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First published online February 9, 2007; 10.1104/pp.106.092585 Plant Physiology 144:703-716 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The MtMMPL1 Early Nodulin Is a Novel Member of the Matrix Metalloendoproteinase Family with a Role in Medicago truncatula Infection by Sinorhizobium meliloti1,[W],[OA]Laboratoire des Interactions Plantes Micro-organismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, 31326 Castanet Tolosan cedex, France
We show here that MtMMPL1, a Medicago truncatula nodulin gene previously identified by transcriptomics, represents a novel and specific marker for root and nodule infection by Sinorhizobium meliloti. This was established by determining the spatial pattern of MtMMPL1 expression and evaluating gene activation in the context of various plant and bacterial symbiotic mutant interactions. The MtMMPL1 protein is the first nodulin shown to belong to the large matrix metalloendoproteinase (MMP) family. While plant MMPs are poorly documented, they are well characterized in animals as playing a key role in a number of normal and pathological processes involving the remodeling of the extracellular matrix. MtMMPL1 represents a novel MMP variant, with a substitution of a key amino acid residue within the predicted active site, found exclusively in expressed sequence tags corresponding to legume MMP homologs. An RNA interference approach revealed that decreasing MtMMPL1 expression leads to an accumulation of rhizobia within infection threads, whose diameter is often significantly enlarged. Conversely, MtMMPL1 ectopic overexpression under the control of a constitutive (35S) promoter led to numerous abortive infections and an overall decrease in the number of nodules. We discuss possible roles of MtMMPL1 during Rhizobium infection.
Rhizobium infection and nodulation are the two key developmental processes involved in establishing the nitrogen-fixing symbiosis between legume plants and the appropriate soil microsymbiont. Both of these processes require Nod factors, lipo-chito-oligosaccharidic molecules that are synthesized by rhizobia in response to root-secreted flavonoids and play an essential role in the activation of symbiotic genetic programs in specific host plants (for review, see Oldroyd and Downie, 2004  ; Riely et al., 2004; Stacey et al., 2006). The plant responses to Nod factors and rhizobia integrate various internal cues, notably nutritional (particularly regarding carbon and nitrogen status), developmental (only specific root regions are responsive), and autoregulatory signals. Indeed, both the location and the number of nodules and infections are tightly controlled by the plant by at least two mechanisms, either ethylene dependent or independent, which lead to a superinfection/supernodulation phenotype when altered by mutations (skl, affected in ethylene perception [Penmetsa and Cook, 1997; Penmetsa et al., 2003]; har1 and sunn, affected in a Leu-rich repeat receptor kinase gene [Krusell et al., 2002; Schnabel et al., 2005]).
In Medicago truncatula, as in many other temperate legumes, Rhizobium infection takes place via plant cell structures called infection threads (ITs), which form in curled, infected root hairs in the presence of Rhizobium, probably following Nod factor-dependent cytoskeleton reorganization (for review, see Brewin, 2004
In legumes with indeterminate nodules, like M. truncatula, ITs first grow inward as a branched network from the root hairs to the newly divided cortical cells of the nodule primordium, following the pathway created by preformed cytoplasmic columns (named pre-ITs) in aligned activated cells. Once the nodule meristem differentiates within this primordium (about 3–4 d postinoculation [dpi] in M. truncatula A17) and the nodule starts to grow continuously out from the root, new IT branches grow outward and behind this apical meristem (Monahan-Giovanelli et al., 2006
A number of infection-defective mutants have been identified in the macro- and microsymbionts, showing that both partners are involved in this process. Plant mutants affected in early infection stages (IT initiation or progression) have been reported in pea (Pisum sativum; Tsyganov et al., 2002
Other sources of information rely on the identification of plant genes whose expression is up-regulated during Rhizobium infection in roots or in the nodule infection zone, such as MtLEC4 (Mitra and Long, 2004
MMPs are structurally related zinc (Zn)-containing endopeptidases thought to play a key role in the breakdown of the ECM. Their importance is well documented in animals in the context of many normal biological processes involving remodeling of connective tissues (e.g. embryonic development, organ morphogenesis, angiogenesis, wound healing, and apoptosis), as well as pathological processes (notably arthritis, cancer metastasis, and inflammation; for review, see Nagase and Woessner, 1999
Several MMPs have been described in plants, with different but still unclear functions and without determining their physiological substrates. The soybean (Glycine max) SMEP1 protein was the first plant MMP to be purified (Graham et al., 1991 Considering the importance of ECM and tissue remodeling during nodulation and infection, we decided to further characterize MtMMPL1 as an interesting example of a plant MMP and to make use of reverse genetics tools recently made available for M. truncatula to explore its possible function during symbiosis.
MtMMPL1: A Novel Variant of the MMP Family
MtMMPL1 is a M. truncatula nodulin gene that was first identified by a subtractive screen of a young nodule cDNA library (designated at that time MtN9; Gamas et al., 1996
In all known MMP catalytic domains, a Glu residue is found next to the first His residue. This Glu is essential for protease activity because it acts as a nucleophilic group promoting attack of a water molecule on the substrate. A Gln was found instead of a Glu in MtMMPL1, and this is why MtN9 was renamed MtMMPL1 (for MMP-like 1). A Glu-to-Gln substitution generated by site-directed mutagenesis in animal MMPs (human gelatinase A and MMP-9) resulted in a dramatic decrease in protease activity (Crabbe et al., 1994 ; Rowsell et al., 2002). We therefore verified that the Glu-to-Gln substitution in MtMMPL1 did not result from a cloning or a sequencing artifact. We examined MtMMPL1 ESTs from independent cDNA libraries as well as MtMMPL1 genomic DNA sequences, obtained both by PCR amplification and from the ongoing M. truncatula genome sequencing program. Although the sequencing and annotation of the bacterial artificial chromosome carrying the MtMMPL1 gene region (CR962135, chromosome 5) was not completed (phase 2 clone, see TIGR Web site, http://www.tigr.org/tdb/e2k1/mta1/), it was possible to conclude that MtMMPL1 comprises a single exon. Moreover, we discovered that MtMMPL1 belongs to a cluster of four related genes that all contain the Glu-to-Gln substitution, present within a region of about 13 kb flanked by transposon-like sequences (see Supplemental Fig. S1 for an alignment of the corresponding predicted proteins).
Because the Glu-to-Gln variant had been confirmed in several M. truncatula MMP-like proteins, we then looked for similar variants in other species, using public nucleotide and protein databases. We first conducted a ScanProsite search on the SP and TrEMBL databases (release 50.2 and release 33.2, respectively) to identify proteins containing the following motif: [VAI]-A-[AMLTV]-H-Q-[FLIV]-G-H-[ALVIS]-L-G-[LM]-X-H-S. All four hits found (for an approximate no. of expected random matches of 3.6 e–08) were from M. truncatula, and three as ESTs (MtC40019 = MtMMPL1, MtD00669, and MtD00924 clusters, containing ESTs from various cDNA libraries; see Supplemental Fig. S2). By comparison, when performing a search with the same sequence except with the Gln residue replaced by a Glu residue, 288 hits were obtained in a variety of plant and animal species. To explore a larger range of species, we then carried out a TBLASTN search on dbEST (36,649,443 sequences), using a 31-amino acid sequence centered on the MMP active site (WDLETVAMHQIGHLLGLDHSSDVESIMYPTI). A total of 502 hits were found in ESTs from 52 species (seven animals and 45 plants, including trees, monocots, and dicots), among which 101 corresponded to the variant (Gln) site. These Gln variants were found exclusively in legume species (one from Trifolium pratense, 14 from M. truncatula, and 86 from soybean). These came from 17 cDNA libraries (one from T. pratense, 10 from M. truncatula, and six from soybean) and often, but not always, corresponded to stress responses (e.g. response to salicylic acid, with 19 soybean ESTs). Legume MMP ESTs with a nonvariant (Glu) site were also found but overall with a lower frequency (one from T. pratense; six from M. truncatula, but not in the libraries where the Gln variants were found; 31 from soybean). In addition, two other active site variants were found at a very low frequency among soybean ESTs, with one Glu-to-Pro and one Glu-to-Asp substitution.
A phylogenetic tree analysis was carried out on all predicted plant MMP proteins or protein fragments containing at least the C switch and the catalytic domain (in total, 38 sequences from 18 plant species). In addition, this analysis included eight representatives of animal MMPs belonging to the same subfamily (M10B in PROSITE nomenclature). To compare fragments of similar lengths and therefore generate a more reliable tree, only sequences spanning the region from the Cys switch to the Met turn were considered. Figure 2
presents the resulting tree, showing clearly that the eight animal MMPs form a subgroup separated from the plant MMPs and that MtMMPL1 stands in a group of closely related proteins, which contains the six (Gln) legume MMP variants as well as GmMMP2. The fact that the soybean SMEP1 protein does not belong to this group indicates that this group does not simply correspond to legume sequences and suggests a very distinct function for SMEP1. None of the proteins of this group possesses the binding site for a calcium ion and a second Zn ion, known as structural Zn, found in many MMPs (Massova et al., 1998
Using quantitative reverse transcription (qRT)-PCR analysis, we compared MtMMPL1 transcript levels in symbiotic and nonsymbiotic conditions. MtENOD11, a repetitive Pro-rich protein gene induced by purified Nod factors and by S. meliloti infection (Journet et al., 2001
Consistent with MtMMPL1 EST distribution, we could not detect significant MtMMPL1 transcription in M. truncatula shoots, stems, flowers, or seed pods (data not shown), while we confirmed a strong MtMMPL1 induction in young nodules (Fig. 3A
). Using this sensitive method, MtMMPL1 expression could not be detected in Nod factor-treated root samples, in contrast to MtENOD11 (data not shown). MtMMPL1 transcripts were detected in wild-type M. truncatula roots at 3 dpi with S. meliloti, while practically undetectable at 1 dpi and in noninoculated roots (Fig. 3B). MtMMPL1 transcripts were about 5 to 14 times less abundant than MtENOD11 mRNA in infected root and nodule samples. Figure 3B also shows that, at 3 dpi, MtMMPL1 expression was much more strongly induced in the hypernodulating M. truncatula mutants sunn (TR122 allele; Sagan et al., 1995
To determine more precisely the spatio-temporal pattern of MtMMPL1 expression, we then carried out in situ hybridizations (ISHs) with 35S-radiolabeled MtMMPL1 antisense riboprobes (Fig. 4 ). As expected, hybridization with the MtMMPL1 sense probe control did not give a detectable signal (data not shown). To study early infection stages, we carried out ISH on sections of roots that had been spot inoculated with S. meliloti and harvested at 2 dpi. At this stage, under our experimental conditions, ITs just begin to form in infected roots hairs, concomitantly with cell divisions in the inner root cortex. Figure 4, A and B, shows an example of an epidermal cell bearing an IT at an early stage of development, to which MtMMPL1 transcripts were found to be specifically associated in several serial sections. No MtMMPL1 hybridization signal was observed in noninfected dividing cortical cells. Once the sequence of the MtMMPL1 genomic region became available, we could confirm this expression pattern in infected roots, using a promoter::GUS fusion generated with a 2.4-kb region upstream of the MtMMPL1 start codon and introduced in M. truncatula by Agrobacterium rhizogenes-mediated root transformation (Fig. 4C).
In young nodules, MtMMPL1 transcripts located to inner nodule tissues where ITs develop and ramify within newly divided cells (Fig. 4, D–F), as previously observed with several other early nodulin genes (de Carvalho Niebel et al., 1998 ; Gamas et al., 1998). In older, well-differentiated nodules, MtMMPL1 transcripts were only detected in the infection zone II where bacteroids are liberated from ITs and it could clearly be seen at high magnification that the hybridization signal was closely and exclusively associated with ITs (Fig. 4, G–I).
To confirm that MtMMPL1 expression is tightly correlated to S. meliloti infection, we then analyzed situations where infection was impaired due to various mutations in bacterial or plant symbiotic genes/locus. Thus, we used an exoA mutant of S. meliloti, defective for EPS production, which shows IT abortion in root hairs and elicits small empty nodules (Yang et al., 1994
We also carried out qRT-PCR analysis of MtMMPL1 expression in three early symbiotic M. truncatula mutants altered in the infection process, namely, nsp1 (B85 allele), hcl (B56 allele), and lin (D8 allele). The nsp1 mutant is unable to trigger cortical cell division and is defective in root hair curling and IT formation in response to S. meliloti (ccd–, hac–, and inf– phenotype, respectively), but exhibits normal root hair deformation and very limited MtENOD11 induction in response to Nod factors (Catoira et al., 2000 ). The hcl mutant is hac– ccd+ (Catoira et al., 2001) and the lin mutant is ccd+ hac+, but all infections are arrested within root hairs where ITs fail to elongate (Kuppusamy et al., 2004). We found that, following S. meliloti inoculation, MtENOD11 was induced in lin and hcl roots (Table I) but not in nsp1. Using the same samples, we were unable to detect MtMMPL1 expression in nsp1 and hcl, while in lin expression was around 10-fold lower than in wild-type M. truncatula and similar to the level obtained in wild-type M. truncatula in response to S. meliloti exoA (Table I). Taken together, these results lead us to conclude that MtMMPL1 expression is specifically associated with S. meliloti infection and triggered at the onset of IT formation.
We further investigated the functional role of MtMMPL1 by an RNAi approach. An RNAi construct, covering most of the MtMMPL1 translated and 3' untranslated regions, was cloned into pRedRoot II. This vector, derived from pRedRoot (Limpens et al., 2004
The symbiotic competence of MtMMPL1 RNAi roots was compared with that of control roots (empty vector transformed). Similar kinetics of nodulation and nodule numbers were observed (Fig. 6A ). MtMMPL1 RNAi nodules were clearly functional because plants grew vigorously for several weeks in a medium without combined nitrogen. In fact, these nodules were slightly larger (about 1.3-fold) than control nodules in three out of the four experiments performed. However, while the overall structure of nodules appeared normal, vibratome (50 or 70 µm) and semi-thin (4 µm) nodule sections revealed that, within the infection zone, ITs were on average significantly larger in diameter in comparison to those found in control nodules (Fig. 7, A and B ). These enlarged ITs were not accompanied by obvious signs of plant defense reactions such as autofluorescence and thickening of IT walls. Electron microscopic observations of ultra-thin sections (Fig. 7, C and D) confirmed the presence of very abundant (but otherwise apparently normal) bacteria filling these enlarged ITs. Bacteroid differentiation and bacterial release appeared normal in the nodule zones II and III, associated with type 1 to type 4 bacteroids (Vasse et al., 1990 ).
To explore whether IT enlargement was accompanied by a difference in bacterial viability, we counted the bacteria recovered from crushed nodules (following surface sterilization). Using nodules harvested at 21 or 44 dpi, we found a substantial increase in the number of bacterial colonies recovered from MtMMPL1 RNAi nodules in comparison to control nodules (Table II ).
We also examined the phenotype of transgenic roots expressing MtMMPL1 constitutively under the control of the 35S promoter, which resulted in a more than a 1,000-fold increase in MtMMPL1 transcript accumulation in noninoculated roots (Fig. 5B). Under these conditions, the number of S. meliloti-induced nodules was decreased by about 60% (Fig. 6B). These nodules were fully functional and indistinguishable from control nodules in terms of structure or number of viable rhizobia. However, small regions of the root displayed a large number of infections aborting in the epidermis, as shown in Figure 7E, which was never seen in control roots (Fig. 7F). It thus seems that expressing MtMMPL1 before the initiation of S. meliloti infection leads to a decrease in the number of productive infections.
In this study, we investigated the possible role in the nitrogen-fixing symbiosis of a nodulin gene, MtMMPL1, which encodes a member of a family of proteins frequently involved in ECM and tissue remodeling.
The combined use of ISH, a promoter::GUS fusion, and infection-defective mutants allowed us to establish that MtMMPL1 transcription accompanies IT development from root hairs to the nodule primordium and then subsequently in the nodule infection zone. MtMMPL1 was not found to be induced by purified Nod factors, and during the preinfection stage, unlike MtENOD11, a well-characterized early nodulin gene also activated by rhizobial infection. MtENOD11 is activated before the formation of ITs (Boisson-Dernier et al., 2005 MtMMPL1 thus shows both common and distinct features in comparison to MtENOD11 and is therefore quite complementary, e.g. for characterizing the phenotype of early symbiotic plant mutants or for identifying cis- and trans-regulatory elements controlling infection-induced genes.
Nodules with enlarged ITs and more viable rhizobia were observed when MtMMPL1 expression was decreased by RNAi. The bacteria recovered on plates from crushed nodules probably corresponded mostly to those present in ITs because differentiated bacteroids found in indeterminate nodules are unable to divide when cultured (Mergaert et al., 2006
There are several reports of enlarged ITs, either due to Rhizobium or plant host mutations. Thus, alterations in the EPSs or in the LPSs of various rhizobia (R. leguminosarum bv viciae, S. meliloti, Azorhizobium caulinodans) lead to the formation of enlarged ITs with thickened cell walls, often associated with plant defense reactions, and to the production of ineffective nodules in their plant host (pea, M. truncatula, and S. rostrata, respectively; Niehaus et al., 1993
Enlarged ITs due to MtMMPL1 RNAi differed strikingly from these cases because they were found in fully functional nodules with an increased Rhizobium accumulation and without any sign of plant defense responses. It could be argued that the MtMMPL1 RNAi used here corresponded to a weak mutant allele, for which an altered IT structure was perhaps the most easily observed phenotype. Bacterial accumulation in ITs could thus result from nonoptimal bacterial release but without problems of viability because these bacteria were wild type. Bacterial surface components are indeed likely to be important for Rhizobium protection (D'Haeze and Holsters, 2004
Considering the preproprotein structure of MtMMPL1 and the localization of the corresponding transcript, MtMMPL1 is probably exported into the IT cell wall or lumen. The lumen matrix shares many components with the ECM (Rae et al., 1992
First, MtMMPL1 could play a positive role in infection, contributing to the formation of the IT cell wall or ECM. Decreasing MtMMPL1 expression could lead to a modified IT structure with an indirect effect on bacterial accumulation. Major proteins in the IT ECM are extensin-like Hyp-rich glycoproteins (HRGPs), hypothesized to play a role both in polar growth of the IT and in the control of bacterial divisions, which takes place only at the IT tip where the ECM is not rigidified by HRGP cross-linking (Wisniewski et al., 2000
Second, the primary role of MtMMPL1 could be to control the number of infecting bacteria, for two reasons: first, no obvious defects in IT growth or bacterial release were observed in MtMMPL1 RNAi roots, and, second, the phylogenic tree analysis showed a higher level of homology with an MMP associated with a stress/defense reaction (Gm-MMP2) than with other plant MMPs involved in developmental processes (e.g. At2-MMP). In this respect, we note that MtMMPL1-like ESTs come from legume stress-response libraries (such as the response to salicylic acid in soybean). The legume host may control not only the number of productive S. meliloti infections (via ethylene-dependent and -independent pathways) but also the number of infecting bacteria within ITs. As mentioned above, various plant proteins induced during nodulation could have a defense-related function and be involved in this kind of control, notably extensin-like proteins and antimicrobial peptides (defensins) that could inhibit cytokinesis and induce bacteroid differentiation (Mergaert et al., 2006
The key amino acid residue (Glu) known to be involved in the metalloproteinase activity of MMPs is replaced by a Gln residue in MtMMPL1. This Glu-to-Gln substitution prevents protease activity in human MMPs (Crabbe et al., 1994
One hypothesis is that there is a compensatory mutation elsewhere in MtMMPL1-like genes enabling some protease activity to be restored. We could not find a collagenase activity in an MtMMPL1 extract synthesized in vitro (data not shown), but this does not, of course, rule out possible protease activity with natural plant substrates and under the particular conditions existing in ITs. Indeed, Dow et al. (1998)
This study highlights MtMMPL1 as a particularly useful gene for studying various aspects of the infection process, including IT structure. Furthermore, MtMMPL1 represents a novel variant of the MMP family. Even though its precise role remains to be elucidated, this is the first member of this biologically important protein family with a clear function in plant-microbe symbiotic associations.
Biological Material Sinorhizobium meliloti RCR2011 pXLGD4 (GMI 6526) and S. meliloti RCR2011 exoA– pXLGD4 (GMI 3072) were grown at 30°C in tryptone yeast medium supplemented with 6 mM calcium chloride and 10 µg mL–1 tetracycline.
Medicago truncatula seeds were surface sterilized and germinated on inverted agar plates in the dark for 3 d at 8°C and 1 d at 20°C. Plants used for qRT-PCR were grown in aeroponic caissons. Plant growth chamber conditions were the following: temperature, 22°C; 75% hygrometry; light intensity, 200 µE m–2 s –1; light-dark photoperiod, 16 h/8 h. Control root (T0) and nodule samples from wild-type M. truncatula Gaertn ‘Jemalong’ genotype A17 were prepared as described by El Yahyaoui et al. (2004)
RNA was extracted using the SV total RNA extraction kit (Promega) according to the manufacturer's recommendations. RNA quality was checked using a Bioanalyser (Agilent Technologies), and the absence of DNA contamination was verified by a PCR reaction with EF1-
RACE PCR was carried out using a 5'/3' RACE kit, 2nd generation (Roche Diagnostics). The resulting MtMMPL1 full-length cDNA sequence is accessible in the EMBL database (accession no. Y18249).
For the RNAi construct, we followed the procedure described by Limpens et al. (2004)
For the promoter-GUS fusion, we used a P-green (www.pgreen.ac.uk)-based vector with a modified polylinker, pPex (L. Sauviac, unpublished data), and a GUS cassette (Combier et al., 2006
Roots were transformed using Agrobacterium rhizogenes, following essentially the procedure described by Boisson-Dernier et al. (2005)
GUS staining (using only 5-bromo-4-chloro-3-indolyl-
Histology of the nodule was performed after fixation in 2.5% of glutaraldehyde buffered in 0.1 M sodium phosphate buffer, pH 7.2, dehydrated in an alcohol series and embedded in Technovit 7100 resin (Hereaus Kulzer). Sections (4 µm thick) were observed after counterstaining in a 0.2% aqueous toluidine blue solution. For electron microscopy studies, following the glutaraldehyde fixation step, nodules were postfixed in 1% osmium phosphate buffer solution; we then proceeded to epon embedding steps as previously described (Vasse et al., 1993
ISHs were carried out as described by de Billy et al. (2001)
Nodules were cut from roots at the indicated times. They were surface sterilized using the following procedure: immersion for 30 s in 70% ethanol, wash with distillate water (five times), immersion for 30 s in 25% (v/v) commercial bleach, and final wash with water (six times). Sterilized nodules were ground with a glass tube in 100 µL of water. Aliquots of serial dilutions (10- to 105-fold) were spread on tryptone yeast plates supplemented with 6 mM calcium chloride.
Protein segments of similar lengths were manually defined, as indicated. Amino acid sequences were then aligned using ClustalW (http://clustalw.genome.ad.jp/) and analyzed with the PHYLIP software package (http://www.csc.fi/molbio/progs/phylip/doc/main.html). Bootstrap values were obtained from 1,000 replicates. The branch lengths indicate the frequency of the corresponding clade in the set of bootstrap trees. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number Y18249.
The following materials are available in the online version of this article.
We thank Jérôme Gouzy, Thomas Ott, and Ton Timmers (LIPM) for their help in bioinformatics, phylogenic tree analysis, and phenotype assessment, respectively. We are also very grateful to David Barker, Clare Gough, and Ton Timmers for their critical reading of the manuscript and to D. Barker for English reviewing. We thank René Geurts (Wageningen University) for providing us with the pRedRoot vectors and Jean-Marie Prosperi (INRA Montpellier) for M. truncatula seeds. Received November 6, 2006; accepted January 20, 2007; published February 9, 2007.
1 This work was supported by the Sixth Framework Programme Grain Legume Integrated Project (postdoctoral grant to J.-P.C.), by the Institut National de la Recherche Agronomique (Département Santé des Plantes et Environnement; postdoctoral grant to F.E.Y.), by the French Research Ministry (doctoral grant to R.M.), and by the European Union/Centre National de la Recherche Scientifique (Fonds Social Européen; doctoral grant to T.V.). 
2 These authors contributed equally to the article.
3 Present address: PROTENIA SAAl, Akhawayn University International, 53000 Ifrane, Morocco.
4 Present address: GEVES-SNES, Rue Georges Morel, BP 24, 49071 Beaucouze, France. The author 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) is: Pascal Gamas (pascal.gamas{at}toulouse.inra.fr).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.092585 * Corresponding author; e-mail pascal.gamas{at}toulouse.inra.fr; fax 33–561–28–50–61.
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housekeeping gene as an internal control. The error bar depicts the variation between two biological repetitions and two technical repeats.
-galactosidase activity detection (note the numerous abortive infections in E, in comparison to F). Transverse and longitudinal sections of ITs are shown by pink and black arrows, respectively (note the enlarged ITs in B and D); green arrowheads point to nucleoli. Bars = 50 µm, except in C and D (5 µm).
-ray mutagenesis. Plant Sci 111: 63–71
