The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Glycerolipid transfer for the building of membranes in plant cells

Membranes of plant organelles have specific glycerolipid compositions. Selective distribution of lipids at the levels of subcellular organelles, membrane leaflets and membrane domains reflects a complex and finely tuned lipid homeostasis. Glycerolipid neosynthesis occurs mainly in plastid envelope and endoplasmic reticulum membranes. Since most lipids are not only present in the membranes where they are synthesized, one cannot explain membrane specific lipid distribution by metabolic processes confined in each membrane compartment. In this review, we present our current understanding of glycerolipid trafficking in plant cells. We examine the potential mechanisms involved in lipid transport inside bilayers and from one membrane to another. We survey lipid transfers going through vesicular membrane flow and those dependent on lipid transfer proteins at membrane contact sites. By introducing recently described membrane lipid reorganization during phosphate deprivation and recent developments issued from mutant analyses, we detail the specific lipid transfers towards or outwards the chloroplast envelope.     

Disruption of the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2 in Arabidopsis reveals the existence of an additional enzyme of galactolipid synthesis

Two genes (DGD1 and DGD2) are involved in the synthesis of the chloroplast lipid digalactosyldiacylglycerol (DGDG). The role of DGD2 for galactolipid synthesis was studied by isolating Arabidopsis T-DNA insertional mutant alleles (dgd2-1 and dgd2-2) and generating the double mutant line dgd1 dgd2. Whereas the growth and lipid composition of dgd2 were not affected, only trace amounts of DGDG were found in dgd1 dgd2. The growth and photosynthesis of dgd1 dgd2 were affected more severely compared with those of dgd1, indicating that the residual amount of DGDG in dgd1 is crucial for normal plant development. DGDG synthesis was increased after phosphate deprivation in the wild type, dgd1, and dgd2 but not in dgd1 dgd2. Therefore, DGD1 and DGD2 are involved in DGDG synthesis during phosphate deprivation. DGD2 was localized to the outer side of chloroplast envelope membranes. Like DGD2, heterologously expressed DGD1 uses UDP-galactose for galactosylation. Galactolipid synthesis activity for monogalactosyldiacylglycerol (MGDG), DGDG, and the unusual oligogalactolipids tri- and tetragalactosyldiacylglycerol was detected in isolated chloroplasts of all mutant lines, including dgd1 dgd2. Because dgd1 and dgd2 carry null mutations, an additional, processive galactolipid synthesis activity independent from DGD1 and DGD2 exists in Arabidopsis. This third activity, which is related to the Arabidopsis galactolipid:galactolipid galactosyltransferase, is localized to chloroplast envelope membranes and is capable of synthesizing DGDG from MGDG in the absence of UDP-galactose in vitro, but it does not contribute to net galactolipid synthesis in planta.     

Can digalactosyldiacylglycerol substitute for phosphatidylcholine upon phosphate deprivation in leaves and roots of Arabidopsis?

To explore the role of digalactosyldiacylglycerol (DGDG) in plants the dgd1 mutant of Arabidopsis thaliana was grown in the presence and absence of inorganic phosphate. Phosphate deficiency in the dgd1 mutant causes a strong decrease in all phospholipids accompanied by an increase in DGDG and sulpholipid. Moreover, a significant DGDG accumulation was found in roots upon phosphate deprivation as well. Our data indicate that DGDG accumulation upon phosphate deprivation is due to the activation of a specific eukaryotic dgd1-independent biosynthetic pathway. We propose that DGDG may substitute for phosphatidylcholine upon phosphate deprivation.     

Arabidopsis type B monogalactosyldiacylglycerol synthase genes are expressed during pollen tube growth and induced by phosphate starvation

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) constitute the major glycolipids of the thylakoid membranes in chloroplasts. In Arabidopsis, the formation of MGDG is catalyzed by a family of three MGDG synthases, which are encoded by two types of genes, namely type A (atMGD1) and type B (atMGD2 and atMGD3). Although the roles of the type A enzyme have been intensively investigated in several plants, little is known about the contribution of type B enzymes to MGDG synthesis in planta. From our previous analyses, unique expression profiles of the three MGDG synthase genes were revealed in various organs and developmental stages. To characterize the expression profiles in more detail, we performed histochemical analysis of these genes using beta-glucuronidase (GUS) assays in Arabidopsis. The expression of atMGD1::GUS was detected highly in all green tissues, whereas the expression of atMGD2::GUS and atMGD3::GUS was observed only in restricted parts, such as leaf tips. In addition, intense staining was detected in pollen grains of all transformants. We also detected GUS activity in the pollen tubes of atMGD2::GUS and atMGD3::GUS transformants grown in wild-type stigmas but not in atMGD1::GUS, suggesting that type B MGDG synthases may have roles during pollen germination and pollen tube growth. GUS analysis also revealed that expression of atMGD2 and atMGD3, but not atMGD1, are strongly induced during phosphate starvation, particularly in roots. Because only DGDG accumulates in roots during phosphate deprivation, type B MGDG synthases may be acting primarily to supply MGDG as a precursor for DGDG synthesis.     

Polar lipid composition of a plastid ribosome-deficient barley mutant

Green and white leaves of the barley mutant line `albostrians' were compared for their polar lipid content and fatty acid composition. The mutant plastids of the white leaves have a double-layered envelope, but in contrast with the normal chloroplasts, lack 70 S ribosomes and thylakoids. In the green leaves, the amount of monogalactosyldiacylglycerol (MGDG) consistently exceeds the amount of digalactosyldiacylglycerol (DGDG) and the amount of galactolipids exceeds the amount of phospholipids. In contrast, in white leaves the amount of DGDG exceeds the amount of MGDG and the amount of phospholipids exceeds the amount of galactolipids. In white leaves, the galactolipid composition reflects the plastid envelope composition which is rich in DGDG, whereas in green leaves the galactolipid composition reflects the thylakoid composition which is rich in MGDG. These results demonstrate the likelihood that all the enzymes involved in galactolipid, sulfolipid and fatty acid synthesis are coded by the nuclear genome.     

Chloroplast envelope membranes: a dynamic interface between plastids and the cytosol

Chloroplasts are bounded by a pair of outer membranes, the envelope, that is the only permanent membrane structure of the different types of plastids. Chloroplasts have had a long and complex evolutionary past and integration of the envelope membranes in cellular functions is the result of this evolution. Plastid envelope membranes contain a wide diversity of lipids and terpenoid compounds serving numerous biochemical functions and the flexibility of their biosynthetic pathways allow plants to adapt to fluctuating environmental conditions (for instance phosphate deprivation). A large body of knowledge has been generated by proteomic studies targeted to envelope membranes, thus revealing an unexpected complexity of this membrane system. For instance, new transport systems for metabolites and ions have been identified in envelope membranes and new routes for the import of chloroplast-specific proteins have been identified. The picture emerging from our present understanding of plastid envelope membranes is that of a key player in plastid biogenesis and the co-ordinated gene expression of plastid-specific protein (owing to chlorophyll precursors), of a major hub for integration of metabolic and ionic networks in cell metabolism, of a flexible system that can divide, produce dynamic extensions and interact with other cell constituents. Envelope membranes are indeed one of the most complex and dynamic system within a plant cell. In this review, we present an overview of envelope constituents together with recent insights into the major functions fulfilled by envelope membranes and their dynamics within plant cells. Special Issue of Photosynthesis Research in honor of Andrew A. Benson.     

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.     

Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein

The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.     

Green light for galactolipid trafficking

Galactolipids not only play a crucial role in photosynthesis but are also important for the adaptation of membrane-lipid composition in plants to phosphate-limiting conditions. The enzymes of galactolipid assembly have been localised to the envelope membranes of chloroplasts. Lipid trafficking is essential for galactolipid synthesis and redistribution because lipid precursors originate from two compartments, the endoplasmic reticulum (ER) and the plastid, and because galactolipids have to be transported to extraplastidial membranes during phosphate deprivation. Analysis of Arabidopsis mutants that are impaired in galactolipid synthesis (i.e. dgd1 and dgd2) or in ER-to-plastid lipid transport (i.e. tgd1) has resulted in the identification of a processive galactosyltransferase whose function is still enigmatic.     

Mutation of a mitochondrial outer membrane protein affects chloroplast lipid biosynthesis

Lipid biosynthesis in plant cells is associated with various organelles, and maintenance of cell lipid homeostasis requires nimble regulation and coordination. In plants, environmental cues such as phosphate limitation require readjustment of the lipid biosynthetic machinery to substitute phospholipids by non-phosphorous glycolipids. Biosynthesis of the galactoglycerolipids predominant in plants proceeds by a constitutive and an alternative pathway that is known to be induced in response to phosphate deprivation. Plant lipid galactosyltransferases involved in both pathways are associated with the plastid envelope membranes and are encoded by nuclear genes. To identify mechanisms governing the activity of the alternative galactoglycerolipid pathway, a genetic suppressor screen was conducted in the background of the digalactolipid-deficient dgd1 mutant of Arabidopsis. A suppressor line that partially restored digalactoglycerolipid content in the dgd1 background carries a point mutation in a mitochondrial protein, which was tentatively designated DGD1 SUPPRESSOR 1 (DGS1). Presumed orthologs of this protein are present in plants, algae and fungi, but its molecular function is not yet known. In the dgd1 dgs1 double mutant, expression of nuclear genes encoding enzymes of the alternative galactoglycerolipid pathway is increased and hydrogen peroxide levels are elevated. This increase in hydrogen peroxide is proposed to be the reason for activation of the alternative pathway in the dgd1 dgs1 double mutant. Accordingly, hydrogen peroxide and treatments producing reactive oxygen also activate the alternative pathway in the wild-type. These results likely implicate the production of reactive oxygen in the regulation of the alternative galactoglycerolipid pathway in plants.     

Zur Frage einer Neubildung von Mitochondrien aus Plastiden

Summary In the cells of the foot of young sporophytes ofPolytrichum commune, plastids form buds which may separate entirely from the mother plastid. In ultra-thin sections these bodies may be easily mistaken for mitochondria. However, with the silver staining method ofMarinozzi, the matrix of these bodies has the same density as the matrix of the plastids and is markedly less dense than the matrix of the mitochondria. Similarly, after silver staining the envelope of these bodies resembles the plastid envelope and is distinctly different from the mitochondrial envelope. Thus, there is no evidence that mitochondria originate from plastids, as some authors believe.     

Long- and short-term phosphate deprivation in bean roots: plasma membrane lipid alterations and transient stimulation of phospholipases

In a long-term experiment bean (Phaseolus vulgaris L.) seedlings were grown for 18 days in hydroponics in either phosphate-sufficient (+P) or phosphate-deficient (-P) nutrient solutions. Phosphate deprivation halved the phosphorous content of roots. In plasma membrane (PM) fractions isolated from -P roots the phospholipid (PL) level was reduced from 35 to 21 mol%, while PL composition and degree of unsaturation were hardly altered. Digalactosyldiacylglycerol (DGDG) accumulated up to 26% of total PM lipids, replacing PL to a large extent. Molecular species and fatty acid compositions of DGDG in root PM were different compared to DGDG present in the -P plastids. In a short-term study, bean seedlings were grown for 18 days in hydroponics with a complete nutrient solution containing phosphate and then incubated in a -P medium for increasing time ranging from 1 up to 96 h. At the end of the starvation period phosphorous content of -P roots was reduced by 30% compared to +P ones. An activation of phospholipase D and phospholipase C was observed after 1 and 2h of phosphate deprivation, respectively. Maximal phosphatidic acid accumulation was detected after 4h of phosphate deprivation, when also DGDG started to accumulate in PM of bean roots. The fatty acid composition of PLD-derived phosphatidylbutanol resembled that of phosphatidylcholine.     

Plastids and glycolipids in the stemwood of Pinus sylvestris L.

Summary The ultrastructure of plastids in xylem ray parenchyma cells of Pinus sylvestris L. was studied and compared with the glycolipid composition of the stemwood. Seasonal changes of the ultrastructure were studied by taking samples regularly throughout the year. The plastids resemble amyloplasts. They usually have one large starch grain, and considerable variation in structure and starch content was observed, especially in the innermost sapwood and in the sapwood-heartwood transition zone. Electron-dense deposits were observed attached to the plastid membranes and envelopes, especially in the transition zone, from April to November. The plastids were aggregated near the nucleus and the starch disappeared during the winter (January–March). The glycolipids, monogalactosyldiacylglycerol (MGDG) and di-galactosyldiacylglycerol (DGDG), were present only in the sapwood, in trace amounts. The glycolipid content was slightly greater in the outer sapwood than in the sapwood-heartwood transition zone. DGDG was the dominant lipid of the two.     

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VIII. Temperature effects and a perspective on the curious case of Karenia mikimotoi as a producer of the unusual, ‘green algal’ fatty acid hexadecatetraenoic acid [16:4(n-3)]

Previous studies have shown that dinoflagellates with different plastid ancestries have distinct differences in the fatty acid compositions and regiochemistries of their chloroplast-associated galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), thus reflecting plastid origin as a major factor in plastid membrane composition. Specifically, dinoflagellates with aberrant plastids (e.g. Karenia brevis, Kryptoperidinium foliaceum and Lepidodinium chlorophorum) possess certain MGDG- and DGDG-associated fatty acids which are not found in peridinin-containing dinoflagellates (the largest group of photosynthetic dinoflagellates with a red algal plastid ancestry which is thought to be an evolutionary precursor to aberrant plastids), but which are common to other algal groups. For example, hexadecatetraenoic acid (16:4(n-3)) is common to green algae and is found in the MGDG and DGDG of L. chlorophorum, which agrees with its green algal plastid ancestry, while hexadecatrienoic acid (16:3) and hexadecadienoic acid (16:2) are found in the MGDG and DGDG of K. foliaceum, which agrees with its diatom plastid ancestry. Notably, 16:4 has been found by others in the total fatty acids and galactolipids of Karenia mikimotoi, but in no other examined members of the Kareniaceae (all of which have plastids of haptophyte origin). However, these findings lack information as to the regiochemistry of 16:4. We have utilized positive-ion electrospray ionization/mass spectrometry (ESI/MS) and ESI/MS/MS to demonstrate that 16:4, which aside from L. chlorophorum is not found conclusively in the MGDG and DGDG of any other dinoflagellates examined to date irrespective of plastid ancestry, is found in K. mikimotoi as 18:5/16:4 (sn-1/sn-2 regiochemistry) MGDG and DGDG, and that its presence is not modulated (i.e. does not become more saturated) with an increase in growth temperature. Considering an aberrant pigment composition as described by others, we present a perspective where galactolipid-associated 16:4 in K. mikimotoi indicates a plastid ancestry more convoluted than for other members of the Kareniaceae.     

Adaptation of Synechococcus sp. PCC 7942 to phosphate starvation by glycolipid accumulation and membrane lipid remodeling

Organisms use various adaptive strategies against phosphate stress, including lipid remodeling. Here, the response of major membrane lipids to phosphate stress was analyzed in Synechococcus sp. PCC 7942. Unlike plants and eukaryotic microalgae, no significant increases in neutral lipids were found, whereas glycolipids content increased to as high as 6.13% (of dry cell weight, DCW) and phospholipids decreased to 0.34% (of DCW) after 16 days of cultivation without phosphate. Glycolipids accumulation were mainly attributed to the significant increase of digalactosyldiacylglycerol (DGDG) by 50% and sulfoquinovosyldiaclglycerol (SQDG) by 90%, both of which acted as complementary lipids for phosphatidylglycerol (PG) in the cyanobacterial membrane. Also, a notable increase in content (by 48%) of C18 fatty acids (especially C18:1) was observed in all glycolipids at the expense of C12 and C14 (72%). These changes may contribute to membrane fluidity and photosynthetic activity for basic cell metabolism and phosphate stress adaptation. Lipidomic analyses showed the reduction of PG 18:1/16: 0 (by 52%) with the increase of DGDG 18:1/16:0 (133%) and SQDG 18:1/16:0 (245%), strongly suggesting a direct conversion of PG to DGDG and SQDG. Moreover, the decreasing amount of monogalactosyldiacylglycerol (MGDG) 16:1/16:0 (22%) was consistent with the increase of free fatty acids (125%) on day 2 of phosphate absence, which suggested that MGDG is more likely to provide a pool of fatty acids for de novo synthesis of glycolipids. This study provides valuable insight into cyanobacteria adaptation strategies to phosphate stress by membrane lipid remodeling and unveils the underlying acyl chain fluxes into glycolipids.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, the three MGDG synthases are classified into type A (atMGD1) and type B MGD isoforms (atMGD2 and atMGD3). atMGD1 is an inner envelope membrane-associated protein of chloroplasts and is responsible for the bulk of galactolipid biosynthesis in green tissues. MGD1 function is indispensable for thylakoid membrane biogenesis and embryogenesis. By contrast, type B atMGD2 and atMGD3 are localized in the outer envelopes and have no important role in chloroplast biogenesis or plant development under nutrient-sufficient conditions. These type B MGD genes are, however, strongly induced by phosphate (Pi) starvation and are essential for alternative galactolipid biosynthesis during Pi starvation. MGD1 gene expression is up-regulated by light and cytokinins. By contrast, Pi starvation-dependent expression of atMGD2/3 is suppressed by cytokinins but induced through auxin signaling pathways. These growth factors may control the functional sharing of the inner envelope pathway by atMGD1 and the outer envelope pathway by atMGD2/3 according to the growth environment.     

Microfilaments and microtubules control the morphology and movement of non-green plastids and stromules in Nicotiana tabacum

Plastid stromules are stroma-filled tubular extensions of the plastid envelope membrane. These structures, which have been observed in a number of species, allow transfer of proteins between interconnected plastids. The dramatic shape of stromules and their dynamic movement within the cell provide an opportunity to study the control of morphology and motion of plastids. Using inhibitors of actin and tubulin, we found that both microfilaments and microtubules affect the shape and motility of non-green plastids. Actin and tubulin control plastid and stromule structure by independent mechanisms, while plastid movement is promoted by microfilaments but inhibited by microtubules. The presence or absence of stromules does not affect the motility of plastids. Photobleaching experiments indicate that actin and tubulin are not necessary for the bulk of green fluorescent protein (GFP) movement between plastids via stromules.