are restricted to Erythrina; (5) paraveinal mesophyll is widespread in the tribe; and (6) peculiar, paired epidermal crystals occur in Canavalia.

Leaf Morphology and Anatomy in Relation to CO2‐exchange Rate of Soybean Leaves1

To learn more about factors limiting leaf photosynthesis of soybeans [Glyclne max (L.) Merr.], we examined the relationships among leaf morphological and anatomical characteristics and CO2‐exchange rate (CER). Infrared gas analysis of the most recent, fully expanded, attached leaf of outdoor, pot‐cultured plants was measured in the laboratory under saturating irradiance, after which stomatal impressions were taken and leaf sections were prepared for anatomical measurementsSpecific leaf weight (SLW), which seemed a function of thickness and not deusity, and leaf thickness (LT), itself, were correlated with CER. The thickness differeuces were most strongly expressed in the upper palisade and paraveinal mesophyll layers. Vapor phase diffusion resistance to CO2 accounted for 22 and 13% of the variation in CER in each of 2 years, and only in the former year, when aperature on the adaxial surface was strongly correlated with resistance and CER, were stomatal characteristics related to CER. Residual (mesophyll, carboxylation) resistance accounted for 15 and 53% of the variation in CER the 2 years. Both CER and residual resistance were related to the thickness and cellular volume of the several leaf tissue layers, but they were poorly related to cell diameters, exposed cell surface area, and cell surface to volmne ratio. We conclude that characteristics internal to the cell, as opposed to resistances related to stomata, intercellular space, or cell surfaces, were regulating CER in these soybean leaves.

Observations on the Development of the Paraveinal Mesophyll of Soybean Leaves

The development of the paraveinal mesophyll of soybeans is described. Its origin can be traced to the third layer from the adaxial protoderm layer, and its development is characterized by increased vacuolation and the formation of conspicuous intercellular air spaces within the layer. Certain morphological features of the development and mature structure of this layer and previous studies on the kinetics of photosynthate movement in soybean leaves are discussed in relation to the possible function(s) of this type of mesophyll.

Kinetics of C-14 Translocation in Soybean

A kinetic study was made of the translocation of (14)C-photosynthate through soybean stems following pulse labeling and during steady state labeling of the first trifoliolate leaf. The translocation profile proceeded down the stem with little or no change in shape. Following pulse labeling, sucrose accounted for 90 to 95% of the radioactivity in the stem at all times up to 2 hours, at which time less than 3% of the activity was in an insoluble form. Kinetic data on the relative specific activities of sucrose in the leaf and petiole indicated that two-thirds of the petiolar sucrose was in the translocation stream and the remaining one-third was in a stationary pool which slowly accumulated sucrose from the translocation stream. With this assumption, the rate of sucrose efflux from the leaf was calculated to be 22 micrograms per minute, which was equivalent to a sucrose mass flux in the sieve tubes of 20 grams per square centimeter per hour.It is concluded that translocation proceeded without a net radial velocity distribution and with slight loss of translocate, mainly as sucrose, from the translocation stream. The kinetics of (14)C translocation must, therefore, be determined primarily by factors operating within the leaf rather than in the stem. One of these factors may be the presence in the soybean leaf of a specialized paraveinal mesophyll. Since there was no spreading of the profile, the results were inconsistent with an "active diffusion" mechanism of translocation (i.e., a mechanism describable by diffusion equations). The data and conclusions are in substantial agreement with those of Moorby, Ebert, and Evans.

Kinetics of C-14 Translocation in Soybean

The kinetics of (14)C-assimilates in the soybean leaf were studied in pulse labeling and steady state labeling experiments. (14)C-Sucrose apparently served as the ultimate source, at least, of translocated (14)C-sucrose. However, since the specific activity of leaf sucrose reached a maximum within 5 minutes after pulse labeling, whereas that of exported sucrose did not reach a maximum until at least 20 minutes, it appeared that there were two sucrose compartments in the leaf. A possible physical basis for the two compartments may be the mesophyll (a photosynthetic compartment) and a specialized "paraveinal mesophyll" (a nonphotosynthetic compartment), through which photosynthate must pass on its way to the veins.The (14)C kinetics of sterol glucoside, and probably esterified sterol glucoside, were similar to those for (14)C-sucrose export. Sterol glucoside was labeled only in its glucose moiety and was the only stem lipid which became strongly labeled during (14)C-sucrose translocation. These sterol derivatives may act as membrane carriers of sucrose between the translocation stream and surrounding cells.The kinetics of (14)C-sucrose and its movement to the veins are discussed with reference to compartmentation within the leaf and metabolic exchange with other compounds, particularly with starch. Although a simple compartmental model gave a fairly accurate description of (14)C-sucrose kinetics, an entirely accurate model could not be provided, primarily because of loss of (14)C from sucrose, at an unknown rate, to starch.