Preeti Dahiya

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

SummaryTransfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.

A fasciclin-domain containing gene, ZeFLA11, is expressed exclusively in xylem elements that have reticulate wall thickenings in the stem vascular system of Zinnia elegans cv Envy

The vascular cylinder of the mature stem of Zinnia elegans cv Envy contains two anatomically distinct sets of vascular bundles, stem bundles and leaf-trace bundles. We isolated a full-length cDNA of ZeFLA11, a fasciclin-domain-containing gene, from a zinnia cDNA library derived from in vitro cultures of mesophyll cells induced to form tracheary elements. Using RNA in situ hybridization, we show that ZeFLA11 is expressed in the differentiating xylem vessels with reticulate type wall thickenings and adjacent parenchyma cells of zinnia stem bundles, but not in the leaf-trace bundles that deposit spiral thickenings. Our results suggest a function for this cell-surface GPI-anchored glycoprotein in secondary wall deposition during differentiation of metaxylem tissue with reticulate vessels.

Transcription of a Gene Encoding a Lectinlike Glycoprotein Is Induced in Root Cells Harboring Arbuscular Mycorrhizal Fungi in Pisum sativum

The gene PsNlec1, which encodes a lectinlike glycoprotein, is strongly expressed in pea nodule tissue. Using gene-specific polymerase chain reaction (PCR) primers, in situ hybridization probes, and specific antisera derived from the PsNlec1 sequence, we investigated gene expression associated with the arbuscular mycorrhizal (AM) symbiosis of pea roots. With the use of reverse transcription (RT)-PCR and cold in situ hybridization, strong expression of the transcript was demonstrated not only in root nodules but also in mycorrhizal roots when the cells were colonized by the endomycorrhizal fungus Glomus versiforme. No transcript was detectable in uninfected pea roots. With an antiserum raised against PsNLEC-1 polypeptide, a single antigenic band (25 kDa) was observed following gel electrophoresis of extracts from mycorrhizal roots. However, the amount of antigen was apparently too low to be detected by immunogold localization in tissue sections of pea mycorrhizal roots.

Immunolocalization of PsNLEC-1, a Lectin-Like Glycoprotein Expressed in Developing Pea Nodules

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.

Symbiotic Gene Sym31 Controls the Presence of a Lectinlike Glycoprotein in the Symbiosome Compartment of Nitrogen-Fixing Pea Nodules

The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as inclusion bodies in the...

A RING Domain Gene Is Expressed in Different Cell Types of Leaf Trace, Stem, and Juvenile Bundles in the Stem Vascular System of Zinnia

The in vitro zinnia (Zinnia elegans) mesophyll cell system, in which leaf mesophyll cells are induced to transdifferentiate into tracheary elements with high synchrony, has become an established model for studying xylogenesis. The architecture of the stem vascular system of zinnia cv Envy contains three anatomically distinct vascular bundles at different stages of development. Juvenile vascular strands of the subapical region develop into mature vascular strands with leaf trace segments and stem segments. Characteristic patterns of gene expression in juvenile, leaf trace, and stem bundles are revealed by a molecular marker, a RING domain-encoding gene, ZeRH2.1, originally isolated from a zinnia cDNA library derived from differentiating in vitro cultures. Using RNA in situ hybridization, we show that ZeRH2.1 is expressed preferentially in two specific cell types in mature zinnia stems. In leaf trace bundles, ZeRH2.1 transcript is abundant in xylem parenchyma cells, while in stem bundles it is abundant in phloem companion cells. Both of these cell types show wall ingrowths characteristic of transfer cells. In addition, ZeRH2.1 transcript is abundant in some phloem cells of juvenile bundles and in leaf palisade parenchyma. The complex and developmentally regulated expression pattern of ZeRH2.1 reveals heterogeneity in the vascular anatomy of the zinnia stem. We discuss a potential function for this gene in intercellular transport processes.

Tissue distribution and subcellular localization of carbonic anhydrase in mature soybean root nodules indicates a role in CO2 diffusion

Abstract Tissue distribution of carbonic anhydrase (CA; EC 4.2.1.1) and phospho enol pyruvate carboxylase (PEPC; EC 4.1.1.31) was examined in developing soybean ( Glycine max ) nodules using an immunohistological approach. The data obtained indicate that in young nodules both CA and PEPC proteins are present in the parenchymatous cells, and at much lower levels, in the central nodular region. In mature nodules, high levels of CA were exclusively present in 2-3 cell layers of the inner cortical region, whereas high levels of PEPC were present both in infected and uninfected cells. Immunogold localization indicated that, in mature nodules, CA was localized in the cytoplasm of the inner cortical cells and the cell walls of the endodermal cells. These results considered together suggest that in mature nodules, CA may facilitate the diffusion of the excessive CO 2 , derived from the respired bacteroids, in the rhizosphere. The distribution of CA was examined in mature nodules of soybean, grown hydroponically, in either limiting or non-limiting phosphate concentrations. The data indicated that in plants growing on non-limiting phosphate concentrations, an additional strong signal was found in cortical cells surrounding the nodule vascular bundles.

Localized expression of cathepsin B-like sequences from root nodules of pea (Pisum sativum).

Cathepsin B is an ancient family of eukaryotic cysteine proteases. We describe PsCat1, a plant cathepsin B-like transcript, identified as an expressed sequence in Rhizobium-induced, nitrogen-fixing root nodules of pea. In situ hybridization studies in root nodules showed strong, extremely localized expression of PsCat1 in individual cells associated with the central infected tissue. Restriction fragment polymorphism mapping of the PsCat1 locus in pea shows no correlation with existing mutant lines defective in symbiosis.

Sym 31, a Mutation Affecting Protein and Vesicle Targetting in Pea Nodule Symbiosomes

We have investigated protein and vesicle targetting in nodules formed by the pea plant mutant Sprint2Fix- (Sym 31). In contrast to wildtype symbiosomes, in the mutant several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane (PBM), or were found within a vacuole-like compartment.

A Lectin-Like Glycoprotein PsNLEC-1 is Absent from Symbiosomes in the Symbiotically Defective Pea Mutant sym31

Because nodule development is mediated by signals from both the plant and the microbe, the study of symbiotically defective mutants of either symbiont can help to elucidate important developmental pathways. However, the cytological and biochemical study of plant mutants with clear blocks in nodule development is still very limited. An interesting case is the pea mutation in sym31, which determines abnormal symbiosome structure and reduced morphological differentiation of bacteroids (Borisov et al., 1992).