Soybean Root Cells Research Articles

A chromosomal assembly of the soybean cyst nematode genome.

The soybean cyst nematode (Heterodera glycines) is a sedentary plant parasite that exceeds billion USD annually in yield losses. This problem is exacerbated by H.glycines populations overcoming the limited sources of natural resistance in soybean and by the lack of effective and safe alternative treatments. Although there are genetic determinants that render soybeans resistant to nematode genotypes, resistant soybeans are increasingly ineffective because their multiyear usage has selected for virulent H.glycines populations. Successful H.glycines infection relies on the comprehensive re-engineering of soybean root cells into a syncytium, as well as the long-term suppression of host defences to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H.glycines into host root tissues. The mechanisms that control genomic effector acquisition, diversification, and selection are important insights needed for the development of essential novel control strategies. As a foundation to obtain this understanding, we created a nine-scaffold, 158Mb pseudomolecule assembly of the H.glycines genome using PacBio, Chicago, and Hi-C sequencing. A Mikado consensus gene prediction produced an annotation of 22,465genes using short- and long-read expression data. To evaluate assembly and annotation quality, we cross-examined synteny among H.glycines assemblies, and compared BUSCO across related species. To describe the predicted proteins involved in H.glycines' secretory pathway, we contrasted expression between preparasitic and parasitic stages with functional gene information. Here, we present the results from our assembly and annotation of the H.glycines genome and contribute this resource to the scientific community.

T-SNAREs bind the Rhg1 α-SNAP and mediate soybean cyst nematode resistance.

Soybean cyst nematode (SCN; Heterodera glycines) is the largest pathogenic cause of soybean yield loss. The Rhg1 locus is the most used and best characterized SCN resistance locus, and contains three genes including one encoding an α-SNAP protein. Although the Rhg1 α-SNAP is known to play an important role in vesicle trafficking and SCN resistance, the protein's binding partners and the molecular mechanisms underpinning SCN resistance remain unclear. In this report, we show that the Rhg1 α-SNAP strongly interacts with two syntaxins of the t-SNARE family (Glyma.12G194800 and Glyma.16G154200) in yeast and plants; importantly, the genes encoding these syntaxins co-localize with SCN resistance quantitative trait loci. Fluorescent visualization revealed that the α-SNAP and the two interacting syntaxins localize to the plasma membrane and perinuclear space in both tobacco epidermal and soybean root cells. The two syntaxins and their two homeologs were mutated, individually and in combination, using the CRISPR-Cas9 system in the SCN-resistant Peking and SCN-susceptible Essex soybean lines. Peking roots with deletions introduced into syntaxin genes exhibited significantly reduced resistance to SCN, confirming that t-SNAREs are critical to resisting SCN infection. The results presented here uncover a key step in the molecular mechanism of SCN resistance, and will be invaluable to soybean breeders aiming to develop highly SCN-resistant soybean varieties.

GunA of Sinorhizobium (Ensifer) fredii HH103 is a T3SS-secreted cellulase that differentially affects symbiosis with cowpea and soybean

The symbiosis between Sinorhizobium fredii HH103 and its host legumes is influenced by the type 3 secretion system (T3SS), which delivers proteins (effectors) directly into the host cells to promote infection. GunA, one of the predicted HH103 effectors, potentially codes for a cellulase. In this work we tried to characterise GunA and elucidate its role in symbosis with soybean and cowpea. A GunA::HA fusion protein was constructed to study T3SS-dependent secretion. Cellulase activity of GunA was measured and gunA::uidA-GFP and gunA::cyA fusions were constructed to monitor gunA expression in nodules and to study translocation to the host cells, respectively. Finally, the symbiotic performance of a gunA mutant was studied in soybean and cowpea. GunA from S. fredii HH103 shows cellulase activity and is secreted through the T3SS in response to the inducer flavonoid genistein. Interestingly, at the beggining of the symbiotic process, GunA was partially responsible for the induction of the expression of the soybean GmPR1 gene, a gene used as a marker for plant defense responses. However, GunA was also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea. Secretion of GunA through the S. fredii HH103 T3SS clearly and differentially impacts the symbiotic performance of this strain with soybean and cowpea. GunA, or its cellulase activity, is recognised by soybean root cells very early in the symbiotic process but, curiously, its secretion can also be detected in mature nodules. This suggests different symbiotic roles at different symbiotic stages that need to be further elucidated.

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.

Microbe-Independent Entry of Oomycete RxLR Effectors and Fungal RxLR-Like Effectors Into Plant and Animal Cells Is Specific and Reproducible.

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, a...

Microbe-Independent Entry of Oomycete RxLR Effectors and Fungal RxLR-Like Effectors Into Plant and Animal Cells Is Specific and Reproducible

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H+ extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H+. Previous results in Fe-deficient cucumber roots showed that high H+ extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover 31P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H+ and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4- predominating in the vacuole and HPO42- in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of the two H+ pumps in Pi fluxes between cytoplasm and vacuole in Fe-deficient cucumber and soybean root cells is discussed.

Rust Secreted Protein Ps87 Is Conserved in Diverse Fungal Pathogens and Contains a RXLR-like Motif Sufficient for Translocation into Plant Cells

BackgroundEffector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.Methodology/Principal FindingsYeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.Conclusions/SignificanceThe results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.

Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: Modelization of the in vitro biochemical response

Secondary metabolites are mostly involved in the relationship of the organism with its environment, e.g. in plant defence mechanisms or as signal compound; their synthesis is often induced by different kinds of biotic and/or abiotic stresses. In general the major strategies to improve their productivity are: the optimization of growth and production media or induction of secondary metabolite pathways by elicitors and metabolic engineering. From this point of view, elicitation is largely employed as a strategy to mimic stress effects, which then lead to a physiological response involving secondary metabolite synthesis and accumulation into the hairy root (HR). Thus, studies on the response to elicitation for HR in accordance with nutritional status are necessary. In the present study, which focuses on tropane alkaloid production in Datura stramonium L. hairy roots, the effects of minerals in the B5 culture medium [O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158] and elicitor (jasmonic acid) are studied using a mathematical model approach (screening analysis) and factorial design 2 3 was carried out to study the response to elicitation in accordance with nutritional status of HR for [NO 3 −], [H 2PO 4 −] and [Ca 2+]. The modelization of different minerals effects showed a response to elicitation only after the 8th day of cultivation. The study of models coefficients revealed that nitrate [NO 3 −], phosphorus [H 2PO 4 −] and calcium [Ca 2+] levels and combinations [NO 3 −/Ca 2+] and [NO 3 −/H 2PO 4 −/Ca 2+] were positive with elicitation; however, interaction effects [NO 3 −/H 2PO 4 −] and [H 2PO 4 −/Ca 2+] were negative. The coefficients position on the 28th day of cultivation, with the Pareto chart allowed to adjust the mathematical model by eliminating the no significant effects [NO 3 −/H 2PO 4 −], [H 2PO 4 −/Ca 2+] and [NO 3 −/H 2PO 4 −/Ca 2+]. As a result, we show that simulation with the adjusted model allows predicting the response to elicitation in accordance with the nutritional state of HR in nitrate and calcium. The measured values on the 28th day were statistically identical with calculated values obtained by mathematical models. These results revealed that HR, with limited nitrate, showed lower response to elicitation by jasmonic acid. Moreover, the adjusted model, of first degree, was not predictably beyond the experimental domain. As a general conclusion, NO 3 − (62.5 mM) must be added to the B5 medium at the beginning of the culture. The lack of these compounds reduced alkaloid metabolism, even in combination with elicitor treatments like jasmonic acid. The same trend was observed with Ca 2+ (11.1 mM), H 2PO 4 − (6.06 mM) and nitrate/calcium ratio (62.5 mM/11.1 mM), which also increased alkaloid production when combined with jasmonic acid 10 mM. These positive effects appeared at the 2nd week after initiation of the culture.

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER-containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

The structure and function of a soybean beta-glucan-elicitor-binding protein.

beta-Glucan elicitor (GE), released from the cell wall of the phytopathogenic fungus Phytophthora megasperma by soybean glucanases, causes defense reactions in soybean. A GE-binding protein (GEBP) was purified from the membrane fraction of soybean root cells, and its cDNA was isolated. Expression of the cDNA clone in tobacco suspension cultured cells and in Escherichia coli conferred GE-binding activity to both. An antibody against the recombinant protein was found to inhibit the GE binding with the soybean cotyledon membrane fraction as well as the resulting accumulation of phytoalexin. Immunolocalization assays indicated that the GEBPs are located in the plasma membrane of root cells. These results suggest that the cDNA encodes a GE receptor and may mediate the signaling of the elicitor.

Auxins and Cytokinins as Antipodal Modulators of Elasticity within the Actin Network of Plant Cells.

The cytoskeleton of plant and animal cells serves as a transmitter, transducer, and effector of cell signaling mechanisms. In plants, pathways for proliferation, differentiation, intracellular vesicular transport, cell-wall biosynthesis, symbiosis, secretion, and membrane recycling depend on the organization and dynamic properties of actin- and tubulin-based structures that are either associated with the plasma membrane or traverse the cytoplasm. Recently, a new in vivo cytoskeletal assay (cell optical displacement assay) was introduced to measure the tension within subdomains (cortical, transvacuolar, and perinuclear) of the actin network in living plant cells. Cell optical displacement assay measurements within soybean (Glycine max [L.]) root cells previously demonstrated that lipophilic signals, e.g. linoleic acid and arachidonic acid or changes in cytoplasmic pH gradients, could induce significant reductions in the tension within the actin network of transvacuolar strands. In contrast, enhancement of cytoplasmic free Ca2+ resulted in an increase in tension. In the present communication we have used these measurements to show that a similar antipodal pattern of activity exists for auxins and cytokinins (in their ability to modify the tension within the actin network of plant cells). It is suggested that these growth substances exert their effect on the cytoskeleton through the activation of signaling cascades, which result in the production of lipophilic and ionic second messengers, both of which have been demonstrated to directly effect the tension within the actin network of soybean root cells.

Aluminum Induces Rigor within the Actin Network of Soybean Cells.

Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins.

Carbohydrate binding activities of Bradyrhizobium japonicum III. Lectin expression, bacterial binding, and nodulation efficiency

In previous studies, evidence that the Bradyrhizobium japonicum lectin, designated BJ38, mediated the observed carbohydrate-specific binding activities of the bacteria, including the saccharide-specific adhesion to soybean root cells was presented. In the present study, it is found that both B. japonicum, as well as the purified BJ38, bind predominantly to young emergent root hairs of soybean roots and, to a much lesser extent, to the root cap, mature root hairs, epicotyl or hypocotyl regions. Thus, the region of preferential binding for both the bacteria and the isolated lectin coincide with the region of the soybean root most susceptible to B. japonicum infection. The importance of bacterial binding for the nodulation process was studied by comparing the nodulation efficiency of binding-deficient mutants N4 and N6 to the wild-type. These mutants had been shown to be defective in carbohydrate recognition, as represented by their diminished ability to bind to soybean roots. BJ38 was immunolocalized to one pole of the cell surface of wild-type B. japonicum, but no surface labeling could be detected on either mutant. Moreover, both N4 and N6 showed a substantial decrease in nodulation activity, relative to the wild-type. These results provide additional evidence that the carbohydrate binding activity of B. japonicum, most probably mediated by BJ38, may play an important role(s) in the initial phases of the infection process.

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells.

Intercellular communication between plant cells for low molecular weight hydrophilic molecules occurs through plasmodesmata. These tubular structures are embedded in the plant cell wall in association with the plasmalemma and endoplasmic reticulum (ER). Transmission electron microscopy has provided strong evidence to support the view that both the ER and plasmalemma are structurally continuous across the wall at these sites. In experiments to be described, the technique of fluorescence redistribution after photobleaching was used to examine the lateral mobility and intercellular transport capability of a number of fluorescent lipid and phospholipid analogs. These probes were shown by confocal fluorescence microscopy to partition in either the ER or plasmalemma. Results from these measurements provide evidence for cell communication between contiguous cells for probes localized predominantly in the ER. In contrast, no detectable intercellular communication was observed for probes residing exclusively in the plasmalemma. It was of particular interest to note that when 1-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)aminoacylphosphatidylcholine was utilized as a potential reporter molecule for phospholipids in the plasmalemma, it was quickly degraded to 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminoacyldiglyceride (NBD-DAG), which then appeared predominantly localized to the ER and nuclear envelope. This endogenously synthesized NBD-DAG was found to be capable of transfer between cells, as was exogenously incorporated NBD-DAG. Results from these investigations provide support for the following conclusions: (1) ER, but apparently not the plasmalemma, can form dynamic communication pathways for lipids across the cell wall between connecting plant cells; (2) the plasmodesmata appear to form a barrier for lipid diffusion through the plasmalemma; and (3) lipid signaling molecules such as diacylglycerol are capable of transfer between contiguous plant cells through the ER. These observations speak to issues of plant cell autonomy for lipid synthesis and mechanisms of intercellular signaling and communication.

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells

Tissue-Specific Expression of the Alternative Oxidase in Soybean and Siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20-70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20-140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.

Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.

An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene product demonstrated immunological crossreactivity with all three reagents. The cDNA from this clone contained 1171 base pairs and coded for a protein of 280 amino acids with a calculated molecular mass of 32,339 Da (migrates as a 29-kDa polypeptide in SDS/polyacrylamide gels). The sequence homology observed between the 32-kDa polypeptide of Arabidopsis and connexin 32 from rat liver, in conjunction with observed similarities in predicted number and distribution of hydrophobic domains, sites for posttranslational modification, and basic pI, provides strong evidence that the biological range for connexin-type proteins may now be considered to include the plant kingdom.

Auxin Transport in Suspension-Cultured Soybean Root Cells

The kinetic parameters of auxin transport in suspension-cultured soybean (Glycine max [L.] Merr.) root cells were investigated. The same processes that are responsible for polar indoleacetic acid (IAA) transport in other plant tissues were found to occur in soybean root cells. These include (a) passive diffusion of the undissociated auxin molecule across the plasma membrane, (b) uptake via a specific, saturable carrier, and (c) phytotropin-sensitive efflux. Metabolism of exogenously added IAA was rapid; at the end of a 15-minute uptake period >80% of the IAA taken up had been converted to other compounds. The time course of [(14)C]IAA uptake in the first 90 seconds revealed two phases, the first corresponding to a rate of uptake approximately twice as large as the second phase. The transition to the second phase was delayed in the presence of the phytotropins triiodobenzoic acid or naphthylphthalamic acid, suggesting that an increase in the efflux of label as IAA accumulates in the cytoplasm is responsible for the transition. Carrier-mediated uptake contributes between 50 and 60% to the total rate of auxin uptake from a 0.28 micromolar IAA solution, with passive diffusion accounting for the remainder. Kinetic analysis of carrier-mediated uptake revealed a pH optimum of 5.0 and a Michaelis-Menten constant of 0.4 micromolar at pH 5.5. Because phytotropins had no effect on the initial rate of uptake, the efflux carrier does not appear to be involved in the uptake process.