Jeffrey J. Volenec

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).

Impact of climate change on crop nutrient and water use efficiencies

Implicit in discussions of plant nutrition and climate change is the assumption that we know what to do relative to nutrient management here and now but that these strategies might not apply in a changed climate. We review existing knowledge on interactive influences of atmospheric carbon dioxide concentration, temperature and soil moisture on plant growth, development and yield as well as on plant water use efficiency (WUE) and physiological and uptake efficiencies of soil-immobile nutrients. Elevated atmospheric CO(2) will increase leaf and canopy photosynthesis, especially in C3 plants, with minor changes in dark respiration. Additional CO(2) will increase biomass without marked alteration in dry matter partitioning, reduce transpiration of most plants and improve WUE. However, spatiotemporal variation in these attributes will impact agronomic performance and crop water use in a site-specific manner. Nutrient acquisition is closely associated with overall biomass and strongly influenced by root surface area. When climate change alters soil factors to restrict root growth, nutrient stress will occur. Plant size may also change but nutrient concentration will remain relatively unchanged; therefore, nutrient removal will scale with growth. Changes in regional nutrient requirements will be most remarkable where we alter cropping systems to accommodate shifts in ecozones or alter farming systems to capture new uses from existing systems. For regions and systems where we currently do an adequate job managing nutrients, we stand a good chance of continued optimization under a changed climate. If we can and should do better, climate change will not help us.

Nitrogen Pools in Taproots of Medicago sativa L. After Defoliation

Summary Alfalfa ( Medicago sativa L.) accumulates organic reserves in taproots that are thought to be used as substrates for newly developing shoots after defoliation. Two experiments were conducted to determine if specific N pools in taproot tissues undergo depletion and reaccumulation following defoliation. In Exp. 1, bark tissues of taproots of ‹Hi-Phy› alfalfa had higher concentrations of total N, soluble NH 2 -N and buffer-soluble protein than did wood tissues. Concentrations of these N pools declined in both tissues after defoliation and then reaccumulated after 21 d of regrowth. In Exp. 2, two genotypes differed in concentration of N-containing pools, although trends following defoliation of both genotypes were similar to those observed in Exp. 1. ASP + ASN were the most prevalent of the amino acids found in bark and wood tissues, together comprising approximately 50% of the total amino acid pool. Concentration of the ASP + ASN pool declined markedly in roots following defoliation, while concentrations of other amino acids (LEU, ILE, TYR, and PHE) increased. Characterization of buffer-soluble proteins using SDS-PAGE indicated that specific proteins with molecular masses of 15 and 19 kDa were depleted, especially in bark tissues, as soluble protein concentrations declined. The depletion of specific amino acids and certain buffer-soluble proteins from taproots during regrowth of defoliated alfalfa suggests that these N-pools may be utilized as a source of N during foliar regrowth after defoliation.

Taproot nitrogen accumulation and use in overwintering alfalfa (Medicago sativa L.)

Summary Alfalfa ( Medicago sativa L.) taproots accumulate organic reserves that are important for winter survival and subsequent growth in spring. Our objective was to determine if specific nitrogen (N) pools accumulate in taproot tissues prior to winter that may subsequently be used during initiation of herbage growth in spring. Taproots were obtained at approximately monthly intervals during fall and winter, and biweekly in early spring. Taproots were separated at the cambium into bark and wood tissues. Bark tissues consistently contained higher N concentrations than did wood tissues. N concentrations of both tissues gradually increased between early and late fall and declined in early spring when growth was initiated. Both soluble amino-N and buffer-soluble proteins increased during autumn and declined extensively during early spring in both tissues. A nonwinterhardy alfalfa line accumulated less soluble protein in taproot tissue when compared to a hardy genotype. Specific proteins with molecular masses of 32, 19, and 15 kDa were identified as major components of the buffer-soluble protein pool. These proteins rapidly disappeared from taproot tissues in spring as buffer-soluble protein concentrations declined. Protease activity in bark tissues declined gradually during late autumn and winter before increasmg over two-fold in early spring. Protease activity in wood tissues was approximately one-half that of bark tissues and also increased in spring when growth resumed. Our results indicate that high concentrations of soluble amino compounds and specific proteins accumulate in taproots during autumn and early winter. These N pools decline markedly in spring coincident with the onset of herbage growth.

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.

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.

Perennial rhizomatous grasses as bioenergy feedstock in SWAT: parameter development and model improvement

The Soil and Water Assessment Tool (SWAT) is increasingly used to quantify hydrologic and water quality impacts of bioenergy production, but crop‐growth parameters for candidate perennial rhizomatous grasses (PRG) Miscanthus × giganteus and upland ecotypes of Panicum virgatum (switchgrass) are limited by the availability of field data. Crop‐growth parameter ranges and suggested values were developed in this study using agronomic and weather data collected at the Purdue University Water Quality Field Station in northwestern Indiana. During the process of parameterization, the comparison of measured data with conceptual representation of PRG growth in the model led to three changes in the SWAT 2009 code: the harvest algorithm was modified to maintain belowground biomass over winter, plant respiration was extended via modified‐DLAI to better reflect maturity and leaf senescence, and nutrient uptake algorithms were revised to respond to temperature, water, and nutrient stress. Parameter values and changes to the model resulted in simulated biomass yield and leaf area index consistent with reported values for the region. Code changes in the SWAT model improved nutrient storage during dormancy period and nitrogen and phosphorus uptake by both switchgrass and Miscanthus.

Watershed-scale impacts of bioenergy crops on hydrology and water quality using improved SWAT model.

Cellulosic bioenergy feedstock such as perennial grasses and crop residues are expected to play a significant role in meeting US biofuel production targets. We used an improved version of the Soil and Water Assessment Tool (SWAT) to forecast impacts on watershed hydrology and water quality by implementing an array of plausible land‐use changes associated with commercial bioenergy crop production for two watersheds in the Midwest USA. Watershed‐scale impacts were estimated for 13 bioenergy crop production scenarios, including: production of Miscanthus × giganteus and upland Shawnee switchgrass on highly erodible landscape positions, agricultural marginal land areas and pastures, removal of corn stover and combinations of these options. Water quality, measured as erosion and sediment loading, was forecasted to improve compared to baseline when perennial grasses were used for bioenergy production, but not with stover removal scenarios. Erosion reduction with perennial energy crop production scenarios ranged between 0.2% and 59%. Stream flow at the watershed outlet was reduced between 0 and 8% across these bioenergy crop production scenarios compared to baseline across the study watersheds. Results indicate that bioenergy production scenarios that incorporate perennial grasses reduced the nonpoint source pollutant load at the watershed outlet compared to the baseline conditions (0–20% for nitrate‐nitrogen and 3–56% for mineral phosphorus); however, the reduction rates were specific to site characteristics and management practices.