Alfalfa Gene Research Articles

Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein.

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

A Gene That Encodes a Proline-Rich Nodulin with Limited Homology to PsENOD12 Is Expressed in the Invasion Zone of Rhizobium meliloti-Induced Alfalfa Root Nodules

To define the early stages of the interaction between Rhizobium and host legumes, we have cloned and characterized three early nodulin-encoding sequences from an alfalfa (Medicago sativa L.) cDNA library by probing with a fragment of a cDNA clone for PsENOD12, an infection-related nodulin from pea (Pisum sativum L.). Although the coding regions of the three clones are 95 to 98% homologous to each other, they are only 43% homologous to the pea clone. However, the putative signal peptide encoded by the alfalfa cDNA clones is 100% homologous to the PsENDO12 signal peptide. The spatial and temporal expression patterns of PsENOD12 and the alfalfa clones were compared. In situ hybridization experiments detected RNA transcripts in the invasion zone of mature nitrogen-fixing nodules, the same site where PsENOD12 mRNAs are found. Transcripts were also found by in situ hybridization in cells of Rhizobium meliloti exoH mutant-induced nodules penetrated by infection threads, but northern analysis did not detect transcripts in inf- (infection thread minus) nodules elicited by R. meliloti exoB nodules or in pseudonodules elicited by treatment with the auxin transport inhibitor N-1-(naphthyl)phthalamic acid. In addition, the alfalfa gene represented by these cDNA clones exhibited a temporal expression pattern that differed from that of PsENOD12, which is transiently expressed. These data, plus information derived from Southern blot analysis, indicate that we have isolated cDNA clones for a novel early nodulin, which we have designated MsENOD10 (Medicago sativa Early Nodulin 10).

Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis

The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35,400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

Aspartate Aminotransferase in Alfalfa Root Nodules

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.

Identification of two groups of leghemoglobin genes in alfalfa (Medicago sativa) and a study of their expression during root nodule development

Differential screening of an alfalfa root nodule cDNA library with either root or nodule mRNA resulted in the isolation of two groups of leghemoglobin cDNA which differ significantly in sequence. Analysis of one member of each group revealed a divergence within the coding region of 15% at the nucleotide level and 14% at the amino acid level. The 3' non-coding sequences are 25% divergent but are highly conserved over a stretch of 54 nucleotides which contains two sequence motifs common to leghemoglobin genes from other plant species. Southern blotting analysis with exon-specific probes has shown that there are approximately twice as many leghemoglobin gene copies in the alfalfa genome corresponding to one type of cDNA as compared with the other. Using the same criterium of DNA sequence relatedness these two distinct groups of leghemoglobin genes have also been identified in the genomes of the diploid annual Medicago truncatula and the closely related genus, Melilotus. Transcripts corresponding to both groups of leghemoglobin genes are first detected in alfalfa nodules 9-10 days after Rhizobium inoculation. Thereafter, mRNA levels increase rapidly and synchronously, reaching a maximum approximately 2 days later. There is a 2-3 fold difference in the steady-state levels of the two mRNA populations and this is maintained throughout the subsequent two weeks of nodule growth. The absence of any detectable transcription during the early stages of nodule development and the apparent co-ordinate expression of leghemoglobin genes in alfalfa contrasts with the situation in soybean and suggests that important differences in leghemoglobin gene regulation exist between these two distantly related legume species.

Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.

An Association between Resistance to Bacterial Wilt and Nitrogen Fixation in Alfalfa1

Bacterial wilt (BW), a major disease of alfalfa (Medicago saliva L.), is caused by the bacterium Corynebacterium insidiosum (McCull.) H. L. Jens. Symbiotic nitrogen (N) fixation of alfalfa involves the bacterium Rhizobium meliloti Dang. The objective of this research was to determine if factors conditioning resistance to BW were associated with plant characteristics related to N2‐fixation in alfalfa. In one study, alfalfa gene pools segregating for different types of resistances to BW were grown in nil‐nitrate greenhouse sandbenches. Each plant was evaluated for acetylene reduction rate, top dry weight, nodule mass score, nodule number score, fibrous root score, and secondary root score. The same plants were then inoculated with C. insidiosum and later evaluated for resistance to BW. In two other studies, breeding lines that had been selected in the greenhouse for acetylene reduction rate, top dry weight, nodule mass, and fibrous root mass were evaluated in the field for resistance to BW.Correlation coefficients between resistance to BW and acetylene reduction rate and between BW and nodule mass were low but significant in one gene pool where resistance to BW was conditioned by an additive gene system. In the second gene pool where a dominant gene conditioned resistance to BW, there was no apparent association between BW resistance and N2‐fixation. In the field studies, lines selected for high levels of N2‐fixation‐associated characteristics, especially nodule mass and top weight, were generally more susceptible to BW than lines selected for low levels of N2‐fixation. Selection for high levels of N2‐fixation did not affect either fall dormancy response, resistance to Phytophthora root rot caused by Phytophthora megasperma Drechs., or resistance to Fusarium wilt caused Fusarium oxysporum Schlecht. f. sp. medicaginis (Weimer) Synd. & Hans. Breeding for high levels of N2‐fixation could increase susceptibility to BW unless plant breeders monitor resistance or utilize the dominant gene (BW1) source of resistance.

Inheritance of Resistance to Bacterial Wilt in Two Alfalfa Gene Pools: Qualitative Analysis1

Bacterial wilt caused by Corynebacterium insidiosum (McCull.) H. L. Jens. is a serious disease of alfalfa (Medicago sativa L.). Inheritance studies were conducted with germplasm from two diverse gene pools, MSA and MSB. Two sets of six‐parent diallels with reciprocals and selfs were produced within each gene pool. Two sets of parents from one gene pool were crossed with two sets of parents from the other. Eleven‐week‐old seedlings of progenies were inoculated with C. insidiosum and transplanted to the field. Fifteen weeks later the taproot of each plant was cross‐sectioned and scored for the amount of yellow‐brown discoloration.MSA contained one dominant gene, BW1, for resistance. In the absence of other genes for resistance, plants with the genotype BW1… were classified as resistant and bw1bw1bw1bw1 plants were classified as susceptible. In addition to the BW1 gene, we believe that MSA contained two other genes, BW2 and BW3 with additive and smaller gene effects than those from BW1. The BW3 gene had a larger effect than the BW2 gene. The MSB gene pool did not contain resistant alleles at the BW1 locus, but it did contain resistant alleles at the BW2 and BW3 loci. Based on the genetic origins of the two gene pools, we suggest that the BW1 gene originated from ‘Turkistan’ and/or ‘Ladak.’ The BW2 and BW3 genes were probably from Chilean origin.

Inheritance of Resistance to Bacterial Wilt in Two Alfalfa Gene Pools: Response to Selection and Quantitative Analysis1

Bacterial wilt (BW) caused by Corynebacterium insidiosum (McCull.) H. L. Jens. is a serious disease of alfalfa (Medicago sativa L.). High levels of resistance to BW in alfalfa are available, but the inheritance of resistance is unclear. Objectives were to study the response to selection for resistance to BW and conduct a quantitative genetic analysis of inheritance of resistance to BW in two unrelated alfalfa gene pools. In the study of response to selection, the original gene pool plus six cycles of selection in the MSA gene pool and the original plus eight cycles of selection in the MSB gene pool were evaluated simultaneously. In the genetic study three sets of six‐parent diallels, including reciprocals and selfs, were produced within each gene pool. Two sets of six‐parent Design II's were produced by crossing parents of one gene pool with parents of the other.The results from the selection study suggested different modes of inheritance in the two gene pools. The response to selection in the MSA gene pool was quadratic, with a rapid increase in resistance in early cycles of selection; the response in MSB was linear and less rapid. In the genetic study variation due to reciprocals was nonsignificant in both gene pools and specific combining ability was significant only in MSB. Assuming a fixed model, all variances of genetic effects were greater in MSB than in MSA. General combining ability effects also suggested that inheritance was different in the two gene pools. The different types of inheritance suggested why the two gene pools responded differently in the selection study.