Eric J. Haas

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.

Eicosanoids: Exploiting Insect Immunity to Improve Biological Control Programs.

Insects, like all invertebrates, express robust innate, but not adaptive, immune reactions to infection and invasion. Insect immunity is usually resolved into three major components. The integument serves as a physical barrier to infections. Within the hemocoel, the circulating hemocytes are the temporal first line of defense, responsible for clearing the majority of infecting bacterial cells from circulation. Specific cellular defenses include phagocytosis, microaggregation of hemocytes with adhering bacteria, nodulation and encapsulation. Infections also stimulate the humoral component of immunity, which involves the induced expression of genes encoding antimicrobial peptides and activation of prophenoloxidase. These peptides appear in the hemolymph of challenged insects 6–12 hours after the challenge. Prostaglandins and other eicosanoids are crucial mediators of innate immune responses. Eicosanoid biosynthesis is stimulated by infection in insects. Inhibition of eicosanoid biosynthesis lethally renders experimental insects unable to clear bacterial infection from hemolymph. Eicosanoids mediate specific cell actions, including phagocytosis, microaggregation, nodulation, hemocyte migration, hemocyte spreading and the release of prophenoloxidase from oenocytoids. Some invaders have evolved mechanisms to suppress insect immunity; a few of them suppress immunity by targeting the first step in the eicosanoid biosynthesis pathways, the enzyme phospholipase A2. We proposed research designed to cripple insect immunity as a technology to improve biological control of insects. We used dsRNA to silence insect genes encoding phospholipase A2, and thereby inhibited the nodulation reaction to infection. The purpose of this article is to place our view of applying dsRNA technologies into the context of eicosanoid actions in insect immunity. The long-term significance of research in this area lies in developing new pest management technologies to contribute to food security in a world with a rapidly growing human population.

Switchgrass contains two cinnamyl alcohol dehydrogenases involved in lignin formation.

Lignin content of switchgrass (Panicum virgatum L.), a bioenergy species, is a critical determinant of biomass quality since it can negatively impact conversion of biomass into liquid fuels via biochemical platforms. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. Here, we have shown that cv. Kanlow switchgrass contains at least two closely related CAD genes (PviCAD1 and PviCAD2) that code for proteins containing highly conserved domains and residues that identify them as bona fide CADs. Both recombinant proteins displayed substrate kinetics consistent with their presumed role in cell wall lignification. Proteomic and immunoblotting detected CAD containing spots in internode protein extracts, and proteomic analyses demonstrated that both CADs were expressed. In planta CAD activity, CAD protein levels were observed at all stages of tiller development. A real-time qPCR analysis of the two CADs and one CAD-like sequence indicated that transcripts coding for PviCAD1 were present in greater abundance than those coding for PviCAD2. Transcripts for a third CAD-like sequence (PviAroADH) were present at intermediate levels as compared to PviCAD1 and CAD2. The predicted protein sequence of PviAroADH indicated that it was an enzyme unrelated to lignification based on phylogenetic and protein modeling data.

Two distinct waxy alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxya; wxa) or presence (waxyb; wxb) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wxa, the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wxb, the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wxb, GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wxa. SEM showed that endosperm architecture was very similar in both wxa and wxb alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wxa and the wxb alleles to aid in molecular breeding of low amylose sorghum.

Switchgrass PviCAD1: Understanding Residues Important for Substrate Preferences and Activity

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis. Although plants contain numerous genes coding for CADs, only one or two CADs appear to have a primary physiological role in lignin biosynthesis. Much of this distinction appears to reside in a few key residues that permit reasonable catalytic rates on monolignal substrates. Here, several mutant proteins were generated using switchgrass wild type (WT) PviCAD1 as a template to understand the role of some of these key residues, including a proton shuttling HL duo in the active site. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme, suggesting that key residues important for efficient catalysis had been identified. We have also shown that a sorghum ortholog containing EW, instead of HL in its active site, displayed negligible activity against monolignals. These results indicate that lignifying CADs require a specific set of key residues for efficient activity against monolignals.

Secreted Phospholipase A2s

The family of secretory phospholipase A2 enzymes (sPLA2) were the first known phospholipase A2s (PLA2s), and were originally discovered over one hundred years ago in human pancreatic juice and a snake venom. In the early years, PLA2s were discovered in pancreatic fluids of other mammals and in many other venoms. It was thought the predominant biological significance of sPLA2 lay in their digestive actions. Later work revealed that sPLA2s act in a large number of physiological functions, including release of lipid mediators, host defense, pathophysiology and venom toxicity. Although it was reasonable to suppose that all these PLA2-mediated events followed from the catalytic actions of the …

Cellular Phospholipase A2

The cellular phospholipases A2(cPLA2s) hydrolyze the fatty acids associated with the sn-2 position of phospholipids. Compared to secretory PLA2s, cPLA2s are large proteins(mw >60 kDa) associated with the cytosolic (and sometimes membrane) fractions of cells. Two distinct types of large, intracellular PLA2s are known and the following comments are meant to establish clear terms. Some large, intracellular PLA2s lack a calcium-binding domain and are catalytically independent of calcium. These are called …

Calcium Independent Phospholipase A2

A calcium-independent phospholipase A2 (PLA2) was first isolated and described from a rodent cell line and then cloned from several cell lines, including human lymphocytic cells. These are large, 85-88 kDa …