Phosphatidylglycerophosphate synthases from Arabidopsis thaliana.

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N-terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity.     

The galactolipid monogalactosyldiacylglycerol (MGDG) contributes to photosynthesis-related processes in Arabidopsis thaliana

Processes putatively dependent on the galactolipid monogalactosyldiacylglycerol (MGDG) were recently studied using the knockdown monogalactosyldiacylglycerol synthase 1 (mgd1-1) mutant (∼40% reduction in MGDG). Surprisingly, targeting of chloroplast proteins was not affected in mgd1-1 mutants, suggesting they retain sufficient MGDG to maintain efficient targeting. However, in dark-grown mgd1-1 plants the photoactive to photoinactive protochlorophyllide (Pchlide) ratio was increased, suggesting that photoprotective responses are induced in them. Nevertheless, mgd1-1 could not withstand high light intensities, apparently due to impairment of another photoprotective mechanism, the xanthophyll cycle (and hence thermal dissipation). This was mediated by increased conductivity of the thylakoid membrane leading to a higher pH in the thylakoid interior, which impaired the pH-dependent activation of violaxanthin de-epoxidase (VDE) and PsbS. These findings suggest that MGDG contribute directly to the regulation of photosynthesis-related processes.Key words: conductivity, galactolipid, light stress, photosynthesis, plastid, xanthophyllThe galactolipid monogalactosyldiacylglycerol (MGDG), the major lipid in plastids,¹ is mainly synthesised in inner plastid envelopes,² where monogalactosyldiacylglycerol synthase 1 (MGD1) catalyses the last step of its production.³ Two MGDG-deficient mutants are known: the knockdown mgd1-1 mutant, which accumulates ∼40% less MGDG than wild type,⁴ and the null mutant mgd1-2, which displays extremely severe defects in chloroplast and plant development.⁵ Thus, the mgd1-1 mutant is more suitable for assessing putative roles of MGDG in processes such as protein targeting and photoprotection.There are conflicting indications regarding the involvement of galactolipids in chloroplast protein targeting: some suggest they play an important role,^6–10 but not all.^11,12 The data recently collected for mgd1-1 do not support MGDG''s involvement in protein targeting, since (inter alia) the level of MGDG in mgd1-1 mutants is clearly sufficient for efficient targeting.¹³ Further, the galactolipid associated with the TOC complex¹² is digalactosyldiacylglycerol (DGDG) and the digalactosyldiacylglycerol synthase 1 (dgd1) mutant,¹⁴ which has ∼10% of wild-type levels of DGDG, has impaired import efficiency.^15,16 Hence, this may indicate that DGDG is relatively more important for chloroplast import than MGDG.The prolamellar bodies (PLBs) of etioplasts have high lipid-to-protein ratios compared to thylakoids. Their major lipid and protein are MGDG and NADPH:Pchlide oxidoreductase (POR), respectively,¹⁷ and MGDG putatively plays an important role, interactively with POR, in the formation of PLBs.^18–20 The transformation of PLBs into thylakoids involves phototransformation of photoactive Pchlide (F656), a precursor of chlorophyll. Non-photoactive Pchlide (F631) is susceptible to photooxidative damage, but POR is believed to suppress this.^21,22 After excitation at 440 nm, mgd1-1 mutants display distinctly higher fluorescence emission peaks corresponding to photoactive Pchlide than wild type counterparts and (hence) higher photoactive:non-photoactive Pchlide ratios.¹³ These changes may be photoprotective responses that favour formation of photoactive Pchlide and optimize the plants'' opportunities to use light for chlorophyll production, enabling the transformation of etioplasts into chloroplasts.Interestingly,the xanthophyll cycle, another photoprotective mechanism, is impaired in mgd1-1.¹³ Normally, the xanthophyll cycle pigment violaxanthin is de-epoxidized into antheraxanthin, and then into zeaxanthin, by the enzyme VDE (Fig. 1), which is dependent on MGDG.²³ MGDG is also an integral component of photosynthetic complexes.^24–26 Thus, since mgd1-1 mutants have reduced total amounts of xanthophyll and chlorophyll pigments, but increased chlorophyll a/b ratios, their photosynthesis capacity is unsurprisingly reduced, even though the organization of their electron transport chains is not strongly affected by the MGDG deficiency.¹³Open in a separate windowFigure 1Reactions of the xanthophyll cycle (adapted from ref. ²⁹). VDE, violaxanthin de-epoxidase; ZE, zeaxanthin epoxidase.During short-term high light stress, antheraxanthin and zeaxanthin are thought to facilitate dissipation of excess light energy in the PSII antenna bed by non-photochemical quenching.^27,28 Upon high light stress the pH decreases, triggering photoprotective mechanisms via changes in the PSII antenna system. The PsbS protein, which is involved in thermal dissipation, is protonated and initiates a conformational change in the PSII antenna bed. This change is further stabilized by the de-epoxidation of violaxanthin to zeaxanthin by the luminal VDE.²⁸ However, the thermal dissipation is impaired in mgd1-1 mutants at high light intensities (>1000 µmol m⁻² s⁻¹) making them more susceptible to light stress. Surprisingly, this is not mediated by direct effects on VDE and PsbS activities, but by changes in the proton conductivity of the thylakoid membrane.¹³The steady-state capacity of the xanthophyll cycle is reduced in mgd1-1 mutants, due to a ∼40% reduction in the proton motive force (pmf) across their thylakoid membranes, indicating that they have impaired capacities to energize these membranes. Nevertheless, the pmf is more or less equal to wild type under light-limited conditions (200 µmol m⁻² s⁻¹ light); it is only the increase in pmf in high light intensities that is impaired in the mutants.¹³ This leads to the thylakoid lumen being less acidic in mgd1-1 than in wild type, hampering full activation of VDE and PsbS. Thus, the thylakoid lumen pH is above the threshold level required for full activation of PsbS and VDE under steady-state conditions and so de-epoxidation rates are retarded and the equilibrium between zeaxanthin and violaxanthin starts to shift slightly towards violaxanthin (Fig. 2).¹³ Thus, increased conductivity of the thylakoid membranes is probably responsible for the diminished non-photochemical quenching in mgd1-1, and the findings strongly indicate that MGDG is required for efficient photosynthesis and photoprotection, in addition to being a physical membrane constituent.Open in a separate windowFigure 2Schematic diagram illustrating the normal mode of action of the xanthophyll cycle. In standard light conditions, V is bound to the photosynthetic complexes and harvests light. In strong light, V is released from the complexes and converted to Z by VDE, which is unable to access V when it is associated with the photosynthetic complexes. The newly formed Z then binds to the photosynthetic complexes (at the PsbS protein), where it dissipates excess energy through NPQ. V, violaxanthin; A, antheraxanthin; Z, zeaxanthin; VDE, violaxanthin de-epoxidase; ZE, zeaxanthin epoxidase. Arrows indicate the directions of reactions.     

Functional analysis of two solanesyl diphosphate synthases from Arabidopsis thaliana

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.     

Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.     

Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells

Summary. Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation. Correspondence and reprints: Department of Gene Technology, Tallinn, University of Technology, Akadeemia tee 15, 19086 Tallinn, Estonia.     

Chemical induction of rapid and reversible plastid filamentation in Arabidopsis thaliana roots

Plastids assume various morphologies depending on their developmental status, but the basis for developmentally regulated plastid morphogenesis is poorly understood. Chemical induction of alterations in plastid morphology would be a useful tool for studying this; however, no such chemicals have been identified. Here, we show that antimycin A, an effective respiratory inhibitor, can change plastid morphology rapidly and reversibly in Arabidopsis thaliana. In the root cortex, hypocotyls, cotyledon epidermis and true leaf epidermis, significant differences in mitochondrial morphology were not observed between antimycin‐treated and untreated tissues. In contrast, antimycin caused extreme filamentation of plastids in the mature cortices of main roots. This phenomenon was specifically observed in the mature root cortex. Other mitochondrial respiratory inhibitors (rotenone and carbonyl cyanide m‐chlorophenylhydrazone), hydrogen peroxide, S‐nitroso‐N‐acetylpenicillamine [a nitric oxide (NO) donor] and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea did not mimic the phenomenon under the present study conditions. Antimycin‐induced plastid filamentation was initiated within 5 min after the onset of chemical treatment and appeared to complete within 1 h. Plastid morphology was restored within 7 h after the washout of antimycin, suggesting that the filamentation was reversible. Co‐applications of antimycin and cytoskeletal inhibitors (demecolcine or latrunculin B) or protein synthesis inhibitors (cycloheximide or chloramphenicol) still caused plastid filamentation. Antimycin A was also effective for plastid filamentation in the chloroplast division mutants atftsZ1‐1 and atminE1. Salicylhydroxamic acid, an alternative oxidase inhibitor, was solely found to suppress the filamentation, implying the possibility that this phenomenon was partly mediated by an antimycin‐activated alternative oxidase in the mitochondria.     

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.     

Metallochaperone-like genes in Arabidopsis thaliana

A complete inventory of metallochaperone-like proteins containing a predicted HMA domain in Arabidopsis revealed a large family of 67 proteins. 45 proteins, the HIPPs, have a predicted isoprenylation site while 22 proteins, the HPPs, do not. Sequence comparisons divided the proteins into seven major clusters (I-VII). Cluster IV is notable for the presence of a conserved Asp residue before the CysXXCys, metal binding motif, analogous to the Zn binding motif in E. coli ZntA. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 in Cluster IV were studied in more detail. All but HIPP21 could rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and atx1 mutants. In Arabidopsis, single and double mutants did not show a phenotype but the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type suggesting the HIPPs can have a role in Cd-detoxification, possibly by binding Cd. Promoter-GUS reporter expression studies indicated variable expression of these HIPPs. For example, in roots, HIPP22 and HIPP26 are only expressed in lateral root tips while HIPP20 and HIPP25 show strong expression in the root vasculature.     

UV-B-induced photomorphogenesis in Arabidopsis thaliana

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290–320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.     

Chromosome painting in Arabidopsis thaliana

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C(-1)). Pools of contiguous 113-139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

Systemic Endopolyploidy in Arabidopsis thaliana

Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant.     

Prenylcysteine methylesterase in Arabidopsis thaliana

Prenylated proteins undergo a series of post-translational modifications, including prenylation, proteolysis, and methylation. Collectively, these modifications generate a prenylcysteine methylester at the carboxyl terminus and modulate protein targeting and function. Prenylcysteine methylation is the only reversible step in this series of modifications. However, prenylcysteine -carboxyl methylesterase (PCME) activity has not been described in plants. We have detected a specific PCME activity in Arabidopsis thaliana membranes that discriminates between biologically relevant and irrelevant prenylcysteine methylester substrates. Furthermore, we have identified an Arabidopsis gene (At5g15860) that encodes measurable PCME activity in recombinant yeast cells with greater specificity for biologically relevant prenylcysteine methylesters than the activity found in Arabidopsis membranes. These results suggest that specific and non-specific esterases catalyze the demethylation of prenylcysteine methylesters in Arabidopsis membranes. Our findings are discussed in the context of prenylcysteine methylation/demethylation as a potential regulatory mechanism for membrane association and function of prenylated proteins in Arabidopsis.     

Salt-avoidance tropism in Arabidopsis thaliana

The orientation of plant root growth is modulated by developmental as well as environmental cues. Among the environmental factors, gravity has been extensively studied because of its overpowering effects in modulating root growth direction. However, our knowledge of the effects of other abiotic signals that influence root growth direction is largely unknown. Recently, we have shown that high salinity can modify root growth direction by inducing rapid amyloplast degradation in root columella cells of Arabidopsis thaliana. By exploiting salt overly sensitive (sos) mutants and PIN2 expression analyses, we have shown that the altered root growth direction in response to salt is mediated by ion disequilibrium and is correlated with PIN2 mRNA abundance and expression and localization of the protein. Our study demonstrates that the SOS pathway may mediate this process. Here we discuss our data from broader perspectives. We propose that salt-induced modification of root growth direction is a salt-avoidance behavior, which is an active adaptive mechanism for plants grown under saline conditions. Furthermore, high salinity also stimulates alteration of gravitropic growth of shoots. These findings illustrate that plants have a fine and sophisticated sensory and communication system that enable plants to dynamically and efficiently cope with rapidly changing environment.Key words: abidopsis, adaptation, avoidance, root, salt stress, tropic growthOwing to their sessile nature, plant roots are constantly bombarded with various environmental stimuli from the soil, such as gravity, physical obstacles and imbalanced distribution of water and/or nutrients and high salinity. Where to grow is an important developmental decision in the life cycle of a plant that is crucial for its adaptation and the subsequent reproductive success. The proper orientation of root growth is shaped by both the developmental inputs and external signals.^1,2 The overwhelming environmental factor that modulates root growth direction is gravity, and plant primary roots grow downward toward the gravity vector. This directed growth of root in response to gravity is named as tropic growth to gravity or gravitropism. Studies of gravity perception and signaling pathway of the root cap at the primary root of Arabidopsis strongly support the starch statolith hypothesis.³ In this hypothesis, the columella cells in the root cap, which contain sedimentable amyloplasts, are the gravity-perceptive site in roots. The inner columella cells of the second tier have been proposed as making the greatest contribution to root gravitropism.⁴ Upon gravity stimulation, cytosolic ions such as Ca²⁺ and rapid cytoplasmic alkalization may be involved in gravity signal transduction.^5–7 Asymmetric distribution of auxin in roots caused by basipetal transport mainly through the auxin efflux carrier PIN-FORMED2 (PIN2), which is distributed asymmetrically within the cells, results in gravitropic root response of the root elongation zone.^8,9In contrast to our understanding of gravitropism of root, our knowledge of tropistic responses of root to other major environmental stimuli, such as water availability, imbalanced nutrient distribution and high salinity, and the interplay between these stimuli in determining the directional growth of root remains enigmatic. Recent studies have confirmed the existence of hydrotropism and the molecular genetic basis of the tropistic growth of root to water in determining the final direction of root growth starts to be deciphered.^10–12 Hydrotropic growth of roots is an important trait for plants to actively find water and to optimize their fitness under drought condition. Salinity is another major constraint to root system development, and limits the productivity of agricultural crops and the distribution of plant species.^13–15 It is known that salt stress-induced disturbed balance of ions is the primary cause for inhibition of plant growth and subsequent yield reduction. How does root minimize entrance of harmful ions and subsequently avoid salt injury? Does plant have capacity to sense salt signal, and prevent potentially harmful ions reaching root and shoot?Previous studies have shown that plant use different strategies to avoid salt injury at various levels. After Na⁺ enters the root cells, the Casparian strip can restrict the movements of the harmful ion into the xylem.¹⁶ Root cells also avoid salt injury by extruding Na⁺ actively back to the outside solution. This energy-dependent ion efflux from cytosol across the plasma membrane is mediated by SOS1 gene, a Na⁺-H⁺ antiporter, which is regulated by at least other genes, SOS3 (calcium binding protein) and SOS2 (serine/threonine kinase). This is the well characterized SOS (Salt Overly Sensitive) signaling pathway.^17,18 Another way for plant root cells to avoid ion injury is to accumulate Na⁺ into vacuole. Vacuolar compartmentation of Na⁺ is also in part regulated by Na⁺-H⁺ antiporters, such as AtNHX1.¹⁹ These findings reveal mechanisms of how plants avoid Na⁺ injury after passive entrance of sodium ions into root cells. We questioned whether a plant is capable of actively preventing the harmful ions from reaching root cells or escape from high salinity in the environment, and how plant roots respond to changing salt conditions, because salt distribution is unbalanced under natural saline conditions, especially after rain and irrigation. With a new assay that allows us to specifically address how plant roots respond to changing salt levels, we discovered an alternative adaptive mechanism for plant root to avoid salt injury.²⁰We set up a two-layer medium assay in which a sodium ion gradient would be generated. A normal nutrient agar medium is at the top of the growth bottle and an agar with salt-stressed medium is in the bottom of the bottle. This simple assay allows us to monitor root growth and orientation. The roots of the wild type seedlings penetrated the interface of the layers and grew straight downwards exhibiting gravitropism, when both layers were MS media. In contrast, when the bottom medium contained NaCl, roots of seedlings grew downward first, and then curved and grew upward toward the lower levels of salt. Roots started to bend upward at an early stage even before contacting high-salt medium (250 mM NaCl) on the bottom. The results indicate that roots can sense ion gradients in the growing environment and transduce the signal, combine with internal signals to make decisions that enable roots to stay away from high salt.^21,22 Here, we would like to propose this salt-induced tropic growth as a salt-avoidance tropism, which is an important adaptive behavior for plant roots to avoid salt injury and direct them toward their goal of optimal fitness.²³ Because salt stress inhibits root elongation, we tested impact of salt-induced negative gravitropism on the root growth. The results showed that inhibitory effect of salt on root growth was largely alleviated with this tropic curve (Fig. 1), further verifying our hypothesis that the salt-induced developmental plasticity is a salt-avoidance behavior (Fig. 2).Open in a separate windowFigure 1Effects of salt on root elongation of Arabidopsis thaliana seedlings from different salt treatments. The inhibitory effect of salt stress on root growth was greatly alleviated in the wild type (Col-0) when root growth of the seedlings was analyzed using a two-layer medium assay (black bars). The MS nutrient medium is on the top, and NaCl concentrations in the media on the bottom are 0, 150 and 250 mM. More severe inhibition of root growth of the seedlings by various levels of NaCl in a root bending assay (white bars) was observed. Data represents means of measurements from >40 individuals from three independent experiments. Bars represent standard error.Open in a separate windowFigure 2An illustrative model of the sensing and response by the plant root when grown under different saline conditions. This model proposes two major mechanisms of salt responses by plants, where salt tolerance is the ability to function while stressed; Salt avoidance is the capacity to stay away from salt stress when growing in changing saline conditions.Another important point that we would like to bring out based on our observation in this work is that salinity also stimulated shoot positive gravitropism or negative phototropism. The observation implicates long-distance communication from root to shoot during plant salt response in the stressed plants. The exact biological function of shoot tropic growth, the signals in this long-distance communication, and underlying molecular mechanism still remains unknown.In conclusion, our study has revealed a novel complex adaptive mechanism that provides plants a capacity for avoiding injury from salt. The hypothesis we have proposed here should provide novel insights into plant stress avoidance. Further analysis using a combinatorial approach, mutant analysis and genomics, is required to decipher the molecular network underlying this salt-avoidance behavior.