Paraveinal Mesophyll Research Articles

Anatomical determinants of gas exchange and hydraulics vary with leaf shape in soybean.

Leaf shape in crops can impact light distribution and carbon capture at the whole plant and canopy level. Given similar leaf inclination, narrow leaves can allow a greater fraction of incident light to pass through to lower canopy leaves by reducing leaf area index, which can potentially increase canopy-scale photosynthesis. Soybean has natural variation in leaf shape which can be utilized to optimize canopy architecture. However, the anatomical and physiological differences underlying variation in leaf shape remain largely unexplored. In this study, we selected 28 diverse soybean lines with leaf length to width ratios (leaf ratio) ranging between 1.1 and 3.2. We made leaf cross-sectional, gas exchange, vein density and hydraulic measurements and studied their interrelationships among these lines. Our study shows that narrow leaves tend to be thicker, with an ~30 µm increase in leaf thickness for every unit increase in leaf ratio. Interestingly, thicker leaves had a greater proportion of spongy mesophyll while the proportions of palisade and paraveinal mesophyll decreased. In addition, narrow and thicker leaves had greater photosynthesis and stomatal conductance per unit area along with greater leaf hydraulic conductance. Our results suggest that selecting for narrow leaves can improve photosynthetic performance and potentially provide a yield advantage in soybean.

Dew absorption by the leaf trichomes of Combretum leprosum in the Brazilian semiarid region

Dew formation is common in several regions of the world, but in arid and semiarid regions dew may represent an important water source for plants. In this study, we tested whether the leaves of a common shrub in the semiarid region of Brazil, Combretum leprosum Mart. (Combretaceae) absorb dew. We also identified the leaf anatomical structures that are involved in this process and tested whether dew absorption favours hydration of leaf tissues in both field and greenhouse experiments. The translucent and shiny leaf trichomes of C. leprosum absorbed dew. Leaf water absorption through hydrophilic trichomes and the presence of hydrophilic polysaccharides in C. leprosum increased its leaf water status. The presence of paraveinal mesophyll and the role of this tissue in the redistribution of water in the leaf are shown for the first time in Combretaceae.

Cell Type-Specific Transcriptome Analysis of the Soybean Leaf Paraveinal Mesophyll Layer

The paraveinal mesophyll (PVM) layer of soybean leaves, which contains cells with various unique ultrastructural properties, has been studied for decades, and several hypotheses regarding its functional role have been developed. Here, we describe a method for obtaining PVM cells using laser capture microdissection and pressure catapulting, subsequent isolation of RNA from these cells, and downstream microarray analysis. A cell type-specific transcriptome analysis was used to compare the gene expression patterns in PVM cells with those of a mesophyll cell type (palisade parenchyma) as a reference. Transcripts related to vegetative storage protein (Vsp) and certain vegetative lipoxygenase (Vlx) isoforms were significantly enriched in PVM cells, which is in accordance with prior work that demonstrated an accumulation of the corresponding proteins. Potential roles of Vsp and Vlx in phosphate mobilization and defense responses, respectively, are discussed. In addition, we found an enrichment of several transport-related genes in PVM cells. Building on our transcriptome data, we provide a fresh discussion of the hypothesis that the PVM plays a role in photoassimilate mobilization and translocation.

Soybean vegetative lipoxygenases are not vacuolar storage proteins.

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A-E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

Roles of the bundle sheath cells in leaves of C3 plants

This review considers aspects of the structure and functions of the parenchymatous bundle sheath that surrounds the veins in the leaves of many C(3) plants. It includes a discussion of bundle sheath structure and its related structures (bundle sheath extensions and the paraveinal mesophyll), its relationship to the mestome sheath in some grasses, and its chloroplast content. Its metabolic roles in photosynthesis, carbohydrate synthesis and storage, the import and export of nitrogen and sulphur, and the metabolism of reactive oxygen species are discussed and are compared with the role of the bundle sheath in leaves of C(4) plants. Its role as an interface between the vasculature and the mesophyll is considered in relation to the movement of water and assimilates during leaf development, export of photosynthates, and senescence.

Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.

The functional status of paraveinal mesophyll vacuoles changes in response to altered metabolic conditions in soybean leaves.

Antibodies raised against tonoplast intrinsic proteins (TIPs) were used to probe the functional status of the soybean [Glycine max (L.) Merr.] paraveinal mesophyll (PVM) vacuole during changes in nitrogen metabolism within the leaf. Young plants grown under standard conditions had PVM vacuoles characterised by the presence of γ-TIP, which is indicative of a lytic function. When plants were then subjected to shoot tip removal for a period of 15 d, forcing a sink-limited physiological condition, the γ-TIP marker diminished while the δ-TIP marker became present in the PVM vacuole, indicating the conversion of the PVM vacuole to a storage function. When the shoot tips were allowed to regrow, the γ-TIP marker again became dominant demonstrating the reversion of these PVM vacuoles back to a lytic compartment. The changes in TIP markers correlated with the accumulation of vegetative storage proteins and vegetative lipoxygenases, proteins implicated in nitrogen storage and assimilate partitioning. This research suggests that the PVM vacuole is able to undergo dynamic conversion between lytic and storage functions and further implicates this cell layer in assimilate storage and mobilisation in soybeans.

Idioblasts and other unusual internal foliar secretory structures in Scrophulariaceae

Leaf samples of mostly herbarium specimens (237 species of 172 genera) were cleared. Internal secretory structures of large size or unusual shape were detected and observed. Selected samples were processed into resin and sectioned for light microscopy or prepared for scanning electron microscopy. Adding results from two earlier publications, our survey includes 365 species from 174 genera. Five types of internal secretory structure, mostly unknown previously in the traditional Scrophulariaceae, occur in only nine genera: 1) single-celled subepidermal idioblasts, empty at maturity, in Scrophularia and Verbascum (Lersten and Curtis 1997), 2) 2–16 (or more)-celled nodules, with fibrous contents, in mid-mesophyll strata of Graderia and Radamaea, 3) epithelium-lined oil cavities in Leucophyllum (Lersten and Beaman 1998) and Capraria, 4) Kranz-type enlarged bundle sheath in Anticharis, and 5) paraveinal mesophyll (PVM) in Picria and Bonnaya.

Activity of Soybean Lipoxygenase Isoforms against Esterified Fatty Acids Indicates Functional Specificity

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coli. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and α-linolenic acid, and the reaction products with the substrates linoleic acid, α-linolenic acid, arachidonic acid , γ-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.

Specific lipoxygenase isoforms accumulate in distinct regions of soybean pod walls and mark a unique cell layer.

Developing seeds constitute a strong sink for the plant and rely on the turnover and mobilization of carbon and nitrogen assimilates to supply the nutrients needed for their maturation. In large part these nutrients emanate from the vegetative organs including leaves and pod walls. Vegetative lipoxygenases (VLXs) accumulate in the paraveinal mesophyll cell layer of soybean (Glycine max L.) leaves where individual isoforms are proposed to play a role(s) as active enzymes or as transient storage proteins. VLXs also are prominent proteins in soybean pod walls, representing approximately 12% of the total soluble protein. Examining the temporal, tissue, and subcellular patterns of individual VLX isoform accumulation and of lipoxygenase activity through pod wall development indicates that VLXD is the principal VLX isoform playing a role in storage in this organ. The major accumulation of VLXD occurs just prior to seed fill within the endocarp middle zone, and protein extracted from this region shows relatively low levels of lipoxygenase activity, suggesting the middle zone may act as a storage tissue. Three other VLX isoforms, VLXA, VLXB, and VLXC colocalize to the cytoplasm of a single discrete cell layer in the mesocarp. Thus, the patterns of VLX cellular and subcellular localization in pod walls suggest independent functions for these different isoforms while also serving as specific markers for a novel cell layer in the pod wall.

The paraveinal mesophyll: a specialized path for intermediary transfer of assimilates in legume leaves

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 The distance between sites of synthesis of assimilates and the site of phloem loading can be large, and specialized leaf cell layers such as the paraveinal mesophyll (PVM) might act to enhance the efficiency of transport. A number of techniques were used to analyse PVM of legume leaves with respect to a hypothesized function in transfer of assimilates between tissues. Of 39 legume species examined, PVM was found in 22. Leaves of all PVM-containing species had multiple palisade parenchyma layers, while non-PVM species generally had only one distinct palisade layer. Morphometric analysis identified a significant correlation between PVM presence and greater numbers of palisade cells per unit leaf surface area. Comparison of photosynthetic rates of four PVM and four non-PVM species showed the PVM species had higher rates on a leaf area basis than all but one of the non-PVM species. Microautoradiography of 14CO2 pulse–chase studies in soybean demonstrated PVM is an intermediary tissue in transfer of assimilates to vascular bundles. In addition, PVM cells but not mesophyll cells, were enriched in a sucrose binding protein previously found to be associated with sucrose-transporting tissues. The structural, positional and transport data support the hypothesis that the PVM acts as a transport pathway between the vascular system and photoassimilatory cells of the leaf, and has probably evolved to overcome diffusion limitations imposed by multiple palisade layers.

Protein dynamics, activity and cellular localization of soybean lipoxygenases indicate distinct functional roles for individual isoforms.

Vegetative lipoxygenases (VLXs) in soybean are hypothesized to function in nitrogen storage and partitioning. Isoform-specific antibodies for four of the five known VLX isoenzymes were used to investigate the influence of source-sink status on protein levels, as well as to analyze the tissue and subcellular localization of the different isoforms. VLXD responded most strongly to sink limitation, although the levels of VLXA, B and C increased as well. After sink limitation, VLXD and the vegetative storage protein, VSPalpha, accumulated in the vacuoles of bundle sheath and paraveinal mesophyll cells, while VLXA, B and C localized to the cytosol of these cells. All five known VLX isoenzymes were active with both linoleic and linolenic acid substrates after expression in Escherichia coli. The strong upregulation of VLXD levels after sink limitation as well as the localization of this isoform to the vacuoles of paraveinal mesophyll and bundle sheath cells (where VSPs are found) strongly suggest that VLXD should be considered as a major storage protein in soybean leaves. Furthermore, since VLXA, B and C also accumulate in sink-limited soybean leaves, are located in the cytosol of paraveinal mesophyll cells and are active at pH values typically found in this compartment, their activities may well contribute to lipid metabolism in this tissue. This multi-gene family is thus ideally poised to play a pivotal role in the balance of N deposition relative to lipid-based storage, defense or signaling, by modulating contributions to these processes in the transient storage cells of the paraveinal mesophyll.

Aspects of leaf anatomy of kudzu (Pueraria lobata, Leguminosae-Faboideae) related to water and energy balance

Kudzu is a cover crop that has escaped cultivation in some subtropical and warm temperate regions. Kudzu has previously demonstrated broad intraspecific physiological plasticity while colonizing new environments. The objective of this paper was to investigate characteristics of kudzu leaflet anatomy that might contribute to its successful growth in climatically distinct environments, and to escape cultivation as well. Fresh and fixed leaflet strips of field-grown plants were analyzed. The lower epidermis of kudzu showed a higher frequency of stomata (147 ± 19 stomata mm-2) than the upper epidermis (26 ± 17 stomata mm-2). The average number of trichomes per square milimeter was 8 for both the upper and the lower epidermis. The average trichome length was 410 ± 200 mum for the upper epidermis and 460 ± 190 mum for the lower epidermis. Cuticle thickness was not considerably different between lower and upper epidermis. The leaflet blade consisted basically of two layers (upper and lower) of unicellular epidermis, two layers of palisade parenchyma and one layer of spongy parenchyma. One layer of paraveinal mesophyll was found between palisade and spongy parenchyma. In conclusion, leaflets of kudzu present anatomical characteristics that might contribute to the broad physiological plasticity shown by kudzu.

Specific soybean lipoxygenases localize to discrete subcellular compartments and their mRNAs are differentially regulated by source-sink status

Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA-vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

Anatomy and distribution of foliar idioblasts in Scrophularia and Verbascum (Scrophulariaceae)

Internal secretory structures have rarely been reported from Scrophulariaceae, and foliar idioblasts only once before, in 1887. Presented here are the first unambiguous descriptions of subepidermal foliar idioblasts in the family, from Scrophularia and Verbascum, genera regarded as closely allied on other grounds. Leaf samples from 183 mostly herbarium specimens (128 species, with 55 replicates) were cleared and stained, which revealed idioblasts in 62 (69.7%) of 89 Scrophularia species and 13 (33.3%) of 39 Verbascum species. We then chose 14 representative species to examine by resin sectioning and scanning electron microscopy. Idioblasts occurred both adaxially and abaxially. Most were conspicuous, in some species penetrating to vasculature level. Idioblasts had a thin primary wall and were empty at maturity. Verbascum and Scrophularia species with and without idioblasts were scattered among the subgeneric taxa without taxonomic clustering; likewise, both types occurred in approximately proportionate numbers throughout the geographic range, except that 14 of 15 North American Scrophularia species had idioblasts. Petals of two species had abundant idioblasts. The 1887 report illustrated a huge idioblast in S. deserti and we also found the largest in either genus in this species. We also noted trichome and stomata types and report that paraveinal mesophyll and foliar endodermis with casparian strip were both absent.

Mechanism of transport of vegetative storage proteins to the vacuole of the paraveinal mesophyll of soybean leaf

Microscopy techniques were used to identify the pathway of transport of soybean leaf vegetative storage proteins (VSPα/β and VSP94) to the vacuoles of a specialized cell type, the paraveinal mesophyll (PVM), where they accumulate. PVM cells are enriched in endoplasmic reticulum and Golgi bodies relative to surrounding mesophyll cells. The margins of medial and trans Golgi cisternae had attached or closely associated noncoated vesicles with densely staining membranes and lumenal contents of the same appearance as material that accumulated in the vacuole. These vesicles appeared to be transported preferentially to the tonoplast, where fusion with the membrane released the granular contents into the vacuole. Cytochemical staining with phosphotungstic acid and silver methenamine supported this interpretation as both the Golgi vesicles and the tonoplast stained intensely with these reagents, unlike the tonoplast of mesophyll cells which do not accumulate VSP. Immunocytochemical localization for VSPα/β labeled the Golgi bodies and associated vesicles, and vacuolar material in PVM cells, but not in mesophyll. Similar labeling was seen in PVM of another legume species previously found to accumulate antigenically similar VSPs. Immunolocalization for VSP94, a lipoxygenase, labeled the PVM cytosol and material in the PVM vacuole, but not the Golgi or vesicles. The results of this study demonstrate that the Golgi pathway is utilized for transport of VSPα/β in the PVM, which follows the mechanism of deposition demonstrated for certain seed storage proteins. VSP94 appeared to follow a separate path for accumulation in PVM vacuoles.

Leaf anatomy ofDombeya andNesogordonia (Sterculiaceae), emphasizing epidermal and internal idioblasts

We studied leaf anatomy, using clearings, resin sections, and scanning electron microscopy, from mostly herbarium specimens of 123 species ofDombeya and 11 species ofNesogordonia (Sterculiaceae). Species were placed in seven idioblast categories, ranging from those without any to single and bicelled epidermal forms to multicelled nodules and single mesophyll idioblasts. Idioblast contents are possibly mucilaginous, but were not identified. In these two genera the range of foliar idioblast morphology surpasses that known previously for the entire family. Leaves are dorsiventral with mostly abaxial anomocytic stomata and typical palisade and spongy layers; paraveinal mesophyll is lacking. Miniature glandular (clavates, capitates) and nonglandular (mostly stellate) trichomes occur. Prismatic crystals predominate; epidermal prismatics and mesophyll druses are rare.

Tomato (Lycopersicon esculentum cv. pik-red) leaf carboxypeptidase: identification, N-terminal sequence, stress-regulation, and specific localization in the paraveinal mesophyll vacuoles.

Wounding of tomato (Lycopersicon esculentum L.) leaves causes systemic induction of a serine-type carboxypeptidase activity. We find this activity to be present in several isoforms. Antibodies raised against the leaf carboxypeptidase inhibited the enzyme activity and the immunoprecipitates were resolved into a 69-kDa polypeptide and a doublet of 35/37-kDa proteins on SDS-PAGE. Immunoblot analysis of the leaf proteins also immunodecorated the 69-kDa and 35/37-kDa proteins. Amino acid sequence analysis of the amino-terminus of the tomato leaf 69-kDa carboxypeptidase I [Sorenson et al. (1986) Carlsberg Res. Commun. 51: 475], sharing Ala as the N-terminus and the sequences, AlaProGln and LeuProGlyPhe. Superimposition of a chemical stress (copper treatment) on wounding apparently lowered wound-induced carboxypeptidase activity in the leaf, suggesting that cupric ions might interact with the wound signal. Immunogold electron microscopy indicated that the leaf carboxypeptidase was specifically localized within the inclusions of vacuoles of vascular parenchyma cells. In cupric ion-treated tissues, carboxypeptidase was found redistributed to other parts of the cell, indicating that this treatment, but not wounding, causes general vacuolar membrane damage.

Immunogold Localization and Quantitative Distribution of Two Proteins of the Glycine Decarboxylase Complex in Developing Leaflets of Pea (Pisum sativum L.)

We investigated the distribution of the P- and H-proteins of the glycine decarboxylase complex (EC 2.1.2.10) in developing pea (Pisum sativum L.) leaflets using immunogold techniques. Antibodies to these two proteins did not bind to young, nonphotosynthetic cells. Once the leaflet turned green, the labeling density in the mitochondria of the mesophyll cells increased substantially. Few particles were found in the mitochondria of the paraveinal mesophyll and guard cells. None were found either in the epidermis or in the parenchyma of the vascular tissue. Thus leaflets not yet exposed to light, and nonphotosynthetic cells, do not express these two proteins needed for photorespiration. The process of photorespiration seems to be correlated with the photosynthetic state of the cell and of the organ. It was also found to be positively linked with the integrity of the chloroplast since the highest label density obtained in the mitochondria was found just before the onset of ultrastructural senescence in the chl...

Characterization of a New Lectin of Soybean Vegetative Tissues

Lectins are carbohydrate-binding proteins that occur widely among plants. Lectins of plant vegetative tissues are less well characterized than those of seeds. Previously, a protein of soybean (Glycine max [L.] Merr.) leaves was shown to possess properties similar to the seed lectin. Here we show that the N-terminal amino acid sequence of this protein shares 63% identity with the seed lectin. Immunoblot analysis indicated that the protein occurs in leaves, petioles, stems, and cotyledons of seedlings but not in seeds. These observations prompted designation of the protein as a soybean vegetative lectin (SVL). Immunohistochemical localization in leaves indicated that SVL was localized to the vacuoles of bundle-sheath and paraveinal mesophyll cells. Removal of sink tissues or exposure to atmospheric methyl jasmonate caused increased levels of SVL in leaves and cotyledons. Co-precipitation of SVL and the soybean vegetative storage protein (VSP) during purification suggested an interaction between these proteins. SVL-horseradish peroxidase conjugate bound to dot blots of VSP or SVL, and binding was inhibited by porcine stomach mucin and heparin but not simple carbohydrates. Binding between SVL and VSP and similarities in localization and regulation support a possible in vivo interaction between these proteins.