Final Report for Tufts Institute of the Environment Graduate Fellowship

Transcription

1 Final Report for Tufts Institute of the Environment Graduate Fellowship TITLE: Using Jasmonates to Enhance Long-Term Sequestration of Atmospheric Carbon. Benjamin A. Babst, Ph. D. Department of Biology, Tufts University, Medford, Massachusetts Present Address: School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, Michigan Faculty Advisor: Colin M. Orians Multiple approaches will be necessary to mitigate increasing global atmospheric carbon dioxide, including greater conservation and efficiency, alternative energy sources, and increasing carbon sequestration. The research conducted during this TIE graduate fellowship was designed to test the feasibility of manipulating plants using a class of plant signal molecules, jasmonates, to increase carbon dioxide sequestration. Plants remove carbon dioxide from the atmosphere during photosynthesis, and much of the acquired carbon becomes plant biomass. Due to climate change effects, soil will be an important site for improvement of the total carbon sequestering capacity of a forest [Makipaa, 1999 #612], for example roots, organic compounds exuded from roots, dead wood, and fallen leaves. However, the rapid breakdown of organic compounds in the soil may pose a barrier to using plants for carbon sequestration [Taneva, 2006 #1163], because most chemical components of plant tissue are short-lived in the soil. Polyphenolics, such as tannins, which are defensive compounds, and lignin, which strengthens cell walls in woody stems and roots, have potential to be good carbon sinks, because they are long-lived in the soil [Camiré, 1991 #603;Schweitzer, 2004 #578] and make up a large percentage of plant carbon mass (3-45% dry mass for lignin, and 1-5% dry mass for tannins) [Amthor, 2003 #605;Poorter, 1992 #606;Driebe, 2000 #576]. My recent studies using carbon-11 tracer, demonstrate the potential for increased carbon partitioning to roots following exposure of plants to jasmonates or damage by insect herbivores. I proposed that changing the supply of carbon to roots of fast growing plants, such as poplar trees, may modulate output from the phenylpropanoid biosynthetic pathway, increasing the production of soil-stable polyphenolics (i.e., tannin and lignin) in the roots. I used weak, chronic treatments of methyl jasmonate or herbivory by gypsy moth caterpillars to test whether increased carbon partitioning to roots can be sustained by plants over time. Overall, neither herbivory nor jasmonate treatment affected stem height or diameter growth during the course of the three week experiment (fig. 1). Jasmonate had no effect on final biomass, and for plants damaged by herbivores there was a small decrease in biomass only in rapidly expanding leaves (fig. 2). Previous research has demonstrated that poplar trees can compensate for leaf tissue loss much greater than that imposed in my experiment [Reichenbacker, 1996 #951]. Since total biomass accumulation by poplars is not affected by moderate herbivory or jasmonate treatment, any increased allocation of carbon to polyphenolics should result in a net increase in sequestered carbon. Condensed tannins were undetectable in poplar tree tissue early in the season, when this experiment was conducted. Preliminary analysis of leaf samples from mid-summer suggests that tannin biosynthesis is under the control of a developmentally or seasonally activated switch.

2 B. A. Babst 2 This seasonality will be an important consideration for future research, but also for timing manipulations in the field to maximize tannin production, while minimizing cultural inputs. The simpler salicylate phenolic glycosides, such as salicortin were present in all leaves and in stems. There was a trend for increased salicortin in leaves following herbivore damage, which was statistically significant in developing (i.e., transition) leaves (fig. 3a). On the contrary, salicortin decreased in stems of herbivore-damaged plants. Generally, salicortin was unaffected by methyl jasmonate treatment. Several other phenolic glycosides were present in smaller amounts, including tremuloidin, and a compound that may be nigracin. Tremuloidin appeared to follow the same pattern as salicortin in mature leaves (fig. 3b), but was present at much lower concentrations in younger leaves, in some cases too low to measure. The simple phenolic glycosides are not long-lived in the soil, but they are of interest because they provide some insight into the phenylpropanoid pathway. Based on the phenolic glycoside measurements, it appears that herbivory strongly upregulates phenylpropanoid biosynthesis, but jasmonate treatment does not. I was able to test this hypothesis using cdna microarrays in collaboration with Stefan Jansson and Andreas Sjödin at the Umeå Plant Science Center in Sweden. The microarrays developed at UPSC measurement of gene expression for most transcribed genes in poplars, allowing a broad view across all metabolic pathways for plant responses. For this experiment, plants were treated in mid-summer, when tannin biosynthesis should be occuring in these poplars. I was able to spend two weeks at UPSC during my fellowship, beginning to analyze this extensive data set. The analysis supports the general pattern observed for phenolic glycosides. Herbivory strongly upregulated many genes in the phenylpropanoid pathway, while jasmonic acid upregulated only a subset of those genes (fig. 4). Interestingly, the alterations in gene expression appeared to be geared toward the biosynthesis of tannins, rather than lignin or flavonoids. The microarray cannot address the salicylate phenolic glycosides, because the biosynthetic pathway for salicylates in Populus has not yet been determined. A surprising outcome from the microarray was that, in addition to genes within the phenylpropanoid pathway, genes involved in carbohydrate and amino acid metabolism appeared to be upregulated in a manner that might provide more substrate for phenylpropanoid biosynthesis (fig. 5). That is, the entire metabolism of the plant may be reorganized in response to herbivory to favor accumulation of phenylpropanoids, which can also serve as deterrents to herbivores. Again, jasmonic acid treatment elicited a narrower and weaker response. Although, jasmonate signaling has long been associated with plant responses to herbivory, this microarray data suggests that herbivory elicits responses through one or more additional signaling pathways. Further, the increase in phenylpropanoid biosynthesis following herbivory appears to be regulated by a non-jasmonate signal. In sum, a low level of herbivory in mid-summer, when tannin biosynthesis occurs may increase the carbon sequestration capacity of poplar trees. In addition to these findings, during this fellowship I was able to develop a micro-scale technique for measuring lignin, which reduces hazardous waste byproducts by about 5-10 fold. Dr. Romualdo Fukushima, a lignin expert, is helping to extract a pure lignin standard for future work. Because lignin is difficult to measure, lignin concentrations have often been calculated without a standard, or more recently, using ground homogenized pine needles, obtained from NIST, which NIST is no longer distributing. When ready, the new lignin standard will provide more robust measurements of lignin content. Recently, I was able to obtain a small aliquot of the pine needle standard from another researcher, which will allow a comparison between the new and old standards. Lignin analysis is ongoing. Approximately 200 samples are half-way through the preparatory extractions that are necessary to remove from the plant tissue other biochemicals, 2

3 B. A. Babst 3 which may interfere with measurement of the lignin. Additionally, about 48 Root samples are ready for the final analysis. Although analyses are in progress, the phenolic glycoside and microarray data suggest that jasmonates alone will not be the key to enhancing carbon sequestration. Microarray analysis, particularly, highlighted the striking differences between plant responses to jasmonates and plant responses to actual herbivory. Further study of the non-jasmonate signaling mechanisms by which plants perceive and respond to herbivory may lead to new means of manipulating phenylpropanoid biosynthesis. Further study should also be directed at understanding which of the multitude of genes that were upregulated following herbivory are critical to increasing phenylpropanoid biosynthesis. At Michigan Technological University, where I just began working as a postdoctoral scientist, our research group is working to understand the phenylpropanoid biosynthetic pathways. By understanding the pathways, and understanding how plants regulate the pathways, we will eventually be able to optimize growth and phenylpropanoid biosynthesis to produce renewable biofuels from wood, while enhancing carbon sequestration in leaves and roots, which are not harvested. Part of my fellowship I proposed to spend preparing a model to scale measurements up to the stand level. Because carbon sequestration by plants is a function of both growth and production of soil-stable carbon compounds, and is affected by multiple environmental factors, scaling up to the stand level is not a simple matter. I was able to assemble a list of factors that may influence carbon sequestration, including, at the individual plant level, growth rate and the rate of biosynthesis of soil stable carbon compounds in leaves and roots (assumes trunks will be harvested), which may have two-way negative interactions, and at the stand level, factors such as tree species, plant density, soil type, climate, including rainfall, air temperature, soil temperature, and average light levels. Ultimately, any carbon sequestration strategy developed in small scale experiments will need to be tested in the field in order to accurately scale to the stand level, or to the global level. Student Mentoring: During my fellowship, I also encountered several mentoring opportunities. I taught lignin analysis to a visiting graduate student (Vincent von Vordzogbe), so he could take the technique back with him to Ghana to study how forestry practices in Africa could enhance or reduce carbon sequestration by favoring certain species over others. I also assisted a new master s student (Minda Berbeco) to learn analysis of condensed tannins and lignin, and introduced her to carbon sequestration research. 3

4 B. A. Babst 4 B Figure 1. Stem size before and after the initiation of treatments. Points are means±se for N=8 to 13 per treatment. Treatments were begun at day 19, and repeated every 3 days for 21 days. Relative growth rates were calculated based on the most recent previous measurement. Repeated measures ANOVA indicated no differences among treatments for stem height or diameter. 4

5 B. A. Babst 5 Figure 2. Dry weights of plant organs after 3 weeks of chronic low level (A) gypsy moth leaf consumption or treatment with empty clip-cages and (B) 50µM MeJA or control solution. Bars are means±se for N=8 to 13 per treatment. P-values are for t-tests for differences in means between treatments and their respective controls (NS=not significant). 5

6 B. A. Babst 6 A salicortin (mg/g) P=0.11 P=0.03 cage control gypsy moth P= mature transition apical B 15 salicortin (mg/g) 10 5 P=0.52 P=0.47 control solution MeJA P= mature transition apical leaf type Figure 3. Concentrations of salicortin in leaves after three weeks of gypsy moth herbivory (A), or treatment with 50µmol methyl jasmonate. P-values are for t-tests for differences in mean between treated plants and their respective controls (N=4). 6

7 Phenylalanine PAL Cinnamic acid C4H p-coumaric acid Upregulated genes Downregulated genes OMT CAH CCR (0.43) p-coumaroyl-coa (1.83) CHS CAD chalcone CHI monolignols flavanone FHT flavanol F3H DFR ANS lignan lignin FNS flavones IFS isoflavonoids Proanthocyanidins (condensed tannins) Figure 4 A

8 B. A. Babst 2 O Phenylalanine CO 2 Me PAL Cinnamic acid C4H p-coumaric acid Upregulated genes Downregulated genes OMT CAH CCR p-coumaroyl-coa CHS CAD chalcone CHI monolignols X flavanone FHT flavanol F3H DFR ANS lignan lignin FNS flavones IFS isoflavonoids Proanthocyanidins (condensed tannins) Figure 4 B. Phenylpropanoid genes upregulated in Populus in response to gypsy moth herbivory (A), and jasmonic acid (B). Upregulated genes are boxed in red. Downregulated genes are boxed in blue. 2

9 A upregulate downregulate Sucrose Export Sucrose Starch fats Glyoxylate cycle Hexose Hexose Phosphate Cell wall Triose Phosphate Triglycerides Nucleic Acids Aromatic Amino Acids Lignin Polyphenols Pyruvate Acetyl-CoA Terpenes Fatty Acids Fatty acid biosynthesis Malate Citric acid cycle Amino Acids (glutamate) α-ketoglutarate B O COOH Sucrose Export Sucrose Starch fats Glyoxylate cycle Hexose Hexose Phosphate Cell wall Triose Phosphate Triglycerides Nucleic Acids Aromatic Amino Acids Lignin Polyphenols Pyruvate Acetyl-CoA Terpenes Fatty Acids Fatty acid biosynthesis Malate Citric acid cycle Amino Acids (glutamate) α-ketoglutarate Figure 5. Gene expression changes in carbohydrate metabolism in Populus leaves in response to (A) gypsy moth herbivory, and (B) jasmonic acid. A red circle indicates upregulation of a gene at a particular point in the pathway. A blue X indicates downregulation of a gene. Where there is both a circle and X, the enzyme is encoded by a multi-gene family, and one locus was upregulated, while another was downregulated. Carbohydrate metabolic pathways diagram modified from Buchanan et al. (2001).