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     

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     

Multiple signaling pathways control nitrogen-mediated root elongation in maize

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition.Key words: nitrogen, root growth, auxin, cytokinin, NONitrogen acquisition is determined by N demand for plant growth. At low N stress, N demand for maximum plant growth rate is not matched by plant N uptake. To acquire adequate N, plants may increase root length density to explore a larger soil volume and/or increase N uptake activity. High root density is also an important root trait for competition with soil microorganisms.¹ Since nitrate is a highly mobile, non-adsorbing ion, theoretic analysis predicts that its uptake is not limited by transport through soil, and a small root system is sufficient for nitrate acquisition.^2–4 In field conditions, however, genotypes that are efficient in N acquisition generally had a larger root system and higher root length density.^5,6 Under conditions of insufficient N supply, N mass flow to roots may not be adequate to meet the N demand for plant growth. Even in N-sufficient soils, various soil constraints (low water content, etc) may reduce the N mass flow rate. In these cases, large root size and high density will be very important for the utilization of the spatially distributed N, especially newly mineralized N, and the competition for organic N with soil microorganisms.^7,8The development of lateral roots in Arabidopsis in response to nitrate supply has been widely studied.⁹ Less attention has been paid to primary root growth in response to N, possibly because root elongtion is insensitive to increased N supply in Arabidopsis.^10,11 In maize, however, root elongation was sigificantly promoted by suboptimal N supply, and inhibited by overdose supply of N (Fig. 1).^12,13 Until recently less is known about the underlying physiological mechansms. It is well documented that cytokinin is a root-to-shoot signal communicating N availability in addition to nitrate itself.¹⁴ Exogenous cytokinin application suppresses the elongation of primary roots.¹⁵ Recent work in Arabidopsis overexpressing cytokinin synthase (IPT) demonstrate that long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem.¹⁶ By comparing two maize inbred lines whose root elongation had a differential response to low N stress, it was found that the change of cytokinin content in roots was closely related to low-N induced root elongation.¹³ In the N-sensitive genotype 478, cytokinin (Zeatin + Zeatin riboside) content was significantly lower at low N condition. While in N-insensitive genotype Wu312, cytokinin content was hardly affected at various N supplies. Higher N supply shortened the distance from root apex to the first visible lateral roots, a phenomenen similar to that caused by exogenous cytokinins. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. All the data suggests that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots.Open in a separate windowFigure 1Root elongation is inhibited at high nitrate supply.Auxin regulates many cellular responses crucial for plant development. Auxin plays a key role in establishing and elaborating patterns in root meristems.^17,18 Root elongation of Arabidopsis is enhanced by exogenous auxin at low concentrations, but is inhibited at high concentrations.¹⁹ In an earlier report, a high external nitrate supply (8 mM) did cause a 70% decrease in the auxin concentration of the root in soybean.²⁰ In maize, inhibition of root growth by high nitrate was found closely related to the reduction of IAA levels in roots and exogenous NAA and IAA restored primary root growth in high nitrate concentrations.²¹ Interesting, it was found that auxin concentrations in phloem exudates were reduced by a greater nitrate supply, suggesting that shoot-to-root auxin transport may be inhibited by high N supply. Considering the antagonism between auxin and cytokinin.²² it was possible that, by increasing the cytokinin level and decreasing the auxin level, high nitrate supply may have negative influences on root apex activity so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex.Nitric oxide (NO) is emerging as an important messenger molecule associated with many biochemical and physiological processes in plants. The involvement of NO in IAA-induced adventitious root development has also been reported.²³ Given that nitrate is a substrate for NR-catalysed NO production, and root development and growth are closely related to NO, it is expected that NO may play a role in nitrate-dependent root growth. Surprisingly, endogenous levels of NO in the root apices of maize seedlings grown in high nitrate solution were much lower than those in apices grown in low nitrate. The nitrate-induced inhibition of root elongation in maize was markedly reversed by treatments of the roots with a NO donor (SNP) and IAA.²⁴ These data suggest that the arrest of root elongation by high levels of external nitrate concentrations may result from an alteration of endogenous NO levels in root apical cells. NR mediated NO production is unlikely to be involved in the nitrate-dependent NO production and root elongation because NR activity is lower at low N supply. A NO synthase (NOS) inhibitor reduced root elongation in maize plants grown in the low-nitrate medium, suggest that NOS activity may be inhibited in plants grown in high-nitrate solution, thus leading to a reduction of the endogenous NO levels.Taken together, high nitrogen supply increases cytokinin level, but decreases auxin and NO levels in roots of maize. Besides, it was well documented ethylene has a negative effect on root elongation of various plants.^25–27 Exogenous supply of cytokinin increase ethylene production (Stenlid 1982; Bertell et al., 1990). Recently, it was demonstrated in Arabidopsis that auxin transport from the root apex via the lateral root cap is required for ethylene-mediated inhibition of root growth.²⁸ Therefore, a complex multiple siganlling pathways may be involved in N-mediated root elongation (Fig. 2). Further study is required to understand how these pathways interact with each other to reduce root elongation in response to high nitrate supply.Open in a separate windowFigure 2A simplified model explaining nitogen-mediated root elongation in maize.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

Plant Parasitic Oomycetes Such as Phytophthora Species Contain Genes Derived from Three Eukaryotic Lineages

Fungi and the oomycetes include several groups of plant pathogenic microbes. Although these two eukaryotic groups are unrelated they have a number of phenotypic similarities suggested to have evolved convergently. We have recently shown that gene transfer events have occurred from fungi to the oomycetes. These gene transfer events appear to be only one part of a complex and chimeric ancestry for the oomycete genome, which has also received genes from a red algal endosymbiont.Key Words: horizontal gene transfer, osmotrophy, phototrophy, biotrophy, endosymbiosis, fungi, Magnaporthe griseaAs genomic sampling increases, a persistent pattern of horizontal gene transfer (HGT) between microbial lineages is becoming evident.^1,2 So far, patterns of horizontal gene transfer have been identified in four main forms: (A) gene transfer between prokaryote lineages, such that a large proportion of many prokaryote genomes are likely to be chimeric,^3,4 (B) gene transfer from the prokaryote progenitors of the mitochondrion and the plastid organelles to a host eukaryote nuclear genome (e.g., refs. ^5–7), (C) gene transfer from prokaryote genomes to eukaryote microbes, often involving phagocytic eukaryotes and microbes that share similar habitats⁸ and (D) gene transfer from a eukaryotic endosymbiont to their host eukaryotic genomes.^9,10 This fourth form of gene transfer includes secondary and tertiary endosymbiotic events and has so far provided our best examples of eukaryote-to-eukaryote gene transfer.^11,12 Secondary and tertiary endosymbiotic events are typified by the engulfment of a photosynthetic eukaryote by another eukaryote followed by the reduction of the consumed photosynthetic eukaryote and transfer of genes from the endosymbiont to the host nuclei with some retargeting of the transferred gene products back to the remnant organelle.^9,10Gene transfer events can be identified using phylogenetic analysis when an individual gene tree topology contradicts a known species relationship. HGT can only be seriously considered, however, if the gene phylogeny shows that the putative HGT is nested within a donor clade with strong bootstrap support.² Endosymbiosis typically leads to multiple cases of nuclear-encoded genes demonstrating endosymbiotic ancestry, with the candidate genes grouping within a clade representing the lineage that gave rise to the progenitor of the endosymbiont.⁵ There have been multiple cases of both secondary and tertiary endosymbiosis within the eukaryotes, making the evolutionary reconstruction of phototrophy in the eukaryotes highly complex.⁹ Secondary and tertiary endosymbiotic remnant organelles are often identified by the presence of three or more membranes surrounding the organelle body.¹⁰ However, secondary endosymbiotic events have led to a range of different combinations of cell apparatus, from the total loss of the endosymbiont-derived organelle^13,14 to the maintenance of the organelle compartment¹⁰ and the possession of a remnant nucleus as a nucleomorph.¹⁵The oomycetes include the plant pathogenic Phytophthora spp. and are heterokonts (sometimes called Stramenopiles).¹⁶ The heterokonts also encompass numerous groups of photosynthetic algae (e.g., Bolidomonas, Diatoms, Xanthophyceae, Phaeophyceae and Chrysophyceae) and are proposed to be derived from an ancestrally photosynthetic cell that obtained its plastid by engulfment of a red alga.¹⁶ Cytological studies of the oomycetes have so far failed to identify a relic plastid organelle but the recent publication of the Phytophthora sojae and Phytophthora ramorum genomes identified 855 genes putatively originating from the genome of a photosynthetic microbe consistent with a phototrophic ancestry for the oomycetes.¹³Phytophthora plant pathogens include the causal agents of sudden oak death (P. ramorum), potato blight (P. infestans) and, P. sojae which causes serious root and stem rot of soybean plants. Initially, P. infestans was identified as a fungal pathogen and the causal agent of the great 1845 Irish potato famine by Rev. Miles J. Berkeley,¹⁷ due to life cycle similarities and an apparently homologous mode of plant infection to ascomycete plant pathogens. It was only with the use of molecular phylogenetic methods starting with small subunit rDNA analysis¹⁶ followed by multiple concatenated gene phylogenies¹⁸ that the oomycetes were demonstrated to group within the heterokont radiation. With the apparent phylogenetic origins of the oomycetes pinpointed it left the apparent similarities in pathogenic mechanism and infective lifecycle between the filamentous ascomycetes and the oomycetes a mysterious case of convergent evolution.¹⁹During the evolutionary analyses of the predicted proteome of the filamentous plant pathogenic ascomycete Magnaporthe grisea²⁰ we detected a series of unexpected similarities in the genomes of plant pathogenic ascomycetes and the oomycete genomes.¹³ We followed up this observation by further investigation using phylogenetic methods combined with comparative genomic analysis, which revealed a series of HGT events. We subjected our datasets to a range of tests: (A) to test that the level of support for the tree topology seen was robust given random resampling of the sequence alignments used to reconstruct the gene phylogenies; (B) to ensure that the possibility that similar topologies with the oomycete/filamentous ascomycete relationship removed could be rejected at the 0.05 confidence level and; (C) to test for alternative patterns of gene evolution including hidden paralogy (duplication with differential patterns of gene loss) were unlikely. Four of the datasets tested in this way held up to our scrutiny and were thus proposed as fungi-to-oomycete horizontal gene transfers.²¹ The predicted function of three of the four genes (CodB, a purine permease, AraJ, a sugar transporter and a PcaH an extracellular dioxygenase) could conceivably be useful for an osmotrophic microbe living in a plant associated habitat (biotrophy), suggesting that these HGT events could in-part explain the convergently evolved similarities in osmotrophy and filamentous growth habit seen in the oomycetes and fungi. Our analyses also suggested that three of these HGTs originated from a genome closely related to the last common ancestor of the Magnaporthe and Aspergillus evolutionary branches. Although the specific branching position of the transferred lineage could not be pinpointed in the fourth analysis, the same point of origin could not however be excluded. This suggests that the four HGTs we identified could be derived from the same source, a phenomenon similar in pattern (if not involving the same lineages) to that seen for phylogenetic tree topologies used to investigate the endosymbiotic events discussed above. Although these analyses do not shed any light on the circumstances in which these transfers occurred, it is possible that an intimate association between a fungus and a heterokont has led to genetic exchange and demonstrates that eukaryote-to-eukaryote gene transfers are not just associated with the acquisition of phototrophy by secondary/tertiary endosymbiosis.Our published study was conducted using only published genome sequences as a seed for comparative genomic analyses.²¹ However, with the very recent publication of two Phytophthora genomes¹³ it is possible that further analyses will identify additional candidate Phytophthora-Fungi HGT events when they are carried out. These tests may determine how pervasive the pattern of HGT is within the oomycetes.The oomycetes have been classified within the phylum Pseudofungi¹⁶ which comprises a number of microbial lineages with phenotypic similarities to true fungi, including hyphae-like structures and osmotrophy. Originally, the term Pseudofungi was used to group together ‘water-moulds’ possessing mastigonemes (tubular tri-partite hairs) on one flagellum. Currently the phylum Pseudofungi comprises the biotrophic oomycetes including parasites of plants and brown algae, the phagotrophic Developayella and the biotrophic hyphochytrids, including the diatom ectoparasite Pirsonia.¹⁶ It will be interesting to ascertain at what point within the diversification of the Pseudofungi the HGTs that are identified²¹ became fixed and how the acquisition of these phenotypes relates to the evolution of Pseudofungi phenotypes within the heterokonts. Independent of the specific ancestry of the gene transfer events within the Pseudofungi it is clear that P. sojae and P. ramorum have chimeric genomes, originating from three separate eukaryotic lineages, the ancestral heterokont nuclear genome, the red algal endosymbiont and at least four genes of fungal ancestry donated to an oomycete nuclear genome.     

CLE genes may act in a variety of tissues/cells and involve other signaling cascades in addition to CLV3-WUS-like pathways

CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA3-WUSCHEL (CLV3-WUS) and CLV3-WUS-like signaling pathways to regulate meristem activity in plant. Although some CLE genes are expressed in meristems, many CLE genes appear to express in a variety of tissues/cells. Here we report that CLE14 and CLE20 express in various specific tissues/cells outside the shoot/root apical meristem (SAM/RAM), including in highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles and involve other signaling cascades in addition to the CLV3-WUS and CLV3-WUS-like pathways.Key words: CLE, CLAVATA3-WUSCHEL, cell signaling and development, root apical meristem, arabidopsisIntercellular communication and coordination between adjacent cell populations are critical for cell-fate specification, as well as for meristem organization and maintenance. In the shoot apical meristem (SAM), local signaling, which involves the CLAVATA3-WUSCHEL (CLV3-WUS) negative feedback loop, controls stem cell homeostasis and SAM activity.¹ As well, it has been suggested that a CLV3-WUS-like negative feedback pathway operates to control root apical meristem (RAM) activity. This view is supported by the facts that a WUS-related homeobox gene, WOX5, is expressed in cells of the quiescent center (QC) in the RAM, and that loss-of-function of WOX5 in the QC leads to the differentiation of the adjacent root cap initials (RCI), whereas gain-of-function blocks the differentiation of derivatives of the RCI in the root.² Additional support for the function in the RAM of a CLV3-WUS-like pathway, comes from observations that CLE genes (collectively referred to as the CLV3/ESR-relate gene family) are not only expressed in the RAM,^3,4 but also, that overexpression of some CLE genes triggers premature termination of the RAM.⁵ In this regard it has been recently reported that CLE40, which expresses in the differentiating daughter cells of the distal root stem cells, restricts WOX5 expression and promotes differentiation of stem cells in the RAM.⁶ Taken together these data suggest a CLV3-WUS-like feedback loop acts to negatively regulate RAM activity in plants.Our previous results have shown that CLE14 and CLE20 express in specific cells of roots, and that overexpression of CLE14 or CLE20 in Arabidopsis triggers early termination of the RAM in a CLAVATA1 (CLV1)-independent, but CLAVATA2 (CLV2)-dependent manner.^7,8 We also showed that both CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the RAM.⁷ CLV2 and CRN (a receptor-like protein kinase, also known as SOL2, isolated as a suppressor of root-specific overexpression of CLE19) are required for CLE14 and CLE20 peptide functions in vitro.^9,10 Using computational modeling approaches we further demonstrated that 12-amino-acid CLE14 and CLE20 peptides may function through a potential heterodimer/heterotetramer CLV2-CRN complex.⁷CLV3 expresses exclusively in the stem cells of the SAM, and it has been consistently shown that the CLV3 peptide is required for homeostasis of the stem cells and for the maintenance of the SAM.¹ Although some CLE genes are found to express in meristems, many CLE genes appear to express in an array of tissues and cells, including highly differentiated tissues/cells.^3,4 In this report we show that CLE14 and CLE20 express in specific tissues outside the RAM and SAM of Arabidopsis, including highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles in regulating the developmental fate of cells, which includes, but is not limited to, stem cells, and also may be involved in other signaling cascades in addition to the CLV3-WUS pathway.     

AtAGP18, a lysine-rich arabinogalactan protein in Arabidopsis thaliana,functions in plant growth and development as a putative co-receptor for signal transduction

Arabinogalactan-proteins (AGPs) are a class of hyperglycosylated, hydroxyproline-rich glycoproteins that are widely distributed in the plant kingdom. AtAGP17, 18 and 19 are homologous genes encoding three classical lysine-rich AGPs in Arabidopsis. We observed subcellular localization of AtAGP18 at the plasma membrane by expressing a translational fusion gene construction of AtAGP18 attached to a green fluorescent protein (GFP) tag in Arabidopsis plants. We also overexpressed AtAGP18 without the GFP tag in Arabidopsis plants, and the resulting transgenic plants had a short, bushy phenotype. Here we discuss putative roles of AtAGP18 as a glycosylphosphatidylinositol (GPI)-anchored protein involved in a signal transduction pathway regulating plant growth and development.Key words: Arabidopsis thaliana, arabinogalactan-proteins, co-receptor, glycosylphosphatidylinositol, lipid rafts, overexpressionArabinogalactan-proteins (AGPs) are plant cell surface glycoproteins or proteoglycans which are thought to play important roles in various aspects of plant growth and development, such as somatic embryogenesis, cell proliferation and elongation, pattern formation and hormone signaling.¹ The lysine-rich classical AGP subfamily in Arabidopsis contains three members: AtAGP17, 18 and 19. The subcellular localization of AtAGP17 and AtAGP18 was previously studied in our laboratory by expressing GFP-AtAGP17/18 fusion proteins in tobacco cell cultures.^2,3 In a recent report, we used Arabidopsis plants to overexpress GFP-AtAGP17/18/19 fusion proteins to observe subcellular localization of the lysine-rich AGPs in planta, in contrast to our previous plant cell culture work.⁴ Moreover, the lysine-rich AGPs alone (i.e., AtAGP17/18/19 without the GFP tag) were overexpressed in Arabidopsis plants, and only AtAGP18 overexpressors had a distinctive phenotype. This phenotype included shorter stems, more branches and less seeds, indicating a role for AtAGP18 in plant growth and development.⁴ In this addendum, we further discuss the putative biological role of AtAGP18 on a molecular level and its possible mode of action in cellular signaling.Classical AGPs are frequently predicted to have a glycosylphosphatidylinositol (GPI) anchor, which would allow for the localization of such AGPs to the outer surface of the plasma membrane. Biochemical analyses were carried out to support this hypothesis in tobacco, pear,⁵ rose⁶ and Arabidopsis.⁷ The lysine-rich classical AGPs, AtAGP17 and 18, were predicted to have a GPI anchor.⁸ To test this idea, tobacco cell cultures expressing GFP-AtAGP17/18 fusion proteins were plasmolyzed and GFP fluorescence was observed on the plasma membrane.^2,3 To corroborate this finding in planta, GFP-AtAGP17/18 were expressed in Arabidopsis plants and leaf trichome cells were plasmolyzed. Enhanced GFP fluorescence was observed at the plasma membrane of these transgenic trichome cells, indicating the presence of GFP-AtAGP17/18 at the plasma membrane.⁴ The localization of these lysine-rich classical AGPs at the plasma membrane suggests possible biological roles in sensing extracellular signals. They are likely associated with lipid rafts involved in cell signaling for the following reasons. In plants as well as animals, there are sterol-enriched, detergent-resistant plasma membrane microdomains called lipid rafts. Lipid rafts are known to be involved in signal transduction and are enriched in transmembrane receptors and GPI-anchored proteins, including AGPs.^9–11 The accumulation of these proteins in such microdomains may allow for interactions between these proteins in sensing extracellular signals which lead to various intracellular events. Interestingly, a recent study shows that lipid rafts from hybrid aspen cells contain callose synthase and cellulose synthase, and these enzymes are active since in vitro polysaccharide synthesis by the isolated detergent-resistant membranes was observed. These results demonstrate that lipid rafts are involved in cell wall polysaccharide biosynthesis.¹² In addition, an Arabidopsis pnt mutant study shows GPI-anchored proteins are required in cell wall synthesis and morphogenesis.¹³ These observations, coupled with previous observations that cellulose synthases as well as AGPs interact with microtubules, suggest that AGPs in lipid rafts may have a role in signal events, including those regulating cellulose and/or callose biosynthesis or deposition.^14,15To examine the role of LeAGP-1, a lysine-rich AGP in tomato, transgenic tomato plants were produced which expressed GFP-LeAGP-1 under the control of the cauliflower mosaic virus 35S promoter.¹⁶ The tomato LeAGP-1 overexpressors and Arabidopsis AtAGP18 overexpressors both have a bushy phenotype similar to transgenic tobacco plants overproducing cytokinins.^4,16,17 Cytokinins are an important class of plant hormones involved in many plant growth and development processes, such as cell growth and division, differentiation and other physiological processes.¹⁸ Therefore, Sun et al. proposed that LeAGP-1 might function in concert with the cytokinin signal transduction pathway.¹⁶ Since the overexpression phenotypes of AtAGP18 are similar to those of LeAGP-1, AtAGP18 is also likely associated with the cytokinin signal transduction pathway. The prevailing model for cytokinin signaling in Arabidopsis is similar to the two-component system in bacteria and yeast. In this model, the cytokinin receptor contains an extracellular domain, a kinase domain and a receiver domain. When the cytokinin receptor senses cytokinin signals, it auto-phosphorylates at a His residue in the kinase domain. The phosphoryl group is then transferred to an Asp residue in the receiver domain. Subsequently, the phosphoryl group is transferred to a His residue in the histidine phosphotransfer protein (Hpt) and the Hpt translocates to the nucleus and transfers the phosphoryl group to an Asp residue in a downstream response regulator to activate it.¹⁹ This model is consistent with our hypothesis since the cytokinin receptor in this model is a receptor kinase located in the plasma membrane with an extra-cellular domain that can potentially interact with AtAGP18. AtAGP18 may function as a co-receptor that first binds to cytokinins, then either directly interacts with cytokinin receptors or brings the cytokinins to cytokinin receptors in the plasma membrane. The first scenario is analogous to the interaction of contactin and contactin-associated protein (Caspr) in neurons. In this model, contactin is a GPI-anchored protein on the cell surface that binds to signal molecules and interacts with the transmembrane receptor Caspr to transmit signals to the cell interior.²⁰ The second scenario is analogous to fibroblast growth factor (FGF) signal activation in which heparan sulfate proteoglycans bind to FGF molecules and bring them to the FGF receptor.²¹Based on all the above observations and findings, a hypothetical model for AtAGP18 function is proposed in Figure 1. The model shows AtAGP18 located on the outer surface of the plasma membrane in lipid rafts where it could act as a co-receptor to sense extracellular signals (such as cytokinin) and interact with transmembrane proteins, possibly receptor kinases or ion channels, in the lipid rafts to initiate signaling by triggering various intracellular events. Interestingly, receptor tyrosine kinases and ion channels are known to be present in lipid rafts.^9,22 Moreover, AGPs are likely associated with ion channels since addition of the AGP-binding reagent Yariv phenylglycoside resulted in elevated cytoplasmic calcium concentrations in tobacco cells and lily pollen tubes.^15,23,24 Clearly, additional work will be required to verify such a model, and to better understand how AtAGP18 might sense extracellular signals and interact with the transmembrane proteins in the lipid rafts.Open in a separate windowFigure 1Model for atAGP18 functioning in cellular signaling to control plant growth and development. In this model, lipid rafts are enriched in glycosphingolipids, sterols, transmembrane proteins (such as receptors, receptor kinases and ion channel proteins) and GPI-anchored proteins including AtAGP18. (a) AtAGP18 acts as a co-receptor by binding to signaling molecules and directly interacting with transmembrane proteins in the lipid rafts. (B) AtAGP18 acts as a co-receptor by binding to signaling molecules and bringing the signaling molecules to transmembrane proteins in the lipid rafts. Upon activation by the extracellular signals, the transmembrane proteins initiate signaling and lead to various intracellular events (e.g., phosphorylation similar to the two-component signaling system, influx of calcium ions). The different components of the AtAGP18 molecule and the various lipid components of lipid rafts and plasma membrane are shown in the boxed inset. Hpt, histidine phosphotransfer protein.     

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.     

Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis

In our recent paper in the Plant Journal, we reported that Arabidopsis thaliana lysophospholipase 2 (lysoPL2) binds acyl-CoA-binding protein 2 (ACBP2) to mediate cadmium [Cd(II)] tolerance in transgenic Arabidopsis. ACBP2 contains ankyrin repeats that have been previously shown to mediate protein-protein interactions with an ethylene-responsive element binding protein (AtEBP) and a farnesylated protein 6 (AtFP6). Transgenic Arabidopsis ACBP2-overexpressors, lysoPL2-overexpressors and AtFP6-overexpressors all display enhanced Cd(II) tolerance, in comparison to wild type, suggesting that ACBP2 and its protein partners work together to mediate Cd(II) tolerance. Given that recombinant ACBP2 and AtFP6 can independently bind Cd(II) in vitro, they may be able to participate in Cd(II) translocation. The binding of recombinant ACBP2 to [¹⁴C]linoleoyl-CoA and [¹⁴C]linolenoyl-CoA implies its role in phospholipid repair. In conclusion, ACBP2 can mediate tolerance to Cd(II)-induced oxidative stress by interacting with two protein partners, AtFP6 and lysoPL2. Observations that ACBP2 also binds lysophosphatidylcholine (lysoPC) in vitro and that recombinant lysoPL2 degrades lysoPC, further confirm an interactive role for ACBP2 and lysoPL2 in overcoming Cd(II)-induced stress.Key words: acyl-CoA-binding protein, cadmium, hydrogen peroxide, lysophospholipase, oxidative stressAcyl-CoA-binding proteins (ACBP1 to ACBP6) are encoded by a multigene family in Arabidopsis thaliana.¹ These ACBP proteins are well studied in Arabidopsis in comparison to other organisms,^1–4 and are located in various subcellular compartments.¹ Plasma membranelocalized ACBP1 and ACBP2 contain ankyrin repeats that have been shown to function in protein-protein interactions.^5,6 ACBP1 and ACBP2 which share 76.9% amino acid identity also confer tolerance in transgenic Arabidopsis to lead [Pb(II)] and Cd(II), respectively.^1,5,7 Since recombinant ACBP1 and ACBP2 bind linolenoyl-CoA and linoleoyl-CoA in vitro, they may possibly be involved in phospholipid repair in response to heavy metal stress at the plasma membrane.^5,7 In contrast, ACBP3 is an extracellularly-localized protein⁸ while ACBP4, ACBP5 and ACBP6 are localized to cytosol.^9,10 ACBP1 and ACBP6 have recently been shown to be involved in freezing stress.^9,11 ACBP4 and ACBP5 bind oleoyl-CoA ester and their mRNA expressions are lightregulated.^12,13 Besides acyl-CoA esters, some ACBPs also bind phospholipids.^9,11,13 To investigate the biological function of ACBP2, we have proceeded to establish its interactors at the ankyrin repeats, including AtFP6,⁵ AtEBP⁶ and now lysoPL2 in the Plant Journal paper. While the significance in the interaction of ACBP2 with AtEBP awaits further investigations, some parallels can be drawn between those of ACBP2 with AtFP6 and with lysoPL2.