MscS-like proteins control plastid size and shape in Arabidopsis thaliana

BACKGROUND: Mechanosensitive (MS) ion channels provide a mechanism for the perception of mechanical stimuli such as sound, touch, and osmotic pressure. The bacterial MS ion channel MscS opens in response to increased membrane tension and serves to protect against cellular lysis during osmotic downshock. MscS-like proteins are found widely in bacterial and archaeal species and have also been identified in fission yeast and plants. None of the eukaryotic members of the family have yet been characterized. RESULTS: Here, we characterize two MscS-like (MSL) proteins from Arabidopsis thaliana, MSL2 and MSL3. MSL3 can rescue the osmotic-shock sensitivity of a bacterial mutant lacking MS-ion-channel activity, suggesting that it functions as a mechanosensitive ion channel. Arabidopsis plants harboring insertional mutations in both MSL3 and MSL2 show abnormalities in the size and shape of plastids, which are plant-specific endosymbiotic organelles responsible for photosynthesis, gravity perception, and numerous metabolic reactions. MSL2-GFP and MSL3-GFP are localized to discrete foci on the plastid envelope and colocalize with the plastid division protein AtMinE. CONCLUSIONS: Our data support a model wherein MSL2 and MSL3 control plastid size, shape, and perhaps division during normal plant development by altering ion flux in response to changes in membrane tension. We propose that MscS family members have evolved new roles in plants since the endosymbiotic event that gave rise to plastids.     

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1–green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backward. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1–GFP-labeled plastids, thus validating the use of FtsZ1–GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.     

Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Early development and gravitropic response of lateral roots in Arabidopsis thaliana

Root system architecture plays an important role in determining nutrient and water acquisition and is modulated by endogenous and environmental factors, resulting in considerable developmental plasticity. The orientation of primary root growth in response to gravity (gravitropism) has been studied extensively, but little is known about the behaviour of lateral roots in response to this signal. Here, we analysed the response of lateral roots to gravity and, consistently with previous observations, we showed that gravitropism was acquired slowly after emergence. Using a lateral root induction system, we studied the kinetics for the appearance of statoliths, phloem connections and auxin transporter gene expression patterns. We found that statoliths could not be detected until 1 day after emergence, whereas the gravitropic curvature of the lateral root started earlier. Auxin transporters modulate auxin distribution in primary root gravitropism. We found differences regarding PIN3 and AUX1 expression patterns between the lateral root and the primary root apices. Especially PIN3, which is involved in primary root gravitropism, was not expressed in the lateral root columella. Our work revealed new developmental transitions occurring in lateral roots after emergence, and auxin transporter expression patterns that might explain the specific response of lateral roots to gravity.     

Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis

Plastid isoprenoids are synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE. In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene. The plant polypeptide, GCPE, contains two structural domains that are absent in the E. coli protein: an N-terminal extension and a central domain of 30 kDa. We demonstrate that the N-terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis. Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE-defective E. coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product.     

Telomere rapid deletion regulates telomere length in Arabidopsis thaliana

Telomere length is maintained in species-specific equilibrium primarily through a competition between telomerase-mediated elongation and the loss of terminal DNA through the end-replication problem. Recombinational activities are also capable of both lengthening and shortening telomeres. Here we demonstrate that elongated telomeres in Arabidopsis Ku70 mutants reach a new length set point after three generations. Restoration of wild-type Ku70 in these mutants leads to discrete telomere-shortening events consistent with telomere rapid deletion (TRD). These findings imply that the longer telomere length set point is achieved through competition between overactive telomerase and TRD. Surprisingly, in the absence of telomerase, a subset of elongated telomeres was further lengthened, suggesting that in this background a mechanism of telomerase-independent lengthening of telomeres operates. Unexpectedly, we also found that plants possessing wild-type-length telomeres exhibit TRD when telomerase is inactivated. TRD is stochastic, and all chromosome ends appear to be equally susceptible. The frequency of TRD decreases as telomeres shorten; telomeres less than 2 kb in length are rarely subject to TRD. We conclude that TRD functions as a potent force to regulate telomere length in Arabidopsis.     

Nitrate or NaCl regulates floral induction in Arabidopsis thaliana

Flowering, the transition from the vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Exposure to an extended period of stress (such as low nitrate or NaCl) can also promote flowering in many species, but little is known about how these forms of stress regulate floral induction. In this study, we found that stress induced by low concentrations of nitrate or NaCl activated the biosynthesis of gibberellin (GA) as evidenced by increased expression of the GA biosynthetic enzyme GA1. Expression of CO and SOC1 were also enhanced, leading to an acceleration of flowering. The effects of nitrate and NaCl on the photoperiod pathway were distinct, however. Two genes related to the photoperiod pathway, CCA1 and LHY, were repressed only under low NaCl treatment, while expression was unaltered by nitrate. Therefore, we suggest that the biosynthesis of gibberellin (GA) may play an important role in integrating signals induced by exogenous stress to regulate flowering in Arabidopsis.     

Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.     

Arabidopsis thaliana hairy roots for the production of heterologous proteins

To evaluate the ability of Arabidopsis thaliana hairy roots to produce heterologous proteins, hypocotyls were transformed with Rhizobium rhizogenes harbouring a green fluorescent protein gene (gfp) fused to a plant signal peptide sequence. Hairy root transgenic lines were generated from wild-type or mutant genotypes. A line secreted GFP at 130 mg/l of culture medium. Unlike as was previously found with turnip hairy roots, a His-tag was still attached to approximately 50?% of the protein. Control of the pH and addition of a protease inhibitor to the culture medium resulted in up to 87?% of the GFP retaining the His-tag. A. thaliana hairy roots expressing the human serpina1 (α-1-antitrypsin) gene secreted the protein, which was visible on a PAGE gel. Protein activity in the culture medium was demonstrated using an elastase inhibition assay. A. thaliana hairy roots can now be considered for the production of heterologous proteins, making it possible to mine the numerous genetic resources for enhancing protein production and quality.