Glutathione transport: a new role for PfCRT in chloroquine resistance.

AIMS: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. RESULTS: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. INNOVATION: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. CONCLUSION: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses.

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

The D-type cyclin CYCD4;1 modulates lateral root density in Arabidopsis by affecting the basal meristem region.

Root cell division occurs primarily in the apical meristem, from which cells are displaced into the basal meristem, where division decreases and cell length increases before the final differentiation zone. The organization of the root in concentric files implies coordinated division and differentiation of cell types, including the xylem pole pericycle cells, which uniquely can resume division to initiate lateral roots (LR). Here, we show that D-type cyclin CYCD4;1 is expressed in meristematic pericycle protoxylem poles and is required for normal LR density. Cycd4;1 mutants also show a displacement of the apical/basal meristem boundary in the pericycle and longer pericycle basal meristem cells, whereas other cell layers and overall meristem size and root growth are unaffected. Auxin is proposed to separately prepattern and stimulate LR initiation. Stimulation is unimpaired in cycd4;1, suggesting CYCD4;1 requirement for normal spacing but not initiation. Both pericycle cell length and LR density phenotypes of cycd4;1 are rescued by low concentrations of applied auxin, suggesting that the basal meristem has a role in determining LR density. We further show CYCD4;1 is rate-limiting for sucrose-dependent LR formation, since CYCD4;1 expression is sucrose-dependent and wild-type roots fully phenocopy cycd4;1 in sucrose absence. We conclude that CYCD4;1 links meristem pericycle cell behavior to LR density consistent with a basal meristem prepatterning model and that D-type cyclins can confer division potential of defined cell types through cell-specific expression patterns.

Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses.

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

A greenprint for growth: signalling the pattern of proliferation.

The shoot and root apical meristems (SAM and RAM, respectively) of plants serve both as sites of cell division and as stem cell niches. The SAM is also responsible for the initiation of new leaves, whereas the analogous process of lateral root initiation occurs in the pericycle, a specialized layer of cells that retains organogenic potential within an otherwise non-dividing region of the root. A picture is emerging of how cell division, growth, and differentiation are coordinated in the meristems and lateral organ primordia of plants. This is starting to reveal striking parallels between the control of stem cell maintenance in both shoots and roots, and to provide information on how signalling from developmental processes and the environment impact on cell behaviour within meristems.

D-type cyclins activate division in the root apex to promote seed germination in Arabidopsis.

Seeds provide survival and dispersal capabilities by protecting the dormant mature plant embryo. Germination and resumption of development under favourable conditions requires the reinitiation of cell growth and division through poorly understood processes. Here we show that four phases of cell division activation during germination in Arabidopsis are related to external morphological changes. Cell division initiates in the root apical meristem (RAM) before root protrusion, followed by sequential activation of cell division in the cotyledons, shoot apical meristem (SAM), and secondary meristems. Major changes in transcript levels of >2,000 genes precede root emergence, including expression peaks of six D-type (CYCD) and two A-type cyclins. Two further CYCDs are activated later with the SAM. Early activated CYCDs play key roles in regulating the extent of cell division, because loss-of-function alleles of early CYCDs display reduced division activation and consequential delayed root emergence. Conversely, elevation of early CYCDs increases cell cycle activation in the RAM and promotes embryonic root (radicle) protrusion, whereas a later-acting CYCD does not. These phenotypes, together with their overlapping expression domains, support a cumulative action of a subset of CYCDs in cell cycle reactivation, rather than a complete functional redundancy. This analysis reveals a phenotype associated with loss-of-function of a plant cyclin and demonstrates that D-type cyclins regulate cell cycle reentry during meristem activation to promote successful germination and early seedling growth.

The developmental context of cell-cycle control in plants.

Plant growth is characterised both by continued growth and organogenesis throughout development, as well as by environmental influences on the rate and pattern of these processes. This necessitates a close relationship between cell cycle control, differentiation and development that can be readily observed and studied. The sequencing of the Arabidopsis genome has revealed the full complexity of cell cycle regulators in plants, creating a challenge to understand how these genes control plant growth and differentiation, and how they are integrated with intrinsic and external signals. Here, we review the control of the cell cycle and examine how it is integrated with proliferative activity within meristems, and during the differentiation processes leading to leaf and lateral root formation.