Journal of Experimental Botany, Vol. 52, No. 90001, pp. 459-468,
March 2001
© 2001 Oxford University Press
Gene expression profiling of two related maize inbred lines with contrasting root-lodging traits
1 Pioneer Hi-Bred Intl., Inc., 7300 NW 62nd Avenue, Johnston, IA 50131-1004, USA
2 Curagen Corp., New Haven, CT. 06511, USA
Received 23 March 2000; Accepted 23 July 2000
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Abstract |
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To assist breeding for increased resistance to root lodging in maize, an attempt was made to identify genes that are associated with root lodging by profiling mRNA expression from two inbreds with contrasting root-related traits. These two inbreds were derived from a common F2 pool, selfed for several generations and showed 75% relatedness based on 106 genetic markers. Under field conditions, the two inbreds exhibited significant differences in root morphology and resistance to root lodging. Whole root tissue was collected at two developmental stages from inbred 100 and 101 grown in 2 years. RNA was isolated from both the V8 and V12 stages, a few weeks prior to flowering. The RNA samples from the 1997 growing season were analysed by GeneCalling analysis, an open-ended mRNA profiling method. From over 13 500 cDNA fragments detected from each of the V8- and V12-stage samples, 229 and 325 cDNA fragments, respectively, showed greater than 2-fold differences between the two inbred lines. A total of 69 cDNA fragments that showed 2-fold or greater differences for both inbred lines were observed at both developmental stages. The gene identity and expression differences of several cDNA fragments were determined and confirmed by RNA gel blot analysis. Two genes out of five identified were homologous to a cytochrome P450 and the impedance-induced protein, both showing high levels of expression in the roots of lodging resistant lines and low levels in the sensitive lines. These data provide the first clues of genes expressed in the roots during the formative stages of root development associated with root-lodging resistance.
Key words: mRNA profiling, edaphic, adventitious, anchorage, Zea mays.
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Introduction |
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Breeding programmes strive to improve the appropriate traits while countering negative environmental influences that reduce crop yield. Some agronomic characteristics such as plant height or flowering time are more easily bred while others such as improved root-related traits are selected indirectly. Since root lodging, which is a failure of plants to maintain an upright stature, can drastically reduce harvestable yield in numerous crops (e.g. Zea mays; Carter and Hudelson, 1988
), it is an important trait for breeding programmes. While improving yield, attempts are made to apply some selection pressure to improve root-lodging resistance in hybrids, usually scored as the percentage of the plants that root lodged. However, this measurement is difficult to reproduce due to the reliance on adverse weather conditions (e.g. high winds) revealing contrasts in root lodging scores. Many studies incorporated deliberate tests whereby hybrid and inbred lines with varying resistances to root lodging were scored following mechanical perturbances under a variety of field conditions (Beck et al., 1987; Guingo and Hébert, 1997; Kato and Koinuma, 1999). Furthermore, the underlying genetic complexity observed with root-lodging susceptibility complicates the selection for any one root morphological trait (Hébert et al., 1992) and a combination of individual traits is required in order to provide positive selection in breeding programmes.
The relationship between root lodging and root or aerial morphological traits has been examined in numerous studies. Root mass, root volume, root numbers, diameter of individual roots, angle of root growth from the stem, stalk diameter, ear height to plant height ratios, and length of base internodes were all shown to correlate with either natural or artificial root-lodging resistance (Hébert et al., 1992; Stamp and Kiel, 1992; Ennos et al., 1993; Crook et al., 1994; Seo et al., 1996; Guingo and Hébert, 1997; Baker et al., 1998; Kato and Koinuma, 1999). Another study demonstrated that soil components affect root development characteristics and these traits correlated with root lodging (Goodman and Ennos, 1999). Based on such studies, it is clear that associating any one trait with resistance to root lodging is difficult. In fact, it has been demonstrated that combinations of at least three root traits (diameter, the number of roots on the upper-tiered nodes and angle of root growth) correlated well with the stiffness coefficient, a measure of root-lodging resistance (Guingo and Hébert, 1997). Remarkably, very few studies on mapping quantitative trait loci (QTL) for maize root lodging per se exist, underscoring the difficulty in securing reproducible environmental conditions to measure this trait. However, QTLs for root morphological traits have been mapped in a few crop species (O' Toole and Bland, 1987; Lebreton et al., 1995; McCouch and Doerge, 1995; Guingo et al., 1998). These studies demonstrate the complexity of heritable root traits that may provide the foundation for resistance to root lodging. Understanding the molecular mechanisms underlying the phenotypic expression of this trait would greatly benefit breeding programmes reducing or eliminating the reliance on environmental conditions for selection.
To this end, the authors chose to investigate the differences of steady-state mRNA levels of genes between two related inbreds that showed contrasting root traits and root-lodging scores. These inbreds were extensively field tested over several years as hybrids from crosses with common tester parents. Since these two lines showed both contrasting root-lodging scores and root morphology, it was predicted that certain genes are differentially expressed in the root tissues between these two lines. These genes may directly or indirectly influence root development, thus affecting the soil anchoring properties of the respective lines. A mRNA profiling analysis was used to identify genes whose differential expression between the two inbred lines may suggest a role in root-lodging resistance in maize. Whole root tissue from the two inbreds was used in the GeneCalling analysis which is an open-architecture, gel-based assay that reproducibly measures changes in the levels of tens of thousands of cDNA fragments simultaneously (Shimkets et al., 1999; Bruce et al., 2000). This analysis provides a means of comparing cDNA fragment profiles from different RNA samples and associating the cDNA fragments to genes whose expression levels modulate between treatments. GeneCalling analysis has been used successfully to identify gene members of the known flavonoid pathway that were induced by appropriate transcription factors under inductive controls (Bruce et al., 2000).
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Materials and methods |
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Inbred lines
The maize (Zea mays L.) inbreds 100 and 101 originated from an F3 pool generated from a segregating F2 population of a cross between two Pioneer proprietary élite inbreds R23xM10. Two plants were selfed six generations before undergoing trait evaluations. The inbreds 100 and 101 were analysed with 106 RFLP, isoenzyme and SSR markers (essentially as described in Beavis et al., 1994
). 80 markers were identical between inbreds 100 and 101 suggesting that the genomes of these two lines were 75% homologous (data not shown). Regions of marker differences were distributed throughout the genome. These two inbred lines were crossed to several common testers and the resulting F1 hybrids were evaluated in single or two row plots at a variety of locations in North America and Europe. Agronomic trait data such as grain yield and percentage lodging were collected from the hybrids grown between 1993–1995. For root-lodging resistance, the number of replicates examined exceeded 200 where indicated. Root-lodging scores were determined as a percentage of the plants lodged per replicate.
The H2 and AC7 lines were generated from an introgression backcrossing programme for root-lodging resistance into the parental inbred 105 and showed contrasting root-lodging scores (PD and S Openshaw, unpublished results). During 1998 and 1999 at two European locations (designated FP and FE), the mechanical root-lodging data were collected for the lines 105, H2 and AC7 either directly (‘FP98’, ‘FE98’ and ‘FE99-1’) or in hybrid combinations with two different testers (‘FE99-2’ and ‘FE99-3’) as shown in Fig. 1B. Since there was a lack of sufficient wind damage, natural root lodging could not be measured. Ten to twelve plants per replicate for three replicates per location were pushed by hand using a 1-m-long wooden rod placed just below the primary ear. The plants were evaluated just after extensive irrigation. The H2 and AC7 lines were grown in the same location as inbreds 100 and 101 in 1999 for tissue harvesting and RNA gel blot analysis as described below.
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Root morphology analysis
Whole roots were carefully excavated from the two inbreds (100 and 101) at the V8 and V12 developmental stages (the stages where the ligule of the 8th and 12th leaf emerges, respectively; Ritchie et al., 1997
) from 1997 and 1999. Soil was removed by two gentle washes in water and patted dry with paper towels. For RNA isolations, roots from two plants per replicate were frozen within 10 min of excavation in 50 ml conical tubes using a CP300 cryoshipper (Taylor-Wharton, Theodore, AL) containing liquid nitrogen. Morphological measurements were taken on the V8-stage roots as previously described (Guingo and Hébert, 1997). The number of roots on the seventh node (top-most node of the developing roots) and the diameter of five randomly chosen 7th nodal roots per plant were measured using an electronic caliper (Fred V Fowler Co., Inc., Newton, MA). The angle of 7th nodal root growth from the vertical was measured on five roots per plants using a protractor. Stem diameter of the internode above the eighth node where adventitious roots were emerging was measured. At least six plants per replicate from two replicates in 1997 and one in 1999 were recorded. These data were averaged across both years since ANOVA for year to year effects showed no significant difference.
DNA, RNA isolation and RNA gel blot analysis
Total RNA was isolated from V8- and V12-stage whole roots from inbred lines 100 and 101 at 1997 and from all four lines (100, 101, H2, and AC7) in 1999 using a protocol previously described (Dehesh et al., 1990). RNA gel blot analysis was conducted using 10 µg of total RNA per gel lane (as described by Bruce et al., 2000). Randomly primed 32P-labelled probes generated from the Pioneer/DuPont maize expressed sequence tag (EST) database were as follows: Ef1
(cssaq52), TrpA (czaal73), Hsp70 (cgeuk42), impedance-induced protein (crtba20), and CYP71C2 (cebae55). The blots were successively probed, exposed to X-ray film from 1–4 d for visualizing signals and re-stripped using the Strip-EZ kit (Ambion, Inc., Austin, TX) according to the manufacturer's protocol.
Genomic DNA was isolated from root tissue of the inbreds 100 and 101 (Doyle and Doyle, 1990) and used in a PCR reaction with the Hot Star Taq Polymerase kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The following primers were used to amplify at least three independent identical fragments of the maize TrpA gene (Kramer and Koziel, 1995): 5'-GAGGCCCGCTCTTGCTATAAACGAGGCAGC-3' and 5'-GGATCGATCTCGGCCGGCTAGCTAGCAG-3'. The resultant fragments were cloned using the pCR2.1TOPO kit (Invitrogen, Carlsbad, CA), sequenced to 4x coverage and aligned using the PC software of Sequencher 4.05 (Gene Codes Corporation, Ann Arbor, MI).
GeneCalling analysis
The GeneCalling analysis comprehensively surveys cDNA populations in a highly sensitive manner allowing for detection of both expression levels and restriction fragment length polymorphisms (RFLP) associated with individual cDNA fragments. It was used here to compare quantitatively the amounts of restriction enzyme-digested cDNA fragments generated from whole root tissue of the two contrasting inbreds at two developmental stages. This analysis was performed according to Shimkets et al. (Shimkets et al., 1999), except that poly (A)-enriched RNA was isolated from 50 µg of total RNA and at least 41 restriction enzyme pairs were used. The GeneCalling gel trace data from four to six replicate cDNA samples derived from two plants per inbred for the inbred 101 were compared to those of inbred 100 for both developmental stages.
RNA was purified, converted to cDNA and fragmented using pairs of restriction enzymes. Adapters were then ligated to the ends of the fragments and PCR amplified. Since one of the PCR primers was labelled with a fluorescent tag, fluorescamine (FAM), amplified fragments were visualized during electrophoresis. For each restriction enzyme-pair reaction per sample, electronic images of gel lane traces were collected and placed in a sample trace database. Comparisons of the trace databases revealed specific expression differences that were characterized by length of the amplified fragment and restriction enzyme sequence information. The identity of each differentially expressed gene fragment was established either by a GeneCalling search in a sequence database, or by cloning and sequencing the desired cDNA fragment. The identity of the cDNA fragment was confirmed by competitive PCR in which the original PCR reaction was re-amplified in the presence or absence of an excess of an unlabelled, gene-specific PCR primer. Further characterization of known and novel sequences, identified in the GeneCalling analysis as differentially expressed, was obtained by BLASTX and BLASTN analysis (Altschul et al., 1990) against public and proprietary databases. Those with little or no match to public databases having an expected probability value less than 1.0x10–5 were subjected to BLAST 2.0 searches in the Pioneer/Dupont maize EST database. EST sequences that matched a novel cDNA fragment sequence with significant similarities were then used in BLASTX searches to determine likely gene identities.
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Results |
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Root lodging and morphology
The inbreds 100 and 101 were tested for numerous agronomic traits in hybrid combinations. One of these traits, the average percentage of lodged hybrids per replicate based on the two inbreds crossed to 5–20 testers is shown in Fig. 1A
. Over 200 replicates in 3 years were conducted across North American and European locations for hybrids derived from either inbred line. Inbred 100 produced hybrids with a significantly higher percentage of root lodging than hybrids from inbred 101. It was evident that the root-lodging resistance phenotype can be manifested in heterozygous genotypes from a number of tester backgrounds.
The differences in root-lodging scores between hybrids from inbreds 100 and 101 may be due in part to differences in root morphological characteristics in the parental inbreds similar to what was observed previously (Guingo and Hébert, 1997). Figure 2 shows examples of root clumps excavated for both inbreds at the V8 stage and Table 1 shows the characteristics of both root and stem for both inbreds averaged over the two years. Based on analysis of variance, the root morphology for each line did not vary significantly (P>0.63) between the two years of measurements in contrast to observations made by Hébert et al. (Hébert et al., 1992). The 7th node was chosen for measurements based on the conclusion that upper nodal roots correlated more strongly with the strength of plant anchorage than lower older roots (Guingo and Hébert, 1997). Inbred 101 showed nearly a 30% increase in the number and diameter of 7th nodal roots over inbred 100. The angle of root growth from the vertical axis for inbred 101 (30.6±9.73°) was nearly 50% more than that for inbred 100 (19.83±4.31°; Table 1). These data support the observations that plants with larger root numbers, thicker roots and larger angled root systems correlated significantly with the strength of plant anchorage (Hébert et al., 1992; Ennos et al., 1993; Guingo and Hébert, 1997).
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mRNA profiling
Using GeneCalling analysis with root RNA samples from both inbred lines, 13 630 and 13 544 cDNA fragments were detected at the V8 and V12 developmental stages, respectively. From a comparison of the cDNA fragment trace data between inbreds, 229 and 325 cDNA fragments showed greater than or equal to a 2-fold difference in the V8 and V12 developmental stages, respectively. Gel trace data comparisons of the cDNA fragments showing a 2-fold or greater difference between the inbreds revealed 69 cDNA fragments in common for both developmental stages as shown in Fig. 3. Nearly half of these cDNA fragments were expressed at higher levels in the inbred 101 than in inbred 100. There was also a good linear correlation (r2=0.72) in N-fold difference between the two developmental stages (data not shown). Seven of the 69 cDNA fragments were selected for further analysis based on their match to publicly known genes. Five cDNA fragments were used for direct competitive PCR confirmations with three known sequences while the remaining two fragments were cloned, sequenced and confirmed by competitive PCR. The seven fragments corresponded to five known genes, tryptophan synthase (TrpA; Kramer and Koziel, 1995), heat shock protein 70 (Hsp70; Rochester et al., 1986), elongation factor 1 (Ef1; Cao et al., 1997), cytochrome P450-dependent monooxygenase (CYP71C2; Frey et al., 1995), and an impedance-induced protein (Huang et al., 1998). The oligonucleotide primers designed for these public genes used in the competitive PCR reaction successfully competed for the original cDNA fragment amplification as demonstrated for the TrpA gene in Fig. 4.
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The competitive PCR reaction provided a sensitive method for determining the proportion of a target cDNA fragment present in a band within the gel trace data. It can also readily reveal intra-fragment polymorphisms that exist especially between different genotypes as noted for three cDNA fragments corresponding to the TrpA gene (Fig. 4
), two of which were part of the original seven fragments targeted for further analysis. The gel trace data revealed that these three cDNA fragments from inbred 101 were approximately nine (±1.5) nucleotides shorter than the corresponding fragments for inbred 100 (Fig. 4B, C). However, there was little change in the trace peak height values for the three fragments suggesting small or no difference in TrpA expression levels between the inbred lines. All three fragments overlapped the same region in a portion of the 3'-translated and 3'-untranslated region of the transcript sequence (data not shown).
To investigate further the potential allelic TrpA gene differences between inbreds 100 and 101, a genomic fragment from each inbred line was cloned and sequenced. Figure 5 shows the alignment of the TrpA gene between the two inbreds both at the nucleotide and amino acid level. The TrpA of inbred 100 shows a three base insertion relative to that from inbred 101 resulting in a 15 amino acids change in the N-terminal region of the gene. Also the TrpA gene was mapped to a locus on chromosome 1L (Davis et al., 1999) very close to where there is a polymorphic marker between the two inbred lines (data not shown), confirming the differences detected both by GeneCalling and sequencing.
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Contrasting levels of gene expression between inbred samples for the other cDNA fragments were observed as shown in Fig. 6
. However, no evidence of polymorphisms for these cDNA fragments was detected (data not shown). These results do not rule out the possibility of polymorphisms affecting the restriction enzyme sites used in fragmenting the cDNA samples. Therefore, the cDNA fragments were examined further by RNA gel blot analysis, shown in Fig. 7, to confirm the expression differences detected by GeneCalling (Figs 4, 6). Supporting the GeneCalling data, the TrpA showed little difference in RNA levels between the inbred lines. In contrast, the CYP71C2 gene and the impedance-induced gene showed significantly higher steady-state levels of transcripts in the root samples harvested at both years for inbred 101 compared to inbred 100. The levels of change for these two genes also agreed with that determined by GeneCalling (Fig. 7). The mRNA levels of a Hsp70 gene showed a small difference between the inbred lines for samples from both years in the RNA gel blot analysis while the GeneCalling analysis showed nearly a 6-fold difference, a greater difference observed than that by the RNA gel blot analysis. The Hsp70 gene family constitutes several highly conserved members (Bates et al., 1994; Rochester et al., 1986). Coupled with the fact that the GeneCalling analysis is sensitive to polymorphic differences (Fig. 4), the apparent discrepancy in fold change between the two analyses is likely due to either problems with cross-hybridization between the members of this gene family during RNA gel blot analysis or polymorphisms in the GeneCalling analysis. The same may be true for the Efl gene (Davis et al., 1999). Ef1 was more actively expressed in inbred 100 than 101 by a factor of five based on GeneCalling whereas the RNA gel blot analysis showed a mild difference between the inbred lines albeit in the same direction.
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RNA from a second pair of maize lines (H2 and AC7) derived from a backcrossing programme of introgressing root-lodging resistance into a root-lodging-susceptible parent (inbred 105) was also used in the RNA gel blot analysis (Fig. 7
). H2 exhibited significantly higher root-lodging resistance relative to a control line, AC7 either directly or in one of the two hybrid combinations tested (Fig. 1B). Since both the H2 and AC7 lines were grown in the same location a few rows away from the inbreds 100 and 101 for RNA sampling in 1999, it was expected that H2 and AC7 experienced the same environmental influences as 100 and 101. Four of the five genes showed similar RNA patterns as that of inbreds 100 and 101 while the Hsp70 gene showed a converse pattern. At least for four of the identified genes, these data help to confirm the expression differences observed with inbreds 100 and 101.
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Discussion |
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Two related inbreds showing contrasting root-lodging traits were characterized for root morphological differences and subjected to GeneCalling analysis, a method where over 13 500 cDNA fragments from root samples were compared between the inbreds at two developmental stages. Higher levels of expression for several genes identified by GeneCalling were associated with either one or the other inbred suggesting a relationship between these genes and root anchorage traits.
Three root morphological traits and the diameter of the lower stem at the V8 stage were measured and showed significant differences between the two inbreds. The roots for the 7th node were targeted because these roots were predominant at the V8 stage and that the 8th nodal roots were just emerging (Fig. 2). It was noted from three years of observations that a divergence in overall root traits between inbreds 100 and 101 was manifested between the V6 and V10 stage of development (W Bruce, unpublished results). The upper nodal roots (5th–8th) for most maize lines grown in the north to central US corn belt initiate around the V8 stage and are essentially developed by the V15 stage (Ritchie et al., 1997). Guingo and Hébert also demonstrated a correlation between upper nodal root traits and a measure of root anchorage (Guingo and Hébert, 1997). Taken together, it was anticipated that any actively transcribed genes contributing to the success in root anchorage in inbred 101 or failures in inbred 100 would be evident within the V8–V12 developmental stages. Measurements of root number, root angle and root diameter for the 7th node revealed that inbred 101 had at least a 30% increase in root diameter and a nearly 50% increase in root numbers and root growth angle over inbred 100. These increases correlated well with the observed hybrid performance in root lodging (Fig. 1) and support the conclusions that such traits in the upper nodal roots contribute to root anchorage (Hébert et al., 1992; Ennos et al., 1993; Guingo and Hébert, 1997).
With the GeneCalling analysis, Shimkets et al. demonstrated that they could theoretically detect cDNA fragments for over 80% of the expressed genes in maize RNA samples with at least two cDNA fragments per single gene using 48 restriction enzyme pairs (Shimkets et al., 1999). Since over 13 500 cDNA fragments using at least 41 restriction enzyme pairs were detected in root RNA samples from the two inbreds at both developmental stages, it can be estimated that cDNA fragment modulations of 4000–6000 genes were detected. Although 229 and 325 bands from whole roots at the V8 and V12 stages, respectively, were detected as showing 2-fold or greater difference between the two inbreds, only 69 differentially expressed cDNA fragments were observed at both developmental stages. It is predicted that these cDNA fragments correspond to genes that relate to the overall architectural differences between the inbreds rather than those that respond to environmental fluctuations occurring between the two developmental stages. Therefore representatives of the 69 cDNA fragments were examined further.
Nearly half of the 69 cDNA fragments that were in common to both developmental stages were expressed at higher levels in the inbred 101 than inbred 100, whereas the other half had the converse expression pattern (Fig. 3). Although, a few cDNA fragments showed contrasting expression differences between the two developmental stages, a high correlation of N-fold difference between inbreds was observed for both developmental stages. Selected cDNA fragments were subjected to either competitive PCR or cloning, sequencing and competitive PCR depending on the match to known gene sequences. This resulted in seven of these fragments corresponding to five previously identified genes. To confirm the expression differences detected by GeneCalling, RNA gel blot analysis was conducted which has previously been demonstrated to correlate well with the modulations of the levels of cDNA fragments detected by GeneCalling (Bruce et al., 2000). The differentially expressed cDNA fragments identified for the inbreds 100 and 101 by competitive PCR method were matched to corresponding EST clones from the Pioneer/DuPont EST collection and these clones were used as probes for the RNA gel blot analysis.
Two of the differentially expressed genes identified, the CYP71C2 and the impedance-induced protein transcripts, were much more abundant in root tissues of two maize lines with high root-lodging resistance (101 and H2) as compared to those with low resistance, confirmed by RNA gel blot analysis (Fig. 7). The CYP71C2 (alias Bx3) is a cytochrome P450-dependent mono-oxygenase, involved in the 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) synthetic pathway in maize (Frey et al., 1995, 1997). The CYP71C2 is part of a pathway of converting indole-3-glycerol phosphate into DIMBOA and the indole-3-glycerol phosphate is an important intermediate for numerous secondary metabolic pathways including tryptophan and indole acetic acid biosynthesis (Frey et al., 1997). Interestingly, the first committed enzyme in the DIMBOA pathway is Bx1, involved in indole production, and is highly homologous to the TrpA gene (Frey et al., 1997), suggesting a correlation between expression differences of members of the DIMBOA pathway including CYP71C2 and differences between the inbreds showing contrasting root traits.
The impedance-induced gene was previously shown to be rapidly induced when elongating roots encounter physical impedance (Huang et al., 1998). This gene product is very similar to Zrp3 observed to be expressed in a subset of cortical cells near the expansion zone of root tips (John et al., 1992) and may be part of a multigene family in maize. The impedance-induced gene product may also function in the early stages of stress-inducible responses (Huang et al., 1998) and act as an indicator of the state of root growth under non-ideal soil conditions especially for the contrasting maize lines.
Two genes, Ef1 and Hsp70, were identified by GeneCalling analysis as showing 5–6-fold differences between the two inbreds, not entirely agreeing with differences detected in the RNA gel blot analysis. Fragment size modulations were not detected in the PCR confirmations for either of these genes, reducing the possibility of polymorphic differences. Since both genes belong to very highly conserved multigene families (Rochester et al., 1986; Bates et al., 1994; Berberich et al., 1995) and because the detection of cDNA-fragment-gel-trace differences is more gene-specific than RNA gel blot analysis, it is suspected that cross hybridization may be occurring in the RNA gel blot analysis. Other more discriminatory methods of expression detection may be necessary to clarify the difference between GeneCalling and RNA gel blot analysis of the Efl and Hsp70 genes.
Based on the GeneCalling data, a Hsp70 gene family member was potentially associated with differences in root morphology or root lodging. Hsp70 proteins have been shown to interact with DnaJ proteins via the DnaJ's ‘J-domain’ (Zuber, 1998). One plant DnaJ protein was recently described as being encoded by the ARG1 gene. When this gene is mutated in Arabidopsis, altered gravitropic responses in roots and hypocotyl were observed without pleiotropic phenotypes (Sedbrook et al., 1999). It is intriguing to speculate that the Hsp70 gene product showing differential expression in the two maize inbreds may influence gravitropic responses via a protein–protein interaction with the gene product of an orthologous maize ARG1 (W Bruce, unpublished results). This interaction may generate differences in the angle of root growth that is important in root-lodging resistance. This will need further confirmation, possibly using the yeast two-hybrid approach.
One of the genes identified by GeneCalling as showing apparent expression differences was TrpA. The cDNA fragment for TrpA was first detected with an apparent 17-fold higher expression level in inbred 101 than in 100. However, this difference was due to a polymorphism based on the competitive PCR reaction with both inbred samples. The calculated ratio was 1.2 between inbred 101 to inbred 100, suggesting no difference in expression levels between the inbreds, as confirmed by the RNA gel blot analysis (Fig. 7). Upon further sequence analysis of the TrpA gene from inbreds 100 and 101, a significant polymorphism was detected resulting in a three-nucleotide change leading to a 15 amino acids difference in the N-terminal region of the TrpA gene product of inbred 100 relative to 101 (Fig. 6). The maize TrpA sequence was shown to be 84 amino acids longer in this N-terminal region than the longest of four fungal and bacterial TrpA genes used in a protein alignment (Kramer and Koziel, 1995). This suggests that the enzyme activity is relegated to the remaining portion of the maize protein. Therefore, changes in the N-terminal region as seen between inbred 100 and 101 TrpA alleles may not necessarily affect the catalytic activity. However, it is not clearly known whether this N-terminal region plays a regulatory, intracellular targeting or factor-binding role. The possibility that the 15 amino acid difference may indeed affect overall enzyme function requires further determination.
It has been demonstrated that mutations in the Arabidopsis TrpA gene (trp3-1) caused greater compressions in the root waving phenotype on tilted agar surfaces (Rutherford et al., 1998). The root waving phenomenon may be a result of integrating gravitropic and impedance avoidance stimuli with circumnutation-like growth. These authors postulated that localized reduction in free L-tryptophan in the roots affected root tip rotation and the circumnutation-like growth in a gravitropism-independent manner (Rutherford et al., 1998). It is possible that maize genotypes showing differential resistance to root lodging may be producing varied levels of tryptophan-related metabolic enzyme activities (TrpA and CYP71C2) in root tissues, ultimately affecting root growth and architecture.
Only a small number of the cDNA fragments identified by GeneCalling were analysed in some detail. It is likely that many of the remaining fragments may overlap with each other and reduce the possible number of genes associated with root-lodging resistant and sensitive lines. It should be noted that some of the identified genes may either be differentially expressed in the contrasting maize lines coincidentally or be directly involved in root development thus affecting root-lodging-related traits. The expression pattern of such genes may also provide a measure of the state of root tissue growth and metabolism as in the example of the EF1 gene involved in protein translation. Regardless of the function, if the expression modulations of these genes show a strong correlation with contrasting root-lodging traits across many genotypes, these genes and their corresponding transcription factors become useful targets for breeding programmes. Using a powerful molecular screening tool, the first steps have been taken in identifying the genes possibly linked to root-lodging resistance. By revealing the identity of the remaining cDNA fragments, there will be a better understanding of the interplay between gene activity and an important but little understood agronomic trait.
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Acknowledgments |
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We wish to thank Marcie Vaughn, Brian Zeka and Leo Koster in assisting with root tissue harvests. We also thank Michael Turner Jr and Jessica Galbraith for technical assistance. An expression of gratitude is extended to Dr Veera Padmanabhan for critical review of the manuscript. A special thanks goes to Dr Gary Weber and his crew for generating the two inbreds, 100 and 101 and to Dr Tom Barker and David Ritland for assistance with the field management for these lines. We especially thank the GeneCalling and PCR Confirmation teams (Curagen Corporation, New Haven, CT) under the direction of Dr Michael McKenna for their excellent contributions.
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Notes |
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3 To whom correspondence should be addressed. Fax: +1 515 334 4788. E-mail: brucewb{at}phibred.com
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