Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA₂₀ and GA₈, in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.Semidwarfism is a valuable trait in many crop species and agricultural environments. In cereal crops, it can result in decreased lodging, increased yield, and improved harvest index (Dalrymple, 1985; Hedden, 2003). Therefore, it was a critical foundation of the “Green Revolution” that resulted in large improvements of yield in wheat (Triticum aestivum) and rice (Oryza sativa; Hargrove and Cabanilla, 1979; Perovic et al., 2008). Semidwarfism has had substantial benefits for fruit tree production, where it enables earlier fruit bearing, higher yields, and easier harvests in orchards (Battisini and Battisini, 2005). Semidwarf woody species are also extensively used in ornamental horticulture, where they allow more compact forms to be fit into small areas around homes and on streets and reduce the need for pruning to avoid interference with structures and transmission lines (Busov et al., 2003).Although against the current orthodoxy of forest tree breeding, where height growth is emphasized, semidwarfism might also have benefits for wood and biomass production (Bradshaw and Strauss, 2001). Such trees could be useful if they were less prone to wind throw due to their shorter, stockier forms and expected greater allocation to roots. Reduced stature could also result in less bending and slanting of trunks in the face of wind and gravity on hillslopes and thus reduce the extent of reaction wood formation, which degrades the performance and value of solid wood and pulp products. Reduced height and increased allocation of growth to roots might enhance stress tolerance, soil nutrient uptake, bioremediation, and carbon sequestration.Semidwarfism can be achieved by the modification of several types of genes and physiological mechanisms, but the most prevalent and advanced forms in agriculture affect GAs or their signaling (for review, see Busov et al., 2008). GAs are endogenous plant hormones that influence several aspects of plant growth and development, including seed germination, leaf expansion, shoot growth, cell division, flower induction, and fruit development (Sun and Gubler, 2004; Fleet and Sun, 2005; Swain and Singh, 2005). With respect to shoot growth, the most obvious effect of GA is its promotion of stem elongation by stimulating both cell elongation and division (Marth et al., 1956). GA modification also has significant effects on plant biochemistry, changing the amounts and distribution of a wide variety of metabolites in shoots and roots (Rossetto et al., 2003; Chen et al., 2004; Busov et al., 2006).Little is known about how semidwarfism affects belowground growth. GA has been shown to play a controlling role in lateral root development (Gou et al., 2010), and GA and ethylene synergistically promote both the initiation and growth of adventitious roots (Osmont et al., 2007). In tomato (Solanum lycopersicum), isogenic GA-deficient mutants (gib) allocate more biomass to roots compared with shoots (Nagel et al., 2001). In poplar (Populus spp.), semidwarf transgenic plants grown in vitro had a lower shoot-to-root ratio, which was at least partly due to proliferation of lateral roots (Busov et al., 2006; Gou et al., 2010).As a domestication trait, semidwarfism has been proposed as a means for mitigating the spread of transgenic plants within and outside of crop environments (Al-Ahmad et al., 2005). The genetic dominance of most semidwarfism transgenes would cause reduced height growth in transgene-containing progeny, reducing their ability to compete for light. Moreover, because of the close linkage of the semidwarfism genes to other genes that were cointroduced on the same plasmid, they would also powerfully retard their spread or introgression, even in cases where the linked transgene would, on their own, impart a selective advantage. However, there have been very few plant species where this concept has been explicitly tested (Al-Ahmad and Gressel, 2006; Gressel and Valverde, 2009), and we know of no examples in woody or perennial plants.To study the effects of semidwarfism genes in a woody plant grown under field conditions, we inserted a number of dominant GA-modifying transgenes into hybrid poplar (Populus tremula × Populus alba), the widely recognized model woody plant for genomics and biotechnology (Herschbach and Kopriva, 2002; Brunner et al., 2004a; Tuskan et al., 2004). Most of the genes studied were overexpressed forms of GA 2-oxidase, GA-Insensitive (GAI), or Repressor of GAI-Like (RGL), all known to cause semidwarfism in other plant species. GA 2-oxidase is a major GA catabolic enzyme in plants, and GAI and RGL are negative regulators of the GA signal transduction pathway (Appleford et al., 2007; Busov et al., 2008). The transgenic trees were first analyzed in the greenhouse (Busov et al., 2006) and then assayed for their effect on height growth in a 2-year field trial (Zawaski et al., 2011), from which we selected 10 transgenic events that grew at approximately three-quarters the rate of wild-type trees. The goal was to select semidwarf trees whose phenotype was not so severe as to be irrelevant to possible crop uses but strong enough to give a clear phenotype in a field study. In this study, we analyzed changes in a number of morphological, physiological, and growth traits and investigated the prospect for semidwarfism to be used as a mitigation tool to reduce the frequency of spread of transgenic and exotic species.     

Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors

B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi.     

Development of genomic simple sequence repeat markers for linseed using next-generation sequencing technology

Linseed (Linum usitatissimum L.) is regarded as a cash crop of tomorrow because of the presence of nutraceutically important ??-linolenic acid (ALA) and lignan. However, only limited breeding progress has been made in this crop, mainly due to the lack of sufficient genetic and genomic resources. Among these, simple sequence repeats (SSR) are useful DNA markers for diversity analysis, genetic mapping and tagging traits because of their co-dominant and highly polymorphic nature. In order to develop SSR markers for linseed, we used three microsatellite isolation methods, viz., PCR Isolation of Microsatellite Arrays (PIMA), 5??-anchored PCR method, and Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). The amplified products from these methods were pooled and sequenced using the 454 GS-FLX platform. A total of 36,332 reads were obtained, which assembled into 2,183 contigs and 2,509 singlets. The contigs and the singlets contained 1,842 microsatellite motifs, with dinucleotide motifs as the most abundant repeat type (54%) followed by trinucleotide motifs (44%). Based on this, 290 SSR markers were designed, 52 of which were evaluated using a panel of 27 diverse linseed genotypes. Among the three enrichment methods, the 5??-anchored PCR method was most efficient for isolation of microsatellites, while FIASCO was most efficient for developing SSR markers. We show the utility of next-generation sequencing technology for efficiently discovering a large number of microsatellite markers in non-model plants.     

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.     

The association between Barrett's esophagus and Helicobacter pylori infection: a meta-analysis

Objective: The effect of Helicobacter pylori on Barrett’s esophagus is poorly understood. We conducted a meta‐analysis to summarize the existing literature examining the effect that H. pylori has on Barrett’s esophagus. Design: We performed a comprehensive search to identify studies pertaining to the association between H. pylori and Barrett’s esophagus. We conducted meta‐regression analyses to identify sources of variation in the effect of H. pylori on Barrett’s esophagus. Results: Our analysis included a total of 49 studies that examined the effect of H. pylori on Barrett’s esophagus and seven studies that examined the effect of cag A positivity on Barrett’s esophagus. Overall, H. pylori, and even more so cag A, tended to be protective for Barrett’s esophagus in most studies; however, there was obvious heterogeneity across studies. The effect of H. pylori on Barrett’s esophagus varied by geographic location and in the presence of selection and information biases. Only four studies were found without obvious selection and information bias, and these showed a protective effect of H. pylori on Barrett’s esophagus (Relative risk = 0.46 [95% CI: 0.35, 0.60]). Conclusions: Estimates for the effect of H. pylori on Barrett’s esophagus were heterogeneous across studies. We identified selection and information bias as potential sources of this heterogeneity. Few studies without obvious selection and information bias have been conducted to examine the effect of H. pylori on Barrett’s esophagus, but in these, H. pylori infection is associated with a reduced risk of Barrett’s esophagus.     

Single-cell census of mechanosensitive channels in living bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering.     

Tissue biomarkers for prostate cancer radiation therapy

Prostate cancer is the most common cancer and second leading cause of cancer deaths among men in the United States. Most men have localized disease diagnosed following an elevated serum prostate specific antigen test for cancer screening purposes. Standard treatment options consist of surgery or definitive radiation therapy directed by clinical factors that are organized into risk stratification groups. Current clinical risk stratification systems are still insufficient to differentiate lethal from indolent disease. Similarly, a subset of men in poor risk groups need to be identified for more aggressive treatment and enrollment into clinical trials. Furthermore, these clinical tools are very limited in revealing information about the biologic pathways driving these different disease phenotypes and do not offer insights for novel treatments which are needed in men with poor-risk disease. We believe molecular biomarkers may serve to bridge these inadequacies of traditional clinical factors opening the door for personalized treatment approaches that would allow tailoring of treatment options to maximize therapeutic outcome. We review the current state of prognostic and predictive tissue-based molecular biomarkers which can be used to direct localized prostate cancer treatment decisions, specifically those implicated with definitive and salvage radiation therapy.     

Group I mGluR5 metabotropic glutamate receptors regulate proliferation of neuronal progenitors in specific forebrain developmental domains

Major classical neurotransmitters including GABA and glutamate play novel morphogenic roles during development of the mammalian CNS. During forebrain neurogenesis, glutamate regulates neuroblast proliferation in different germinal domains using receptor subtype-specific mechanisms. For example, ionotropic N -methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors mediate distinct proliferative effects in ventral or dorsal forebrain germinal domains, and regulate the correct number of neurons that populate the striatum or cerebral cortex. Recent work suggests metabotropic receptors may also mediate glutamate's proliferative effects. Group I mGluR5 receptor subtypes are highly expressed in forebrain germinal zones. Using in vitro and in vivo methods, we demonstrate mGluR5 receptor activation plays an important role in neuroblast proliferation in the ventral telencephalon, and helps determine the complement of striatum projection neurons. mGluR5 receptor-mediated effects on striatal neuronal progenitors are restricted mainly to early cycling populations in the ventricular zone, with little effect on secondary proliferative populations in the subventricular zone. In contrast to proliferative effects in the ventral telencephalon, mGluR5 receptors do not modulate proliferation of dorsal telencephalon-derived cortical neuroblasts. Heterogeneous domain-specific proliferative effects of glutamate-mediated by specific receptor subtypes provide an important developmental mechanism allowing generation of the correct complement of neuronal subtypes that populate the mammalian forebrain.