The natural compound trans-chalcone induces programmed cell death in Arabidopsis thaliana roots

Chalcone (1,3-diphenyl-2-propen-1-one) is an aromatic ketone precursor of important molecules in plants such as flavonoids or anthocyanins. Its phytotoxicity has been demonstrated on different plant species, but to date little is known about the mechanisms of action of this secondary metabolite at plant cellular level. Detailed analysis by light and transmission electron microscopy (TEM) was conducted to examine the root meristems' ultrastructure of control and chalcone-treated Arabidopsis seedlings. Mitochondrial dysfunction was analysed by measuring mitochondrial membrane potential with JC-1 fluorochrome. Finally, acridine orange/ethidium bromide staining was used for the detection of programmed cell death. Microscopy revealed tissue alterations, inhibition of root hair formation and important changes after 7 and 14 d at the chalcone IC(50) value. Chalcone-treated cells showed signs of programmed cell death such as mitochondrial condensation, disruption of organelles and chromatin fragmentation. Acridine orange/ethidium bromide staining confirmed the programmed cell death, which could be induced by the reduction of mitochondrial transmembrane potential (ΔΨ(m)) that was detected after chalcone treatment. These results confirm the phytotoxic activity of chalcone on Arabidopsis seedlings, the alteration of mitochondrial membrane potential and the induction of programmed cell death.     

Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.     

Between Xylem and Phloem: The Genetic Control of Cambial Activity in Plants

Abstract: Post-embryonic development is controlled by two types of meristems: apical and lateral. There has been considerable progress recently in understanding the function of root and shoot apical meristems at the molecular level. Knowledge of analogous processes in the lateral, or secondary, meristems, i.e. the vascular cambium or cork cambium, is, however, rudimentary. This is despite the fact that much of the diversity in the plant kingdom is based on the differential functions of these meristems, emphasizing the importance of lateral meristems in the development of different plant forms. The vascular cambium is particularly important for woody plants, but it also plays an important role during the development of various herbaceous species, such as Arabidopsis thaliana. In this review, we focus on the two basic functions of cambial activity: cell proliferation and pattern formation.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

Modulation of Arabidopsis CYCB1 expression patterns by polyamines and salt stress

Polyamines are new plant growth regulators that participate in various physiological processes modulating cell division and differentiation, stimulating secondary metabolite production and in stress responsiveness. In the present study, we evaluated the effect of polyamine application on CYCB1-GUS reporter line in Arabidopsis, in order to monitor changes in cell division. We observed that polyamines modulate the expression of CYCB1-GUS, most likely in an amine-specific manner. In particular, spermidine and spermine induced significant increases in CYCB1-GUS expression in shoot apex and root meristems. According of this view, mainly the higher polyamines stimulate the lateral root formation in Arabidopsis. Furthermore, the application of d-arginine and methylglyoxal bis-(guanylhydrazone) polyamine inhibitors drastically reduced Arabidopsis CYCB1-GUS root growth and plant fresh weight, as well as CYCB1-GUS expression. Another key point on this study was to analyze the effect of polyamines on CYCB1-GUS expression under salt stress. Salt stress treatments repressed CYCB1-GUS expression in a concentration dependent manner; this negative effect was ameliorated by polyamine application, in particular by spermidine and spermine, even at 125 mM NaCl, allowing the maintenance of CYCB1-GUS levels under salt stress. This work is one more contribution on the role of polyamines in cell cycle modulation and abiotic stress protection.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.     

Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings

Changes in root architecture are one of the adaptive strategies used by plants to compensate for nutrient deficiencies in soils. In this work, the temporal responses of Arabidopsis (Arabidopsis thaliana) root system architecture to low boron (B) supply were investigated. Arabidopsis Col-0 seedlings were grown in 10 μM B for 5 days and then transferred to a low B medium (0.4 μM) or control medium (10 μM) for a 4-day period. Low B supply caused an inhibition of primary root (PR) growth without altering either the growth or number of lateral roots (LRs). In addition, low B supply induced root hair formation and elongation in positions close to the PR meristem not observed under control conditions. The possible role of auxin and ethylene in the alteration of root system architecture elicited by low B supply was also studied by using two Arabidopsis reporter lines (DR5:GUS and EBS:GUS) and two Arabidopsis mutants with impaired auxin and ethylene signaling (aux1-22 and ein2-1). Low B supply increased auxin reporter DR5:GUS activity in PR tip, suggesting that low B alters the pattern of auxin distribution in PR tip. Moreover, PR elongation in aux1-22 mutant was less sensitive to low B treatment than in wild-type plants, which suggests that auxin resistant 1 (AUX1) participates in the inhibition of PR elongation under low B supply. From all these results, a hypothetical model to explain the effect of low B treatment on PR growth is proposed. We also show that ethylene, via ethylene-insensitive 2 (EIN2) protein, is involved in the induction of root hair formation and elongation under low B treatment.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Role of hormones in the induction of iron deficiency responses in Arabidopsis roots

In "strategy I" plants, several alterations in root physiology and morphology are induced by Fe deficiency, although the mechanisms by which low Fe levels are translated into reactions aimed at alleviating Fe shortage are largely unknown. To prove whether changes in hormone concentration or sensitivity are involved in the adaptation to suboptimal Fe availability, we tested 45 mutants of Arabidopsis defective in hormone metabolism and/or root hair formation for their ability to increase Fe(III) chelate reductase activity and to initiate the formation and enlargement of root hairs. Activity staining for ferric chelate reductase revealed that all mutants were responsive to Fe deficiency, suggesting that hormones are not necessary for the induction. Treatment of wild-type plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid caused the development of root hairs in locations normally occupied by non-hair cells, but did not stimulate ferric reductase activity. Ectopic root hairs were also formed in -Fe roots, suggesting a role for ethylene in the morphological responses to Fe deficiency. Ultrastructural analysis of rhizodermal cells indicated that neither Fe deficiency nor 1-aminocyclopropane-1-carboxylic acid treatment caused transfer-cell-like alterations in Arabidopsis roots. Our data indicate that the morphological and physiological components of the Fe stress syndrome are regulated separately.     

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

Background and Aims Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Methods Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Key Results Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 μg mL(-1) MCY-LR, accelerated cell cycle at 10 μg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. Conclusions MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.     

Nitric oxide is essential for vesicle formation and trafficking in Arabidopsis root hair growth

The functions of nitric oxide (NO) in processes associated with root hair growth in Arabidopsis were analysed. NO is located at high concentrations in the root hair cell files at any stage of development. NO is detected inside of the vacuole in immature actively growing root hairs and, later, NO is localized in the cytoplasm when they become mature. Experiments performed by depleting NO in Arabidopsis root hairs indicate that NO is required for endocytosis, vesicle formation, and trafficking and it is not involved in nucleus migration, vacuolar development, and transvacuolar strands. The Arabidopsis G'4,3 mutant (double mutant nia1/nia2) is severely impaired in NO production and generates smaller root hairs than the wild type (WT). Root hairs from the Arabidopsis G'4,3 mutant show altered vesicular trafficking and are reminiscent of NO-depleted root hairs from the Arabidopsis WT. Interestingly, normal vesicle formation and trafficking as well as root hair growth is restored by exogenous NO application in the Arabidopsis G'4,3 mutant. All together, these results firmly support the essential role played by NO in the Arabidopsis root-hair-growing process.     

Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency

Plant root sensing and adaptation to changes in the nutrient status of soils is vital for long-term productivity and growth. Reactive oxygen species (ROS) have been shown to play a role in root response to potassium deprivation. To determine the role of ROS in plant response to nitrogen and phosphorus deficiency, studies were conducted using wild-type Arabidopsis and several root hair mutants. The expression of several nutrient-responsive genes was determined by Northern blot, and ROS were quantified and localized in roots. The monitored genes varied in intensity and timing of expression depending on which nutrient was deficient. In response to nutrient deprivation, ROS concentrations increased in specific regions of the Arabidopsis root. Changes in ROS localization in Arabidopsis and in a set of root hair mutants suggest that the root hair cells are important for response to nitrogen and potassium. In contrast, the response to phosphorus deprivation occurs in the cortex where an increase in ROS was measured. Based on these results, we put forward the hypothesis that root hair cells in Arabidopsis contain a sensing system for nitrogen and potassium deprivation.     

MGOUN3, an Arabidopsis gene with TetratricoPeptide-Repeat-related motifs, regulates meristem cellular organization

In order to understand the functioning of apical meristems in Arabidopsis more clearly, a new mutant, mgoun3 (mgo3), affected in the structural organization and the functional regulation of both shoot and root meristems has been isolated. mgo3 plants display perturbations in leaf morphogenesis, in the spatial and the temporal formation of primordia, and frequent fasciation of the inflorescence stem. Cellular analysis showed that both cellular organization and cell identity patterning are impaired in the mutant meristems. The MGO3 gene has been isolated by positional cloning. The protein deduced from the cDNA sequence contains TetratricoPeptide Repeats (TPR) and Leucine-Rich Repeats (LRR), two motifs that are thought to act in protein-protein interactions. This gene appears to be unique in the Arabidopsis genome. Although the MGO3 protein presents TPR as in the Arabidopsis proteins HOBBIT and SPINDLY, the MGO3 motifs are more similar to those present in LGN-related proteins, which are regulators for some of the asymmetric cell divisions in animal development. These features suggest a key role for MGO3 in meristematic cell divisions and would be of interest for the comparison between plant and animal development.