Cryogenic imaging of the water conducting tissues of plants (the “xylem”) is a powerful tool for addressing fundamental questions in plant physiology. Examination of material frozen under natural conditions provides insight into the physical and physiological processes associated with sub zero temperatures, while rapid freezes can be used to “fix” in place the spatial distribution of water within plant tissues, thus allowing the effects of xylem tensions on cell walls to be studied. However, substantial skill is needed to obtain high quality images of frozen plant material, and thus at present very few groups are able to take full advantage of this approach. This Seed Money Project will allow my lab group to develop and demonstrate this expertise. A major factor contributing to the success of the studies outlined here is that they will be carried out in close collaboration with Dr. Marilyn Ball, Research School of Biological Sciences, Australian National University. Ball and members of her research group are the world leaders in both cryogenic imaging of plant tissues and the physics of freeze-tolerance in plants. Ball has agreed to help train members of my research group in the use of cryo-SEM for imaging plant tissues, allowing us to transfer the expertise that they have developed over the past decade back to Harvard where we can then make use of the cryo-SEM equipment recently acquired by CNS.

The proposed study has three major objectives: the first scientific, the second technical, and the third educational. The scientific goal is to use cryogenic imaging to address basic issues in xylem physiology. Two projects that require cryogenic imaging are outlined below. The second objective is to develop the technical expertise at Harvard for cryogenic imaging of xylem tissues. This includes both cryo-SEM and epi-florescence of frozen material in which autofluorescence of lignified cell walls allows the outline of the conducting cells to be determined. Cryo-SEM provides the greatest resolution of structure, as well as the potential for elemental analysis when combined with EDS capability. However, it is also relatively time-consuming and equipment intensive. In contrast, epi-fluorescence is relatively simple in principle, but requires specialized tissue holders and can only be used to look at lignified tissues. Both techniques will be developed here for a wide range of plant materials. The key steps in cryo-SEM relate to sample preparation (sectioning and coating); a component of this project is to visit the microscopy group at ANU so as to work side-by-side with their cryo-SEM expert, Dr. Cheng Huang. The final component of the study is to involve students in this work. Freezing represents a major environmental stress for plants in Massachusetts and the timing of the Harvard academic calendar makes freezing an appropriate topic for student research projects. During Fall semester 2004, I taught a graduate course on freezing in plants (OEB 212r, Advanced Topics in Plant Physiology) that required that students conduct an independent research project that involved cryo-imaging. It is anticipated that several honors thesis projects will result from the activities supported by this Seed Money Project.

Project Research

The water transport system in vascular plants can be “damaged” by freeze-thaw events if the air forced out solution during freezing expands to block xylem conduits. Whether or not bubbles expand during thawing is primarily a function of the radius (r) of the bubble formed and the difference between the pressure within the bubble (P_b) and the pressure in the xylem sap (P_x):

P_x = P_b – (2T/r)

where T is the surface tension of water (Young-Laplace equation). Because the absolute pressures within the xylem (P_x) are typically < 0 (as low as –2.5 MPa in some cases during freeze-thaw events), even small bubbles nucleated during freezing can result in the formation of an air emboli upon thawing.

The goal of this study is to understand how the size (diameter) of xylem conduits influences the size of gas voids formed during freezing. Although there is a fair amount of evidence to suggest that smaller diameter conduits are less sensitive to freeze-thaw induced cavitation, there has been no attempt to image frozen conduits to determine the size and orientation of gas voids. In this study we will freeze stems collected from species that have a range of conduit diameters and then image the still-frozen material using cryo-SEM. We will compare our empirical measurements of air spaces with values predicted from the speed of ice propagation as a function of conduit size and degree of super cooling, as well as the estimated distance that gases could diffuse through the xylem sap during the same period of time. This will be the first study that looks directly at what happens to dissolved gases during freezing, and thus is likely to advance significantly our understanding of how freezing influences xylem function. Once we have established the relation between conduit diameter and bubble size for fully hydrated material, we will explore interactions between conduit diameter and xylem tension (P_x) on bubble formation and freeze-thaw cavitation.

Project #2: Effects of xylem tensions on conduit wall deformation in leaves

Water transport through the xylem occurs along gradients in tension generated by the evaporation of water from leaf surfaces. Because of the substantial friction involved in moving large amounts of water through a series of narrow conduits, the gravitation potentials that must be overcome in lifting water from soil to leaves, and the work that must be done in extracting water from the soil matrix – absolute pressures within the xylem are typically well below zero (i.e., true negative pressures). Water in the xylem is maintained in this metastable state with a fair degree of stability due to the relative impermeability of the xylem walls to air, as well as the energy barrier for the de novo nucleation of a bubble of sufficient size that it will expand rather than collapse.

One of the most dramatic demonstrations of the existence of negative pressures in xylem conduits is the deformation of xylem conduits when the tensions within the xylem sap are large. Using either cryo-SEM or epi-fluorescence, one can examine the shape of conduits frozen under different xylem tensions to determine the mechanical properties of the wall material, as well as assess whether losses in xylem transport capacity under low water availability (= high xylem tensions) are due to conduit collapse or to cavitation. Leaves are at particular risk of xylem failure due to the high xylem tensions occurring at the distal end of the pathway, as well as the need to have relatively thin-walled conduits so as to facilitate the delivery of water to photosynthetic cells. However, conduit collapse has so far been demonstrated only in the leaves of conifers. We are interested in whether this occurs widely across vascular plants and thus the central objective of the study will be to determine the failure mode (i.e., collapse vs. cavitation) of conduits in leaves of a wide variety of plant material (e.g., ferns, cycads, conifers, etc.). We will image fine veins of leaves dried to different xylem tensions. Using cryo-SEM we can determine if conduits were water filled at the time the samples were flash-frozen or if they had already embolized. We will also use measurements of the degree of conduit deformation to estimate the material strength of the walls.

This project will allow my research group to become proficient in the use of cryo-SEM and to establish credibility in the area of freeze-thaw effects on xylem physiology. Because of the difficulties associated with obtaining high quality cryo-SEM images of xylem, it is essential to demonstrate that we have the capability to obtain such images at Harvard before we will be competitive for outside funding in this area. The opportunity to receive training in sample preparation by the very best plant cryo-SEM group makes me confident that we can achieve a great deal during the upcoming year. According to Ball, there are a number of small modifications associated with both the cryo-planing and coating that substantially improve image quality. The ANU microscopy group (in collaboration with the Canberra CSIRO folks) have spent the past 10 + years perfecting their cryo-SEM skills. Thus, the travel to Canberra will greatly speed our ability to make good use of the newly purchased cryo-SEM capability at CIMS. In addition, this visit will also allow us to interact closely with the Ball research group, with whom we will collaborate with on the conduit diameter/cavitation study. The supported work will also open many opportunities for student research projects, something that I hope to encourage by focusing OEB 212r on this topic.

Participants: Dr. Brendan Choat, Fulton Rockwell