Specific roles of AtEXPA1 in plant growth and stress adaptation

Expansins are a group of proteins that appear to be involved in the disruption of the noncovalent bonds within the cell wall. The distinctly expressed expansin genes can independently regulate cell expansion in place and time, and their diverse expression patterns suggest their distinct effects on plant growth. In this paper, we analyzed the effects of excessive expansin AtEXPA1 on plant growth and plant adaptation to NaCl and ABA stresses by overexpressing its gene in Arabidopsis plants. The AtEXPA1 overexpressing plants exhibited stunted shoot growth, mainly during the early phase of vegetative growth, and the growth of transgenic seedlings was also impaired. Comparing with their growth under normal growth condition, the AtEXPA1 overexpressing plants showed alleviated impairment under salt and ABA stress conditions. These results suggest that, although excessive AtEXPA1 could disturb cell wall organization and lead to growth reduction, it inversely helped enhancing cell wall organization under stress conditions and thus helped plant better to adapt to adverse environment.     

Cytological expression of early response to infection by Heterodera glycines Ichinohe in resistant PI 437654 soybean.

The soybean PI 437654 is resistant to all known races of the soybean cyst nematode (SCN) in the U.S.A. and became a new source of resistance genes in cultivar development. Race 3, a wide-ranging nematode pathotype, was used to examine root cells of PI 437654 and susceptible 'Essex', 2, 3, and 5 days after inoculation (DAI). In initial response to SCN, both genotypes formed syncytia by cell wall dissolutions. Hypertrophy of syncytium component cells and hyperplasia of cells near syncytia were observed. At 2 DAI, incompatible response of PI 437654 to SCN was exhibited: limited cell hypertrophy, inhibition of syncytium growth, initiation of necrosis, and wall appositions. At 3 DAI, cellular events appeared to be a sum of the operative mechanisms for SCN resistance: irregular wall thickening, pronounced wall appositions, necrosis, and nuclear breakdown followed by cytoplasmic collapse. The cells surrounding the syncytia showed necrosis, wall apposition, and accumulation of electron-dense bodies. By 5 DAI, syncytia and neighboring cells were totally devoid of ground plasma and the degeneration process was completed. The normal route for early syncytium development in 'Essex' (increased number of organelles, intense vacuolization, accumulation of dense deposits in vacuoles, and wall ingrowths) suggests the involvement of portions of the developmental pathway of differentiating tissues in organogenesis. Early onset of SCN resistance 2 DAI in PI 437654 suggests rapid activation of genes in a cascade reaction leading to cell death. Key words : soybean, nematode, syncytium, cell death.     

Changes in the structure ofArabidopsis thaliana during female development of the plant-parasitic nematodeHeterodera schachtii

Summary The beet cyst nematodeHeterodera schachtii is able to establish a feeding structure (syncytium) in the vascular tissue of roots and shoots ofArabidopsis thaliana. Histological and ultrastructural studies were performed to assess plant responses during the development of juvenile females under monoxenic conditions. After destructively invading a root the nematode selects and pierces a single procambial cell with its stylet and transforms it into an initial syncytial cell (ISC) by secretory activity. The first most obvious changes in the ISC occur in the vacuolar system and at the wall. Differentiation of a central vacuole is impeded resulting in the formation of numerous small vacuoles. Multivesicular and paramural bodies are formed. An electron translucent material is deposited on the cell wall. Partial dissolution of the cell wall leads to the formation of a syncytium. At the juveniles' last pre-adult developmental stage the syncytium attains its maximum longitudinal and radial extension, occupying a major part of the central cylinder. Its features are indicative of a very high level of metabolic activity. The hypertrophied syncytium is ensheathed by a peridermal cover in which secondary xylem and phloem elements are interspersed. When females die the syncytia degenerate. The ultrastructural and histological features of syncytia described from roots are also found in syncytia induced in aerial parts of the plant.     

Syncytia in plants: cell fusion in endosperm—placental syncytium formation in Utricularia (Lentibulariaceae)

The syncytium formed by Utricularia is extremely unusual and perhaps unique among angiosperm syncytia. All typical plant syncytia (articulated laticifers, amoeboid tapetum, the nucellar plasmodium of river weeds) are formed only by fusion of sporophytic cells which possess the same genetic material, unlike Utricularia in which the syncytium possesses nuclei from two different sources: cells of maternal sporophytic nutritive tissue and endosperm haustorium (both maternal and paternal genetic material). How is this kind of syncytium formed and organized and is it similar to other plant syncytial structures? We used light and electron microscopy to reconstruct the step-by-step development of the Utricularia syncytia. The syncytia of Utricularia developed through heterotypic cell fusion involving the digestion of the cell wall, and finally, heterokaryotic multinucleate structures were formed, which possessed different-sized nuclei that were not regularly arranged in the cytoplasm. We showed that these syncytia were characterized by hypertrophy of nuclei, abundant endoplasmic reticulum and organelles, and the occurrence of wall ingrowths. All these characters testify to high activity and may confirm the nutritive and transport functions of the syncytium for the developing embryo. In Utricularia, the formation of the syncytium provides an economical way to redistribute cell components and release nutrients from the digested cell walls, which can now be used for the embryo, and finally to create a large surface for the exchange of nutrients between the placenta and endosperm.     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Antagonistic relationship between AtRALF1and brassinosteroid regulates cellexpansion-related genes

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.     

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled “Parthenon pillars.” In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.     

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

Regulation of root hair initiation and expansin gene expression in Arabidopsis

The expression of two Arabidopsis expansin genes (AtEXP7 and AtEXP18) is tightly linked to root hair initiation; thus, the regulation of these genes was studied to elucidate how developmental, hormonal, and environmental factors orchestrate root hair formation. Exogenous ethylene and auxin, as well as separation of the root from the medium, stimulated root hair formation and the expression of these expansin genes. The effects of exogenous auxin and root separation on root hair formation required the ethylene signaling pathway. By contrast, blocking the endogenous ethylene pathway, either by genetic mutations or by a chemical inhibitor, did not affect normal root hair formation and expansin gene expression. These results indicate that the normal developmental pathway for root hair formation (i.e., not induced by external stimuli) is independent of the ethylene pathway. Promoter analyses of the expansin genes show that the same promoter elements that determine cell specificity also determine inducibility by ethylene, auxin, and root separation. Our study suggests that two distinctive signaling pathways, one developmental and the other environmental/hormonal, converge to modulate the initiation of the root hair and the expression of its specific expansin gene set.