Ana Saballos

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.

Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench).

Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. The conversion of cellulose to monomeric sugar units is affected by lignin content and composition. Sorghum has attracted the attention of the scientific and industrial community as a promising source of biomass for bioenergy due to its great yield potential and tolerance to stresses. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue associated with altered lignin content. Twenty-eight bmr mutants have been identified since the late 1970s, but the allelic relationships have not been fully established, and the function of only one of the Bmr loci has been unequivocally established. In this study, we combined genetic and chemical approaches to establish that there are mutations at least four independent bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 groups. Since each allelic group presents unique staining characteristics, rapid classification of emerging bmr lines into the existing groups can be achieved using phloroglucinol-HCl as a histochemical stain. In addition, pyrolysis-gas chromatography-mass spectrometry, enabled the characterization of changes in subunit lignin composition in each of the allelic groups, to help predict the genes underlying the mutations. Enzymatic saccharification of stover from plants representing each allelic bmr group demonstrated that lignin changes in lines belonging to the bmr2, bmr6 and bmr12 groups can increase glucose yields, up to 25% compared to wild-type isolines. In order to expedite the selection of the bmr mutant alleles in breeding populations, we have developed molecular markers specific for bmr7 and bmr25, two novel mutant alleles of the gene encoding caffeic acid O-methyl transferase. Based on the results from this study, we propose to rename the bmr mutants in a manner that reflects the number of independent loci.

Brown midrib2 (Bmr2) encodes the major 4-coumarate:coenzyme A ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench)

Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation.

Development and Utilization of Sorghum as a Bioenergy Crop

Sorghum is not a newcomer crop. World-wide, it is the fifth most important cereal crop, after wheat rice, corn, and barley. Sorghum is unique in that is can grow in some of the harshest environments, where many other crops cannot be produced. It is highly resistant to drought, but can also withstand water logging much better than most of the other cereals. Sorghum’s large root system allows it to obtain nutrients from poor soils or, when fertilizers are applied, to use those nutrients very efficiently. Even though the species originated from semi-arid regions of Africa, it has been adapted to a wide variety of climates, including temperate and humid environments. Ironically, it may have been its superior tolerance to the marginal environments, common in many developing countries that hindered its wider adoption in the developed countries, as sorghum was labeled a poor-man’s crop. Additional factors that contributed to an underutilization of sorghum were the lower nutritional value of the grain and the lower yield compared to corn under good growing conditions. Although breeding efforts have produced dramatic improvements in grain and forage sorghum varieties, the amount of resources invested in the study of the crop have traditionally been minuscule compared to those invested in many other species. As a consequence, most of the abundant genetic diversity of cultivated and wild sorghums remains untapped. The renewed interest in biofuel production offers the possibility to exploit the useful traits of this species. Sorghum represents an ideal dedicated energy crop, as grain quality is now considered less important, and concerns about the environmental cost of high-input agriculture and food security grow. The lower need for fertilizers and pesticides may make it an ecologically attractive crop, especially when combined with conservation-agriculture production systems.

Identification and characterization of four missense mutations in brown midrib 12 (Bmr12), the caffeic O-methyltranferase (COMT) of sorghum.

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.

Characterization of Novel Sorghum brown midrib Mutants from an EMS-Mutagenized Population

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.

Genetic Enhancement of Sorghum for Biomass Utilization

Biomass produced from sorghum can be utilized as forage and silage to feed ruminant animals and as feedstock for biofuels and bio-based products. The efficiency of biomass utilization is a function of biomass composition and plant architecture. This chapter provides a description of the cell wall polymers that make up the bulk of sorghum biomass, along with information on the genes involved in their biosynthesis. The close evolutionary relationships among the grasses makes it possible to infer gene function across species. Newly developed genomics and bioinformatics resources offer exciting opportunities for the genetic enhancement of sorghum as a biomass crop.

Using Genotyping by Sequencing to Map Two Novel Anthracnose Resistance Loci in Sorghum bicolor

Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar ‘Bk7’, a biparental mapping population of F3:4 and F4:5 sorghum lines was generated by crossing ‘Bk7’ with the susceptible inbred ‘Early Hegari-Sart’. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from ‘Bk7’. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. Genetic analysis following an independent selection experiment of lines derived from a cross between ‘Bk7’ and sweet sorghum ‘Mer81-4’ narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases.

QTLs Associated with Crown Root Angle, Stomatal Conductance, and Maturity in Sorghum

QTLs for crown root angle, stomatal conductance, and maturity were identified in two field studies through the construction of a high‐density bin map. The QTL for stomatal conductance was associated with reduced leaf transpiration but not reduced net assimilation rate. Candidate genes are proposed based on the physical location of the QTLs and the function of known genes in those locations.