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.     

Piriformospora indica mycorrhization increases grain yield by accelerating early development of barley plants

Root colonization by the basidiomycete fungus Piriformospora indica induces host plant tolerance against abiotic and biotic stress, and enhances growth and yield. As P. indica has a broad host range, it has been established as a model system to study beneficial plant-microbe interactions. Moreover, its properties led to the assumption that P. indica shows potential for application in crop plant production. Therefore, possible mechanisms of P. indica improving host plant yield were tested in outdoor experiments: Induction of higher grain yield in barley was independent of elevated pathogen levels and independent of different phosphate fertilization levels. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. Analysis of plant development and yield parameters indicated that positive effects of P. indica on grain yield are due to accelerated growth of barley plants early in development.Key words: mycorrhiza, barley development, Piriformospora indica, phosphate uptake, grain yield, pathogen resistanceThe wide majority of plant roots in natural ecosystems is associated with fungi, which very often play an important role for the host plants'' fitness.¹ The widespread arbuscular mycorrhizal (AM) symbiosis formed by fungi of the phylum Glomeromycota is mainly characterized by providing phosphate to their host plant in exchange for carbohydrates.^2,3 Fungi of the order Sebacinales also form beneficial interactions with plant roots and Piriformospora indica is the best-studied example of this group.⁴ This endophyte was originally identified in the rhizosphere of shrubs in the Indian Thar desert,⁵ but it turned out that the fungus colonizes roots of a very broad range of mono- and dicotyledonous plants,⁶ including major crop plants.^7–9 Like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner¹⁰ and promotes growth in several tested plant species.^6,11,12 The root endophyte, moreover, enhances yield in barley and tomato and increases in both plants resistance against biotic stresses,^7,9 suggesting that application in agri- and horticulture could be successful.     

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     

Antennal responses of an oligolectic bee and its cleptoparasite to plant volatiles

Cleptoparasitic or cuckoo bees lay their eggs in nests of other bees, and the parasitic larvae feed the food that had been provided for the host larvae. Nothing is known about the specific signals used by the cuckoo bees for host nest finding, but previous studies have shown that olfactory cues originating from the host bee alone, or the host bee and the larval provision are essential. Here, I compared by using gas chromatography coupled to electroantennographic detection (GC-EAD) the antennal responses of the oligolectic oil-bee Macropis fulvipes and their cleptoparasite, Epeoloides coecutiens, to dynamic headspace scent samples of Lysimachia punctata, a pollen and oil host of Macropis. Both bee species respond to some scent compounds emitted by L. punctata, and two compounds, which were also found in scent samples collected from a Macropis nest entrance, elicited clear signals in the antennae of both species. These compounds may not only play a role for host plant detection by Macropis, but also for host nest detection by Epeoloides. I hypothesise that oligolectic bees and their cleptoparasites use the same compounds for host plant and host nest detection, respectively.Key words: Macropis fulvipes, Epeoloides coecutiens, Lysimachia punctata, oligolectic oil-bee, floral scent, dynamic headspace, GC-EAD, cuckoo bee, host nest findingBees are the most important animal pollinators worldwide, and guarantee sexual reproduction of many plant species.^1,2 This is especially true for female bees, which collect pollen and mostly nectar for their larvae and frequently visit flowers. For finding and detection of suitable flowers, bees are known to use, besides optical cues,^3,4 especially olfactory signals.^5–8 However, c. 20% of bees do not collect pollen for their larvae by their own, but enter nests of host bees and lay eggs into the broodcells.^1,9 The parasitic larvae subsequently feed the food that had been provided for the host larvae. These so called cuckoo or cleptoparasitic bees can be generalistic, indicating that they use species of several other bee groups as host, whereas others can be highly specialized, laying eggs in cells of only few host species.¹ Until now little is known about the cues used by the cuckoo bees for finding host nests. Nevertheless, Cane¹⁰ and Schindler¹¹ demonstrated that parasitic Nomada bees use primarily visual cues of the nest entrance holes for finding possible nests, and olfactory cues for detection of suitable host nests. The chemical cues used by the cleptoparasites originate from the host bee^10,11 and also pollen,¹⁰ the main larval provision. In most bee species, pollen is mixed together with nectar as larval provision, and both floral resources are known to emit volatiles.^12,13 It is unknown, whether cuckoo bees in search for host nests also use volatiles originating from nectar. While the odours of the host bee used as signal by the cleptoparasites, e.g., cuticiular hydrocarbons and glandular secretions, are often species-specific,¹⁴ the chemical cues from the larval provision may just indicate the presence of pollen in the nest without more specifity. As a consequence cuckoo bees could use species-specific host odours to detect nests of a suitable host, and odours released from the larval provision could indicate to them that broodcells are foraged. However, especially those cuckoo bees with oligolectic hosts foraging pollen only on few closely related plant species,¹ may also use the olfactory signals from host broodcell supplies as more specific cue for host nest detection. Thus the same signal from certain flowers may be used for different informations: for the host bee for host plant and for the cuckoo bee for host nest detection.In this concern I tested oligolectic Macropis (Melittidae, Melittinae) and its specific cuckoo bee, Epeoloides (Apidae, Apinae) by using gas chromatography coupled to electroantennographic detection (GC-EAD) on floral scent of Lysimachia (Myrsinaceae). Macropis is highly specialized on Lysimachia, because it is not only collecting pollen from plants of this genus, but also floral oil. Both floral products are the only provision for the larvae.^1,15 Recently, we have shown that the oil bee Macropis is strongly attracted to floral scent of its oil host Lysimachia though the compounds used for host plant finding are still unknown.⁷ Macropis is the only host of Epeoloides, and larvae of this cleptoparasite only feed on the Lysimachia pollen-oil mixture provided for the larvae of Macropis. Worldwide, there are only 2 species of this genus, one in North America and the other in Europe/Asia.^1,16,17 I hypothesized that both bee species respond to specific Lysimachia compounds, which may be used for host plant as well as host nest detection.The measurements with M. fulvipes (F.) and E. coecutiens (F.) antennae demonstrate that both bees, host as well as cuckoo bee, respond to some scent compounds emitted by inflorescences of Lysimachia punctata L. (Fig. 1), a plant being an important pollen and oil source for M. fulvipes. Macropis responded to much more Lysimachia compounds compared to the cuckoo bee, however, two compounds elicited clear signals in the antennae of both bee species: the benzenoid 1-hydroxy-1-phenyl-2-propanone, and the fatty acid derivative 2-tridecanone. Interestingly, both compounds are also emitted from the floral oil of this plant,⁷ and both compounds were also detected in scent samples collected by dynamic headspace in the entrance of a Macropis nest (Dötterl, unpublished data). Therefore, an Epeoloides female being in search for a host nest can detect volatiles emitted from the provision of the host bee at the entrance of a bee nest, and may use these specific compounds for detection of a Macropis nest provisioned with Lysimachia pollen and oil.Open in a separate windowFigure 1Coupled gas chromatographic and electroantennographic detection of a Lysimachia punctata headspace scent sample using antennae of a female oligolectic Macropis fulvipes and a female cleptoparasitic Epeoloides coecutiens bee. (1) 1-hydroxy-1-phenyl-2-propanone, (2) 2-tridecanone.Present results show that an oligolectic oil-bee as well as its cleptoparasite detects volatiles originating from the host plant of the pollen collecting bee, and that oligolectic bees as well as their cuckoo bees may use the same specific signals for host plant and host nest finding, respectively. Biotests are now needed to test this hypothesis.     

Self-Incompatibility Involved in the Level of Acetylcholine and cAMP

Elongation of pollen tubes in pistils after self-pollination of Lilium longiflorum cv. Hinomoto exhibiting strong gametophytic self-incompatibility was promoted by cAMP and also promoted by some metabolic modulators, namely, activators (forskolin and cholera toxin) of adenylate cyclase and inhibitors (3-isobutyl-1-methylxanthine and pertussis) of cyclic nucleotide phosphodiesterase. Moreover, the elongation was promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, L-α-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. A potent inhibitor (neostigmine) of acetylcholinesterase (AChE) as well as acetylcholine also promoted the elongation. cAMP enhanced choline acetyltransferase (ChAT) activity and suppressed AChE activity in the pistils, suggesting that the results are closely correlated with self-incompatibility in L. longiflorum. In short, it came to light that cAMP modulates ChAT (acetylcholine-forming enzyme) and AChE (acetylchoine-decomposing enzyme) activities to enhance the level of ACh in the pistils of L. logiflorum after self-incompatible pollination. These results indicate that the self-incompatibility on self-pollination is caused by low levels of ACh and/or cAMP.Key Words: pollen tubes, self-incompatibility, Lilium longiflorum, cAMP, acetylcholie, AChE, ChATCyclic AMP (cAMP) is an essential signaling molecule in both prokaryotes and eukaryotes.¹ The existence of cAMP in higher plants was questioned by some reviewers^2–4 in the mid 1970''s, so that many workers were discouraged from studying roles in plant biology. However, its presence was confirmed by mass spectrometry⁵ and infrared spectrometry⁶ in the early 1980''s and increasing evidence^7–12 now suggests that cAMP makes important contributions in plant cells, as in animals.Lily (Lilium longiflorum) exhibits strong gametophytic self-incompatibility.^13,14 Thus, elongation of pollen tubes in the pistil after self-incompatible pollination in L. longiflorum cv. Hinomoto stops halfway, in contrast to the case after cross-compatible pollination (cross with cv. Georgia).¹⁴ This self-incompatibility appears to be associated with the stress and self-incompatible pollination on stigmas of lilies results in activation and/or induction of enzymes such as NADH- and NADPH-dependent oxidases, xanthine oxidase, superoxide dismutase (SOD), catalase and ascorbate peroxidase in the pistils.¹⁵ The activities of NADH- and NADPH-dependent oxidases (O₂⁻-forming enzymes), however, are known to be suppressed by cAMP¹⁶ and increase in the level of cAMP in guinea pig neutrophils results in their decreased expression.¹⁷ The level of O₂⁻ reactions with SOD is also decreased by cAMP.¹⁸ In the case of the lily, inhibition of NADH- and NADPH-dependent oxidases by cAMP was found to be noncompetitive with NAD(P)H.¹⁶ We hypothesized that decrease in active oxygen species such as O₂⁻ and suppression of stress enzyme activities in self-pollinated pistils of lily by cAMP might cause elongation of pollen tubes after self-pollination and this proved to be the case. Namely, elongation of pollen tubes after self-incompatible pollination in lily was promoted by exogenous cAMP at a concentration as low as 10 nM, a conceivable physiological level.¹³ Moreover, similar elongation could be achieved with adenylate cyclase activators [forskolin(FK) and cholera toxin] and cAMP phosphodiesterase inhibitors [3-isobutyl-1-methylxanthine (IBMX) and pertussis toxin].^14,19 These phenomena led us to examine the involvement of endogenous cAMP in pistils after self-incompatible or cross-compatible pollination. As expected, the level of endogenous cAMP in pistils after self-pollination was approximately one half of that after cross-pollination. Furthermore, this was associated with a concomitant decrease in adenylate cyclase and increase in cAMP phosphodiesterase.¹⁹Many researchers in the field of plant biology have been unsuccessful in attempts to estimate the quantity of cAMP and to detect activities of adenylate cyclase and cAMP phosphodiesterase. On major difficulty is the presence of proteases and we have overcome this problem by using protease inhibitors, such as aprotinin and leupeptin.¹⁹In 1947, acetylcholine (ACh) of higher plants was first reported in a nettle (Urtica urens) found in the Himalaya mountain range.²⁰ In 1983, its existence in plants was confirmed by mass spectrometry of preparations from Vigna seedlings.²¹ In our preliminary studies, CCC (chlorocholinechloride), a plant growth retardant (specifically an anti-gibberellin), enhanced the elongation of the pollen tubes in pistils after self-incompatible pollination in lilies. This led us to investigate whether other choline derivatives cause similar effects and positive findings were obtained with ACh, acetylthiocholine and L-α-phosphatidlylcholine.²² Moreover, the elongation was also promoted by neostigmine, an inhibitor of acetylcholine esterase (AChE) activity. In line with these results, choline acetyltransferase (ChAT) demonstrated low and AChE high activity in pistils after self-incompatible pollination.The positive influence of cAMP^14,19 and ACh²² in pistils of L. longiflorum after self-incompatible pollination encouraged us to examine the involvement of these two molecules in regulation of pollen tube elongation of lily after self-incompatible and cross-compatible pollination. As a result, it was revealed that cAMP promotes ChAT and suppresses AChE activity in pistils after both self- and cross-pollination. In other words, the self-incompatibilty in pistils of L. longiflorum appears to be due to levels of ACh and/or cAMP below certain threshold values.Hitherto, these substances have not been recognized to play important roles in the metabolic systems of higher plants. However, given their conservation through evolution, it is natural that such central metabolic substances make essential contributions, regardless of the organism. We have succeeded in establishing physiological functions of cAMP and ACh in pistils of lily^14,19,22 and this points to use of plant reproductive organs such as research materials. The exact responsibilities of the two molecules may depend on differences in tissues or organs of plants and further molecular biological studies in this area are clearly warranted. This issue is currently being investigated.     

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     

The importance of floral signals in the establishment of plant-ant mutualisms

Visual and olfactory floral signals are essential for the establishment of plant-pollinator mutualisms. Different batteries of floral features attract different pollinators and may achieve specific relationships that are essential for the immediate plant reproductive success, and at an evolutionary time scale have been of vital importance in the radiation of Angiosperms. We have found that mutualistic services by ants, insects traditionally considered ineffective pollinators, are essential for the pollination of Cytinus hypocistis (Cytinaceae), a Mediterranean root holoparasitic plant. Diverse floral signals, mainly nectar characteristics and floral scent could be playing a key role in the attraction of different species of ants, which pollinate effectively the flowers. Surprisingly, the abundance of other insects foraging in this parasite was very low and, although this scarcity could be due in part to the presence of ants, we suggest that different floral features exhibited by C. hypocistis could be evolving for attracting ants. Based on some current findings, we suspect that the study of floral signals in Cytinaceae is critical in the understanding the divergence of pollination systems in this fascinating family of parasitic plants.Key words: ant, Cytinus hypocistis, Cytinaceae, floral signal, nectar, plant-animal interaction, scentThe flowers of angiosperms exhibit an amazing variety of floral and nectar colors,^1,2 floral morphology and displays,^3,4 floral scents⁵ and nectar characteristics,^6,7 that may influence pollinator type and pollination quality. The existence of those signals help to the establishment of interactions among plants and pollinators, that range from drastic generalists, when flowers are visited by an elevated number of pollinators, to extreme specialists, being the plant pollinated by only one or a few species of pollinators.^8,9In the majority of terrestrial ecosystems, ants stand out as one of the most common floral visitors.¹⁰ In spite of such ubiquity, ants have been largely considered as ineffective pollinators, mainly due to their small size, erratic behaviour and the presence of metapleural glands that produce antibiotic secretions reducing pollen viability.^11,12 Moreover, it has been postulated that ants act as nectar thieves and reduce visitation by other potential pollinator.¹⁰ However, new findings are highlighting that their role on pollination is not fully understood. Recently we have described, combining a four-year field observation study with experimental pollination treatments at six study sites, a new ant-plant mutualism, between the holoparasitic plant Cytinus hypocistis (Cytinaceae) and different ant species,¹³ joining a growing body of evidence stressing the prominent role that ants are playing in some plant-pollinator systems.^14–17 Nevertheless, in contrast to preceding studies where only one ant species pollinated the plants (but see refs. ¹⁷ and ¹⁸) in C. hypocistis as many as ten ant species behave as true pollinators. Moreover, our study has shown that the different ant species that pollinate C. hypocistis differ in their effectiveness as pollinators, as commonly observed in other pollinator guilds.^19,20 We therefore suggest that generalizations about the importance and quality of ants as pollinators should be avoided or made with caution unless backed by careful field measurements.Our study system was a monoecious rootless, stemless and leafless holoparasitic plant with inflorescences appearing at ground level. Although C. hypocistis does not exhibit typical features of the purported ‘ant-pollination syndrome’,²¹ a number of floral signals could be playing key roles in the establishment of the plant-ant interactions. Among them, the low stature of plants that make flowers to stay a ground level, in combination with sweet scented flowers that offer concentrated nectar and large quantity of pollen, are signals that may enhance the attractiveness of C. hypocistis to diurnal and nocturnal ants that pollinate flowers efficiently. Flying insect visits were unexpectedly low, and only a fly was a predictable visitor. The presence of ants can discourage flying pollinators from visiting the flowers,²² but we suspect that this low number of visits can be explained by the floral features exhibited by C. hypocistis which, although probably still no specialized, may be becoming more important for attracting ants.It has been postulated that different species of animals are attracted by diverse sets of visual (colour and floral shape) and chemical (floral scent, nectar features) floral characteristics.^5,6,9 These features are critical for plant reproductive success,^23,24 and at an evolutionary time scale they have been of vital importance in the radiation of Angiosperms.²⁵ Although our study does not specifically deal with the relative importance of different floral signals, circumstantial evidence suggests that in the family Cytinaceae various signalling-related features could be essential for the establishment of plant-pollinator mutualisms. More specifically, floral scent seems to be an important channel of communication between these flowering plants and their pollinators and has been a likely key factor in the evolution of pollination mechanisms in this outstanding family of parasitic plants. In the only three studies conducted on plant-pollinator systems in the family Cytinaceae a remarkable diversity of mutualisms has been observed. First, in Mexico, the yeasty and unpleasant scent produced by the dark flowers of Bdallophyton bambusarum has been described to attract carrion flies,²⁶ a common pollinator guild in other parasitic plants such as Rafflesiaceae or Hydnoraceae, with a characteristic pollination system by deceit with flowers mimicking insect mating or egg-laying habitats.²⁷ Second, in South Africa, the crimson flowers of Cytinus visseri emit a strong scent dominated by aliphatic compounds that play a central role in attracting small mammals, the only flower visitors and pollinators of this species.²⁸ And third, our recent findings on C. hypocistis in Spain and Morocco point to an important effect of floral scent in attracting ants, its main pollinators. Although research on the importance of floral signals to attract pollinators in this family is still in its infancy, recent findings make of Cytinaceae an ideal model to study at an intrafamily level the influence of divergent selection from pollinators on the evolution of high floral signal diversity. Mechanisms acting on plant-animals signalling obviously deserve further studies to provide an accurate picture of the importance of visual and chemical cues on the evolution of pollination systems in Angiosperms.     

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.     

Plasmodesmata transport of GFP and GFP fusions requires little energy and transitions during leaf expansion

Plasmodesmata (Pd) are symplastic channels between neighboring plant cells and are key in plant cell-cell signaling. Viruses of proteins, nucleic acids, and a wide range of signaling macromolecules move across Pd. Protein transport Pd is regulated by development and biotic signals. Recent investigations utilizing the Arrhenius equation or Coefficient of conductivity showed that fundamental energetic measurements used to describe transport of proteins across membrane pores or the nuclear pore can also apply to protein movement across Pd. As leaves continue to expand, Pd transport of proteins declines which may result from changes in cell volume, Pd density or Pd structure.Key words: plasmodesmata, diffusion, GFP, viral transport, PVX, triple gene blockResearchers have argued for the last decade that movement of proteins and other macromolecules across Pd is regulated by development, stress and biotic signals. There are four current models describing different mechanisms of Pd transport. First is the non cell autonomous protein (NCAP) pathway that carries ribonucleoprotein complexes across Pd. NCAPs often carry RNA in a ribonucleoprotein complex to the Pd.^1–4 This mode of transport involved targeted movement, meaning that a set of proteins must dock within the Pd to gate it open to enable transfer between cells. Proteins which are normally too large to move across Pd can gate open Pd to enable its own transfer into neighboring cells. This is contrasted by nontargetted movement, which is passive movement of proteins that are sufficiently small enough to pass between cells.^5,6 The green fluorescent protein (GFP) has been described as a protein whose movement is non-targeted, meaning that it can diffuse across Pd. Reasons that we do not see continuous movement of small proteins between cells include protein compartmentalization or subcellular targeting signals. For example proteins may be synthesized and modified via the ER and Golgi networks and then transferred into vesicles and transported within cells to their destination. These proteins would not be free in the cytosol for diffusion across Pd. Alternatively, proteins which have dominant subcellular targeting signals which direct them to certain organelles such as the nucleus, peroxisome, or other destination would not be free to move across Pd.^5,6 A third model represents proteins in the ER that move laterally along the membrane or through the ER lumen into neighboring cells. This transport is quite rapid and investigations are ongoing to determine how this is regulated.^7–11 Finally, there is vesicle transport which deliver cargo to Pd.^12,13 The origin of these vesicles is still under investigation. Much more research has been accomplished toward defining non-targeted movement and the NCAP pathway while the ER and vesicle transport pathways are only recently described and very little is known about the regulatory mechanisms underlying these pathways.Pd permeability is governed in part by architecture, but also by key regulatory factors that determine Pd conductivity. Factors such as mysoin VIII, actin and calreticulin were identified in Pd which likely regulate expansion and contraction.^14–19 In addition calcium, ATP and plant hormones can downregulate Pd permeability during development and stress.^20,21 The tools for measuring Pd permeability has been to study the transport of fluorescently tagged proteins, fluorescent dextran beads, GFP or GFP fusions following microinjection or biolistic delivery to the cytoplasm of one cell.^22–26 Then video imaging or captured still images at select time intervals are used to characterize Pd transport. Until recently researchers quantified movement by the frequency they observed a certain type of movement. Therefore our ability to describe Pd permeability has been limited.Evidence that ATP impacts Pd conductivity has led investigations to explore the energy requirements for macromolecular transport across Pd. By understanding the energy requirements for transport of various proteins and nucleic acids we can better characterize passive or active transport processes. Toward this end two recent studies detailed quantitative approaches that can be employed to describe the developmental and energy requirements cell-to-cell transport of cytosolic proteins. Both papers used biolistic bombardment to deliver plasmids expressing GFP or GFP fusions to tobacco leaf epidermal cells and then captured still images of GFP fluorescence in neighboring cells. We employed the Arrhenius equation to characterize transport of GFP or GFP fused to the Potato virus X (PVX) TGBp1 movement protein. PVX TGBp1 was selected to compare with GFP alone since it is known to gate open Pd and has ATPase activity.⁴⁵ We predicted that the abilities of GFP alone and GFP-TGBp1 to move across Pd might be different and were surprised to learn that the energy for transport of both proteins was similar. This project established the principle that GFP and GFP-TGBp1 transport is temperature dependent showing a linear relationship between protein movement and the temperatures at which leaves were incubated.Green fluorescent sites on bombarded leaves were scored for the movement or no movement. Movement is defined as evidence of fluorescence in 2 or more cells at 24 h and no movement is when fluorescence is in single cells. These were then presented as a percentage of the total. So by digitizing the representation of movement we were able to represent a linear relationship between movement and temperature. Representing movement in this way also enabled us to represent movement values on a logarithm scale necessary for a classic Arrhenius plot. The activation energy (Ea) was calculated by fitting the data to the Arrhenius equation:% movement = A exp(-Ea/RT); and the Ea for GFP and GFP-TGBp1 was approximately 38 kJ/mol and 29 kJ/mol. These low activation energies are comparable to the reported 30 kJ/mol calculated for temperature dependence of protein transport through the cytosol. Evidence that GFP movement across Pd requires slightly more energy than through the cytoplasm suggests there may be some resistance within the pore. The lower energy for GFP-TGBp1 suggests that movement is facilitated, which likely reflects Pd gating by TGBp1, enabling greater transfer between cells.Liarzi and Epel define a new coefficient of conductivity of Pd.⁴² This study also concluded that cell-to-cell transport of GFP in nontransgenic or transgenic N. benthamiana plants expressing the Tobacco mosaic virus (TMV) movement protein (MP) is temperature dependent. The method was to measure the exponential decay, which is a measure of the impedance to diffusion driven cell-to-cell movement of fluorescence. The exponential decay factor? was determined by calculating the ratio of GFP fluorescence in bombarded cell 0 and neighboring cells. This was presented as a measure of fluorescence transfer from cell 0 to cell 1 to cell 2. A coefficient for conductivity C(Pd), 1/? for GFP was reflective of diffusion. Interestingly the (TMV) MP did not increase conductivity of GFP between neighboring leaf epidermal cells indicating that movement was already maximal. Considering prior reports that the TMV MP shows preferential spread into mesophyll rather than epidermal tissues during virus infection, it is possible that preferential spread into mesophyll cells would prevent experimental efforts to achieve improved conductivity of GFP between epidermal cells.^27,28 In which case the absence of a trans effect of TMV MP on GFP conductivity in the epidermis may not be surprising. In fact, prior investigations of TMV MP gating activities were conducted in mesophyll cells.^29,30 The best explanation for the combined studies is that cytosolic GFP can diffuse across Pd , however viral proteins which gate Pd enable their own low energy transfer into neighboring cells without allowing other proteins to flood into neighboring cells. Therefore viral movement proteins, such as PVX TGBp1 and TMV MP, which gate Pd provide themselves with an energetic advantage for transport into neighboring cells which is essential for rapid dissemination of virus into further tissues.These studies provide an interesting contrast between PVX TGBp1 and TMV MP. Both proteins gate open Pd for virus cell-to-cell transport, but there seems to be differences in how these proteins function in epidermal cells. This is likely due to their different roles in promoting virus cell-to-cell movement. PVX TGBp1 protein is also a suppressor of RNA silencing. We recently proposed a model in which TGBp1 rapidly moves from cell-to-cell ahead of virus infection, to suppress the cell''s RNA degradation machinery, as a means to promote infection.³¹ The TMV MP on the other hand is reported to bind viral RNA for transfer into neighboring cells.^32,33 Therefore, the different observations of PVX TGBp1 and TMV MP transport between epidermal cells likely reflect their functional differences. Both proteins are required for virus cell-to-cell movement, but their exact roles in virus movement are not identical.As mentioned earlier, Pd permeability is downregulated during plant development. Research tracking GFP diffusion through Pd in embryonic cells, in young emerging leaves, and in fully expanded leaves showed that fluorescence is highly mobile between cells in young tissues but is restricted during maturation. Viral movement proteins such as Cucumber mosaic virus 3a, and PVX TGBp1 remain highly mobile in mature leaves because they gate open Pd under conditions that normally restrict movement of GFP.^34,35 Schoenknecht et al., undertook a straightforward investigation of leaf maturation describing Pd transport in relationship to leaf area expansion. The outcome of this study was evidence that GFP movement between cells declines as leaves expand.It is reasonable to consider that simultaneous changes in gene expression and physiology is reflected in a downward trend in Pd conductivity and an increased requirement for Pd gating to enable selected transport of macromolecules between cells. In Arabidopsis embryos there is an obvious transition between developmental stages which are also represented by a decline in the ability for GFP to diffuse across Pd.^36,37 A detailed analysis of Pd structure in source and sink tissues revealed that Pd are simple single channeled structures in sink tissues while source tissues contain predominantly “H” shaped branching Pd structures. The change in Pd structure has been correlated with changes in conductivity and is often correlated with changes in sink to source metabolism.^38,39 The sink-to-source transition in leaf development is typically monitored using phloem loading of carboxyfluorescein diacetate. Leaves where CF dye unloads are defined as sink leaves and leaves that were restricted in dye unloading were defined as source leaves. Then biolistic bombardment of GFP expressing plasmids to sink and source leaves revealed that GFP readily diffuses across Pd in sink leaves but is more often restricted in source leaves.^{26,34,40–42}Leaf development is typically defined as a transition from juvenile to adult which is represented by homeotic transformations as well as vegetative phase changes.^43,44 Source and sink regions of a leaf have been shown to correlate with changes in Pd structure and conductivity during leaf expansion. However, in our study we found that N. tabacum leaves identified as source during week 2 or 3 would continue to expand over an 8 week period to twice or three times the leaf area which provides a real indication that the source designation may not entirely reflect final leaf maturation or completion of leaf development.⁴⁵> For example, as cells transition from sink to source physiology it is suggested that the frequency of single channeled Pd declines while the frequency of branched Pd increases.³⁹ It is possible that even after leaves transition into photosynthetic sources that Pd architecture continues to change and there is a further decline in the proportion of single channel to branched channels. Therefore either the change in cell volume or Pd architecture or both can slow-down diffusion of GFP between cells.Researchers often point to the ER continuity between cells as a driving force for Pd formation and function. During cell division the cell wall is laid down and forms around the ER creating Pd channels.⁴⁶ However, it is also worth noting that the actin cytoskeleton is also present in Pd and is central to organ and reproductive development.^19,47 Actin and actin binding proteins are necessary for a number of plant processes determining the cell division plane, cell polarity, cell elongation, cytoplasmic streaming, transporting mRNAs and proteins, and defense.^48–51 Overexpression of ACT1 in Arabidopsis leaves can lead to changes in epidermal leaf shape and cause dwarfism in plants.⁵² Actin binding proteins are also necessary for organizing and remodeling the F-actin network which drives normal development of specific cell types and organs.⁵³ Actin filament bundling and remodeling are also seen in nonhost defense responses.⁵⁴ We do not know the effects of overexpressing certain actin homologues or actin remodeling on Pd formation or conductivity. Because the F-actin network is also central to Pd trafficking of proteins and macromolecules between cells it is worth considering F-actin as an early factor contributing to Pd formation which may be necessary to ensure cell-to-cell communication when cell polarity and elongation as well as defense machinery are being established.In summary, the novel quantitative tools developed for measuring protein movement across Pd reveal the temperature dependence of protein trafficking. Both the use of Arrhenius equation and C(Pd) provide new opportunities to measure the energy requirements for protein transport. These tools will enable researchers to quantify effects of environmental and developmental conditions on Pd conductivity, as well as comparing differences in Pd conductivity between plant species or induced by genetic mutations.     

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.     

Auxin acts independently of DELLA proteins in regulating gibberellin levels

Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA₁, play critical roles in the control of elongation,^1–3 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.^4,5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,⁶ since auxin upregulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and downregulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA₁.⁷ In our recent paper,¹ we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,^8,9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA₁ synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.¹⁰ However, our recent results,¹ in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.Key words: auxin, gibberellins, DELLA proteins, interactions, elongation     

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.     

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