Plant Signaling & Behavior 6:3, 382-392; March 2011; ©2011 Landes Bioscience
Global expression pattern comparison between low phosphorus insensitive 4 and WT Arabidopsis reveals an important role of reactive oxygen species and jasmonic acid in the root tip response to phosphate starvation Alejandra Chacón-López,1,2 Enrique Ibarra-Laclette,2 Lenin Sánchez-Calderón,3 Dolores Gutiérrez-Alanís2 and Luis Herrera-Estrella2,* Departamento de Ingeniería Genética de Plantas; 2Laboratorio Nacional de Genómica para la Biodiversidad; Centro de Investigación y Estudios Avanzados; Campus Guanajuato; Irapuato Guanajuato; 3Unidad Académica de Biología Experimental; Universidad Autónoma de Zacatecas; Zacatecas, México
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Key words: arabidopsis, phosphate-deprivation, jasmonic acid, redox state, ethylene, meristem exhaustion, transcription profiling Abbreviations: WT, wild type; Pi, phosphate; lpi, low phosphate insensitive; L, low Pi; H, high Pi; QC, quiescent center; ROS, reactive oxygen species; AA, ascorbic acid; DAB, 3,3-diaminobencidine; JA, jasmonic acid; ACC, aminocyclopropane-1-carboxylic acid; AVG, 2-aminoethoxyvinyl glycine; dag, days after germination
Plants are exposed to several biotic and abiotic stresses. A common environmental stress that plants have to face both in natural and agricultural ecosystems that impacts both its growth and development is low phosphate (Pi) availability. There has been an important progress in the knowledge of the molecular mechanisms by which plants cope with Pi deficiency. However, the mechanisms that mediate alterations in the architecture of the Arabidopsis root system responses to Pi starvation are still largely unknown. One of the most conspicuous developmental effects of low Pi on the Arabidopsis root system is the inhibition of primary root growth that is accompanied by loss of root meristematic activity. To identify signalling pathways potentially involved in the Arabidpsis root meristem response to Pi-deprivation, here we report the global gene expression analysis of the root tip of wild type and low phosphorus insensitive4 (lpi4) mutant grown under Pi limiting conditions. Differential gene expression analysis and physiological experiments show that changes in the redox status, probably mediated by jasmonic acid and ethylene, play an important role in the primary root meristem exhaustion process triggered by Pi-starvation.
Introduction Plants as sessile organisms acquired during evolution different adaptive mechanisms to cope with unfavorable environmental conditions. Among the environmental stresses that plants face in both natural and agricultural ecosystems, low phosphate (Pi) availability represents one of the most common constraints for growth and reproduction.1 Pi is a component of many biological molecules, such as DNA, RNA and phospholipids, and plays a vital role in energy transfer and metabolic regulation. Although the total concentration of Pi in the soil can be high, in many cases its availability for plant uptake is limited because of its low mobility in the soil solution and its rapid conversion to organic forms that are not readily available to be assimilated by plants.2 To overcome low Pi availability, plants evolved a wide array of morphological, physiological, molecular and biochemical adaptive strategies to
optimize Pi uptake from the soil and to improve Pi use efficiency.3,4 Among the adaptive responses to low Pi availability that have been characterized in Arabidopsis, several low Pi-induced modifications in root architecture have been reported, such as the inhibition of primary root elongation and an increase in length and density of root hairs, as well as an increase in lateral root formation.5-7 In the last decade, there has been an important progress in the knowledge of the molecular mechanisms by which plants cope with Pi deficiency. The use of genetic strategies and global expression analysis allowed the identification of several important components of the Pi starvation network.8-13 However, the sensing mechanisms that trigger specific responses to Pi starvation are still largely unknown. It has been postulated that the root apical meristem is one of the sites sensing environmental conditions that modulate root growth and development. For instance, primary root growth is arrested when the root tip contacts low
*Correspondence to: Luis Herrera-Estrella; Email:
[email protected] Submitted: 10/20/10; Revised: 11/08/10; Accepted: 11/09/10 DOI: 10.4161/psb.6.3.14160 382
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research paper
Previous studies have shown that in Arabidopsis, Pi deficiency induces a determinate developmental root growth program in which meristematic cells have a limited number of divisions, undergo early cellular differentiation and have a gradual reduction of cell elongation zone, resulting in a short root phenotype characterized by exhaustion of the meristem.18 With the purpose of identifying potential components in the low-Pi response, Sánchez-Calderón et al.19 isolated a group of Arabidopsis low phosphorus insensitive (lpi1 to lpi4) mutants with altered root architecture responses to low Pi. In limiting Pi conditions, these mutants show an indeterminate root growth leading to a long primary root, providing further genetic evidence of the existence of a sensing mechanism at the primary root meristem that regulates root growth and elongation. Based on the different mutants that do not respond to low Pi in terms of changes in root architecture, it can be assumed that a dynamic behavior in the root apical meristem integrates environmental signals and developmental clues to define a root developmental program that regulates primary root elongation in an appropriate manner to cope with adverse environmental conditions. Several studies have provided evidence of the important role that hormones, such as auxin, cytokinins and ethylene, play to integrate environmental signals to produce root adaptive responses.20-23 To identify signaling pathways potentially involved in the Arabidopsis root meristem responses to Pi-deprivation, here we report the global gene expression analysis of the root tip of wild type and lpi4 plants grown under Pi limiting conditions. Analysis of differentially expressed genes between the wild type and lpi4 Pi-deprived seedlings indicate that oxidative responses as well as jasmonic acid and ethylene signaling could be involved in the low Pi response. We also carried out experiments to determine the potential role of redox balance, jasmonic acid and ethylene in primary root meristem maintenance related to the Pi-deprivation response. Finally, we discuss the potential mechanisms involved in triggering the signaling and metabolic processes that ultimately affect the maintenance of primary root meristem in response to Pi stress. Figure 1. Phenotype of wild type and lpi4 mutant grown in contrasting Pi conditions. Photograph of WT (col0 ecotype) and lpi4 growing on the surface of agar plates containing 0.1x MS medium and high (1 mM) (A) or low (5 μM) (B) Pi. (C) Means values (±SE) of primary root length of WT and lpi4 grown in high and low Pi (n = 20 seedlings). Micrographs of primary root meristem structure of WT in high (D) and low (F) Pi and lpi4 in high (E) and low (G) Pi conditions.
Pi medium, even if the rest of the plant is exposed to high Pi medium.14 Arabidopsis mutants defective in Low Phosphate Response1 (LPR1, a gene encoding a multicopper oxidase expressed in the primary root meristematic region) do not arrest primary root growth when exposed to low Pi medium, providing genetic evidence that, at least in part, sensing of Pi availability takes place at the root tip. Moreover, the Arabidopsis phosphate deficiency response (pdr2) mutant, affected in a P5 type ATPase, is defective in local Pi sensing showing hypersensitivity to low Pi in terms of the inhibition of primary root growth.15-17
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Results The lpi4 mutant is defective in the low-Pi response. We previously reported the lpi (lpi1–lpi4) mutants group,19 however only the lpi1 and lpi2 were characterized in detail. For this study we selected lpi4 because among the lpi mutants it shows the longest primary root phenotype in low Pi medium. To characterize in more detail the lpi4, we germinated wild type (WT) and lpi4 seeds in 0.1x MS media containing either 5 μM (L medium) or 1 mM (H medium) Pi. Both WT and lpi4 seedlings grown in H medium have a similar root phenotype, consisting of a long primary root with few lateral roots near the shoot-root junction (Fig. 1A). However, in contrast to the root phenotype observed for WT seedlings grown in L medium, which show a short primary root phenotype with lateral root forming close to the root tip, under this conditions, the lpi4 seedlings present a long primary root phenotype quite similar to that observed in WT seedlings grown in H medium (Fig. 1B). When primary root length
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was measured, it was observed that the roots of WT seedlings achieve in average a maximum length of 2 cm and stops growing 8 days after germination (dag), whereas in H medium root elongation does not stop and roots reach an average length of approximately 5.5 cm at 14 dag (Fig. 1C). The lpi4 seedlings present a root growth kinetics and maximal length in both L and H media that is quite similar to that observed for the WT in H medium (Fig. 1C). As expected, in H medium, both WT and lpi4 seedlings show normal meristem morphology, with well defined cell types and no signs of differentiation (Fig. 1D and E). In contrast to the WT seedlings grown in L medium, that show the previously reported meristem exhaustion, characterized by the lack of well defined cell types and an early differentiation process including the formation of root hairs and even some lateral roots very close to the root tip (Fig. 1F), lpi4 seedlings under this conditions show a root tip phenotype that resembles that observed for the WT in H medium (Fig. 1G). The phenotypic differences observed between the wild type and lpi4 suggest that, at least some, of the transcriptional responses triggered by Pi deprivation in WT plants that lead to the arrest of primary root growth and meristem exhaustion, are not activated in lpi4 mutant. Transcription profiling. Since in contrast to the WT, lpi4 does not show an arrest of primary root growth and meristem exhaustion in response to Pi-deprivation, a global gene expression comparison between these two genotypes could provide important information about potential signaling and metabolic events involved in Arabidopsis root tip responses to Pi deprivation. The idea being that since in L medium lpi4 does not show a decrease in root growth and the subsequent meristem inactivation, and keeps a similar growth to the WT in H medium, those genes that are differentially expressed, either up or downregulated, in the WT during root response to low Pi, could be identified as oppositelyregulated in lpi4 when comparing gene expression levels between these two genotypes in L medium. With this aim, we carried out a global gene expression analysis using gene chip arrays and mRNA extracted from a 300 μm long section of the root tip from WT and lpi4 seedlings grown under Pi deficient conditions. Our experiment was designed considering three sampling points: 1, 4 and 7 dag (see materials and methods). Differentially expressed genes between WT and lpi4 at the different sampling points, as well as the gene expression differences caused by the Pi deprivation by time interaction (Time x Pi effect) were determined. According to our stringency levels (FDR ≤ 0.15 and Fold ±2), a total of 846 genes showed differential expression between WT and lpi4 in at least one of the three sampled time points (Fig. 2A; Sup. Table S1). Figure 2B shows a complete linkage agglomerative hierarchical clustering24 for differential genes based on their expression patterns. For both induced and repressed genes between WT and lpi4 plants, the fold-change in expression of Pi-responsive genes gradually increased from day 1 to 7 dag under Pi stress. Interestingly, most of the differential expressed genes were downregulated in lpi4 compared to the WT or are activated in WT respect to lpi4 plants in response to Pi deprivation. Only 34.3% of these differentially expressed genes had been previously indentified in microarray studies as Pi-responsive genes in Arabidopsis plants (Sup. Table S2).10-13 As expected, the
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Figure 2. Overview of Pi-starvation responsive genes in Arabidopsis root tips. (A) Venn diagrams showing common or distinct regulated genes over the sampled time points. (B) Expression patterns identified by agglomerative hierarchical clustering. The Fold change (Ratio; r) values were used for the analysis. Each column represents a single time (1, 4 or 7 days), each row a gene. Clustering was performed using the Pearson correlation (around zero) and average linkage clustering. Green indicates ≤0.5 values, red ≥2 values and black one, as shown on the color scale at the bottom of the figure.
expression pattern for most of these genes in lpi4 is the opposite as previously reported for Pi-responsive genes (downregulated genes in lpi4 mutant as compared to the WT, have been reported as upregulated genes in response to Pi-starvation). The higher proportion of novel Pi-starvation responsive genes indentified in this study could be explained because we only used Arabidopsis root tips, in contrast to previously microarray studies where whole plants or at most separated shoots and roots were used and/or to the different experimental conditions used in previous global expression studies of the Arabidopsis Pi response. The differential expression pattern observed in the microarray experiment was validated for 15 genes using qRT-PCR. All genes showed a similar expression pattern in both assays. Differences were observed at the quantitative level, the qRT-PCR analysis showed in general a higher fold of induction or repression than
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the microarray analysis (Sup. Fig. S1). Similar quantitative differences between qRT-PCR and microarray data have been previously reported in reference 13 and 25. To determine whether the hypothesis that differentially expressed genes, identified as induced or repressed in lpi4 respect to the WT, indeed correspond to genes that are repressed or induced in the WT because the lpi4 mutant is insensitive in terms of the primary root response to Pi deprivation, we examined the relative mRNA levels, respect to the actin (At3g18780) transcript level expressed as the 40-ΔCT (similar to Bari et al.26), of the 15 genes chosen to validate the microarray in WT and lpi4 seedlings. We found that the relative mRNA levels of these genes significantly increase or decrease in the WT, whereas in lpi4 seedlings their relative mRNA levels showed a minor change compared to that observed in WT seedlings (Sup. Fig. S2). These data show that our hypothesis that comparing the changes in the transcriptome of an insensitive mutant to an environmental factor with those of the WT can be very useful to identify genes involved in a particular response to environmental factor. Genes that are still responsive to both the mutant and the WT will not appear as differentially expressed genes since their changes in mRNA should be similar in both genotypes. Functional classification of differential genes: lpi4 mutant vs. wild type plant. Differential genes were classified in functional categories according to the Munich Information Center for Protein Sequences classification (MIPS),27 using the FunCat database. Overrepresented MIPS categories were identified using the BioMaps tool from the Virtual Plant database (virtualplant. bio.nyu.edu). Based on this bioinformatics tool, differential genes could be grouped into the following sets of functional categories: Metabolism, Protein with binding function or cofactor requirement, Cellular transport, Cell rescue, Defense and virulence, Interaction with the environment, Systemic interaction with the environment and Cell fate, each comprising their respective subcategories (Sup. Table S3). Among the most overrepresented and highly downregulated genes in lpi4, respect to the WT, are those belonging to categories “plant hormonal regulation,” “oxygen and radical detoxification” and “biogenesis of cellular components” (Sup. Table S4). Among the strongly downregulated genes in lpi4 in the category of plant hormonal regulation, the largest group belongs to the subcategory of JA synthesis and signaling pathway, which include encoding genes key enzymes of JA biosynthesis such as three lipases (two GDSL-motif lipases and the EDS1 Lipase), the lipoxygenase LOX3, three allene oxide synthases (AOS, AOC2 and AOC3) and the 12-oxophytodienoate reductase OPR3,28 (Sup. Fig. S4 and Table S4), as well as three ZIM-domain transcription factors (JAZ1, JAZ2 and JAZ6) that are important components of the JA signaling pathway,29-31 (see Sup. Table S4). These results suggest that JA synthesis and signaling is probably involved in the Arabidopsis root tip response to Pi-deprivation. A combinatorial action of JA and ethylene has been reported in defense and root development signaling which is coordinated through the induction of genes that encode key ethylene transcription factors (ERFs).32 Interestingly, two ethylene transcription factors were also identified as downregulated genes in lpi4,
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namely ERF2 and ERF5 (Sup. Table S4). Additionally 37 genes, classified as involved in oxidative stress responses, are downregulated in lpi4, which include genes encoding 14 putative Class III peroxidases, nine Glutathione S-transferases, two catalase genes (CAT1 and CAT3) and the MDHAR (monodehydroascorbate reductase) (Sup. Fig. S3 and Table S4), suggesting that oxidative stress or redox balance maintenance could be important for the root tip responses to low Pi. We also found that 23 differentially expressed genes (most of them upregulated) in lpi4 belong to the cell wall subcategories of the biogenesis of cellular components category (Sup. Tables S3 and S4). This is probably not surprising since cell division and elongation during root growth implicates significant changes in the composition and structure of the cell wall, and these classes of genes should be more actively expressed in lpi4 in order to maintain root growth respect to the WT, which shows primary root arrest during Pi-deprivation. An appropriate redox status is critical for primary root meristem maintenance. The large number of genes related to oxidative stress (including one member of the ascorbate oxidase gene family) that are downregulated in the root tip of lpi4 plants compared to WT, suggests that under Pi starvation an imbalance in the root meristem redox status might be occurring and that this redox alteration could play an important role in the loss of root elongation and meristem maintenance of WT plants in response to Pi-deprivation. To determine whether Pi availability alters the concentration of reactive oxygen species (ROS) in the Arabidopsis root tip, WT and lpi4 seedlings grown in H and L media were stained with the H2O2 specific dye 3,3-Diaminobenzidine (DAB). We analyzed DAB staining at 4, 7 and 10 dag. In WT seedlings grown in H medium, DAB staining was detected in most cells with a higher intensity in the central cylinder with a maximum around the quiescent center (QC) region and elongation zone, at all time points analyzed (Fig. 3A–C), which agrees with previous reports in reference 33. Interestingly, in 4-day-old WT seedlings grown in L medium, DAB staining was similar to that observed for seedlings germinated in H medium (Fig. 3D), however in 7- and 10-day-old seedlings DAB staining was significantly reduced (Fig. 3E and F). When lpi4 seedlings were analyzed, it was observed that in both H (Fig. 3G–I) and L media (Fig. 3J–L) the DAB staining was similar to that observed in the WT grown in H Pi medium (Fig. 3A–C). These results suggest that dramatic changes in H2O2 levels occur during the meristem exhaustion process that WT seedlings undergo in response to Pi-deprivation. It is has been reported that in addition to the role of AA as a oxygen radical scavenger, it also plays a role in the regulation of cell division34,35 and cell elongation.36 Since both of these two processes are affected during the Pi-starvation response in Arabidopsis, we decided to test the effect of AA treatment on the root meristem exhaustion process. With this aim, WT and lpi4 seeds were germinated in H and L media supplemented with AA at concentrations ranging between 0.01 and 0.25 mM, and primary root length and root phenotype scored. It was observed that in H medium, no significant differences in primary root growth were observed for both WT and lpi4 at AA concentrations
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Figure 3. ROS expression pattern in root tips of WT and lpi4 seedlings. Kinetics of DAB staining in root tip of WT seedlings at 4, 7 and 10 days after germination grown in high (A–C) and low (D–F) Pi conditions. ROS pattern in root tip of lpi4 mutant seedling grown in high (G–I) and low (J–L) Pi conditions. Note the DAB staining decrease in Col0 seedlings grown in Pi deficiency from 7 dag. Scale bar = 50 μM.
between 0.01 to 0.05 mM compared with their respective control without AA (data not shown). Higher concentrations of AA (0.25 and 0.50 mM) caused a reduction in primary root growth for both WT and lpi4 seedlings. In L medium, 0.01 and 0.05 mM AA had no significant effect on lpi4 primary root growth, whereas treatment with the same concentrations resulted in 25% longer primary roots in WT plants compared to untreated controls (Fig. 4A). Higher AA concentrations resulted in a root growth inhibition in L medium for both lpi4 and WT seedlings. Since changes in, at least some, ROS occur during the meristem exhaustion process in WT plants, and treatment with AA promoted longer primary roots in Pi-deprived WT seedlings, we decided to analyze the effect of AA treatment on ROS distribution and meristem morphology in WT and lpi4 seedlings germinated in H and L media. In seedlings grown in H medium supplemented with concentrations of 0.01, 0.05 and 0.1 mM AA, the root tip of Pi-sufficient WT and lpi4 seedlings had a similar morphology and DAB staining to that of untreated seedlings,
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showing a DAB maximum staining in the central cylinder region around the QC (Fig. 4C–E compared with 4B, H–J compared with G). At a concentration of 0.25 mM DAB staining is more evenly distributed in all root cell types and the maximum around the QC is no longer observed (Fig. 4F and K). As previously shown in WT seedlings grown in L medium, DAB staining is much lower than in H seedlings (Fig. 4L compared with B), however treatment with 0.01 and 0.05 mM AA resulted in an increase in DAB staining and in a more typical root tip morphology that does not show the lack of organization and differentiation observed in Pi-deprived seedlings, i.e., root hairs are no longer formed near the root tip (Fig. 4M and N compared with L). Treatment of Pi-deprived WT seedlings with higher AA concentrations (0.1 and 0.25 mM) did not rescue the low Pi root tip phenotype or DAB staining (Fig. 4O and P compared with L). In the case of lpi4 seedlings grown in L medium, as expected, the ROS pattern and root tip morphology was in most cases similar to that observed in WT seedlings grown in H medium, except in seedlings treated with 0.25 mM AA in which DAB staining was significantly lower (Fig. 4U compared with B, G and Q). These data together suggest that changes in the level and distribution of ROS at the root tip correlate with meristem exhaustion process induced by Pi-deprivation and that treatment with low concentrations of AA can partially rescue the root tip phenotype of Pi deprived WT seedlings. Jasmonic acid partially reverts the long root phenotype of lpi4. Other important group of deregulated genes in lpi4 mutant unveiled from our microarrays results, are the genes involved in JA biosynthesis, such as LOX3, AOC2, AOC3, OPR3 (Sup. Table S4). Since in L medium the most conspicuous difference between lpi4 and WT is the absence of meristem exhaustion in lpi4, we tested the effect of treatment with different concentrations of JA (1, 5, 10 and 20 μM) on root tip morphology and root meristem maintenance in WT and lpi4 seedlings grown in H and L media. It was observed that in H medium, JA induced a similar dose-dependent reduction in primary root elongation in both the WT and lpi4 seedlings (Fig. 5A). Interestingly, in L medium, JA had a greater inhibitory effect on root elongation for lpi4 than that observed for the WT; whereas in the WT root length was not significantly affected by treatment with 1 and 5 μM JA, these treatments resulted in a significant reduction in lpi4 root length (approximately 40% and 60%, respectively) (Fig. 5B). Higher JA concentrations in L medium further reduced primary root length in the case of lpi4 seedlings, whereas for the WT only very high JA concentrations (20 μM) had an observable effect of primary root growth. These results suggest that JA seems to be involved in the Arabidopsis Pi starvation root tip response, since lpi4 seedlings recover the wild type phenotype after JA addition. Moreover, lpi4 was more susceptible to the primary root elongation inhibition by JA than the WT seedlings. It has been reported that low Pi induces several changes in the Arabidopsis root system morphology, such as inhibition of primary root length and primary root meristem consumption which is preceded by the lost of QC and cell division.18 Therefore, we decided to analyze the effect of JA treatment on primary root meristem maintenance on WT and lpi4 seedlings. We also examined
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Figure 4. Effect of ascorbic acid on root elongation and ROS pattern at the root tip. (A) Primary root length of WT and lpi4 mutant with ascorbic acid concentrations ranging between 0.01 and 0.5 mM. Photographs of primary root meristem at 10 days after germination showing DAB staining of root tip of WT seedlings grown in high (B–F) and low (L–P) Pi, lpi4 mutant seedling also grown high (G–K) and low (Q–U) Pi media supplement with the indicate concentration of AA. Different letters in (A) are used to indicate means that differ significantly (p < 0.05 n = 20). Scale bar = 50 μM.
the effect of JA treatment on the expression of the CycB1;1:uidA and QC46:GUS markers in WT seedlings. With this aim seedlings were germinated in H and L media supplemented with 10 μM JA. As expected, in H medium without JA the root meristem maintain its organization in both WT (Fig. 5C) and lpi4 (Fig. 5D) seedlings. In WT seedlings in H medium, expression of the cell cycle marker CycB1;1:uidA indicates normal cell proliferation (Fig. 5E) and expression of QC46::GUS shows an intact QC (Fig. 5F). In H medium supplement with 10 μM JA, no significant changes in meristem organization were observed in both WT (Fig. 5G compared with C) and lpi4 (Fig. 5H compared with D) neither in the expression of QC46 marker (Fig. 5J
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compared with F). However, it was observed that JA treatment drastically reduced cell division according to the reduction in CycB1;1::uidA marker expression (Fig. 5I compared with E). As previously reported, in L medium, WT seedlings show meristem disorganization (Fig. 5K), early differentiation (as indicated by the formation of root hairs very close to the root tip), decreased cell proliferation (Fig. 5M) and disorganization of the QC (Fig. 5N). In contrast to previously observed, Pi deficiency had no effect on lpi4 root meristem, as it maintained a normal organization and early differentiation is not observed (Fig. 5L). Treatment of WT seedlings with JA in L medium did not have a drastic effect (Fig. 5O compared with K), besides the acceleration of meristem exhaustion as indicated by the complete loss of cell proliferation and QC activity (Fig. 5Q and R respectively). Interestingly, JA treatment in L medium had a drastic effect on lpi4 root meristem, as its normal organization was lost and early differentiation occurred in a similar fashion to that observed in WT seedlings grown in L medium (Fig. 5P). These results suggest that JA plays an important role in the meristem exhaustion process observed in Pi-deprived Arabidopsis seedlings. However, since JA was not sufficient to trigger meristem exhaustion neither lpi4 or WT seedlings grown in H medium (Fig. 5G and H), indicates that this process not depend solely on JA which suggest that exist other factors involved in the root tip response to Pi deficiency. Ethylene effect on primary root meristem in Pi contrasted conditions. Previously it has been reported that the interaction between JA and ethylene plays an important role in response to both biotic and abiotic stresses.37,38 Thus we decided to investigate the effect of ethylene on the primary root meristem consumption process induced by Pi-deprivation. QC46::GUS seedlings were germinated in H and L Pi media supplement with 10 μM of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), 0.2 μM of ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG) and 20 μM of AgNO3 inhibitor or ethylene perception. We analyzed the root meristem zone at 10 dag by Nomarski microscopy. As expected, the primary root meristem of seedlings grown in H medium alone maintained its organization and typical expression of the QC marker was observed (Fig. 6A), whereas in L medium seedlings presented meristem consumption and the expression of the QC marker was not detectable (Fig. 6E). By contrast, in L medium supplement with either AVG (Fig. 6G) or AgNO3 (Fig. 6H), meristem organization is maintained and the expression of the QC marker is similar to that observed in seedlings grown in H medium (Fig. 6G compared with C and H compared with D). Seedlings treated with ACC showed a drastic effect of it on meristem organization both in H (Fig. 6B) and L medium (Fig. 6F). However, root meristem organization is not completely lost in H medium supplemented with ACC (Fig. 6B). These results show that ethylene has an important role in changes that occur in Arabidopsis root tip in response to Pi starvation but it is not sufficient to induce meristem exhaustion by itself because the root meristem of seedlings grown in H medium containing ACC were not completely exhausted. JA and ethylene act in a synergic manner in the Pi-deficiency response. Since neither JA and ethylene were able to emulate in
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H medium the changes in the root tip induced in response to Pi-starvation, and considering previous reports about the interaction between JA and ethylene in response both biotic and abiotic stresses,37,38 we decided to explore the effect of JA and ACC together on primary root meristem maintenance in seedlings grown in H media. With this aim, WT and lpi4 seedling were germinated in H medium supplemented with 10 μM JA and 10 μM ACC. It was observed that the combined treatment with JA and ACC in H medium produced a primary root meristem phenotype in WT and lpi4 seedlings quite similar to that observed for the WT grown in L medium (Fig. 7B and D). And as expected, the primary root meristem of WT and lpi4 seedlings grown in H medium without JA/ACC maintained its organization (Fig. 7A and C respectively). Moreover, lpi4 seedlings grown in L medium supplemented with JA/ACC also produced a phenotype that resembles to WT in L medium (Fig. 7F). In contrast, lpi4 seedlings grown in L medium without JA/ACC show an organized meristem (Fig. 7E) as we have already shown before. These results suggest that both JA and ethylene are necessary to trigger, in seedlings grown in H medium, the meristem exhaustion process characteristic of the Arabidopsis root tip response to Pi deprivation. Discussion It has been previously reported that among the different physiological, molecular and cellular responses of Arabidopsis to Pi-deprivation, there are important alteration in the postembryonic development of the root system.39 Three main changes in root architecture occur in Arabidopsis in response to low Pi availability as compared to plants grown in Pi sufficient conditions: (1) root hair length and density increases, (2) lateral root formation is also increased and (3) primary root growth is inhibited that is accompanied by loss of root meristematic activity. Several lines of evidence show that the increase in lateral root formation is mediated by an increase in auxin sensitivity and concentration in the roots of Pi deprived seedlings.7,40 However, very little information is available about the hormones and signaling pathways that mediate the increase in density and length of root hairs and in the arrest of primary root growth, although both processes seem to be independent of auxin signaling.41 The site of Pi sensing that mediates primary root growth arrest and meristem exhaustion have propose to occur at the root tip, in which two members of the a multicopper oxidase participate.14 To explore the pathways involved in the inhibition of primary root growth and the loss of root meristematic activity in Arabidopsis as a response to Pi-deprivation, we carried out global gene expression analysis of root tips exposes to Pi limiting conditions. To have more direct information about the signaling and metabolic pathways involved in these processes, in this global gene expression analysis we compared the differences between the WT and lpi4, assuming that those genes involved in the inhibition of primary root growth and meristem exhaustion, which expression is induced or repressed in the WT would be not affected in lpi4 and therefore, these genes could be identified as differentially expressed when comparing the expression profile of WT and lpi4.
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Figure 5. Effect of JA on primary root length and primary root meristem maintenance in Pi-deprived seedlings. JA was applied at indicate concentrations in the growing medium. Primary root length of WT and lpi4 seedlings grown in H (A) and L medium (B). Photographs of the root tip from 10 dag seedlings grown in H medium without (C–F) and with 10 μM JA (G–J) of WT (C and G), lpi4 (D and H), CycB1;1::uidA (E and I) and QC46::GUS (F and J). Photographs of the root tip from 10 dag seedlings grown in L medium without (K–N) and with 10 μM JA (O–R) of WT (K and O), lpi4 (L and P), CycB1;1::uidA(M and Q) and QC46::GUS(N and R). Note the inhibitor effect of JA on cell cycle and the drastic effect on root meristem organization in low Pi conditions. Scale bar = 50 μM.
From the microarray analysis, we decided to focus mainly on two metabolic processes, oxidative stress and signaling pathway mediated by hormones such as JA and ethylene. The production of ROS during abiotic stresses was long considered as toxic by-products of aerobic metabolism, however, in recent years, this concept has changed and it is now generally accepted
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that ROS not only are toxic compounds produced when plants are subjected to different types of stress, but also that ROS can play an important role as key regulators of many biological processes such as growth, cell cycle, programmed cell death, hormone signaling, biotic and abiotic cell responses and development.42-44 Among the differentially expressed genes between the root tips of WT and lpi4 Pi-deprived seedlings, we identified several genes that are upregulated in WT seedlings that play important roles in the homeostasis of redox status, such as superoxide dismutase, glutathione reductase, ascorbate peroxidase, dehydroascorbate reductase and catalase (Sup. Fig. S3). This suggests that during the root tip meristem exhaustion process an imbalance in redox status might occur. DAB staining of the root tip of WT seedlings grown in L medium showed a gradual decrease in H2O2, which correlates with the induction of peroxiFigure 6. Effect of ethylene on root meristem maintenance in high and low dase genes in Pi-deprived WT seedlings. Moreover, it Pi media. Photographs of QC46::GUS primary root meristem 10 dag grown in was found that treatment of WT seedlings germinated high Pi: control (A), medium supplement with 10 μM ACC (B), 0.2 μM AVG (C) and 20 μM of AgNO3 (D). Root tip of 10 dag QC46::GUS seedlings grown in low in L medium with low concentrations of AA promoted Pi: control (E), medium containing 10 μM ACC (F), 0.2 μM AVG (G) and 20 μM of primary root growth (Fig. 4A), the maintenance of the AgNO3 (H). Scale bar = 50 μM. primary root meristem and the ROS pattern. It has been reported that AA has a role stimulating cell division and cell elongation in the Arabidopsis root meristem.34-36 Our results show that in the WT ascorbate oxidase is induced in response to Pi-deprivation, which would lead to an oxidation of AA into monodehydroascorbate (MDHA) that inhibits cell division and a concomitant decrease in H2O2, trough the coupling with the reduction of H 2O2 to H2O. The loss of the hydrogen peroxide maximum in the root tip could be involved in the process of meristem exhaustion. Therefore, it is possible that treatment with exogenous AA, the ratio of AA to MDHA leads to the partial restoration in the concetration of cellular AA and of the redox status required to sustain cell division, which in turn directly or indirectly would result in an increase in H2O2 levels in the RAM, which is reflected in the increase in primary root elongation of Pi-deprived seedlings treated with AA. Further experiments are needed to demostrate the direct link of AA and ascorbate oxidase levels with the maintenance of the redox status necessary to maintain the QC and cell division in the root apical meristem. Nevertheless, our results support the notion that redox status plays an important role in the regulation of RAM organization and maintanance in response to environmental factors. It has been reported that there is a close interplay between hormones such jasmonic acid and ethylene and the production of ROS and redox balance in some plant tissues.45 Moreover, it has been shown that treatment of JA on Arabidopsis seedlings causes an inhibition of primary root growth.46,47 Moreover, our data indicates that the low Pi-induced alterations in root develFigure 7. Synergistic effect of JA and ethylene on root tip morphology. opment and morphology correlate with the induction of genes The pictures show the morphology of primary root from WT seedlings involved in JA synthesis and signaling pathways (Sup. Fig. S4 grown in Pi sufficient media alone (A) and supplement with 10 μM and Table S4), suggesting that JA could play a role in the root JA/10 μM ACC (B). Primary root tip of lpi4 seedlings grown in high media without (C) and with 10 μM JA/10 μM ACC (D) and grown in low Pi mearchitecture changes observed in Pi-deprived seedlings. We dia without (E) and supplemented with 10 μM JA/10 μM ACC (F). also found that treatment of Pi-deprived lpi4 seedlings with JA resulted in a reduction of primary root growth and root meristem
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exhaustion similar to that observed for WT controls grown in the same media, suggesting that indeed JA plays an important role in the inhibition of primary root growth induce by low Pi. However, since treatment of WT and lpi4 seedlings with JA in H medium inhibited primary root growth but did not caused the loss of meristematic activity, suggest that and increase in JA synthesis and the activation of its signaling pathway is necessary but not sufficient to trigger the meristem exhaustion process observed in Pi-deprived Arabidopsis seedlings. Some reports have demonstrated that several JA-activated processes involve positive and negative cross-talks between JA and ethylene signaling pathways48,49 through the induction of genes that encode key transcription factors like ERF1 in Arabidopsis.31 The microarray data revealed that genes involved in biosynthesis and signaling of plant hormones are induced in the WT in response to low Pi (observed as repressed in lpi4), including key genes in the crosstalk between ethylene and JA, e.g., ERF2 that acts as an activator of JA-responsive defense genes.50 It has also been proposed that ethylene is part of a signaling pathway that modulates cell division in the QC during the development of the root system.51 Previously it has been reported that Pi starvation induce cell division in the QC,18 so we decided to test the effect of ethylene on primary root meristem of WT and lpi4 seedlings grown in H and L media. Ethylene inhibitors such as AVG an AgNO3 prevent meristem exhaustion in Pi-deprived seedlings (Fig. 6G and H) and ethylene precursor ACC show a drastic effect on meristem organization both in H and L media. However, in H medium the organization of the meristem is not completely lost (Fig. 6B). Therefore, we decided to test JA and ACC together in WT seedling grown in H media and lpi4 grown both H and L media, in all three cases, the primary root meristem was exhausted quite similar to that of WT seedling grown in L media. These results show that JA and ACC act synergistically and have an important role in the root tip response to Pi deprivation. All together our results show that interplay of alterations of redox status and hormone signaling plays an important role in the Arabidopsis root tip response to Pi-deprivation. Hormones act as chemical messengers in the modulation of physiological and molecular processes regulating growth and development. To successfully complete their life cycle, plants heavily depend upon the proper physiological and developmental adjustments that determine their ability to secure edaphic resources. Therefore, hormones probably play an essential role as integrators of environmental signals with developmental processes. The finding that Pi availability regulates the expression of genes involved in JA and ethylene synthesis and signaling in the root tip of Arabidopsis seedlings, illustrates how the interaction of hormone signaling and environmental cues can impact developmental processes that alter postembryonic development to produce root system architectures that enable plants to adapt to nutrient limiting conditions. Our results also show the utility of global gene expression profiling to compare the responses of different mutant or ecotype background as a tool to identify genes and pathways involved in the responses of plants to environmental signals.
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Materials and Methods Plant material and growth conditions. Arabidopsis thaliana ecotype Col-0 was used as a WT type control for all experiments. The Arabidopsis previously reported transgenic lines CycB1;1:uidA,52 QC46:GUS53 and the mutant line lpi4,19 were used in these experiments. Seeds were surface sterilized by sequential treatments with 95% (v/v) ethanol for 5 min, with 20% (v/v) bleach for 7 min and rinsed five times with distilled water. Sterilized seeds were stored at 4°C in sterile water were for 48 hours and then were germinated on agar plates containing low (5 μM) or high (1 mM) NaH2PO4 in 0.1x MS medium, as described by López-Bucio et al.7 Plates were placed vertically in a plant growth chamber (Percival Scientific, Perry, IA), under a photoperiod of 16 h light/8 h darkness, and temperature of 22–24°C. Experimental design and microarray platform. For microarray analyses, a loop design was implemented in order to contrast gene expression differences between lpi4 and WT Arabidopsis root tips. Root tip sections of approximately 300 μm in length were collected from 1, 4 and 7 dag of seedlings grown in L medium. Four biological replicates representing each sampling point were obtained by pooling the whole root tips of at least 800 randomly chosen plants. This experiment involved a total of 12 sets of microarray hybridizations, including direct and dye swap comparisons between treatments as well as across time points for the same treatment. This design allowed us to determine differences in gene expression between Pi-depleted and control meristem roots (Pi availability effect) and whether the differences were time dependent (Pi x time effect). The Arabidopsis Oligonucleotide Microarrays (AOM) from ag.arizona.edu/microarray/was used to carry out this study. The AOM contains about 32,000 individual spots and putatively contains all Arabidopsis genes identified to date. RNA isolation, labeling, hybridization and image processing. RNA isolation, fluorescent labeling of probes, slide hybridization and washing were performed as described previously in reference 25. Slides were scanned with an Axon GenePix 4100 scanner at a resolution of 10 μm adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. Spot intensities were quantified using Axon GenePix Pro 5.1 image analysis software. Normalization and data analysis. Raw data from the 12 slides was imported into the R 2.2.1 software (http://www.R-project. org) and background correction was carried out using the Robust Multichip Analysis (RMA) method.54 Normalization of the corrected signal intensities within slides was carried out using the “printtiploess” method55 using the LIMMA package (at www. bioconductor.org).56 Similar as described previously in reference 25, normalized data were fitted into mixed model ANOVA’s57,58 using the Mixed procedure (SAS 9.0 software, SAS Institute Inc., Cary, NC USA) with two sequenced linear models considering as fixed effects the dye, time, Pi-treatment and time x Pi-treatment. Array and array x dye were considered as random effects. The Type 3 F-tests and P-values of the time x Pi-treatment and P-treatment model terms were explored and significance levels for those terms were adjusted for by the False Discovery Rate (FDR)
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method.59 Estimates of the expression differences were calculated using the mixed model. Based on these statistical analyses, the spots with an FDR value of less than or equal to 0.15 and with changes in signal intensity between WT and lpi4 RNA samples of 2.0-fold or higher were considered as differentially expressed. Functional classification of differential genes. Functional classifications of differential genes were performed according to the Functional Catalogue (FunCat) version 2.1 available at http://mips.gsf.de/proj/funcatDB.26 Over-represented functional categories in the two lists (up and downregulated genes) as compared to the complete Arabidopsis genome were obtained according to the MIPS classification using BioMaps tool from the VirtualPlant webpage (virtualplant.bio.nyu.edu). Hypergeometric method and Bonferroni correction were used for the analysis with a p value cutoff of 0.05. qRT-PCR. Genes whose expression was considered as regulated by Pi deficiency in the microarray analysis were selected with the aims of validating the expression patterns found. Primer design (Tm, 60–65°C) was performed according the guidelines recommended in the Primer Express Software, Version 3 (Applied Biosystems) using as template the original target sequences from which the oligonucleotides printed in the array were designed. Oligonucleotide sequences for qRT-PCR are shown in Supplemental Table S5. cDNA templates for PCR amplification were prepared from each condition by using reverse specific primers for each gene to evaluate (Sup. Table S5) and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Each reaction contained cDNA template from 10 μg total RNA, 1x SYBR Green PCR Master Mix (Applied Biosystems) and 500 nM forward and reverse primers. Real-time PCR was performed in an ABI PRISM 7500 sequence detection system (Applied Biosystems) under the following thermal cycling conditions: 10 min at 95°C followed by a total of 40 cycles of 30 s at 95°C, 30 s min at 60°C and 40 s at 72°C. For qRT-PCR, relative transcript abundance was calculated and normalized with respect to ACTIN 1 transcript levels. Data shown represents mean values and standard error obtained from at least three independent amplification reactions. All calculations and analyses were References 1. Schachtman DP, Reid RJ, Ayling SL. Phosphorus uptake by plants: from soil to cells. Plant Physiol 1998; 116:447-53. 2. Holford ICR. Soil phosphorus: Its measurement and its uptake by plants. Aust J Soil Res 1997; 35:227-39. 3. Raghothama KG. Phosphate Acquisition. Annu Rev Plant Phys 1999; 50:665-93. 4. Vance CP, Uhde-Stone C, Allan DL. Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytol 2003; 157:423-47. 5. Bates TR, Lynch JP. Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant Cell Environ 1996; 19:529-38. 6. Williamson L, Ribrioux S, Fitter A, Leyser O. Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiol 2001; 126:1-8.
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performed using 7500 Software v2.0.1 (Applied Biosystems) and the 2-ΔΔCt method with a relative quantification (RQ)min/ RQmax confidence set at 95%.60 The error bars display the calculated maximum (RQmax) and minimum (RQmin) expression levels that represent SE of the mean expression level (RQ value). Amplification efficiency (87.3% to 97.8%) for the primer sets was determined by amplification of cDNA dilution series (1:5). Specificity of the RT-PCR products was followed by a melting curve analysis with continual fluorescence data acquisition during the 65–95°C melt. ROS Detection. For H2O2 localization we used the DAB staining method as described by Orozco and Ryan 1999.61 Histochemical analysis. For histochemical analysis of GUS activity, Arabidopsis seedlings were incubated overnight at 37°C in a GUS reaction buffer (0.5 mg/mL of 5-bromo-4-chloro-3-indolyl-b-D-glucuronide in 100 mM sodium phosphate, pH 7). Histological Analysis WT and mutant seedlings were cleared using the method previously described by Malamy and Benfey.62 At least 10 transgenic plants were analyzed. A representative plant was chosen for each Pi treatment and imaged using Nomarski optics on a Leica DMR microscope. Hormone treatments. L (5 μM) or H (1 mM) NaH2PO4 in 0.1x MS medium before pouring into plates was supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) from a 1 mM stock solution of ACC dissolved in water, with jasmonic acid (JA) from a 1 mM stock solution dissolved in ethanol 20%. The final hormone concentrations in plates were ACC 10 μM and JA 10 μM. Both ACC and JA were obtained from SigmaTM (ACC catalogue # A3903; JA catalogue # J2500). Acknowledgements
A.C.L. is indebted to CONACyT (Mexico) for a Ph.D. fellowship. This work was supported in part by grants from the Howard Hughes Medical Institute (Grant 55005946) and CONACyT (299/43979 and 106725) to L.H.E. Note
Supplemental materials can be found at: www.landesbioscience.com/journals/psb/article/14160
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