Two independent loss-of-function mutations in anthocyanidin synthase homeologous genes are responsible for the all-green phenotype of sweet basil.

Sweet basil, Ocimum basilicum L., is an important culinary herb grown worldwide. Although basil is green, many landraces, breeding lines, and exotic cultivars have purple stems and flowers. This anthocyanin pigmentation is unacceptable in traditional Italian basil used for Pesto sauce production. In the current study, we aimed to resolve the genetics that underlines the different colors. We used the recently published sweet basil genome to map quantitative trait loci (QTL) for flower and stem color in a bi-parental F2 population. It was found that the pigmentation is governed by a single QTL, harboring an anthocyanidin synthase (ANS) gene (EC 1.14.20.4). Further analysis revealed that the basil genome harbors two homeologous ANS genes, each carrying a loss-of-function mutation. ObANS1 carries a single base pair insertion resulting in a frameshift, and ObANS2 carries a missense mutation within the active site. In the purple-flower parent, ANS1 is functional, and ANS2 carries a nonsense mutation. The functionality of the ObANS1 active allele was validated by complementation assay in an Arabidopsis ANS mutant. Moreover, we have restored the functionality of the missense-mutated ObANS2 using site-directed activation. We found that the non-functional alleles were expressed to similar levels as the functional allele, suggesting polyploids invest futile effort in expressing non-functional genes, offsetting their advantageous redundancy. This work demonstrated the usefulness of the genomics and genetics of basil to understand the basic mechanism of metabolic traits and raise fundamental questions in polyploid plant biology.

Genome-based high-resolution mapping of fusarium wilt resistance in sweet basil.

Fusarium wilt of basil is a disease of sweet basil (Ocimum basilicum L.) plants caused by the fungus Fusarium oxysporum f. sp. basilici (FOB). Although resistant cultivars were released > 20 years ago, the underlying mechanism and the genes controlling the resistance remain unknown. We used genetic mapping to elucidate FOB resistance in an F2 population derived from a cross between resistant and susceptible cultivars. We performed genotyping by sequencing of 173 offspring and aligning the data to the sweet basil reference genome. In total, 23,411 polymorphic sites were detected, and a single quantitative trait locus (QTL) for FOB resistance was found. The confidence interval was < 600 kbp, harboring only 60 genes, including a cluster of putative disease-resistance genes. Based on homology to a fusarium resistance protein from wild tomato, we also investigated a candidate resistance gene that encodes a transmembrane leucine-rich repeat - receptor-like kinase - ubiquitin-like protease (LRR-RLK-ULP). Sequence analysis of that gene in the susceptible parent vs. the resistant parent revealed multiple indels, including an insertion of 20 amino acids next to the transmembrane domain, which might alter its functionality. Our findings suggest that this LRR-RLK-ULP might be responsible for FOB resistance in sweet basil and demonstrate the usefulness of the recently sequenced basil genome for QTL mapping and gene mining.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death.

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.

Short De-Etiolation Increases the Rooting of VC801 Avocado Rootstock.

Dark-grown (etiolated) branches of many recalcitrant plant species root better than their green counterparts. Here it was hypothesized that changes in cell-wall properties and hormones occurring during etiolation contribute to rooting efficiency. Measurements of chlorophyll, carbohydrate and auxin contents, as well as tissue compression, histological analysis and gene-expression profiles were determined in etiolated and de-etiolated branches of the avocado rootstock VC801. Differences in chlorophyll content and tissue rigidity, and changes in xyloglucan and pectin in cambium and parenchyma cells were found. Interestingly, lignin and sugar contents were similar, suggesting that de-etiolated branches resemble the etiolated ones in this respect. Surprisingly, the branches that underwent short de-etiolation rooted better than the etiolated ones, and only a slight difference in IAA content between the two was observed. Gene-expression profiles revealed an increase in ethylene-responsive transcripts in the etiolated branches, which correlated with enrichment in xyloglucan hydrolases. In contrast, transcripts encoding pectin methylesterase and pectolyases were enriched in the de-etiolated branches. Taken together, it seems that the short de-etiolation period led to fine tuning of the conditions favoring adventitious root formation in terms of auxin-ethylene balance and cell-wall properties.

Myosin XI-K is involved in root organogenesis, polar auxin transport, and cell division.

The interplay between myosin- and auxin-mediated processes was investigated by following root development in the triple myosin knockout mutant xi-k xi-1 xi-2 (3KO). It was found that the 3KO plants generated significantly more lateral and adventitious roots than the wild-type plants or the rescued plant line expressing functional myosin XI-K:yellow fluorescent protein (YFP; 3KOR). Using the auxin-dependent reporter DR5:venus, a significant change in the auxin gradient toward the root tip was found in 3KO plants, which correlated with the loss of polar localization of the auxin transporter PIN1 in the stele and with the increased number of stele cells with oblique cell walls. Interestingly, myosin XI-K:YFP was localized to the cell division apparatus in the root and shoot meristems. In anaphase and early telophase, XI-K:YFP was concentrated in the midzone and the forming cell plate. In late telophase, XI-K:YFP formed a ring that overlapped with the growing phragmoplast. Myosin receptors MyoB1 and MyoB2 that are highly expressed throughout the plant were undetectable in dividing cells, suggesting that the myosin function in cell division relies on distinct adaptor proteins. These results suggest that myosin XIs are involved in orchestrating root organogenesis via effects on polar distribution of auxin responses and on cell division.

Expression of MAX2 under SCARECROW promoter enhances the strigolactone/MAX2 dependent response of Arabidopsis roots to low-phosphate conditions.

MAIN CONCLUSION: MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partially sufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions. Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.

Analysis of Microtubule-Associated-Proteins during IBA-Mediated Adventitious Root Induction Reveals KATANIN Dependent and Independent Alterations of Expression Patterns.

Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs.

Dissecting the contribution of microtubule behaviour in adventitious root induction.

Induction of adventitious roots (ARs) in recalcitrant plants often culminates in cell division and callus formation rather than root differentiation. Evidence is provided here to suggest that microtubules (MTs) play a role in the shift from cell division to cell differentiation during AR induction. First, it was found that fewer ARs form in the temperature-sensitive mutant mor1-1, in which the MT-associated protein MOR1 is mutated, and in bot1-1, in which the MT-severing protein katanin is mutated. In the two latter mutants, MT dynamics and form are perturbed. By contrast, the number of ARs increased in RIC1-OX3 plants, in which MT bundling is enhanced and katanin is activated. In addition, any1 plants in which cell walls are perturbed made more ARs than wild-type plants. MT perturbations during AR induction in mor1-1 or in wild-type hypocotyls treated with oryzalin led to the formation of amorphous clusters of cells reminiscent of callus. In these cells a specific pattern of polarized light retardation by the cell walls was lost. PIN1 polarization and auxin maxima were hampered and differentiation of the epidermis was inhibited. It is concluded that a fine-tuned crosstalk between MTs, cell walls, and auxin transport is required for proper AR induction.

Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling.

Strigolactones (SLs) are plant hormones that regulate the plant response to phosphate (Pi) growth conditions. At least part of SL-signalling execution in roots involves MAX2-dependent effects on PIN2 polar localization in the plasma membrane (PM) and actin bundling and dynamics. We examined PIN2 expression, PIN2 PM localization, endosome trafficking, and actin bundling under low-Pi conditions: a MAX2-dependent reduction in PIN2 trafficking and polarization in the PM, reduced endosome trafficking, and increased actin-filament bundling were detected in root cells. The intracellular protein trafficking that is related to PIN proteins but unassociated with AUX1 PM localization was selectively inhibited. Exogenous supplementation of the synthetic SL GR24 to a SL-deficient mutant (max4) led to depletion of PIN2 from the PM under low-Pi conditions. Accordingly, roots of mutants in MAX2, MAX4, PIN2, TIR3, and ACTIN2 showed a reduced low-Pi response compared with the wild type, which could be restored by auxin (for all mutants) or GR24 (for all mutants except max2-1). Changes in PIN2 polarity, actin bundling, and vesicle trafficking may be involved in the response to low Pi in roots, dependent on SL/MAX2 signalling.

Role of plant myosins in motile organelles: is a direct interaction required?

Plant organelles are highly motile, with speed values of 3-7 µm/s in cells of land plants and about 20-60 µm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant's ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins. Most plants express multiple members of two main classes: myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.

Gene expression profiling in juvenile and mature cuttings of Eucalyptus grandis reveals the importance of microtubule remodeling during adventitious root formation.

BACKGROUND: The ability to form adventitious roots (AR) is an economically important trait that is lost during the juvenile-to-mature phase change in woody plants. Auxin treatment, which generally promotes rooting in juvenile cuttings, is often ineffective when applied to mature cuttings. The molecular basis for this phenomenon in Eucalyptus grandis was addressed here. RESULTS: A comprehensive microarray analysis was performed in order to compare gene-expression profiles in juvenile and mature cuttings of E. grandis, with or without auxin treatment on days, 0, 1, 3, 6, 9 and 12 post AR induction. Under these conditions AR primordia were formed only in auxin-treated juvenile cuttings. However, clustering the expression profiles revealed that the time after induction contributed more significantly to the differences in expression than the developmental phase of the cuttings or auxin treatment. Most detected differences which were related to the developmental phase and auxin treatment occurred on day 6, which correlated with the kinetics of AR-primordia formation. Among the functional groups of transcripts that differed between juvenile and mature cuttings was that of microtubules (MT). The expression of 42 transcripts annotated as coding for tubulin, MT-associated proteins and kinesin motor proteins was validated in the same RNA samples. The results suggest a coordinated developmental and auxin dependent regulation of several MT-related transcripts in these cuttings. To determine the relevance of MT remodeling to AR formation, MTs were subjected to subtle perturbations by trifluralin, a MT disrupting drug, applied during auxin induction. Juvenile cuttings were not affected by the treatment, but rooting of mature cuttings increased from 10 to more than 40 percent. CONCLUSIONS: The data suggest that juvenile-specific MT remodeling is involved in AR formation in E. grandis.

Profiling microRNAs in Eucalyptus grandis reveals no mutual relationship between alterations in miR156 and miR172 expression and adventitious root induction during development.

BACKGROUND: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. RESULTS: We characterized the population of miRNAs of Eucalyptus grandis and compared the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. CONCLUSIONS: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability.

The availability of filament ends modulates actin stochastic dynamics in live plant cells.

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament-filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls.

Strigolactone analog GR24 triggers changes in PIN2 polarity, vesicle trafficking and actin filament architecture.

Strigolactones (SLs) are plant hormones that regulate shoot and root development in a MAX2-dependent manner. The mechanism underlying SLs' effects on roots is unclear. We used root hair elongation to measure root response to SLs. We examined the effects of GR24 (a synthetic, biologically active SL analog) on localization of the auxin efflux transporter PIN2, endosomal trafficking, and F-actin architecture and dynamics in the plasma membrane (PM) of epidermal cells of the primary root elongation zone in wildtype (WT) Arabidopsis and the SL-insensitive mutant max2. We also recorded the response to GR24 of trafficking (tir3), actin (der1) and PIN2 (eir1) mutants. GR24 increased polar localization of PIN2 in the PM of epidermal cells and accumulation of PIN2-containing brefeldin A (BFA) bodies, increased ARA7-labeled endosomal trafficking, reduced F-actin bundling and enhanced actin dynamics, all in a MAX2-dependent manner. Most of the der1 and tir3 mutant lines also displayed reduced sensitivity to GR24 with respect to root hair elongation. We suggest that SLs increase PIN2 polar localization, PIN2 endocytosis, endosomal trafficking, actin debundling and actin dynamics in a MAX2-dependent fashion. This enhancement might underlie the WT root's response to SLs, and suggests noncell autonomous activity of SLs in roots.

Enantioselective effects of (+)- and (-)-citronellal on animal and plant microtubules.

Citronellal is a major component of Corymbia citriodora and Cymbopogon nardus essential oils. Herein it is shown that whereas (+)-citronellal (1) is an effective microtubule (MT)-disrupting compound, (-)-citronellal (2) is not. Quantitative image analysis of fibroblast cells treated with 1 showed total fluorescence associated with fibers resembling that in cells treated with the MT-disrupting agents colchicine and vinblastine; in the presence of 2, the fluorescence more closely resembled that in control cells. The distribution of tubulin in soluble and insoluble fractions in the presence of 1 also resembled that in the presence of colchicine, whereas similar tubulin distribution was obtained in the presence of 2 and in control cells. In vitro polymerization of MTs was inhibited by 1 but not 2. Measurements of MT dynamics in plant cells showed similar MT elongation and shortening rates in control and 2-treated cells, whereas in the presence of 1, much fewer and shorter MTs were observed and no elongation or shrinkage was detected. Taken together, the MT system is suggested to be able to discriminate between different enantiomers of the same compound. In addition, the activity of essential oils rich in citronellal is affected by the relative content of the two enantiomers of this monoterpenoid.

Microarray analysis revealed upregulation of nitrate reductase in juvenile cuttings of Eucalyptus grandis, which correlated with increased nitric oxide production and adventitious root formation.

The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), which is involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO was observed upon excision, which was higher in juvenile cuttings than in mature ones. Treatment with an NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production in intact plants. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to increased ability to produce NO and to form adventitious roots. Arabidopsis transgenic plants constantly expressing EgNIA did not exhibit a significantly higher lateral or adventitious root formation, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root differentiation.

Myosin XIK is a major player in cytoplasm dynamics and is regulated by two amino acids in its tail.

It has recently been found that among the 17 Arabidopsis myosins, six (XIC, XIE, XIK, XI-I, MYA1, and MYA2) have a major role in the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum. Here, the same dominant negative tail fragments were also found to arrest the movement of Gogi bodies when transiently expressed in Arabidopsis plants. However, when a Golgi marker was transiently expressed in plants knocked out in these myosins, its movement was dramatically inhibited only in the xik mutant. In addition, a tail fragment of myosin XIK could inhibit the movement of several post-Golgi organelles, such as the trans-Golgi network, pre-vacuolar compartment, and endosomes, as well as total cytoplasmic streaming, suggesting that myosin XIK is a major player in cytoplasm kinetics. However, no co-localization of myosin tails with the arrested organelles was observed. Several deletion truncations of the myosin XIK tail were generated to corroborate function with localization. All deletion mutants possessing an inhibitory effect on organelle movement exhibited a diffuse cytoplasmic distribution. Point mutations in the tail of myosin XIK revealed that Arg1368 and Arg1443 are essential for its activity. These residues correspond to Lys1706 and Lys1779 from mouse myosin Va, which mediate the inhibitory head-tail interaction in this myosin. Therefore, such an interaction might underlie the dominant negative effect of truncated plant myosin tails and explain the mislocalization with target organelles.

Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica.

Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.

Differential effects of prenylation and s-acylation on type I and II ROPS membrane interaction and function.

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.

Glutathione peroxidase regulation of reactive oxygen species level is crucial for in vitro plant differentiation.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme's catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a beta-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point.