Vascular plants use the phloem to transport photoassimilates via sieve elements, which are cells with modified anatomy to accommodate long distance transport. Mature sieve elements lack a majority of cell organelles to reduce friction in the tube system, but some organelles like the endoplasmic reticulum, mitochondria, phloem-proteins and sieve element plastids remain. Most of the functions of sieve element organelles are poorly characterized. This leads to the fact that sieve element plastids are often omitted from textbooks as a plastid type, although they apparently derive from proplastids. As they are ultrastructurally diverse, sieve element plastids have been used for taxonomical purposes, but functional information is scarce. Here we review the currently available literature on the structure and function of sieve element plastids.

The movement of molecules through plasmodesmata (PD) is a fundamentally important process in plant tissues. Several decades of investigations have provided deeper insights into the basics of diffusive and advective flow through PD. However, PD still represent one of the least understood plant structures of major importance. This is based on their complex variable shape-composed of proteins, membranes, and wall material at nanoscale. We critically discuss approaches to study movement through PD, including microinjection/microdiffusion, expression of fluorescent proteins and photoactivatable probes. In addition, we highlight physical principles that form the basis for movement through PD.

Most plant tissues are symplasms in which cells are connected by plasmodesmata, membrane-lined cytosplasmic bridges that enable diffusive and/or advective cell-to-cell movements of components of the cytosol. Current models assume that hydrodynamic radius alone governs the mobility of molecules through plasmodesmata. In contrast, physical theory predicts that nm-sized pores with electrically charged walls are selective for counterions. Narrow plasmodesmal pores lined by membranes-which carry negative surface charges-therefore should be permselective for cations. Quantifying cell-to-cell movements of fluorophores varying in molecular mass and electrical charge in cell types with differently sized plasmodesmata, we confirmed the applicability of physical theory to plasmodesmata. Surprisingly, narrow plasmodesmata were permselective for anions rather than cations, suggesting a major flaw in our current understanding of plasmodesma structure. We hypothesize that structural proteins known to exist in plasmodesmata may establish the cationic electrostatic environment required to explain our findings.

Plants respond to biotic and abiotic stresses through complex and dynamic mechanisms that integrate physical, chemical, and biological cues. Here, we present a multi-physics platform designed to systematically investigate these responses across scales. The platform combines a six-axis micromanipulator with interchangeable probes to deliver precise mechanical, electrostatic, optical, and chemical stimuli. Using this system, we explore calcium signaling in Arabidopsis thaliana, thigmonastic motion in Mimosa pudica, and chemical exchange via microinjection in Rosmarinus officinalis L. and Ocimum basilicum. Our findings highlight stimulus-specific and spatially dependent responses: mechanical and electrostatic stimuli elicit distinct calcium signaling patterns, while repeated electrostatic stimulation exhibited evidence of response fatigue. Thigmonastic responses in Mimosa pudica depend on the location of perturbation, highlighting the intricate bi-directional calcium signaling. Microinjection experiments successfully demonstrate targeted chemical perturbations in glandular trichomes, opening avenues for biochemical studies. This open-source platform provides a versatile tool for dissecting plant stress responses, bridging the gap between fundamental research and applied technologies in agriculture and bioengineering. By enabling precise, scalable, and reproducible studies of plant-environment interactions, this work offers new insights into the mechanisms underlying plant resilience and adaptability.

The ever increasing breadth of biological knowledge has led to recent efforts to combine information from various fields into cell– or tissue atlases. Anatomical features are the structural basis for such efforts, but unfortunately large scale analysis of subcellular anatomical traits is currently a missing feature. Similarly, small phenotypic alterations of organelle– or cell-specific anatomical traits, such as an increase of the total volume or the number of mitochondria in response to certain stimuli, are currently hard to quantify. To provide tools to extract quantitative information from available 3D microscopic datasets generated with methods such as serial block face scanning electron microscopy we a) developed much improved fixation and embedding protocols for plants to drastically reduce processing artifacts and b) generated an easy-to-use AI tool for quantitative analysis and visualization of large-scale data sets. We make this tool available as open source.

While plant cells are enclosed by rigid cell walls that counteract intracellular hydrostatic pressure[1][1], their plasma membrane, cytosol, and endoplasmic reticulum (ER) remain connected through plasmodesmata, nanoscopic cell wall pores[2][2]. Plasmodesmal cell-to-cell transport occurs in the cytosolic sleeve between the plasma membrane and the ER membrane[3][3]–[5][4], and is generally thought to be limited by the size of the moving particle alone[6][5]. Given that biological membranes carry negative electric surface charges[7][6]–[9][7], this steric notion conflicts with physical theory of ion diffusion in nanometer-sized pores with charged walls[10][8]. Quantifying the movements of differently sized and charged fluorescent dyes in Tradescantia stamen hairs, we found that anionic fluorophores of up to 1 kDa traversed plasmodesmata whereas much smaller cationic ones did not. While this agrees with theoretical expectations of different size exclusion limits for cations and anions, it questions current dogma concerning plasmodesma function and also structure, as it implies positively rather than negatively charged surfaces within plasmodesmal pores. Our findings call for re-evaluations of current models of symplasmic transport, especially of charged molecules like the phytohormone auxin (indole-acetic acid) and certain amino acids. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7 [7]: #ref-9 [8]: #ref-10

Cassava is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilate unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and timepoints to better understand source-sink carbon allocation. We find that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we show that tissue-specific changes in anatomy and NSC contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically, and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

A micro-cantilever technique applied to individual leaf epidermis cells of intact Arabidopsis thaliana and Nicotiana tabacum synthesizing genetically encoded calcium indicators (R-GECO1 and GCaMP3) revealed that compressive forces induced local calcium peaks that preceded delayed, slowly moving calcium waves. Releasing the force evoked significantly faster calcium waves. Slow waves were also triggered by increased turgor and fast waves by turgor drops in pressure probe tests. The distinct characteristics of the wave types suggest different underlying mechanisms and an ability of plants to distinguish touch from letting go.

Epidermal trichomes function as mechanosensors, but how trichome-less plants perceive mechanical forces remains unclear. Touching epidermal pavement cells with micro-cantilevers, we discovered distinct cytosolic calcium waves upon application and release of small forces. Thus, not only do plants perceive forces independently of trichomes, they may also distinguish touch from letting go.

Molecular diffusion in bulk liquids proceeds according to Fick's law, which stipulates that the particle current is proportional to the conductive area. This constrains the efficiency of filtration systems in which both selectivity and permeability are valued. Previous studies have demonstrated that interactions between the diffusing species and solid boundaries can enhance or reduce particle transport relative to bulk conditions. However, only cases that preserve the monotonic relationship between particle current and conductive area are known. In this paper, we expose a system in which the diffusive current increases when the conductive area diminishes. These examples are based on the century-old theory of a charged particle interacting with an electrical double layer. This surprising discovery could improve the efficiency of filtration and may advance our understanding of biological pore structures.

Analysis of proteins often requires generation of transgenic plants to gain knowledge of their in planta function. The acquisition of such plants represents a significant investment in terms of workload, time, and space. Here we describe an easy-to-use alternative system by transforming Brassica napus cv. Jet Neuf suspension cells derived from microspores. The cells can be transformed with high efficiency, and even double transformations with different transgenes are possible. In addition, we describe a procedure that allows for long-term storage to maintain diverse transgenic cell lineages with low effort, to build up cell suspension libraries for research purposes. Overall, the work can be helpful for scientists that want to use B. napus as a research organism in a reduced time-frame without the need for generating whole plants and the requirement of larger greenhouse spaces. This study establishes a protocol for generation of stable Brassica napus transgenic cell cultures, their long-term storage, and contains examples how these cell cultures can be used in studying gene function in planta without generating whole plants.

Phloem transport of photoassimilates from leaves to non-photosynthetic organs, such as the root and shoot apices and reproductive organs, is crucial to plant growth and yield. For nearly 90 years, evidence has been generally consistent with the theory of a pressure-flow mechanism of phloem transport. Central to this hypothesis is the loading of osmolytes, principally sugars, into the phloem to generate the osmotic pressure that propels bulk flow. Here we used genetic and light manipulations to test whether sugar import into the phloem is required as the driving force for phloem sap flow. Using carbon-11 radiotracer, we show that a maize sucrose transporter1 (sut1) loss-of-function mutant has severely reduced export of carbon from photosynthetic leaves (only ~4% of the wild type level). Yet, the mutant remarkably maintains phloem pressure at ~100% and sap flow speeds at ~50-75% of those of wild type. Potassium (K+) abundance in the phloem was elevated in sut1 mutant leaves. Fluid dynamic modelling supports the conclusion that increased K+ loading compensated for decreased sucrose loading to maintain phloem pressure, and thereby maintained phloem transport via the pressure-flow mechanism. Furthermore, these results suggest that sap flow and transport of other phloem-mobile nutrients and signalling molecules could be regulated independently of sugar loading into the phloem, potentially influencing carbon-nutrient homoeostasis and the distribution of signalling molecules in plants encountering different environmental conditions.

Although the phloem is a highly specialized tissue, certain pathogens, including phytoplasmas, spiroplasmas, and viruses, have evolved to access and live in this sequestered and protected environment, causing substantial economic harm. In particular, Candidatus Liberibacter spp. are devastating citrus in many parts of the world. Given that most phloem pathogens are vectored, they are not exposed to applied chemicals and are therefore difficult to control. Furthermore, pathogens use the phloem network to escape mounted defenses. Our review summarizes the current knowledge of phloem anatomy, physiology, and biochemistry relevant to phloem/pathogen interactions. We focus on aspects of anatomy specific to pathogen movement, including sieve plate structure and phloem-specific proteins. Phloem sampling techniques are discussed. Finally, pathogens that cause particular harm to the phloem of crop species are considered in detail.

In most plant tissues, threads of cytoplasm, or plasmodesmata, connect the protoplasts via pores in the cell walls. This enables symplasmic transport, for instance in phloem loading, transport and unloading. Importantly, the geometry of the wall pore limits the size of the particles that may be transported, and also (co-)defines plasmodesmal resistance to diffusion and convective flow. However, quantitative information on transport through plasmodesmata in non-cylindrical cell wall pores is scarce. We have found conical, funnel-shaped cell wall pores in the phloem-unloading zone in growing root tips of five eudicot and two monocot species, specifically between protophloem sieve elements and phloem pole pericycle cells. 3D reconstructions by electron tomography suggested that funnel plasmodesmata possess a desmotubule but lack tethers to fix it in a central position. Model calculations showed that both diffusive and hydraulic resistance decrease drastically in conical and trumpet-shaped cell wall pores compared with cylindrical channels, even at very small opening angles. Notably, the effect on hydraulic resistance was relatively larger. We conclude that funnel plasmodesmata generally are present in specific cell-cell interfaces in angiosperm roots, where they appear to facilitate symplasmic phloem unloading. Interestingly, cytosolic sleeves of most plasmodesmata reported in the literature do not resemble annuli of constant diameter but possess variously shaped widenings. Our evaluations suggest that widenings too small for unambiguous identification on electron micrographs may drastically reduce the hydraulic and diffusional resistance of these pores. Consequently, theoretical models assuming cylindrical symmetries will underestimate plasmodesmal conductivities.

Long-distance transport of photoassimilates in the phloem of vascular plants occurs as bulk flow in sieve tubes. These tubes are arrays of cells that lose nuclei, cytoskeleton, and some organelles when they differentiate into mature sieve elements. Symplasmic continuity is achieved by perforations that turn the cell walls between adjoining sieve elements into sieve plates. These structural features are interpreted as adaptations that reduce the resistance sieve tubes offer to cytoplasmic bulk flow. According to the common reading of Ernst Münch's pressure-flow theory, the driving forces for these flows are osmotically generated gradients of hydrostatic pressure along the sieve tubes. However, the significance of pressure gradients in the flow direction has also been questioned. Münch himself stated that no detectable pressure gradients existed between the linked osmotic cells that he used to demonstrate the validity of his ideas, and the earliest explanation of osmotically driven flows by Wilhelm Pfeffer, on which Münch based his theory, explicitly claimed the absence of pressure gradients. To resolve the apparent contradiction, we here reconstruct the history of the idea that osmotically driven transport processes in organisms necessarily require steps or gradients of hydrostatic pressure along the transport route. Our analysis leads us to conclude that some defects of overly simplifying interpretations of Münch's ideas (such as the sieve plate fallacy) could be avoided if our descriptions of his theory in textbooks and the scientific literature would follow the logics of the theory's earliest formulations more closely.

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins (SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (BK2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, BK2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning. Funding Information The research was supported by US National Science Foundation Plant Genome Research Program grants (IOS-1025976 and IOS-1444448) to DMB.