Scott E. Sattler

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination.

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.

Characterization of Tocopherol Cyclases from Higher Plants and Cyanobacteria. Evolutionary Implications for Tocopherol Synthesis and Function

Tocopherols are lipophilic antioxidants synthesized exclusively by photosynthetic organisms and collectively constitute vitamin E, an essential nutrient for both humans and animals. Tocopherol cyclase (TC) catalyzes the conversion of various phytyl quinol pathway intermediates to their corresponding tocopherols through the formation of the chromanol ring. Herein, the molecular and biochemical characterization of TCs from Arabidopsis (VTE1 [VITAMIN E 1]), Zea mays (SXD1 [Sucrose Export Deficient 1]) and Synechocystis sp. PCC6803 (slr1737) are described. Mutations in the VTE1, SXD1, or slr1737 genes resulted in both tocopherol deficiency and the accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), a TC substrate. Recombinant SXD1 and VTE1 proteins are able to convert DMPBQ to γ-tocopherol in vitro. In addition, expression of maize SXD1 in a Synechocystis sp. PCC6803 slr1737 knockout mutant restored tocopherol synthesis, indicating that TC activity is evolutionarily conserved between plants and cyanobacteria. Sequence analysis identified a highly conserved 30-amino acid C-terminal domain in plant TCs that is absent from cyanobacterial orthologs. vte1-2 causes a truncation within this C-terminal domain, and the resulting mutant phenotype suggests that this domain is necessary for TC activity in plants. The defective export of Suc in sxd1 suggests that in addition to presumed antioxidant activities, tocopherols or tocopherol breakdown products also function as signal transduction molecules, or, alternatively, the DMPBQ that accumulates in sxd1 disrupts signaling required for efficient Suc export in maize.

Highly Divergent Methyltransferases Catalyze a Conserved Reaction in Tocopherol and Plastoquinone Synthesis in Cyanobacteria and Photosynthetic Eukaryotes

Tocopherols are lipid-soluble compounds synthesized only by photosynthetic eukaryotes and oxygenic cyanobacteria. The pathway and enzymes for tocopherol synthesis are homologous in cyanobacteria and plants except for 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ MT), which catalyzes a key methylation step in both tocopherol and plastoquinone (PQ) synthesis. Using a combined genomic, genetic, and biochemical approach, we isolated and characterized the VTE3 (vitamin E defective) locus, which encodes MPBQ/MSBQ MT in Arabidopsis. The phenotypes of vte3 mutants are consistent with the disruption of MPBQ/MSBQ MT activity to varying extents. The ethyl methanesulfonate–derived vte3-1 allele alters tocopherol composition but has little impact on PQ levels, whereas the null vte3-2 allele is deficient in PQ and α- and γ-tocopherols. In vitro enzyme assays confirmed that VTE3 is the plant functional equivalent of the previously characterized MPBQ/MSBQ MT (Sll0418) from Synechocystis sp PCC6803, although the two proteins are highly divergent in primary sequence. Sll0418 orthologs are present in all fully sequenced cyanobacterial genomes, Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana but absent from vascular and nonvascular plant databases. VTE3 orthologs are present in all vascular and nonvascular plant databases and in C. reinhardtii but absent from cyanobacterial genomes. Intriguingly, the only prokaryotic genomes that contain VTE3-like sequences are those of two species of archea, suggesting that, in contrast to all other enzymes of the plant tocopherol pathway, the evolutionary origin of VTE3 may have been archeal rather than cyanobacterial. In vivo analyses of vte3 mutants and the corresponding homozygous Synechocystis sp PCC6803 sll0418::aphII mutant revealed important differences in enzyme redundancy, the regulation of tocopherol synthesis, and the integration of tocopherol and PQ biosynthesis in cyanobacteria and plants.

Nonenzymatic Lipid Peroxidation Reprograms Gene Expression and Activates Defense Markers in Arabidopsis Tocopherol-Deficient Mutants

Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels

This review focuses on the potential advantages and disadvantages of forages such as switchgrass (Panicum virgatum), and two small grains: sorghum (Sorghumbicolor), and wheat (Triticum aesitvum), as feedstocks for biofuels. It highlights the synergy provided by applying what is known from forage digestibility and wheat and sorghum starch properties studies to the biofuels sector. Opportunities therefore, exist to improve biofuel qualities in these crops via genetics and agronomics. In contrast to cereal crops, switchgrass still retains tremendous exploitable genetic diversity, and can be specifically improved to fit a particular agronomic, management, and conversion platform. Combined with emerging studies on switchgrass genomics, conversion properties and management, the future for genetic modification of this species through conventional and molecular breeding strategies appear to be bright. The presence of brown-midrib mutations in sorghum that alter cell wall composition by reducing lignin and other attributes indicate that sorghum could serve as an important model species for C4-grasses. Utilization of the brown-midrib traits could lead to the development of forage and sweet sorghums as novel biomass crops. Additionally, wheat crop residue, and wheat and sorghum with improved starch content and composition represent alternate biofuel sources. However, the use of wheat starch as a biofuel is unlikely but its value as a model to study starch properties on biofuel yields holds significant promise.

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.

Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum.

Sorghum (Sorghum bicolor (L.). Moench) BMR-6 and BMR-12 encode cinnamylalcohol dehydrogenase and caffeic acid-O-methyltransferase, respectively. We have evaluated the impact of two bmr alleles, bmr-6 and bmr-12, respectively, on soluble and wall-bound aromatics in near isogenic, wild-type (WT), bmr-6, bmr-12 and double-mutant (DM; bmr-6 and bmr-12) plants in two genetic backgrounds, RTx430 and Wheatland. Immunoblots confirmed that COMT protein was essentially absent in bmr-12 and DM plants, but was present in bmr-6 and WT plants. In contrast, although CAD activity was not detected in bmr-6 and DM plants, proteins crossreacting to anti-CAD sera were found in stem extracts from all genotypes. In both sorghum backgrounds, WT plants had lowest amounts of free aromatics, higher levels of cell wall-bound pCA and FA esters and guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) lignins. Soluble aromatics and cell wall phenolic ester content in Wheatland DM plants resembled that of Wheatland bmr-6 plants, whereas in the RTx430 background, levels of these components in the DM plants more closely resembled those observed in bmr-12 plants. In both backgrounds, bmr-6 plants exhibited reduced levels of G, S, and H lignins relative to WT, and increased incorporation of G-indene into lignin. In bmr-12 plants, there was greater incorporation of G- and 5-hydroxyguaiacyl (5-OHG) lignin into cell walls. Histochemical staining of internode sections from Wheatland plants indicated that apparent lignification of cortical sclerenchyma and vascular bundle fibers was greatest and most uniform in WT plants. Relative staining intensity of these tissues was decreased in bmr-6, followed by bmr-12 plants. DM plants exhibited poor staining of cortical sclerenchyma and vascular bundle fibers.

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.

Modifying lignin to improve bioenergy feedstocks: strengthening the barrier against pathogens?

Lignin is a ubiquitous polymer present in cell walls of all vascular plants, where it rigidifies and strengthens the cell wall structure through covalent cross-linkages to cell wall polysaccharides. The presence of lignin makes the cell wall recalcitrant to conversion into fermentable sugars for bioenergy uses. Therefore, reducing lignin content and modifying its linkages have become major targets for bioenergy feedstock development through either biotechnology or traditional plant breeding. In addition, lignin synthesis has long been implicated as an important plant defense mechanism against pathogens, because lignin synthesis is often induced at the site of pathogen attack. This article explores the impact of lignin modifications on the susceptibility of a range of plant species to their associated pathogens, and the implications for development of feedstocks for the second-generation biofuels industry. Surprisingly, there are some instances where plants modified in lignin synthesis may display increased resistance to associated pathogens, which is explored in this article.

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.