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INVESTIGATOR: Slewinski, T.

PERFORMING INSTITUTION:
CORNELL UNIVERSITY
ITHACA, NEW YORK 14853

ROLE OF PHLOEM LOADING ON GROWTH RATES IN POPLAR

NON-TECHNICAL SUMMARY: Commodities production from trees represents a significant portion of agricultural, manufacturing and energy business in the United States. Despite the critical importance of tree-based products in our daily lives, very little is known about the underlying physiology and genetics that govern the growth and resource allocation traits important for agricultural production. Phloem loading and transport plays a critical function in carbon allocation throughout the plant. Recent evidence indicates that phloem loading in poplar, and in many other tree species, is passive and therefore is not regulated by sucrose transporters. Rather, we propose that the synthesis of raffinose-family oligosaccharides (RFOs) plays an important regulatory role. The primary aim of this investigation is to determine the roles of RFOs in phloem loading and carbon transport in poplar (Populus trichocarpa), an important biofuels crop. By doing so, we test a new mechanistic model for passive phloem loading and the hypothesis that upregulating RFO synthesis in the phloem will increase growth rate and biomass accumulation. Therefore, results of this study could directly impact biofuels production. This is a fundamental research project that will advance our understanding of basic plant biology and has the potential for broad application in the renewable biofuels industry. This project will also advance the scientific, technical, and professional training of the postdoctoral fellow Dr. Thomas L. Slewinski.

OBJECTIVES: Goals: To determine the specific role of raffinose family oligosaccharides in phloem loading in poplar. Specific Hypotheses and Objectives: 1) RFO synthesis plays an important and previously unrecognized role in phloem transport in poplar. 2) More specifically, downregulation of RFO synthesis will severely inhibit phloem loading in poplar leading to reduced photosynthesis and growth rates. 3) Upregulating RFO synthesis in the phloem of poplar will stimulate photosynthesis by reducing mesophyll sucrose concentrations and will also increase carbohydrate transport to growing leaves. Increasing new leaf surface area will have an additional stimulatory effect, leading to substantially higher growth rates. 4) Facilitated export in poplar by upregulation of RFO synthesis will allow plants to take better advantage of additional photosynthate in high CO2 atmospheres. (This hypothesis is long-term and will not be addressed during the grant period.) By testing this new model of phloem loading, we could potentially increase the photosynthetic, growth, and biomass accumulation rates in poplar, which is an important biofuels crop.

APPROACH: Experimental- This study will investigate the role of Raffinose family oligosaccharides (RFOs) in phloem loading in the model tree species poplar. The specific genes that regulate RFO synthesis will be determined by quantitative expression analysis using Q-PCR on cDNA derived from multiple tissues at different stages of development. Candidate genes will be determined from expression profiling to construct RNA silencing constructs that will be transformed into poplar. The specific location of synthesis of RFOs will be determined by tissue dissection and carbohydrate analysis as well as 14C-CO2 pulse chase labeling studies. Transgenic poplar lines will be constructed to eliminate, as well as up-regulate, the production of ROFs in poplar. The impacts on phloem loading and whole plant physiology will be assayed by photosynthetic measurements, carbohydrate analysis, 14C-CO2 pulse-chase labeling studies, and determination of biomass accumulation rates.

PROGRESS: 2011/08 TO 2013/08
Target Audience: Academic community with primary focus on basic plant science. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This grant has given me the opportunity to design, execute, and analyze experimentation as a primary investigator as well as develop novel hypotheses for the evolution of phloem loading. This grant has given me the opportunity to design, execute, and analyze experimentation as a primary investigator as well as develop novel hypotheses. I also had the opportunity to participate in community outreach teaching as part of the Cornell Prison Education Program at Auburn Maximum Security Prison – broadening my teaching skills and audience. How have the results been disseminated to communities of interest? The major results of this project have been published or submitted to scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2011/08 TO 2013/08
What was accomplished under these goals? Over the two year duration of the grant we determined that phloem loading in poplar does have a significant symplastic component and partially involves the synthesis of ROFs. One of the major findings of this project is that poplar, as well as the majority of angiosperms, have structurally and functionally heterogeneous loading and transport phloem tissues. This finding was published and elaborated in Slewinski et al. 2013, where we describe the apparently wide-spread the structural and physiological patterns seen in the more exaggerated example of phloem heterogeneity - appear to be conserved throughout vascular plants, just manifesting in more subtle forms. Thus If minor vein phloem heterogeneity is as widespread as we suggest, then the emergence of a dominant form in one group or another in the present era likely occurs as an elaboration, rather than the de novo fabrication, of specific mechanisms.This new hypothesis for the physiology and evolution of phloem loading opens up many new avenues of investigation and engineering.

PUBLICATIONS (not previously reported): 2011/08 TO 2013/08
1. Type: Journal Articles Status: Published Year Published: 2013 Citation: Slewinski TL, Zhang C, Turgeon R. (2013)Structural and functional heterogeneity in phloem loading and transport. Front Plant Sci. 2013 Jul 5;4:244. doi: 10.3389/fpls.2013.00244. eCollection 2013
2. Type: Journal Articles Status: Published Year Published: 2013 Citation: Ruan YL, Patrick JW, Shabala S, Slewinski TL.(2013)Uptake and regulation of resource allocation for optimal plant performance and adaptation to stress. Front Plant Sci. 2013 Nov 14;4:455. doi: 10.3389/fpls.2013.00455. eCollection 2013.
3. Type: Journal Articles Status: Published Year Published: 2013 Citation: Slewinski, TL. (2013)Using evolution as a guide to engineer kranz-type c4 photosynthesis. Front Plant Sci. 2013 Jul 1;4:212. doi: 10.3389/fpls.2013.00212. eCollection 2013.
4. Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Cankui Zhang, Tomas L. Slewinski, Jianlei Sun, Jing Zhang, Zengyu Wang, and Robert Turgeon (2014)Symplastic loading in poplar (submitted)

PROGRESS: 2011/08/15 TO 2012/08/14
OUTPUTS: Within the first year of funding all of the proposed DNA constructs were generated and are currently being transformed into poplar. Several lines of transformed poplar are currently under analysis. Physiological analysis of the carbohydrate complement of the poplar leaf and phloem sap is underway. Presence of raffinose and stachyose in the phloem has been confirmed by 14C labeling studies. We are currently determining the specific location of the raffinose and stachyose synthesis within the cells of the phloem. RNAseq analysis of leaf tissues from Poplar, Tobacco and Maize grown under different regimes of sink source manipulation were performed. Genes related to carbohydrate metabolism and transport are specifically targeted. The data have been process and is currently being analyzed and confirmed. PARTICIPANTS: Participants Thomas L. Slewinski PI of the project Robert Turgeon Post Doctoral Adviser Alyssa Anderson Undergraduate researcher Robert Turgeon Advised Thomas L. Slewinski in the experimental design of the project as well as the writing of three published manuscripts. Thomas L. Slewinski has been acting as primary adviser to Alyssa Anderson over the last year. As a result of Alyssa's work, she was second author on one publication so far. It is anticipated that more publications will result from her current work. TARGET AUDIENCES: The findings of this project are applicable to the general audience of basic plant and evolution scientists as well as applied biofuels scientists. PROJECT MODIFICATIONS: RNA Seq analysis was substituted for QPCR due to the shift in gene expression technology from the time of proposal submission to the time the experiment was conducted.

IMPACT: 2011/08/15 TO 2012/08/14
The findings of this work are leading to a new mechanistic model of phloem loading in plants. It is likely that most plants are capable of "mixed loading" derived from functional heterogeneity within the cells that populate the companion cell sieve element complex. This new model has broad impacts for basic plant research and well as evolutionary biology. An invited review paper describing this model, entitled "Functional Heterogeneity in phloem loading and transport", is currently under construction and will be submitted in the spring of 2013. In the summer of 2013 the PI took the opportunity to participate in volunteer outreach teaching in the Cornell Prison Education Program as a Genetics instructor. The gold of this outreach was to teach a college-level scientific curriculum to a select group of students at Auburn Maximum Security Correctional Facility, supporting incarcerated persons academic ambitions and preparation for successful re-entry into society.

PUBLICATIONS: 2011/08/15 TO 2012/08/14
1. Thomas L. Slewinski, Alyssa Anderson, Cankui Zhang, and Robert Turgeon, 2012 Scarecrow plays a role in establishing kranz anatomy in maize leaves. Plant Cell and Physiology doi 10.1093 pcp pcs147
2. Thomas L. Slewinski, 2012 Non-structural carbohydrate partitioning in grass stems A target to increase yield stability, stress tolerance, and biofuels production. Invited Darwin Review Journal of Experimental Botany 63 4647 70