Discussion

Light-responsive photosynthate translocation to fruits

Photosynthate translocation is directly or indirectly influenced by environmental factors such as light intensity, air temperature, drought stress and CO2 concentration. In protected horticulture, it is important to understand the response of photosynthate translocation to different environments in order to establish an environmental control system supporting high yield and high-quality fruit production. Many studies have therefore assessed the relationship between different environmental factors and dynamics of photosynthate translocation. Regarding air temperature, Yoshioka (1986) reported an increased translocation speed of photosynthates with an increasing temperature in petioles of tomato plants. Pickard & Minchin (1990) showed the inhibition of phloem translocation by abrupt 10°C temperature drops in the stems of Phaseolus vulgaris . Makino & Mae (1999) reviewed the effects of elevated CO2 levels on photosynthesis and plant growth. It was found that short-term CO2 enrichment stimulated the rate of leaf photosynthesis and enhanced plant biomass. In contrast, prolonged exposure to CO2 enrichment generally reduced the initial stimulation of photosynthesis and often suppressed leaf photosynthesis by means of secondary responses (e.g. accumulation of carbohydrates, decrease in leaf nitrogen and Rubisco concentration). Sung & Krieg (1979) described a reduction in sensitivity of photosynthetic assimilation and translocation to drought stress in leaves of cotton and sorghum. Furthermore, several studies describe the relationship between photosynthate translocation and light conditions because of their direct link through photosynthesis (Troughton et al. 1977; Lemoineet al. 2013; Lanoue et al. 2018) since light provides energy for the synthesis of sugars. Troughton et al. (1977) and Lanoue et al. (2018) evaluated the effects of light intensity and light quality on photosynthate translocation in leaves of maize and tomato, respectively, by using 11C- and14C-labelled CO2, respectively. Both of these studies found the straightforward result that photosynthate translocation increases as light intensity increases. Whereas all of the above-mentioned studies reported the influence of several factors on the photosynthate translocation within stem and leaf parts, we investigated the translocation dynamics to fruits. Therefore, both source organ (i.e. leaves) and sink organ (e.g. roots, young shoots, developing seeds and in this study in particular fruits) are important. Phloem mass flow between these organs is generally believed to be driven by an osmotically generated pressure gradient, a mechanism known as Münch’s flow (Münch 1930; Nobel 2009). Hereby, photosynthates generated in herbaceous crop leaves are mainly loaded into the phloem by active transport (Hammond & White 2008). As the photosynthate concentration in the phloem increases, its osmotic water potential decreases (more negative). Consequently, water moves from the xylem into the phloem by osmosis increasing the turgor pressure of the phloem (Lalonde et al. 2003). This pressure pushes the phloem’s content down to the sink organs where photosynthates are unloaded by active transport (Maynard & Lucas 1982; Lalonde et al. 1999; Hölttä et al. 2006). Hence, the photosynthate concentration of the phloem in sink organs is decreased, which increases the overall water potential of the phloem. Water molecules are hereby released from the phloem and return to the xylem (De Schepper et al. 2013).
We investigated the effect of light intensity and application of Munch’s theory to our study, while taking into account the positive correlation between light intensities and translocation in leaves as described earlier by Troughton et al. (1977) and Lanoue et al.(2018). We assumed an increasing amount of photosynthates to be loaded into the phloem tissue under high light intensity compared to low light intensity. This causes more water to flow into the phloem from xylem resulting in a high turgor pressure of the phloem. Finally, a high-pressure gradient is generated and photosynthate translocation is promoted from sources to sinks. Summarised, by altering the light intensity above the source leaf, we expected a linear increase in relative photosynthate translocation into strawberry fruits. However, we did not obtain a relationship between light intensity and relative photosynthate translocation rate (Fig. 4B). This finding can be attributed to the environment inside the exposure bag during PET measurements. During each PET experiment, the controlled air was supplied to the exposure bag at a rate of 400 mL min-1. This flow rate was too low to renew the air inside the exposure bag resulting in high relative humidity conditions of 94.61 ± 0.97 %. This environment created small water droplets at both the exposure bag and leaf surfaces, the former lowering the amount of light reaching the leaf surface, and the latter inhibiting the gas exchange through stomata (Ishibashi & Terashima 1995). This is shown by the lower photosynthesis measured during the PET measurements (Fig. 4A; grey dots) with respect to the light response curve of the same leaf.

Transpiration as a key effector on photosynthate translocation to fruits

Transpiration rate was found to be the main factor affecting relative11C-photosynthate translocation to strawberry fruits. This outcome is assumed to result from the decrease in xylem water potential at the source leaf. When transpiration rate increases, leaf xylem water potential becomes more negative (Alarcón et al. 2003; Dodd et al. 2009; Steppe et al. 2015). This decrease in water potential lowers water flow into the phloem from the xylem, causing a decrease in phloem turgor pressure. Less turgor pressure creates a decreasing pressure gradient between source and sink organs, thus generating less pressure flow in the phloem (Sung & Krieg 1979). This eventually results in a negative correlation between transpiration rate and relative photosynthate translocation rate. In other words, suppression of photosynthate translocation into fruits is caused by the promotion of transpiration rate at the source leaf.
Because of the difference in translocation distance that depends on plant size, the total amount of translocated11C-tracer to the fruits through the PET measurement was smaller in large plants compared to small plants. However, there was no difference in the relationship between the relative translocation rate and transpiration rate for larger plants (“Red-27mm-L” and “Red-18mm-L”) and smaller plants (i.e. “Red-27mm-S”, “White-22mm-S” and “Green-18mm-S”). On the other hand, for the same transpiration rate, a larger relative translocation rate is observed in larger plants. Larger plants can hence maintain translocation of photosynthates under higher transpiration rates (>c. 0.15 mmol H2O m-2s-1, i.e. the point from where zero photosynthate flow is observed in small plants) than smaller plants. For the biggest fruits on the large plants (i.e. “Red-27mm-L”) a less negative slope is observed compared to all other fruits. This could be related to the developmental stage of these fruits since they were already fully grown (bright red colour) at the beginning of the experiment whereas the other fruits, both red, white and green, were still growing. A linear increase of the tracer concentration with time was observed for the last 40 min of each TTC representing a constant flow of the tracer into the fruits.
Generally, the negative correlation between transpiration rate and relative photosynthate translocation rate was similarly observed in every PET measurement regardless of fruit’s developmental stage (colour and size). Therefore, it is considered that suppression of photosynthate translocation caused by the promotion of transpiration is a physical phenomenon associated with xylem water potential. The physiological factor related to fruit’s developmental stages, such as sink strength (Marcelis 1996), did not affect the mechanism of photosynthate translocation suppression associated with transpiration. This suggests that, in protected horticulture, the transpiration management of source leaf by environmental, for example vapour pressure deficit, control is an effective method which can similarly control the photosynthate translocation into fruits at all development stages. Furthermore, this also suggests the possibility that the rate of photosynthate translocation into fruits is promoted during night-time when transpiration in a leaf is supressed.

Future potential of PET to investigate photosynthate translocation

Positron emission tomography or PET was originally developed as a key diagnostic tool used clinically to follow-up and treat diseases by making use of positron-emitting radioisotopes. Given the in vivonature of this technique, its use has been extrapolated to plant science to measure the transport of nutrients as well as phytohormones. Despite the limited number of studies on plants, this imaging technique has shown its applicability to investigate photosynthate translocation to fruits (Kikuchi et al. 2008; Kawachi et al. 2011; Yamazakiet al. 2015; Hidaka et al. 2019). Nevertheless, the full potential of 11C-positron emission tomography remains largely unexploited (Hubeau & Steppe 2015). This can be related to the fact that important aspects of whole-plant carbon allocation occur late in development, i.e. when the plants are large. Most PET devices used in research have not been adjusted to support experiments on bigger plants which has restricted their use in plant biology (Karve et al.2015). Studies of large, full-grown plants are especially important because much of our food is produced as seed late in development, i.e. during the reproductive stage. In case of our study, it would have been advantageous if, aside from the fruits, also the source leaf and other leaves could have been imaged at the same time. As such it could be determined which fraction of the assimilated11CO2-tracer is used for storage, respired to the atmosphere or ends up in the fruits/other leaves. These difficulties could be overcome by making use of clinical PET, which is developed for human imaging, as these systems have two main advantages. Firstly, these imaging devices allow visualisation of bigger objects since they are characterised by a transverse and axial field of view (FOV) up to 85 and 26 cm, respectively (Vandenberghe & Marsden 2015; Vandenberghe et al. 2016). This is generally larger than the ones of small animal PET scanners (transverse and axial FOV of 10 and 7.5 cm in this study). Besides, clinical PET scanners are equipped with a moving bed on which the plant can be placed which enables the visualisation of bigger plants. A drawback of clinical PET systems is the lower spatial resolution (c. > 5 mm (Vandenberghe et al. 2016)) compared to the sub-millimetre resolution of small animal PET scanners (España et al. 2014; Fineet al. 2014). Hence, visualisation of smaller sized plant structures (e.g. petioles and peduncles) will be complicated using clinical PET systems in contrast to bigger sized fruits and whole leaves. The second advantage of the clinical PET systems is the fact that they are nearly exclusively used in combination with structural imaging like computed tomography (CT) or magnetic resonance imaging (MRI). Consequently, the functional information about the radiotracer under study provide by PET is reinforced with anatomical data provided by CT or MRI. Despite the intensive occupancy of these clinical PET systems, we believe that studies making use of these imaging devices will make a major contribution to reveal complicated in vivointeractions in plants (e.g. link between xylem and phloem tissue in larger trees).

Conclusion

Non-invasive, real time 11C-photosynthate translocation from source leaf to strawberry fruits was successfully dynamically visualised by making use of PET. To our understanding this study is the first to report dynamic tracing of photoassimilates in 3D. By manipulating the light intensity at the source leaf, we expected to affect the translocation rate. However, we found that photosynthesis is not the main driver for the photosynthate translocation to fruits since our results suggest transpiration being the key effector. This is an important finding with regard to the optimisation of commercial strawberry production. Furthermore, we acknowledge the advantage ofin vivo experiments based on radiotracers and suggest that plant sciences could benefit from using clinical PET devices to study intact plants.