The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarization of the plasma membrane, which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarization and wall expansion, the activation of the plasma membrane P-type H⁺-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyze long-distance BL signaling in Arabidopsis seedlings.Key words: BRI1, fluorescence lifetime, membrane potential, P-ATPase, cell wall expansionUsing spectro-microscopic technologies, we recently started the quantitative analysis of the properties and subcellular function of GFP fusion of the plasma membrane-localized brassinosteroid (BR) receptor, BRI1, in living plant cells of Arabidopsis thaliana and tobacco (Nicotiana benthamiana) leaf cells.^1,2 Brassinosteroids, such as brassinolide (BL), are involved in responses to biotic and abiotic stresses and developmental processes, including cell elongation.³ The present model of the BR response pathway includes the binding of BRs to BRI1, resulting in the autophosphorylation of the receptor and the subsequent recruitment of the co-receptor BRI1-associated receptor kinase 1 (BAK1). This association is followed by trans-phosphorylation between BRI1 and BAK1 and results in the activation of downstream BR signaling processes leading to differential gene expression and, finally, to the execution of the specific responses.⁴ However, the molecular events that take place in the plasma membrane immediately after the perception of BL and initiate cell elongation still have to be included in this model.⁵ We recently reported a rapid BRI1-GFP-dependent cell wall expansion in Arabidopsis seedlings, which is attributed to wall loosening and water incorporation into the wall, and precedes cell elongation.^1,2 This expansion response was accompanied by a change in the FLT of BRI1-GFP, which reflects an alteration in the plasma membrane potential (E_m).^2,6 For both the FLT change in BRI1-GFP and the wall expansion, the activity of the plasma membrane P-ATPase is crucial. Notably, H⁺-pump activation was shown to depend on the kinase activity of BRI1.² This suggests a fast BRI1-dependent response pathway in the plasma membrane which links BL perception via P-ATPase activation and E_m hyperpolarization to wall expansion. In this report, we demonstrate that the phosphorylation of a conserved threonine in the auto-inhibitory domain of AHA1 is not required for the enzymatic activation by BRI1 suggesting a novel mechanism by which BRI1 may initiate the activation of the P-ATPase. Furthermore, we show that the FLT of BRI1-GFP is a useful and senstitive probe for the non-invasive analysis of systemic signaling processes in living plants.     

Brassinosteroid-independent functions of the BRI1-associated kinase BAK1/SERK3

Eukaryotes have evolved programmed cell death (PCD) mechanisms that play important roles in both, development and immunity.^1–3 We demonstrated a requirement for the Arabidopsis thaliana leucine-rich repeat receptor-like kinase (LRR-RLK), BAK1/SERK3 (BRI1-Associated receptor Kinase 1/Somatic Embryogenesis Receptor Kinase 3) in regulating the containment of microbial infection-induced necrosis. BAK1-deficient plants showed constitutive expression of defense-related genes and developed spreading cell death upon infection by necrotizing pathogens that result in enhanced susceptibility to necrotrophic pathogens. This reaction was not inducible by exposition of bak1 mutants to general stresses but appeared to be solely inducible by necrotizing pathogen infection. BAK1 is known to interact with the brassinosteroid receptor, BRI1, and thereby facilitates plant growth and development in a brassinolide (BL)-dependent manner.^4,5 Surprisingly, the cell death-related phenotype in bak1 mutants is brassinolide-independent. In this addendum we want to present recent new data on BAK1 and discuss its role as a general regulator in plant processes being as diverse as brassinosteroid signaling in development, perception of pathogen associated molecular patterns (PAMPs), and cell-death control in innate immunity.Key words: LRR-RLK, cell-death control, immunity, brassinosteroids, BAK1, SERK3, BRI1, FLS2     

LeEix1 functions as a decoy receptor to attenuate LeEix2 signaling

The receptors for the fungal elicitor EIX (LeEix1 and LeEix2) belong to a class of leucine-rich repeat cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Both receptors are able to bind the EIX elicitor while only the LeEix2 receptor mediates defense responses. We show that LeEix1 acts as a decoy receptor and attenuates EIX induced internalization and signaling of the LeEix2 receptor. We demonstrate that BAK1 binds LeEix1 but not LeEix2. In plants where BAK1 was silenced, LeEix1 was no longer able to attenuate plant responses to EIX, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires the kinase activity of BAK1.Key words: LRR-RLP, LeEix, Bak1, decoy receptor, endocytosisLeucine-rich-repeat receptor proteins (LRR-RLPs) have been linked with defense response signaling in plants.^1–5 The tomato Cf genes which mediate resistance to Cladosporium fulvum encode LRR-RLPs. Additional LRR-RLPs include the tomato Verticillium (Ve) resistant proteins^6,7 and the LeEix proteins.⁸ The Eix receptors (LeEix1 and LeEix2) contain a signal for receptor-mediated endocytosis, which we have previously shown to be essential for proper induction of defense responses.^9,10 Both receptors are able to bind Eix, but only LeEix2 mediates EIX-induced defense.⁸ In a recent work we demonstrate that LeEix1 attenuates Eix-induced internalization and signaling, and heterodimerizes with LeEix2 upon application of Eix.¹¹ Our work further shows that the brassinosteroid co-receptor Bri-Associated Kinase 1 (BAK1) binds LeEix1 but not LeEix2. In BAK1-silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation and leading to the hypothesis that LeEix1 functions as a decoy receptor for LeEix2.¹¹     

Tissue-type specific systemin perception and the elusive systemin receptor

Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate systemin binding protein that does not activate the systemin signaling pathway. Here we provide evidence that systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to systemin, while they responded to other elicitors. We hypothesize that the elusive systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the systemin precursor prosystemin. Binding of systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.Key words: systemin, systemin receptor, brassinosteroids, BRI1, BRL, protoplastsSystemin is thought to be processed from its precursor prosystemin upon insect attack and wounding of tomato leaves. Strong evidence has been gathered for an important role of (pro-)systemin in the activation of defenses against insects, and the underlying signaling pathway has been studied in detail.¹ However, the perception of systemin is controversial. Meindl et al.² and Scheer and Ryan³ identified high affinity, saturable, reversible and specific cell surface binding sites on Solanum peruvianum suspension-cultured cells which are known to be highly sensitive to systemin.⁴ A purification approach using a photoaffinity systemin analog identified a 160 kDa protein as the systemin receptor (SR160).⁵ Follow-up studies showed that overexpression of tomato 35S::SR160 in systemin-insensitive tobacco plants conferred systemin sensitivity to tobacco.⁶ Surprisingly, SR160 turned out to be the tomato homolog of the brassinosteroid receptor BRI1,⁷ which raised many questions as to the functionality of a receptor for two structurally and functionally diverse ligands. It was then shown in two independent papers that a null mutant for tomato BRI1, cu-3, exhibited a normal response to systemin.^8,9 This was strong evidence that SR160/BRI1 does not represent the functional systemin receptor. Our recent data added a peculiar twist to this story. We found that overexpression of tomato BRI1 in tobacco suspension-cultured cells resulted in binding of a fluorescently labeled systemin to the plasma membranes of the transgenic tobacco cells, but not to wild type cells. Surprisingly, this did not result in BRI1-dependent signal transduction and activation of a defense response, although we detected weak BRI1-independent signaling responses to systemin.¹⁰ Together with the identification of BRI1 as the systemin receptor by Scheer and Ryan,⁵ the simplest explanation for this phenomenon is that BRI1 is a systemin binding protein, but not the physiological systemin receptor.Therefore and for other reasons, we suggested that the true systemin receptor may be a protein with very similar properties as BRI1, e.g., a homolog of the BRI1-like (BRL) proteins. The purification strategy employed by Scheer and Ryan5 may have resulted in binding of a photoaffinity-systemin derivative to BRI1 and one or more BRL proteins. Since BRLs and BRI1 have a very similar MW, multiple bands on a SDS-PAGE would not be detectable.Here, we would like to add another aspect of systemin perception. We provide evidence for tissue-specific systemin sensitivity and discuss how this may affect systemin binding to BRI1 and the elusive systemin receptor. Prosystemin is only present in phloem parenchyma cells.¹¹ It can be surmised that the systemin receptor is located close to these cells. Systemin perception results in JA synthesis in companion cells of vascular bundles.¹² Since JA or a JA derivative is the most likely phloem-mobile candidate for a systemic long-distance wound signal, it is thought that JA is moving from companion cells into sieve cells to reach distant parts of the plant for upregulation of wound response genes in leaf cells, including mesophyll cells.^13–15Here, we tested the hypothesis that mesophyll cells lack systemin perception. We generated mesophyll protoplasts from tomato leaf material as well as protoplasts from S. peruvianum suspension-cultured cells, the same cell line that had been used for the purification of SR160/BRI1 and is known to be highly sensitive to systemin. Mesophyll protoplasts showed increased phosphorylation of MAP kinases (MPKs) in response to the elicitors flg22 and chitosan, bacterial and fungal MAMPs, respectively. However, they did not respond to systemin. In contrast, the S. peruvianum protoplasts did respond to systemin and to flg22, demonstrating that the protoplasting procedure did not compromise the systemin perception mechanism (Fig. 1). Immunocomplex kinase assays with specific antibodies against tomato MPK2 produced similar results (data not shown). Since flg22, chitosan and systemin activated the same MPKs (Fig. 1), our data indicate that systemin perception is absent in mesophyll protoplasts. Our leaf protoplasting protocol is a modification of the protocol by Yoo et al. which results in the generation of mesophyll protoplasts.¹⁶ In contrast, suspension-cultured cells do not normally represent specific cell types and it is not known why the S. peruvianum cells are highly sensitive to systemin.Open in a separate windowFigure 1Absence of systemin-induced MPK phosphorylation in mesophyll cells. Protoplasts were generated (protocol available upon request) from S. peruvianum suspension-cultured cells and from S. lycopersicum cv. MicroTom leaves. After a 1.5 hour recovery phase on ice, protoplasts were resuspended in WI medium (0.5 M mannitol, 5 mM ME S pH 5.7, 20 mM KCl), recovered for 1 hour in non-stick tubes with constant rotation on a rotary shaker at room temperature, and then treated with either water (con), 10 nM systemin (sys), 100 nM flg22, or 2.5 µg/ml chitosan (from crab shells—chi) for 10 min at room temperature. Protoplasts were analyzed for MPK phosphorylation by immunoblotting using an anti-phospho-ER K antibody (phospho-p44/42 MA PK (Erk1/2) (Thr202/Tyr204); D13.14.4E; Cell Signaling Technology) at a dilution of 1:2,000. This antibody recognizes MPKs that are phosphorylated on either the Thr and Tyr or on only the Thr within the TE Y phosphorylation motif which is conserved among plant and metazoan MPKs. It is known to recognize the tobacco MPKs SIPK and WIPK²¹ and Arabidopsis MPK6 and MPK3,²² the orthologs of tomato MPK1/2 and MPK3.²³ Bands were visualized as described.¹⁰ Proteins on membranes were stained with Ponceau S to demonstrate equal loading.Intriguingly, BRL1, BRL2 and BRL3 are expressed in the vasculature and function in vascular pattern formation in Arabidopsis, while BRI1 is ubiquitously expressed in dividing and elongating cells. BRL3 is even specifically expressed in phloem cells.¹⁷ This matches the highly specific localization of prosystemin in the phloem parenchyma cells.^11,18 The highest BRI1 expression is found in growing parts of young leaves^17,19 while prosystemin is specifically present in the phloem parenchyma cells throughout all developmental stages.¹¹ In this context, it is also interesting to note that application of systemin to tomato plants via the cut stem results in rapid and strong MPK activation. In this assay, systemin is delivered to leaf cells via the transpiration stream and therefore present in vascular tissue.²⁰Based on the combined evidence, we propose that the true systemin receptor is a BRL or similar protein which is expressed in phloem cells in the vicinity of the parenchyma cells that express prosystemin, but not in mesophyll cells. Because of the similarity between BRLs and BRI1, BRI1 was erroneously identified as the systemin receptor. Inappropriate binding of systemin to BRI1 is consistent with the high similarity between BRI1 and BRLs. However, because of the tissue-specificity of the systemin signaling pathway, inappropriate binding of systemin to BRI1 may rarely occur in wild type plants and may not pose an interference problem for either systemin or brassinosteroid signaling.     

Cell Migration from Baby to Mother

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

RALFs: Peptide regulators of plant growth

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

A Novel MAPKK Involved in Cell Death and Defense Signaling

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

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two-cell stage with inappropriate marking of the pre-apical patch (PAP )—the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.Key words: lumen, N-WASP, tuba, E-cadherin, pre-apical patchMany epithelial tissues are organized as hollow tubes whose open lumina connect the body with its external environment.^1,2 These tubes consist of a monolayer of polarized cells that envelope the central lumen. Lumen formation is thus a key process in epithelial morphogenesis that depends upon cell polarity to establish three cell surface domains: a basal surface adherent to the extracellular matrix, a lateral surface between cells, and an apical surface that is exposed to the luminal fluids. Of note, the apical membrane is biochemically and morphologically distinct from the baso-lateral surfaces and effectively defines the luminal surface.^3,4For a lumen to form, cells must first mark the site at which apical membrane is to be inserted, something that is achieved at the first cell division.⁵ Targeted trafficking of apical membrane constituents defines a pre-apical patch (PAP), the precursor to the definitive lumen.⁵ Such insertion of apical membrane must presumably be coordinated with the assembly of apical junctions to segregate nascent apical from lateral membrane domains.² Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement.⁶ Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract,⁷ or by apoptosis or autophagy of the central cells as is observed in mammary gland development.^8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.^5,6Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process,^2,10 with downstream consequences that include control of mitotic spindle orientation,⁵ which itself influences PAP placement⁵ and potentially regulation of cell-cell junctions. Like other Rho family GTPases, the subcellular location of Cdc42 signaling is determined by the action of upstream proteins, notably guanine nucleotide exchange factors (GEFs).^11,12 Of these, Tuba, a Cdc42-specific GEF,¹³ has emerged as a regulator of lumenal morphogenesis that controls PAP placement through mitotic spindle orientation.¹⁰Tuba is also a scaffolding protein¹³ capable of linking the actin assembly machinery with trafficking pathways. Not only is Tuba required for Cdc42 activation to direct spindle orientation,⁵ it also has the potential to interact with phosphoinositides that define the PAP.¹⁴ Additionally, Tuba binds directly to the actin regulator N-WASP, a key molecule in the organization of actin and itself a Cdc42 effector.¹⁵ Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior.¹⁶ Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions.¹⁷ In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.¹⁸ Therefore, Tuba has the potential to play a central role in coordinating the molecular complexes required for productive polarization of epithelial cells and placement of the PAP during lumenogenesis. However, whether other protein interactions contribute to the morphogenetic impact of Tuba remain to be assessed.Three-dimensional cell culture systems are being utilized to identify critical components in lumen formation. In particular, Madin-Darby canine kidney cells (MDCK) and Caco-2 gastrointestinal cells are commonly used to study cyst and/or tubule formation. MDCK cells undergo both cyst and tubule growth, apoptosis being primarily responsible for the final step in lumen formation,¹⁹ while Caco-2 cells primarily utilize fluid influx to expand cysts.⁵ Cyst culture systems replicate aspects of in vivo organogenesis²⁰ providing tangible, powerful models to analyze and dissect the coordinated cellular mechanisms and processes that occur during epithelial morphogenesis.In this study we examined the relationship between Tuba and N-WASP in early epithelial lumenogenesis using Caco-2 three dimensional cyst cultures. Both Tuba and N-WASP RNAi cell lines result in mature cysts with multiple lumina, and at the two-cell stage, formed multiple PAPs. Interestingly, N-WASP KD perturbed Tuba localization at the PAP, however, N-WASP localization to the PAP was not affected to the same extent by Tuba KD. Taken together, these results suggest a complex interrelationship between Tuba and N-WASP for the coordinated formation of a single hollow lumen.     

Autophosphorylation desensitizes phytochrome signal transduction

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.Key words: phytochrome, autophosphorylation, phosphorylation, protein kinase, protein degradation, light signaling, signal desensitizationHigher plants continually adapt to their light environments to promote photosynthesis for optimal growth and development. Natural light conditions are monitored by various plant photoreceptors, including red (R)/far-red (FR) photoreceptor phytochromes.^1,2 Phytochromes are dimeric chromoproteins covalently linked to tetrapyrrole chromophore phytochromobilin, and exist as two photo-interconvertible species, red-light absorbing Pr and far-red-light absorbing Pfr forms. Phytochromes are biosynthesized as the Pr form in the dark, and are transformed to the Pfr form upon exposure to red light. This photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants.^3,4 Recently, phosphorylation and dephosphorylation have been suggested to play important roles in phytochrome-mediated light signaling;^5,6 for instance, a few phytochrome-associated protein phosphatases have been shown to act as positive regulators of phytochrome signaling.^7–9 However, the functional roles of phytochrome phosphorylation remain to be explored.     

Why have chloroplasts developed a unique motility system?

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

Hypoxia: A novel function for VIN3

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