Step by Step: Deciphering Ion Transport in the Root Xylem Parenchyma

Proton pumps produce electrical potential differences and differences in pH across the plasma membrane of cells which drive secondary ion transport through sym- and antiporters. We used the patch-clamp technique to characterize an H⁺-pump in the xylem parenchyma of barley roots. This cell type is of special interest with respect to xylem loading. Since it has been an ongoing debate whether xylem loading is a passive or an active process, the functional characterization of the H⁺-pump is of major interest in the context of previous work on ion channels through which passive salt efflux into the xylem vessels could occur. Cell-type specific features like its Ca²⁺ dependence were determined, that are important to interpret its physiological role and eventually to model xylem loading. We conclude that the electrogenic pump in the xylem parenchyma does not participate directly in the transfer of KCl and KNO₃ to the xylem but, in combination with short-circuiting conductances, plays a crucial role in controlling xylem unloading and loading through modulation of the voltage difference across the plasma membrane. Here, our recent results on the H⁺ pump are put in a larger context and open questions are highlighted.Key Words: plant nutrition, H⁺-ATPase, anion conductance, K⁺ channel, electrophysiology, signaling networkThe root xylem parenchyma is of major interest with respect to nutrient (and signal) traffic between root and shoot. One of its main functions appears to be xylem loading. However, the cell walls of the vascular tissue provide apoplastic paths between xylem and phloem that represent the upward and downward traffic lanes, allowing nutrient circulation¹ (Fig. 1). Therefore mechanisms for ion uptake and for ion release must exist side by side. In the last 15 years major progress has been made in the investigation of transport properties of xylem-parenchyma cells, and both uptake and release channels and transporters were identified. Today, we have good knowledge on the role of K⁺ and anion conductances in xylem loading with salts.² Note, that from the functionally well characterized conductances only the molecular structure of K⁺ channels is known. In contrast, many transporters are identified on the molecular level, but functional data are scarce.Open in a separate windowFigure 1Distribution of tissues in the periphery of the stele. The stippled area marks the region from which early metaxylem protoplasts originated. E, Endodermis with Casparian strip; eMX, ‘early’ metaxylem vessel; IMX, ‘late’ metaxylem vessel; Mph, metaphloem (sieve tube); Pph, protophloem (sieve tube); P, pericycle; Cx, cortex. Symplasmic and apoplasmic transport routes are indicated in red and black, respectively. The Casparian strip prevents apoplastic transport into the stele. Plasmodesmata are shown exemplarily for the indicated symplastic pathway. All cells of the symplast are connected via plasmodesmata. Sites of active uptake into the root symplast and of release into the stelar apoplast are indicated by a black and an orange arrow. Modified from Wegner and Raschke, 1994.³A challenging question to deal with was the dispute about xylem loading with ions being a passive or active process. While it is clear that energy through electrogenic H⁺ efflux is needed to take up nutrient ions from the soil against their electrochemical gradient into the cortical symplast, it has been a matter of debate if ion release into xylem vessels also is energy-linked or if the electrochemical potentials of ions are raised high enough to allow a thermodynamically passive flux.^2,3 The Casparian strip prohibits apoplastic transport of nutrients into the stele and electrically insulates the stelar from the cortical apoplast. Therefore the electrical potential difference of the cells in the xylem parenchyma could be independent from the cortical potential difference but be subject to control, for instance, from the shoot.⁴ Indeed, evidence points to xylem loading as a second control point in nutrient transfer to the shoot.^5,6 The identification and characterization of K⁺ and anion conductances clearly showed that release of KCl and KNO₃ into the xylem can be passive through voltage-dependent ion channels.^2,3,7–9 No need appeared for a pump energizing the transfer of salts to the xylem.However, H⁺ pumps are ubiquitous. H⁺-ATPases are encoded by a multigene family and heterologous expression in yeast showed that isoforms have distinct enzymatic properties.^10,11 As the example of the amino acid transporter AAP6 from the xylem parenchyma shows, a cell-type specific functional characterization of transporters is essential to draw conclusions on their physiological role. AAP6 is the only member of a multigene family with an affinity for aspartate in the physiologically relevant range. The actual apoplastic concentration of amino acids and the pH will determine what is transported in vivo.^12,13 Xylem-parenchyma cells of barley roots were strongly labelled by antibodies against the plasma membrane H⁺-ATPase.¹⁴ In a recent publication in Physiologia Plantarum we report the functional analysis of the electrogenic pump from the plasma membrane of xylem parenchyma from barley roots that was done with the patch-clamp technique after specific isolation of protoplasts from this cell type. It displayed characteristics of an H⁺-ATPase: current-voltage relationships were characteristic for a ‘rheogenic’ pump¹⁵ and currents were stimulated by fusicoccin or by an enlarged transmembrane pH gradient and inhibited by dicyclohexylcarbodiimide (DCCD). Importantly, it also showed distinct characteristics. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Noteworthy, K⁺ and anion conductances from the same cell type are controlled by intracellular [Ca²⁺]^7,9 (Fig. 2). It was proposed that the effect of abscisic acid (ABA) on anion conductances is mediated via an increase in the cytosolic Ca²⁺ concentration.¹⁶ Very likely stelar H⁺ pumps are stimulated by ABA.¹⁷ Thus, a Ca²⁺ independent control has to be hypothesized in this case.Open in a separate windowFigure 2Control of ion conductances in the plasma membrane of xylem-parenchyma cells. Arrowheads indicate stimulation and bars indicate inhibition by an increase in cytosolic [Ca²⁺],^7,9,16 by ABA,^16,17,21 by cytosolic and apoplastic acidification,^4,22 by G-proteins²³ and by an increase in apoplastic [K⁺]⁷ and [NO₃⁻].²⁴ Apoplastic [K⁺] and [NO₃⁻] modify the voltage dependence exerting negative feedback on K⁺ efflux and a positive feedback on NO₃⁻ efflux. Abscisic acid has an immediate effect on ion channel activity, most likely via [Ca²⁺], and causes a change in gene expression as indicated by circles (up) and bars (down). ABA perception is not clear. A Ca²⁺ influx could occur through a hyperpolarization activated cation conductance (HACC).^16,25 Cation transporters are NORC, nonselective cation conductance, KORC, K⁺-selective outwardly rectifying conductance (=SKOR⁸), and KIRC, K⁺-selective inwardly rectifying conductance, and anion conductances with different voltage-dependencies and gating characteristics are X-QUAC, quickly activating anion conductance, X-SLAC, slowly activating anion conductance, and X-IRAC, inwardly rectifying anion channel.^2,3,9,16,26 Transported ions and direction of flux are plotted.To date, we know that besides Ca²⁺ and abscisic acid also the pH, nonhydrolyzable GTP analogs and extracellular NO₃⁻ and K⁺ affect membrane transport capacities of root xylem-parenchyma cells (Fig. 2). Other control mechanisms by metabolites, the redox potential and phytohormones have to be included, especially if they represent signals in xylem loading or root-shoot communication. The composition of the xylem sap changes during the course of a day, depending on nutrient supply and various stresses, and the apoplastic ion concentration is considered to be an important factor in ion circulation.^6,18,19 ABA is such a signal. It is known to increase solute accumulation within the root by inhibiting release of ions into the xylem.¹⁷ Any change in transport activity has an impact on the membrane potential. This again determines whether salt release or uptake takes place. Passive salt release is restricted to a limited range of membrane potentials in which conductances for anions and cations are active simultaneously, that is with depolarization. Negative membrane voltages will be required for reabsorption of NO₃⁻ by a putative NO₃⁻/H⁺-symporter and for the uptake of K⁺ and amino acids.^3,13 As shown in our recent paper, the balance between the activities of the H⁺-pump and the anion conductances could affect the position between a depolarized and a hyperpolarized state of the parenchymal membrane. Thus, H⁺ pump activity is crucial in membrane voltage control. Furthermore, the simultaneous activities of H⁺ pumps and anion conductances make the generation of a high pH gradient possible, whilst maintaining electroneutrality. The proton gradient could be used for ion transport through cotransporters and antiporters as suggested for the loading of borate into the xylem through the boron transporter BOR1.²⁰ So we are on the way to decipher xylem loading in roots and this exciting field will also provide information about small-scale nutrient cycling and root-shoot communication. To determine how the activities of pumps, channels and transporters are adjusted among each other is the next challenge. Further insight has to be obtained by experimentation as well as by biophysical modeling.     

The TRPA1 channel and oral hypoglycemic agents

Diabetes mellitus type 2 (DM2) results from the combination of insulin unresponsiveness in target tissues and the failure of pancreatic β cells to secrete enough insulin.¹ It is a highly prevalent chronic disease that is aggravated with time, leading to major complications, such as cardiovascular disease and peripheral and ocular neuropathies.² Interestingly, therapies to improve glucose homeostasis in diabetic patients usually involve the use of glibenclamide, an oral hypoglycemic drug that blocks ATP-sensitive K⁺ channels (K_ATP),^3,4 forcing β cells to release more insulin to overcome peripheral insulin resistance. However, sulfonylureas are ineffective for long-term treatments and ultimately result in the administration of insulin to control glucose levels.⁵ The mechanisms underlying β-cell failure to respond effectively with glibenclamide after long-term treatments still needs clarification. A recent study demonstrating that this drug activates TRPA1,⁶ a member of the Transient Receptor Potential (TRP) family of ion channels and a functional protein in insulin secreting cells,^7,8 has highlighted a possible role for TRPA1 as a potential mediator of sulfonylurea-induced toxicity.     

Voltage,reactive oxygen species and the influx of calcium

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca²⁺ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca²⁺ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca²⁺ influx, suggesting that these enzymes are involved in sensing Ca²⁺ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) encodes plant cell signals.¹ Reactive oxygen species (ROS) are potent regulators of the PM Ca²⁺ channels implicated in signalling and developmental increases in [Ca²⁺]_cyt.^1,2 Plasma membrane (PM) voltage (V_m) also plays a significant part in generating specific [Ca²⁺]_cyt elevations through the opening of voltage-gated Ca²⁺-permeable channels, allowing Ca²⁺ influx.^1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca²⁺ channels (HACCs),⁴ ROS-activated HACCs⁵ and depolarisation-activated Ca²⁺ channels (DACCs).⁶ The DACC characterisation pointed to the presence of a Cl⁻-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.⁶ This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.⁷ Previous studies have shown that root hair PM also harbors K⁺ channels (mediating inward or outward flux)^8–10 and a H⁺-ATPase.¹¹ A key problem to address now is how these transporters interact to generate and be influenced by PM V_m, thus gating and in turn being regulated by their companion Ca²⁺ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between V_m and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.¹² In the “pump state” the H⁺-ATPase predominates, there is net H⁺ efflux and the hyperpolarized V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 and oscillations between these states may explain the temporal changes in H⁺ flux recently observed at the apex of growing Arabidopsis root hairs.¹⁵ Peaks of H⁺ influx may reflect a depolarized V_m that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca²⁺ influx it permits could be amplified through intracellular release.The PM H⁺-ATPase traditionally lies at the core of models of voltage and ionic flux^14,16 but in terms of [Ca²⁺]_cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.⁵ AtrbohC is implicated in the transition to polar growth at normal extracellular pH⁵ and also osmoregulation.¹⁷ NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e⁻ efflux systems,¹⁸ their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.¹⁹ Two H⁺ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e⁻ efflux by animal NADPH oxidases is fairly constant over negative V_m and decreases at very depolarized V_m.²⁰ AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H⁺ flux oscillations.¹⁵ The latter suggests that AtrbohC function does in some way affect V_m, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.²¹NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency^22,23 to signal stress conditions. Blockade of PM Ca²⁺ channels by lanthanides increases superoxide production in tobacco suspension cells.²⁴ This suggests that NADPH oxidases are involved in sensing the cell''s Ca²⁺ status and the prediction would be that extracellular Ca²⁺ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).^25,26 Lowering extracellular Ca²⁺ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca²⁺ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca²⁺]_cyt²⁷ (Fig. 1). However, reduction of Ca²⁺ influx should promote voltage hyperpolarisation (just as block of K⁺ influx causes hyperpolarisation in root hairs²⁸) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity⁵ to increase Ca²⁺ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl₂, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ to increase [Ca²⁺]_cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to V_m, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca²⁺]_cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing V_m and [Ca²⁺]_cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.²⁹ While there is still much to do on the “inventory” of PM transporters involved in Ca²⁺ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action.     

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     

Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake

The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.Key words: silicon, efflux transporter, pumpkin, cucumber, bloomSilicon (Si) is the second most abundant elements in earth''s crust.¹ Therefore, all plants rooting in soils contain Si in their tissues. However Si accumulation in the shoot differs greatly among plant species, ranging for 0.1 to 10% of dry weight.^1–3 In higher plants, only Poaceae, Equisetaceae and Cyperaceae show a high Si accumulation.^2,3 Si accumulation also differs with cultivars within a species.^4,5 These differences in Si accumulation have been attributed to the ability of the roots to take up Si.^6,7Genotypic difference in Si accumulation has been used to produce bloomless cucumber (Cucumis sativus L.).⁸ Bloom (white and fine powders) on the surface of cucumber fruits is primarily composed of silica (SiO₂).⁹ However, nowadays, cucumber without bloom (bloomless cucumber) is more popular in Japan due to its more attractive and distinctly shiny appearance. Bloomless cucumber is produced by grafting cucumber on some specific pumpkin (Cucurbita moschata Duch.) cultivars. These pumpkin cultivars used for bloomless cucumber rootstocks have lower silicon accumulation compared with the rootstocks used for producing bloom cucumber.⁹Our study showed that the difference in Si accumulation between bloom and bloomless root stocks of pumpkin cultivars results from different Si uptake by the roots.¹⁰ Si uptake has been demonstrated to be mediated by two different types of transporters (Lsi1 and Lsi2) in rice, barley and maize.^11–15 Lsi1 is an influx transporter of Si, belonging to a NIP subfamily of aquaporin family.^10,11,13,14 This transporter is responsible for transport of Si from external solution to the root cells.¹¹ On the other hand, Lsi2 is an efflux transporter of Si, belonging to putative anion transporter.¹² Lsi2 releases Si from the root cells towards the xylem. Both Lsi1 and Lsi2 are required for Si uptake by the roots.^11,12 To understand the mechanism underlying genotypic difference in Si uptake, we have isolated and functionally characterized an influx Si transporter CmLsi1 from two pumpkin cultivars used for rootstocks of bloomless and bloom cucumber.¹⁰ Sequence analysis showed only two amino acids difference of CmLsi1 between two pumpkin cultivars. However, CmLsi1 from bloom rootstock [CmLsi1(B⁺)] showed transport activity for Si, whereas that from bloomless rootstock [CmLsi1(B⁻)] did not.¹⁰ Furthermore, we found that loss of Si transport activity was caused by one amino acid mutation at the position of 242 (from proline to leucine).¹⁰ This mutation resulted in failure to be localized at the plasma membrane, which is necessary for functioning as an influx transporter. The mutated protein was localized at the ER.¹⁰ Here, we report isolation and expression analysis of Si efflux transporters from two pumpkin cultivars contrasting in Si uptake and accumulation to examine whether Si efflux transporter is also involved in the bloom and bloomless phenotypes.     

Potassium flux in the pollen tubes was essential in plant sexual reproduction

Potassium channels are controlling K⁺ transport across plasma membrane and thus playing a central role in all aspects of osmolarity as well as numerous other functions in plants, including in sexual reproduction. We have used whole-cell and single-channel patch-clamp recording techniques investigated the regulation of intracellular free Ca²⁺-activated outward K⁺ channels in Pyrus pyrifolia pollen tube protoplasts. We have also showed the channels could be inhibited by heme and activated carbon monoxide (CO). In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO. Considered the oxygen concentration approaching zero in the ovary, the heme will inhibit the K⁺ outward flux from the intracellular of pollen tube, increasing the pollen tubes osmolarity, inducing pollen tube burst. Here we discuss the putative role of K⁺ channels in plant sexual reproduction.Key words: pear, pollen, K⁺ channels, heme, carbon monoxideIon channels in the pollen tube play critical roles in mediation pollen germination and pollen tube growth.^1–3 Early studies were focus on the plasma membrane calcium channel regulation and cytosolic free calcium concentration variation in the pollen tube reason by which was one of the most important second messengers in plants.^3–7 However, reports have also showed that the potassium channels in the pollen tubes were also involved in several important steps of plant sexual reproduction.^8–19 Recently, more reports further demonstrated this phenomena.^20–24 In the report by Lu et al. they demonstrated that two cation/proton exchangers (CHX), CHX21 and CHX23, are essential for pollen tube growth guidance in Arabidopsis.²² chx21 chx23 double mutant induces the fertility impaired, but which is unchanged in both single chx21 or chx23 mutants. They have also found that the double mutant pollen grains germination and pollen tube growth in the transmitting tract were not difference with the wild-type, however, the double mutant pollen tubes fail to turn toward ovules.²² Protein localization experiments show CHX23 is expressed in the endoplasmic reticulum of pollen tubes; functional analysis results showed that CHX23 as a K⁺ transporter mediates K⁺ uptake in a pH-dependent manner. So, these protein affect the signal transduction pathway of pollen tube growth toward to the ovule by controlling the cation balance and pH in the pollen tube.²² Amien et al. identified a signaling ligand of defensin-like (DEFL) protein, ZmES4, which expressed in maize synergid. ZmES4 activates the maize pollen tube tip plasma membrane K⁺ Shaker channel KZM1.²⁰ This finding is also very interesting. Pollen tube bursting suggested to be based on the osmotic stress; the influx of K⁺ mediated by ZmES4-activated KZM1 will trigger rapid plasma membrane depolarization, which induced the pollen tube tip burst.²⁰ Furthermore, the osmotic increasing induced by too much K⁺ in the cytosolic of pollen tube was not only resulted by inward K⁺ channel activation, but also resulted by outward K⁺ channel inhibition in the pollen tube plasma membrane. In our report, we find a intracellular Ca²⁺-sensitive outward K⁺ channel in pear pollen tube plasma membrane, which could be inhibited by heme and activated by heme oxidative production, carbon monoxide (CO), may play a functional role in the pollen tube brusting.²³In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO.²⁵ Early reports showed that oxygen plays an important role in plant sexual reproduction. Pollen tubes grow through the style toward the ovary with high speed, a process that consumes tremendous amounts of energy and requires rapid oxygen uptake by pollen tubes.²⁶ Pollen grains have roughly 20 times the level of mitochondria and respire 10 times faster than vegetative tissue.^12,27–29 Furthermore, oxygen has been proposed as a possible cue for pollen-tube guidance.³⁰ Indeed, the existence of an oxygen gradient in the unpollinated style has been shown in some species such as Hipeastrum hybridum. Oxygen pressure is high in the stigma and style but suddenly decreases at the base of the style, approaching zero in the ovary. Moreover, pollen-tube growth itself creates hypoxic regions within the style.³¹ Therefore, we suggest that the outward K⁺ channel inhibited by heme is dominant compared with which activated by CO when pollen tubes reach the ovary, based on where the hypoxic condition (Fig. 1). However, the gene encode the outward K⁺ channel in the pear pollen tube remains to be determined in the further study.Open in a separate windowFigure 1Reciprocal regulation of heme and carbon monoxide in putative Ca²⁺-activated outward K⁺ channel. Under normal condition, in the presence of NADPH, heme is metabolized by hemeoxygenase to generate carbon monoxide (CO), which activates outward K⁺ channel. However, without the oxygen, heme cannot be metabolized. The accumulated heme acts as an inhibitor of outward K⁺ channel, even in the presence of NADPH. The accumulated K⁺ in the cytosolic of pollen will induced the pollen tube depolarized, then burst.     

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     

The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection.     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Strigolactones as mediators of plant growth responses to environmental conditions

Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant''s response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants'' adaptive adjustment to growth conditions.Key words: strigolactones, light, nutrient status, root, shoot, branching, lateral roots, root hairsStrigolactones (SLs) are carotenoid-derived terpenoid lactones suggested to stem from the carotenoid pathway¹ via the activity of various oxygenases.^2,3 SLs production has been demonstrated in both monocotyledons and eudicotyledons (reviewed in ref. ⁴), suggesting their presence in many plant species.⁵ SLs are synthesized mainly in the roots and in some parts of the stem and then move towards the shoot apex (reviewed ref. ⁷).^6,8,9SLs were first characterized more than 40 years ago as germination stimulants of the parasitic plants Striga and Orobanche and later, as stimulants of arbuscular mycorrhiza hyphal branching as well (reviewed in ref. ^{4, 10–13}). Recently, SLs or derivatives thereof, have been identified as a new group of plant hormones, shown to play a role in inhibition of shoot branching,^2,3,8,9 thereby affecting shoot architecture; more recently they have also been shown to affect root growth by affecting auxin efflux.¹⁴Plants have developed mechanisms that allow adaptive adjustments to a variety of different habitats by employing plasticity in their growth and development.¹⁵ Shoot architecture is affected by environmental cues, such as light quality and quantity and nutrient status.^16–19 Root-system architecture and development are affected by environmental conditions such as nutrient availability (reviewed in ref. ^{20, 21}). At the same time, plant hormones are known to be involved in the regulation of plant growth, development and architecture (reviewed in ref. ^22–24) and to be mediators of the effects of environmental cues on plant development; one classic example is auxin''s role in the plant''s shade-avoidance response (reviewed in ref. ²⁵).The ability of SLs to regulate shoot and root development suggests that these phytohormones also have a role in the plant''s growth response to its environment. To play this putative role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward enhancing its adaptive adjustment. The present review examines the SLs'' possible role in adaptive adjustment of the plant''s response to growth conditions, by discussing their effect on plant development and the possible associations and feedback loops between SLs and two environmental cues: light and nutrient status.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

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.