Phloem anatomy predicts berry sugar accumulation across 13 wine-grape cultivars.

Introduction: Climate change is impacting the wine industry by accelerating ripening processes due to warming temperatures, especially in areas of significant grape production like California. Increasing temperatures accelerate the rate of sugar accumulation (measured in °Brix) in grapes, however this presents a problem to wine makers as flavor profiles may need more time to develop properly. To alleviate the mismatch between sugar accumulation and flavor compounds, growers may sync vine cultivars with climates that are most amenable to their distinct growing conditions. However, the traits which control such cultivar specific climate adaptation, especially for °Brix accumulation rate, are poorly understood. Recent studies have shown that higher rates of fruit development and sugar accumulation are predicted by larger phloem areas in different organs of the plant. Methods: Here we test this phloem area hypothesis using a common garden experiment in the Central Valley of Northern California using 18 cultivars of the common grapevine (Vitis vinifera) and assess the grape berry sugar accumulation rates as a function of phloem area in leaf and grape organs. Results: We find that phloem area in the leaf petiole organ as well as the berry pedicel is a significant predictor of °Brix accumulation rate across 13 cultivars and that grapes from warm climates overall have larger phloem areas than those from hot climates. In contrast, other physiological traits such as photosynthetic assimilation and leaf water potential did not predict berry accumulation rates. Discussion: As hot climate cultivars have lower phloem areas which would slow down brix accumulation, growers may have inadvertently been selecting this trait to align flavor development with sugar accumulation across the common cultivars tested. This work highlights a new trait that can be easily phenotyped (i.e., petiole phloem area) and be used for growers to match cultivar more accurately with the temperature specific climate conditions of a growing region to obtain satisfactory sugar accumulation and flavor profiles.

Coordination Between Phloem Loading and Structure Maintains Carbon Transport Under Drought.

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.

Seasonal patterns of callose deposition and xylem embolism in five boreal deciduous tree species.

PREMISE: Phloem tissue allows for sugar transport along the entirety of a plant and, thus, is one of the most important anatomical structures related to growth. It is thought that the sugar-conducting sieve tube may overwinter and that its cells persist multiple seasons in deciduous trees. One possible overwintering strategy is to build up callose on phloem sieve plates to temporarily cease their function. We tested the hypothesis that five deciduous tree species produce callose on their sieve plates on a seasonal basis. METHODS: Young shoots of five deciduous tree species were sampled periodically between April 2019 and February 2020 in Edmonton, Alberta, Canada. After enzymatic digestion of cytoplasmic constituents, cross sections were imaged using scanning electron microscopy to observe and quantify the level of callose deposition at monthly intervals, and sieve plate pore size was measured. Using a conductivity apparatus, we measured xylem native embolism during these sampling periods. RESULTS: Contrary to past work on some of the same species, we found little evidence that sieve tubes overwinter by becoming occluded with callose. Instead, we found that most sieve plates remain open. Xylem embolism was minimal during the peak growing season, but increased over winter. CONCLUSIONS: Many species had been assumed to deposit callose on sieve plates over winter, though anatomical and phenological phloem data were sparse. Our data do not support this notion.

Super-Resolution Microscopy of Phloem Proteins.

Super-resolution microscopy bridges the gap between light and electron microscopy and gives new opportunities for the study of proteins that contribute to phloem function. The established super-resolution techniques are derived from fluorescence microscopy and depend on fluorescent dyes, proteins tagged with GFP variants or fluorochrome-decorated antibodies. Compared with confocal microscopy they improve the resolution between 2.5 and 10 times and, thus, allow a much more precise (co-) localization of membranes, plasmodesmata, and structural proteins. However, they are limited to thin tissue slices rather than intact plant organs and can only show immobilized or only slowly moving targets. Accordingly, the first super-resolution micrographs of the phloem were recorded from fixed tissue which was sectioned using a vibratome or microtome. As with transmission electron microscopy, preparation of phloem tissue for super-resolution microscopy is challenged by the sudden pressures release when dissecting the functional tissue (see Chapter 2 ).This chapter describes a protocol for investigation of proteins in the plasma membranes of sieve elements and companion cells. It illustrates how high-resolution fluorescence imaging can provide information that could not be obtained with confocal or electron microscopy. Further, a brief introduction outlines the theoretical background of super-resolution techniques suitable for phloem imaging and summarizes the findings of the first available super-resolution studies on the phloem. The protocol focusses on the crucial steps for super-resolution microscopy of immunolocalized phloem proteins, adjusted to the use of three-dimensional structured illumination microscopy (3D-SIM).

Aquaporins Respond to Chilling in the Phloem by Altering Protein and mRNA Expression.

Previous experiments using heat exchangers (liquid cooled blocks) to chill a portion of plant stem have shown a transient stoppage in phloem translocation and an increase in measured phloem pressure. Although a chilled-induced stoppage of phloem transport has been known for over 100 years, the mechanism of this phenomenon is still poorly understood. Recently, work has highlighted that aquaporins occur within the plasma membrane of the sieve tubes along the entire source-to-sink pathway, and that isoforms of these water channel proteins may change dynamically. Aquaporins show regulatory roles in controlling tissue and cellular water status in response to environmental hardships. Thus, we tested if protein localization and mRNA transcript abundance changes occur in response to chilling in balsam poplar (Populus balsamifera) using immunohistochemistry and qrtPCR. The results of the immunolocalization experiments show that the labeling intensity of the sieve elements treated for only 2 min of chill time significantly increased for PIP2. After 10 min of chilling, this signal declined significantly to lower than that of the pre-chilled sieve elements. Overall, the abundance of mRNA transcript increased for the tested PIP2s following cold application. We discuss the implication that aquaporins are responsible for the alleviation of sieve tube pressure and the resumption of flow following a cold-induced blockage event.

Integrating Prolonged Field Care Into Rough Terrain and Mountain Warfare Training: The Mountain Critical Care Course.

Current prolonged field care (PFC) training routinely occurs in simulated physical locations that force providers to continue care until evacuation to definitive care, as based on the staged Ruck-Truck-House-Plane model. As PFC-capable teams move further forward into austere environments in support of the fight, they are in physical locations that do not fit this staged model and may require teams to execute their own casualty evacuation through rough terrain. The physical constraints that come specifically with austere, mountainous terrain can challenge PFC providers to initiate resuscitative interventions and challenge their ability to sustain these interventions during lengthy, dismounted movement over unimproved terrain. In this brief report, we describe our experience with a novel training course designed for PFC-capable medical teams to integrate their level of advanced resuscitative care within a mountainous, rough terrain evacuation-training program. Our goals were to identify training gaps for Special Operations Forces medical units tasked to operate in a cold-weather, mountain environment with limited evacuation resources and the challenges related to maintaining PFC interventions during dismounted casualty movement.

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients.

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.

Are phloem sieve tubes leaky conduits supported by numerous aquaporins?

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.

Effects of phyllotaxy on biomechanical properties of stems of Cercis occidentalis (Fabaceae).

PREMISE OF THE STUDY: Phyllotaxy, the arrangement of leaves on a stem, may impact the mechanical properties of woody stems several years after the leaves have been shed. We explored mechanical properties of a plant with alternate distichous phyllotaxy, with a row of leaves produced on each side of the stem, to determine whether the nodes behave as spring-like joints. METHODS: Flexural stiffness of 1 cm diameter woody stems was measured in four directions with an Instron mechanical testing system; the xylem of the stems was then cut into node (former leaf junction) and nonnode segments for measurement of xylem density. KEY RESULTS: Stems had 20% greater flexural stiffness in the plane perpendicular to the original leaf placement than in the parallel plane. The xylem in the node region was more flexible, but it had significantly greater tissue density than adjacent regions, contradicting the usual correlation between wood density and stiffness. CONCLUSIONS: Nodes can behave as spring-like joints in woody plants. For plagiotropic shoots, distichous phyllotaxy results in stems that resist up-and-down bending more than lateral back-and-forth movement. Thus, they may more effectively absorb applied loads from fruits, animals, wind, rain, and snow and resist stresses due to gravity without cracking and breaking. Under windy conditions, nodes may improve damping by absorbing vibrational energy and thus reducing oscillation damage. The effect of plant nodes also has biomimetic design implications for architects and material engineers.