Proteomic Analysis of Root Meristems and the Effects of Acetohydroxyacid Synthase-Inhibiting Herbicides in the Root of Medicago Truncatula Peta Holmes,† Ryan Farquharson,‡ Prudence J. Hall,§ and Barry G. Rolfe*,† ARC Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, Research School of Biological Sciences, Australian National University, Canberra ACT 2601, Australia Received February 24, 2006
Quantitative proteome analyses of meristematic and nonmeristematic tissues from Medicago truncatula primary and lateral roots and meristem tissues from plants treated with acetohydroxyacid synthaseinhibiting herbicides were made. The accumulation of 81 protein spots changed in meristematic and nonmeristematic tissues and 51 protein spots showed significant changes in accumulation in herbicidetreated meristems. Identified proteins indicate two trends, (i) increased accumulation of cell division and redox-mediating proteins in meristems compared to nonmeristematic tissues and (ii) increased accumulation of pathogenesis-related and decreased accumulation of metabolic proteins in herbicidetreated roots. Keywords: root • meristem • acetolactate synthase • PR10
Introduction The root and shoot apical meristems (RAM and SAM) are established during embryogenesis and serve as a source of stem cells for plant growth and organogenesis.1 The primary RAM produces all the tissues of the main root by a highly defined pattern of cell divisions.2 Cells produced by the meristem undergo proliferative cell divisions as they are added to files of different cell types and their fate is determined by positional information.3,4 Stem cells of the root are maintained by the quiescent center (QC),5,6 which is maintained by auxin.7 Jiang et al.8 demonstrated the highly oxidized state of the QC cells in Zea mays and proposed that the QC is established and regulated by reactive oxygen species (ROS) generated by auxin. ROS production is thus a downstream component of an auxin mediated signaling pathway in the root.9 The root meristemless1 mutant of Arabidopsis shows that activation of cell division in the root meristem following germination requires the activity of a glutathione-dependent developmental pathway,10 possibly to mediate redox stress generated by auxin. Lateral roots arise through the post-embryonic development of a meristem,11 and then follow the same developmental pattern as the primary root. Auxin is also fundamental for the establishment of lateral root primordia and activation of the lateral root meristem.12-14 The root and lateral root can be viewed along a developmental gradient. The simple classical model of root structure separates the root tip into distinct, but overlapping zoness * To whom correspondence should be addressed. Tel: +61 (02) 6125 4054. Fax: +61 (02) 6125 0754. E-mail
[email protected]. † Australian National University. ‡ CSIRO Land and Water, Glen Osmond SA 5064, Australia. § Departments of Biology and Chemistry, Hiram College, Hiram, OH44234 U.S.A.. 10.1021/pr0600677 CCC: $33.50
2006 American Chemical Society
meristem, elongation, and maturation regions.15 Studies with Arabidopsis showed that the seedling root consists of three partly overlapping developmental zones: the division, elongation, and differentiation zones.16 Studies with Arabidopsis have also shown that seven files of root cell layers arise from the root region containing the quiescent center and the adjacent initials.3,17 Groot et al.,18 have shown that this model does not clearly apply to all plants, and they describe the organization of the root apical meristem as open or closed. Arabidopsis has a closed meristem in which the initials of the structural layers of the root are clearly apparent; the initials in an open meristem are not clearly apparent. Legumes such as peas (Pisum sativa) have this type of RAM. This different RAM organization is usually reflected in different patterns of tissue origin.18 Thus, legumes may have a different, or a less stringent program for differentiation from the root meristem. In addition to lateral roots, legumes also develop nondeterminate root nodules post-embryonically that have an active meristem. The nodule may share common developmental pathways with the lateral root. A Medicago truncatula mutant that affects nodule and root development has been isolated and characterized. Lateral roots of the M. truncatula mutant lateral root organ defective (latd) are initiated but remain short while the primary root develops like a wild-type root but gradually ceases growth and forms an abnormal deformed root tip.19 Similarly, a relationship between root morphology and nodulation was observed with different hypernodulating mutants of M. truncatula and Lotus japonicus.20,21 Acetohydroxyacid synthase (commonly called acetolactate synthase) is the target of a group of herbicides that are divided into three main classes based on their chemical structure; sulfonylureas like chlorsulfuron, metsulfuron methyl, and Journal of Proteome Research 2006, 5, 2309-2316
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research articles triasulfuron; imidazolinones like imazethapyr, imazamox, or imazaquin; and triazolopyrimidine sulfonamides including flumetsulam. AHAS catalyses the first common step in the synthesis of the branched chain amino acids, but there is still conjecture over the mechanisms by which AHAS-inhibiting herbicides impede plant growth and eventually cause plant death. Depletion of amino acids and the accumulation of intermediates to toxic levels have been proposed.22 Other perturbations reported include the induction of increased carbohydrate accumulation and enhanced fermentative activities.23,24 Studies have shown that AHAS-inhibiting herbicides can reduce nodulation and nitrogen fixation by some legume species,25-28 though the exact mechanisms have not been determined. Herbicides can potentially affect the legumes rhizobia symbiosis in a number of ways.29 There is some evidence that chlorsulfuron, an AHAS inhibitor, inhibits the progression of pea root cells from G1 to S and G2 to M, which was predicted to deplete the meristem of cycling cells in the longer term.29 Martensson and Nilsson23 reported that chlorsulfuron inhibited lateral root formation and deformed root hairs in M. sativa. It also appears that these herbicides can affect the ability of rhizobia to form effective symbioses with the indeterminate nodulator chickpea (Cicer arietinum).30 There is therefore a need to determine the exact mechanisms by which acetohydroxyacid synthase inhibiting herbicides impact nitrogen fixation by agronomically important legumes. In this study, we have used proteomic analysis of the primary root, lateral root, and roots treated with acetohydroxyacid inhibitors to examine root developmental pathways. We have used a proteomic approach to analyze root tissues and examined and compared the protein profiles of the two regions, the meristem and the nonmeristematic elongation zone of the M. truncatula root. The meristem and elongation zones of the primary root were also compared to those of the lateral root. Furthermore, the proteomic response of these zones of the primary root to flumetsulam and metsulfuron methyl, the two AHAS-inhibiting herbicides, was examined by comparison to the proteomes of untreated roots.
Materials and Methods Plant Materials. Seeds of M. truncatula cv. A17 were scarified, surface-sterilized with 6% hypochlorite solution and washed 7 times with sterile distilled water. Seeds were germinated on nitrogen-free Fåhraeus medium31 on Petri plates in the dark for 24 to 30 h. To provide intact primary roots for sectioning, germinated seeds that lacked any visible signs of microbial contamination were transferred to new Petri plates, 14 to 16 seedlings per plate, and grown for a further 3 days in a growth chamber until the roots had reached a length of 3 to 4 cm and before lateral roots emerged. Lateral root formation was induced by excision of the root tip from germinated seedlings. Excising more than 5 mm of root induced synchronous lateral root formation. The seedlings were plated as described for intact roots, but were grown for a further 6 days, synchronous lateral roots appearing 3 days after excision of the root tip. Plates were kept vertically and the bottom half of each plate was sealed with Nescofilm R. Light was kept from the roots by the insertion of a black sheet between the plates during incubation. An aluminum foil spacer was placed under the lid of the Petri dish to allow gas exchange. Plates were incubated in a growth chamber at 20 °C over a 16 h photope2310
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riod and a photon flux density of 100 mmol m-2 s-1 and 86% relative humidity. To compare meristematic and nonmeristematic root tissues, root sections were harvested from 3 day old plants. Tissue 3 mm from the root tip which contains the meristematic cells and a further 1 cm section from the root containing nonmeristematic differentiating and elongating cells were collected. Lateral roots were harvested as described for primary roots. All harvested plant materials were immediately frozen in liquid nitrogen and stored at -80 °C. Herbicide-Treated Plant Materials. Seeds were sterilized and germinated as above, with the exception that plants were germinated on nitrogen-free Fåhraeus medium containing 200 ng mL-1 flumetsulam (Dow Agrosciences) or 10 ng mL-1 metsulfuron methyl (Dupont), or no herbicide for the control. Herbicide stocks were made in 0.1 N NaOH to ensure that the herbicide was completely dissolved. Germinated seeds were transferred to corresponding new plates (containing the same herbicide or no herbicide) 10 plants per plate. Plates were incubated at 20 °C with 350 mmol m-2 s-1 light intensity over a 16 h photoperiod, at 86% relative humidity. After 5 days, plants were harvested for protein extraction; both meristematic and nonmeristematic tissues were collected as described above. Some plants were kept under these conditions for up to 18 days for root length measurements and morphological observations. The primary roots of 70 plants for each treatment were measured from scans at 6, 9, 12, and 18 days post germination using ImageJ software (http://rsb.info.nih.gov/ij). The relative growth rates (RGR ) (ln(root length at time 2) - ln(root length at time 1))/(time 2 (days) - time 1 (days)) for each treatment and time point were calculated. Protein Extraction and Electrophoresis. Total protein was extracted from root sections using a trichloroacetic acidacetone extraction method.32 The concentration of the soluble proteins extracted from the plant material was determined by the Bradford assay using Bradford reagent (Bio-Rad) and bovine serum albumin as the standard. Total protein was subjected to isoelectric focusing (IEF) on 24 cm Immobiline dry strips with linear pH gradients from 4 to 7 (Amersham) and 2-D gel electrophoresis using a Multiphor II horizontal electrophoresis system and precast ExcelGels SDS gels with a 12-14% acrylamide gradient.33 Silver-stained 2-D gels from IEF gels loaded with 200 µg of protein created from three biological repeats were used for gel image analysis. For protein identification by mass spectroscopy, 800 µg of protein was subjected to IEF and the second-dimension gels were stained with Coomassie Brilliant Blue. Image and Statistical Analysis. Gels were scanned at 600 dots per inch on a UMAX Astra 2400S scanner (UMAX Technologies, Freemont, CA). Gels were scanned and all protein spot volumes were quantified with Imagemaster (Swiss Institute of Bioinformatics, Geneva, Switzerland) for three biological repeats for each treatment. All gels from each treatment were matched to each other and to the other treatments, and spots assigned arbitrary identifiers. To compare differences in protein accumulation among the treatments, the % volume of each spot was subjected to restricted maximum likelihood (REML) analysis as described in Imin et al.34 using GenStat 8.0 (VSN International, Hertz, United Kingdom) to determine whether the volume of a spot varied significantly between treatments. Mass Spectrometry. Proteins were analyzed by MALDI-TOF/ TOF-MS or by tandem LC-MS if they could not be matched to the identified proteins in the proteome reference map of
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Proteomic Analysis of Root Meristems
Figure 1. 2D resolution of the M. trunculata root proteome. (A) The proteome of the primary root meristem, the proteome of the nonmeristematic primary root (B), the meristem (C), and nonmeristematic lateral root (D). The numbered spots are those identified as having altered accumulation that were identified by mass spectrometry.
M. truncatula root or embryogenic cultures.32-34 Proteins were excised from Coomassie stained gels and destained in 50% acetonitrile (AcN) and 25 mM NH4HCO3 at pH 7.8 for 1 h at room temperature on an orbital shaker. The destaining solution was removed and gel pieces were dehydrated in 100% AcN for 20 min and then allowed to air-dry. Gel pieces were rehydrated in 40 µL of porcine trypsin (15 ng µL-1 in 250 µM NH4HCO3) at 4 °C for 1 h, after which 40 µL 25 mM NH4HCO3 was added and the digest incubated at 37 °C overnight. Peptides were acidified by adding 1% trifluoroacetic acid (TFA) (pH < 4) and were then extracted from the gel pieces using ZipTipC18 Reverse-Phase pipet tips (Millipore, Bedford, MA), eluting the peptides in 0.1% TFA for MALDI-TOF/TOF MS/MS analysis or in MeOH and 0.1% formic acid for LC MS analysis. For MALDITOF/TOF MS/MS peptides extracted from gel pieces were spotted onto a sample plate with 1 µL of matrix (R-cyano-4hydroxycinnamic acid, 8 mg mL-1 mL-1 in 70% v/v AcN, 1% v/v TFA) and allowed to air-dry. MALDI mass spectrometry was performed with an Applied Biosystems 4700 or 4800 Proteomics analyzer with TOF/TOF optics in MS mode. Following MALDITOF, the instrument was then switched to MS/MS mode, and the eight strongest peptides from the MS scan were isolated and fragmented by collision-induced dissociation with filtered lab air, then reaccelerated to measure their mass and intensities. LC-MS/MS data acquisition was performed with a LCQ DECA XP Plus (Thermo Electron, San Jose, CA). PMF was performed on a Micromass TofSpec 2E time-of-flight mass spectrometer (Waters, Milford, MA). The data from all mass spectrometers was exported in a format suitable for analysis with Mascot (Matrix Science, London). MS and MS/MS Analysis. Searches were run against the M. truncatula gene index database (MtGI, Release 8.0, January 2005) that contains ∼37 000 minimally redundant tentative clusters (TC) of M. truncatula expressed sequence tags (EST) downloaded from The Institute of Genomic Research (TIGR) (ftp://ftp.tigr.org/pub/data/tgi/Medicago_truncatula/). For the analysis of MS and MS/MS data the search engine Mascot was used to search the MtGI database, for these database Mowse scores greater than 66 are significant (P < 0.05).33 Searches were
conducted using a mass accuracy of (100 ppm and one missed cleavage. In the searches the following mass modifications were allowed: carbamidomethyl modification of cysteine residues by iodoacetamide, oxidation of methionine, and propionamide modification of cysteine by acrylamide. For PMF matches with significant Mowse scores and at least 4 matched peptides, 4 peptides are sufficient to predict a statistically significant match. For MS/MS data, matches with fewer peptides were also examined. Where no matches to MtGI were made the mass data was searched against the Mascot MSDB nonredundant protein database, which includes other plant sequences. Proteins identified from MtGI were subjected to a protein-protein BLAST (BLASTP) search against the Mascot MSDB nonredundant protein database to validate that the correct open reading frame had been predicted by TIGR.
Results and Discussion Meristematic and Nonmeristematic Root Tissues. Twodimensional gels of meristematic and nonmeristematic root sections were used to compare patterns of protein accumulation. No statistically significant differences were observed between the proteomes of the meristems of the primary and lateral roots. Similarly, no differences were observed between the nonmeristematic regions of the primary and lateral roots. However, the same set of changes in protein accumulation between the meristem and nonmeristematic root could be identified from both types of root. From a nonselective statistical analysis, 81 spots were found to differentially accumulate between the meristem and nonmeristematic zones of both primary and lateral roots, including 18 spots that only occur in the differentiating root. No protein spots unique to the meristem could be found on the gels. Examples of the silver stained gels used for quantitative analysis are shown in Figure 1. Silver stained gels were used for protein quantification as reproducibility is better between silver stained gels than those stained with Coomassie Brilliant Blue. More spots could be resolved on silver stained gels with each silver stained gel showing approximately 2400 spots compared to each Coomassie stained gel showing about 1500. Journal of Proteome Research • Vol. 5, No. 9, 2006 2311
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Table 1. Proteins that Differentially Accumulate in the M. truncatula Root Meristem and Nonmeristematic Tissue fold change P spot ID %vol a value b LSDb
389
2.46
