Evidence for an evolutionarily conserved interaction between cell wall biosynthesis and flowering in maize and sorghum

Background  

Factors that affect flowering vary among different plant species, and in the grasses in particular the exact mechanism behind this transition is not fully understood. The brown midrib (bm) mutants of maize (Zea mays L.), which have altered cell wall composition, have different flowering dynamics compared to their wild-type counterparts. This is indicative of a link between cell wall biogenesis and flowering. In order to test whether this relationship also exists in other grasses, the flowering dynamics in sorghum (Sorghum bicolor (L.) Moench) were investigated. Sorghum is evolutionarily closely related to maize, and a set of brown midrib (bmr) mutants similar to the maize bm mutants is available, making sorghum a suitable choice for study in this context.     

Genetic and physical fine mapping of the novel brown midrib gene bm6 in maize (Zea mays L.) to a 180?kb region on chromosome 2

Brown midrib mutants in maize are known to be associated with reduced lignin content and increased cell wall digestibility, which leads to better forage quality and higher efficiency of cellulosic biomass conversion into ethanol. Four well known brown midrib (bm) mutants, named bm1-4, were identified several decades ago. Additional recessive brown midrib mutants have been identified by allelism tests and designated as bm5 and bm6. In this study, we determined that bm6 increases cell wall digestibility and decreases plant height. bm6 was confirmed onto the short arm of chromosome 2 by a small mapping set with 181 plants from a F(2) segregating population, derived from crossing B73 and a bm6 mutant line. Subsequently, 960 brown midrib individuals were selected from the same but larger F(2) population for genetic and physical mapping. With newly developed markers in the target region, the bm6 gene was assigned to a 180?kb interval flanked by markers SSR_308337 and SSR_488638. In this region, ten gene models are predicted in the maize B73 sequence. Analysis of these ten genes as well as genes in the syntenic rice region revealed that four of them are promising candidate genes for bm6. Our study will facilitate isolation of the underlying gene of bm6 and advance our understanding of brown midrib gene functions.     

Genetic analyses of a putative Mutator-induced deletion involving the blue fluorescent1 (bf1) and brown midrib4 (bm4) loci in maize.

A putative Mutator-induced alteration (isolate) involving the blue fluorescent1 (Bf1) and the brown midrib4 (bm4) loci is described. This isolate (Bf1-Mu-044-4) was one of the blue fluorescent1 mutants in a large experiment designed to produce Mutator-induced mutations involving this locus. Evidence is presented supporting the conclusion that this alteration deletes all or part of both the bf1 and bm4 loci. The isolate is infrequently transmitted through the pollen, but is more readily transmitted through the egg, although at less than the expected frequency of 50% for a simple mutation. Heterozygotes of this isolate with the bm4-R allele have brown midribs. These brown midrib plants are shorter than homozygous bm4-R plants and approximately 50% of these Bf1-Mu-044-4/bm4-R heterozygotes do not reach maturity. One putative Mutator-induced bm4 mutation, which occurred in the course of the analysis of the Bf1-Mu-044 isolate, is described. Key words : Mutator, deletion, bf1-bm4 loci.     

The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase.

The brown midrib mutations are among the earliest described in maize. Plants containing a brown midrib mutation exhibit a reddish brown pigmentation of the leaf midrib starting when there are four to six leaves. These mutations are known to alter lignin composition and digestibility of plants and therefore constitute prime candidates in the breeding of silage maize. Here, we show that two independent brown midrib3 (bm3) mutations have resulted from structural changes in the COMT gene, which encodes the enzyme O-methyltransferase (COMT; EC 2.1.1.6), involved in lignin biosynthesis. Our results indicate that the bm3-1 allele (the reference mutant allele) has arisen from an insertional event producing a COMT mRNA altered in both size and amount. By sequencing a COMT cDNA clone obtained from bm3-1 maize, a retrotransposon with homology to the B5 element has been found to be inserted near the junction of the 3' coding region of the COMT gene intron. The second bm3 allele, bm3-2, has resulted from a deletion of part of the COMT gene. These alterations of the COMT gene were confirmed by DNA gel blot and polymerase chain reaction amplification analyses. These results clearly demonstrate that mutations at the COMT gene give a brown midrib3 phenotype. Thus, the gene genetically recognized as bm3 is the same as the one coding for COMT.     

A candidate-gene approach to clone the sorghum <Emphasis Type="Italic">Brown midrib </Emphasis>gene encoding caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.     

The influence of ageing on cell wall composition and degradability of three maize genotypes

Stem segments of the maize (Zea mavs L.) hybrids LGH, Eta Ipho (EI) and a brown midrib mutant. INRA 260 bm3 (bm3) were freeze-dried, ground and analysed for cell wall content, hemicellulose, cellulose, lignin and in vitro cell wall degradability with rumen fluid. Stem cross-sections, stained with acid phloroglucinol (AP) and chlorine sulphite (CS) showed an increased intensity in staining during maturation, but no considerable difference in staining intensity was observed between genotypes. The lignin content increased during maturation with evidently less lignin in bm3 than in EI and LGH. However, cell wall degradability did not differ between the older stem segments of EI and bm3, although the amount of lignin in LGH was twice that of bm3.

It can be concluded that an increase in lignin content occurs simultancously with a decrease in cell wall degradability within a genotype. However, between different genotypes the lignin content is not an indicator of degree of cell wall degradability.     

Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants

The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.     

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.     

Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2K b mutants

Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bml and bm3 mutant mice that were not lytic for virus-infected targets expressing the K^b and D^b MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to K^bm1 and K^bm3, rather than to D^b. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bm3 and bmll mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bm3 are the most cross-reactive pair in their presentation of vaccinia. The bm1 and bm10 pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bm1 and bm3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, K^bm1 and K^bm10 differ for three amino acids, but bm1 T cells are highly lytic for bm10 virus-infected targets. However, there is no cross-reactivity for K^bm1 and K^b, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the K^bm1, K^bm10, and K^b glycoproteins.     

Mutations in the Sup-38 Gene of Caenorhabditis Elegans Suppress Muscle-Attachment Defects in Unc-52 Mutants

Mutations in the unc-52 locus of Caenorhabditis elegans have been classified into three different groups based on their complex pattern of complementation. These mutations result in progressive paralysis (class 1 mutations) or in lethality (class 2 and 3 mutations). The paralysis exhibited by animals carrying class 1 mutations is caused by disruption of the myofilaments at their points of attachment to the cell membrane in the body wall muscle cells. We have determined that mutations of this class also have an effect on the somatic gonad, and this may be due to a similar disruption in the myoepithelial sheath cells of the uterus, or in the uterine muscle cells. Mutations that suppress the body wall muscle defects of the class 1 unc-52 mutations have been isolated, and they define a new locus, sup-38. Only the muscle disorganization of the Unc-52 mutants is suppressed; the gonad abnormalities are not, and the suppressors do not rescue the lethal phenotype of the class 2 and class 3 mutations. The suppressor mutations on their own exhibit a variable degree of gonad and muscle disorganization. Putative null sup-38 mutations cause maternal-effect lethality which is rescued by a wild-type copy of the locus in the zygote. These loss-of-function mutations have no effect on the body wall muscle structure.     

Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification

Analysis of lignification in rice has been facilitated by the availability of the recently completed rice genome sequence, and rice will serve as an important model for understanding the relationship of grass lignin composition to cell wall digestibility. Cinnamyl-alcohol dehydrogenase (CAD) is an enzyme important in lignin biosynthesis. The rice genome contains 12 distinct genes present at nine different loci that encode products with significant similarity to CAD. The rice gene family is diverse with respect to other angiosperm and gymnosperm CAD genes isolated to date and includes one member (OsCAD6) that contains a peroxisomal targeting signal and is substantially diverged relative to other family members. Four closely related family members (OsCAD8A–D) are present at the same locus and represent the product of a localized gene duplication and inversion. Promoter-reporter gene fusions to OsCAD2, an orthologue of the CAD gene present at the bm1 (brown midrib 1) locus of maize, reveal that in rice expression is associated with vascular tissue in aerial parts of the plant and is correlated with the onset of lignification. In root tissue, expression is primarily in the cortical parenchyma adjacent to the exodermis and in vascular tissue.     

A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis . Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.     

EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

We have characterized Arabidopsis esd1 mutations, which cause early flowering independently of photoperiod, moderate increase of hypocotyl length, shortened inflorescence internodes, and altered leaf and flower development. Phenotypic analyses of double mutants with mutations at different loci of the flowering inductive pathways suggest that esd1 abolishes the FLC-mediated late flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. We found that ESD1 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of esd1 in a flc-3 null genetic background and the downregulation of other members of the FLC-like/MAF gene family in esd1 mutants suggest that flowering inhibition mediated by ESD1 occurs through both FLC-and FLC-like gene-dependent pathways. The ESD1 locus was identified through a map-based cloning approach. ESD1 encodes ARP6, a homolog of the actin-related protein family that shares moderate sequence homology with conventional actins. Using chromatin immunoprecipitation (ChIP) experiments, we have determined that ARP6 is required for both histone acetylation and methylation of the FLC chromatin in Arabidopsis.     

The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine‐mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu‐induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one‐carbon (C1) metabolism to polyglutamylate substrates of folate‐dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS‐deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild‐type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.     

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. IV. Self-determinants recognized by wild-type anti-H-2Kb and H-2Db-restricted cytotoxic T cells specific for sulfhydryl and amino-reactive haptens are absent in certain H-2 mutant strains

Virus-specific H-2-restricted cytotoxic T cells (CTL) have been found to discriminate between wild-type and mutant class I molecules. The only results reported concerning a hapten-self model, however, indicate that TNP-specific CTL do not discriminate between wild-type and mutant self determinants (7). In the present study, hapten-specific CTL generated against N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine-modified syngeneic cells (AED-self) were used to determine whether a hapten that is known to react with different cell surface sites than TNP can induce CTL that distinguish mutant H-2K and D molecules from those of wild type. The findings of this study indicate that H-2Kb-AED-self cytotoxic effector cells can discriminate between self-determinants of H-2Kb wild-type and the H-2bm1 and H-2bm11 mutants, but not between wild-type and the H-2bm6 and H-2bm9 mutants. H-2Db-AED-self effector cells were also found to discriminate between self-determinants of H-2Db wild-type and the H-2bm13 and H-2bm14 mutants. Furthermore, cold target competition experiments indicated that the bm1 and bm11 Kb products also lack some determinants recognized by anti-wild-type Kb TNP-specific CTL. These findings provide the first demonstration that hapten-self-specific effectors can detect alterations in H-2 mutant class I molecules. The results in the present report also support the hypothesis that haptens do not have to derivatize H-2 molecules in order to form antigens recognized by H-2-restricted CTL. These findings are discussed with respect to the involvement of self-determinants on MHC and non-MHC cell surface molecules.     

Arabidopsis late-flowering fve mutants are affected in both vegetative and reproductive development

The development of wild-type Arabidopsis thaliana (L.) Heyhn and two late-flowering fve mutants has been analysed under different environmental conditions. In wild-type plants, short-day photoperiods delay the floral transition as a consequence of lengthening all the developmental phases of the plant. Moreover, short days also alter the inflorescence structure by reducing the internode elongation and delaying the establishment of the floral developmental programme in the lateral meristems of the inflorescence and co-florescences. Mutations at the FVE locus cause a delay in flowering time, and a change in the inflorescence structure, similar to the effect of short photoperiods on wild-type plants. However, the effect of the fve mutations is additive to the effect of short days, and all the aspects of the Fve phenotype are corrected by vernalization. These results seem to indicate that FVE is not simply involved in timing the transition from vegetative to reproductive growth, but that it could play a role during all stages of plant development.