GEM,a Novel Factor in the Coordination of Cell Division to Cell Fate Decisions in the Arabidopsis Epidermis

Cell division and cell fate decisions are highly regulated processes that need to be coordinated both spatially and temporally for correct plant growth and development. Gaining a deeper molecular and cellular understanding of these links is especially relevant for plant biology since, unlike in animals, formation of new organs is a process that takes place after embryogenesis and continues throughout the entire plant lifespan. The recent identification of a novel factor, GEM, has provided a molecular framework that coordinates cell division to cell fate in the Arabidopsis epidermis. GEM is an inhibitor of cell division through interacting with CDT1, a DNA replication protein. It also inhibits the expression of the homeobox GLABRA2 (GL2) gene that determines the hair/non-hair fate and the pavement/trichome fate in the root and leaf epidermis, respectively. GEM seems to be crucial in controlling the balance of activating/repressing histone modifications at its target promoters.Key Words: cell division, cell cycle, cell fate, GEM, GLABRA2, CDT1, DNA replication, chromatin, histone methylation, gene expression, root hair, Arabidopsis, plant     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

Cell Elongation and Division of Chloroplasts

Before undertaking the permanent culture of chloroplasts, theirpower to divide in vitro was investigated. The results suggestthat factors or conditions which govern cell elongation alsocontrol chloroplast division.     

Al Binding in the Epidermis Cell Wall Inhibits Cell Elongation of Okra Hypocotyl

Al inhibits root elongation at micromolar concentrations, butthe mechanisms leading to this process are unknown. In thesestudies, Al-induced inhibition of cell elongation was examinedusing hypocotyl of okra (Abelmoschus esculentus Moench cv. ClemsonSpineless) as an experimental model. One-h exposure to Al (0.5mM A1Cl₃) in the presence of 10 µM auxin in 0.5 mM CaCl₂,pH 4.0 significantly inhibited auxin-induced cell elongationof okra hypocotyl segments. Elongation was further suppressedwith increasing Al concentrations up to 1 mM. Treatment of thehypocotyl with 1 mM citrate for 10 minutes after 2-h exposureto Al resulted in significant recovery of elongation. The amountof Al in the cell wall relative to the total in the tissue was96.0, 96.2, and 85.4%, respectively, following 1-, 2-, and 3-hexposure to the Al solution. The total and cell wall Al contentwas decreased by half after the citrate desorption treatment.Further-more, 95% of Al was found in the epidermis, and 95%of the Al in the epidermis was associated with the cell wall.Experiments using split hypocotyl segments showed that Al exposureincreased the outward bending of hypocotyl segments, suggestingthat the epidermis elongation was specifically inhibited byAl. Al inhibited the autolysis of epidermis by about 20%, buthad little effect on the autolysis of core tissue. Taken together,these results suggest that Al binding in the epidermal cellwall inhibits critical components in cell wall loosening mechanism,resulting in inhibition of cell elongation.     

POLYGALACTURONASE INVOLVED IN EXPANSION1 Functions in Cell Elongation and Flower Development in Arabidopsis

Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1’s involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.     

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.     

Herkogamy and Its Effects on Mating Patterns in Arabidopsis thaliana

The evolution of mating systems, which exhibit an extraordinary diversity in flowering plants, is of central interest in plant biology. Herkogamy, the spatial separation of sexual organs within flowers, is a widespread floral mechanism that is thought to be an adaptive trait reducing self-pollination in hermaphroditic plants. In contrast with previous studies of herkogamy that focused on plants with relatively large floral displays, we here characterized herkogamy in Arabidopsis thaliana, a model plant with a strong selfing syndrome. Developmental features, reproductive consequences, and genetic architecture of herkogamy were exploited using naturally variable A. thaliana accessions, under both greenhouse and natural conditions. Our results demonstrate that the degree of herkogamy can strongly influence the mating patterns of A. thaliana: approach herkogamy can effectively promote outcrossing, no herkogamy is also capable of enhancing the opportunity for outcrossing, and reverse herkogamy facilitates efficient self-pollination. In addition, we found that the expression of herkogamy in A. thaliana was environment-dependent and regulated by multiple quantitative trait loci. This study reveals how minor modifications in floral morphology may cause dramatic changes in plant mating patterns, provides new insights into the function of herkogamy, and suggests the way for dissecting the genetic basis of this important character in a model plant.     

Cell Proliferation,Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex

Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2''-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.     

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion.The actin cytoskeleton provides tracks for the deposition of cell wall materials and plays important roles during many cellular processes, such as cell expansion and morphogenesis, vesicle trafficking, and the response to biotic and abiotic signals (Baskin, 2005; Smith and Oppenheimer, 2005; Szymanski and Cosgrove, 2009; Ehrhardt and Bezanilla, 2013; Rounds and Bezanilla, 2013). Plant cells respond to diverse internal and external stimuli by regulating the turnover and rearrangement of actin cytoskeleton networks in the cytoplasm (Staiger, 2000; Pleskot et al., 2013). How these actin rearrangements sense the cellular environment and what accessory proteins modulate specific aspects of remodeling remain an area of active investigation (Henty-Ridilla et al., 2013; Li et al., 2014a, 2015).Using high spatial and temporal resolution imaging afforded by variable-angle epifluorescence microscopy (VAEM; Konopka and Bednarek, 2008), we quantified the behavior of actin filaments in Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells (Staiger et al., 2009). There are two types of actin filament arrays in the cortical cytoplasm of epidermal cells: bundles and single filaments. Generally, actin bundles are stable with higher pixel intensity values, whereas individual actin filaments are fainter, more ephemeral, and constantly undergo rapid assembly and disassembly through a mechanism that has been defined as “stochastic dynamics” (Staiger et al., 2009; Henty et al., 2011; Li et al., 2012, 2015). Elongating actin filaments in the cortical cytoskeleton originate from three distinct locations: the ends of preexisting actin filaments, the side of filaments or bundles, and de novo in the cytoplasm. Plant actin filaments elongate at rates of 1.6 to 3.4 μm/s, which is the fastest assembly reported in eukaryotic cells. Distinct from the mechanism of treadmilling and fast depolymerization in vitro, however, the disassembly of single actin filaments occurs predominately through prolific severing activity (Staiger et al., 2009; Smertenko et al., 2010; Henty et al., 2011). A commonly held view is that the dynamic actin network in plant cells is regulated by the activities of conserved and novel actin-binding proteins (ABPs). Through reverse-genetic approaches and state-of-the-art imaging modalities, we and others have demonstrated that several key ABPs are involved in the regulation of stochastic actin dynamic properties in a wide variety of plants and cell types (Thomas, 2012; Henty-Ridilla et al., 2013; Li et al., 2014a, 2015). Through these efforts, the field has developed a working model for the molecular mechanisms that underpin actin organization and dynamics in plant cells (Li et al., 2015).Profilin is a small (12–15 kD), conserved actin-monomer binding protein present in all eukaryotic cells (dos Remedios et al., 2003). Profilin binds to actin by forming a 1:1 complex with globular (G-)actin, suppresses spontaneous actin nucleation, and inhibits monomer addition at filament pointed ends (Blanchoin et al., 2014). The consequences of profilin activity on actin filament turnover differ based on cellular conditions and the presence of other ABPs. In vitro studies show that the profilin-actin complex associates with the barbed ends of filaments and promotes actin polymerization by lowering the critical concentration and increasing nucleotide exchange on G-actin (Pollard and Cooper, 1984; Pantaloni and Carlier, 1993). When barbed ends are occupied by capping protein, profilin acts as an actin-monomer sequestering protein. These opposing effects of profilin might be a regulatory mechanism for profilin modulation of actin dynamics in cells. In addition to actin, profilin interacts with Pro-rich proteins, as well as polyphosphoinositide lipids in vitro (Machesky et al., 1994). Formin is an ABP that mediates both actin nucleation and processive elongation using the pool of profilin-actin complexes (Blanchoin et al., 2010). The primary sequence of formin includes a Pro-rich domain, named Formin Homology1 (FH1). Evidence from fission and budding yeast shows that profilin can increase filament elongation rates by binding to the FH1 domain (Kovar et al., 2003; Moseley and Goode, 2005; Kovar, 2006). The FH1 domain of Arabidopsis FORMIN1 (AtFH1) is also reported to modulate actin nucleation and polymerization in vitro (Michelot et al., 2005). Recently, two groups reported that profilin functions as a gatekeeper during the construction of different actin networks generated by formin or ARP2/3 complex in yeast and mammalian cells (Rotty et al., 2015; Suarez et al., 2015). These studies highlight the importance of profilin regulation in coordinating the different actin arrays present in the same cytoplasm of eukaryotic cells. However, direct evidence for how profilin facilitates formin-mediated actin nucleation or barbed end elongation in cells remains to be established.Genomic sequencing and isolation of PROFILIN (PRF) cDNAs from plants reveal that profilin is encoded by a multigene family. For example, moss (Physcomitrella patens) has three isovariants (Vidali et al., 2007) and maize (Zea mays) has five (Staiger et al., 1993; Kovar et al., 2001). In Arabidopsis, at least five PRF genes have been identified (Christensen et al., 1996; Huang et al., 1996; Kandasamy et al., 2002). Studies in maize show that the biochemical properties of profilin isoforms differ in vitro (Kovar et al., 2000). Moreover, the localization of profilin isoforms reveals organ-specific expression patterns. Detection of protein levels in vivo with isovariant-specific profilin antibodies demonstrate that Arabidopsis PRF1, PRF2, and PRF3 are constitutively expressed in vegetative tissues, whereas PRF4 and PRF5 are expressed mainly in flower and pollen tissues (Christensen et al., 1996; Huang et al., 1996; Ma et al., 2005).Several genetic studies on the functions of profilin in plants have been conducted. Reduction of profilin levels in P. patens results in the inhibition of tip growth, disorganization of F-actin, and formation of actin patches (Vidali et al., 2007). Moreover, it was shown that the interaction between profilin and actin or Pro-rich ligands is critical for tip growth in moss. Arabidopsis PRF1 has been demonstrated to be involved in cell elongation, cell shape maintenance, and control of flowering time through overexpression and antisense PRF1 transgenic plants, and further, the reduction of PRF1 inhibits the growth of hypocotyls (Ramachandran et al., 2000). However, investigation of a prf1-1 mutant, which contains a T-DNA insertion in the promoter region of the PRF1 gene, indicates that cell expansion of seedlings is promoted and that protein levels of PRF1 are regulated by light (McKinney et al., 2001). Recently, Müssar et al. (2015) reported a new Arabidopsis T-DNA insertion allele, prf1-4, that shows an obvious dwarf seedling phenotype. To date, however, there has not been a critical examination of the impact of the loss of profilin on the organization and dynamics of bona-fide single actin filaments in vivo.Here, we use a combination of genetics and live-cell imaging to investigate the role of PRF1 in the control of actin dynamics and its effect on axial cell expansion. We observed a significant decrease in the overall filament nucleation frequency in prf1 mutants, which is opposite to expectations if profilin suppresses spontaneous nucleation. Through a pharmacological approach, we found that nucleation frequency in wild-type cells treated with a formin inhibitor, SMIFH2, phenocopied prf1 mutants. We also analyzed the dynamic turnover of individual filaments in prf1 mutants and observed a significant decrease in the rate of actin filament elongation and maximum length of actin filaments. Specifically, we found that PRF1 favors the growth of a subpopulation of actin filaments that elongate at rates greater than 2 μm/s and similar results were obtained in cells after SMIFH2 treatment. Our results provide compelling evidence that Arabidopsis PRF1 contributes to stochastic actin dynamics by modulating formin-mediated actin nucleation and filament elongation during axial cell expansion.     

Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca²⁺ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca²⁺ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.     

MreB-Dependent Inhibition of Cell Elongation during the Escape from Competence in Bacillus subtilis

During bacterial exponential growth, the morphogenetic actin-like MreB proteins form membrane-associated assemblies that move processively following trajectories perpendicular to the long axis of the cell. Such MreB structures are thought to scaffold and restrict the movement of peptidoglycan synthesizing machineries, thereby coordinating sidewall elongation. In Bacillus subtilis, this function is performed by the redundant action of three MreB isoforms, namely MreB, Mbl and MreBH. mreB and mbl are highly transcribed from vegetative promoters. We have found that their expression is maximal at the end of exponential phase, and rapidly decreases to a low basal level upon entering stationary phase. However, in cells developing genetic competence, a stationary phase physiological adaptation, expression of mreB was specifically reactivated by the central competence regulator ComK. In competent cells, MreB was found in complex with several competence proteins by in vitro pull-down assays. In addition, it co-localized with the polar clusters formed by the late competence peripheral protein ComGA, in a ComGA-dependent manner. ComGA has been shown to be essential for the inhibition of cell elongation characteristic of cells escaping the competence state. We show here that the pathway controlling this elongation inhibition also involves MreB. Our findings suggest that ComGA sequesters MreB to prevent cell elongation and therefore the escape from competence.     

Specific Leaf Weight in Zones of Cell Division, Elongation and Maturation in Tall Fescue Leaf Blades

Patterns of change in specific leaf weight (SLW), water-solublecarbohydrate (WSC) content and leaf width were used to delineatethe region of secondary cell wall accumulation, and determinethe rate of increase in structural material along a developingleaf blade of tall fescue (Festuca arundinacea Schreb.). Structuralspecific leaf weight (SSLW) was determined by subtracting WSCmass from dry weight to emphasize structural material. Becausemeristematic activity, cell elongation, and cellular maturationare arranged successively in the grass leaf, these patternsrepresent a developmental sequence through which each segmentof the leaf blade passes. Patterns were generally similar fortwo genotypes, one selected for high (HYT) and the other forlow (LYT) yield per tiller, for a single genotype grown at 17or 25 C, and for two field-grown populations which differedin leaf area expansion rate (LAER). In all three studies, the elongation zone of the developingleaf had 31 to 39 per cent WSC on a dry weight basis. The LYTgenotype had a higher SLW at all stages of development whengrown at 17 than at 25 C, due to greater WSC accumulation.At 20 C, the HYT genotype had a higher SLW all along the elongatingleaf blade than the LYT genotype. This difference was due toa difference in SSLW, while WSC content was similar. The LERwas 64 per cent higher in the high population than the low,but elongation zones were similar in WSC. In all cases, SSLWwas high in the meristematic region, lowest near the distalend of the cell elongation zone, then increased linearly astissue matured. Rate of increase in SSLW was 8.5 and 5.2 g m^–2d^–1 for the HYT and LYT genotypes, respectively, and 7.6and 6.7 g m^–2 d^–1 for the high and low LAER populations,respectively. Festuca arundinacea Schreb., tall fescue, specific leaf weight, leaf width, water-soluble carbohydrates, leaf elongation rate     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.     

Stimulation of Cell Elongation by Tetraploidy in Hypocotyls of Dark-Grown Arabidopsis Seedlings

Plant size is largely determined by the size of individual cells. A number of studies showed a link between ploidy and cell size in land plants, but this link remains controversial. In this study, post-germination growth, which occurs entirely by cell elongation, was examined in diploid and autotetraploid hypocotyls of Arabidopsis thaliana (L.) Heynh. Final hypocotyl length was longer in tetraploid plants than in diploid plants, particularly when seedlings were grown in the dark. The longer hypocotyl in the tetraploid seedlings developed as a result of enhanced cell elongation rather than by an increase in cell number. DNA microarray analysis showed that genes involved in the transport of cuticle precursors were downregulated in a defined region of the tetraploid hypocotyl when compared to the diploid hypocotyl. Cuticle permeability, as assessed by toluidine-blue staining, and cuticular structure, as visualized by electron microscopy, were altered in tetraploid plants. Taken together, these data indicate that promotion of cell elongation is responsible for ploidy-dependent size determination in the Arabidopsis hypocotyl, and that this process is directly or indirectly related to cuticular function.     

Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots

We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development.