Xyloglucan deficiency leads to a reduction in turgor pressure and changes in cell wall properties, affecting early seedling establishment.

Xyloglucan is believed to play a significant role in cell wall mechanics of dicot plants. Surprisingly, Arabidopsis plants defective in xyloglucan biosynthesis exhibit nearly normal growth and development. We investigated a mutant line, cslc-Δ5, lacking activity in all five Arabidopsis cellulose synthase like-C (CSLC) genes responsible for xyloglucan backbone biosynthesis. We observed that this xyloglucan-deficient line exhibited reduced cellulose crystallinity and increased pectin levels, suggesting the existence of feedback mechanisms that regulate wall composition to compensate for the absence of xyloglucan. These alterations in cell wall composition in the xyloglucan-absent plants were further linked to a decrease in cell wall elastic modulus and rupture stress, as observed through atomic force microscopy (AFM) and extensometer-based techniques. This raised questions about how plants with such modified cell wall properties can maintain normal growth. Our investigation revealed two key factors contributing to this phenomenon. First, measurements of turgor pressure, a primary driver of plant growth, revealed that cslc-Δ5 plants have reduced turgor, preventing the compromised walls from bursting while still allowing growth to occur. Second, we discovered the conservation of elastic asymmetry (ratio of axial to transverse wall elasticity) in the mutant, suggesting an additional mechanism contributing to the maintenance of normal growth. This novel feedback mechanism between cell wall composition and mechanical properties, coupled with turgor pressure regulation, plays a central role in the control of plant growth and is critical for seedling establishment in a mechanically challenging environment by affecting shoot emergence and root penetration.

Actin is involved in pollen tube tropism through redefining the spatial targeting of secretory vesicles.

In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood. We designed an experimental setup using a microscope-mounted galvanotropic chamber that allowed us to induce the redirection of in vitro pollen tube growth through a precisely timed and calibrated external signal. Actin destabilization, reduced calcium concentration in the growth medium and inhibition of calcium channel activity decreased the responsiveness of the pollen tube to a tropic trigger. An increased calcium concentration in the medium enhanced this response and was able to rescue the effect of actin depolymerization. Time-lapse imaging revealed that the motion pattern of vesicles and the dynamics of the subapical actin array undergo spatial reorientation prior to the onset of a tropic response. Together these results suggest that the precise targeting of the delivery of new wall material represents a key component in the growth machinery that determines directional elongation in pollen tubes.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Seedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook. This auxin maximum induces PP2C.D1 expression, leading to asymmetrical H+-ATPase activity across the hypocotyl that contributes to the differential cell elongation underlying hook development. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, thereby driving hypocotyl elongation. Our findings demonstrate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.

Spatial and temporal expression of actin depolymerizing factors ADF7 and ADF10 during male gametophyte development in Arabidopsis thaliana.

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.

Galvanotropic Chamber for Controlled Reorientation of Pollen Tube Growth and Simultaneous Confocal Imaging of Intracellular Dynamics.

Successful fertilization and seed set require the pollen tube to grow through several tissues, to change its growth orientation by responding to directional cues, and to ultimately reach the embryo sac and deliver the paternal genetic material. The ability to respond to external directional cues is, therefore, a pivotal feature of pollen tube behavior. In order to study the regulatory mechanisms controlling and mediating pollen tube tropic growth, a robust and reproducible method for the induction of growth reorientation in vitro is required. Here we describe a galvanotropic chamber designed to expose growing pollen tubes to precisely calibrated directional cues triggering reorientation while simultaneously tracking subcellular processes using live cell imaging and confocal laser scanning microscopy.

Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen.

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5 degrees C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical.

Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry.

Fast directional growth is a necessity for the young seedling; after germination, it needs to quickly penetrate the soil to begin its autotrophic life. In most dicot plants, this rapid escape is due to the anisotropic elongation of the hypocotyl, the columnar organ between the root and the shoot meristems. Anisotropic growth is common in plant organs and is canonically attributed to cell wall anisotropy produced by oriented cellulose fibers. Recently, a mechanism based on asymmetric pectin-based cell wall elasticity has been proposed. Here we present a harmonizing model for anisotropic growth control in the dark-grown Arabidopsis thaliana hypocotyl: basic anisotropic information is provided by cellulose orientation) and additive anisotropic information is provided by pectin-based elastic asymmetry in the epidermis. We quantitatively show that hypocotyl elongation is anisotropic starting at germination. We present experimental evidence for pectin biochemical differences and wall mechanics providing important growth regulation in the hypocotyl. Lastly, our in silico modelling experiments indicate an additive collaboration between pectin biochemistry and cellulose orientation in promoting anisotropic growth.

High Light Intensity Leads to Increased Peroxule-Mitochondria Interactions in Plants.

Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas, stress mitigation favors peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 min of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further, it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell.