Heather I. McKhann 1 and Ann M. Hirsch*. Department of Biology ... Received 27 August 1993; accepted in revised form 12 January 1994. Key words: chalcone ...
Plant Molecular Biology 24: 767-777, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium.
767
Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips Heather I. McKhann 1 and Ann M. Hirsch*
Department of Biology, 405 Hilgard Ave., University of California, Los Angeles, Los Angeles, CA 90024, USA (* author for correspondence); 1Present address: Institut des Sciences V@Otales,Avenue de la Terrasse, 91198 Gif-sur- Yvette, France Received 27 August 1993; accepted in revised form 12 January 1994
Key words: chalcone synthase, chalcone isomerase, alfalfa, Rhizobiurn, nodule
Abstract
Flavonoids are involved in several different interactions between plants and microorganisms. In the
Rhizobium-legume symbiosis, they play an important role as inducers of rhizobial nodulation (nod) genes. We have identified from an alfalfa c D N A library four clones for chalcone synthase (CH S) and two clones for chalcone isomerase (CHI); CHS and CHI are key enzymes in flavonoid biosynthesis. In Medicago sp., CHS is encoded by 8-12 genes, and CHI is encoded by 1-2 genes. Here we report the D N A sequence of these clones as well as their relatedness to other legume CHS and CHI clones. In addition, we report on the expression patterns of two CHS gene family members as well as the CHI gene in M. sativa cv. Iroquois. While CHS and CHI transcript levels are high in root tips and entire young roots, they are low in effective nodules elicited by wild-type strains of Rhizobium meliloti and very low in aerial portions of the plant (stems, leaves, flowers). However, wounding the cotyledons results in a rapid increase in transcript levels of both chalcone synthase and chalcone isomerase genes in these organs.
Introduction
Flavonoids, products of the phenylpropanoid pathway, play a critical role in nodulation of legumes by functioning as inducers of Rhizobium and Bradyrhizobium nodulation (nod) genes [7, 9, 17, 20, 27, 31, 35, 38, 45]. Other postulated roles for flavonoids include chemoattraction of rhizobia to roots [1, 4] and growth enhancement of
rhizobia cultured in minimal media [14]. Evidence for the importance of flavonoids in the nodulation process has been extensively documented in two different nodulating systems. In Vicia sativa, which forms indeterminate nodules, a positive feedback occurs whereby Rhizobium leguminosarum bv. viciae brings about an increase in flavonoid synthesis upon inoculation [36, 37, 42]. In soybean, which forms determinate
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers U01018 (MsCHS4-1), U01019 (MsCHS4-2), U01020 (MsCHS6-4), U01021 (MsCHS12-1), M91079 (MsCHI1) and M91080 (MsCHI2)
768 nodules, inoculation with compatible Bradyrhizobium japonicum strains brings about the expression of specific members of the phenylalanine ammonia lyase and chalcone synthase gene families, whereas inoculation with incompatible rhizobia induces the expression of other gene family members [8]. Furthermore, flavonoid phytoalexins have been reported to accumulate in soybean nodules following inoculation with certain ineffective Bradyrhizobium strains [33, 34, 44]. As part of a continuing investigation to study the plant's response to inoculation with Rhizobium meliloti, we have cloned four chalcone synthase and two chalcone isomerase cDNAs from alfalfa (Medicago sativa L.) nodules. Chalcone synthase (EC 2.3.1.74), a key enzyme in the phenylpropanoid biosynthetic pathway, catalyzes the condensation of malonyl-CoA with 4-coumaryl CoA to yield naringinen chalcone. Chalcone isomerase (EC 5.5.1.6) catalyzes the isomeriz ation of chalcones to their corresponding (-)-flavanones. Here we report the D N A and derived amino acid sequence of these clones and their homology to other clones. The pattern of expression in different plant organs was also examined.
Materials and methods
Growth conditions Medicago sativa L. cv. Iroquois or M. truncatula L. cv. Jemalong seeds were sterilized and plants were grown as previously described [15]. Seeds for time course experiments were sown in sterilized plastic pans as described by LONer and Hirsch [25]. Three days after germination, the seedlings were inoculated or were harvested ('time zero'). Whole root systems were harvested into liquid nitrogen and stored at -70 °C until use. Cultures of wild-type Rhizobium meliloti strain Rml021 [29], grown in Rhizobium Defined Medium (RDM [42]) to an OD6oo -- 0.2 to 0.5, were used to inoculate alfalfa seedlings.
Library construction and screening A c D N A library was constructed in 2gtl i from poly(A) + RNA of 21-day-old nodules induced by wild-type R. meliloti strain 1021 [24]. The library was screened sequentially according to Maniatis etal. [26] with the 1.4 kb Eco RI insert from pCHS 1 [39] and with the 808 bp insert ofpCHI1 [3, 30] of Phaseolus vulgaris. Five-hundred thousand plaques were screened. The bean c D N A clones used as probes were obtained from C. Lamb (Salk Institute, La Jolla, CA). Seven putative CH S and two putative CHI clones were isolated from the library and subcloned using PCR [32] into pUC19.
DNA sequencing Double-stranded sequencing was performed using the dideoxy method with the Sequenase kit from USB. Sequence analysis was done using G C G [ 5 ]. Synthetic oligonucleotide primers were used as necessary to obtain the complete sequences.
DNA isolation and Southern analysis Genomic D N A was prepared from leaf tissue of M. sativa and M. truncatula as described previously [23], cut with the appropriate restriction enzyme, subjected to electrophoresis in 0.7% agarose, and blotted onto Nytran (Schleicher & Schuell) following the manufacturer's instructions. Ca. 2 #g of restricted genomic D N A were loaded per lane. Restriction fragments used as probes were labeled by random priming using 32p-dCTP. The blots were hybridized at 37 °C overnight in 6 x SSPE, 50% formamide, 1% SDS, 0.4 x Denhardt's solution, and 10% dextran sulfate. High-stringency washes were performed in 0.2 x SSC at 50 °C.
RNA isolation and northern analysis Total RNA was isolated from alfalfa roots, nodules, stems, leaves, flowers, or cotyledons using
769 the protocol of Goldberg et al. [ 11] or using RNA-Stat 60 (Tel-Test, Friendswood, TX) following the manufacturer's instructions. RNA was subjected to electrophoresis on a formaldehyde gel [26], and blotted onto Nytran following the instructions of Schleicher & Schuell. Restriction fragments used as probes were labeled by random priming using 32p-dCTP. For the CHI probe, the entire Eco RI insert of CHI-1 was used. The Msc27 c D N A clone from M. sativa, used as a control for determination of equal loading, was obtained from D. Dudits [19] and is a 710bp Pst I fragment.
ACCGTTGTTCTCCA-1259 and the pUC19 reverse primer. CHS6-4P is 148 bp in length and was cloned using the primers 856-CTCCGG A T C C G T G G C T C T A T G A G A T G C - 8 7 2 and 1005-TTGAATTCAAATAACATAGTATATTC-986. An internal fragment of the clone Msc27 was also obtained by PCR using the primers 248-AAGGATCCGTCGACATTGTTGACG-268 and 367-GCGAATTCTCTTGTTTCTCTGCATCTAG-346 to generate Msc27P, which is 105 bp long.
Wounding RNase protection studies RNase protection was performed following the protocol of Gilman [10] with the following changes. Probes were run over a Sephadex G50 column (NICK column, Pharmacia) to remove unincorporated label and then precipitated following two phenol/chloroform/isoamyl alcohol extractions. Approximately 1.2 x 106 cpm of probe RNA were added to each sample. The three probes, CHS4-1P, CHS6-4P, and Msc27P (see below), were in the same reaction mixture. Hybridization was carried out at 42 °C. Digestion took place for 60 min at room temperature in 300 #1 of digestion buffer containing 40 #g/ml RNase A and 330 U RNase T1. A PhosphorImager (Molecular Dynamics) was used to quantify transcript levels. The transcript levels corresponding to the two CHS clones increased linearly with increasing RNA concentration [28]. Both clones gave specific protected bands [28]. The transcript levels were corrected for loading differences by expressing the transcript level as the ratio between CHS and Msc27 (relative transcript levels). To look at gene-specific patterns of CHS expression, fragments corresponding to the 3'untranslated region of clones CHS4-1 and CHS6-4 were generated by PCR and subcloned into the vector p G E M 4 Z (Promega). CH $4-1P is 175 bp in length. The primers used to clone this fragment were 1248-CCGGATCCATTGAA-
Cotyledons of 7-day-old plants were collected and either left untreated or were wounded by pinching with a pair of large forceps. After treatment, the cotyledons were kept in sterile Pyrex dishes on moistened filter paper and collected 3 and 6 h after wounding. They were then harvested into liquid nitrogen and stored at -70 °C until RNA was extracted.
Results
Isolation of cDNA clones Seven chalcone synthase clones, obtained from screening a nodule c D N A library, were selected for sequencing and further characterization. Sequence analysis revealed that, of the seven CHS clones identified, four were distinct and probably represent different gene family members. The three remaining CHS clones were partially sequenced and found to correspond to one of the four completely sequenced clones, but were not full-length. The four distinct clones were designated CHS4-1, CHS4-2, CHS6-4, and CHS12-1. CHS4-1 was 1423 bp in length and contained the coding region for the entire 389 amino acids of chalcone synthase. Both CHS4-2 and CHS6-4 were truncated at the 5' end. CHS4-2 was 1268 bp in length and encoded 370 of the predicted 389 amino acids, whereas CHS6-4 was 1043 bp in length and encoded 285 of the pre-
770 dicted 389 amino acids. CHS12-1 was a fulllength cDNA clone, consisting of 1380 bp. The homologies among these clones and with other alfalfa CHS cDNAs are shown in Table 1. Based on Southern blot analysis (Fig. 1A) and the isolation of other CHS clones from alfalfa, CHS appeared to be encoded by a relatively large multigene family in alfalfa. Of the 11 cDNAs for chalcone synthase already isolated from alfalfa and reported in the literature, we found that only one was identical in D N A sequence to one of the clones described in this report, that is, the CHS12-1 c D N A clone and CHS4A isolated by Junghans etal. [18] from elicitor-treated alfalfa suspension cultures (Table 1). Clones CHS4-2 and CHSI ([7a]; see Table 1) were 99~o identical. The major difference between these two clones was a stretch of 5 nucleotides immediately before the poly(A) tail that is present in CHS4-2, but not in CHSI (not shown). This variation may be due to cultivar or allelic differences or to the tissue from which the c D N A was made. The per cent identity among the remaining clones ranged from 86.0 to about 98.0 ~ . It is expected that the alfalfa CH S clones will be differentially expressed both developmentally and in response to environmental stimuli, as has been found for CHS gene family members in P. vulgaris [40].
The homologies of the alfalfa cDNAs to CHS clones from pea (PsCHS, PsCHS2, PsCHS3), soybean (GmCHS), and bean (PvCHS) are shown in Table 2. Chalcone synthase is highly conserved among the legumes, with 82-90~o sequence identity among genera. We isolated two chalcone isomerase c D N A clones from the alfalfa c D N A library. MsCHI-1 was a full-length c D N A consisting of 666 bp of coding sequence, 34 bp of 5'-untranslated region, 238 bp of 3'-untranslated region (with a potential polyadenylation site from 911 to 916bp) and a short poly(A) tail. MsCHI-2 was truncated at the 5' end and was 845 bp long. It consisted of 591 bp of coding region, 238 bp of 3'-untranslated region, and a potential polyadenylation site at 806811. Although MsCHI-2 showed slight sequence divergence with MsCHI-1 (not shown), the derived amino acid sequences for the two cDNAs were identical. On the basis of their sequence similarity and the Southern analysis (see below), it is likely that these two cDNAs represent different alleles of one gene. A bestfit analysis [5] showed that the sequence of MsCHI-1 is 79.5 ~o similar at the D N A level and 89.95~o similar at the amino acid level with that of CHI from Phaseolus vulgaris reported by Blyden et al. [3].
Table 1. D N A sequence homologies between alfalfa chalcone s y n t h a s e c D N A clones. 4-1 4-1 a 4-2 a 6-4 a 12-1 a
4-2
6-4
12-1
I
II
1A
2A
4A
8A
9A
87.5
94.2 86.7
88.4 94.0 87.0
86.8 99.0 86.7 92.5
86.0 96.1 87.6 93.0 96.1
97.3 87.4 94.9 88.0 86.1 87.0
89.1 93.1 88.7 92.9 92.6 92.7 88.5
88.6 93.9 87.3 100 93.6 93.1 88.1 93.6
88.1 97.3 87.2 93.7 96.2 97.5 87.8 93.0 94.0
97.9 88.2 95.9 89.1 86.8 88.0 96.7 89.8 89.2 88.9
Ib
IIb 1A c 2A ° 4A c 8A c 9A c Accession numbers U01018-U01021. b A c c e s s i o n n u m b e r s X 6 8 1 0 6 - X 6 8 1 0 7 [7a]. c A c c e s s i o n n u m b e r s L 0 2 9 0 1 - L 0 2 9 0 5 [ 18].
771 Table 2. Homologies between CHS cDNA clones of alfalfa and other legumes.
PsCHS [ 16] PsCHS2 [16] PsCHS3 [ 16] GmCHS [8] PvCHS [39]
CHS4-1
CHS4-2
CHS6-4
CHS12-1
88.27 87.20 89.11 83.88 83.54
89.52 88.76 86.73 82.53 81.58
88.83 86.22 89.75 84.11 82.22
90.55 88.60 86.06 83.10 81.60
Southern analysis
Fig. 1. Southern analysis of chalcone synthase and chalcone isomerase in alfalfa (Medicago sativa cv. Iroquois) and Medicago truncatula L. cv. Jemalong. Duplicate genomic Southern blots probed with full-length CHS4-1 probe (A) and CHI-1 probe (B). Restriction enzymes used are as indicated. Ca. 2 #g DNA were loaded in each lane.
Southern analysis was performed to estimate the n u m b e r of genes encoding chalcone synthase and chalcone isomerase. Figure 1A shows the hybridization o f the full-length CHS4-1 to genomic D N A from M. sativa cv. Iroquois, an out-crossing tetraploid Medicago, and from M. truncatula, a self-fertilizing, diploid Medicago. The latter was included to help discriminate between alleles or gene family members. A large n u m b e r of C H S hybridizing bands w a s seen in the M. truncatula lanes, especially in the lane containing B c I I digested D N A , indicating that there are multiple genes ( 8 - 1 2 genes) for C H S . Several lower molecular weight bands are also evident in the Bcl I digest of M. sativa D N A , some of which are the same size as the BclI-digested fragments of M. truncatula D N A . In the other digests o f M . sativa D N A , the higher-molecular-weight bands are not clearly distinguishable. This may be due to the large size of the restriction fragments that hybridize to the chalcone synthase clone. Southern analysis using M s C H I - 1 showed that C H I is encoded by 1 to 2 genes in alfalfa (Fig. 1B). One hybridizing b a n d was observed in the M. truncatuIa lanes whereas two bands were present in the M. sativa lanes, indicating that there is either a single gene or tightly linked genes in alfalfa.
Northern and RNase protection analysis T o determine the expression patterns of the C H S and C H I m R N A s that were represented by the
772 cDNA clones isolated from a nodule c D N A library, we compared the transcript levels of these genes in different organs of alfalfa. Two CHS clones (CHS4-1 and CHS6-4) were chosen to examine patterns of differential expression of different gene family members by generating genespecific probes for RNase protection analysis.
RNase protection analysis for following CHS gene expression rather than northern analysis was used because it allowed us to distinguish between gene family members more effectively. The transcript levels were corrected for loading differences by expressing the transcript level as the ratio between CHS and Msc27 (relative transcript level).
Fig. 2. CHS and CHI in alfalfa organs. A. RNase protection analysis using CHS4-1P and CHS6-4P as probes. B. Northern analysis using CHI-1 as a probe. C. Northern blot in B probed with Msc27. Ten #g total RNA were loaded for each organ type. Inset shows relative transcript levels in stems, leaves, and flowers. S, stems; L, leaves; F, flowers; R, root tips of 22-day-old plants; N-I, N-2, N: 21 day-old nodules induced by wild-type R. rneliloti 1021.
773 Although CHS4-1 and CHS6-4 transcript levels were nearly identical in leaves, the level of CH S 6-4 transcript in stems was nearly three times higher than that of CHS4-1 whereas CHS4-1 transcript levels were higher than CHS6-4 levels in flowers. However, very low levels of both CHS and CHI mRNAs were detected in stems, leaves, and flowers compared to nodules and roots (Fig. 2A and 2B). The level of CHS and CHI transcripts detected in mature nodules was low relative to root tips, but was still 14- to 16-fold higher than in leaves (Fig. 2). Root tip s contained the highest amount of CH S and CHI transcripts (Fig. 2; R). Levels of transcripts corresponding to CHS6-4 and CHS4-1 were ca. 50 and 40 times higher, respectively, in the root tips than in leaves. To examine the ac-
cumulation of CHS and CHI m R N A in roots with time, the expression patterns of CHS and CHI genes were determined in RNA isolated from entire root systems. The highest levels observed were in young roots at 0 to 4 days, with the level decreasing significantly at 6 days (Fig. 3). The transcript levels then remained relatively steady up to 26 d. As found for the other aerial organs of the plants, 7-day-old cotyledons contained low levels of CHS and CHI mRNAs (Fig. 4). The levels in untreated cotyledons were approximately the same as those found in leaves. A nearly linear increase in CHS and CHI transcript levels was seen following wounding, however (Fig. 4). Wounding caused greater accumulation of CHS4-1 than CHS6-4, with a ca. 8-fold increase.
Fig. 3. CHS and CHI in alfalfa roots. A. RNase protection analysis using CHS4-1P and CHS6-4P as probes. B. Northern analysis using CHI-1 as a probe. Ten/~g total RNA were loaded for each tissue type. Numbers indicate days after time zero.
774 5;:!:!:!:!:!:!:!: i:!:!:!:i:i:i:
4-
.........,..., .:.:.:.:-:,:.1-.
3-
..,...........
[ ] 4-1hrtsc
".'.'.'.'.v. ".'.v.'.','.'
g
,-:+1.:.:.:.:, 2'~':4":@:'.
[ ] 6-4/msc
.,;.:-:,:.:.:..
• ....- . . . . . . . ;
0
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3
6
Time (hours post-wounding)
Fig. 4. Effect of wounding on CHS and CHI transcript levels in cotyledons. RNase protection analysis using CH $4-1P and CHS6-4P as probes. Ten/zg total RNA were loaded for each tissue type. Numbers refer to hours after wounding.
CHS6-4, on the other hand, increased about 5-fold, but the maximum level was less than half that of CHS4-1. Discussion
Flavonoids play multiple roles in a plant's response to the environment and especially to other organisms [6]. Legumes possess a wide variety of flavonoids and have the special feature of being able to accumulate a series of structurally and biosynthetically interrelated compounds [ 13]. This ability may reflect the fact that flavonoids have multiple functions in the interactions between legumes and microbes, both beneficial and pathogenic. The presence of a large gene family may allow fine tuning of the plant responses to multiple environmental cues [6]. To begin an examination of the environmental cues, particularly those related to Rhizobium that trigger the synthesis of flavonoids, we identified four distinct clones of chalcone synthase and one of chalcone isomerase from a nodule c D N A library of M . sativa. Three of the four chalcone synthase c D N A clones that we identified were unique, whereas CHS12-1 was identical to CHS4A identified by Junghans etal. [18]. The clone CHS4-2 was 99~o identical to CHSI [7a].
Thus, we focused our studies on the two chalcone synthase gene family members, CHS4-1 and CHS6-4, that remained. When we examined the transcript levels of these CHS gene family members and chalcone isomerase in different organs of alfalfa, we found that only the underground portions of the plant accumulated significant levels of CHS and CHI transcripts. Our results revealed that young root tissue, the cells of which have recently undergone cell division and were still elongating, contained the highest levels of CHS and CHI transcripts. Young seedling roots and root tips of plants 22 days after the start of the experiment contained very high levels of transcripts. The lower, but steady level of transcript accumulation observed after 6 days probably reflects the increasing proportion of mature root tissue in the root system. Although CHS and CHI are expressed in the aerial portions of the plant, transcript levels are very low in stems, leaves, cotyledons, and flowers. However, a dramatic increase in CHS m R N A levels in the cotyledons was observed when these tissues were wounded. Our results are in accordance with other studies that have examined CHS and CHI expression patterns in legumes, but with some differences. The alfalfa CHS clones identified by Junghans et al. [18] also showed the highest level of transcript accumulation in roots with the next highest level in root nodules. However, one of the clones they identified hybridized strongly to floral tissue. Earlier, Schmid et al. [41], by using a bean CHS promoter fused to G U S in tobacco, found that CHS was most highly expressed in roots and in the pigmented tissue of flower petals. Leaves, stems and other flower parts had 10- to 100-fold less activity. Lambais and Mehdy [21] also found relatively high levels of chalcone isomerase transcripts in bean roots, indicating that high transcript levels of CHS and CHI in roots may be a common feature in legumes. Because most studies of flavonoid synthesis and gene expression focus on aerial portions of the plant or on cell cultures, it is not known if legumes are unique in their ability to synthesize high levels offlavonoids in the root.
775 Induction of CHS and CHI by wounding or by elicitor treatment also appears to be a general phenomenon among CHS and CHI genes in legumes. All the CHS transcripts studied by Junghans etal. [18] were induced in elicitor-treated suspension cultures and in wounded roots, although not in leaves. Mehdy and Lamb [29] observed that CHI transcripts were not detectable in excised segments of bean hypocotyl, but that treatment with elicitor induced CHI expression within 1 h. Because CHS and CHI induction have been shown to be caused by wounding as well as by elicitor treatment [2, 30, 46], another explanation for the higher levels of CHI m R N A in roots may be because roots are in constant contact with the soil and soil microorganisms. Wingender et al. [46] observed that wound induction of CHS is larger and more rapid in roots than in cotyledons, indicating that roots may be more sensitive to wounding than other portions of the plant. Wounding clover roots also causes a rapid increase in CHS transcript accumulation (J. Weinman, personal communication). The fact that the highest transcript levels were seen in roots of young seedlings may also be directly related to the role of flavonoids in nodulation. Immediately after germination, it is advantageous for the seedling to produce flavonoids that will attract the rhizobia, enhance rhizobial growth in the rhizosphere, and induce Rhizobium nod genes. Flavonoid nod gene inducers have been isolated from the rhizosphere of alfalfa [22] and methanolic extracts of alfalfa rhizosphere soil contain higher levels of nod gene inducers than non-rhizosphere soil. We have found that inoculated alfalfa roots contain higher levels of CH S 6-4 and CHS4-1 transcripts 2 to 4 days after inoculation in comparison to uninoculated roots (H.I. McKhann, Y. Fang, N.L. Paiva, R.A. Dixon, and A.M. Hirsch, manuscript in preparation). In contrast to young roots, mature nodules elicited by wild-type R. meIiloti have low CHS and CHI transcript levels. Because higher levels of CHS and CHI are seen in certain ineffective associations [12; H.I. McKhann, N.L. Paiva, R.A. Dixon, and A.M. Hirsch, manuscript in preparation], it appears that phenylpropanoid biosyn-
thetic gene expression differs between effective and ineffective associations. The clones reported here will provide a means for examining differential expression of two chalcone synthase gene family members and chalcone isomerase gene expression in alfalfa, in response to Rhizobium meliloti, in both effective and ineffective associations.
Acknowledgements We thank C. Lamb for the Phaseolus vulgaris CH S and CHI clones and D. Dudits for Msc27. We also thank Judy Brusslan for assistance with RNase protection assays and Richard Gaynor and the members of his laboratory, especially Jacob S. Seeler, at Southwestern Medical Center (Dallas, TX) for additional help. Jeremy Weinman is thanked for communicating research results before publication and for helpful discussions. Gratitude is also extended to Agway, Syracuse, NY, for supplying the M. sativa cv. Iroquois seeds, and is Dr Rebecca Dickstein of Drexel University (Philadelphia, PA) for M. truncatula cv. Jemalong seeds. H.I.M. was supported by a U S D A training fellowship and a California Biotechnology training fellowship. A Sigma Xi grant in-aid-ofresearch of H.I.M. helped fund portions of this work. A grant from the U S D A (CRGO 91-373076603) to A.M.H. also supported portions of this work.
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