Agronomic Characterization of the 20 Inbred Line Panel
The subset of inbred lines evaluated showed variation in the agronomic traits tested. The range obtained for days to silking and anthesis indicates that all inbred lines included in the panel develop appropriately and were well adapted to the growing conditions at the Pontevedra location (42°25′ N, 8°38′ W and 20 m above sea level). Inbred lines also differed for grain, forage and stover yield, the last two being positively correlated. In this sense, it is important to highlight two facts from the yield evaluations: (i) the value obtained for each inbred refers to the maximum-potential yield, not to the yield in a plot; and (ii) these values are for inbred lines, not being the standard material to be tested for yield, as it would be hybrid combinations.
Studying the Variability in the Cell Wall Composition
The relative quantities of secondary cell wall components in model grasses are: 35-45% cellulose, 40-50% hemicellulose and ~20% lignin(44). We studied the composition of the second internode below the main ear in a panel of 20 inbred lines of maize. We observed variation for cellulose, ranging from 29 to 51%; hemicellulose 19-28%, and lignin ranging from 19 to 33%. The internode represents a more uniform tissue than the whole plant or stover, allowing to obtain more accurate determination of the stalk composition.
As previously noted, the inbred lines included in this study show differences in cellulose, lignin, and hydroxycinnamates, but not in hemicellulose. The only hemicellulose related trait that showed significant differences in the set of inbred lines was the neutral sugars concentration.
We do not find variation in the monosaccharide composition among the 20 genotypes evaluated despite the germplasm variability. This may be because either, they are not diverse enough, or because there is no variability for those traits in maize, or at least not in the types of maize represented in this set. Besides, we are working with secondary cell wall, which is less variable than primary cell walls. (45)However, the lack of differences for monosaccharide composition in the second internode does not exclude variation between the tissues that constitute the internode (pith and rind). (46) Barros-Rios et al. (46) previously showed differences in polysaccharides in the pith (791 mg/g of cell wall) and the rind (772 mg/g of cell wall) of maize internodes..
Regarding lignin polymer, we found variation not only for lignin content, but also for lignin monomeric composition. In concordance with previous studies, S subunit represented the largest proportion, and H subunits the lowest proportion (47). Variation for hydroxicinnmamic acids among the inbred lines was also found in accordance with other studies.(3,15) For cell wall bound hydroxycinnamates we found that p-coumaric acid was the most abundant, which is also consistent with previous studies (5,15). The cell wall composition of some particular inbred lines in the current panel set has been previously established. In this sense, Santiago et al. (5) studied the relationship between the concentration of cell wall phenylpropanoids in the pith tissues (PCA, FA, DFA 8-O-4, DFA 8-5-b, DFA 8-5-l) and the level of resistance to corn borers in a set of 8 inbred lines (A509, EP42, EP47, F437 and PB130 were included). Some of the amounts described in the study from Santiago et al. (5) are kept in the present study. Inbred line A509 in both studies was the inbred that showed greater concentration in PCA content, not differing from F473 in this study; EP47 shows in both studies the greater concentration for DFA 8-5-l, and PB130 and EP42 show the lowest concentration of DFA 8-O-4 in both studies. Similarly, Barros-Rios et al. (46) determined the lignin and hydroxycinnamates (PCA, FA, and DFAT) concentrations in four inbred lines of maize (A509, EP39, EP42 and EP47) in the pith and rind tissues separately. The results showed larger concentration of cell wall components in the rind. In our case, analysing the complete internode, we also found higher concentrations of lignin and hydroxycinnamates than in core pith tissues for A509, EP42 and EP47.
In relation to the correlation analysis between cell wall components, ferulic acid dimers were positively and strongly correlated, and showed covariation in agreement with previous research (46,48). However, unlike in previous studies, individual and total diferulates did not show covariation with FA monomers (46,48). These discrepancies may be due to the type of tissue analysed. Barros-Rios et al. (46) studied the correlation between cell wall components independently in pith and rind tissues, and found that in pith tissues total diferulates and ferulic acid co-variate, although this covariation was not significant in rind tissues. In the same study, Barros-Rios et al. found greater concentration of both ferulic acid and total diferulates in the pith than in the rind. Similarly, positive correlations among ferulic acid, and individual and total dimers are described by López-Malvar et al. (48), where determinations were made in pith tissue. This reinforces the importance of the tissue selected for analysis, since it seems demonstrated that both the hydroxycinnmamic acid content and the relationship between hydroxycinnamates is different within the tissue.
Cell wall fibres were strongly correlated indicating that main cell wall polymers (cellulose, hemicellulose and lignin) covariate together (49). Cell wall fibres were strongly and positive correlated with PCA monomer which is in accordance with previous studies, as PCA is considered an indicator of lignification and secondary cell wall deposition (50,51). The strong positive correlations observed between individual and total diferulates and fibres are explained by the linkages between cell wall polymers and diferulates. Ferulic acid dimers crosslink hemicellulose chains binding specifically to arabinose. (52) Thus, this positive correlation may be caused by the linkage of diferulates and arabinoxylans, as the strongest correlations are among diferulates and hemicellulose. Moreover, ferulic acid and diferulic esters form cross-links through the etherification of the phenolic hydroxyl group to lignin polymers. Finally, the increasing amount of cell wall fibres is indicative of secondary cell wall deposition that is also correlated with an increasing cross-linkage. We found significant correlations among cell wall fibres and monosaccharides, both positive (arabinose, fucose) and negative (rhamnose) in agreement with previous studies. (53) The opposite correlations may be explained by dilution of primary cell wall material by the accumulation of secondary wall material. Furthermore, maize present type II cell walls characterized by a low rhamnose content (45).
In the cluster analysis, where it is reported the classification of inbred lines according to their variability in cell wall composition, 2 of the 4 groups were represented by just one inbred line: EP53 and EP86. EP53 was the most distinctive line due to deeper differences in cell wall composition from the other 19, including the highest value for lignin content. Lignification has been shown as the most detrimental component for both cell wall digestibility and biofuel production (4,47). Indeed, inbred line EP53 was included in the low digestibility and saccharification groups obtained after the variance analysis, even presenting the lowest saccharification value. Furthermore, in the multivariate analysis using the principal components approach it stands out for presenting low value for the PRIN2 (PRIN2 was mainly determined by low lignin concentration). Thus confirming the detrimental role that lignin plays on biomass quality, making less accessible cell wall polysaccharides to hydrolytic enzymes.
Inbred EP86, like EP53, was clustered alone in one group. For this inbred line we can highlight the high concentration of PCA and low concentration of individual and total dimers of FA. As previously mentioned, the structural reinforcement (lignin and cross-link) of the secondary cell walls is directly linked with low degradability values (54). Moreover, in the principal component analysis inbred line EP86 presented high values for PRIN2 and 3, and a low value for PRIN1. PRIN1 was mainly represented by individual and total dimers, indicating their influence in EP86 cell wall organization. This low concentration of total dimers and high concentration of PCA may also contribute in the susceptibility of this inbred line to corn borer attack (5). In accordance with its cell wall composition, this inbred line was classified in the analysis of variance in the low digestibility and borer susceptible group. Cell walls richer in crosslinking mediated by diferulates have been suggested to be a deterrent to borer attack and larvae development, presenting limited digestibility (55).
From e breeder point of view, EP86 and EP53 come from European Landraces, representing a reservoir for variability. These inbred lines present characteristics that make them different from the other eighteen inbred lines (Figure 2) and may be valuable for breeding purposes.
Another group that deserves attention is the group composed by inbred lines EC212, EP42, C103, CO348 and PB130. All the inbred lines included in this group were characterized by lower amounts of diferulates and lignin, and presented a highly degradable cell wall; in opposition to the two inbred lines previously mentioned (46,47,56).
Finally, in the correlation analysis we observed that the determinations of digestibility and saccharification of the cell walls in the internode and the whole plant did not show any significant correlation, indicating that results obtained for the internode cannot be extrapolated in or correlated to the whole plant. From a breeding standpoint, the aforementioned results indicate that to improve stover quality it seems more appropriate to evaluate stover. However, if the goal is to perform genetic analyses to find candidate genes that alter cell wall composition and digestibility of specific plant components, focusing on specific tissues would be more appropriate; as determinations performed in specific and more homogeneous tissues are more accurate. In addition, the sample uniformity in this last tissue makes this material more suitable for high-throughput robotic evaluations, which are generally based on replications of a few milligrams of biomass.
Relationship among Cell Wall Components and R.E.D. Traits
Resistance to corn borer (R.) has been associated to shorter tunnel length of galleries in the stalks (2). Taking into account this previous classification, groups of inbred lines classified as resistant or susceptible to corn borer showed differences in the percentage of hemicellulose content, being larger in inbred lines susceptible to borers attack, as well as in p-coumaric acid concentration, also in higher amounts in the cell walls of susceptible inbred lines.
Krakowsky et al. (57) suggested a positive correlation between European corn borer stalk tunnelling and stalk and sheath NDF adjusted for ADF. NDF adjusted represents the relatively digestible hemicellulosic fraction. Additionally, Terra et al. (58) found that hemicellulose is partly digested by the larvae of Erinnyis ello feeding on Euphorbia pulcherrima leaves, as opposed to other cell wall components. A larger proportion of hemicellulose could favour the attack of the insect, while other structural components involved in tissue strengthening, such as lignin, or cellulose, deter the larvae advance (59).
Susceptible and resistant inbred lines differed in PCA amounts; and susceptible lines have a higher concentration of p-coumaric acid. This result appears contradictory with previous results, as an increasing concentration of p-coumaric acid has been related to resistance to corn borer attack (5). Most p-coumarate accretion occurs in tandem with lignification and its accumulation can be considered a relevant indicator of lignin deposition. In maize, lignins are acylated (primarily syringyl units) at the γ-position by p-coumarates (60). Acylation has a marked influence on the bonding mode of S lignin units, on the spatial organization of lignins and consequently on their capacity to interact with polysaccharides. It is known that syringyl type lignin forms a more linear structure, (61) with little or no branching and with a lesser degree of polymerization. Although S lignin has been involved in defence against biotic stresses (62,63), in the case of corn borer resistance S-type lignin, indirectly associated to more PCA acetylation, may favours borer susceptibility, at least in the materials evaluated in the current study. In this sense, the type of tissues analysed has to be further considered in the establishment of clear relationships.
Differences in high and low saccharification efficiency (E.) obtained in the contrasts analysis were mainly determined by the ferulic acid dimers concentrations, showing larger values in the high saccharification efficiency group. Supporting these results, in the multiple linear regression analysis we identified cellulose and DFA 8-5-b as significantly involved in the variability for saccharification. Lignocellulosic polysaccharides, mainly cellulose (40%), serve as main substrate for fermentation of cell wall sugars in ethanol (40), Thereby, cell walls richer in cellulose have more sugars to potentially be fermented. Improving the relative content of cellulose was already one of the main strategies towards the development of advanced lignocellulosic feedstocks (64).
The rigid structure of the cell wall makes cellulosic materials inaccessible to hydrolytic enzymes, making a pre-treatment essential before the saccharification in order to enhance the effectivity of those enzymes. Among the several types of pre-treatments that could be used, alkaline pre-treatment is appropriate for corn stover and other monocots due to its particular cell wall composition (65). The cell walls of graminaceous monocots are known to contain alkali-labile ferulate ester cross-links within the hemicellulose (66), as well as high phenolic hydroxyl contents in their lignins, resulting in increased alkali solubility (67), rendering the cell wall highly susceptible to delignification by alkaline pretreatments (6). As a consequence, mild alkali pre-treatment of grasses such as maize has shown substantial advantages, as these can be employed for both fractionating biomass and generating a pre-treated biomass that is highly amenable to enzymatic hydrolysis (66).
Our findings indicate that a greater concentration of ester diferulates can be associated with an enhanced saccharification efficiency of the maize stalk. This was unexpected since a more cross-linked cell wall has been suggested to be more recalcitrant to enzyme deconstruction (68). This may involve cell wall plasticity, so a greater amount of crosslinking elements in the cell wall may replace the deposition of other structural elements, such as lignin, finally favouring deconstruction (69). A cell wall where the structural reinforcement is mainly due to a greater crosslinking effect mediated by dimers could represent an improvement in the process of obtaining ethanol, considering that among the pre-treatments of the samples for saccharification the saponification is usually included and would eliminate these compounds (70). Li et al. (6) found in a study of a subset of 26 inbred lines of maize that the pre-treated cell-wall hydrolysis yields were positively correlated with the ferulate released by alkali pre-treatment, indicating that breaking of ferulate cross-links between cell- wall polymers is an important outcome of pre-treatment. This result reinforces the hypothesis that cell walls where the structural support is based in a higher cross-linkage by diferulates could be more favourable to sugar release, but only after the corresponding alkaline pre-treatment. The role of crosslink is still negative for saccharification if not pre-treated. On the other hand, a greater proportion of ester-link diferulates may indicate a lesser proportion of ether-linked diferulates bound to lignin, and therefore less recalcitrant cell walls. However, ether-linked diferulates cannot be precisely determined under the current wet chemistry procedures.
Regarding to forage feedstocks (D.), highly degradable lines presented lower concentration of PCA, DFA 5-5, DFA 8-5-l and DFAT, lower proportion of glucuronic acid, and greater proportions of G subunits. In addition, we obtained that DOM was negatively affected by the content in NDF, whereas positively affected by FA and G subunits.
High concentration of fibres directly affects digestibility by the limitation of the energy intake by the animals (71). In agreement with our results, Wolf et al. (71) analysed two maize populations (with high and low concentrations of NDF, ADF, lignin, and silica) and foundthat the population exhibiting low range of NDF also showed greater digestibility.
During lignification the ferulic acid and diferulic esters form cross-links through the etherification of the phenolic hydroxyl group to lignin polymers, (72) forming a polysaccharide-lignin matrix that renders cell wall more recalcitrant to enzymatic hydrolysis (72). The results obtained in the contrast analysis are consistent with this statement, however, in the multiple linear regression analysis, we observed that 6% of the variation for DOM was positively explained by FA concentration. As this was unexpected, we next performed a simple regression analysis between DOM and FA in order to test if the positive effect was repeated, and it was not (data not shown). Overall, we suggest that this could be an effect of the multiple linear regression model. When constructing a multiple regression, the coefficient for the first independent traits coincides with that of the multiple regression for that trait. For the rest of the traits included in the analysis the regression is done with the residuals for each genotype, unless traits are totally independent. This may cause a change in the effect as in the case of FA.
In relation to the role of the p-coumaric acid in the DOM, cell walls presenting lower PCA content were more digestible. p-coumaric acid is mainly esterified to the y-position of the side chains of S units in lignin (60). The role of p-coumarate-monolignol conjugates, is to help control the three-dimensional organization of grass lignin. As previously mentioned Syringyl-type lignin is more linear in structure and extends further into the secondary wall, protecting a larger proportion of the polysaccharides in the wall from digestion; thus reducing cell wall digestibility (61). In our study, lines included in the high digestibility group also showed greater proportion of G subunits. Mechin et al. (3) observed a reduced proportion of subunits S in bm3 mutants, characterized by its improved stover digestibility. They also observed that the decrease in the proportion of S subunit was balanced by an increase in proportion of G. A greater proportion of subunit G implies a decrease of resistant C-C interbonds, explaining the increase in degradability in bm3 mutants.
Finally, a greater concentration of glucuronic acid was detrimental for DOM. Hemicellulose could be a potential repository of fermentable sugars (73), but unlike cellulose, hemicelluloses are not chemically homogeneous. Corn ﬁbre xylan is one of the complex heteroxylans containing β-(1,4)-linked xylose residues. (74) About 80% of the xylan backbone is highly substituted with monomeric side-chains of arabinose or glucuronic acid linked to O-2 and/or O-3 of xylose residues. The effect of hydrolytic enzymes used in processes such as DOM are influenced by the variation of the primary structure of the arabinoxylans (75). Based on the works of Van Eylen et al. (76) and Appeldoorn et al.(77) reductions in the frequency of acetic acid, uronic acid and arabinose side groups in glucuronoarabinoxylans would concurrently lead to a reduction in the cell wall recalcitrance. The presence of more substitutions in the arabinoxylan chain, particularly glucuronic acids, could interfere the specific mode of action of hydrolytic enzymes, limiting this way the DOM.
Promising Genotypes to be Used in R.E.D Improvement
Regarding saccharification efficiency, the 20 inbred lines showed significant differences and could be divided into three groups: high (101.58-108.96 nmol mg material-1 hour-1), intermediate (101.42-94.72 nmol mg material-1 hour-1) and low (93.67-80.18 nmol mgmaterial-1 hour-1) saccharification. Other researchers have studied the saccharification efficiency of several crops species following the same method and pre-treatment as in this study: Acevedo et al. (78) studied the cell wall composition and saccharification efficiency of internodes of seven Sorghum sudanense accessions; they obtained significant differences across the accessions and an average level of 53.63 nmol mg-1 hour-1 of reducing sugars. Similarly, Whitehead et al. (79) tested Miscanthus sinensis and Arabidopsis thaliana obtaining average values of reducing sugars of 60 nmol mg-1 hour-1 and 80 nmol mg-1 hour-1 for Arabidopsis and Miscanthus respectively. In a study using Brachypodium stem material Whitehead et al. (80) obtained variability in the saccharification efficiency ranging from 28.83 nmol mg-1 hour-1 to 46.67 nmol mg-1 hour-1. Finally, Liu et al. (81) studied the genetic variability of saccharification efficiency in straw samples from a rice RIL population and they observed a wide range of saccharification values, ranging from 59 nmol mg-1 hour-1 to 116 nmol mg-1 hour-1, with the highest saccharification plant stems producing 97% more reducing sugar than the lowest saccharification line in this population. Species like Miscanthus, switchgrass or sugarcane, are promising candidates for the industrial production of biofuel as they present high biomass yields (15-25 Mg/ha), broad geographic adaptation, superior carbon sequestration and efficient nutrient utilization.(82) However, these species cannot be readily implemented on a wide-commercial scale. In contrast, maize is the most important crop in terms of production, 1300 million tons of dry maize stover are produced worldwide ;(83) and can potentially supply vast amounts of lignocellulose in the form of agricultural residues (5 Mg/ha) (82). In view of the results obtained for saccharification efficiency in contrast to other crops, maize arises as an outstanding model able to contribute significantly to the energy industry’s feedstock both in quality and quantity.
Among the set of inbreds, the sugar corn lines C103, CO348, CO384, CO444 and CO442 were included because of their promising performance to be used in hybrid combination for ethanol production (25). However, this consideration was noted according to the soluble sugar content in the stalk of their hybrids, but without considering the sugars coming from the cell wall polysaccharides. In this study, we just can highlight the inbred line CO442 (Iodent) as the only one included in the high saccharification efficiency group. The high saccharification efficiency of this inbred line may be due to a cell wall rich in diferulate crosslinking that responds better to alkaline pre-treatment. Conversely, some other inbred lines identified as potential candidates as base material in breeding programs could be A654, EC212, EP17, EP42 and F473. Four of these inbred lines are included in the European Flint Heterotic Group (EC212, EP17, EP42 and F473) and the last one in the Reid Group (A654), so that it is possible to obtain good heterosis for grain production, while getting high saccharification efficiency.
The results from DOM were not included on the variance analysis by the reasons previously mentioned, but we can revise the rank obtained for this trait. DOM values in this research varied from 53.4% to 63.8%. This rank is in agreement with the previous values published for maize in vitro digestibility (84). Compared with other crops, digestibility of the organic matter was lower in sorghum than in maize, with values ranging between 40% and 45%; for rice straw differences in in vitro digestibility of the organic matter have been reported and ranged from 23.6% to 36.9%;(85) similarly Capper et al. (86) reported means of 32 to 35% in Barley stems. In search for a forage ideotype in cereals, it is necessary to improve the enzymatic digestibility and intake of the forage. DOM and cell wall digestibility are one of the major target for the improvement of feeding value in cereal plants. This subset of inbred lines represents a rich genetic background for improving R.E.D traits, since itincluded variation for cell wall composition and the well adapted genoytypes (87).
To respond to the question Is it Possible to Identify a Particular Ideotype Useful in the R.E.D.?, our results show that it is very difficult to find a maize ideotype presenting a cell wall composition that fits the three requirements of the R.E.D, as one combination of components would favour one use and negatively affect the other. For example, that is the case of the diferulic acid content, that affects cell wall digestibility and saccharification efficiency, or borer resistance and saccharification, both in opposite ways. Therefore, we could aim to obtain a putative genotype from this subset of lines with the best combination of cell wall compositional traits to be used in a particular usage. Despite this, among this subset of inbred lines, we found inbred lines that present high saccharification efficiency and high digestibility of the organic matter: EP42 and EC212; however, we must note that both are susceptible to corn borer attack.
According to these results, we can propose a maize cell wall ideotype to each specific area to be improved: (i) borers resistance in the internode may involve cell walls with low p-coumaric acid concentration and hemicellulose content; (ii) saccharification efficiency must be improved by increasing the presence of cellulose and diferulates; (iii) and to improve digestibility cell wall must have poor neutral detergent fiber and diferulate cross-links, combined with a lignin polymer richer in G subunits.