Antisense Down-Regulation of 4CL Expression Alters Lignification,Tree Growth,and Saccharification Potential of Field-Grown Poplar

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.Composed of diverse layers of cellulose microfibrils and amorphous hemicelluloses within a matrix of pectins, proteins, and lignin, the secondary cell walls of plants are diverse in their morphology, chemistry, and physiological functions. Lignification is of particular interest, as it exhibits highly predictable temporal and spatial patterning and is the last major step in the structural reinforcement of cell walls before the protoplast is dissolved (Donaldson, 2001). To gain detailed insights into cell wall assembly, mutant or transgenic perturbations to lignin biosynthesis have been employed to alter native lignin content and monomer compositions (i.e. to shift ratios of syringyl [S], guaiacyl [G], and p-hydroxyphenyl [H] lignins; Porter et al., 1978; Miller et al., 1983; Baucher et al., 1996; Kajita et al., 1996; Lee et al., 1997; Anterola and Lewis, 2002; Davin et al., 2008a, 2008b; Patten et al., 2010a). In addition, such perturbations give needed insight into the role of lignin in providing resistance to mechanical (Mark, 1967; Niklas, 1992; Gindl and Teischinger, 2002) and biotic (Dixon and Paiva, 1995) stresses. Lignin affects xylem conductance and protects the vasculature from embolism by imparting a barrier between water under transpiration-induced tension in the xylem and the atmosphere (Raven, 1977; Boyce et al., 2004) and retards tissue digestion and decomposition by pathogens and herbivores. Economic incentives have also helped drive research on lignin reductions in wood because lignin is considered the principal cause of recalcitrance to chemical pulping and to simultaneous saccharification and fermentation to produce liquid biofuels (Huntley et al., 2003; Schubert, 2006; Jørgensen et al., 2007; Davin et al., 2008a, 2008b; Foust et al., 2008; Li et al., 2008; Yang and Wyman, 2008).Because each of the major cell wall biopolymers has different functions, changes in one component should induce “compensatory” shifts in concentrations or compositions of the others. Indeed, altering lignin composition and content has been shown to have wide-ranging effects on cell wall morphology, including specification of cell identity and plant form (Davin et al., 2008a, 2008b). An early study of aspen (Populus tremuloides) down-regulated for 4-coumarate:coenzyme A ligase (4CL) reported that young trees had up to 45% less lignin, increased cellulose contents, and increased growth (Hu et al., 1999). These results led Hu and coworkers (1999) to hypothesize that enhanced growth and compensatory deposition of cell wall polysaccharides resulted from reduced carbon demand for lignin synthesis. However, these results were questioned on both analytical and biochemical grounds (Anterola and Lewis, 2002). Subsequent studies of greenhouse-grown aspen (Li et al., 2003; Hancock et al., 2007, 2008) and Chinese white poplar (Populus tomentosa; Jia et al., 2004) containing transgenes that suppress RNA expression of 4CL found no comparable growth enhancement.4CL is generally considered to be the third step in the phenylpropanoid pathway. Consisting of a multigene family (Costa et al., 2005), 4CL is important for monolignol biosynthesis as well as for the generation of other secondary metabolites for plant defense in leaves and stem xylem tissues (Tsai et al., 2006). However, little is known about how down-regulation of 4CL can differentially affect the production of secondary metabolites and whether or not the types and amounts of the defense compounds produced may differ depending on the level of environmental stresses perceived by growing plants.Because of the large differences in plant physiological behavior under field versus laboratory or greenhouse conditions, and the complex development of xylem in growing trees, field studies are essential to understand the level of lignin modification that might be economically useful yet also preserve tree health and productivity. Previous field studies with other forms of lignin modification have suggested that some kinds of perturbations might be tolerated (Pilate et al., 2002). However, comparable studies have not been reported on trees with lignin modifications induced by 4CL inhibition.In this study, we report that 4CL down-regulation via antisense RNA was effective in reducing lignin contents of wood in field-grown trees. In agreement with more recent work (Li et al., 2003; Hancock et al., 2007) and in contrast to an early study (Hu et al., 1999), these changes did not promote increased growth rate. High levels of lignin reduction observed in approximately one-third of the transgenic events led to reduced growth and serious physiological abnormalities. In these low-lignin transgenic events, we identified and quantified significant nonlignin phenolic depositions and utilized a novel combination of cryofixation and confocal microscopy to visualize the in vivo distribution of these compounds within the wood. Finally, we determined that reductions in lignin content did not increase wood processability that would benefit fermentation to produce liquid biofuels.     

Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and ¹⁵N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with ¹⁵N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.     

Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue

The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using alpha-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.     

Calcium-dependent changes in the flexibility of the regulatory domain of troponin C in the troponin complex

With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca(2+). We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide (15)N relaxation times were measured in the presence of Ca(2+) or EGTA/Mg(2+). T(1) relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide (15)N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T(2) relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T(2) times in the presence of Ca(2+) could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg(2+), relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca(2+), whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca(2+), the N-domain of TnC moves independently from the rest of the complex.     

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289-8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124-31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence of the switch peptide [Gagné et al. (1995) Nat. Struct. Biol. 2, 784-789]. The switch peptide is bound in a shallow and complementary hydrophobic surface cleft largely defined by helices A and B and also has key ionic interactions with sNTnC. These results show that bifunctional rhodamine probes can be attached to surface helices via suitable pairs of solvent-accessible residues that have been mutated to cysteines, without altering the conformation of the labeled domain. A set of such probes can be used to determine the orientation and motion of the target domain in the cellular environment [Corrie et al. (1999) Nature 400, 425-430; Ferguson et al. (2003) Mol. Cell 11(4), in press].     

Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.     

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.