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J Exp Bot. 2010 May; 61(9): 2479–2490.
Published online 2010 April 21. doi:  10.1093/jxb/erq093
PMCID: PMC2877902

Phenotypic plasticity in cell walls of maize brown midrib mutants is limited by lignin composition

Abstract

The hydrophobic cell wall polymer lignin is deposited in specialized cells to make them impermeable to water and prevent cell collapse as negative pressure or gravitational force is exerted. The variation in lignin subunit composition that exists among different species, and among different tissues within the same species suggests that lignin subunit composition varies depending on its precise function. In order to gain a better understanding of the relationship between lignin subunit composition and the physico-chemical properties of lignified tissues, detailed analyses were performed of near-isogenic brown midrib2 (bm2), bm4, bm2-bm4, and bm1-bm2-bm4 mutants of maize. This investigation was motivated by the fact that the bm2-bm4 double mutant is substantially shorter, displays drought symptoms even when well watered, and will often not develop reproductive organs, whereas the phenotypes of the individual bm single mutants and double mutant combinations other than bm2-bm4 are only subtly different from the wild-type control. Detailed cell wall compositional analyses revealed midrib-specific reductions in Klason lignin content in the bm2, bm4, and bm2-bm4 mutants relative to the wild-type control, with reductions in both guaiacyl (G)- and syringyl (S)-residues. The cellulose content was not different, but the reduction in lignin content was compensated by an increase in hemicellulosic polysaccharides. Linear discriminant analysis performed on the compositional data indicated that the bm2 and bm4 mutations act independently of each other on common cell wall biosynthetic steps. After quantitative analysis of scanning electron micrographs of midrib sections, the variation in chemical composition of the cell walls was shown to be correlated with the thickness of the sclerenchyma cell walls, but not with xylem vessel surface area. The bm2-bm4 double mutant represents the limit of phenotypic plasticity in cell wall composition, as the bm1-bm2-bm4 and bm2-bm3-bm4 mutants did not develop into mature plants, unlike the triple mutants bm1-bm2-bm3 and bm1-bm3-bm4.

Keywords: brown midrib, cell wall, lignin, maize, SEM, Zea mays

Introduction

The chemical composition and physical properties of materials are intricately associated. In the case of polymers, the nature of the monomer(s), parameters such as the degree of crosslinking, and the average chain length determine physical properties such as elasticity, glass transition temperature, and thermostability. This applies both to synthetic polymers (Bicerano, 2002), and biological polymers such as starch (Kortstee et al., 1998; Groth et al., 2008) and pectins (Willats et al., 2001).

The cell wall polymer lignin is a complex aromatic polymer present in the cell walls of plants, in particular, secondary cell walls of the xylem and sclerenchyma. Lignin provides structural support and facilitates water transport and is also important in the defence against pests and pathogens. Lignin is formed via the oxidative coupling of monolignols, synthesized via the shikimic acid and phenylpropanoid pathways (reviewed by Boerjan et al., 2003; Ralph et al., 2004). The three main monolignols in maize and other grasses are p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, which give rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) residues, respectively.

In the cell walls of grasses such as maize, lignin is chemically cross-linked with glucuronoarabinoxylans (GAXs) via ferulate and diferulate bridges (Ralph et al., 1995; Hatfield et al., 1998; Zhang et al., 2009). The GAXs in turn are associated with cellulose microfibrils through hydrogen bonding and Van der Waals forces, resulting in a complex matrix (Carpita and Gibeaut, 1993).

There is considerable variation in lignin subunit composition between species, between tissues within the same species, and between different developmental stages (Terashima et al., 1993; Müsel et al., 1997; Joseleau and Ruel, 1997; Vermerris and Boon, 2001), which is possibly a reflection of different demands placed on lignified tissues (Boyce et al., 2004). The plant must somehow be able to control this spatio-temporal variation in lignin composition, but the precise mechanism is unclear. Chemical control has been proposed, whereby the composition of the surrounding matrix affects the reaction kinetics (Hatfield and Vermerris, 2001). An opposing view is biological control, which would require the involvement of proteins that stipulate the formation of the different interunit linkages (Davin and Lewis, 2005; Davin et al., 2008).

Regardless of the mechanism the plant employs to establish a certain lignin subunit composition, the physical properties of the lignified cell wall, including wall strength and water conductivity, ultimately depend on the chemical composition. Defining the relationship between structure and function of cell wall components is important for understanding the evolution of land plants and plays a key role in the development of new crops with cell wall properties that are more amenable to down-stream applications, such as the production of green chemical feedstocks (Yang and Wyman, 2004; Vermerris et al., 2007; Saballos et al., 2008).

Variation in lignin subunit composition can be introduced through the use of mutations in lignin biosynthetic genes or through transgenic down-regulation of gene expression (Boerjan et al., 2003; Vanholme et al., 2008). A group of cell wall mutants in maize is known as the brown midrib (bm) mutants. These mutants, bm1, bm2, bm3, and bm4, have reddish-brown vascular tissue in the leaves and stems as a result of changes in cell wall composition. The bm1 mutant has reduced activity of the lignin biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD) (Halpin et al., 1998). Consequently, the cell walls of this mutant contain increased levels of cinnamaldehydes (Halpin et al., 1998; Marita et al., 2003). The bm1 mutation is unlikely to be in the CAD gene itself (Guillaumie et al., 2007a) and the function of Bm1 remains to be elucidated, as is the case for Bm2 and Bm4. The bm2 mutant contains fewer guaiacyl residues (Chabbert et al., 1994b) and has a disturbance in the tissue-specific patterns of lignification (Vermerris and Boon, 2001). NMR analyses of lignin isolated from bm4 stems harvested just before anthesis did not reveal major changes relative to the wild-type control (Marita et al., 2003). The Bm3 gene encodes the enzyme caffeic acid O-methyl transferase (Vignols et al., 1995; Morrow et al., 1997). Mutations in this gene result in lignin with a higher G/S ratio and the incorporation of 5-hydroxyconiferyl alcohol (Lapierre et al., 1988; Chabbert et al., 1994a), which can form benzodioxane structures (Marita et al., 2003).

Phenotyping and chemical and structural analyses on the bm2, bm4, and bm2-bm4 mutants were performed to gain a better understanding of how changes in lignin subunit composition affect plant development, possibly through changes in the ultrastructure of the conductive and supportive tissues (Nakashima et al., 2008). From these analyses it is apparent that lignin subunit composition can vary considerably without affecting the overall performance of the maize plant, but that once a certain threshold is crossed, the plant is no longer able to develop and function normally.

Materials and methods

Plant material

The development of the bm1, bm2, bm4, and bm1-bm2 near-isogenic lines (NILs) in inbred line A619 following a minimum of six backcrosses has been described by Vermerris and McIntyre (1999). The bm2bm4 double mutant was created by crossing a bm2 and a bm4 NIL. The F1 progeny was self-pollinated and putative double mutants were identified in the F2 population, self-pollinated, and subjected to test crosses to both bm2 and bm4 single mutants to confirm the presence of the two mutations. The same approach was used to create the remaining three possible bm double mutant NILs: bm1-bm3, bm2-bm3, and bm3-bm4. The fact that these different mutant lines are all in the same genetic background makes it possible to attribute any phenotypic differences directly to the mutations.

The bm1-bm2-bm4 triple mutant was generated by crossing bm1-bm2 with bm2-bm4, followed by self-pollination of the F1 progeny. Since both parents were homozygous for bm2, the F2 progeny was homozygous for bm2, but segregating for bm1 and bm4. Sixty seeds from each of three F2 families were planted. Based on the reduced height of the bm2-bm4 double mutant and normal height of the bm1-bm4 double mutant, DNA from all small plants plus wild-type and bm1 controls was isolated using the Plant Red ExtractNAmp kit (Sigma, St Louis, MO), and used to determine the genotype for the dupSSR10 simple sequence repeat marker, which is tightly linked to the bm1 locus (www.maizegdb.org). The PCR was based on the manufacturer's instructions, and was performed in a 15 μl volume containing 50 ng of each primer (forward 5′-AGA AAA TGG TGA GGC AGG-3′, reverse 5′-TAT GAA ATC TGC ATC TAG AAA TTG-3′) using a three-step program consisting of an initial denaturation of 2 min at 94 °C, followed by 35 cycles of 10 s at 94 °C, 20 s at 55 °C and 40 s at 72 °C. PCR products were separated on a 4% SFR agarose gel (Amresco) containing ethidium bromide.

The triple mutants bm1-bm2-bm3 and bm1-bm3-bm4 were generated by crossing confirmed bm1-bm2 and bm2-bm3 double mutants, self-pollinating the F1 progeny and selection in the F2 progeny for triple mutants based on the molecular marker dup10SSR (see above) and a PCR-based detection of the bm3-ref allele (Vignols et al., 1995) based on the presence of the B5 retrotransposon using the primers B5-29 (5′-GTT CTC TTC CTC GCC TTC CTC AG-3′) and bm3-L1522 (5′-ACG GGA TGA CGG CGT TCG AGT A-3′; Vermerris and Bout, 2003) using a three-step PCR program as described above with an annealing temperature of 64 °C. The genotype of the putative triple mutants was then confirmed by testcrosses to the individual single bm mutants. The bm1-bm3-bm4 triple mutant was generated similarly after crossing bm1-bm4 and bm3-bm4. Attempts to generate a bm2-bm3-bm4 triple mutant by crossing bm2-bm4 with bm3-bm4 and by crossing bm2-bm4 with bm2-bm3 did not result in viable triple mutant plants.

Single and available double bm mutants along with the wild-type inbred line A619 were planted in the field in 5 m rows, spaced 0.76 m apart using a randomized complete block design with two replicates of 12 plants of each genotype. The field was located at the Agronomy Center for Research and Education near West Lafayette, IN. Plant height at anthesis was recorded for all plants. In addition, from each replicate three randomly selected plants wild-type, bm2, bm4, and bm2-bm4 plants were sampled for chemical and ultrastructural analyses. The same field design was used the following year in a nearby field at the same location, with additional double mutants that had become available.

Scanning electron microscopy

Leaf six (the ear leaf) was harvested from wild-type, bm2, bm4, and bm2-bm4 plants in the field after anthesis. The leaves were placed in water, and transported to the laboratory for fixation. A 1.5 cm strip containing the midrib was cut from each of the leaves at approximately 4 cm from the base of the leaf blade. The strips were immersed in primary fixative (4% glutaraldehyde in 0.1 M potassium phosphate buffer, pH 6.8) and cut into 2 mm sections. The sections were fixed overnight and then washed four times in buffer. The samples were subsequently fixed in 2% (w/v) OsO4 in 0.1 M potassium phosphate buffer, pH 6.8, for 4 h followed by buffer and water washes (2× each). They were dehydrated in an ethanol series, critical-point dried (LADD Research Ind., Williston, VT), mounted, and sputter coated with AuPd prior to imaging.

Digital SEM images of two sections from each of the four genotypes were acquired using a JEOL JSM-840 scanning electron microscope (JEOL USA, Inc., Peabody, MA) operating at 5 kV. An overview image was generated for each sample at a magnification of ×200. Close-up images of the individual xylem vessels and sclerenchyma walls were generated at magnifications of ×750 and ×1000, respectively. Magnifications were calibrated using a carbon replica grid having line spacings of 0.463 μm.

Image analysis

The SEM images were imported into Photoshop® 7.0 (Adobe Systems, San Jose, CA) and xylem vessel lumina were selected and filled with white to facilitate the quantification of area. The image analysis program IP Lab version 3.6 (Scanalytics, Fairfax,VA) was calibrated using the SEM images of the carbon replica grating. The images were imported into IP Lab and the thickness of sclerenchyma and xylem cell walls and xylem luminal area were measured.

Preparation of cell wall samples

The midribs were isolated by removal of the leaf blade tissue, using the remainder of the leaves used for SEM sample preparation. The midribs were dried for 4 d in an oven set at 50 °C, ground in a Wiley mill to pass a 1 mm screen, and extracted in 50% (v/v) ethanol to remove soluble sugars and phenolics. The ear leaf of three independent plants per genotype was collected and used for the dissection of the sclerenchyma on the abaxial side of the midrib, as described by Vermerris et al. (2002). Stem tissue from three plants per genotype was collected at physiological maturity.

Histochemical reactions

Several pieces of dissected midribs were treated with acid phloroglucinol (Wiesner reagent) as described by Vermerris et al. (2002), and scanned on a flat-bed scanner. In order to investigate the origin of the brown colour in the mutants, midrib samples were incubated overnight in 6 ml 5:1 v/v DMSO:NH4OH containing 1.7% (w/v) NaBH4. This results in the reduction of aldehydes and ketones to their corresponding alcohols. The excess NaBH4 was removed by adding 0.1 ml glacial acetic acid, followed by rinsing the samples twice in water and once in acetone. This protocol was based on procedures described by Tsai et al. (1998) and Carpita et al. (2001).

Pyrolysis-GC-MS

This technique relies on the thermal degradation of tissue samples under anoxic conditions. The pyrolysate contains volatile compounds that are separated by gas chromatography and identified with mass spectrometry. This method provides information on the composition of aromatic compounds in the cell wall. Information is also obtained on the composition of cell wall polysaccharides, but this information is limited because of degradation of the original compounds. A major advantage of Py-GC-MS is that it only requires small amounts of tissue (Boon, 1989; Ralph and Hatfield, 1991; Fontaine et al., 2003). Like most analytical techniques that are used to analyse lignin composition, including thioacidolysis (Lapierre, 1993) and ‘derivatization followed by reductive cleavage’ (Lu and Ralph, 1997), Py-GC-MS provides compositional information on the fraction of the lignin in which the monomers are cross-linked via β-O-4 linkages (Lapierre, 1993), which represents the most common linkage in maize (Marita et al., 2003). Py-GC-MS was performed as described by Bout and Vermerris (2003), using 1 mg samples of dissected midrib tissue. In order to evaluate the content of p-coumarate and ferulate, the midrib samples were trans-methylated as described by Vermerris and Boon (2001) and subjected to Py-GC-MS.

Klason lignin analysis

Klason lignin is defined as the ash-corrected residue remaining after the cell wall polysaccharides have been removed via acid hydrolysis. The procedure was based on the method described by Theander and Westerlund (1986), with the modifications of Hatfield et al. (1994). One hundred milligrams of ground midrib tissue or ground stem tissue that had been extracted in warm 50% (v/v) ethanol for 30 min was used for this analysis.

Cellulose determination

Cellulose content was determined as acetic-nitric acid resistant material obtained from 50 mg dried and ground midrib tissue, assayed spectrophotometrically using the anthrone reagent (Updegraff, 1969). α-Cellulose (SigmaAldrich, St Louis, MO) was used as a standard.

Statistical analyses

It was first determined whether height and peak area percentages in the pyrograms and cell wall characteristics showed evidence for differences between the wild type and the mutants. The following fixed effects model was used: Yij =μ+ giij, where Yij is the peak area percentage or cell wall characteristic, the parameter μ is the overall mean, gi the genotypic effect i from individual j and ϵij the experimental error. To determine whether genotypes were statistically significantly different, pair-wise means were compared using Tukey's least significant difference at a threshold of 5% (Steel et al., 1996). For those measurements that showed evidence of at least one pair-wise difference, the compounds were compared to each other using Pearson's correlation (Steel et al., 1996).

In order to facilitate the visualization of differences among genotypes, the average peak area percentage was calculated across replicates. Averages were standardized for each peak area and a hierarchical clustering analysis was performed. In addition, a linear discriminant analysis (LDA) was performed to determine whether the four genotypes could be distinguished based on their chemical profiles. Compounds included in the LDA had to show statistically significant differences in at least one pair-wise comparison between genotypes. The statistical analyses were performed using SAS and JMP (SAS Institute, Cary, NC).

Results

Single bm mutants and most double bm mutants are well within the normal range

Single bm mutations have reported changes in flowering time (Vermerris and McIntyre, 1999; Vermerris et al., 2002) and, in the case of bm3, increased susceptibility to Fusarium rot (Zuber et al., 1977) and reduced yield (Gentinetta et al., 1990). These mutants have been receiving interest as a potential source of lignocellulosic biomass for fuel production, as they can yield more fermentable sugars upon enzymatic saccharification compared to the same amount of biomass from a wild-type control (Vermerris et al., 2007). Important for biomass production, the height differences in the single mutants compared with the wild type are modest (Fig. 1; see Supplementary Table S1 at JXB online). When independent bm mutations are combined, some phenotypic differences are identified. The height of the double mutants bm1-bm2, bm3-bm4, and bm2-bm4 is statistically significantly different from the wild type (Fig. 1). Additional differences in height exist, such as between bm1-bm2 and bm2-bm3 (see Supplementary Table S1 at JXB online). All plants with the exception of bm2-bm4, however, flowered and set seed.

Fig. 1.
Height at anthesis of wild-type inbred A619 and near-isogenic single and double bm mutants in two consecutive years (Year 1, grey bars; Year 2, black bars). Not all double mutants were available in the first year. Error bars indicate 1 SD.

The bm2-bm4 double mutant is radically different from wild type and the single mutants bm2 and bm4

Figure 2 illustrates the drastically reduced height of a mature bm2-bm4 mutant and its very dark brown midribs which, in contrast to the bm2 and the bm4 single mutants, are consistently visible from the adaxial side of the leaf. While the double mutant displayed in this image was viable and ultimately produced seed, the majority (>80%) of the bm2-bm4 plants never reach maturity. Instead, their development is often arrested prior to or at the four-leaf stage. Observational evidence indicates that the size of the leaves and midribs tends to correlate with the height of the bm2-bm4 plants. The bm2-bm4 seed has been planted over a period of five years in different geographical regions in the United States (North Carolina, Indiana; summer) and in Puerto Rico (winter), and in different soils and in the greenhouse. Intense irrigation and application of fertilizer were of no avail. The bm2-bm4 double mutant has been generated independently several times and displays a consistent phenotype, so that the developmental differences cannot be attributed to a parent with one or more additional spontaneous mutations, or to maternal effects during seed production. The two triple mutants that include bm2 and bm4, namely bm1-bm2-bm4 and bm2-bm3-bm4 are unable to develop into mature plants under the conditions tested. Plants shown to be bm1-bm2-bm4 triple mutants stopped growing at a very early seedling stage, wilted, and died (Fig. 2). Based on the inability to identify bm2-bm3-bm4 seedlings among segregating progeny, the combination of those three mutations appears to arrest growth even prior to seedling emergence.

Fig. 2.
(A) The bm2-bm4 double mutant (blue arrow) is significantly shorter and thinner than the single mutants bm2 (shown to the right) and bm4. (B) The vascular tissue of the bm2-bm4 mutant is dark-brown. (C) Six-week old, field-grown seedlings of inbred A619 ...

Why is bm2-bm4 so different from the bm2 and bm4 single mutants and other double mutants?

The brown colour of the vascular tissue in mutants or transgenics in which lignin biosynthesis was compromised has been attributed to the accumulation of coniferaldehyde (Higuchi et al., 1994; Mackay et al., 1997; Tsai et al., 1998; Vermerris et al., 2002). The lignin of the bm1 mutant is indeed known to accumulate high levels of coniferaldehyde end-groups (Halpin et al., 1998; Vermerris et al., 2002). To test the hypothesis that coniferaldehyde is responsible for the brown colour of the midribs in bm mutants other than bm1, dissected midribs from the mutants bm2, bm4, and bm2-bm4 were stained with acid phloroglucinol, with wild-type and bm1 midribs as controls. The unstained bm2-bm4 midribs are the darkest, followed by the bm2, bm4, and bm1 midribs (Fig. 3). After staining with phloroglucinol the bm1 midribs produce the expected, intense staining, whereas the bm4, bm2, and bm2-bm4 samples display much less coloration, indicating that they contain fewer rather than more coniferaldehyde end-groups. If the brown colour in the vascular tissue of the bm mutants is the result of the accumulation of aldehydes and/or ketones, as suggested by Tsai et al. (1998), reduction with NaBH4 would be expected to eliminate the brown colour. Figure 3 shows that the NaBH4 treatment results in a yellowing of the wild-type and bm1 midribs after the complete reduction of the coniferaldehyde-end groups. The reduction with NaBH4 lessens but does not completely eliminate the brown colour of bm2, bm4, and bm2-bm4 midribs, indicating a different chemical origin of the brown color in these mutants compared to bm1 plants.

Fig. 3.
Colour differences between dissected vascular tissue of the brown midrib mutants. (A) Dissected vascular bundles of inbred A619 and the near-isogenic brown midrib mutants bm1 (on far right), bm2, bm4, and bm2-bm4. The samples were prepared from leaf 6 ...

To characterize the phenotype of the bm2, bm4 and bm2-bm4 mutants further, Klason lignin content in the leaf midribs and mature stem sections was determined (Table 1). The effect of these mutations on Klason lignin content in the mature stems is small and not statistically significant and confirms the limited effect of bm2 and bm4 on stem tissue harvested prior to flowering time reported by Marita et al. (2003). In contrast, in the midribs the bm2 and bm4 mutations, each cause an 11% reduction in Klason lignin content, and this effect is additive in the bm2-bm4 mutant.

Table 1.
Klason lignin content (mean and standard deviation) of midribs and stems from leaf 6 of wild-type, bm2, bm4, and bm2-bm4 plants

Since the Klason lignin indicated that the bm2 and bm4 mutations primarily affect the leaves, the cell wall composition of the wild-type, bm2, bm4, and bm2-bm4 samples was analysed in more detail using pyrolysis-GC-MS. The average peak areas of a set of diagnostic compounds were identified from the different cell wall constituents (Table 2). The pyrograms do not contain evidence for the appearance of novel residues that could be associated with the brown colour of the vascular tissue. The compound 3-(4-hydroxyphenyl)-3-oxopropanal, derived from H-residues, was not detected in all samples, making statistical comparisons invalid. This compound was, therefore, not considered in further analyses. The remaining compounds were tested for pairwise differences (Table 3). Many compounds derived from lignin are reduced in the bm2, bm4, and bm2-bm4 mutants compared with the wild type, whereas compounds corresponding to breakdown products from cell wall carbohydrates are increased in intensity. The midribs of both the bm2 and bm4 mutants show overall similar and statistically significant reductions in compounds derived from guaiacyl and syringyl residues in the lignin (Table 3). There are no statistically significantly different differences between bm2 and bm2-bm4, whereas bm4 and bm2-bm4 are different in both coniferaldehyde and acetosyringone, indicating that the composition of the bm2-bm4 double mutant is more similar to the bm2 mutant than the bm4 mutant (Fig. 4). The compounds 4-vinylphenol and 4-vinylguaiacol are abundant pyrolysis products that can be derived from both lignin (H and G residues, respectively) and hydroxycinnamic acids (p-coumarate and ferulate, respectively). Based on the pyrograms, only the midribs of the bm4 mutant showed a reduction in 4-vinylphenol, 35% less than the wild type. By contrast, the levels of 4-vinylguaiacol were reduced in the bm2 and bm4 mutants, and dramatically further in the bm2-bm4 double mutant. Trans-methylated midrib samples were analysed with Py-GC-MS in order to determine to what extent these changes were attributable to changes in the levels of hydroxycinnamic acids. This indicated that both the bm4 and the bm2-bm4 samples contained 30% less (of the dimethyl ester of) p-coumarate than the wild type, whereas the bm2 sample did not differ from the wild type. These data are consistent with the increase in H-derived compounds observed in the bm2-bm4 mutant. The level of (the dimethyl ester of) ferulate was reduced by 10, 30, and 40% in the bm2, bm4, and bm2-bm4 mutants, respectively, which largely explains the reductions in 4-vinylguaiacol.

Table 2.
Chemical composition of dissected midribs from leaf 6 of wild-type, bm2, bm4 and bm2-bm4 plants obtained with Py-GC-MS
Table 3.
P-values for pair-wise comparisons among genotypes for chemical composition
Fig. 4.
For each genotype the average value of the measured chemical composition was calculated. Each compound's average across genotypes was subtracted from the genotypic mean to centre the data. A heatmap based on a hierarchical cluster analysis performed on ...

Given the considerable pyrolytic degradation of fragments derived from cell wall carbohydrates, cellulose content was determined via wet chemical analysis. No significant variation in cellulose content was observed among the different genotypes (Table 2). Given the decrease in Klason lignin and the increase in pentose-derived pyrolysis fragments, the cell walls of the mutants must contain more hemicellulosic polysaccharides that compensate for the reduction in lignin.

The similarities in chemical changes in the bm2 and bm4 single mutants as determined by Py-GC-MS, and the apparent additive effects on Klason lignin could be the result of both mutations affecting the same metabolic or developmental process(es) independently. If Bm2 and Bm4 act sequentially in a linear process, the double mutant would reflect the phenotype associated with a mutation in the upstream gene. The score plot resulting from a linear discriminant analysis of the pyrolysis data (Fig. 5) shows that all four genotypes can be clearly separated from each other based on the chemical composition of their midribs. This suggests that Bm2 and Bm4 have different functions that are not part of a linear pathway or process.

Fig. 5.
Discriminant function score plot displaying the different genotypes based on the chemical composition of the cell wall based on Py-GC-MS. Canonical 1 explains 83% of the variance; Canonical 2 explains 12% of the variance. The dark dots represent the three ...

Does variation in cell wall composition translate into ultrastructural variation?

Scanning electron microscopy was performed to determine how the observed changes in cell wall composition affected the ultrastructure of the cell walls in the vascular tissue of the midrib. Figure 6 shows representative cross-sections of the midrib in the different genotypes. The most striking difference can be observed in the sclerenchyma, which is the supportive tissue between the xylem and the epidermis. The walls of the sclerenchyma cells are much thicker in the mutants, particularly in the double mutant, compared to the wild type. Higher magnification (×1000) images clearly show that the secondary wall no longer adheres to the primary wall. While this could be a result of the sectioning as opposed to being reflective of the in vivo situation, it does indicate that the physical characteristics of the sclerenchyma in the double mutant are very different from those of the wild type. A quantitative analysis was performed to assess variation in cell wall thickness between the different genotypes. The results of this analysis are shown in Table 4. This table does not include the double mutant because, due to the loss of adherence, it was not possible to make accurate measurements of wall thickness. It is apparent that the cell walls of the bm2 and bm4 mutant are statistically significantly different (P <0.001), and, on average, 2.3 and 1.8 times, respectively, thicker than the wild-type sclerenchyma walls.

Table 4.
Cell wall thickness of sclerenchyma cells and surface area of the xylem vessels (mean and standard deviation) measured in SEM images from wild-type, bm2, bm4, and bm2-bm4 plants
Fig. 6.
SEM images of cross sections through the midrib. (A, E) Wild type, (B, F) bm2, (C, G) bm4, and (D, H) bm2-bm4. (A–D) represent overview images, whereas (E–H) represent close-up views of the sclerenchyma. The magnification in images (A–C) ...

Changes in the size of the xylem vessels were evaluated by measuring individual xylem vessel area in cross-sections from the different genotypes (Table 4). The area of xylem vessels in the bm2 mutant was smaller, but not statistically significantly different from the wild-type xylem vessels. The area of the xylem vessels in the bm4 mutant was significantly larger. By contrast, the bm2-bm4 double mutant had xylem vessels that were significantly smaller than the wild type and the two single mutants. In addition, a ‘stringy’ material can be observed on the cut surface of the vascular tissue in the double mutant. The chemical nature of this material is unknown, but its appearance may be a result of the chemical differences described above.

Discussion

Defining the relationship between the structure and function of complex polymers is important in understanding the chemical basis for physical properties. This can provide insights into how species evolved and diverged, and is of help in attempts to improve field performance of crop plants (resistance to biotic and abiotic stress), and to tailor the composition of biological materials to enhance down-stream applications through the use of genetic modification (Chen and Dixon, 2007; Vermerris et al., 2007; Saballos et al., 2008).

The Klason lignin and pyrolysis analyses indicate that the bm2 and bm4 mutations primarily affect the cell wall composition of the leaves, as opposed to the stems and thus act in a tissue-specific manner. This explains why the chemical composition of the stalks of these two mutants was similar to that of the wild-type control, in sharp contrast to what was observed for the bm1 and bm3 mutants (Marita et al., 2003; Barrière et al., 2004). The leaf-specific effects of the bm2 and bm4 mutations also explain why the yield of fermentable sugars obtained from enzymatic saccharification of bm2 and bm4 stover, which is composed primarily of stem tissue, was not significantly different from that of wild-type stover (Vermerris et al., 2007), and why there is no brown coloration in the vascular tissue of the stem.

The SEM images reveal significant changes in ultrastructure in both the xylem and the sclerenchyma cells, with the increase in cell-wall thickness of the sclerenchyma in all three mutants being unexpected. A number of mutants and transgenic plants in which lignin biosynthetic genes were down-regulated had very thin secondary walls that could not withstand the negative pressure in the xylem. Consequently, the xylem cells collapsed (Piquemal et al., 1998; Jones et al., 2001; Franke et al., 2002; Day et al., 2009). Down-regulation of both the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) genes in tobacco resulted in thinner and more irregular xylem walls (Chabannes et al., 2001), and mutations in the sorghum genes encoding cinnamyl alcohol dehydrogenase2 (Saballos et al., 2009; Sattler et al., 2009) and caffeic acid O-methyltransferase (Bout and Vermerris, 2003) were shown to result in thinner sclerenchyma cell walls (Palmer et al., 2008). The difference between these studies and the data reported here could be the result of differences between species, the impact of changes in gene expression on specific tissues (e.g. sclerenchyma versus xylem vessels), or specific changes in cell wall composition.

There are several possible explanations for the increased cell wall thickness in the sclerenchyma. One possibility is that the reduction in lignin content has caused the cell walls to be more water-permeable. The cell wall carbohydrates, which are hydrophilic in nature, may have swollen as a result of the absorption of water. The lack of adherence of the cell walls may also be a result of the swelling. This effect may be enhanced by the apparent increase in the content of hemicellulosic polysaccharides. Alternatively, the reduction in ferulic acid levels in the walls of the mutants may reduce the degree of crosslinking between lignin and glucuronoarabinoxylans, resulting in a less compact wall structure that is not as well adhered to the primary wall.

The increase in the content of hemicellulosic polysaccharides may reflect the existence of a compensatory mechanism that the plant activates in response to a reduction in lignin content or certain changes in lignin composition. The presence of compensatory mechanisms was proposed by Hu et al. (1999) based on observations of poplar plants in which lignin content had been reduced as a result of down-regulation of the 4-coumarate-CoA ligase (4CL) gene. In these plants the reduction in lignin content resulted in a concomitant increase in cellulose content. Furthermore, the rice brittle culm1 mutant, which has reduced cellulose content, was shown to contain more lignin based on staining with phloroglucinol and Klason lignin determination (Li et al., 2003). Similar observations were reported for the maize brittle stalk2 mutant (Ching et al., 2006; Sindhu et al., 2007). The latter study also provided evidence for an increase in GAX in response to a reduction in cellulose. These studies, combined with the results presented here, provide evidence for an interdependence between the different cell wall polymers.

The altered structure of the midrib in the bm mutants is likely to impact the function of the conducting tissues in terms of both water and mineral transport. The drought symptoms and reduced growth of the bm2-bm4 mutant is probably the result of the reduced diameter of the xylem vessels, which not only limits the flux, but also requires a higher pressure to transport xylem sap. The reduced hydrophobicity as a result of the lower lignin content further exacerbates these effects, as hydraulic properties of xylem tissue are influenced by the degree of lignification and mineral content of the xylem sap (Zwieniecki et al., 2001; Boyce et al., 2004). By extrapolation, the changes in xylem vessel structure in the bm1-bm2-bm4 mutant may be even more extreme, thereby completely preventing normal growth. An additional factor that may play a role is variation in torsion resulting from torque applied by the wind. Thin-walled hollow tubes experience less torsion than a solid cylinder of the same dimensions. The thin-walled sclerenchyma cells of the wild type may experience less torsion than the thick-walled sclerenchyma cells of the bm2-bm4 mutant. Increased torsion in the double mutant may stress or damage adjoining cells, hence compromising cell function and ultimately growth and development. These hypotheses warrant further investigations by follow-up biophysical studies, ideally in a way that also allows separation of effects of the mutations on the sclerenchyma versus xylem vessels.

Based on the combined data presented here, it is apparent that variation in cell wall composition affects the physical parameters of the secondary cell walls in sclerenchyma tissue, with reductions in G- and S-content and a concomitant increase in hemicellulosic polysaccharides leading to thicker walls. The changes in chemical composition of the bm2-bm4 double mutant, which may be a result of fundamental changes in gene expression regulation (Guillaumie et al., 2007), are severe enough to compromise growth and development. The addition of the bm1 mutation to create a triple mutant bm1-bm2-bm4 resulted in a genotype in which this limit was apparently exceeded as evidenced by the failure to develop to a point where tissue could be sampled for chemical analysis. Given that all of the other bm double mutants and the triple mutants that do not contain bm2 and bm4 are appreciably more vigorous, these observations demonstrate that there is a limit to the metabolic plasticity of cell wall composition which should be taken into consideration when engineering plants for down-stream processing.

Supplementary data

Supplementary data can be found at JXB online.

Supplementary Table S1. Height measurements in the wild type (inbred A619) and all single and double bm mutants.

Supplementary Material

[Supplementary Data]

Acknowledgments

The authors thank William Foster and Phil Devillez for excellent assistance in the field, Javier Campos and Stephany Esposito for assistance with the lignin analyses, and Drs Nicholas Carpita, Michael Ladisch, and Paul Sisco for their support. Part of this research was supported by The Consortium for Plant Biotechnology Research, Inc. by DOE Prime Agreement No. DEFG36-02GO12026 (to WV). This support does not constitute an endorsement by DOE or by The Consortium for Plant Biotechnology Research, Inc. of the views expressed in this publication. Additional financial support from the National Science Foundation Plant Genome Research Project 0821954 (to LMM), the Showalter Foundation, Dow AgroSciences, and the University of Florida is gratefully acknowledged.

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