Jeffrey F. Pedersen

A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype

brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.

Structure and Functional Properties of Sorghum Starches Differing in Amylose Content

Starches were isolated from grains of waxy, heterowaxy, and normal sorghum. To study the relationship between starch structure and functionality and guide applications of these starches, amylose content, amylopectin chain-length distributions, gelatinization and retrogradation, pasting properties, dynamic rheological properties, and in vitro enzyme digestion of raw starches were analyzed. Heterowaxy sorghum starch had intermediate amylose content, pasting properties, and dynamic rheological properties. Stress relaxation was a useful indicator of cooked starch cohesiveness. Cooked heterowaxy sorghum starch (10% solids) had a viscoelastic-solid type of character, whereas cooked waxy sorghum starch behaved like a viscoelastic liquid. Amylopectin of normal sorghum starch had a slightly higher proportion of chains with degree of polymerization (DP) of 6-15 (45.5%) compared with amylopectin of heterowaxy starch (44.1%), which had a gelatinization peak temperature 2 degrees C higher than normal sorghum starch. Heterowaxy sorghum starch contained significantly lower rapidly digestible starch (RDS) and higher resistant starch (RS) than waxy sorghum starch.

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.

Association Mapping for Grain Quality in a Diverse Sorghum Collection

Knowledge of the genetic bases of grain quality traits will complement plant breeding efforts to improve the end‐use value of sorghum [Sorghum bicolor (L.) Moench]. Candidate gene association mapping was used on a diverse panel of 300 sorghum accessions to assess marker–trait associations for 10 grain quality traits measured using the single kernel characterization system (SKCS) and near‐infrared reflectance spectroscopy (NIRS). The analysis of the accessions through 1290 genomewide single nucleotide polymorphisms (SNPs) separated the panel into five subpopulations that corresponded to three major sorghum races (durra, kafir, and caudatum), one intermediate race (guinea‐caudatum), and one working group (zerazera‐caudatum). These subpopulations differed in kernel hardness, acid detergent fiber, and total digestible nutrients. After model testing, association analysis between 333 SNPs in candidate genes and/or loci and grain quality traits resulted in eight significant marker–trait associations. A SNP in starch synthase IIa (SSIIa) gene was associated with kernel hardness (KH) with a likelihood ratio‐based R2 (RLR2) value of 0.08, a SNP in starch synthase (SSIIb) gene was associated with starch content with an RLR2 value of 0.10, and a SNP in loci pSB1120 was associated with starch content with an RLR2 value of 0.09.

Evaluation of the single kernel characterization system (SKCS) for measurement of sorghum grain attributes

ABSTRACT The single kernel characterization system (SKCS) has been widely used in the wheat industry, and SKCS parameters have been linked to end-use quality in wheat. The SKCS has promise as a tool for evaluating sorghum grain quality. However, the SKCS was designed to analyze wheat, which has a different kernel structure from sorghum. To gain a better understanding of the meaning of SKCS predictions for grain sorghum, individual sorghum grains were measured for length, width, thickness (diameter), and weight by laboratory methods and by the SKCS. SKCS predictions for kernel weight and thickness were highly correlated to laboratory measurements. However, SKCS predictions for kernel thickness were underestimated by ≈20%. The SKCS moisture prediction for sorghum was evaluated by tempering seven samples with varying hardness values to four moisture levels. The moisture contents predicted by SKCS were compared with a standard oven method and, while correlated, SKCS moisture predictions were less than moistur...

In vitro starch disappearance procedure modifications

Four in vitro experiments evaluated the effects of ruminal fluid inoculum:artificial saliva ratios, grinder type, grind size, and diet of ruminal fluid donor on in vitro starch disappearance. Experiment 1 examined rates of starch disappearance and coefficients of determination obtained by linear regression of starch disappearance using five grain sorghum (Sorghum bicolor (L.) Moench) lines, a corn (Zea mays L.) control, and a wheat (Triticum aestivum L.) control. Grains were incubated for 4, 8, 12, 16, and 20 h with inoculum varying in proportion of ruminal fluid and artificial saliva (1:1, 1:2, 1:3, and 1:4). In vitro rates of starch disappearance and coefficients of determination were similar for the 1:2, 1:3, and 1:4 inoculum dilutions and were higher (quadratic, P 0.10) by grain type fed; however, absolute rates of digestion varied among inoculate sources. Grinder, grind size, and ruminal fluid inoculum:artificial saliva ratios affected rate of starch disappearance in samples digested in vitro. Diet of ruminal fluid donor affected the rate of starch digestion, but not the relative ranking of the grains. If rates are to be compared across in vitro runs for different grains, these processing and dietary factors must be kept constant.

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.

Alteration in Lignin Biosynthesis Restricts Growth of Fusarium spp. in Brown Midrib Sorghum

To improve sorghum for bioenergy and forage uses, brown midrib (bmr)6 and -12 near-isogenic genotypes were developed in different sorghum backgrounds. The bmr6 and bmr12 grain had significantly reduced colonization by members of the Gibberella fujikuroi species complex compared with the wild type, as detected on two semiselective media. Fusarium spp. were identified using sequence analysis of a portion of the translation elongation factor (TEF) 1-alpha gene. The pathogens Fusarium thapsinum, F. proliferatum, and F. verticillioides, G. fujikuroi members, were commonly recovered. Other frequently isolated Fusarium spp. likely colonize sorghum asymptomatically. The chi(2) analyses showed that the ratios of Fusarium spp. colonizing bmr12 grain were significantly different from the wild type, indicating that bmr12 affects colonization by Fusarium spp. One F. incarnatum-F. equiseti species complex (FIESC) genotype, commonly isolated from wild-type and bmr6 grain, was not detected in bmr12 grain. Phylogenetic analysis suggested that this FIESC genotype represents a previously unreported TEF haplotype. When peduncles of wild-type and near-isogenic bmr plants were inoculated with F. thapsinum, F. verticillioides, or Alternaria alternata, the resulting mean lesion lengths were significantly reduced relative to the wild type in one or both bmr mutants. This indicates that impairing lignin biosynthesis results in reduced colonization by Fusarium spp. and A. alternata.

Reaction of Sorghum Lines Genetically Modified for Reduced Lignin Content to Infection by Fusarium and Alternaria spp.

Two genes conferring the brown midrib (bmr) trait had been backcrossed into six elite sorghum lines, resulting in reduced lignin in the bmr lines when compared with the wild-type parent. Seed and leaf tissue from field-grown plants, planted at two locations, were screened for Alternaria spp. and Fusarium spp. on semi-selective media. The results suggest that bmr lines do not have increased susceptibility to colonization by Alternaria spp. However, significantly fewer colonies of Fusarium spp., including Fusarium moniliforme, were recovered from seed of reduced lignin lines from two genetic backgrounds. That the bmr trait in some genetic backgrounds might enable increased resistance to colonization by F. moniliforme was further supported by greenhouse experiments in which peduncles of developing heads were inoculated with F. moniliforme. Mean lesion measurements on bmr lines were significantly lower than those resulting from inoculations on wild-type lines. Analysis of near-isogenic lines revealed that mean lesion lengths on bmr lines were significantly less than those produced on their wild-type counterparts in four of the six genetic backgrounds. These results suggest that reduced lignin lines exhibit, in some cases, increased resistance to Fusarium spp., including F. moniliforme.

Characterization of waxy grain sorghum lines in relation to granule-bound starch synthase

The waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in sorghum (Sorghum bicolor L. Moench) since 1933. Although variants of the waxy gene are well characterized in other cereals, the waxy trait has been assumed to be controlled by a single allele, wx, in sorghum. Recent improvements in technologies encourage re-examination of the waxy sorghums. The objectives of this research were therefore to identify and characterize sorghum lines with differing waxy alleles and to describe the actions of those alleles in crosses. Grain of eight waxy sorghum lines (BTxARG1, BTx630, Tx2907, B.9307, 94C274, 94C278, 94C289, 94C369), three wild-type checks (BWheatland, RTx430, BN122), and F2 families from crosses among a subset of these lines were evaluated for presence or absence of granule-bound starch synthase (GBSS), the gene product of the wx locus, and wild-type vs. waxy endosperm. The F2 segregation ratios were tested for fit to a 3:1 ratio using Chi-square analyses. Two distinctly different naturally occurring waxy alleles were identified: One with no GBSS (GBSS−), and one with apparently inactive GBSS present (GBSS+). We propose that the waxy allele with no GBSS be designated wxa, and that waxy allele with apparently inactive GBSS present be designated wxb. These two alleles are located in close proximity on the waxy locus. The wxb allele is dominant to the wxa allele in terms of GBSS production, and both are recessive to the wild-type Wx in terms of amylose content.