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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Proteomic Analysis of Different Mutant Genotypes of Arabidopsis Led to the Identification of 11 Proteins Correlating with Adventitious Root Development^1,[W]

Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France (C.S., C.B.); Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umea, Sweden (C.S., G.S., C.B.); and Unité Mixte de Recherche de Génétique Végétale, Institut National de la Recherche Scientifique/Centre National de la Recherche Scientifique/Université Paris-Sud/Institut National Agronomique Paris-Grignon, 91190 Gif-sur-Yvette, France (L.N., T.B., H.C., M.-P.J., M.D., M.Z.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.

While the physiology of adventitious root formation is reasonably well known, the genetic and molecular mechanisms involved are still poorly understood. The few genetic and molecular studies of adventitious rooting that have been undertaken have shown that it is a heritable character, and quantitative trait loci analyses of adventitious rooting in trees (Han et al., 1994; Marques et al., 1999) and in Arabidopsis (Arabidopsis thaliana; King and Stimart, 1998) showed that it is a quantitative genetic trait as expected for a character controlled by multiple factors. Recently, Brinker et al. (2004) investigated gene expression during adventitious root formation in Pinus contorta hypocotyls pulse treated with auxin and harvested at different developmental time points of root development. More than 200 genes were differentially expressed during the different phases analyzed. These genes were related to auxin transport, photosynthesis, cell replication, or cell wall synthesis (Brinker et al., 2004). In Arabidopsis, nine temperature-sensitive mutants have been isolated that are altered in different stages of adventitious root formation from hypocotyl explants (Konishi and Sugiyama, 2003). One of the mutations has been identified. It is in the MOR1/GEM1 gene that encodes a microtubule-associated protein, suggesting that microtubule organization is important during root meristem initiation. Most model systems have compared explants treated with exogenous auxin with non-auxin-treated material, making it difficult to distinguish events specifically associated with adventitious rooting from those resulting from exogenous auxin application. We took advantage of two classes of Arabidopsis mutants affected in adventitious root formation for identifying molecular markers associated with the initiation and developmental phases of adventitious rooting. The superroot1 (sur1) and superroot2 (sur2) mutants are auxin overproducers that spontaneously develop adventitious roots on the hypocotyl (Boerjan et al., 1995; Delarue et al., 1998). The superroot genes have been identified, and both were shown to encode enzymes of the indole-glucosinolate pathway (Bak et al., 2001; Mikkelsen et al., 2004). SUR2 encodes CYP83B1 (Barlier et al., 2000), which converts indole-3-acetaldoxime in 1-aci-nitro-2 indolyl-ethane, and SUR1 encodes the C-S lyase that acts one step later, catalyzing the conversion of S-alkylthiohydroximate to thiohydroximate (Bak et al., 2001; Mikkelsen et al., 2004). The blockage of either of these steps results in the redirection of indole-3-acetaldoxime toward indole-3-acetic acid (IAA) biosynthesis (Bak et al., 2001; Mikkelsen et al., 2004).

The argonaute1 (ago1) mutant was first characterized as a leaf developmental mutant (Bohmert et al., 1998). We showed recently that both the null allele ago1-3 and hypomorphic alleles were altered in their ability to produce adventitious roots (Sorin et al., 2005). AGO1 is the founding member of a gene family that is conserved among eukaryotes (Bohmert et al., 1998) and has been shown to be involved in the regulation of posttranscriptional gene silencing (Fagard et al., 2000; Morel et al., 2002). More recently, the AGO1 gene was shown to play a crucial role in the regulation of gene expression through the microRNA (miRNA) pathway, where it is potentially required for miRNA-directed mRNA cleavage (Vaucheret et al., 2004). ago1 has a pleiotropic developmental phenotype, some aspects of which can be explained by the deregulation of transcription factors targeted by miRNAs (Jones-Rhoades and Bartel, 2004). We recently showed that the defect in adventitious rooting in ago1 mutants can at least partially be explained by the accumulation of the auxin response factor ARF17 (Sorin et al., 2005), whose expression is regulated by the miRNA mir160 (Jones-Rhoades and Bartel, 2004; Mallory et al., 2005). A transgenic line overexpressing ARF17 was shown to produce less adventitious roots than the wild type, suggesting that ARF17 could be a major regulator of adventitious rooting. To identify other genes involved in adventitious root development, we coupled genetic and physiological analysis with a proteomic characterization of different single and double mutants.

This approach has already been successfully used for the characterization of Arabidopsis developmental mutants (Santoni et al., 1994, 1997) and the identification of proteins correlated to a developmental process. The first characterization of two-dimensional (2-D) protein patterns of Arabidopsis mutants led to the identification of proteins positively correlated with hypocotyl elongation, one of which was actin (Santoni et al., 1994). Biochemical distance indices between Arabidopsis mutants could be estimated from 2-D electrophoresis data and correlated with phenotypic and physiological analysis of the mutants (Santoni et al., 1997). More recently, proteome analysis has become a major approach for functional characterization of plants (for review, see Canovas et al., 2004). It has proven useful for the identification of proteins associated with a range of developmental or physiological processes (de Vienne et al., 1999; Plomion et al., 2000; Bae et al., 2003; Gallardo et al., 2003; Mang et al., 2004; Schiltz et al., 2004; Vincent et al., 2005).

In this article, we describe the analysis and comparison of 2-D protein profiles of hypocotyls of ago1-3, sur1-3, sur2-1, and sur2-1ago1-3 Arabidopsis mutants during initiation of adventitious roots. This showed that, although AGO1 is involved in the regulation of gene expression through the miRNA pathway, its mutation does not induce more variations in hypocotyls than a mutation in SUR1 or SUR2. We also showed significant differences between the sur1 and sur2 protein profiles, although the two genes act in the same biosynthesis pathway. Finally, we identified 11 proteins, including three auxin-inducible GH3-like proteins, whose content correlated either positively or negatively with early and/or late phases of adventitious root development.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Analysis of 2-D Protein Patterns of the Different Genotypes

This work describes proteomic variations between the ago1-3, sur1-3, and sur2-1 Arabidopsis mutants and the sur2-1ago1-3 double mutant, with particular reference to the early developmental events associated with adventitious root initiation in the hypocotyl. All four genotypes behave differently in terms of adventitious root development. The auxin overproducers sur1-3 and sur2-1 spontaneously develop adventitious roots on the hypocotyl either in the light (Boerjan et al., 1995; Delarue et al., 1998) or when etiolated prior to transfer to the light (Sorin et al., 2005). In contrast, the ago1-3 mutant and the sur2-1ago1-3 double mutant were unable or barely able to produce adventitious roots when grown directly in the light or after being etiolated prior to transfer to the light (Sorin et al., 2005).

Seeds were germinated in the dark and young seedlings were etiolated until they were about 5 mm and then transferred to the light for 48 h, as described previously (Sorin et al., 2005). Although wild-type, ago1, and superroot mutants have very different adult phenotypes, they are phenotypically almost identical to each other at this developmental stage, that is, before any adventitious root primordia have emerged from the hypocotyls of wild-type or superroot mutants (Sorin et al., 2005). Microscopic observation of cleared hypocotyls showed that, in all genotypes, adventitious roots initiated from pericycle cells opposite to xylem poles, as described previously (Boerjan et al., 1995; Delarue et al., 1998; Takahashi et al., 2003; Sorin et al., 2005). Nevertheless, a clear difference in the number of very early root primordia could be observed between the genotypes as shown by the early differential expression of the CyclinB1:uidA reporter gene that was used to monitor adventitious root initiation in sur2-1 mutants and sur2-1ago1-3 double mutants (Sorin et al., 2005). This developmental stage was therefore suitable for protein profiling. Soluble proteins were prepared from dissected hypocotyls and subjected to 2-D PAGE (Fig. 1 ) as described in "Materials and Methods." The proteomic data were then analyzed for correlation of the abundance of individual proteins with the number of adventitious roots and/or the endogenous auxin content among the different genotypes. In this way, genes whose expression was correlated to adventitious root formation could be identified.

View larger version (123K): [in this window] [in a new window]   Figure 1. Representative 2-D gel pattern of hypocotyl proteins from Arabidopsis seedlings etiolated and transferred to light for 48 h. A typical gel of the genotype sur2-1 (RG) is presented as a representative 2-D gel. The spots that could be identified are indicated. Rings indicate spots that cannot be seen on this gel. The spots underlined have been correlated to one or more parameters (Table III; Fig. 2) The GH3-like proteins (square boxes) are spots 1,157 (GH3-6/DFL1), 1,166 (GH3-5/AtGH3a), and 1,253 (GH3-3). Spots in triangle, 2,230 is CORI-7/AtST5a; 1,871 and 1,875 are two putative isoforms of the JR1 lectin (At3g16470). Proteins were prepared as described in "Materials and Methods," subjected to 2-D PAGE, and the resulting gels were then silver stained.

View larger version (100K): [in this window] [in a new window]   Figure 2. Example of the differential accumulation of seven correlated spots identified in the comparative analysis of protein profiles of etiolated Arabidopsis seedling transferred to light for 48 h. Spots 1,157 (At5g28540), 1,166 (At1g79930), and 1,253 (At5g20890), three GH3-like proteins, were positively correlated with the number of mature adventitious roots (A). Spot 2,230 (At1g74100, CORI-7/AtST5) was positively correlated with the free IAA content and the number of adventitious root primordia 2 d after transfer to light. Spots 2,274 (At3g18490) and 4,742 (At4g38970) were negatively correlated with the free IAA content and the number of adventitious root primordia 2 d after transfer to light (B). Proteins were prepared as described in "Materials and Methods," subjected to 2-D electrophoresis, and the resulting gels were then silver stained.

We also compared variations observed in ago1-3 versus wild type (Col-0) to those in sur1-3 versus wild type (Col-0) and in sur2-1 versus wild type (Ws) after removal of the spots variable between the two wild-type ecotypes Ws and Col-0 (Table I). Interestingly, we could see that 37 spots were commonly affected by the ago1-3 and sur1-3 mutations, which represents on average 50% of the variable spots in ago1-3 and sur1-3 compared to their wild types. Three spots were absent in the wild type (Col-0) and appeared in both sur1-3 and ago1-3, and one spot varied in opposite ways in sur1-3 and ago1-3. In the comparison between ago1-3/wild type (Col-0) and sur2-1/wild type (Ws), 14 spots were commonly affected by sur2-1 and ago1-3 mutations. This indicates that there are more than 50% less common variable spots in ago1-3/wild type (Col-0) and sur2-1/wild type (Ws) than in ago1-3/wild type (Col-0) and sur1-3/wild type (Col-0). Three of them varied in opposite ways in sur2-1 and ago1-3 compared to their respective wild types.

The sur2-1ago1-3 double mutant derived from a cross between a homozygote sur2-1 plant in a Ws ecotype and a plant heterozygote for the ago1-3 mutation in a Col-0 background. This recombinant genetic background will be referred to as recombinant genotype (RG) in the remainder of the article. The characterization of the double mutant sur2-1ago1-3 (RG) showed that they produced more auxin than the wild type and the single ago1-3 mutant, but less than sur2-1 (RG) and developed no, or very few, adventitious roots (Table II; Sorin et al., 2005). We thus investigated the protein variation in the double mutant sur2-1ago1-3 (RG). The 2-D protein pattern of sur2-1ago1-3 (RG) was compared to that of siblings homozygous for sur2-1 in the same genetic background (i.e. RG). Quantitative and qualitative variations are summarized in Table I. Only 4% of the spots were shown to be variable between sur2-1ago1-3 (RG) and sur2-1 (RG), although 7.4% were variable in sur2-1 (Ws) compared to Ws and 7.1% in ago1-3 (Col-0) compared to Col-0. A possible reason is that the ago1 mutation has no effect on the level of accumulation of several proteins in a sur2-1 background, whereas it has in a wild-type background. After elimination of spots significantly variable between genetic backgrounds, seven were shown to be variable only in the sur2-1ago1-3 (RG) versus sur2-1 (RG) comparison and neither in the ago1-3 (Col-0) versus wild-type (Col-0) comparison nor in the sur2-1 (Ws) versus Ws comparison. These variations reflect the effects of the ago1-3 mutation that can only be seen in a sur2-1 genetic background. Nine other spots were significantly affected by the ago1-3 mutation in a wild-type background and a sur2-1 background. This result fits with the previously observed phenotypic effect of ago1 in a sur2-1 background. Indeed, the double mutant sur2-1ago1-3 displays an ago1-3-like phenotype in the aerial parts of the plant (Sorin et al., 2005). Thus, groupings of variable spots can be made according to their behavior.

To identify proteins associated with adventitious root formation, we computed Pearson correlations of spot intensities with free IAA content, number of adventitious root primordia 2 d after seedling transfer to light, and number of adventitious roots 7 d after transfer to light. These different parameters are reported in Table II and were defined either based on a previous characterization of the mutants (Sorin et al., 2005) or as described in "Materials and Methods."

Because sur1-3 and ago1-3 are sterile, the double mutant sur1-3ago1-3 could only be unambiguously identified in the 2-week-old progeny of double heterozygotes grown in light (Camus, 1999). It was impossible to distinguish the phenotype of the double mutant sur1-3ago1-3 from the single mutant ago1-3 at the young stages of development used for proteomic analysis (data not shown). In addition, as a consequence of the strong phenotype of sur1-3, it was impossible to determine the number of adventitious roots 7 d after transfer to light because there were too many. Therefore, we preferred to eliminate sur1-3 from the correlation analysis. We are in the process of creating an inducible sur1 mutant that hopefully should allow easier identification of double mutants and completion of this analysis.

Only the spots that were shown to be significantly variable after variance analysis were used for correlation analysis. Pearson correlation coefficients were computed between the mean of the relative intensity in the six genotypes and the different parameters. The results of the correlations are reported in Table III. Among the variable proteins that were affected by at least one mutation, 18 showed a significant correlation (P < 0.01) with at least one parameter. Positively or negatively correlated spots were in approximately the same amount for each parameter.

Identification of Variable Spots and Correlated Spots

From the 192 spots that were significantly variable, only 110 were identifiable on preparative gels stained with SYPRO ruby protein gel stain (see "Materials and Methods") for identification via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among these 110, several did not give enough protein for proper sequencing or were degraded. After LC-MS/MS analysis, only 50 could be unambiguously identified (Table IV). For several spots, several proteins matched the sequence data and were not reported in Table IV. Nevertheless, they can be found in Supplemental Table I, where detailed sequence data are provided. These data will be made available together with quantitative data on the freely accessible database PROTICdb (Ferry-Dumazet et al., 2005; http://moulon.inra.fr/bioinfo/PROTICdb), designed for storage and analysis of plant proteome data obtained by 2-D PAGE and MS.

Four (At3g01500, ATCG00490, At2g21330, and At1g53240) out of the 10 proteins related to energy and carbon metabolism were up-regulated in ago1-3, and one (At2g21330) was up-regulated in sur2-1ago1-3. At1g31390, a putative fructokinase, was also found up-regulated in sur2-1ago1-3, but since the induction level (1.45) was just below the cutoff level of 1.5, it appears in Table IV as nonsignificantly variable. In contrast, five proteins (At1g06680, At1g32060, At2g21330, At2g38970, and At1g31390) of the same category were down-regulated in sur2-1.

Twelve proteins were significantly variable in sur1-3 compared to Col-0 but were not affected by the sur2-1 mutation, whereas 18 proteins were significantly variable in sur2-1 compared to Ws but were not affected by the sur1-3 mutation. Seven proteins were affected by both mutations and were always modified in the same way (i.e. either up-regulated or down-regulated) in both mutant genotypes. At1g62380 and At3g11830 (a putative 1-aminocyclopropane-1-carboxylic acid oxidase and a putative T-complex protein 1 -subunit, respectively) were also up-regulated in sur1-3, but since their induction levels were just below the cutoff level (1.35 and 1.45, respectively), they appear in Table IV as nonsignificantly variable.

Interestingly, two lectins (At3g16450 and At3g16470) similar to myrosinase-binding proteins were identified. One of them (At3g16470) may have been posttranslationally modified as it was found in two different locations (see spots 1,871 and 1,875 in Fig. 1). They were both significantly variable in ago1-3, but only At3g16450 was significantly down-regulated in sur2-1ago1-3 (RG). At3g16450 was significantly down-regulated in sur1-3 compared to Col-0, but no significant variation was observed in sur2-1 compared to Ws. Both putative At3g16470 isoforms were down-regulated in ago1-3 compared to Col-0. Interestingly, the isoform corresponding to spot 1,875 was down-regulated in sur1-3 compared to Col-0 but not in sur2-1 compared to Ws, and it was the opposite for spot 1,871.

Three different auxin-inducible GH3-like proteins (GH3-3/At2g23170, GH3-5/AtGH3a/At4g27260, and GH3-6/DFL1/At5g54510) were up-regulated in sur2-1 and down-regulated in sur2-1ago1-3 (RG; Table IV; Figs. 2A and 3B ). Surprisingly, only one of these GH3-like proteins appeared to significantly accumulate in sur1-3 (Table IV).

View larger version (19K): [in this window] [in a new window]   Figure 3. GH3-like protein and RNA level in wild-type and mutant seedlings 2 d after transfer to light. A, Analysis of expression of the GH3 genes by RT-PCR in wild type (Col-0), ago1-3 (Col-0), sur2-1 (RG), and sur2-1ago1-3 (RG). Total RNAs were extracted from hypocotyls of wild-type (Col-0), ago1-3 (Col-0), sur2-1 (RG), and sur2-1ago1-3 (RG) siblings etiolated in the dark until the hypocotyl reached a 5-mm length and then transferred to light for 48 h. The relative expression of the genes was estimated by semiquantitative RT-PCR using 18S RNA as an internal control. For each genotype, expression of each gene was presented as a ratio between the value for the gene and the value of the corresponding 18S RNA. Units are arbitrary. Errors bars = SD of three PCR reactions. The experiment was performed on two independent biological replicates. B, Differential accumulation of the three GH3 proteins in different genotypes. Proteins were prepared as described in "Materials and Methods" and subjected to 2-D PAGE. Gels were analyzed as described in "Materials and Methods," and the volume value was estimated for each protein spot. Histograms were performed with the mean value of the volume for each genotype (mean of three or four values depending on the genotype) and for each spot. When a spot was absent in one of the gels, a value of zero was attributed to the volume of the spot (relative amount of protein) in the corresponding gel. Error bars = SE.

Out of the 18 correlated spots, 11 could be identified (Tables III and IV; Fig. 2). Five of these were negatively correlated with at least one parameter (Table III). Two proteins from the energy/carbon metabolism group (At1g32060 and At4g38970) were negatively correlated to adventitious root number. At4g38970 was also negatively correlated to the free IAA content. This was also the case for one protein related to protein degradation (At3g18490), also annotated as a putative chloroplast nucleoid DNA-binding protein. At1g32060 was negatively correlated to the three parameters as it correlated also with free IAA content and the number of adventitious root primordia. Two proteins related to stress responses were negatively correlated, the first one (At4g25100) to the number of primordia 2 d after transfer to light and the second (At5g20630) to the number of adventitious roots 7 d after transfer to light.

Among the identified proteins, six were positively correlated with one or more parameters. One protein related to glucosinolate metabolism (CORI-7/AtST5a/At1g74100), which was up-regulated in both sur1-3 and sur2-1, was positively correlated with both free IAA content and adventitious root primordia 2 d after transfer to light. Two putative chaperone proteins were positively correlated to the number of primordia 48 h after transfer to light. The three GH3-like proteins were all positively correlated with the number of adventitious roots 7 d after germination but were surprisingly not correlated with free IAA content, although they are known to be auxin-inducible proteins.

Expression of GH3-Like Genes Is Down-Regulated in the ago1-3 Background But Still Inducible by Exogenous Auxin

Because GH3-like proteins (GH3-3/At2g23170, GH3-5/AtGH3-a/At4g27260, and GH3-6/DFL1/At5g54510) that were positively correlated with adventitious root number belong to an auxin-inducible protein family and were recently shown to be auxin-conjugating enzymes (Staswick et al., 2002, 2005), we decided to complete the analysis by checking their expression at the transcriptional level in the wild type (Col-0), ago1-3 (Col-0), sur2-1 (RG), and sur2-1ago1-3 (RG) using a semiquantitative reverse transcription (RT)-PCR technique (Fig. 3A). All three genes were down-regulated in ago1-3 compared to the wild type (Col-0; P < 0.001 for GH3-6/DFL1 and GH3-5/AtGH3a and P < 0.01 for GH3-3). Although no significant difference in protein accumulation could be detected between ago1-3 (Col-0) and wild type (Col-0; Fig. 3B), most likely because the spot intensity was at the limit of the detection level (Fig. 2A), this suggests that difference in protein accumulation might occur between the two genotypes. GH3-5/AtGH3a, GH3-3, and GH3-6/DFL1 transcription was down-regulated in the double mutant sur2-1ago1-3 (RG) compared to the sur2-1 (RG) mutant (P < 0.001). Indeed, we found that the mRNA level of these three genes was almost the same in sur2-1ago1-3 (RG) as in ago1-3 (Col-0) and were significantly lower than in Col-0 (P < 0.05). Nineteen GH3 genes have been described in Arabidopsis (Hagen and Guilfoyle, 2002) and they are clustered in three groups based on their sequence homology (Staswick et al., 2002). There are eight GH3 proteins in group II, seven of which have been shown to adenylate IAA in vitro (Staswick et al., 2002, 2005). These include GH3-5/AtGH3a, GH3-3, GH3-6/DFL1, as well as GH3-2/YDK1/At4g37390 (Takase et al., 2004) and GH3-4/At1g59500. We checked the transcription level of the latter two by semiquantitative PCR and showed that they were also down-regulated in the double mutant sur2-1ago1-3 (RG) compared to the sur2-1 (RG) mutant (Fig. 4A ). Nevertheless, no significant difference in RNA levels between wild type (Col-0) and ago1-3 could be detected for GH3-4 or GH3-2/YDK1. Although the sur2-1ago1-3 (RG) double mutant contains less endogenous auxin than sur2-1 (RG), it still contains more auxin than the wild type. Since all these genes were induced by exogenous auxin (Nakazawa et al., 2001; Tanaka et al., 2002; Tian et al., 2002; Zhao et al., 2003; Takase et al., 2004), it was important to check whether or not a mutation in the AGO1 gene would modify auxin inducibility of these GH3-like genes. We therefore checked their induction by exogenously applied 1-naphthalene acetic acid in the wild type and in the ago1-3 mutant and showed that all five genes were normally induced in the ago1-3 mutant by exogenous auxin (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Figure 4. GH3-like genes are still inducible by auxin in the ago1-3 mutant. Total RNAs were extracted from hypocotyls of wild-type (Col-0) and ago1-3 (Col-0) siblings etiolated in the dark until the hypocotyl reached a 5-mm size, then transferred to light for 48 h with or without an auxin treatment as described in "Materials and Methods." The relative expression of the genes was estimated by semiquantitative RT-PCR using 18S RNA as an internal control. For each genotype, expression of each gene was presented as a ratio between the value for the gene and the value of the corresponding 18S RNA. Units are arbitrary. Errors bars = SD of three PCR reactions. The experiment was performed on two independent biological replicates.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The protein pattern of the different mutants was first compared to their respective wild types, and this led to interesting observations regarding the effect of their mutations. Comparing 2-D gels, the ago1-3 mutant and its wild type (Col-0) revealed that less than 10% of the spots varied. Since AGO1 is involved in the regulation of gene expression through the miRNA pathway (Vaucheret et al., 2004), we a priori expected to see many more proteins affected in this mutant background. One explanation could be that only the most abundant proteins are revealed on a 2-D gel and that variations affecting the less abundant proteins might not be detected. Nevertheless, the proportion observed was similar to that for the superroot mutants that are affected in enzymes involved sequentially in the same biosynthesis pathway leading to auxin overproduction (Barlier et al., 2000; Bak et al., 2001; Mikkelsen et al., 2004). Considering that auxin would also modify the expression of many genes, it is likely that the proportion of variable proteins observed in ago1-3 compared to Col-0 reflects the effect of the mutation. This suggests that the pleiotropic phenotype of argonaute mutants observed at later stages (Bohmert et al., 1998) might be due to a cascade of events occurring downstream of the direct targets of AGO1.

Identification of variable proteins showed that several cellular functions were affected by ago1-3. One of the interesting features was the up-regulation of several proteins involved in energy and carbon metabolism. Up-regulation of some of the proteins linked directly or indirectly to photosynthesis in the ago1-3 mutant is in agreement with the ago1-3 mutant being hypersensitive to light (Sorin et al., 2005). The accumulation of some photosynthetic proteins is regulated by light. For example, carbonic anhydrase is regulated at the mRNA level (Fett and Coleman, 1994), and there are interactions between mitochondrial metabolism and photosynthetic carbon assimilation (Raghavendra and Padmasree, 2003). Consequently, up-regulation of proteins linked to photosynthesis and to the TCA cycle (such as malate dehydrogenase) fits with the light hypersensitivity of the ago1-3 mutant.

The analysis of the sur1-3 and sur2-1 mutants interestingly showed that, although both mutants were altered in the same biosynthesis pathway and displayed very similar phenotypes in young seedlings (Boerjan et al., 1995; Delarue et al., 1998), they displayed differences in protein profiles. A similar number of polypeptides was affected by the two mutations; nevertheless, only 30% of the variable proteins in sur1-3 and sur2-1 were commonly variable. For example, three proteins of the GH3 family (GH3-3/At2g23170, GH3-5/AtGH3-a/At4g27260, and GH3-6/DFL1/At5g54510) were significantly up-regulated in the sur2-1 mutant compared to the wild type (Ws), but only GH3-6/DFL1 accumulation increased significantly in sur1-3 compared to the corresponding wild type (Col-0). Since these three GH3 genes are induced by auxin (this work; Nakazawa et al., 2001; Tanaka et al., 2002; Tian et al., 2002; Zhao et al., 2003; Takase et al., 2004), we would expect to observe as strong an accumulation of all of them in the sur1-3 mutant as in a sur2-1 background because SUR1 acts downstream of SUR2. This suggests that auxin is not the only factor controlling the expression of GH3 genes, but that other mechanisms are also involved in regulating their expression. A second interesting differential effect of the sur1-3 and sur2-1 mutations is highlighted by the two lectins similar to myrosinase-binding proteins that have been suggested to play roles in glucosinolate degradation (Rask et al., 2000). At3g16450 accumulation was affected in sur1-3 but not in sur2-1. One isoform of At3g16470 was affected in sur1-3 and the other one was affected in sur2-1. Both sur1 and sur2 are affected in glucosinolate biosynthesis but not to the same extent. In 5-week-old sur1-3 mutants, glucosinolates were not detectable, whereas in sur2-1 the glucosinolate content was decreased by 50% (Mikkelsen et al., 2004). This could explain the differential expression of glucosinolate degradation enzymes in these mutants. The fact that two isoforms of the same enzyme differentially accumulated in the sur1 and sur2 mutants suggests that they may target different types of glucosinolates. Interestingly, the CORI-7/AtST5a (At1g74100) protein that was recently proposed to be involved in the last step of indole-glucosinolate biosynthesis downstream of the SUR2 and SUR1 proteins (Piotrowski et al., 2004) accumulated normally in sur2-1 and sur1-3 mutants. While it is not surprising to see down-regulation of glucosinolate degradation enzymes in mutants that contain less or no glucosinolates, it is intriguing to see accumulation of a biosynthetic enzyme in the absence of precursors. It suggests that feedback loop regulation of this gene might exist (i.e. a low level of glucosinolates would stimulate the accumulation of enzymes of the biosynthetic pathway). Proteins of energy/carbon metabolism were also differentially affected by sur1 and sur2. Out of 10 proteins, five were down-regulated in the sur2-1 background, whereas only two (that were not affected by sur2-1 mutation) were differentially expressed in the sur1-3 background. One possible explanation for this could be the different behavior of sur1 and sur2 mutants toward light perception and/or signaling. Indeed, sur2 was recently shown to be allelic to red1, a mutant that has an abnormal de-etiolation response in continuous red light but not in continuous far-red light (Hoecker et al., 2004). Nevertheless, although we were careful to eliminate variations due to ecotype effect (i.e. variable between Col-0 and Ws) from the comparison between sur1-3 and sur2-1, we cannot rule out that some of the differences we observed between sur1-3 and sur2-1 may indeed be due to an ecotype effect. Indeed, this would also be supported by the fact that ago1-3 and sur1-3, which are in the same ecotype, share more similarities than sur2-1 and sur1-3 or sur2-1 and ago1-3, which are in different ecotypes. Since a sur2 allele is now available in a Col-0 background (Smolen and Bender, 2002), we will be able to verify the differences described here.

The proteins that varied significantly were submitted to correlation analysis in order to find those that were related to adventitious root formation. A Pearson correlation analysis of spot intensity with the three physiological parameters (free IAA content, mean number of adventitious root primordia after 2 d of light, and mean number of adventitious roots after 7 d of light) was performed. Eleven proteins were correlated with at least one parameter.

As one could expect in the case of a developmental process regulated by auxin, proteins involved in the control of auxin homeostasis were identified. The three GH3 proteins, GH3-3/At2g23170, GH3-5/AtGH3-a/At4g27260, and GH3-6/DFL1/At5g54510, were significantly positively correlated with adventitious root number 7 d after transfer to light. Expression of these GH3 genes was lower in the ago1-3 mutant compared to Col-0 and in the sur2-1ago1-3 (RG) mutant compared to sur2-1 (RG). The lower level of expression in the ago1-3 background could be explained by the lower level of endogenous free IAA (Table II; Sorin et al., 2005). Nevertheless, the level of expression was never above the wild-type level in the double mutant sur2-1ago1-3 (RG), although it contained twice as much auxin as the wild type. In addition, we have shown that the five GH3-like genes analyzed were still induced by exogenous auxin in ago1-3 mutant hypocotyls under the conditions described in "Materials and Methods." Therefore, if GH3-like gene expression was strictly linked to the level of auxin, we would have expected to observe an up-regulation of GH3-like gene expression in the sur2-1ago1-3 double mutant compared to the wild type. This was not the case, and our results suggested that the down-regulation in the ago1-3 background was independent of the auxin level and likely due to other regulatory mechanisms. We have recently shown that the auxin response factor ARF17, a repressor of auxin-inducible genes, is regulated by a miRNA and accumulated in ago1-3 hypocotyls (Sorin et al., 2005). We also showed that the expression of three GH3-like genes correlated with adventitious root number was down-regulated in a transgenic line overexpressing ARF17 (Sorin et al., 2005). The up-regulation of ARF17 would be expected to repress GH3-like gene expression in an ago1-3 hypocotyl. Interestingly, the ARF17 overexpresser line was also affected in adventitious root formation, strengthening the hypothesis that GH3-like genes might be involved in the regulation of adventitious root development. GH3-2/YDK1, GH3-6/DFL1, and GH3-5/AtGH3a have also been shown to be associated with the light control of development (Nakazawa et al., 2001; Tanaka et al., 2002; Takase et al., 2004). Since ago1-3 is altered in light sensitivity, we cannot exclude a combined effect on the regulation of the GH3-like genes. The ongoing characterization of mutants altered in the expression of GH3-like and/or ARF17 genes will hopefully lead to a better understanding of the relative importance of the different genes in the regulation of adventitious rooting.

Among the other correlated proteins, two were related to energy and carbon metabolism and they were both negatively correlated to adventitious rooting. This is in agreement with data obtained by microarray analyses from Brinker et al. (2004) during adventitious rooting of hypocotyl cuttings of P. contorta. These authors proposed that the down-regulation of plastid proteins was due to the loss of photosynthetic capacity of hypocotyl cells during adventitious root formation. Indeed, in our case, several plastid-encoded proteins or proteins predicted to be localized to plastids (At3g01500, ATCG00490) accumulated in the genotypes that made less adventitious roots (i.e. in the ago1-3 background). In contrast, several plastid proteins (At1g06680, At1g32060, and At4g38970) were down-regulated in sur2-1, a genotype that makes many adventitious roots, strengthening the hypothesis that light regulates adventitious rooting (Sorin et al., 2005).

Finally, several proteins either positively or negatively correlated were putative chaperones or stress-related proteins. Although it is difficult at this point to discuss the potential role of these proteins in adventitious root development, our results confirm at the protein level the observations made at the transcriptional level by Brinker et al. (2004). Indeed, they showed modification of the expression of a similar family of genes, suggesting a role in adventitious rooting.

Interestingly, and although these proteins were not correlated to adventitious rooting, we could find some overlap among the protein identified here and the genes described to be potentially associated with adventitious root formation in P. contorta (Brinker et al., 2004). This is the case of the 20S proteasome subunits, the putative -tubulin and the tubulin 2/4 chain, and the caffeoyl-CoA methyltransferase-like protein. A PINHEAD/ZWILLE-like gene was found up-regulated in young adventitious rooting stages in P. contorta. Since in Arabidopsis PINHEAD/ZWILLE is the closest gene related to AGO1, this suggests that the function of these genes is at least partially conserved among different plant species.

In conclusion, the proteomic analysis of ago1-3, sur1-3, sur2-1, and the sur2-1ago1-3 double mutant allowed us to identify proteins whose expression was altered by the mutations, particularly in regard to the adventitious rooting process. We identified auxin-related proteins and light-related proteins positively or negatively correlated to adventitious root formation. Several other proteins related to stress responses, protein degradation, or cytoskeleton function were correlated to at least one of the studied parameters. As similar functions were affected during adventitious root formation in P. contorta (Brinker et al., 2004), these results strongly suggest that the identified proteins will be valuable markers for further quantitative genetic analysis of adventitious root development.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

ago1-3, sur1-3, and sur2-1 mutants were identified in Col-0 and Ws Arabidopsis (Arabidopsis thaliana L. Heyhn.) ecotypes, respectively (Boerjan et al., 1995; Delarue et al., 1998). sur2-1ago1-3 double mutant was obtained by crossing a homozygote sur2-1/sur2-1 plant with a heterozygote +/ago1-3 plant followed by several generations of selfing of the sur2-1/sur2-1, +/ago1-3 plants. Double mutants were selected in the progeny of recombinant lines homozygous for the sur2-1 mutation heterozygote for ago1-3. They are in a recombinant Ws/Col-0 genetic background.

ago1-3 and sur1-3 mutants, which are sterile and kept as heterozygotes, were compared to their wild-type siblings, Col-0; sur2-1 was compared to the Ws wild-type ecotype. ago1-3sur2-1 double mutants were compared to their sur2-1 siblings in the recombinant Ws/Col-0 background (RG). For simplification, the background (Col-0, Ws, or RG) will always be indicated in parentheses after the genotype.

Growth Conditions and Preparation of Biological Samples for Protein Extraction

Seeds from the different genotypes were sterilized and sown in vitro as described previously (Santoni et al., 1994). They were kept at 4°C for 72 h to homogenize germination and then transferred to light (130–140 µmol m^–2 s^–1) for 10 h to induce germination. Seedlings were germinated and grown in the dark until the hypocotyl reached an average size of 5 mm. To create darkness, the petri dishes were wrapped in four layers of aluminum foil. Seedlings were then transferred to light, in long-day conditions, for 48 h (130–140 µmol m^–2 s^–1, 16 h light, 20°C average day temperature, 15°C average night temperature). For observation of adventitious root initiation and counting the number of adventitious root primordia 2 d after transfer to light, complete seedlings were cleared overnight with a buffer described by Herr (1982) and observed with a Nikon microphot FXA microscope with Nomarski optics. For proteomic analysis, cotyledons and roots were removed, and hypocotyls were harvested, dried on tissue paper, and frozen in liquid nitrogen. Three to four independent biological replicates (of at least 300 hypocotyls each) were prepared for each genotype.

Protein Extraction

The protein extraction method was derived from Damerval et al. (1986). Frozen hypocotyls were ground into a fine powder in liquid nitrogen. The powder was mixed with cold acetone containing 10% (w/v) TCA and 0.07% (v/v) -mercaptoethanol. Proteins were left to precipitate for 1 h at –20°C. They were then centrifuged at 10,000 rpm for 10 min at –20°C. Pellets were washed twice with cold acetone containing 0.07% (v/v) -mercaptoethanol, then dried, weighed, and solubilized in R2D2 buffer (300 µL mg^–1 dry pellet; R2D2, 5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris (2-carboxyethyl)phosphine, 0.5% carrier ampholytes 4–6.5, 0.25% carrier ampholytes 3–10; Mechin et al., 2003). Cellular debris was eliminated by centrifugation at 12,000 rpm. Protein amounts were estimated with the PlusOne 2D quant kit (Amersham Biosciences).

2-D Electrophoresis

Thirty-five micrograms of protein were mixed together with R2D2 buffer (Mechin et al., 2003) and used for isoelectric focusing (IEF). IEF was performed in 24-cm-long immobilized pH gradient (IPG) strips (pH range from 4–7; Amersham Biosciences). Migration was performed at 20°C with limited amperage of 50 µA on a Protean IEF cell (Bio-Rad) as follows: after an active rehydration at 50 V during 13 h, steps at 200 V, 500 V, and 1,000 V were run for 30 min, 30 min, and 60 min, respectively. Voltage was then increased to 10,000 V and IEF was stopped when 84,000 Vh were reached. After IEF, IPG strips were equilibrated after Görg et al. (1987). Strips were then sealed on top of a 1-mm-thick 2-D gel (24 x 24 cm) with the help of 1% low-melting agarose in electrophoresis buffer. Continuous gels (11% acrylamide gels with potato dextrose agar as a cross-linking agent) were used. SDS-PAGE was carried out with running buffer in a PROTEAN Plus Dodeca cell (Bio-Rad) electrophoresis tank at 14°C at 20 V for 1 h followed by 120 V overnight (limited by a maximum of 30 mA per gel) until the bromphenol blue front reached the end of the gel. A silver-staining procedure was performed according to Mechin et al. (2003). Three to four gels per genotype (i.e. independent biological replicates) were used for the analysis. Stained gels were scanned and gel analysis was performed with Progenesis software (Nonlinear Dynamics). After spot detection, 2-D gels were aligned and matched to a reference gel created by the software on which were identified a total number of 1,174 reproducible spots. Optical density calibration was performed with a Kodak density calibration strip (21-step photographic step tablet, density range 0.05–3.05). After background subtraction, normalization was performed (mode total spot volume).

Protein Identification by LC-MS/MS

For protein identification, a preparative gel containing 150 µg of protein was prepared and stained with SYPRO ruby protein gel stain (Bio-Rad). Staining visualization was performed with the use of a dark reader transilluminator DR190M (blue light source 400–500 nm; Clare Chemical Research). Spots were picked out and the acrylamide pieces were collected in 96-well microplates. In-gel digestion was performed with the Progest system (Genomic Solution) according to a standard trypsin protocol. Briefly, gel pieces were washed and then subsequently digested with 125 ng of modified trypsin (Promega) during 5 h. The peptides were extracted with 30 µL of 5% trifluoroacetic acid (TFA), 10% acetonitrile (ACN), then 30 µL of 0.2% TFA, 83% ACN. Peptide extracts were dried in a vacuum centrifuge and resuspended in 20 µL of 0.1% TFA, 3% ACN. The peptides were separated on HPLC Famos-Switchos II-Ultimate (LC Packings-Dionex). Sample (5 µL) was loaded on a PEPMAP C18 column (5 µm, 75 µm x 15 cm; LC Packings-Dionex) after a 3-min preconcentration step at 5 µL min^–1 on a micro precolumn cartridge (300 µm x 5 mm). The separation was achieved with a linear gradient from 5% to 30% B for 25 min at 200 nL min^–1. Buffers were 0.1% HCOOH, 3% ACN (A) and 0.1% HCOOH, 0.95% ACN (B). The LCQ deca xp+ (Thermofinnigan) was used with a nanoelectrospray interface. Ionization (1.2- to 1.4-kV ionization potential) was performed with liquid junction and a noncoated capillary probe (New Objective). Peptide ions were analyzed by the nth-dependent method as follows: (1) full MS scan (m/z 500–1500); (2) ZoomScan (scan of the two major ions with higher resolution); and (3) MS/MS of these two ions. Data were then analyzed with Bioworks 3.1 and the Arabidopsis protein sequences database downloaded from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) or the plant genome database (http://www.plantgdb.org). Identified tryptic peptides were filtered according to their cross-correlation (Xcorr) score and their charge state (Xcorr > 1.7 for +1 and Xcorr > 2.2 for +2 charge). Annotation of the identified proteins was obtained on http://www.arabidopsis.org/servlets/Search; http://www.tigr.org and http://www.genome.ad.jp/kegg were also used as a complement in protein function identification. Molecular weights were obtained from http://au.expasy.org/sprot and http://mips.gsf.de/proj/thal/db.

Data Analysis

Statistical analysis was performed on 1,147 reproducible spots. ANOVA was performed for each of the 1,147 reproducible proteins. The factor was the combination between genetic background (Col-0, Ws, and RG) and the genotype at the locus of the different studied mutations. Thus, the different levels of the factor were wild type (Col-0), sur1-3 (Col-0), wild type (Ws), ago1 (Col-0), sur2-1 (Ws), sur2-1 (RG), and sur2-1ago1-3 (RG). This analysis allowed the computation of the residual variance of the spots over the complete dataset. For proteins showing a significant variation (P < 0.01), contrasts were computed between the following couples: sur1-3 (Col-0) versus wild type (Col-0), ago1 (Col-0) versus wild type (Col-0), sur2 (Ws) versus wild type (Ws), and sur2ago1 (RG) versus sur2 (RG). Contrasts were considered significant at P < 0.01. Thus, only proteins showing a significant quantitative difference at least between a mutant and its corresponding wild type were selected. Then spots showing an induction factor below 1.5 or above 0.66 were not considered further. The GLM procedure of the SAS software package (SAS Institute, version 8.1) was used for ANOVA and contrast analysis.

Spots were described as showing qualitative variations when they were present (or absent) in all the gels of a mutant genotype and absent (or present) in all the gels of the corresponding wild type or sur2 (in the case of the sur2ago1 [RG] versus sur2 [RG] comparison).

Correlation Analysis

For each protein that was significantly variable, Pearson correlation coefficients (correlation procedure of the SAS package) were computed between means of relative intensity in the six genotypes and IAA (IAA concentration), number of adventitious root primordia 2 d after transfer to light, and number of adventitious roots emerging from the hypocotyl 7 d after transfer to light. When the protein was not detected in a genotype, the genotype was excluded from the computation of the correlation with this protein. When the correlation was significant (P < 0.01), consistence of the correlation with the absence of the protein in this genotype was verified.

Endogenous free IAA levels (pg mg^–1 fresh weight) were previously measured in the apical part of seedlings, 48 h after transfer to light in wild type (Col-0), wild type (Ws), ago1-3 (Col-0), sur2-1 (RG), and sur2-1ago1-3 (RG) (Sorin et al., 2005).

The average number of primordia 2 d after transfer to light was determined in sur2-1 (RG) mutants and sur2-1ago1-3 (RG) double mutants by analyzing the expression of the CyclinB1:uidA reporter gene as described by Sorin et al. (2005). For wild-type Ws, wild-type Col-0, and sur2-1 (Ws), the average number of primordia was determined on seedlings cleared overnight in Herr's buffer (Herr, 1982) and observed with a Nikon microphot FXA microscope with Nomarski optics. An average of 40 seedlings was analyzed in three independent biological replicates.

Adventitious roots emerging from the hypocotyls were counted on seedlings 7 d after transfer to light using a stereomicroscope. An average of 40 seedlings was analyzed in three independent biological replicates.

RNA Analysis

Siblings from Col-0 and ago1-3 or sur2-1 (RG) and sur2-1ago1-3 (RG) were grown in the same conditions as for protein extraction. For auxin induction of GH3-like genes, the seedlings were transferred, after 44 h in the light, into liquid culture medium with or without 10 µM of 1-naphthylacetic acid for 4 h in the same environmental conditions. RNA was extracted from dissected frozen hypocotyls.

For semiquantitative RT-PCR, total RNA was extracted using the RNAqueous kit (Ambion). Five hundred nanograms or 1 µg of RNA was transcribed into cDNA by using iScript reverse transcriptase (Bio-Rad). PCR amplification was carried out in triplicate with each cDNA and primer pairs 5'-CCTATGCTGGGCTTTACAGG-3' and 5'-ACCAGGGGACCATTTAGGAC-3' for GH3-6/DFL1/At5g54510; 5'-AAGTTTGTGCGGAGGAAGAA-3' and 5'-AAAGCGGGCTGAAGTGTGT-3' for GH3-3/At2g23170; 5'-AATGCCAACAATCGAAGAGG-3' and 5'-CTTGCACTCAAATTCCACGA-3' for GH3-5/AtGH3a/At4g27260; 5'-GAAATGACTCGGAACCCTGA-3' and 5'-GCAGAGGATGGCTTCGTTAG-3' for GH3-2/YDK1/At4g37390; and 5'-AGCCATCCTCTGCTGTGACT-3' and 5'-ACTCCTCCATCTCCATCGTG-3' for GH3-4/At1g59500. Quantum 18S RNA internal standard kit (Ambion) served as an internal control. PCR products were analyzed on a 1.5% agarose gel. The experiment was performed on two independent biological replicates.

	ACKNOWLEDGMENTS

 
We thank John D. Bussell, Brian Jones, Thomas Moritz, and Gunnar Wingsle for discussions and critical reading of the manuscript.

Received July 1, 2005; returned for revision November 2, 2005; accepted November 2, 2005.

	FOOTNOTES

 
¹ This work was supported by the Institut National de la Recherche Agronomique (C.B. and M.Z.) and the Swedish Foundation for Strategic Research (C.B.). C.S. received a Ph.D. fellowship from the Ministère de la Recherche et de l'Enseignement Supérieur and from the Swedish Foundation for Strategic Research.

² Present address: Departament de Genètica Molecular, Institut de Biologia Molecular de Barcelona (Consejo Superior de Investigaciones Científicas), 08034 Barcelona, Spain.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Michel Zivy (zivy{at}moulon.inra.fr) and Catherine Bellini (catherine.bellini{at}genfys.slu.se).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.067868.

^* Corresponding author; e-mail catherine.bellini{at}genfys.slu.se; fax 46–90–786–85–61.

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