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Borassi, C.

Publications and source records attributed to Borassi, C..

11 recordsLinked to original sources
bioRxiv2026

Plant Acupuncture: a low-cost and open-source device for local mechanical stimulation

Background: Mechanical signals are important regulators of cellular responses in plants. They guide plant development and can activate defense and repair mechanisms. Yet, the molecular mechanisms by which plants perceive, transduce, and interpret mechanical signals are still poorly understood. This is in part due to the lack of methods to apply local, precise and non-damaging mechanical forces to plant cells. Micro-indentation is highly suitable for this purpose, yet available instrumentation is often expensive and difficult to combine with high-resolution microscopy. Results: We designed an open-source and affordable modular indentation device, consisting of 3D printed elements, 3 commercially available piezo motors, and a variety of indentation needles. Due to its modularity, the setup can be readily adapted to meet experimental requirements and works on all microscopes with bespoke adaptors. We show that the setup can be used to explore both rapid and slower touch responses, exemplified by visualizing calcium waves and actin patches induced by touch. We also show that the setup is compatible with various plant species and tissues and can be combined with high-resolution functional imaging. Conclusions: The simple and flexible design of the indentation device presented in this paper ensures that any lab with a 3D printer can build their own setup at low cost and with minimal time investment. The system has a wide range of applications for live plant tissue, making indentation experiments and thereby plant mechanobiology studies, more accessible.

plant biology↗
bioRxiv2026

ER-located Ca2+ ATPase ACA2 regulates Ca2+ cytoplasmic pool linked to root hair growth in Arabidopsis thaliana

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is regulated by both intrinsic and environmental signals and is associated with the existence of a highly controlled cytoplasmic tip Ca{superscript 2} gradient, whose disruption impairs RH development. The molecular mechanisms underlying the Ca2+ homeostasis fine tuning and the Ca2+ organellar contributions to the cytoplasmic pool remain unclear. In the model plant Arabidopsis thaliana, many efflux routes are present, including those that employ Ca2+-pumps from the Autoinhibited Ca2+-ATPase (ACA) family. Here, we identified that the ER localized ACA2, and to a lower extent ACA7, are crucial ACAs required to control RH growth. By using genetically encoded Ca2+ biosensors we showed that Ca2+-dynamics are compromised in the aca2-2 mutant, having lower cytosolic Ca2+ concentration [Ca2+]cyt and growth rate, showing an altered homeostatic calcium setpoint compared to Col-0. Accordingly, the ACA2 mutation changed the dynamics of [Ca2+]cyt oscillations coupled to growth rate, inducing longer periods and more regular oscillations in the dominant high-frequency range (around 22 s), and slower oscillations (around 1 min) in the low-frequency range. Finally, expression of ACA2 with changes in four putative Ca2+ binding residues (ACA2{Delta}Ca2+) failed to rescue the RH growth phenotype in the aca2-2 mutant. Collectively, our findings indicate that ER-localized ACA2 and possibly ACA7 are crucial for modulating cytoplasmic Ca2+ signals, possibly composing a critical part of a negative feedback loop, and their absence leads to impairments in RH cell elongation.

plant biology↗
bioRxiv2026

Plant actin networks are rapid mechano-adaptive scaffolds

The actin cytoskeleton is among the most highly conserved eukaryotic structures. In animal cells, actin plays a key role in mechanobiology, serving as a structural scaffold that shapes cellular mechanics and a mechanoresponsive network that reorganizes in response to mechanical cues. In plants, these roles are generally attributed to the cell wall and the microtubule cytoskeleton. This leaves the role of plant actin in mechanical responses largely unclear. Here, we ask whether a key actin feature that is essential for animal mechanobiology may also be conserved in plants. We use quantitative live-cell imaging and quantitative image analysis to show that plant actin networks are rapidly mechano-responsive. We find a quantitative correlation between the cellular distribution of cortical actin networks and cellular deformations induced by laser ablation. This process is rapid and completes within 2-4 minutes. We propose that this is an intrinsic physical property of crosslinked filamentous networks, as these findings can be reproduced in a physical model that lacks biological regulation. Moreover, actin networks also respond to different types of mechanical stresses: both osmotic treatment and squeezing lead to distinct changes in actin organization, distribution, and dynamics. Our findings show that plant actin networks form mechano-adaptive scaffolds that sense mechanical deformations and reorganize in response within just a few minutes. This gives rise to a picture of plants having a two-geared cytoskeletal mechano-response system in which microtubules mediate slow responses to subtle developmental stresses, whereas actin provides a rapid response to large and potentially damaging deformations.

plant biology↗
bioRxiv2025

Transgenerational polarity axis inheritance during Ceratopteris embryogenesis

For sexually reproducing organisms to pass on their genetic information, progeny must successfully establish. Various life history strategies have evolved, using either dispersal of large numbers of progeny or intensive nurturing of a few. Most plants use the former strategy, but ferns generate a single embryo in the exact same location as the mother, and it is unknown how progeny success is promoted, or how embryogenesis is adapted. By studying Ceratopteris richardii embryogenesis, we find that maternal tissues guide orientation of the early embryo body axis, thus aligning its root pole towards the homologous maternal rhizoids. We find that axis polarity inheritance is mediated by maternal tissue mechanical patterns, and thus identify a robust mechanism for progeny establishment as a nurturing strategy in plants.

plant biology↗
bioRxiv2023

An elastic proteinaceous envelope encapsulates the early Arabidopsis embryo

Plant external surfaces are often covered by barriers that control the exchange of molecules, protect from pathogens, and offer mechanical integrity. A key question is when and how such surface barriers are generated. Post-embryonic surfaces have well-studied barriers, including the cuticle, and late Arabidopsis embryo was shown to be protected by an endosperm-derived sheath deposited onto a primordial cuticle. Here we show that both cuticle and sheath are preceded by another structure during the earliest stages of embryogenesis. This structure, which we named the embryonic envelope, is tightly wrapped around the embryonic surface but can be physically detached by cell wall digestion. We show that this structure is composed primarily of Extensin and Arabinogalactan O-glycoproteins and lipids, which appear to form a dense and elastic crosslinked embryonic envelope. The envelope forms in cuticle-deficient mutants and in a mutant that lacks endosperm. This embryo-derived envelope is therefore distinct from previously described cuticle and sheath structures. We propose that it acts as an expandable diffusion barrier, as well as a means to mechanically confine the embryo to maintain its tensegrity during early embryogenesis. Summary statementThe early Arabidopsis embryo is surrounded by a proteinaceous envelope that is distinct from the cuticle or embryo sheath

plant biology↗
bioRxiv2022

Arabidopsis pollen Prolyl-hydroxylases P4H4/6 are required for correct hydroxylation and secretion of LRX11 in pollen tubes

Major constituents of the plant cell walls are structural proteins that belong to the Hydroxyproline-rich glycoprotein family. Leucine-rich repeat extensis are contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro(3-5) motifs plausible to be glycosylated. We have demonstrated that pollen-specific LRX8-11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In classical EXTs and likely in LRXs, proline residues are converted to hydroxyproline by Prolyl-4-hydroxylases, thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro(3-5) motifs of LRX8-LRX11. Here, we show the p4h4-1 p4h6-1 double mutant exhibits a significant reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by specific P4Hs inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial relocalization of LRX11-GFP from the pollen tube tip apoplast to the cytoplasm. Finally, IP-MS- MS analysis revealed a decrease in oxidized prolines in LRX11-GFP in the p4h4-1 p4h6-1 background when compared to lrx11 plants expressing pLRX11::LRX11-GFP. Together, these results suggest that P4H4 and P4H6 are required for pollen germination and are also involved in LRX11 hydroxylation necessary for its localization at the cell wall of pollen tubes. One Sentence SummaryPollen-expressed P4H4 and P4H6 are required for pollen germination and for proper hydroxylation and secretion of LRX11 in pollen tubes.

plant biology↗
bioRxiv2021

Apoplastic class III peroxidases PRX62 and PRX69 regulate ROS-homeostasis and cell wall associated extensins linked to root hair growth at low-temperature in Arabidopsis thaliana

Root Hairs (RHs) growth is highly influenced by endogenous as well as by external environmental signals that coordinately regulate its final cell size. RHs actively expand the root surface responsible for nutrient uptake and water absorption. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explored the molecular basis of this strong RH growth response by using the Genome Wide Association Studies (GWAS) approach on Arabidopsis thaliana natural accessions. We identified the poorly characterized PEROXIDASE 62 (PRX62) as a key protein triggering this conditional growth under a moderate low-temperature stress. In addition, we identified the related protein PRX69 as an important factor in this developmental process. The prx62 prx69 double mutant and the PRX62 and PRX69 over-expressing lines showed contrasting RH phenotypes, peroxidase activities and cyt/apoReactive Oxygen Species (ROS) levels. Strikingly, a cell wall protein extensin (EXT) reporter revealed the effect of peroxidase activity on the EXT cell wall association at 10{degrees}C in the RH apical zone. EXT cell wall insolubilization was enhanced at 10{degrees}C, which was completely abolished under the PRX inhibitor salicylhydroxamic acid (SHAM) treatment. Finally, we demonstrated that the Root Hair defective 6-like 4 (RSL4) bHLH family transcription factor directly controls the expression of PRX69. Collectively, our results indicate that both PRX62 and PRX69 are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low-temperature.

plant biology↗
bioRxiv2021

Proline-rich Extensin-like Receptor Kinases PERK5 and PERK12 are involved in Pollen Tube Growth

BackgroundCell wall integrity plays an essential role during polarized cell growth typical of pollen tubes and root hairs. Proline-rich Extensin-like Receptor Kinases (PERK) belong to the hydroxyproline-rich glycoprotein (HRGP) superfamily of cell surface glycoproteins. ResultsHere, we identified two PERKs from Arabidopsis thaliana, PERK5 and PERK12 highly expressed in mature pollen. Pollen tube growth was impaired in the single and double perk5-1 perk12-1 loss of function mutants, with a moderate impact on seed production. When the segregation of self- and reciprocal-crosses of the perk5-1, perk5-2 and perk12-1 single mutants, and reciprocal-crosses of the perk5-1 perk12-1 double mutant were carried out, a male gametophytic defect was found, indicating that perk5-1 and perk12-1 mutants carry defective pollen tubes, resulting in deficient pollen transmission. Furthermore, double perk5-1 perk12-1 mutants show excessive accumulation of pectins and cellulose at the cell wall pollen of the tube tip. In addition, an upregulation of cytoplasmic ROS levels were detected by using 2,7-dichlorofluorescein diacetate probe (H2DCF-DA), and in agreement, similar results were obtained with HyPer, a genetically encoded YFP-based radiometric sensor, which is used to follow the production of hydrogen peroxide (H2O2). Single and double perk5-1 perk12-1 mutants show higher levels of cytoplasmic H2O2 in their pollen tube tips. ConclusionsTaken together, our results suggest that PERK5 and PERK12 are necessary for proper pollen tube growth highlighting their role on cell wall assembly and ROS homeostasis.

plant biology↗
bioRxiv2020

PRX1, PRX44, and PRX73 are Class-III extensin-related peroxidases that modulates root hair growth in Arabidopsis thaliana

O_LIRoot hair cells are important sensors of soil conditions. Expanding several hundred times their original size, root hairs grow towards and absorb water-soluble nutrients. This rapid growth is oscillatory and is mediated by continuous remodelling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins including extensins (EXTs). C_LIO_LIClass-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening, mediated by oxygen radical species, or polymerization of cell wall components, including the Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. C_LIO_LIUsing genetic, biochemical, and modeling approaches, we identified and characterized three root hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. The triple mutant prx01,44,73 and the PRX44 and PRX73 overexpressors had opposite phenotypes with respect to root hair growth, peroxidase activity and ROS production with a clear impact on cell wall thickness. C_LIO_LIModeling and docking calculations suggested that these three putative EXT-PRXs may interact with non-O-glycosylated sections of EXT peptides that reduce the Tyr-to-Tyr intra-chain distances in EXT aggregates and thereby may enhance Tyr crosslinking. These results suggest that these three putative EXT-PRXs control cell wall properties during the polar expansion of root hair cells. C_LI

plant biology↗
bioRxiv2019

A cell surface O-glycosylated peptide, AGP21, acts on the brassinosteroid pathway and modulates root hair cell fate

Root hairs (RHs) develop from specialized epidermal cells called trichoblasts, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling root epidermal cell fate is only partially understood. Root epidermis cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE-4 (RSL4 [Yi et al. 2010]) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence root epidermis cell fate. In the absence of BRs, phosphorylated BIN2 (a Type-II GSK3-like kinase) inhibits a protein complex that directly downregulates GL2 [Chen et al. 2014]. Here, we show that the genetic and pharmacological perturbation of the arabinogalactan peptide (AG) AGP21 in Arabidopsis thaliana, triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O-glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2-dependent manner. These results suggest that perturbation of a cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermal cells. In addition, AGP21 also acts in a BR-independent, AGP-dependent mode that together with BIN2 signalling cascade controls RH cell fate. SignificanceIn the plant Arabidopsis thaliana, the root epidermis forms in an alternating pattern atrichoblasts with trichoblast cells that end up developing root hairs (RHs). Atrichoblast cell fate is directly promoted by the transcription factor GLABRA2 (GL2) while the lack of GL2 allows RH formation. The loss of AGP21 peptide triggers an abnormal RH cell fate in two contiguous cells in a similar manner as brassinosteroid (BRs) mutants. In the absence of BR signaling, BIN2 (a GSK3 like-kinase) in a phosphorylated state, downregulate GL2 expression to trigger RH cell fate. The absence of AGP21 is able to repress GL2 expression and activates the expression of RSL4 and EXP7 root hair proteins.

plant biology↗