PREMISE OF THE STUDY: Aquaporin membrane water channels have been previously identified in the phloem of angiosperms, but currently their cellular characterization is lacking, especially in tree species. Pinpointing the cellular location will help generate new hypotheses of how membrane water exchange facilitates sugar transport in plants. METHODS: We studied histological sections of balsam poplar (Populus balsamifera L.) in leaf, petiole, and stem organs. Immuno‐labeling techniques were used to characterize the distribution of PIP1 and PIP2 subfamilies of aquaporins along the phloem pathway. Confocal and super resolution microscopy (3D‐SIM) was used to identify the localization of aquaporins at the cellular level. KEY RESULTS: Sieve tubes of the leaf lamina, petiole, and stem were labeled with antibodies directed at PIP1s and PIP2s. While PIP2s were mostly observed in the plasma membrane, PIP1s showed both an internal membrane and plasma membrane labeling pattern. CONCLUSIONS: The specificity and consistency of PIP2 labeling in sieve element plasma membranes points to high water exchange rates between sieve tubes and adjacent cells. The PIP1s may relocate between internal membranes and the plasma membrane to facilitate dynamic changes in membrane permeability of sieve elements in response to changing internal or environmental conditions. Aquaporin‐mediated changes in membrane permeability of sieve tubes would also allow for some control of radial exchange of water between xylem and phloem.

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade-off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier-Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.

Maintaining phloem transport under water stress is expected to be crucial to whole-plant drought tolerance, but the traits that benefit phloem function under drought are poorly understood. Nearly half of surveyed angiosperm species, including important crops, use sucrose transporter proteins to actively load sugar into the phloem. Plants can alter transporter abundance in response to stress, providing a potential mechanism for active-loading species to closely regulate phloem loading rates to avoid drought-induced reductions or failures in phloem transport. We developed an integrated xylem-phloem-stomatal model to test this hypothesis by quantifying the joint impacts of transporter kinetics, phloem anatomy, and plant water status on sucrose export to sinks. We parameterized the model with phloem hydraulic resistances and sucrose transporter kinetic parameters compiled from the literature, and simulated loading regulation by allowing loading rates to decline exponentially with phloem pressure to prevent excessive sucrose concentrations from inducing viscosity limitations. In the absence of loading regulation, where loading rates were independent of phloem pressure, most resistance values produced unrealistic phloem pressures owing to viscosity effects, even under well-watered conditions. Conversely, pressure-regulated loading helped to control viscosity buildup and improved export to sinks for both lower and higher resistant phloem pathways, while maintaining realistic phloem pressures. Regulation also allowed for rapid loading and export in wet conditions while maintaining export and viable phloem pressures during drought. Therefore, we expect feedbacks between phloem pressure and loading to be critical to carbon transport in active-loading species, especially under drought, and for transporter kinetics to be strongly coordinated with phloem architecture and plant water status. This work provides an important and underexplored physiological framework to understand the ecophysiology of phloem transport under drought and to enhance the genetic engineering of crop plants.

We studied the seasonal dynamics of xylem and phloem formation in four boreal tree species that differed in leaf phenology (evergreen vs. winter-deciduous) and wood anatomy (angiosperms vs. gymnosperms). We sampled branch cuttings of balsam poplar (Populus balsamifera), trembling aspen (Populus tremuloides), lodgepole pine (Pinus contorta), and Siberian larch (Larix siberica) in bi-weekly intervals from the beginning to the end of the growing season. Cross sections were stained with astra blue and safranin in order to assess the width of the current year's phloem and to distinguish between the current year's mature and enlarging/wall-thickening xylem. After enzymatic clearance of cytoplasmic components, we used scanning electron microscopy to observe seasonal patterns of callose deposition in sieve plates of balsam poplar. The sieve plate pores were not blocked by callose in April (prior to the start of the growing season) and in July. In October, after the end of the growing season, we found only minor callose accumulation, and only in a subset of samples. In three of the four species studied here, phloem formation in spring began before the start of xylogenesis, but the lag was shorter than what was previously reported for temperate environments with longer growing seasons. New xylem cells were first produced in trembling aspen, followed by lodgepole pine, balsam poplar and Siberian larch. Most of the xylem was produced in June and July, and all cells were mature in early September. Phloem production was mostly completed by early August. Balsam poplar had the shortest growing season in terms of cambial activity and leaf presence, suggesting a risk avoidance strategy with regards to frost damage.