Symplastic Phloem Loading in Poplar

Sap is driven through phloem sieve tubes by an osmotically generated pressure gradient between source and sink tissues. In many plants, source pressure results from thermodynamically active loading in which energy is used to transfer sucrose (Suc) from mesophyll cells to the phloem of leaf minor veins against a concentration gradient. However, in some species, almost all trees, correlative evidence suggests that sugar migrates passively through plasmodesmata from mesophyll cells into the sieve elements. The possibility of alternate loading mechanisms has important ramifications for the regulation of phloem transport and source-sink interactions. Here, we provide experimental evidence that, in gray poplar (Populus tremula × Populus alba), Suc enters the phloem through plasmodesmata. Transgenic plants were generated with yeast invertase in the cell walls to prevent Suc loading by this route. The constructs were driven either by the constitutive 35S promoter or the minor vein-specific galactinol synthase promoter. Transgenic plants grew at the same rate as the wild type without symptoms of loading inhibition, such as accumulation of carbohydrates or leaf chlorosis. Rates of photosynthesis were normal. In contrast, alfalfa (Medicago sativa) plants, which have limited numbers of plasmodesmata between mesophyll and phloem, displayed typical symptoms of loading inhibition when transformed with the same DNA constructs. The results are consistent with passive loading of Suc through plasmodesmata in poplar. We also noted defense-related symptoms in leaves of transgenic poplar when the plants were abruptly exposed to excessively high temperatures, adding to evidence that hexose is involved in triggering the hypersensitive response.In the mid-1930s, several laboratories discovered that sugar concentrations are higher in the phloem than in mesophyll cells, where the sugar is synthesized (Crafts, 1961). These findings led to the concept of thermodynamically active phloem loading, in which Suc and other transport compounds are transferred into the sieve tubes against a concentration gradient. The idea was rapidly accepted, in part because it was consistent with the pressure flow hypothesis proposed earlier by Münch (1930). Münch (1930) had suggested that sap is propelled through the sieve tubes by a pressure gradient between the leaves (sources) and sinks (Patrick, 2012; De Schepper et al., 2013; Stroock et al., 2014), and because elevated solute levels increase hydrostatic pressure, it was reasonable to assume that the energy used to load the phloem generates the pressure at the source end of the transport stream needed to drive long-distance transport.However, it is important to note that the hypothesis by Münch (1930) predated the discovery of active phloem loading. Münch (1930) assumed that the upstream pressure is generated in the mesophyll cells and not the phloem and that carbohydrate is carried passively from the mesophyll into the sieve tubes (Münch, 1930). The two hypotheses, active and passive loading, lead to different perspectives on several important aspects of phloem physiology, including the regulated entry of ionic and molecular species into the transport system and the mechanisms of source-sink signaling.We provide evidence here that phloem loading of Suc in poplar (Populus tremula × Populus alba) is passive, as envisioned by Münch (1930). The reason for choosing poplar for study is that there is correlative evidence consistent with a passive loading mechanism in this species. First, the mesophyll cells and minor vein phloem of poplar are linked by plasmodesmata that are much more dense than those at the same interfaces in plants known to load through the apoplast (Russin and Evert, 1985). Second, the osmotic potential of the sieve element-companion cell complex in the minor veins, estimated by plasmolysis, is lower than commonly found in herbaceous plants and in the same range as that of the mesophyll cells (Russin and Evert, 1985). In species that load actively, the osmotic potential in the phloem is generally, but not always, well above that in the photosynthetic cells.Although these data are suggestive, they are only correlative and for several reasons, inconclusive (see “Discussion”). In the studies reported here, we experimentally tested the hypothesis of passive loading in poplar by introducing yeast invertase to the apoplast of transgenic plants. Invertase in the cell walls inhibits apoplastic loading by hydrolyzing Suc en route to the phloem (von Schaewen et al., 1990; Dickinson et al., 1991; Heineke et al., 1992). For comparison, we conducted the same experiments on alfalfa (Medicago sativa), which on the basis of low plasmodesmata numbers in the minor vein phloem (Gamalei, 1991), loads from the apoplast. Invertase-expressing alfalfa exhibited well-documented symptoms of loading inhibition: elevated foliar sugar and starch, leaf chlorosis, and slow growth. In contrast, transgenic poplar grew normally and accumulated little, if any, excess sugar and starch in the leaves, and it did so even under high light conditions, where sugar synthesis is most active and the loading mechanism is most challenged. The results are consistent with passive, symplastic (through plasmodesmata) phloem loading in poplar.