Serge Laberge

A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature.

When alfalfa (Medicago sativa L. cv Apica) plants grown at room temperature are transferred to 2[deg]C, the temperature at which 50% of the plants fail to survive (LT50) decreases from -6 to -14[deg]C during the first 2 weeks but then increases to -9[deg]C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2[deg]C and then transferred to -2[deg]C for another two weeks, the LT50 declines to -16[deg]C. These changes in freezing tolerance are paralleled by changes in transcript levels of cas15 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas15 occurs even when protein synthesis is inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas15 transcripts depend on preexisting gene products, cas15 shows little homology to any known gene at the nucleotide or amino acid level. The deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of cas15 DNA as a fraction of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, its organization in the genome is different. The possible role of cas15 in the development of cold-induced freezing tolerance is discussed.

PRODUCTION OF A DIAGNOSTIC MONOCLONAL ANTIBODY IN PERENNIAL ALFALFA PLANTS

The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs.

Raffinose and Stachyose Accumulation, Galactinol Synthase Expression, and Winter Injury of Contrasting Alfalfa Germplasms

been identified in alfalfa (Mohapatra et al., 1989; McKersie et al., 1993; Monroy et al., 1993, 1998; Wolfraim et Large differences in winter hardiness exist among alfalfa (Medicago al., 1993; Castonguay et al., 1994; Monroy and Dhindsa, sativa L.) cultivars, but the physiological and molecular bases for 1995), the function of these genes in planta and their these differences are not understood. Our objective was to determine how raffinose family oligosaccharide (RFO) accumulation and steady relationship with fall dormancy and winter hardiness is state mRNA levels for galactinol synthase (GaS) in roots relate to not understood. genetic variation in alfalfa winter survival. A GaS cDNA was isolated Several physiological processes also have been associthat possesses over 70% identity with GaS clones from other plant ated with improved winter hardiness of alfalfa. For despecies. Induction of GaS transcripts in crowns of winter hardy alfalfa cades, the accumulation of starch and soluble sugars in cultivars occurred within 8 h of exposure to 2 C, and was intensified roots has been the focus of research (Graber et al., 1927; by exposing plants to 2 C for 2 wk. Galactinol synthase transcripts Grandfield, 1943; Smith, 1964). Initially, it was believed increased in November in crown and root tissues of winter hardy that the accumulation of total nonstructural carbohyalfalfa plants. This increase was accompanied by large increases in drates (TNC, sum of sugar and starch concentrations) root RFO concentrations between October and December. A close in roots was critical to successful overwintering and subpositive association between RFO accumulation in roots in December and genetic differences in winter survival was observed in these alfalfa sequent spring growth of this perennial species. Later, populations. Although roots and crowns of nondormant alfalfa cultiit was shown that soluble sugars accumulated in alfalfa vars accumulated both GaS transcripts and RFO, accumulation was roots and crowns as plants hardened for winter (Bula delayed until December and these cultivars did not survive winter. et al., 1956; Ruelke and Smith, 1956), but how sugar Understanding the mechanisms regulating GaS gene expression and accumulation affected genetic differences in winter harsubsequent RFO accumulation in roots and crowns provides opportudiness was not studied. Recently, we have shown that nity to genetically improve alfalfa winter hardiness. sugar concentrations are consistently lower in roots of nondormant alfalfa cultivars when compared with fall dormant, winter hardy alfalfa cultivars (Cunningham V differences in winter hardiness exist among and Volenec, 1998). alfalfa (Medicago sativa L.) cultivars, but the physiCastonguay et al. (1995) reported that sucrose, stachyological and molecular bases for these differences are ose, and raffinose accumulated in alfalfa roots, while not understood. From a morphological standpoint, fall concentrations of glucose, fructose, and starch declined dormancy reduces alfalfa shoot growth in autumn and during alfalfa cold acclimation. Further, differences in is associated with greater winter survival (Smith, 1961; the maximum level of freezing tolerance between nonStout, 1985; Stout and Hall, 1989; Sheaffer et al., 1992). hardy and winter hardy cultivars were better related to However, fall dormant cultivars have slow shoot rethe capacity of the plants to accumulate stachyose and growth after defoliation, which reduces forage yield and raffinose than to accumulate sucrose. To understand overall agronomic performance in summer. Recent gemechanisms controlling fall dormancy and winter hardinetic evidence suggests that understanding the relationness better, we have studied alfalfa populations selected ship between fall dormancy and winter survival may for contrasting fall dormancy. These populations also enable us to devise schemes to improve winter hardiness differ in winter hardiness and several other traits includwhile simultaneously reducing fall dormancy (Brummer ing root sugar concentrations (Cunningham et al., 1998, et al., 2000). Although several cold-inducible genes have 2001). They permit study of discrete changes in physiology and gene expression associated with selection for contrasting fall dormancy and winter hardiness in a manS.M. Cunningham and J.J. Volenec, Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907-1150 USA; P. Nadeau and Y. Castonguay, ner not possible using traditional cultivars that differ Station de Recherches, Agriculture and Agri-Food Canada, 2560 Hofor many characteristics. We do not know if changes chelaga Blvd., Sainte-Foy, QC, Canada G1V 2J3; S. Laberge, Soils in sugar composition occurred as a result of genetic and Crops Research and Development Centre, Agriculture and Agriselection for contrasting fall dormancy in these germFood Canada, 2560 Hochelaga Blvd., Sainte-Foy, QC, Canada G1V plasms, and if expression of genes for key enzymes in2J3. This work was supported, in part, by USDA-IFAFS grant number 00-52100-9611. Contribution from the Purdue Univ. Agric. Exp. Stn., volved in RFO synthesis, such as galactinol synthase, Journal Series No. 16719. The authors acknowledge the contribution are associated with RFO accumulation and improved of Dr. L.R. Teuber at the University of California, Davis, who provided seed of the contrasting fall dormancy selections used in this Abbreviations: cDNA, complementary DNA; FD, fall dormancy; research. Received 24 Apr. 2002. Corresponding author (jvolenec@ GaS, galactinol synthase; HPLC, high pressure liquid chromatograpurdue.edu). phy; LSD, least significant difference; mRNA, messenger RNA; RFO, raffinose family oligosaccharides. Published in Crop Sci. 43:562–570 (2003).

Control of somatic embryogenesis and embryo development by AP2 transcription factors

Members of the AP2 family of transcription factors, such as BABY BOOM (BBM), play important roles in cell proliferation and embryogenesis in Arabidopsis thaliana (AtBBM) and Brassica napus (BnBBM) but how this occurs is not understood. We have isolated three AP2 genes (GmBBM1, GmAIL5, GmPLT2) from somatic embryo cultures of soybean, Glycine max (L.) Merr, and discovered GmBBM1 to be homologous to AtBBM and BnBBM. GmAIL5 and GmPLT2 were homologous to Arabidopsis AINTEGUMENTA-like5 (AIL5) and PLETHORA2 (PLT2), respectively. Constitutive expression of GmBBM1 in Arabidopsis induced somatic embryos on vegetative organs and other pleiotropic effects on post-germinative vegetative organ development. Sequence comparisons of BBM orthologues revealed the presence of ten sequence motifs outside of the AP2 DNA-binding domains. One of the motifs, bbm-1, was specific to the BBM-like genes. Deletion and domain swap analyses revealed that bbm-1 was important for somatic embryogenesis and acted cooperatively with at least one other motif, euANT2, in the regulation of somatic embryogenesis and embryo development in transgenic Arabidopsis. The results provide new insights into the mechanisms by which BBM governs embryogenesis.

Alfalfa Winter Hardiness: A Research Retrospective and Integrated Perspective*

Abstract Insufficient cold hardiness is a major impediment to reliable alfalfa (Medicago sativa L.) production in northern regions experiencing harsh winter conditions. Numerous studies have documented the morphological and physiological traits associated with the acquisition of freezing tolerance and winter survival in alfalfa. Use of this information as selection criteria to breed cultivars with superior winter hardiness has thus far been met with limited success. This can be attributed to many factors including: the large number of traits affecting winter survival; the multigenic nature of most traits, large environmental interactions, and an undesirable linkage between acquisition of freezing tolerance and fall growth cessation (fall dormancy). In the last two decades, the advent of molecular biology and quantitative genetic techniques has markedly increased our knowledge of the molecular and genetic bases of superior alfalfa winter hardiness. Our understanding of the mechanisms underlying the perception of the low temperature signal and its transduction into morphological and physiological responses leading to cold hardiness has progressed, but still remains fragmentary. Current evidence indicates that cold hardiness of alfalfa relies on tolerance to extensive freeze‐induced desiccation. Low temperature‐induced accumulation of soluble sugars and stress‐related translation products were found to be, in some instances, more abundant in cold‐tolerant cultivars and to be under some level of genetic control. Limited stability of these traits and conflicting reports on their relationship with freezing tolerance preclude their adoption as molecular screening tools. The development of robust screening techniques will require a more complete knowledge of the genetic bases of freezing tolerance. Heritability estimates suggest that independent selection for winter hardiness, freezing injury and autumn growth is possible, and that winter hardiness and autumn growth could be manipulated independently. This creates the opportunity to develop high‐yielding cultivars with improved winter hardiness. A screening test for freezing tolerance performed under controlled conditions recently led to the development of populations with increased freezing tolerance and led to significant improvement in alfalfa winter survival. Unique genetic material, combined with novel gene discovery approaches, could be lead to the identification of genetic polymorphisms associated with freezing tolerance in alfalfa and pave the way to marker‐assisted selection. Based on the current knowledge, we propose a conceptual framework for the genetic determination of cold adaptation of alfalfa.

New cold- and drought-regulated gene from Medicago sativa

Plants are known to differ in their ability to withstand freezing temperatures, but the molecular/genetic basis of this differential freezing tolerance is unclear. Exposure of plants to low, nonfreezing temperatures (cold acclimation) increases their tolerance to subsequent freezing (see recent review by Guy, 1990). Significant biochemical modifications occur during cold acclimation of plants, including changes in gene expression (Thomashow, 1990). Three cold acclimation-specific cDNAs (Mohapatra et al., 1989) and one cDNA responsive to environmental stress (cold and drought) and ABA (Mohapatra et al., 1988) have previously been isolated from alfalfa (Medicago sativa L. cv Apica). DNA sequence determination of four alfalfa cDNAs that were shown to be responsive to environmental stresses (low temperature and drought) and ABA indicated that they are part of a family of genes encoding Gly-rich proteins containing many repeated peptide motifs (Lu0 et al., 1991, 1992). We report here the isolation of a new full-length cDNA clone (MsaciA) that is cold and drought regulated in alfalfa. Plants of the cold-tolerant alfalfa were grown at 21OC for 5 weeks and cold acclimated for 2 weeks at 2OC. A XgtlO library was constructed with mRNA isolated from cold-acclimated crowns. The cDNA of a cold-inducible transcript was isolated by differential hybridization using single-strand cDNA synthesized from cold-acclimated and nonacclimated crowns. The nucleotide sequence of the full-length cDNA and the deduced amino acid sequence of MsaciA have been determined (Table I). MsaciA encodes a putative Gly-rich protein (38%), which contains many repeated motifs. This putative protein shares homology in the range of 68 to 88% (amino acid identity) with the previously isolated environmental stressand ABA-regulated putative proteins from alfalfa (Lu0 et al., 1991, 1992) and, thus, represents a new member of this gene family.

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 μg ml-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.

A cold-induced gene from Medicago sativa encodes a bimodular protein similar to developmentally regulated proteins.

A new cold-regulated (COR) gene, msa CIC, was isolated by differential screening of a cDNA library from cold-acclimated crowns of alfalfa (Medicago sativa L. cv. Apica). Transcripts of msa CIC were not detectable in unacclimated alfalfa and accumulated to higher levels in cold-acclimated plants of the cold-tolerant cv. Apica than in those of the cold-sensitive cv. CUF-101. The DNA sequence analysis of a full-length cDNA clone revealed that msa CIC encodes for a putative protein (MSACIC) of 166 amino acids with distinct proline-rich and hydrophobic domains. Protein sequence comparisons indicated that MSACIC is similar to a group of bimodular proteins that are developmentally regulated in other plant species.

SRAP polymorphisms associated with superior freezing tolerance in alfalfa (Medicago sativa spp. sativa)

Sequence-related amplified polymorphism (SRAP) analysis was used to uncover genetic polymorphisms among alfalfa populations recurrently selected for superior tolerance to freezing (TF populations). Bulk DNA samples (45 plants/bulk) from each of the cultivar Apica (ATF0), and populations ATF2, ATF4, ATF5, and ATF6 were evaluated with 42 different SRAP primer pairs. Several polymorphisms that progressively intensified or decreased with the number of recurrent cycles were identified. Four positive polymorphisms (F10-R14, Me4-R8, F10-R8 and F11-R9) that, respectively, yielded 540-, 359-, 213-, and 180-bp fragments were selected for further analysis. SRAP amplifications with genotypes within ATF populations confirmed that the polymorphisms identified with bulk DNA samples were reflecting changes in the frequency of their occurrence in response to selection. In addition, the number of genotypes cumulating multiple polymorphisms markedly increased in response to recurrent selection. Independent segregation of the four SRAP polymorphisms suggests location at unlinked loci. Homology search gave matches with BAC clones from syntenic Medicago truncatula for the four SRAP fragments. Analysis of the relationship with low temperature tolerance showed that multiple SRAP polymorphisms are more frequent in genotypes that maintain superior regrowth after freezing. These results show that SRAP analysis of bulk DNA samples from recurrent selections is an effective approach for the identification of genetic polymorphisms associated with quantitative traits in allogamous species. These polymorphisms could be useful tools for indirect selection of freezing tolerance in alfalfa.

Differential accumulation of two glycine-rich proteins during cold-acclimation alfalfa.

Two mRNAs, MsaCiA and MsaCiB, encoding for proteins harboring glycine-rich motifs, accumulate in alfalfa during cold acclimation. Fusion polypeptides containing the amino acid sequences deduced from these mRNAs were produced in Escherichia coli and used to raise antibodies. Each antibody cross-reacted specifically with soluble polypeptides, MSACIA-32 and MSACIB, respectively. These polypeptides were detectable only in crowns of cold-acclimated plants, even though MsaCiA mRNA accumulated in both crows and leaves during cold acclimation. The analysis of parietal proteins showed that several MSACIA-related proteins, with a molecular mass of 32, 41 and 68 kDa, did accumulate in leaf cell walls and one of 59 kDa crown cell walls. This diversity is most probably due to a tissue-specific maturation of MSACIA. A discrepancy was found between the time-course of accumulation of MSACIB and the one of the corresponding transcript. These results indicate that timing and localization of MSACIA and MSACIB expression are different, and suggest that this differential expression involves both transcriptional and post-transcriptional events. Comparisons made among six cultivars of contrasting freezing tolerance suggest that low tolerance could be explained by failure to accumulate proteins like MSACIA and MSACIB at a sufficient level.