Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

The conserved fission complex on peroxisomes and mitochondria

Peroxisomes are eukaryotic organelles highly versatile and dynamic in content and abundance. Plant peroxisomes mediate various metabolic pathways, a number of which are completed sequentially in peroxisomes and other subcellular organelles, including mitochondria and chloroplasts. To understand how peroxisomal dynamics contribute to changes in plant physiology and adaptation, the multiplication pathways of peroxisomes are being dissected. Research in Arabidopsis thaliana has identified several evolutionarily conserved families of proteins in peroxisome division. These include five PEROXIN11 proteins (PEX11a to -e) that induce peroxisome elongation and the fission machinery, which is composed of three dynamin-related proteins (DRP3A, -3B and -5B) and DRP''s membrane receptor, FISSION1 (FIS1A and -1B). While the function of PEX11 is restricted to peroxisomes, the fission factors are more promiscuous. DRP3 and FIS1 proteins are shared between peroxisomes and mitochondria, and DRP5B plays a dual role in the division of chloroplasts and peroxisomes. Analysis of the Arabidopsis genome suggests that higher plants may also contain functional homologs of the yeast Mdv1/Caf4 proteins, adaptor proteins that link DRPs to FIS1 on the membrane of both peroxisomes and mitochondria. Sharing a conserved fission machine between these metabolically linked subcellular compartments throughout evolution may have some biological significance.Key words: Arabidopsis, peroxisomal and mitochondrial division, dynamin-related protein (DRP), FISSION1 (FIS1), mitochondrial division 1 (Mdv1), CCR4p-associated factor 4 (Caf4)Peroxisomes are single membrane-delimited organelles involved in a variety of metabolic pathways essential to development.¹ Plant peroxisomes participate in processes such as lipid mobilization, photorespiration, detoxification, hormone biosynthesis and metabolism, and plant-pathogen interaction.^2,3 A number of these metabolic functions, such as photorespiration, fatty acid metabolism and jasmonic acid biosynthesis, are accomplished through the cooperative efforts of peroxisomes and other subcellular compartments, such as mitochondria and chloroplasts.^3–5 The function, morphology and abundance of peroxisomes can vary depending on the organism, cell type, developmental stage and prevailing environmental conditions in which the organism resides.^6,7 It is now believed that in addition to budding from the endoplasmic reticulum (ER), peroxisomes also multiply from pre-existing peroxisomes via division, going through steps including peroxisome elongation/tubulation, membrane constriction and fission.^7,8In the reference plant Arabidopsis thaliana, three evolutionarily conserved families of proteins have been identified as key components of the peroxisome division apparatus. Five integral membrane proteins, named PEX11a to -e, are mainly responsible for inducing the elongation and tubulation of peroxisomes in the early stage of peroxisome division.^9–11 DRP3A and DRP3B are members of a dynamin-related protein family that powers the fission of membranes and FIS1A and FIS1B are homologous proteins believed to anchor the DRP proteins to the membrane.^12–19 Similar to their counterparts in yeasts and mammals, DRP3 and FIS1 are shared by the fission machineries of peroxisomes and mitochondria.^12–19 We recently reported the unexpected finding that DRP5B, a plant/algal-specific DRP distantly related to the DRP3 proteins and originally discovered for its function in chloroplast division, is also involved in the division of peroxisomes. Using co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we further demonstrated that DRP5B and the two DRP3 proteins can homo- and hetero-dimerize and each DRP can form a complex with FIS1A and/or FIS1B and most of the Arabidopsis PEX11 isoforms.²⁰ These results together demonstrate that, despite their distinct evolutionary origins, structures and functions, peroxisomes, mitochondria and chloroplasts use some of the same factors for fission. These data also revealed that, like in yeasts and mammals, the FIS1-DRP complex exits on peroxisomes and mitochondria in plants.DRP5B, a DRP unique in the plant and photosynthetic algae lineages, seems to be the sole component shared by the division of chloroplasts and peroxisomes.²⁰ However, both FIS1 and DRP are found to be required for the division of peroxisomes and mitochondria throughout eukaryotic evolution,^21,22 prompting the question: to what extent is the FIS1-DRP complex conserved among diverse species? In the yeast Saccharomyces cerevisiae, this fission complex also contains an adaptor encoded by two homologous WD40 proteins, Mdv1 and Caf4, which are partially redundant in function with Mdv1 playing the major role. Mdv1 and Caf4 share an N-terminal extension (NTE) domain with two α-helices, a middle coiled-coil domain (CC) and C-terminal WD40 repeat. Both proteins use the NTE to interact with the tetratricopeptide repeat (TPR) domain-containing N-terminus of Fis1, the CC domain to dimerize and the C-terminal WD40 repeat to interact with and recruit the DRP protein, Dnm1.^23,24 The Hansenula polymorpha Mdv1 (Hp Mdv1) also has a dual function in the division of peroxisomes and mitochondria.²⁵ In addition, a Mdv1/Caf4 homolog, Mda1, was identified from the primitive red algae Cyanidioschyzon merolae and found to be involved at least in mitochondrial fission.²⁶ However, higher eukaryotes do not seem to have obvious homologs of Mdv1/Caf4. For example, mammals contain Fis1 and Drp (called DLP1 or Drp1) but no apparent homologs to Mdv1 and Caf4. Instead, a metazoan-specific tail-anchored protein, Mitochondrial Fission Factor (Mff), was recently found to regulate the fission of mitochondria and peroxisomes in a similar manner to Fis1. Mff is essential in recruiting Drp1, at least in mitochondrial division, yet it functions in a Fis1-independent pathway.^27,28To determine whether plants contain structural or functional homologs of Mdv1 and Caf4, we performed blast searches of the Arabidopsis genome, which resulted in the retrieval of ∼300 WD40 proteins. However, just like the search results from mammals, none of these proteins show significant sequence similarity with Mdv1 and Caf4 beyond the WD40 repeats. To identify proteins with similar domain structures with Mdv1/Caf4, we further analyzed these WD40 proteins, using the online Simple Modular Architecture Research Tool (smart.embl-heidelberg.de/). After eliminating proteins apparently inappropriate to be part of this complex, such as kinases and proteins with drastically distinct domain organizations despite of having both WD40 repeats and CC domains, we were able to narrow down to eight proteins. These proteins, which are encoded by At1g04510, At2g32950, At2g33340, At3g18860, At4g05410, At4g21130, At5g50230 and At5g67320, respectively, each contain a central CC domain in addition to the WD40 repeat region and are ranging from 450 to 900 amino acids in length (Fig. 1A). Subcellular localization studies will need to be performed to determine whether some of these proteins are associated with peroxisomes and mitochondria. If such a WD40 protein is proven to be part of the FIS1-DRP complex in Arabidopsis, it will be important to determine whether it simply acts as an adaptor or it also plays other roles, such as to promote and maintain the active structure and conformation of DRP3A/3B at the division site (Fig. 1B). Consistent with the latter scenario, it was found that Sc Mdv1 accumulates at the division sites after Dnm1 assembles and that the mammalian Fis1 and Drp1 proteins physically interact.^29,30 Peroxisomes and mitochondria are functionally linked in a number of metabolic pathways. For example, in plants, they act cooperatively in important processes such as fatty acid metabolism and photorespiration.³ An interesting question to address in the future is whether sharing such a conserved fission machine between peroxisomes and mitochondria throughout evolution has critical biological consequences.Open in a separate windowFigure 1Domain structure of Mdv1/Caf4 and their homologs or putative homologs. (A) Domain structure of Sc Mdv1 and Sc Caf4 from S. cerevisiae, their homologs from H. polymorpha and C. merolae, and the eight Arabidopsis proteins with similar domain organization. Grey boxes indicate the CC domain and black boxes are Wd40 repeats. (B) The putative FIS1-WD40-DRP complex in Arabidopsis. CC, coiled-coil; NTE, N-terminal extension; TPR, tetratricopeptide repeat; TMD, transmembrane domain.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

A Novel MAPKK Involved in Cell Death and Defense Signaling

A high-throughput in planta overexpression screen of a Nicotiana benthamiana cDNA library identified a mitogen activated protein kinase kinase (MAPKK), NbMKK1, as a potent inducer of hypersensitive response (HR)-like cell death. NbMKK1-mediated cell death was attenuated in plants whereby expression of NbSIPK, an ortholog of tobacco SIPK and Arabidopsis AtMPK6, was knocked down by virus-induced gene silencing (VIGS), suggesting that NbMKK1 functions upstream of NbSIPK. In accordance with this result, NbMKK1 phosphorylated NbSIPK in vitro, and furthermore NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. VIGS of NbMKK1 in N. benthamiana resulted in a delay of Phytophthora infestans INF1 elicitin-mediated HR as well as in the reduction of resistance against a non-host pathogen Pseudomonas cichorii. Our data of NbMKK1, together with that of LeMKK4,¹ demonstrate the presence of a novel defense signaling pathway involving NbMKK1/LeMKK4 and SIPK.Key Words: MAPK, defense, cell death, in planta screenMitogen activated protein kinase (MAPK) cascades are highly conserved signaling pathways in eukaryotes, comprising three tiered classes of protein kinase, MAPKKK (MAPKK kinase), MAPKK and MAPK, that sequentially relay phosphorylation signals.² The Arabidopsis genome carries genes for 20 MAPKs, 10 MAPKKs³ and more than 25 MAPKKKs.⁴ In plants, MAPK signaling is known to function in various biotic^4,5 and abiotic⁶ stress responses and cytokinesis.⁷ In defense signaling, extensive research has been carried out for two tobacco MAPKs, SIPK⁸ (salicylic-acid-induced protein kinase; hereafter designated as NtSIPK) and WIPK⁹ (wound-induced protein kinase = NtWIPK), and their orthologs in Arabidopsis¹⁰ (AtMPK6 and ATMPK3, respectively), partly because kinase activities of these two MAPKs are easy to detect by an in gel kinase assay using myeline basic protein (MBP) as substrate.¹¹ Both NtSIPK and NtWIPK are activated by the interaction between host resistance (R)- gene and cognate avirulence gene of pathogen^11,12 and elicitor perception by host cells.^13,14 Shuqun Zhang and his group showed that an upstream kinase of both NtSIPK and NtWIPK is NtMEK2.¹⁵ Transient overexpression of constitutively active NtMEK2 caused phosphorylation of NtSIPK and NtWIPK, resulting in rapid HR-like cell death in tobacco leaves.¹⁵ Later, the same lab showed that overexpression of NtSIPK alone also caused HR-like cell death.¹⁶ The downstream target proteins of NtSIPK and AtMPK6 are being identified and include 1-aminocyclopropane-1-carboxylic acid sythase-6 (ACS-6).^17,18 Although recent studies identified another MAPK cascade (NtMEK1 → Ntf6) involved in defense responses^19,20 we can still say that the current research focus of MAPK defense signaling centers around the cascade comprising [NtMEK2→ NtSIPK/NtWIPK→ target proteins] of tobacco and its orthologous pathways in other plant species.In an effort to search for plant genes involved in HR-like cell death, we have been employing a high-throughput in planta expression screen of N. benthamiana cDNA libraries. In this experimental system, a cDNA library was made in a binary potato virus X (PVX)-based expression vector pSfinx.²¹ The cDNA library was transferred to Agrobacterium tumefaciens, and 40,000 of the bacterial colonies were individually inoculated by toothpicks onto leaf blades of N. benthamiana leaves. The phenotype around the inoculated site was observed 1–2 weeks following the inoculation. This rapid screen identified 30 cDNAs that caused cell death after overexpression, including genes coding for ubiquitin proteins, RNA recognition motif (RRM) containing proteins, a class II ethylene-responsive element binding factor (EREBP)-like protein²² and a MAPKK protein (this work). Such an in planta screening technique has been used before for the isolation of fungal²¹ and oomycete^23,24 elicitors and necrosis inducing genes, but not for isolation of plant genes. Overexpression screening of cDNA libraries is a common practice in prokaryotes, yeast and amimal cells,^25,26 so it is a surprise that this approach has not been systematically applied in plants. Given its throughput, we propose that this virus-based transient overexpression system is a highly efficient way to isolate novel plant genes by functional screen.²⁷ Since overexpression frequently causes non-specific perturbation of signaling, genes identified by overexpression should be further validated by loss-of-function assays, for instance, VIGS.²⁸Overexpression of the identified MAPKK gene, NbMKK1, triggered a rapid generation of H₂O₂, followed by HR-like cell death in N. benthamiana leaves (this work). NbMKK1-GFP fusion protein overexpression also caused cell death, and curiously NbMKK1-GFP was shown to localize consistently in the nucleus. Sequence comparison classified NbMKK1 to the Group D of MAPKKs about which little information is available. So far, a MAPKK, LeMKK4, from tomato belonging to the Group D MAPKKs, was shown to cause cell death after overexpression.¹ Based on amino acid sequence similarity and phylogenetic analyses, LeMKK4 and NbMKK1 seem to be orthologs. To see whether NbMKK1 transduces signals through SIPK and WIPK, we performed NbMKK1 overexpression in N. benthamiana plants whereby the expression of either NbSIPK or NbWIPK (WIPK ortholog in N. benthamiana) was silenced by VIGS. NbMKK1 did not induce cell death in NbSIPK-silenced plants, suggesting that the NbMKK1 cell death signal is transmitted through NbSIPK. Indeed, NbMKK1 phosphorylated NbSIPK in vitro, and NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. These results suggest that NbMKK1 interacts with NbSIPK, most probably with its N-terminal docking domain, and phosphorylates NbSIPK in vivo to transduce the cell death signal downstream.NbMKK1 exhibits constitutive expression in leaves. To determine the function of NbMKK1 in defense, we silenced NbMKK1 by VIGS, and such plants were challenged with Phytophthora infestans INF1 elicitin²⁹ and Pseudomonas cichorii, a non-host pathogen. INF1-mediated HR cell death was remarkably delayed in NbMKK1-silenced plants. Likewise, plant defense against P. cichorii was compromised in NbMKK1-silenced plants. These results indicate that NbMKK1 is an important component of signaling of INF1-mediated HR and non-host resistance to P. cichorii.Together, our analyses of NbMKK1 and independent work from Greg Martin''s lab on LeMKK4¹ suggest that a Group D MAPKK, NbMKK1/LeMKK4, functions upstream of SIPK and transduces defense signals in these solanaceous plants (Fig. 1). In plants as well as in other eukaryotes, it is common that kinases have multiple partners. The work on these kinases fits this concept. A single MAPK (e.g., SIPK) is phosphorylated by multiple MAPKKs (e.g., NtMEK2 and NbMKK1), and a single MAPKK (e.g., NtMEK2) can phosphorylate multiple MAPKs (e.g., NtSIPK and NtWIPK).Open in a separate windowFigure 1Defense signaling through NbMKK1/LeMKK4. Two defense signal pathways involving NtMEK2 (indicated as MEK2) → WIPK/SIPK and NtMEK1(indicated as MEK1) → Ntf6 are well documented. By our and Pedley and Martin''s¹ works, another novel MAPKK, NbMKK1/LeMKK4 was demonstrated to participate in defense signaling by phosphorylation of SIPK.     

Proteome Analysis of Peroxisomes from Etiolated Arabidopsis Seedlings Identifies a Peroxisomal Protease Involved in β-Oxidation and Development

Plant peroxisomes are highly dynamic organelles that mediate a suite of metabolic processes crucial to development. Peroxisomes in seeds/dark-grown seedlings and in photosynthetic tissues constitute two major subtypes of plant peroxisomes, which had been postulated to contain distinct primary biochemical properties. Multiple in-depth proteomic analyses had been performed on leaf peroxisomes, yet the major makeup of peroxisomes in seeds or dark-grown seedlings remained unclear. To compare the metabolic pathways of the two dominant plant peroxisomal subtypes and discover new peroxisomal proteins that function specifically during seed germination, we performed proteomic analysis of peroxisomes from etiolated Arabidopsis (Arabidopsis thaliana) seedlings. The detection of 77 peroxisomal proteins allowed us to perform comparative analysis with the peroxisomal proteome of green leaves, which revealed a large overlap between these two primary peroxisomal variants. Subcellular targeting analysis by fluorescence microscopy validated around 10 new peroxisomal proteins in Arabidopsis. Mutant analysis suggested the role of the cysteine protease RESPONSE TO DROUGHT21A-LIKE1 in β-oxidation, seed germination, and growth. This work provides a much-needed road map of a major type of plant peroxisome and has established a basis for future investigations of peroxisomal proteolytic processes to understand their roles in development and in plant interaction with the environment.Peroxisomes, originally known as microbodies, are small and single-membrane eukaryotic organelles that compartmentalize various oxidative metabolic functions. Most peroxisomal matrix proteins carry a C-terminal tripeptide named PEROXISOME TARGETING SIGNAL TYPE1 (PTS1), and fewer contain an N-terminal nonapeptide, PTS2 (Lanyon-Hogg et al., 2010). PTS1 is further divided into major and minor PTS1s. Major PTS1 tripeptides, such as SKL> and SRL> (> represents the stop codon), are by themselves sufficient to direct a protein to the peroxisome (Reumann, 2004), whereas minor PTS1s are usually found in low-abundance proteins and require additional upstream elements for peroxisomal targeting (Kaur et al., 2009). Peroxisomes are highly variable morphologically and metabolically, as their size, shape, abundance, and enzymatic content can differ depending on the species, tissue and cell type, and prevailing environmental conditions (Beevers, 1979; van den Bosch et al., 1992; Kaur et al., 2009; Hu et al., 2012; Schrader et al., 2012).Plant peroxisomes participate in a wide range of metabolic processes, such as lipid metabolism, photorespiration, detoxification, biosynthesis of jasmonic acid, and metabolism of indole-3-butyric acid (IBA), nitrogen, sulfite, and polyamine (Kaur et al., 2009; Hu et al., 2012). Specific names had been given to certain types of peroxisomes due to their unique metabolic properties. For example, the term glyoxysome was coined when a new type of organelle that contained enzymes of the glyoxylate cycle was identified from the endosperm of castor bean (Ricinus communis; Breidenbach et al., 1968). It was later realized that glyoxysomes are in fact a type of peroxisome, and Beevers (1979) subsequently classified plant peroxisomes into three subtypes based on their primary biochemical functions. Glyoxysomes are located in storage organs such as fatty seedling tissues and play a major role in converting fatty acids to sugar; leaf peroxisomes are involved in photorespiration; and nonspecialized peroxisomes exist in other plant tissues and perform unknown functions.The primary function of leaf peroxisomes is the recycling of phosphoglycolate during photorespiration, a process coordinated by chloroplasts, peroxisomes, mitochondria, and the cytosol. In this pathway, phosphoglycolate produced by the oxygenase activity of Rubisco is ultimately converted to glycerate, which reenters the chloroplastic Calvin-Benson cycle (Foyer et al., 2009; Peterhansel et al., 2010). The peroxisome-localized enzymes glycolate oxidase (GOX), catalase, aminotransferase (serine:glyoxylate aminotransferase [SGT] and glutamate-glyoxylate aminotransferase [GGT]), HYDROXYPYRUVATE REDUCTASE1 (HPR1), and peroxisomal malate dehydrogenase (PMDH) are involved in the process (Reumann and Weber, 2006). On the other hand, lipid mobilization through fatty acid β-oxidation and the glyoxylate cycle is the main function for peroxisomes in seeds and germinating seedlings. In this process, fatty acids are first activated into fatty acyl-CoA esters by the acyl-activating enzyme (AAE)/acyl-CoA synthetase before entering the β-oxidation cycle, during which an acetyl-CoA is cleaved in each cycle by the successive action of acyl-CoA oxidase (ACX), multifunctional protein (MFP), and 3-keto-acyl-CoA thiolase (KAT). Acetyl-CoA, an end product of β-oxidation, is further converted to four-carbon carbohydrates by the glyoxylate cycle, in which isocitrate lyase (ICL) and malate synthase (MLS) are two key enzymes that function exclusively in this pathway. Products of the glyoxylate cycle exit the peroxisome, enter gluconeogenesis, and are further converted to hexose and Suc to fuel the postgerminative development of seedlings (Penfield et al., 2006).Immunocytochemical studies of germinating seeds from pumpkin (Cucurbita pepo), watermelon (Citrullis vulgaris), and cucumber (Cucumis sativus) demonstrated that seed peroxisomes (glyoxysomes) are directly transformed into leaf peroxisomes during greening of the cotyledons without de novo biogenesis of leaf peroxisomes (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). This conversion was illustrated by the import of photorespiratory enzymes and their concomitant presence with glyoxylate cycle enzymes within the same organelle. Furthermore, the increase in abundance of photorespiratory enzymes coincided with the marked decrease, and subsequently the absence, of glyoxylate cycle enzymes (ICL and/or MLS) at the culmination of this process (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). It was suggested that the specific names for plant peroxisomal variants should be eliminated because protein composition between leaf peroxisomes and glyoxysomes may differ by only two proteins (i.e. ICL and MLS) out of the over 100 total proteins in the peroxisome (Pracharoenwattana and Smith, 2008). This prediction needed to be tested. In addition, mutants lacking core peroxisome biogenesis factors or major β-oxidation enzymes are nonviable, suggesting that peroxisomes are essential to embryogenesis and seed germination (Hu et al., 2012). However, how peroxisomes contribute to seed germination and seedling establishment is not completely understood. In the past, studies have been successfully undertaken to catalog the proteome of mitochondria and plastids isolated from different plant tissues, which uncovered unique facets of organelle metabolism in various tissues (van Wijk and Baginsky, 2011; Havelund et al., 2013; Lee et al., 2013). As such, it was necessary to establish a protein atlas for peroxisomes in dark-grown seedlings.Proteomic analyses of leaf peroxisomes and peroxisomes from suspension-cultured, leaf-derived cells followed by protein subcellular localization studies confirmed a total of over 30 new peroxisomal proteins, uncovering additional metabolic functions for leaf peroxisomes (Fukao et al., 2002; Reumann et al., 2007, 2009; Eubel et al., 2008; Babujee et al., 2010; Kataya and Reumann, 2010; Quan et al., 2010). For Arabidopsis (Arabidopsis thaliana), around 100 peroxisomal proteins were shown to be present in leaves or leaf-derived cells. Compared with the over 80 bona fide peroxisomal proteins detected by leaf peroxisomal proteomics (Reumann et al., 2007, 2009), the number of proteins identified from peroxisomal proteomic studies on etiolated seedlings was significantly smaller, with less than 10 known peroxisomal proteins from Arabidopsis (Fukao et al., 2003) and approximately 31 from soybean (Glycine max; Arai et al., 2008a, 2008b). Thus, a more in-depth analysis of the proteome of peroxisomes from these tissues was highly needed.Here, we performed proteomic analysis of peroxisomes isolated from etiolated Arabidopsis seedlings and detected peroxisomal proteins that encompass most of the known plant peroxisomal metabolic pathways. Fluorescence microscopy verified the peroxisomal localization of a number of proteins newly identified in this study or detected from previous proteomics that had not been verified by independent means. Reverse genetic analysis demonstrated the role for a Cys protease in germination, β-oxidation, and growth.     

Fasting and differential chemotherapy protection in patients

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organisms that short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, ten patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical and clinical studies on fasting and cancer therapy.Key words: fasting, cancer, chemotherapy, calorie restriction, stress resistanceAfter decades of slow progress in the identification of treatments effective on a wide range of malignancies, cancer treatment is now turning to personalized therapies based in part on pharmacogenomics. By contrast, aging research is moving in the opposite direction by searching for common ways to prevent, postpone and treat a wide range of age-related diseases, based on the modulation of genetic pathways that are conserved from yeast to mammals.¹ In fact, it may be a solid evolutionary and comparative biology-foundation, which makes this ambitious goal of biogerontologists a realistic or at least a promising one. On the other hand, the progress of biogerontology is viewed by many clinicians as too fundamental and far from translational applications. In most cases, it is not clear how aging research will be translated into FDA approved drugs or treatments that have effects that are superior to those already available or being developed. For example, it is not clear how the long-term 20–30% reduction in calorie intake (dietary restriction, DR) that we and many others before us have shown to be effective in extending the life span of model organisms will make humans live longer or healthier.^1–3 Furthermore, despite the fact that long-term DR was confirmed to reduce cancer and cardiovascular disease in monkeys⁴ and to be effective in preventing obesity, type 2 diabetes, inflammation, hypertension and atherosclerosis, as indicated by the early results in humans studies,⁵ it is highly unlikely to be adopted in its more extreme and effective version by even a small portion of the population. For example, the 20 to 40% chronic reduction in daily calorie intake shown to be effective in retarding cancer growth in mice would not be feasible for cancer therapy for multiple reasons: (1) the effects of chronic DR in patients with a clinically evident tumor is expected to delay but not stop the progression of the disease^6–8 and this delay may only occur for a portion of the malignancies,⁹ (2) although weight loss and cachexia in the early stages of treatment are less prevalent than commonly thought,^10–12 the ∼15% loss of BMI and ∼30% long-term loss of body fat caused by a moderate (20%) calorie restriction¹³ may be tolerated by only a very small portion of cancer patients receiving treatment, (3) Because this long-term restriction is accompanied by delayed wound healing and immunologic impairment in rodents,^1,14,15 it is not clear what risks it may impose on cancer patients receiving treatment.¹⁶ Our studies of DSR, which were triggered by our fundamental findings that switching yeast cells to water protected them against a wide range of toxins, started as a way to address these concerns but also as an attempt to achieve a much more potent therapeutic effect than that achieved by DR.^17,18 Because starvation-induced protection can increase many fold when combined with modulation of pro-aging pathways and since it is in principle blocked by the expression of any oncogene, it has the potential to provide a method to allow common chemotherapy to selectively kill cancer cells, independently of the type of cancer.^19–21 The DSR experiments in mammals were also based on our hypothesis that stress resistance and aging regulatory pathways were conserved from yeast to mammals.We found that fasting for 48 or more hours or in vitro starvation conditions that mimic fasting protected mice and/or normal cells but not cancer cells from various chemotherapy drugs and other deleterious agents.²¹ This effect was shown to depend in part on the reduction of circulating IGF-I and glucose levels.^21,22 Although a differential regulation of cell division in normal and cancer cells^23,24 is likely to contribute to DSR, much of it appears to be dependent on protective systems which are normally maintained in an inactive or low activity state even in non-dividing cells.^1,25 In fact, in non-dividing yeast and mice, deficiencies in glucose or IGF-I signaling that match those observed after starvation promote resistance to doxorubicin, a chemotherapy drug that specifically targets muscle cells in the heart.^21,22As expected, many clinicians were skeptical of our hypothesis that cancer treatment could be improved not by a “magic bullet” but by a “not so magic DSR shield” as underlined by Leonard Saltz, an oncologist at Memorial Sloan-Kettering Cancer Center: “Would I be enthusiastic about enrolling my patients in a trial where they''re asked not to eat for 2.5 days? No.”²⁶ However, ten oncologists did allow their patients, suffering from malignancies ranging from stage II breast cancer to stage IV esophageal, prostate and lung malignancies to undergo a 48–140 hours pre-chemotherapy and a 5–56 hours post chemotherapy water-only fast. The six patients who received chemotherapy with or without fasting reported a reduction in fatigue, weakness and gastrointestinal side effects while fasting²⁷ (Fig. 1). A trend for a reduction of many additional side effects was also reported by the group of patients who always fasted before chemotherapy.²⁷ In those patients whose cancer progression was assessed, chemotherapy was effective and in some cases it was highly effective.²⁷ A clinical trial sponsored by the V-Foundation for Cancer Research, aimed at testing the safety and efficacy of a 24 hour fast in combination with chemotherapy, is in its safety stage. Because it was originally limited to patients diagnosed with bladder cancer the clinical trial progressed slowly. However, its recent expansion to include patients receiving platinum-based chemotherapy (breast, ovarian, lung cancer), is expected to expedite it. Conclusive results for the effect of a 3–4 day fast on chemotherapy-dependent side effects and possibly therapeutic index are not expected to become available for several years. Even if a more modest effect than the 1,000-fold differential protection against oxidative stress and chemotherapy observed in normal and cancer-like yeast cells was achieved in humans, this method could result in long-term survival for many patients with metastatic cancers, particularly those in which malignant cells have not acquired multidrug resistance.Open in a separate windowFigure 1Average self-reported severity of symptoms in patients that have received chemotherapy with or without fasting.