Kimberly D. Cameron

Increased Accumulation of Cuticular Wax and Expression of Lipid Transfer Protein in Response to Periodic Drying Events in Leaves of Tree Tobacco

Cuticular wax deposition and composition affects drought tolerance and yield in plants. We examined the relationship between wax and dehydration stress by characterizing the leaf cuticular wax of tree tobacco (Nicotiana glauca L. Graham) grown under periodic dehydration stress. Total leaf cuticular wax load increased after each of three periods of dehydration stress using a CH2Cl2 extraction process. Overall, total wax load increased 1.5- to 2.5-fold, but composition of the wax was not altered. Homologous series of wax components were classified into organic groups; n-hentriacontane was the largest component (>75%) with alcohols and fatty acids representing <10% of the entire wax load. An increase in density, but no change in the three-dimensional shape, of leaf wax crystals was evident under low-kV scanning electron microscopy after each drying event. Leaves excised from plants subjected to multiple drying events were more resistant to water loss compared to leaves excised from well-watered plants, indicating that there is a negative relationship between total wax load and epidermal conductance. Lipid transfer proteins (LTPs) are thought to be involved in the transfer of lipids through the extracellular matrix for the formation of cuticular wax. Using northern analysis, a 6-fold increase of tree tobacco LTP gene transcripts was observed after three drying events, providing further evidence that LTP is involved in cuticle deposition. The simplicity of wax composition and the dramatic wax bloom displayed by tree tobacco make this an excellent species in which to study the relationship between leaf wax deposition and drought tolerance.

Nitration of a peptide phytotoxin by bacterial nitric oxide synthase

Nitric oxide (NO) is a potent intercellular signal in mammals that mediates key aspects of blood pressure, hormone release, nerve transmission and the immune response of higher organisms. Proteins homologous to full-length mammalian nitric oxide synthases (NOSs) are found in lower multicellular organisms. Recently, genome sequencing has shown that some bacteria contain genes coding for truncated NOS proteins; this is consistent with reports of NOS-like activities in bacterial extracts. Biological functions for bacterial NOSs are unknown, but have been presumed to be analogous to their role in mammals. Here we describe a gene in the plant pathogen Streptomyces turgidiscabies that encodes a NOS homologue, and we reveal its role in nitrating a dipeptide phytotoxin required for plant pathogenicity. High similarity between bacterial NOSs indicates a general function in biosynthetic nitration; thus, bacterial NOSs constitute a new class of enzymes. Here we show that the primary function of Streptomyces NOS is radically different from that of mammalian NOS. Surprisingly, mammalian NO signalling and bacterial biosynthetic nitration share an evolutionary origin.

A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325–660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non‐pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site‐specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram‐positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.

Diversity of cuticular wax among Salix species and Populus species hybrids.

The leaf cuticular waxes of three Salix species and two Populus species hybrids, selected for their ability to produce high amounts of biomass, were characterized. Samples were extracted in CH(2)Cl(2) three times over the growing season. Low kV SEM was utilized to observe differences in the ultrastructure of leaf surfaces from each clone. Homologous series of wax components were classified into organic groups, and the variation in wax components due to clone, sample time, and their interaction was identified. All Salix species and Populus species hybrids showed differences in total wax load at each sampling period, whereas the pattern of wax deposition over time differed only between the Salix species. A strong positive relationship was identified between the entire homologous series of alcohols and total wax load in all clones. Similarly strong relationships were observed between fatty acids and total wax load as well as fatty acids and alcohols in two Salix species and one Populus species hybrid. One Salix species, S. dasyclados, also displayed a strong positive relationship between alcohols and alkanes. These data indicate that species grown under the same environmental conditions produce measurably different cuticular waxes and that regulation of wax production appears to be different in each species. The important roles cuticular waxes play in drought tolerance, pest, and pathogen resistance, as well as the ease of wax extraction and analysis, strongly suggest that the characteristics of the cuticular wax may prove to be useful selectable traits in a breeding program.

Analysis of Biomass Composition Using High-Resolution Thermogravimetric Analysis and Percent Bark Content for the Selection of Shrub Willow Bioenergy Crop Varieties

Rapid determination of biomass composition is critical for the selection of shrub willow varieties with optimized biomass properties for conversion into fuels or chemicals. In order to improve the process for identifying and selecting shrub willow clones with distinct biomass composition, high-resolution thermogravimetric analysis (HR-TGA) was developed as a rapid, low-cost method for analyzing large numbers of willow biomass samples. In order to validate the HR-TGA method, bulk biomass collected from 2-year-old stems of a selected set of 25 shrub willow clones was analyzed using traditional wet chemistry techniques in addition to HR-TGA. The results of the wet chemistry and the HR-TGA method were compared using regression analysis resulting in R-squared values above 0.7 for the three main wood components, cellulose, hemicellulose, and lignin. Bark was removed from duplicate stem samples of the same clones, the proportion of bark was determined, and the debarked wood was used for HR-TGA analysis of composition. While there were significant differences in the proportions of lignin and cellulose in debarked wood compared to bulk biomass, as well as significant differences in bark percentage among clones, there was no correlation between bark percentage and bulk biomass component analysis. This work validates the effectiveness, precision, and accuracy of HR-TGA as a reasonably high-throughput method for biomass composition analysis and selection of shrub willow bioenergy crop varieties.

High-resolution Thermogravimetric Analysis For Rapid Characterization of Biomass Composition and Selection of Shrub Willow Varieties

The cultivation of shrub willow (Salix spp.) bioenergy crops is being commercialized in North America, as it has been in Europe for many years. Considering the high genetic diversity and ease of hybridization, there is great potential for genetic improvement of shrub willow through traditional breeding. The State University of New York—College of Environmental Science and Forestry has an extensive breeding program for the genetic improvement of shrub willow for biomass production and for other environmental applications. Since 1998, breeding efforts have produced more than 200 families resulting in more than 5,000 progeny. The goal for this project was to utilize a rapid, low-cost method for the compositional analysis of willow biomass to aid in the selection of willow clones for improved conversion efficiency. A select group of willow clones was analyzed using high-resolution thermogravimetric analysis (HR-TGA), and significant differences in biomass composition were observed. Differences among and within families produced through controlled pollinations were observed, as well as differences by age at time of sampling. These results suggest that HR-TGA has a great promise as a tool for rapid biomass characterization.

Genetic Improvement of Willow (Salix spp.) as a Dedicated Bioenergy Crop

Fast-growing woody crops are emerging as an attractive source of biomass, because they have the capability to meet many of the agronomic, environmental, and societal parameters associated with successful deployment as a source for energy. Willows, especially those that grow as shrubs with multiple small stems, have many of the characteristics desired in dedicated energy crops, making them an excellent feedstock choice in a number of settings. Wide-scale cultivation of willow bioenergy crops in Europe over the last two decades has encouraged development of efficient mechanized planting and harvesting systems. Vigorous breeding programs have also been active in broadening the genetic base and increasing the yield of varieties that are deployed commercially. Unfortunately, the majority of the fast-growing varieties developed in Europe are highly susceptible to a debilitating insect pest in North America, highlighting the need for region-specific breeding and selection programs. Since 1986, researchers at the State University of New York College of Environmental Science and Forestry in Syracuse have been researching a willow cropping system for North America, including the development of genetically improved willow varieties. This program has involved assembling a large and diverse collection of willow clones, successfully performing controlled pollinations, and selecting new varieties with improved yield, pest and disease resistance across a range of sites in North America. Many of these varieties have been commercially deployed for use in bioenergy plantations in New York and elsewhere.

Correlations of expression of cell wall biosynthesis genes with variation in biomass composition in shrub willow (Salix spp.) biomass crops

We have measured significant genetically determined variation in biomass composition among breeding populations of shrub willow, a biomass feedstock crop. This project was aimed to ask whether patterns of cell wall gene expression can be correlated with genetic variation in biomass composition at harvest, in order to develop assays of early differences in gene expression as indicators of harvestable biomass chemical composition and potentially reduce the time of selection for new willow genotypes. Previous studies have demonstrated that manipulation of expression of cell wall biosynthetic genes results in altered biomass chemical composition. We analyzed genes encoding enzymes involved in lignin biosynthesis and carbohydrate active enzymes selected based on their functional characterization and conservation in Populus trichocarpa and Arabidopsis thaliana. Fragments of 20 genes were cloned from young stem cDNA of Salix sachalinensis and Salix miyabeana. Expression profiling in willow stem apical tissue and developing stem tissue was performed for each isolated gene using probe-based quantitative real-time PCR. Two willow parental genotypes and six progeny within a hybrid family were selected for analysis, and significant differences in expression among the individuals and between tissue types were observed for most of the genes. Significant correlations between patterns of gene expression and variation in the biomass chemical composition of those genotypes provide insight into the genetic regulation of lignocellulosic deposition in this important bioenergy crop and could be utilized as a tool for early selection of new genotypes.

A second member of the Nicotiana glauca lipid transfer protein gene family, NgLTP2, encodes a divergent and differentially expressed protein

Multiple, highly similar members of the lipid transfer protein (LTP) family have been identified in Nicotiana glauca L. Here we describe four new members of the NgLTP gene family and further characterise one member. Three genes were isolated from a guard cell cDNA library and one (NgLTP2) was isolated from a genomic library. These four NgLTPs, as well as one described previously, NgLTP1, share >83% amino acid similarity, but the deduced protein sequence of NgLTP2 lacks the last five residues compared with other LTPs. Since the DNA sequences of the five genes are nearly identical, techniques based on nucleic acid hybridisation or PCR amplification were not sufficient to resolve the expression of the individual genes with confidence. Therefore, we characterised the expression pattern of NgLTP2, the only NgLTP gene that was not found in the guard cell cDNA library, using an NgLTP2 promoter-GUS reporter assay. GUS activity driven by the NgLTP2 promoter was assayed in three species of transgenic plants as an indicator of the endogenous pattern of expression of this gene. GUS was strongly induced upon wounding, whereas NgLTP1 was induced by drought stress. Sequence analysis of the NgLTP2 promoter revealed cis-acting motifs associated with induction by wounding. Differential expression of the NgLTP gene family, revealed by the different expression patterns of NgLTP1 and NgLTP2, is further evidence that these genes have multiple functions in N. glauca.