S. M. Cunningham

Physiological genetics of alfalfa improvement: past failures, future prospects

Abstract The objective of this paper is to assess the effectiveness of alfalfa (Medicago sativa L.) improvement efforts over the last century, and with the advent of molecular biology, identify challenges for alfalfa improvement in the future. Yield trials conducted between 1986 and 1998 from around the US were used to compare yield and persistence of older alfalfa cultivars to those released in the 1990s. First and second harvest forage yield of recently released alfalfa cultivars were not improved over those of older cultivars. New cultivars had higher forage yield at fourth harvest, in early September, possibly due to a reduction in fall dormancy. Efforts to improve alfalfa persistence by breeding for improved disease resistance and greater winter hardiness also have not been effective at most locations. Use of molecular biology for alfalfa improvement depends upon identifying genes that control important agronomic traits that translate into greater yield, improved persistence, and enhanced forage quality. Few such genes have been identified in alfalfa, and their use might be complicated by the polyploid nature of this outcrossing species. The Medicago truncatula genome project is providing large amounts of sequence information, but little is known about the regulation of these genes and the function of their protein products in planta. Uncertainty exists regarding the effectiveness of transferring these genes to alfalfa to obtain a desired phenotype. Much remains to be done to identify key genes that determine agronomic performance of crop plants, including alfalfa, and to clarify mechanisms that regulate the expression of genes and the function(s) of their protein products under field conditions. Future efforts to improve agronomic performance of alfalfa will be enhanced by partnerships between public and private scientists because companies now dominate commercial release of new alfalfa cultivars.

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).

Seasonal carbohydrate and nitrogen metabolism in roots of contrasting alfalfa (Medicago sativa L.) cultivars

Summary Prostrate shoot growth of fall dormant alfalfa ( Medicago sativa L.) cultivars in autumn is positively associated with winter survival. Our objective was to determine how carbohydrate and nitrogen pools in roots of alfalfa cultivars exhibiting contrasting fall dormancy change during winter hardening in autumn and when shoot growth resumes in spring. Sugars, buffer-soluble protein, low molecular weight-N, and vegetative storage proteins (VSPs) increased in roots of all cultivars in autumn, while root starch concentrations declined throughout autumn and winter. Sugar, protein, low molecular weight-N, and VSPs levels declined in spring as shoot growth resumed, then re-accumulated in roots as shoots began to flower on June 2. Defoliation on June 2 resulted in a loss of starch, protein, and VSPs from roots as shoots regrew. Roots of fall dormant, winter hardy cultivars contained higher concentrations of sugars and buffer soluble protein in November and December, whereas higher concentrations of starch and low molecular weight-N were found in roots of nondormant cultivars at these times. Concentrations of total N and VSPs were similar between dormant and nondormant cultivars indicating that N deficiency caused by low dinitrogen fixation during hardening is not a factor contributing to the poor winter survival of nondormant alfalfa. Efforts aimed at understanding fall dormancy and winter hardiness of alfalfa should focus on mechanisms controlling accumulation of sugars and specific (non-VSP) soluble proteins in roots in autumn.

Purification and Characterization of Vegetative Storage Proteins from Alfalfa (Medicago sativa L.) Taproots

Summary Alfalfa ( Medicago sativa L.) accumulates C and N reserves in taproots and utilizes these reserves for shoot growth in spring and for shoot regrowth after defoliation. Three proteins are very abundant in taproots and undergo a cyclic pattern of utilization during early shoot growth followed by reaccumulation during late shoot development. Our objectives were to purify and characterize these putative vegetative storage proteins from alfalfa taproots. The proteins were purified using organic-solvent and ionic-precipitation techniques, gel filtration, and affinity chromatography. Polyclonal antibodies were raised against the purified proteins, and electrophoresis and immunoblotting were utilized to determine protein distribution and relative abundance. These proteins are present in high concentrations in alfalfa taproots, but were not found in seeds, nodules, leaves, or stems of alfalfa. Taproots of all perennial Medicago species examined contained these proteins, whereas roots of annual Medicago species had very low to undetectable amounts of these proteins. Taproots of other forage legume species ( Lotus, Melilotus, and Trifolium ) did not contain proteins that cross-reacted with antibodies raised against the three alfalfa taproot proteins. The three proteins have molecular masses of 15, 19, and 32 ku, are glycosylated, and have epitopes in common. The amino acids asparagine and aspartate make up 15 mole percent of the three alfalfa taproot proteins. These proteins possess features consistent with their role being vegetative storage proteins.

Potassium and nitrogen effects on carbohydrate and protein metabolism in alfalfa roots

Abstract An investigation was conducted to determine the effect of potassium (K) nutrition on alfalfa (Medicago sativa L.) growth and metabolism of root total nonstructural carbohydrates (TNC) and proteins, and to study whether nitrogen (N) fertilization overcomes N deficiency and low root protein concentrations caused by K deficiency. In Experiment 1, nodulated alfalfa plants were grown in plastic pots containing washed quartz sand and provided minus‐N Hoaglands solution containing 0, 0.6, or 6.0 mM K. Shoot and root K concentrations increased with increasing solution K. Root N concentrations were higher in plants receiving 6.0 mM K than in plants receiving 0.6 or 0 mM K, but shoot N concentrations were similar for all treatments. Plant persistence, shoots per plant, and shoot mass increased as solution K levels increased. Root starch concentration and utilization were positively associated with K nutrition. Total amylase activity was higher, but endoamylase activity was lower in roots of plants receivi...

Effects of environmental factors and endogenous signals on N uptake, N partitioning and taproot vegetative storage protein accumulation in Medicago sativa

Our objectives were to study the regulation of N partitioning within tissues of non-nodulated alfalfa (Medicago sativa L.) and N storage in taproots as vegetative storage proteins (VSP) of 15, 19, and 32 kDa and β-amylase (57 kDa) by environmental (photoperiod, temperature, N availability) and endogenous factors (methyl jasmonate). When compared to long-day conditions (LD, 16 h day/8 h night), short-day (SD, 8 h day/16 h night), exposure to low temperature (5˚C) or application of methyl jasmonate (MeJA, 100 M ) for 35 d reduced the biomass shoot/ root ratio and modified the source–sink relationships for N. SD and MeJA treatments resulted in partitioning of N to taproots and a concomitant accumulation of VSPs. In comparison with LD, SD treatment also stimulated β-amylase gene expression 2.5-fold. Although low temperature increased the N partitioning to root tissues and the accumulation of soluble proteins in taproot, VSP concentration and β-amylase mRNA levels remained low. Increasing N concentration from 1 to 5 mM KNO3 doubled the total dry matter but did not affect the N partitioning within the plant, VSP accumulation, or l β-amylase expression. These results suggested that short photoperiod can result in preferential N allocation toward taproots with a concomitant induction of VSP accumulation.

Short-day photoperiod induces changes in N uptake, N partitioning and accumulation of vegetative storage proteins in two Medicago sativa cultivars

Our objective was to study the effect of short-day photoperiod for 28, 42 and 56 d on growth, N uptake and N partitioning, particularly vegetative storage protein (VSP) accumulation in taproots of two alfalfa (Medicagosativa L.) cultivars (Lodi and Europe). For both varieties, the reduction of daylength from 16 h (long day,LD) to 8 h (short day, SD) for 28 d reduced total plant growth by decreasing shoot growth. Nitrogen uptake and N distribution within the plant was determined by 15N labeling. N uptake decreased with SD treatment duration, and was 2- and 3-fold lower for Europe and Lodi, respectively, for 56 d in SD conditions when compared with LD plants. The SD treatment resulted in preferential partitioning of N to taproots in comparison with LD conditions (19vs 9% for Lodi and 12 vs 5% for Europe after 28 d). For both cultivars, the SD-induced changes in N allocation to taproots did not significantly affect taproot soluble protein concentrations during 42 d of daylength treatment. In contrast, VSP accumulation occurred after only 28 d for plants grown in SD conditions (6.2 vs 4.8 mg g-1 DW for Lodi and 5.1 vs 1.4 mg g-1 DW for Europe). SD exposure also increased vsp 57 and vsp 32 mRNA transcript levels in Lodi and Europe (up to 2-fold higher) taproots in SD for 28 d compared with LD conditions. Overall results indicate that photoperiod modulates taproot N accumulation in alfalfa by enhancing both β-amylase (vsp 57) and vsp 32 gene expression and accumulation. The enhanced VSP accumulation by short-day photoperiod may result from altered VSP gene expression / transcript stability or occur indirectly through altered N source-sink relationships. Additionally, when SD treatment included a night break with 15 min illumination with sodium high pressure light or red light, our results suggest that the induction of vsp 57 and vsp 32 gene expressions by SD signal is mediated by the phytochrome system.

Effects of phosphorus nutrition on carbohydrate and protein metabolism in alfalfa roots

Abstract Alfalfa (Medicago sativa L.) root reserves are thought to provide nutrients to regrowing shoots, enhance stress tolerance, and improve plant persistence. Factors affecting carbohydrate and protein accumulation and metabolism in roots are important in alfalfa production. Our objectives were to determine 1) the influence of phosphorus (P) nutrition on alfalfa shoot growth and root carbohydrate and protein metabolism after defoliation and 2) how quickly growth and root carbohydrate and protein metabolism of P‐deficient alfalfa plants responds to supplemental P. In Experiment 1, nodulated alfalfa was grown in quartz sand with minus‐nitrogen (N) Hoaglands solution containing 0,1,2, or 6 mM P. Root P concentrations increased with increasing solution P levels. Phytate P in roots of plants grown with 6 mM P was greater than that of plants grown in 0, 1, or 2 mM P. Shoot mass and shoots per plant were reduced by 67 and 43%, respectively, in plants grown with 0 mM P as compared to plants grown with 6 mM P...

Phosphate nutrition effects on growth, phosphate transporter transcript levels and physiology of alfalfa cells

Phosphorus deficiency reduces forage yield and stand persistence of alfalfa (Medicago sativa L.). Our objectives were to isolate and characterize a high-affinity phosphate-transporter (P-transporter) from alfalfa roots (Medicago sativa L.); determine how phosphorus (P) nutrition impacts P-uptake, growth, and carbohydrate and protein metabolism of alfalfa cells; and learn how expression of the P-transporter is influenced by P nutrition. An 1087-base pair (bp) sequence was isolated using RT-PCR that possessed high nucleotide and amino acid sequence similarity to high-affinity P-transporters. Cultured cells were sampled at 3-day intervals for 9 days while growing in media containing P concentrations ranging from 0 to 10 mM. Media P concentrations declined rapidly in all P treatments by day 6. Low media P concentrations (0, 0.1 and 0.5 mM) reduced cell growth rates compared to higher media P levels (2.5, 5 and 10 mM). Suspension cell cultures supplied 0.5, 2.5, 5, and 10 mM P had lower starch concentrations by day 3 compared to cells cultured in media containing 0 and 0.1 mM P. Steady-state transcript levels for the high-affinity P- transporter were high in P-deprived cells, but declined within 1 day when cells were provided 10 mM P.

Effect of summer irrigation on seasonal changes in taproot reserves and the expression of winter dormancy/activity in four contrasting lucerne cultivars

In the summer dry environment of cool temperate Tasmania, summer irrigation is used to maximise forage production. For lucerne (Medicago sativa L.) this irrigation is likely to interact with winter-dormancy genotypes to influence seasonal changes in taproot reserves and thus, the process of cold acclimation. To test this hypothesis four lucerne cultivars with contrasting levels of winter dormancy (DuPuits: winter-dormant; Grasslands Kaituna: semi winter-dormant; SARDI 7: winter-active: SARDI 10, highly winter-active) were grown in small plots at Elliott, Tasmania, under irrigated or dryland conditions. At each defoliation taproots were sampled and assayed for the concentration of soluble sugars, starch, amino acids, soluble protein, the abundance of vegetative storage proteins (VSP), and the abundance of mRNA transcripts associated with cold acclimation and VSP. Taproot-soluble protein concentrations in DuPuits significantly increased from summer to autumn when plants were grown under dryland conditions. When grown under irrigated conditions, taproot-soluble protein concentrations decreased over summer and increased in autumn for all cultivars. The abundance of VSP increased in summer in all cultivars grown under dryland conditions. Taproot-soluble sugar concentrations increased and starch decreased in autumn for all cultivars grown under both water regimes. Plants grown under dryland conditions showed little change in RNA transcript abundance of cold acclimation genes across all cultivars and sampling dates, while in those plants grown under irrigated conditions, transcript abundance was influenced by sampling date, and for some genes, by cultivar. There was a clear carry-over effect from the exposure of summer drought on the winter-dormancy response. The expression of winter dormancy at an agronomic and molecular level was greater under dryland conditions.