Staff Publications

This thesis deals with Nuclear Magnetic Resonance (NMR) imaging of long distance transport in plants. Long distance transport in plants is an enigmatic process. The theoretical framework that describes its basic properties has been in place for almost a century, yet at the same time only little is known about the dynamics of long distance transport inside the living plant. The latter is caused by the fact that the two pathways in which transport takes place, the xylem and the phloem, are virtually inaccessible to invasive experimentation. As a result a wide range of questions about the dynamics of long distance transport have yet to be answered. Examples of such questions, as addressed in this study, are: how fast does phloem sap move; how variable is the phloem sap flow velocity between species and over the diurnal cycle; what percentage of the potential flow conducting area in xylem tissue is functional. Or with regard to fruits: what percentage of the influx to fruits occurs through the xylem, what percentage through the phloem; does the xylem remain functional throughout fruit development; and does backflow from the fruit to the plant occur. Here, we show that NMR flow imaging provides a non-invasive and quantitative means to answer these intriguing questions.
In order to be able to compare the results from different plants, the flow imaging data need to be independent from anatomical characteristics such as conduit diameter. To quantify flow, the signal from a known quantity of water in a reference object is compared with the signal from flowing water in the plant. When the NMR signals arising from the flowing water in the plant and that of the reference object have different relaxation rates, quantification problems may arise. In porous materials relaxation is influenced by pore diameter. Correspondingly, the relaxation of flowing water in plants will be influenced by conduit diameter. We developed a T2 resolved flow imaging method to measure the T2 relaxation behaviour of the flowing water (chapter 2), and used it to determine how xylem conduit diameter affects T2 relaxation. Furthermore, we investigated whether conduit diameter dependent T2 changes need to be corrected for when quantifying results of NMR flow imaging (chapter 3). We found that conduit diameter indeed affected the T2 of the flowing water. However, because the effects were relatively small, T2 resolved flow imaging was not needed to correct for conduit diameter induced changes in T2. Standard, non flow resolved T2 imaging sufficed. The accuracy of the quantification of volume flow in the xylem in all cases was ±10% or better. The T2 resolved flow imaging sequence that was developed will be of interest for research that deals with liquids moving through microscopic conduits, such as porous media, bioreactors, and biomats.
With regard to the dynamics and basic properties of xylem and phloem transport, three subjects are taken into account: xylem transport, phloem transport, and long distance transport to fruits.
For the xylem, one of the questions to be solved is how much of the potential flow conducting area that is present in the xylem anatomy, is in reality used to conduct flow. Xylem tissue consists of many conduits of varying diameters, ranging from small to large. We investigated the relationship between xylem conduit diameter distribution and flow conducting area in stems of various plant species (chapter 3), and the relation between the diurnal dynamics of xylem sap flow and the flow conducting area (chapter 4). We found that in the stems with the widest conduits only a small proportion of the potential flow conducting area conducted flow (as low as 31%). In stems that only possessed narrow conduits, a much larger part of the total xylem conduit cross sectional area conducted flow (up to 86%). We conclude that when wide conduits are present and are functional, the role of the narrowest conduits in terms of the conductance of water is almost negligible. Secondly, we found that the flow conducting area does not stay constant throughout the diurnal cycle. Decreases in xylem flux at night were accompanied by a decrease in velocity, but also by a decrease in flow-conducting area. It is well known that plant stems exhibit a diurnal pattern of shrinkage and expansion due to changes in xylem pressure. However, the diurnal changes in the flow conducting area were opposite to the changes in stem diameter and thus could not be explained by pressure dependent elastic changes in conduit diameter.
With regard to the phloem we investigated the following questions: how fast does phloem sap move, how variable is the sap flow velocity between species and over the diurnal cycle; and what percentage of the shoot-bound xylem sap is returned to the root system by means of the phloem (chapter 4). Furthermore we investigated how phloem volume flow responds to local cooling (chapter 6). We compared the diurnal phloem and xylem flow dynamics in poplar, tomato, castor bean, and tobacco. In contrast to the highly variable sap flow velocities in the xylem, the sap flow velocities in the phloem remained very constant throughout the diurnal cycle. The differences in the average phloem flow velocity between the four species also were remarkably small (0.25 - 0.40 mm/s). We hypothesize that upper and lower bounds for phloem flow velocity may exist: when phloem flow velocity is too high, wall bound (parietal) organelles may be stripped away from sieve tube walls; when sap flow is too slow or is highly variable, phloem borne signalling could become unpredictable. The phloem to xylem volume flow ratio reflects the amount of xylem water that within the plant is (re)used for phloem transport. It may be indicative for the water use efficiency of a plant. This ratio was surprisingly large at night for poplar, castor bean and tobacco (ranging from 0.19 for poplar to 0.55 in tobacco), but as low as 0.04 in tomato.
With regard to long distance transport to fruits we investigated three long-standing questions: how much of the influx into the fruit occurs by means of the xylem, and how much by means of the phloem; does the xylem remain functional throughout fruit development; and does backflow from the fruit to the plant occur. As a model system we used a tomato truss. We found that xylem transport into the truss remained functional throughout the full 8 weeks of truss growth. During that period at least 75% of the net influx occurred through the xylem, and about 25% through a region that contains both internal phloem and internal xylem (perimedullary region). These results contradict earlier estimates that were made on the basis of indirect measurements. Halfway during truss development a xylem backflow to the plant appeared. However, the influx volume always was larger, implying that there was no net loss of water from the truss to the plant. Interestingly a circulation of xylem sap in the truss stalk remained even after the fruits were removed, probably caused by pressure gradients originating from the main stem. During the experiment about half of the cumulative net influx into the truss was lost to the air due to evaporation.
Because of the extreme sensitivity of xylem and phloem to invasive experimentation, only little is known about the dynamics of long distance transport in the living plant. The fact that the exploratory NMR flow imaging experiments in this study swiftly turned up a number of surprises with regard to the dynamics of flow underscores this observation. We found indications that phloem flow velocity is much more constant in nature than previously assumed, over the course of a day as well as between species. We observed that, depending on the species, at night a significant amount of xylem water can be recycled by means of the phloem, thus helping to maintain xylem circulation during periods of low transpiration. With regard to long distance transport to fruits, we found that during truss growth in tomato the majority of water influx does not occur by means of the phloem, but through the xylem. These results illustrate that the dynamics of xylem and phloem sap flow in the living plant are far from being understood, but also that NMR flow imaging provides an excellent non-invasive tool to help elucidate it.

Pseudotsuga taxifolia type, to which belong many Pinaceae (while the other Pinaceae species belong to a subtype, e.g. the Tsuga canadensis subtype);

Gingko biloba type, to which belong the Cycadaceae, Araucariaceae and many Podocarpaceae and Taxaceae;

Chamaecyparis pisifera type, to which belong the Cupressaceae, Taxodiaceae and the rest of the Taxaceae and Podocarpaceae.

The phylogenetic sequence of the axial system and the reduction of the phloem rays starts in the Ps. taxifolia type with an almost uniform axial system and ray- albuminous cells, and a complex heterogeneous phloem ray, differentiating through the G. biloba type to the C. pisifera type, which has a highly specialized axial system with phloem-albuminous cells and a simpler, reduced homogeneous phloem ray. The reduction of the phloem rays parallels the differentiation of the axial system.