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a USDA-ARS, NPA Wheat, Sorghum, and Forage Research, Dep. of Agronomy, Univ. of Nebraska-Lincoln, Lincoln, NE 68583-0937
b USDA-ARS, NPA Wheat, Sorghum, and Forage Research, Dep. of Plant Pathology, Univ. of Nebraska-Lincoln, Lincoln, NE 68583-0937
* Corresponding author (jfp{at}unlserve.unl.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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From an agricultural viewpoint, Darwinian fitness is often not relevant because artificial selection is practiced. Agronomically favorable traits are not necessarily those that would promote survival of wild species in at least some environments. Life-spans are modified to accommodate growing seasons. Progeny are harvested and utilized before maturity or dissemination. Fecundity or clonal reproduction is controlled. Agriculturally fit plants are those that are the most useful to humans in agricultural systems, and their fitness must be measured with regard to such systems.
In annual cropping systems, agricultural fitness is therefore defined as the ability to produce economically harvestable yields of useful and commercially desirable plant products. In perennial cropping systems, agricultural fitness is defined as the ability to produce economic yields of useful plant product across multiple years. Like the metric components of fitness described by Falconer (1989), agricultural fitness is influenced by many metric factors, including germination, pest resistance, survival, growth rate, lodging, and yield. We will examine the effect of reduced lignin on such measurable components of agricultural fitness. Because of the strong interaction of environment on fitness defined in these terms, we will largely limit our discussion to data derived from the field.
Before examining the effects of reduced lignin on plant fitness, a brief review of lignin functions in whole plants is useful. Walter (1992) simply states "lignin is one of the prerequisites for terrestrial plant life." Campbell and Sederoff (1996) state "lignin, a complex phenolic polymer, is important for mechanical support, water transport, and defense in vascular plants." Lignin provides strength and rigidity to plants and to plant structures such as xylem, which allows for the transport of water; and sclerenchyma and bundle sheath cells, which provide a natural barrier to microorganisms (Bird, 1988). Production of lignin has been demonstrated to be induced in plants in response to mechanical damage or infection by pathogens in numerous species including cucumber (Cucumis sativus L.), carrot [Dacus carota subsp. sativus (Hoffm.) Arcang], narcissus (Narcissus peoticus L.), radish (Raphanus sativus L.), melon (Cucumis melo L.), hop (Humulus lupulus L.), potato (Solanum tuberosum L.), bean (Phaseolus spp.), and grasses (Bird, 1988).
Lignin is integral to plant health, survival, and functions. In classical Darwinian fitness terms, the answer to the question "Does reduced lignin impact plant fitness"? is very probably "Yes," especially with respect to natural populations. For example, glume and caryopses lignin content of the wild relative of sorghum, shattercane [Sorghum bicolor subsp. drummondii (Nees ex Steud.) de Wet & Harlan], is higher than that of cultivated sorghum [Sorghum bicolor (L.) Moench], and lignin content is positively correlated with over winter survival of shattercane seed (Fellows and Roeth, 1992). However, in terms of agronomic fitness, reducing lignin may have positive, neutral, or negative effects. In the following pages, we will explore these possible effects in various crop species.
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Multiple Sources of Reduced Lignin and Implications |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Maize |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Brown Midrib
Four brown midrib mutations are known in maize (bm1, bm2, bm3, and bm4) and are associated with significant alterations in lignin composition and reductions in lignin content in the stover. The scientific literature contains considerable information about the effects of these genes, especially bm3, on plant composition and on agricultural fitness. The bm3 mutation results in reduced caffeic acid O-methyltransferase (COMT) activity (Vignols et al., 1995), while the bm1 mutation reduces cinnamyl alcohol dehydrogenase (CAD) activity (Haplin et al., 1998). The reader is cautioned that the following is a discussion of the effects on fitness parameters of maize in lines containing these lignin reducing genes, not a discussion of the direct effects of reduced lignin on agricultural fitness.
bm3.
Miller et al. (1983) developed a set of S1 lines and S1 x S1 hybrids from three populations segregating for bm3. Brown midrib lines had lowered grain, stover, and fodder yields (averaged 77, 90, and 84% of normal lines, respectively), a 10% reduction in average seedling dry weight, and a 15% reduction in dry matter accumulation across the growing season (Miller et al., 1983; Miller and Geadelmann, 1983). Brown midrib lines also exhibited higher incidence of stalk breakage at maturity, 2- to 4-d-later maturity, and reduced vigor during vegetative growth as compared with normal lines (Miller et al., 1983). There was considerable genetic variation among the bm3 lines, but the authors concluded that although early vigor and growth rates could be improved to acceptable levels through breeding, improvement of bm3 line grain yields to that of normal maize lines was unlikely (Miller and Geadelmann, 1983).
Using a set of 15 bm3 lines and their 15 normal isogenic lines, Lee and Brewbaker (1984) showed average grain yields of the brown midrib lines to be reduced by 20%, and average stover yields to be reduced by 17%. Average reductions associated with bm3 for kernel number per row (12%), filled ear length (10%), ear height (7%), plant height (5%), and stem diameter (5%) were also documented (Lee and Brewbaker, 1984). Again, considerable variation among bm3 lines was noted, but the authors implied that it is unlikely that these reductions in fitness could be overcome by breeding. They also excluded linkage of the bm3 gene and yield-reducing genes as the mechanism for yield reduction and intimate pleiotropism and reduced photosynthesis.
Reduction in dry matter yield (15–20%) in bm3 isolines compared with their normal counterparts has been reported by Inoue and Kasuga (1989). Gentinetta et al. (1990) demonstrated similar yield reductions due to bm3 in 21 hybrids compared with their isolines counterparts but indicated that yield reduction was genotype specific and in one genetic background, the bm3 line had virtually identical yield to its normal counterpart. Lines developed by Weller et al. (1985) did not show yield differences between bm3 and normal isolines, but silking was delayed an average of 3 d in the bm3 isolines. Although a specific brown midrib gene is not identified, Cox and Cherney (2001) confirmed a 20% reduction in yield in commercial brown midrib hybrids when compared with other commercial hybrids, and noted a reduction in early season growth in the brown midrib hybrids.
Increased lodging is generally assumed to be associated with brown midrib maize. However, an increase in lodging attributable to brown midrib was not detected in several studies, possibly because of overriding effects of genetic backgrounds (Weller et al., 1985; Inoue and Kasuga, 1989), maturity (Miller et al., 1983), or environmental or other experimental design factors. However, in a study designed specifically to evaluate stalk strength, Zuber et al. (1977) showed a 17 to 26% decrease in crushing strength in three bm3 hybrids compared with their normal counterparts.
bm1, bm2, bm4.
Although cell wall composition in all four maize brown midrib mutants has now been described (Marita et al., 2003), little has been published describing the effects of bm1, bm2, and bm4 on the agricultural fitness of maize. The bm1 mutation appears to decrease days to flowering while the bm2 mutation appears to increase days to flowering (Vermerris and McIntyre, 1999). Vermerris et al. (2002a) later demonstrated that Bm1/bm1 maize plants flower earlier than their homozygous counterparts. The authors speculate that changes in lignin composition may mimic drought stress and alter plant development, but conclude that there is no evidence that the bm1 gene (heterozygous or homozygous) mimics drought stress or affects early growth and development of maize (Vermerris et al., 2002a). A single study reports no reduction in dry matter yields in bm1 maize hybrids (Barriere et al., 1994).
Transformation
Reduction in lignin content of maize has recently been accomplished by downregulation of OMT (He et al., 2003) and COMT (Piquemal et al., 2002). Both studies utilized individual greenhouse grown plants. He et al. (2003) reported no detectible differences in dry matter yield due to reduced OMT because of highly variable yield data. Piquemal et al. (2002) did not report yield data but indicate that the effect of downregulating COMT on agronomically important traits is currently under investigation.
In conclusion, other than minor shifts in days to flower (1–2 d), only the bm3 mutant of maize has been demonstrated to have appreciable effects on agricultural fitness. These effects are often genotype dependent, but the reduction in yield associated with the bm3 mutant is so significant that when combined with other negative effects it will be difficult to produce maize bm3 hybrids equivalent to normal hybrids in terms of agricultural fitness. Sources of reduced lignin in maize and their effects on agricultural fitness are summarized in Table 1.
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Sorghum |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Insect Resistance
A study was conducted, comparing six lines moderately resistant to stemborer [Chilo partellus (Swinhoe)] and shootfly [Atherigona soccata (Rond.)], and three lines susceptible to these insects (Khurana and Verma, 1983). No relationship between lignin and insect resistance was observed in plants 30 d of age. Although the group of resistant lines had slightly lower average lignin content than the group of susceptible lines (6.3 and 7.3%, respectively) at 50 d of age, the results were inconclusive due to a wide range in lignin content among the resistant lines (5.0% to 8.2%).
In a study examining 42 sorghum conversion1 lines for chemical makeup (crude protein, neutral detergent fiber, tannin, and lignin content) and resistance to the fall armyworm [Spodtera frugiperda (J. E. Smith)], Moussa et al. (1991) observed significantly higher average lignin content in panicles of lines identified as resistant (7.9% lignin) than lines identified as susceptible (5.9% lignin). However, within each resistant or susceptible group of sorghum lines, there was considerable variation in lignin content with susceptible lines ranging from 2.7 to 8.7% lignin, and resistant lines ranging from 3.4 to 13.9% lignin (Moussa et al., 1991). Within the resistant group, lignin concentration was negatively correlated (strength of correlation not reported) with duration of larval stage (Moussa et al., 1991). These seemingly contradictory results lead us to conclude that a relationship between reduced lignin content and susceptibility to insects has yet to be clearly demonstrated.
Brown Midrib
Brown midrib mutants of sorghum were first described by Porter et al. (1978) and are associated with significantly reduced lignin content in affected plants. Three of those mutations, bmr-6, bmr-12, and bmr-18, have been the focus of considerable study on lignin chemistry, cell wall composition, and animal performance, but surprisingly little information is available on their impact on agronomic fitness. The genes bmr-12 and bmr-18 are allelic (Bittinger et al., 1981) but not identical (Bout and Vermerris, 2003). In some instances—especially in extension and popular literature—brown midrib is used to describe a phenotype, and specific mutations are not identified. It has been suggested that "degree of coloration has no meaning" (Butler, 2003). However, in our laboratory, greenhouse, and field studies, we consistently observe earlier expression and more intense coloration in bmr-6 lines than in bmr-12 lines (data not shown). These mutations are quite different in their effect on lignin chemistry. The bmr-6 mutation exhibits reduced CAD activity (Pillonel et al., 1991), while the bmr-12 and bmr-18 mutations exhibit reduced COMT activity (Bout and Vermerris, 2003).
Extension and popular literature give strong indication that brown midrib is associated with lodging in forage sorghum. Our own experience and knowledge of producer experience with brown midrib forage sorghum is in complete agreement.
bmr-6.
Although it is generally believed that the effect of sorghum brown midrib mutations on yield is similar to those reported in maize (Kalton., 1988), only one report of yield differences in normal and brown-midrib counterparts of sorghum has been published. In a multistate forage trial comparing normal and brm-6 ‘Piper’ and normal and brm-6 ‘Greenleaf’ sudangrass [Sorghum bicolor subsp. drummondii (Nees ex Steud.) de Wet & Harlan], Casler et al. (2003) found the brown midrib phenotype as a result of bmr-6 to be environmentally and cultivar sensitive. First harvest yields of bmr-6 Greenleaf were reduced in Wisconsin (15%), but not in Nebraska, while yields of bmr-6 Piper were reduced in both Wisconsin and Nebraska (32 and 27%, respectively) as compared with their normal counterparts (Casler et al., 2003). Reduced tillering, and reduction in plant height were observed in the bmr-6 lines across environments (Casler et al., 2003). Second harvest yields of bmr-6 lines were also reduced across environments and varieties, indicating a limitation in re-growth potential in bmr-6 sorghum (Casler et al., 2003).
Unlike the bm1 CAD mutation of maize which reduces days to flowering by approximately 2 d, the bmr-6 mutation in sorghum has been shown to delay flowering by 5 d when compared with wild-type controls (Vermerris et al., 2002b).
bmr-12 and bmr-18.
Only two reports of the specific effects of bmr-12 and bmr-18 on plant agronomic fitness were found in the literature. Vermerris et al. (2002b) noted an average 7-d reduction in days to flower in two bmr-12 sorghum lines and essentially no change in days to flower in two bmr-18 sorghum lines compared with their wild-type counterparts. In a study using an F2 population segregating for various traits, Kasuga et al. (2001) conclude that the bmr-18 gene has no negative effects on resistance to sheath blight (Rhizoctonia solani Kühn).
In conclusion, naturally occurring variation in sorghum lignin content could not be convincingly correlated with resistance or susceptibility to insects. The brown midrib mutations increased lodging, and reduced yield, regrowth, height, and tillering. Effects of the bmr-6 mutation were shown to be sensitive to both environment and genetic background. The various brown midrib genes affected days to flower by up to 1 wk with bmr-12 being the most severe. Sources of reduced lignin in sorghum and their effects on agricultural fitness are summarized in Table 2.
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Pearl Millet |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Small Grains |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Several studies have attempted to establish a relationship between lodging and lignin content in wheat. Low lignin content was observed in the basal internodes of lodged wheat (Mulder, 1954). However, more recent studies using growth regulators to elicit reduced lodging showed no relationship between lignin content and lodging in wheat (Clark and Fedak, 1977; Knapp et al., 1987), barley (Clark and Fedak, 1977; Stanca et al., 1979) and oat (Clark and Fedak, 1977). Sources of reduced lignin in small grains and their effects on agricultural fitness are summarized in Table 3.
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Tobacco |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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An underlying assumption is that to remain agriculturally fit, plants with reduced lignin would need to remain morphologically similar to commercially desirable unmodified crops. Expected responses to significant biochemical or physiological modification of lignin content are reduced vigor and growth. Indeed, such responses have been noted in attempts to modify lignin in tobacco. For example, Pincon et al. (2001) showed reduced plant size in tobacco transformed with COMT and CCoAOMT (caffeoyl-CoA-O-methyltransferase) antisense sequences reducing lignin by 48%. Conversely, Zhong et al. (1998) demonstrated significantly reduced lignin (34–59%) in tobacco plants transformed with COMT and CCoAOMT antisense sequences compared with wild-type tobacco, but with normal morphological phenotype. Pincon et al. (2001) observed differences in growth rate under in vitro conditions as compared with greenhouse conditions, leading again to the possibility of genotype x environment interactions. Chabannes et al. (2001) reported a synergistic interaction of CAD and CCR (cinnamyl CoA reductase) antisense transgenes leading to severe reduction in lignin content in transformants that had a normal morphological phenotype under controlled conditions, concluding that "expression of a specific transgene may have different impact depending on the genetic background."
To our knowledge, research on modified lignin content in tobacco has been confined to the laboratory or greenhouse, since such tobacco would have no known commercial value at present, other than its important role as a model plant for the study of lignin biosynthesis. However, field tests of lignin-reduced tobacco would be required to determine agricultural fitness, including interactions of lignin reducing mechanisms with environment and genetic background. Sources of reduced lignin in tobacco and their effects on fitness, as determined in laboratory and greenhouse studies, are summarized in Table 3.
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Perennial Forages |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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Smooth Bromegrass
A high-IVDMD, low-lignin population and a low-IVDMD, high-lignin population were selected from a single source population of smooth bromegrass (Bromus inermis Leyss.) (Ehlke et al., 1986). Seedlings from these two populations were transplanted to fields in three states (Nebraska, Iowa, and Wisconsin) and percentage of surviving individuals recorded periodically for 49 mo (Casler et al., 2002). Little mortality was noted, and no differences in agricultural fitness were attributable to differences in IVDMD or lignin content.
Orchardgrass
Similar high-IVDMD and low-IVDMD populations were successfully selected from a source population of orchardgrass (Dactylis glomerata L.) by Rind and Carlson (1988). Seedlings were transplanted to fields in three states (as above), and an inverse relationship of lignin content and IVDMD was assumed, but not measured (Casler et al., 2002). Survival was shown to be related to cycle and direction of selection for IVDMD with 93% survival within the low IVDMD population and 87% survival within the high IVDMD population after four years (Casler et al., 2002). The authors speculate that natural selection in actual high-IVDMD swards could cause a shift to lower average IVDMD in that population over time. Although this research indicates a possible negative relationship of increased digestibility and survival in orchardgrass, the authors did not determine lignin content, nor did they report the strength of the relationship of IVDMD and survival (Casler et al., 2002).
Switchgrass
A source population of switchgrass (Panicum virgatum L.) was also selected for high and low IVDMD resulting in a high-IVDMD population and a low-IVDMD population (Vogel et al., 1981). Two additional cycles of selection were conducted for high-IVDMD and the resulting populations were transplanted in a multistate experiment (Casler et al., 2002). A negative relationship between IVDMD and lignin was established. The low-IVDMD population averaged 47% IVDMD and 7.5% lignin while the high-IVDMD population averaged 53% IVDMD and 6.5% lignin. Plant survival and yield were highly correlated to cycle of selection with the high-IVDMD (low-lignin) population exhibiting poorest survival and yield. All mortality occurred during winter months, and effects were most pronounced in the northern-most environment. Vogel et al. (2002) confirmed the above findings on a space-plant population basis, but noted that differential survival was attributable to survival within families, indicating that it should be possible to increase survival in high-IVDMD populations.
Tall Fescue
Chen et al. (2003) successfully decreased lignin content in transgenic tall fescue (Festuca arundinacea Schreb.) by downregulation of CAD. No differences between control and transgenic plants were observed for maturity, height, growth habit, tillering, seed yield, lodging, or pest or pathogen susceptibility. The development of these lines is too recent to have data on survival, but the authors indicate such studies are planned. For perennial forage grasses we conclude that although most studies do not measure the direct effect of reduced lignin on agricultural fitness, the effects of selection for IVDMD in some species is indicative of a relationship between reduced lignin, high IVDMD, and reduced survival with the latter at least in part affected by environment. The differential effects of increased IVDMD among species, and among populations within species is again indicative of strong interaction of lignin-reducing traits and genetic background of individuals or populations.
Alfalfa
Using alfalfa (Medicago sativa L.) populations divergently selected for high and low lignin content developed by Hill (1981), Kephart et al. (1989) demonstrated associated changes in morphology and yield. The low lignin population exhibited increased leaf to stem ratio. The high lignin population exhibited increased maturity at a fixed date, and increased stem length. Under field conditions, the high lignin population yielded 28% more than the low lignin population. Survivability for 2 yr significantly decreased in the low lignin population versus the high lignin population (34 and 64%, respectively) in a test conducted at a single location (Ames, IA) (Buxton and Casler, 1993).
Transgenic alfalfa with downregulated COMT and caffeoyl CoA 3-O-methyltransferase (CCOMT) exhibits reduced lignin content (Guo et al., 2001), while no reduction in lignin quantity was observed in downregulated CAD transgenic alfalfa but lignin composition was altered (Baucher et al., 1999). Data on fitness of such transgenic alfalfa are not yet available.
On the basis of the survival and yield of two divergently selected alfalfa populations, it appears feasible to conclude that reduced lignin can negatively impact the agricultural fitness of alfalfa. However, readers are reminded that in other species results were often variable and dependent on the genetic background of the populations of lines with reduced lignin. Clearly, more varied studies on lignin modification in alfalfa will need to be done before a definitive conclusion on its effect on agricultural fitness can be drawn. Sources of reduced lignin in perennial forages and their effects on agricultural fitness are summarized in Table 4.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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It is essential that reduction in lignin content, whether resulting from mutations in lignin biosynthesis genes, from selection for traits associated with reduced lignin, or from transformation, be evaluated in diverse genetic backgrounds and gene combinations, and in diverse environments to discover optimal combinations and a true measure of value to, and fitness in agricultural systems.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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1 A backcrossing program in which tropically adapted sorghum accessions are converted to lines suitable in height for harvest by commercial combines and that will reach maturity in temperate latitudes (Stephens et al., 1967). 
Received for publication March 8, 2004.
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TOPNOTESABSTRACTINTRODUCTIONMultiple Sources of Reduced...MaizeSorghumPearl MilletSmall GrainsTobaccoPerennial ForagesCONCLUSIONSREFERENCES |
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