Membrane–wall attachments in plasmolysed plant cells

Summary. Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100–250nm. This suggests that the fibres may be cellulose. After 4h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.Correspondence and reprints: Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.     

Plasmolysis, Plasmodesmata, and the Electrical Coupling of Oat Coleoptile Cells

Ultrastructural studies on oat coleoptile parenchyma cells (Avenasativa L. cv. Victory) reveal that severe plasmolysis eitherbreaks plasmodesmatal connections or leaves the protoplastsstill connected via strands of cytoplasm (Hechtian strands).Plasmolysis also induces the formation of callose around theplasmodesmata. The callose remains for several hours after recoveryof the cells to full turgor. Immediately following recovery of turgor, intercellular electricalcoupling cannot be detected. However, during the next 6 h, somedegree of coupling is restored. These results indicate that,while plasmolysis does not necessarily break all plasmodesmatalconnections, the treatment probably does disrupt them sufficientlyto interfere, at least temporarily, with symplastic transport.     

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Wall-to-membrane linkers in onion epidermis: some hypotheses

Wall-to-wall linkage may help maintain cell integrity and polarity, and focus mechanical stress from wall to mech-anotransductive ion channels within the plasm a lemma. When cells of onion bulb scale epidermis shrink during plasmolysis with CaCl₂, the plasmalemma remains attached to the cell wall by Hechtian strands which we hypothesize might possibly be drawn out from linkages fulfilling the above functions. We show that at least many of the attachment loci are independent of the plasmodesmata. A priori, wall glycoproteins seem good candidates for the wall-to-membrane linkers; therefore, we investigated the distribution in wall and plasmalemma of antigen recognized by antibody to hydroxyproline-rich glycoprotein (HRGP). Using fluorescent secondary antibodies, we showed that polyclonal antibodies prepared against wall HRGP from soybean bind to the onion walls (following mild depectination), but also bind to the plasmalemma after the wall is enzymatically digested. The distribution of the antibodies is punctate. On the plasmalemma, the points tend to be scattered more or less uniformly, but can cluster at termini of large streaming strands (which rarely form in wall-constrained cells.) These streaming strands can be seen to exert tension on the membrane. We hypothesize that (1) the antigen on the surface of the protoplast may correspond to the antigen in the walls, (2) such antigen may be responsible for adhesion of membrane to wall at the linkage sites visualized by CaCl₂ plasmolysis, and (3) the linkage sites may be transmembrane proteins to which cytoskeleton can attach at the inner surface.     

A plasmolytic cycle: The fate of cytoskeletal elements

Summary In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.Abbreviations DIC differential interference contrast - DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Molecular interactions of arabinogalactan proteins with cortical microtubules and F-actin in Bright Yellow-2 tobacco cultured cells

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.     

Plasmolysis and cell wall deposition in wheat root hairs under osmotic stress

We analysed cell wall formation in rapidly growing root hairs of Triticum aestivum under reduced turgor pressure by application of iso- and hypertonic mannitol solutions. Our experimental series revealed an osmotic value of wheat root hairs of 150 mOsm. In higher concentrations (200–650 mOsm), exocytosis of wall material and its deposition, as well as callose synthesis, still occurred, but the elongation of root hairs was stopped. Even after strong plasmolysis when the protoplast retreated from the cell wall, deposits of wall components were observed. Labelling with DiOC₆(3) and FM1-43 revealed numerous Hechtian strands that spanned the plasmolytic space. Interestingly, the Hechtian strands also led towards the very tip of the root hair suggesting strong anchoring sites that are readily incorporated into the new cell wall. Long-term treatments of over 24 h in mannitol solutions (150–450 mOsm) resulted in reduced growth and concentration-dependent shortening of root hairs. However, the formation of new root hairs does occur in all concentrations used. This reflects the extraordinary potential of wheat root cells to adapt to environmental stress situations.     

Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration

Fungal pathogens almost invariably trigger cell wall-associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane-cell wall adhesion as shown by the loss of the thin plasma membrane-cell wall connections known as Hechtian strands, reduced the expression of cell wall-associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall-associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane-cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall-associated defenses, there was no change, or an increase, in plasma membrane-cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall-associated defenses and a localized decrease in plasma membrane-cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane-cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall-associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane-cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713-718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation.     

The apicomplexan parasite Babesia divergens internalizes band 3, glycophorin A and spectrin during invasion of human red blood cells

Plasmodium falciparum invades human red blood cells (RBC), while Babesia divergens infects bovine and, occasionally, human RBC. The mammalian RBC is normally unable to endocytose or phagocytose and the events leading to invasion are incompletely understood. Initially, both parasites are surrounded by the RBC plasma membrane‐derived parasitophorous vacuolar membrane (PVM) that is formed during invasion. In P. falciparum‐infected RBC, the PVM persists at least until parasite replication is completed whereas it has been proposed that the B. divergens PVM is disintegrated soon upon invasion. Here, we have used a B. divergens strain adapted to human RBC to investigate the formation and fate of the PVM. Using ultrastructural analysis and whole‐mount or on‐section immunofluorescence and immunogold labelling, we demonstrate that the initial vacuolar membrane is formed from protein and lipid components of the RBC plasma membrane. Integral membrane proteins band 3 and glycophorin A and the cytoskeletal protein spectrin are associated with the PVM of the B. divergens, but are absent from the PVM of P. falciparum at the ring or the trophozoite stage. Our results provide evidence that the biophysical properties of the RBC cytoskeleton per se do not preclude the internalization of cytoskeletal proteins by invading parasites.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility

Summary Plasmolysis of hyphae of the oomycetesSaprolegnia ferax andAchlya ambisexualis and the ascomyceteNeurospora crassa produced abundant cytoplasmic strands between the retracted cytoplasm and punctate adhesions of the plasma membrane to the cell wall. These strands formed throughout the length of mature hyphae and are the first demonstration of Hechtian strands in hyphae. In contrast to similar strands in various plant cells, the strands inSaprolegnia lacked endoplasmic reticulum but contained F-actin, suggesting similarity between their adhesion sites and focal contacts in animal cells. However, strand adhesion to the wall was insensitive to RGD-containing peptides, suggesting that the trans-membrane adhesion molecules differ from animal integrins. The pattern of plasma membrane-cell wall adhesion varied in different zones along hyphae, with broad, irregular connections in the extreme apex, uniform and continuous connection in a transition zone, and small, punctate adhesions in the mature subapical zone, suggesting differential functions in these different regions. The apical adhesions are important in tip growth, as diverse inhibitors induced concomitant changes in hyphal growth and the adhesions in the apical and transition zones. Plasmolysis also induced cytoplasmic migrations throughout hyphae. Such migrations were dominated by the central cytoplasm, and produced distorted organelles which spanned central and peripheral cytoplasm, thus supporting the idea that the adhesions in mature zones of hyphae anchor the peripheral cytoplasm and facilitate cytoplasmic and organelle migrations.Abbreviations OM organic medium - RP rhodamine phalloidin - DIC differential interference contrast - PIPES piperazine-N,N-bis-2-ethanosulphonic acid     

Osmotic Stress Decreases the Activity of ATPase Associated with the Root Cap Plasmodesmata in Zea mays

With light and electron microscopy the substructural change and the ATPase activity of corn (Zea mays L. ) root cap cells after short-term osmotic stress were studied. Some spoke-like fine strands originating from the departed periplasm and stretching towards cell wall could be observed even after plasmolysis. By observing the precipitation of ATPase activity product (lead phosphate) at plasma membrane and plasmodesmata, it was found that the fine strands were plasma membrane-lined channels surrounding the cytoplasm and that they still firmly connected to the plasmodesmata during plasmolysis. Compared with the control (unstressed), a sharp decrease of ATPase activity in the plasmodesmata of the stressed cells was observed. Inhibition of energy metabolism in these limited locales would affect the physiological activity, maybe including the regulation of permeability and the change of size exclusion limit (SEL) of plasmodesmata.     

渗透胁迫引起玉米根冠处胞间连丝的ATP酶活性下降

利用光镜和电镜研究了短期渗透胁迫下玉米(Zea mays L.)根冠区超微结构和ATP酶活性变化。经历了质壁分离后,在根冠细胞仍然可以观察到许多从“撤退”的周质出发向细胞壁辐射的纤丝。利用ATP酶活性产物(磷酸铅)在原生质膜和细胞壁处沉积的特点,发现这些纤丝是质膜围绕原生质而形成的管状结构;在质壁分离过程中,这些纤丝依然与胞间连丝相连。与对照(未胁迫)相比,受渗透胁迫细胞胞间连丝处ATP酶活性明显下降。能量代谢在局部区段的抑制会影响胞间连丝的生理活性,可能包括胞间连丝的扩散调节能力和分子扩散上限的改变。     

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

Hypertonic conditions trigger transient plasmolysis,growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

Abstract

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5–2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.     

In vivo light‐sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue‐ and organ‐specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light‐sheet and Airyscan confocal microscopy. FSD1‐GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1‐GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1‐GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1‐GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.