Plant growth relies on the activity of stem cells clustered at shoot tips, leaf margins, and root tips, and also stem cell clusters that are quiescent and await activation. The activity of these so-called meristems determines whether plants are tall with few branches, short and bushy, etc. Similarly, the architecture of the root system is determined by the activity of meristems at the tips of primary and lateral roots, and the initial activation of these meristems in root primordia. It is essential that the activity of the plant's many meristems is coordinated to produce a plant whose morphology is optimized for its habitat. Indeed, root and shoot system growth is known to be coordinated, but how this coordination occurs is poorly understood. Previous studies clearly demonstrated that artificially increasing sucrose within a physiological range in the leaves dramatically stimulates lateral root formation, suggesting that the sucrose normally produced by leaves through photosynthesis is regulating root system development. This implicates a long distance mechanism that communicates the sucrose status of the aerial tissues to the root system. Sucrose moves from shoot to root system via the phloem, where it is unloaded at root meristems based on the steep concentration gradient. Interestingly, the plant hormone auxin is also produced in the leaves and moved to the root system via the phloem, This project will investigate how photosynthate from aerial tissues drives phloem allocation to root meristems, delivering auxin and thereby regulating their activity. This project will use transgenic plants that alter the levels of sucrose and auxin translocated in the phloem, together with fluorescent dyes that allow direct visualization of phloem movement and delivery, to test this model. If successful, the results from this project will 1) demonstrate that phloem acts to integrate plant organ development at different locations; and 2) determine the molecular signal that is carried in the phloem. This would be one of very few examples in which coordinated development in the whole-plant is understood mechanistically at the physiological and molecular levels. The broader impacts of this project include training of students at all levels and scientific outreach to the community.
Intellectual Merit: We validated our model that increased photosynthetic sucrose leads to increased lateral root formation, but we did not obtain results to support the hypothesis that this results from an increase in phloem delivery to the LRP. In contrast, we did show that activated lateral root meristems in growing lateral roots have a higher rate of phloem delivery that correlates well with their growth rate. Our data did not support the hypothesis that increased sucrose leads to increased auxin delivery to the lateral root primordia. Our experiments showed that shoot and root growth are tightly coordinated with sucrose levels, and we demonstrated that this coordination is independent of the osmotic effect of the sucrose. However, we also demonstrated that alteration in nitrogen levels was able to break this coordinated growth pattern, again independent of the osmotic effect of the nitrogen. Our experiments identified a novel gene, LRD3. that is essential for early development of the phloem in the seedling root. This gene is expressed exclusively in phloem, and its absence results in abnormal phloem morphology and function. This is the first gene identified to function in early phloem development. Phloem morphological and functional defects spontaneously disappear after 9-11 days of growth, suggesting that there is a redundant pathway that control development of this critical organ. Exogenous auxin also ameliorates the effects of a mutation in LRD3, revealing an unexpected link between auxin and phloem development. Examination of root systems of mutant in LRD3 revealed that when phloem delivery is compromised, there is competition between primary root and lateral root meristems, and even among lateral root meristems for phloem delivery, and the "winner" of this competition becomes the dominant root in the root system. Our data suggest that restricted sucrose availability leads to defects in cell wall formation. Additional data shows that proper cell wall biosynthesis is critical for constraining lateral root formation, revealing a previously unappreciated role for cell wall composition in maintaining correct root system architecture. We have begun to develop a novel method of isolation of phloem sap from Arabidopsis without use of EDTA (which yields contaminated samples) or aphid stylets (which is highly laborious). The methos still needs validation, but if successful will have a tremendous impact on the field. Broader Impacts: This project resulted in 3 research publications, 1 book chapter and 2 Ph.D. theses. 2 high school students, 2 graduate students and 1 post doctoral fellow received training through the three years of the project, and the 2 graduate students earned Ph.D. degrees. The high school students were both women. One REU student, who was a member of an underrepresented minority group, briefly participated in the project as well. The PI has presented her research at other research universities. In addition, she has presented her work several times in the context of a broader explanation of modern plant biology approaches to crop improvement. These talks have been given to civic groups at their request and are intended to inform the listeners about the science underlying genetic modification of plants, and distinguish fact from fiction in the current public controversey on this topic. During the course of this project the PI gave these outreach lectures to regional and national meetings of nutritionists and dieticians, and also to the general public at a local Whole Foods. A lecture to the Dairy Association is scheduled. Slides and teaching materials were made available to sponsoring organizations and attendants for thier use in educational settings. In addition, the PI organized a seminar series for the Genetics, Genomics and Systems Biology graduate program at the University of Chicago. This series of seminars revolved around the theme of Biological Research for the Developing World. In this context the PI invited eminent plant biologist Jonathan Lynch, as well as entrepeneur/pediatrician/plant biologist Mark Manary and hosted dinners to give graduate students an opportunity to interact with these and other speakers in the series. These speakers included scientists working in crop improvement and plant biology, and their visits prompted discussions on genetic modification of plants and associated issues of intellectual property. This forum provided an invaluable opportunity for students to explore the relationship between research science and the complex socio-economic problems that research aims to solve.
