Progress 04/01/06 to 06/30/10
Outputs
OUTPUTS: During this final reporting period, the project resulted in three peer-reviewed publications (Silvera et al., 2009; Silvera et al., 2010a, Silvera et al., 2010b). A total of 12 poster presentations by Katia Silvera and Bahay Gulle at various national and international meetings. Dr. Silvera presented three oral presentations at national and international meetings. Dr. Cushman delivered three oral presentations about the project at the University of Liverpool, the National Autonomous University of Mexico, and the Ohio State University. Three additional research publications on the evolution of gene family structure and gene expression patterns are in progress. PARTICIPANTS: Senior project participants include: Klaus Winter, Senior Investigator, Smithsonian Tropical Research Institute, P.O. Box 0843-03092, Balboa, Ancon, Republic of Panama; Louis S. Santiago, Assistant Professor, Botany & Plant Sciences, University of California, 2150 Batchelor Hall, Riverside, CA 92521. Two graduate students (Katia Silvera and Bahay Gulle) were trained on the project. Both received advising and mentoring from the project director about their various research activities and their career development. One lab manager was trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno, NV 89557, USA. Seven undergraduate student researchers were trained on the project and are still pursuing undergraduate degrees or have entered graduate school to pursue Ph.D. degrees: Leticia Rodriguez (2006-2007), Laura Burke (2007-2008), Abdulrahman Busse (2008-2009), Jessica Grabow (2008-2009), J. Evan Villaluz (2009-present), Jeremiah Smith (2009-2010), and Antionette Gray (2010). Two high-school students, Kayla Taylor (2008) and Chris Castro (2009), participating in UNRs Upward Bound program, was also trained in the lab. TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community, specifically to research scientists and undergraduate and graduate students and post-doctoral fellow conducting research on the molecular mechanisms of CAM evolution. Outreach projects were targeted at K-12 students and the lay public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, with climatic variables and the evolution of epiphytism in tropical regions. This study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae (Silvera et al., 2009). A second manuscript on this survey work was published (Silvera et al., 2010a) along with a review article summarizing the state-of-the-art knowledge about CAM evolution (Silvera et al., 2010b). We have completed deep sampling of cDNA clones for the phosphoenolpyruvate carboxylase (PEPC), PEPC kinase, carbonic anhydrase, and glucose 6-phosphate transporter gene families from leaves and root tissues from four strong CAM species, three weak CAM species, and three C3 photosynthesis species. This work has revealed that weak and strong CAM species, as well as C3 species, recruit one specific isogene for apparent CAM function. Isogenes with CAM functionality are distinct from C3 and weak CAM isoforms indicating that recruitment to CAM function might be associated with discrete amino acid changes and enzymes characteristics. Full-length cDNA transcripts are being recovered in order to validate this hypothesis. Additional plastid genome sequencing of combined ITS, trnL-F and matK regions for species within the subtribe Oncidiinae was also completed to improve the resolution of the molecular phylogeny within the subtribe. We completed 454 sequencing of cDNA prepared from RNA sampling from leaf, flower tissue and pseudobulb tissue of the strong CAM species (Chelyorchis (Oncidium) ampliatum), which resulted in 189 Mb of DNA sequence, 41,115 contigs, and 100,889 singletons. A Nimblegen microarray containing 11 mer probesets for about 39,000 genes has been designed and fabricated. mRNA expression profiling was completed and showed that C3 and weak CAM species had average hybridization intensities that diverged from the strong CAM species by 2 and 3 percent, respectively. More than 2,000 genes showed a greater than five-fold increase in relative transcript abundance in CAM versus C3 species including many transcription factors. We also completed 24 h gas exchange for four C3 photosynthesis species, four weak CAM species, and three strong CAM species. Our research is providing novel insights into molecular mechanisms that occur during evolutionary progression towards CAM. Our mRNA expression studies have revealed that large-scale changes in the expression of thousands of genes in associated with the evolutionary progression from C3 photosynthesis to CAM.
Publications
- Silvera K, Santiago LS, Cushman JC, Winter K. (2010a) Incidence of crassulacean acid metabolism in the Orchidaceae derived from carbon isotope ratios: a checklist of the flora of Panama and Costa Rica. Botanical Journal of the Linnean Society. 163: 194-222.
- Silvera K, Neubig KM, Whitten WM, Williams NH, Winter K, Cushman JC. (2010b) Evolution along the Crassulacan acid metabolism continuum. Function Plant Biology. 37: 995-1010 (Cover article).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: During this reporting period, the project has resulted in two peer-reviewed publications (Silvera et al., 2009; Silvera et al., 2010). Two poster presentations by Katia Silvera and Bahay Gulle were given about the research project at the joint meeting of the American Society of Plant Biologists (ASPB) and the Phycological Society of America (PSA) on July 18-22, 2009 in Honolulu, Hawaii. One poster presentation by Katia Silvera was delivered at the 94th Ecological Society of America Annual Meeting, August 2-7, 2009, Albuquerque, New Mexico. Two poster presentations by Katia Silvera and Bahay Gulle were given about the research project at the First Annual UNR Molecular Biosciences Retreat, October 17, 2009 at the University of Nevada, Reno, NV. Finally, Dr. Cushman delivered three oral presentations about the project at the University of Liverpool, the National Autonomous University of Mexico, and the Ohio State University. PARTICIPANTS: Senior project participants include: Klaus Winter, Senior Investigator, Smithsonian Tropical Research Institute, P.O. Box 0843-03092, Balboa, Ancon, Republic of Panama; Louis S. Santiago, Assistant Professor, Botany & Plant Sciences, University of California, 2150 Batchelor Hall, Riverside, CA 92521. To date, two graduate students (Katia Silvera and Bahay Gulle) were trained on the project. Both are receiving advising and mentoring from the project director about their various research activities and their career development. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno, NV 89557, USA. Six undergraduate student researchers have been trained on the project and are still pursuing undergraduate degrees or have entered graduate school to pursue Ph.D. degrees: Leticia Rodriguez (2006-2007), Laura Burke (2007-2008), Abdulrahman Busse (2008-2009), Jessica Grabow (2008-2009), J. Evan Villaluz (2009-present), and Jeremiah Smith (2009-present). Two high-school students, Kayla Taylor (2008) and Chris Castro (2009), participating in UNRs Upward Bound program, was also trained in the lab. TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community, specifically to research scientists and undergraduate and graduate students and post-doctoral fellow conducting research on the molecular mechanisms of CAM evolution. Outreach projects were targeted at K-12 students and the lay public. PROJECT MODIFICATIONS: None.
Impacts
To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, with climatic variables and the evolution of epiphytism in tropical regions. This study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae (Silvera et al., 2009). A second manuscript on this survey work is in press (Silvera et al., 2010). We have completed deep sampling of cDNA clones for the phosphoenolpyruvate carboxylase (PEPC), PEPC kinase, carbonic anhydrase, and glucose 6-phosphate transporter gene families from leaves and root tissues from four strong CAM species, three weak CAM species, and three C3 photosynthesis species. This work has revealed that weak and strong CAM species, as well as C3 species, recruit one specific isogene for apparent CAM function. The PEPC genes with CAM functionality are phylogenetically distinct from C3 and weak CAM isoforms indicating that recruitment to CAM function is likely associated with discrete amino acid changes and enzymes characteristics. Full-length cDNA transcripts are being recovered in order to validate this hypothesis. Additional plastid genome sequencing of combined ITS, trnL-F and matK regions for species within the subtribe Oncidiinae was also completed to improve the resolution of the molecular phylogeny within the subtribe. We have completed 454 sequencing of cDNA prepared from RNA sampling from leaf, flower tissue and pseudobulb tissue of the strong CAM species (Chelyorchis (Oncidium) ampliatum), which resulted in 189 Mb of DNA sequence, 41,115 contigs, and 100,889 singletons. A Nimblegen microarray containing 11 mer probesets for about 39,000 genes has been designed and fabricated. mRNA expression profiling is now complete and shows that C3 and weak CAM species had average hybridization intensities that diverged from the strong CAM species by 2 and 3 percent, respectively. More than 2,000 genes showed a greater than five-fold increase in relative transcript abundance in CAM versus C3 species including many transcription factors. We have also completed 24 h gas exchange for four C3 photosynthesis species, four weak CAM species, and three strong CAM species. Today, global climate change is expected to impact on the future distribution and habitat selectivity of tropical orchid species. Our research is providing novel insights into molecular mechanisms that occur during evolutionary progression towards CAM. Our mRNA expression studies have revealed that large-scale changes in the expression of thousands of genes in associated with the evolutionary progression from C3 photosynthesis to CAM.
Publications
- Silvera K, Santiago LS, Cushman JC, Winter K. (2009) Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae. Plant Physiol. 149:1838-1847.
- Silvera K, Santiago LS, Cushman JC, Winter K. (2010) Incidence of crassulacean acid metabolism in the Orchidaceae derived from carbon isotope ratios: a checklist of the flora of Panama and Costa Rica. Botanical Journal of the Linnean Society. In press.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During this reporting period, the project has resulted in one peer-reviewed publication (Silvera et al., 2009). Four poster presentations and one oral presentation by Katia Silvera was presented about the research project. For K-12 and public outreach, a educational display about neotropical orchids, CAM, and tropical rainforest ecosystems was developed and presented at 1) the Botany & Plant Biology Joint Congress 2007 meeting of the American Society of Plant Biologists (ASPB) and the Botanical Society of America (BSA) in Chicago, IL, July 2007 and won the 2007 ASPB Education Booth Competition. The display was presented at the C4 and CAM satellite meeting at the University of Cambridge, UK (Summer 2007), the Moorbank Botanic Gardens in Newcastle, UK, University of Liverpool, Liverpool, UK, and the Wilbur May Arboretum, Reno, NV in 2008. PARTICIPANTS: Senior project participants include: Klaus Winter, Senior Investigator, Smithsonian Tropical Research Institute, P.O. Box 0843-03092, Balboa, Ancon, Republic of Panama; Louis S. Santiago, Assistant Professor, Botany & Plant Sciences, University of California, 2150 Batchelor Hall, Riverside, CA 92521. To date, two graduate students (Katia Silvera and Bahay Gulle) were trained on the project. Both are receiving advising and mentoring from the project director about their various research activities and their career development. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project: Rebecca Albion, Staff Research Associate II, University of Nevada, Reno, NV 89557, USA. Four undergraduate researchers were provided training on the project and are still pursuing undergraduate degrees or are about to enter graduate school to pursue Ph.D. degrees: Leticia Rodriguez (2006-2007), Laura Burke (2007-2008), Abdulrahman Busse (2008-present), Jessica Grabow (2008-present). On high-school student, Kayla Taylor (2008), participing in UNRs Upward Bound program, was also trained in the lab. TARGET AUDIENCES: The project results and outcomes were targeted to the scientific community, specifically to research scientists and undergraduate and graduate students and post-doctoral fellow conducting research on the molecular mechanisms of CAM evolution. Outreach projects were targeted at K-12 students and the lay public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, with climatic variables and the evolution of epiphytism in tropical regions. Altitude was the most important predictor of photosynthetic pathway when all environmental variables were taken into account, with CAM being most prevalent at low altitudes. By creating integrated orchid trees to reconstruct ancestral character states, we found that C3 photosynthesis is the ancestral state and that CAM has evolved at least 10 independent times with several reversals. A large CAM radiation event within the Epidendroideae, the most species-rich epiphytic clade of any known plant group, is linked to a Tertiary species radiation that originated 65 million years ago. Our study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae (Silvera et al., 2009). A second manuscript on this survey work is in preparation. We have completed deep sampling of cDNA clones for the phosphoenolpyruvate carboxylase (PEPC) and PEPC kinase gene families from leaves and root tissues from four strong CAM species, three weak CAM species, and three C3 photosynthesis species. This work has revealed that weak and strong CAM species, as well as C3 species, recruit one specific isogene for apparent CAM function. Additional plastid genome sequencing of combined ITS, trnL-F and matK regions for species within the subtribe Oncidiinae has also been completed to improve the resolution of the molecular phylogeny within the subtribe. We have completed 454 sequencing of cDNA prepared from RNA sampling from leaf, flower tissue and pseudobulb tissue of the strong CAM species (Chelyorchis (Oncidium) ampliatum), which resulted in 189 Mb of DNA sequence, 41,115 contigs, and 100,889 singletons. A Nimblegen microarray containing 11 mer probesets for about 39,000 genes has been designed and fabricated for mRNA expression profiling. We have also completed 24 h gas exchange for three C3 photosynthesis species, two weak CAM species, and three strong CAM species. Today, global warming and associated climate changes such as the availability of precipitation, particularly within tropical ecosystems, may have an impact on the future survival and success of orchid species. By studying the evolutionary mechanisms and the effects of water deficit on genetic changes in gene structure and expression that have occurred both over evolutionary time scales and instantaneously, we can better understand the molecular mechanism responsible for CAM evolution.
Publications
- Silvera K, Santiago LS, Cushman JC, Winter K. (2009) Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae. Plant Physiol. In press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Delta-13C isotope analysis was performed on 1,300 species of orchids (180 species within the Oncidiinae) with a goal of completing the analysis of about 5% of all orchid species. In addition, leaf thickness measurements were performed on 214 orchid species and titratable acidity measurements were performed on 173 orchid species. The delta-13C isotope values were mapped to habitat elevation for 466 species and to the terrestrial or epiphytic habitats for 826 species. Dr. Klaus Winter completed 24h gas exchange studies on the following species: CAM: Oncidium ampliatum, Oncidium carthagenense; Weak CAM: Oncidium sphacelatum, Oncidium panamense. Results from isotopic survey studies will be presented in contributed oral or poster presentations at the 2008 meeting of the American Society of Plant Biologists. Approximately 720 species of Oncidiinae were selected for phylogenetic analysis. These species have been vouchered, databased, and total DNA has been extracted. Three regions
were selected to sequence for phylogenetic analyses: nrITS, matK, and ycf1 (3' end). To date, the ITS region was sequenced for 800 accessions, matK for 700 accessions, and ycf1 for 700 accessions. The plastid rbcL and psaB and trnL-F genes were also sequenced for about 200 taxa. The sequence data are being prepared for deposition in GenBank in Spring 2008. All 700 accessions will be sequences at the 5' portion of ycf1 and the trnH-psbA spacer. These will provide additional resolution and support in the phylogeny. Results from phylogenetic studies will be presented in contributed lectures at the 2008 Botanical Society meetings. More than 1600 sequence reads from PEPC cDNA clones have been sampled to investigate the gene family structure evolution in CAM, weak CAM, and C3 species. mRNA expression pattern analysis has begun. RNA from different tissues (leaves, flowers, petioles, inflorescence spikes, pseudobulbs, and roots) has been isolated, and cDNA library construction is currently in
progress in preparation of 454 pyrosequencing sequencing.
PARTICIPANTS: The major outreach activity in the 2007 reporting period was the recruitment of two Hispanic graduate students, Leticia Rodriguez (formerly an undergraduate working on the project) and Leyla Hernandez, and two more underprivileged high school students (Christina Milner (Summer 2006) and Natalie Godinez and Sandra Ochoa (Summer 2007) to the project. Each of these students was trained under our "hands-on" training program and worked under the mentorship of graduate students (Katia Silvera and Leticia Rodriguez). Each student learned about the specific model gene families (either PEPC, CA, PPCK, or GPT) and orchid species under investigation and was trained how to perform RNA isolation, RT-PCR, gene cloning, plasmid DNA preparation, DNA sequencing, database searches and analysis of on-line annotation and information gleaned from scientific literature searches. In the next year, the students will investigate stress-inducible or diel mRNA expression changes using real time
RT-PCR or by data mining of microarray results. We have not yet been able to familiarize the students in 2D PAGE and western blot analysis, but are hoping to accomplish this in 2007. The major outreach activity in the 2007 reporting period was the continued training of graduate student Kurt Neubig. Plastid DNA sequence results generated by Mr. Neubig are being integrated into OrchidTree: Phylogeny of Epiphytes on the Tree of Life http://www.flmnh.ufl.edu/orchidatol/. DNA samples of Oncidiinae are being deposited in liquid nitrogen archival storage at the Florida Museum of Natural History Genetics Resources Repository (http://www.flmnh.ufl.edu/tissues/), with associated voucher specimens mostly deposited in the FLAS herbarium. These DNA samples will be available to other researchers. We are encouraging participation of under-represented and/or under-served groups participation by targeting recruitment from the UNR campus with the aid of three different outreach and mentoring programs
at UNR. We are working with two outreach programs for recruitment of undergraduate researchers: 1) the TRIO Scholars Program and 2) the McNair Scholars Program. To date we have trained one undergraduate underrepresented minority student, Leticia Rodriguez. We have also trained three underprivileged high school student, Christina Milner (Summer 2006), and Natalie Godinez and Sandra Ochoa (Summer 2007), through the Upward Bound's (UB) Summer Workshop Program. This program is designed to increase the rates at which under-represented high school students enroll in and graduate from college by exposing high school students to new information and laboratory experiences in a broad range of science-related areas. Leticia Rodriguez and Katia Silvera attended the Plant Biology 2007 meeting, presented their results as both and oral and a poster presentation, and help host the educational display. Katia Silvera also presented her results at the C4-CAM satellite meeting in the UK on July 2007, and
she is planning on presenting her results at the Isoscape Meeting in Santa Barbara, CA in April 2008, and the ASPB meetings in Merida, Mexico on the Summer 2008.
TARGET AUDIENCES: The completed version of the orchid educational display was presented at 1) the Botany & Plant Biology Joint Congress 2007 meeting of the American Society of Plant Biologists and the Botanical Society of America in Chicago, IL on July 7-11, 2007; 2) The C4 and CAM satellite meeting, Department of Plant Sciences, University of Cambridge, UK on July 17-20, 2007. Since that time, the display was installed for 6 months at the Moorbank Botanic Gardens in Glasgow, UK and is currently installed at the School of Biological Sciences at the University of Liverpool, Liverpool, UK. The display contains panels covering topics of orchid biology and classification, CAM biology and research, and rainforest ecology and conservation (Figure 1). The display won one of two competitive awarded prizes for participation in the ASPB Educational booth in 2007, which provided travel support to Cushman, Silvera, and Rodrguez. Because the materials and shipping costs for the display were
greater than anticipated (especially the shipping costs!), electronic distribution of the display has been made to the University of Florida, the Smithsonian Tropical Research Institute (Panama), and James Cook University (Australia). Dr. Cushman also demonstrated the "CAM kit", a stand-alone teaching module for the determination of CAM in plants using a simple pH indicator dye. A sample "CAM kit" was raffled off using a prize drawing and more than 20 different instructors were given the written lesson plan that outlined the use of the kit in high school or undergraduate plant biology classes. The lesson plan and kit is being used by at least five undergraduate faculty.
Impacts
Of the 1,300 species of orchids (180 species within the Oncidiinae) survey using delta-13C isotope analysis, approximately 11.2% of species (24% within the Oncidiinae) were found to perform CAM. If CAM was verified by titratable acidity measurements, then up to 50% of species were found to perform CAM or weak CAM. Leaf thickness also correlates positively with CAM. CAM is also positively correlated with lower elevations and epiphytic habitats. Among the three plastid genome regions being sequenced for phylogenetic analyses (e.g., nrITS, matK, and ycf1). The ycf1 region has not been utilized previously in phylogenetic studies, and appears to be the most variable gene in the chloroplast. The combined multigene data set is providing a highly resolved and well-supported phylogenetic tree that will be suitable for mapping of CAM-related traits. DNA sequence matrices are being prepared for submission to GenBank. The phylogenetic trees produced from this research are providing
the phylogenetic evidence for a generic reclassification of Oncidiinae by Mark Chase (Kew Gardens). Phylogenetic analysis of PEPC cDNA clone sequences has revealed that CAM species (e.g. Oncidium ampliatum, Oncidium carthagenense, Oncidium nanum and Rossioglossum insleayi) contain up to 5 isogenes, whereas weak CAM species (e.g., Oncidium panamense, Oncidium sphacelatum, Oncidium flexuosum) contain three isogenes, and C3 species (e.g. Oncidium maduroi, Onc. ornithorhynchum, Ticoglossum krameri) contain mainly two isoforms. So far, these observations are consistent with our hypothesis that during CAM evolution, the PEPC gene family has undergone expansion by duplication events with one isogene being recruited to fulfill the photosynthetic demands (nocturnal C4 carbon fixation) of CAM. Within the CAM species, we find that a single isogene accounts for between 70 and 85% of cDNA clones sampled suggesting that this isogene is expressed more strongly than the other isogenes, which only
account for between 3 and 18% of cDNA clones sampled. According to our hypothesis, these strongly expressed isogenes are likely to be the CAM-specific isogenes that have been recruited to perform a role in CAM. Phylogenetic analysis has revealed that the CAM-specific isogenes are more derived and form a separate clade, whereas the non-CAM isogenes are more closely related to ancestral C3 types of PEPC and are distributed across the phylogenetic tree. Interestingly, the most abundant isogenes in roots corresponds to the most abundant isogene in leaves, which provides evidence for this isogene being recruited for root-specific photosynthesis in orchids. Preliminary results suggests that there are several amino acid substitutions that are consistent amongst CAM-specific isoforms when compared to weak CAM and C3 species. This is a novel observation as previous efforts to identify isoform-specific amino acid changes were not possible, due to the phylogenetically diverse sample base of past
sample sets. Our focus on orchid species within the Oncidium clade has made this possible.
Publications
- Williams, N.H., J.D. Ackerman, E.M. Fernandez, and J. Llamacho. 2007 Antillanorchis: Its rediscovery and systematic position. Lindleyana (Orchids) 76: 856-858.
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Progress 01/01/06 to 12/31/06
Outputs
The water-conserving photosynthetic pathway known as Crassulacean acid metabolism (CAM) has evolved multiple times in 33 families and 328 genera comprising more than 6 percent of all vascular plant species making it the second most common mode of photosynthesis among vascular plants. Although the basic metabolic reactions required for CAM are well known, the molecular mechanisms responsible for the evolution of this important photosynthetic adaptation to water-limitation are completely uncharacterized. Current estimates indicate that approximately 50 percent of the 20,000 species within the Orchidaceae, the largest family of vascular plants, exhibit CAM photosynthesis. However, this estimate is based on surveys of less than approximately 3% of all species. Foliar carbon isotopic composition measurements and nocturnal acid accumulation measurements were conducted within this family with over 900 orchid species and more than 90 species within the subtribe Oncidiinae
being sampled. These results showed that CAM exhibits a bimodal distribution with the majority of species having foliar carbon isotopic composition values diagnostic of C3 photosynthesis and weak CAM and a minority of species with values diagnostic of strong CAM. The occurrence of CAM correlated well with lower elevations, greater leaf thickness, and epiphytic growth habit. Surveys for the presence of CAM coupled with a robust molecular phylogeny within the Subtribe Oncidiinae (Orchidaceae) suggest at least eight independent origins of CAM, with several reversals. We have completed the deep sampling of cDNA clones for the phosphoenolpyruvate carboxylase (Ppc) gene family for four strong CAM species (Oncidium ampliatum, Oncidium nanum, Oncidium carthagenese, and Rossioglossum insleayi). In each species, three different isogenes have been identified with one isogene exhibiting from 4-10-fold greater relative transcript abundance than the other 2 isoforms. This abundant isoform is
predicted to be a putative CAM-specific isoform that has been specifically recruited to fulfill the metabolic demands of primary carbon dioxide fixation during the performance of CAM. Additional Ppc gene sampling for weak CAM species (Oncidium panamense, Oncidium sphacelatum, Oncidium flexuosum, Oncidium dichromatichum) and C3 species (Oncidium stenotis, Oncidium cheirophorum, Oncidium maduroi, Ticoglossum kramerii) is in progress. Additional plastid genome sequencing of combined ITS, trnL-F and matK regions for species within the subtribe Oncidiinae has also been completed to improve the resolution of the molecular phylogeny within the subtribe.
Impacts
Increased understanding of the molecular mechanisms responsible for CAM evolution will provide new knowledge about the interaction between phylogenetic relationships and the occurrence of adaptive photosynthetic mechanisms that will likely have broad scientific interest. Water limitation and the resulting limitation of CO2 brought about by stomatal closure and reductions in atmospheric CO2 concentrations may have provided the selective pressures for the evolution of CAM over the last 40-100 million years. Today, global warming and associated climate changes such as the availability of precipitation, particularly within tropical ecosystems, may have an impact on the future survival and success of orchid species. By studying the evolutionary mechanisms and the effects of water deficit on genetic changes in gene structure and expression that have occurred both over evolutionary time scales and instantaneously, we can better understand the molecular mechanism responsible
for CAM evolution. In addition to the laboratory research, a hands-on outreach exhibit of tropical orchids will be developed that will provide an excellent means of outreach and integration of research and education. The exhibit will used in local area high-schools, community colleges, universities, and public venues to conduct community outreach to inform high school, college students, and the general public about the abundance and importance of ecophysiological diversity of terrestrial and epiphytic orchids in the neotropics and the larger role that tropical rain forests play in global climate change.
Publications
- No publications reported this period
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