Lawrence B. Smart

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.

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700‐chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light‐activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild‐type cells showed a 4‐fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700‐apoprotein; greatly reduced whole‐chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA‐C and PSA‐D were not detected in this mutant. ADK9 does assemble near wild‐type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6‐dichloro‐p‐benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site‐directed mutagenesis of the PS I core.

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, FA and FB. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700+ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to FA and FB were absent in the mutant strain, but a reversible Fx signal was present with broad resonances at g=2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g=2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of FA- and FB-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700+ [FA/FB]- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to Fx.

Expression of photosynthesis genes in the cyanobacteriumSynechocystis sp. PCC 6803:psaA-psaB andpsbA transcripts accumulate in dark-grown cells

We have cloned and sequenced thepsaA andpsaB genes from the unicellular cyanobacteriumSynechocystis sp. PCC 6803. These genes are arranged in tandem, are co-transcribed, and are highly homologous to thepsaA andpsaB genes previously characterized. RNA was isolated from light-grown cells, from cells put in total darkness with and without glucose, and from cells grown under light-activated heterotrophic growth (LAHG) conditions. Quantitation of hybridization to northern blots revealed only a slight decrease in the accumulation of thepsaA-psaB transcript in cells grown in complete darkness with glucose and in LAHG cells, relative to light-grown cells. Accumulation of thepsbA transcript steadily declines through dark incubation, with a steady-state level in LAHG cells 28% of that in light-grown cells. Transcripts frompsbD, psaD, andrbcLS accumulate in cells grown in complete darkness and in LAHG cells to approximately the same levels as in light-grown cells. Photosynthesis gene transcripts in cells grown in the dark without glucose were detected, but were highly degraded. Our data prove that transcripts from photosynthesis genes do accumulate in dark-grownSynechocystis 6803, which may allow for synthesis and assembly of photosystem (PS) I and PS II in the dark.

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.

Site-directed conversion of cysteine-565 to serine in PsaB of photosystem I results in the assembly of iron-sulfur [3Fe-4S] and iron-sulfur [4Fe-4S] clusters in Fx. A mixed-ligand iron-sulfur [4Fe-4S] cluster is capable of electron transfer to FA and FB

We reported earlier [Smart, L. B., Warren, P. V., Golbeck, J. H., & McIntosh, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1132-1136] that the site-directed conversion of cysteine-565 to serine (C565S) in PsaB of Synechocystis sp. PCC 6803 leads to an accumulation of photosystem I polypeptides and the low-temperature photoreduction of the terminal electron acceptors FA and FB. In this paper, we report the occurrence of a [3Fe-4S]1 + ,0 cluster in dodecyl maltoside-solubilized photosystem I complexes prepared from the C565S mutant. The [3Fe-4S] cluster is reducible with dithionite at pH 6.5, implying a midpoint potential considerably more oxidizing than either FA or FB. Similar to the behavior of FX, the [3Fe-4S] cluster undergoes partial, reversible photoreduction when the complex is illuminated at 15 K, and complete photoreduction when the sample is illuminated during freezing. Contrary to the result expected in the presence of a relatively high-potential FX, there is significant low-temperature and room temperature photoreduction of FA and FB in the C565S complex. Although the FA and FB resonances are more intense when the complex is frozen during illumination, they still account for < 60% of FA and FB found by chemical reduction. When the FA and FB clusters are prereduced with dithionite at pH 10.0, a new set of resonances appear upon illumination at g = 2.015, 1.941, and 1.811, and disappear on subsequent darkness. The species giving rise to this signal is most likely a mixed-ligand [4Fe-4S]2+,1+ cluster located in the FX site.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.

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.

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality.

BackgroundThere is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency.ResultsWe used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.).ConclusionOur results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.