RESUMEN
Basal progenitor cells serve as a stem cell pool to maintain the homeostasis of the epithelium of the foregut, including the esophagus and the forestomach. Aberrant genetic regulation in these cells can lead to carcinogenesis, such as squamous cell carcinoma (SCC). However, the underlying molecular mechanisms regulating the function of basal progenitor cells remain largely unknown. Here, we use mouse models to reveal that Hippo signaling is required for maintaining the homeostasis of the foregut epithelium and cooperates with p53 to repress the initiation of foregut SCC. Deletion of Mst1/2 in mice leads to epithelial overgrowth in both the esophagus and forestomach. Further molecular studies find that Mst1/2-deficiency promotes epithelial growth by enhancing basal cell proliferation in a Yes-associated protein (Yap)-dependent manner. Moreover, Mst1/2 deficiency accelerates the onset of foregut SCC in a carcinogen-induced foregut SCC mouse model, depending on Yap. Significantly, a combined deletion of Mst1/2 and p53 in basal progenitor cells sufficiently drives the initiation of foregut SCC. Therefore, our studies shed light on the collaborative role of Hippo signaling and p53 in maintaining squamous epithelial homeostasis while suppressing malignant transformation of basal stem cells within the foregut.
Asunto(s)
Carcinoma de Células Escamosas , Transducción de Señal , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Homeostasis , Transducción de Señal/genética , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
How plants balance growth and stress adaptation is a long-standing topic in plant biology. Abscisic acid (ABA) induces the expression of the stress-responsive Asparagine Rich Protein (NRP), which promotes the vacuolar degradation of PP6 phosphatase FyPP3, releasing ABI5 transcription factor to initiate transcription. Whether NRP is required for growth remains unknown. We generated an nrp1 nrp2 double mutant, which had a dwarf phenotype that can be rescued by inhibiting auxin transport. Insufficient auxin in the transition zone and over-accumulation of auxin at the root tip was responsible for the short elongation zone and short-root phenotype of nrp1 nrp2. The auxin efflux carrier PIN2 over-accumulated in nrp1 nrp2 and became de-polarized at the plasma membrane, leading to slower root basipetal auxin transport. Knock-out of PIN2 suppressed the dwarf phenotype of nrp1 nrp2. Furthermore, ABA can induce NRP-dependent vacuolar degradation of PIN2 to inhibit primary root elongation. FyPP3 also is required for NRP-mediated PIN2 turnover. In summary, in growth condition, NRP promotes PIN2 vacuolar degradation to help maintain PIN2 protein concentration and polarity, facilitating the establishment of the elongation zone and primary root elongation. When stressed, ABA employs this pathway to inhibit root elongation for stress adaptation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos , Raíces de Plantas/metabolismoRESUMEN
BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.
Asunto(s)
Saccharomyces cerevisiae , Vacuolas , Animales , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteínas de la Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Polarity is a feature of life. In higher plants, non-autonomous polarity is largely directed by auxin, the morphogen that drives its own polarized flow, Polar Auxin Transport (PAT), to guide patterning events such as phyllotaxis and tropism. The plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers are rate-limiting factors in PAT. In yeasts and metazoans, the STE20 kinases are key players in cell polarity. We had previously characterized SIK1 as a STE20/Hippo orthologue in Arabidopsis and confirmed its function in mitotic exit and organ growth. Here we explore the possible link between SIK1, auxin, PIN, and polarity. Abnormal phyllotaxis and gravitropism were observed in sik1. sik1 was more sensitive to exogenous auxin in primary root elongation and lateral root emergence. RNA-Seq revealed reduced expression in auxin biosynthesis genes and induced expression of auxin flux carriers in sik1. However, normal tissue- and sub-cellular localization patterns of PIN1 and PIN2 were observed in sik1. The dark-induced vacuolar degradation of PIN2 also appeared normal in sik1. An additive phenotype was observed in the sik1 pin1 double mutant, indicating that SIK1 does not directly regulate PIN1. The polarity defects of sik1 are hence unlikely mediated by PINs and await future exploration.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Polaridad Celular , Proteínas de Transporte de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cotiledón/crecimiento & desarrollo , Oscuridad , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Gravitropismo/fisiología , Ácidos Indolacéticos/farmacología , Mutación/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Haz Vascular de Plantas/efectos de los fármacos , Haz Vascular de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive. RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response. CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.
Asunto(s)
Autofagia , Membrana Celular/fisiología , Diglicéridos/metabolismo , Homeostasis , Saccharomyces cerevisiae/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Transporte de ProteínasRESUMEN
A germinating seedling undergoes skotomorphogenesis to emerge from the soil and reach for light. During this phase, the cotyledons are closed, and the hypocotyl elongates. Upon exposure to light, the seedling rapidly switches to photomorphogenesis by opening its cotyledons and suppressing hypocotyl elongation. The E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is critical for maintaining skotomorphogenesis. Here, we report that jasmonate (JA) suppresses hypocotyl elongation and stimulates cotyledon opening in etiolated seedlings, partially phenocopying cop1 mutants in the dark. We also find that JA stabilizes several COP1-targeted transcription factors in a COP1-dependent manner. RNA-seq analysis further defines a JA-light co-modulated and cop1-dependent transcriptome, which is enriched for auxin-responsive genes and genes participating in cell wall modification. JA suppresses COP1 activity through at least two distinct mechanisms: decreasing COP1 protein accumulation in the nucleus; and reducing the physical interaction between COP1 and its activator, SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1). Our work reveals that JA suppresses COP1 activity to stabilize COP1 targets, thereby inhibiting hypocotyl elongation and stimulating cotyledon unfolding in etiolated Arabidopsis seedlings.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Ciclopentanos/farmacología , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Oxilipinas/farmacología , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cotiledón/efectos de los fármacos , Cotiledón/crecimiento & desarrollo , Cotiledón/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Hipocótilo/efectos de los fármacos , Plantones/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The soil-borne fungal pathogen Verticillium dahliae infects a wide range of dicotyledonous plants including cotton, tobacco, and Arabidopsis. Among the effector proteins secreted by V. dahliae, the 16 kDa PevD1 induces a hypersensitive response in tobacco. Here we report the high-resolution structure of PevD1 with folds resembling a C2 domain-like structure with a calcium ion bound to the C-terminal acidic pocket. A yeast two-hybrid screen, designed to probe for molecular functions of PevD1, identified Arabidopsis asparagine-rich protein (NRP) as the interacting partner of PevD1. Extending the pathway of V. dahliae effects, which include induction of early flowering in cotton and Arabidopsis, NRP was found to interact with cryptochrome 2 (CRY2), leading to increased cytoplasmic accumulation of CRY2 in a blue light-independent manner. Further physiological and genetic evidence suggests that PevD1 indirectly activates CRY2 by antagonizing NRP functions. The promotion of CRY2-mediated flowering by a fungal effector outlines a novel pathway by which an external stimulus is recognized and transferred in changing a developmental program.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Bacterianas/genética , Criptocromos/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Verticillium/fisiología , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Criptocromos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Alineación de Secuencia , Nicotiana/genética , Nicotiana/metabolismo , Verticillium/genéticaRESUMEN
How the organ size is determined is a fundamental question in developmental biology. The metazoan Hippo signaling pathway is well established to negatively regulate organ sizes. Recent studies in plants have started to shape an emerging Hippo signaling pathway. In this review, we summarize the studies in the past decade on the two known components of plant Hippo signaling pathway, the Ste20/Hippo homolog SIK1, and the MOB1/Mats homolog MOB1, with a focus on their developmental functions. Then we envision future discoveries that may shape a complete Hippo signaling pathway in plants.
Asunto(s)
Proteínas de Plantas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Tamaño de los ÓrganosRESUMEN
COP1-interacting protein 1 (CIP1, At5g41790) was the first reported interacting protein for CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) of Arabidopsis; however its physiological function has remained unknown for two decades. Here we show that CIP1 is a positive regulator of abscisic acid (ABA) response. CIP1 is mainly expressed in the photosynthetic cells and the vascular tissue, and its promoter activity can be induced by osmotic stress and ABA. The CIP1 protein is localized to the plasma membrane. A T-DNA insertion mutant cip1-1 was then characterized. The mutant is sensitive to osmotic stress and has ABA insensitive phenotypes. RNA sequencing showed that cip1-1 has lower levels of gene expression in abiotic stress response compared with the wild-type. Meanwhile, transcript levels of ABA biosynthesis genes are higher in cip1-1 than in the wild-type. These results suggested that CIP1 is positively involved in ABA response.
Asunto(s)
Ácido Abscísico/administración & dosificación , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Fotosíntesis/fisiología , Estrés Fisiológico/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Presión Osmótica/efectos de los fármacos , Presión Osmótica/fisiología , Fotosíntesis/efectos de los fármacos , Distribución Tisular , Ubiquitina-Proteína LigasasRESUMEN
Multicellular organisms co-ordinate cell proliferation and cell expansion to maintain organ growth. In animals, the Hippo tumor suppressor pathway is a master regulator of organ size. Central to this pathway is a kinase cascade composed of Hippo and Warts, and their activating partners Salvador and Mob1/Mats. In plants, the Mob1/Mats homolog MOB1A has been characterized as a regulator of cell proliferation and sporogenesis. Nonetheless, no Hippo homologs have been identified. Here we show that the Arabidopsis serine/threonine kinase 1 (SIK1) is a Hippo homolog, and that it interacts with MOB1A to control organ size. SIK1 complements the function of yeast Ste20 in bud site selection and mitotic exit. The sik1 null mutant is dwarf with reduced cell numbers, endoreduplication, and cell expansion. A yeast two-hybrid screen identified Mob1/Mats homologs MOB1A and MOB1B as SIK1-interacting partners. The interaction between SIK1 and MOB1 was found to be mediated by an N-terminal domain of SIK1 and was further confirmed by bimolecular fluorescence complementation. Interestingly, sik1 mob1a is arrested at the seedling stage, and overexpression of neither SIK1 in mob1a nor MOB1A in sik1 can rescue the dwarf phenotypes, suggesting that SIK1 and MOB1 may be components of a larger protein complex. Our results pave the way for constructing a complete Hippo pathway that controls organ growth in higher plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas Portadoras/química , Recuento de Células , Proteínas de Ciclo Celular/química , Proliferación Celular , Tamaño de la Célula , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Ploidias , Unión Proteica , Dominios Proteicos , Fracciones Subcelulares/metabolismo , Transcripción GenéticaRESUMEN
In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Dinaminas/metabolismo , Retículo Endoplásmico , Proteínas de Unión al GTP/metabolismo , Fusión de Membrana , Desarrollo de la Planta , Eliminación de Gen , Saccharomyces cerevisiae/metabolismoRESUMEN
After germination, cotyledons undertake the major role in supplying nutrients to the pre-photoautorophy angiosperm seedlings until they senesce. Like other senescence processes, cotyledon senescence is a programmed degenerative process. Nitric oxide can induce premature cotyledon senescence in Arabidopsis thaliana, yet the underlying mechanism remains elusive. A screen for genetic mutants identified the nes1 mutant, in which cotyledon senescence was accelerated by nitric oxide. Map-based cloning revealed that NES1 is allelic to a previously reported mitotic checkpoint family gene, MAD1. The nes1/mad1 mutants were restored to the wild type, in response to nitric oxide, by transforming them with pNES1::NES1. Ectopic expression of NES1 in the wild type delayed nitric oxide-mediated cotyledon senescence, confirming the repressive role of NES1. Moreover, two positive regulators of leaf senescence, the ethylene signalling component EIN2 and the transcription factor ORE1/AtNAC2/ANAC092, were found to function during nitric oxide-induced senescence in cotyledons. The block of ORE1 function delayed senescence and ectopic expression induced the process, revealing the positive role of ORE1. EIN2 was required to induce ORE1. Furthermore, the genetic interaction analysis between NES1 and ORE1 showed that the ore1 loss-of-function mutants were epistatic to nes1, suggesting the dominant role of ORE1 and the antagonistic role of NES1 during nitric oxide-induced cotyledon senescence in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Cotiledón/crecimiento & desarrollo , Óxido Nítrico/farmacología , Receptores de Superficie Celular/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Clonación Molecular , Cotiledón/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Mutación/genética , Fenotipo , Plantas Modificadas Genéticamente , Transducción de SeñalRESUMEN
Starvation- or stress-induced phosphatidylinositol 3-phosphate (PtdIns3P/PI3P) production at the endoplasmic reticulum (ER) subdomains organizes phagophore assembly and autophagosome formation. Coat protein complex II (COPII) vesicles budding from ER exit site (ERES) also contribute to autophagosome formation. Whether any PtdIns3P phosphatase functions at ERES to inhibit macroautophagy/autophagy is unknown. Here we report Myotubularin 2 (MTM2) of Arabidopsis as a PtdIns3P phosphatase that localizes to ERES and negatively regulates autophagy. MTM2 binds PtdIns3P with its PH-GRAM domain in vitro and acts toward PtdIns3P in vivo. Transiently expressed MTM2 colocalizes with ATG14b, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex, and overexpression of MTM2 blocks autophagic flux and causes over-accumulation of ATG18a, ATG5, and ATG8a. The mtm2 mutant has higher levels of autophagy and is more tolerant to starvation, whereas MTM2 overexpression leads to reduced autophagy and sensitivity to starvation. The phenotypes of mtm2 are suppressed by ATG2 mutation, suggesting that MTM2 acts upstream of ATG2. Importantly, MTM2 does not affect the endosomal functions of PtdIns3P. Instead, MTM2 specifically colocalizes with COPII coat proteins and is cradled by the ERES-defining protein SEC16. MTM2 interacts with SEC23A with its phosphatase domain and inhibits COPII-mediated protein secretion. Finally, a role for MTM2 in salt stress response is uncovered. mtm2 resembles the halophyte Thellungiella salsuginea in its efficient vacuolar compartmentation of Na+, maintenance of chloroplast integrity, and timely regulation of autophagy-related genes. Our findings reveal a balance between PtdIns3P synthesis and turnover in autophagosome formation, and provide a new link between autophagy and COPII function.
RESUMEN
Unlike animals, plants and yeasts only have a class III phosphatidylinositol 3-kinase (PI3KC3). Its lipid product, phosphatidylinositol 3-phosphate (PtdIns-3-P, PI3P), organizes intracellular trafficking routes such as autophagosome formation, multivesicular body (MVB) formation, retro-transport from trans-Golgi network (TGN) to late Golgi, and the fusion events between autophagosomes and MVBs and the vacuole. The catalytic subunit of plant PI3KC3 is encoded by the essential gene Vacuolar Protein Sorting 34 (VPS34). Despite the importance of VPS34 in cellular homeostasis and plant development, a VPS34 interactome is lacking. Here we employed TurboID, an enzyme-catalyzed proximity labelling (PL) method, to describe a proximal interactome of Arabidopsis VPS34. TurboID catalyzed spatially restricted biotinylation and enabled VPS34-specific enrichment of 273 proteins from affinity purification coupled with mass spectrometry. The interactome confirmed known functions of VPS34 in endo-lysosomal trafficking. Intriguingly, carbohydrate metabolism was the most enriched Gene Ontology (GO) term, including glycolytic enzymes in the triose portion and enzymes functioning in chloroplast triose export and sucrose biosynthesis. The interaction between VPS34 and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1/2) was validated in planta. Also verified was the interaction between VPS34 and the plasma membrane H+-ATPase AHA2, a primary determinant of membrane potential. Our study links PI3KC3 to carbohydrate metabolism and membrane potential, two key processes that maintain cellular homeostasis.
RESUMEN
As the last stage of leaf development, senescence is a fine-tuned process regulated by interplays of multiple signaling pathways. We have previously identified soybean (Glycine max) SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK), a leucine-rich repeat-receptor-like protein kinase from soybean, as a positive regulator of leaf senescence. Here, we report the elucidation of the molecular mechanism of GmSARK-mediated leaf senescence, especially its specific roles in senescence-inducing hormonal pathways. A glucocorticoid-inducible transcription system was used to produce transgenic Arabidopsis (Arabidopsis thaliana) plants for inducible overexpression of GmSARK, which led to early leaf senescence, chloroplast destruction, and abnormal flower morphology in Arabidopsis. Transcript analyses of the GmSARK-overexpressing seedlings revealed a multitude of changes in phytohormone synthesis and signaling, specifically the repression of cytokinin functions and the induction of auxin and ethylene pathways. Inhibition of either auxin action or ethylene biosynthesis alleviated the senescence induced by GmSARK. Consistently, mutation of either AUXIN RESISTANT1 or ETHYLENE INSENSITIVE2 completely reversed the GmSARK-induced senescence. We further identified a homolog of GmSARK with a similar expression pattern in Arabidopsis and named it AtSARK. Inducible overexpression of AtSARK caused precocious senescence and abnormal floral organ development nearly identical to the GmSARK-overexpressing plants, whereas a T-DNA insertion mutant of AtSARK showed significantly delayed senescence. A kinase assay on recombinant catalytic domains of GmSARK and AtSARK revealed that these two leucine-rich repeat-receptor-like protein kinases autophosphorylate on both serine/threonine and tyrosine residues. We inferred that the SARK-mediated pathway may be a widespread mechanism in regulating leaf senescence.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Glycine max/enzimología , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Citocininas/metabolismo , Etilenos/metabolismo , Flores/efectos de los fármacos , Flores/enzimología , Flores/genética , Flores/fisiología , Glucocorticoides/farmacología , Ácidos Indolacéticos/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Análisis de Secuencia de ADN , Transducción de Señal/fisiología , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/genética , Factores de TiempoRESUMEN
Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Ligasas/metabolismo , Fosfatos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Ligasas/genética , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatos/deficiencia , Ftalimidas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/enzimología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , ARN de Planta/genética , Plantones/crecimiento & desarrolloRESUMEN
The Arabidopsis Serine/Threonine Kinase 1 (SIK1) is a Sterile 20 (STE20)/Hippo orthologue that is also categorized as a Mitogen-Activated Protein Kinase Kinase Kinase Kinase (MAP4K). Like its animal and fungi orthologues, SIK1 is required for cell cycle exit, cell expansion, polarity establishment, as well as pathogenic response. The catalytic activity of SIK1, like other MAPKs, is presumably regulated by its phosphorylation states. Since no crystal structure for SIK1 has been reported yet, we built structural models for SIK1 kinase domain in different phosphorylation states with different pocket conformation to see how this kinase may be regulated. Using computational structural biology methods, we outlined a conduction path in which a phosphorylation site on the A-loop regulates the catalytic activity of SIK1 by controlling the closing or opening of the catalytic pocket at the G-loop. Furthermore, with analyses on the dynamic motions and in vitro kinase assay, we confirmed that three key residues in this conduction path, Lys278, Glu295, and Arg370, are indeed important for the kinase activity of SIK1. Since these residues are conserved in all STE20 kinases examined, the regulatory mechanism that we discovered may be common in STE20 kinases.
RESUMEN
Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice (Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.
RESUMEN
The phytohormones ethylene and abscisic acid (ABA) play essential roles in the abiotic stress adaptation of plants, with both cross-talk of ethylene signalling and ABA biosynthesis and signalling reported. Any reciprocal effects on each other's biosynthesis, however, remain elusive. ACC synthase (ACS) acts as the key enzyme in ethylene biosynthesis. A pilot study on changes in ACS promoter activities in response to abiotic stresses revealed the unique involvement in abiotic stress responses of the only type 3 ACC synthase, ACS7, among all nine ACSs of Arabidopsis. Hence an acs7 mutant was characterized and its abiotic stress responses were analysed. The acs7 mutant germinated slightly faster than the wild type and subsequently maintained a higher growth rate at the vegetative growth stage. Ethylene emission of acs7 was merely one-third of that of the wild type. acs7 exhibited enhanced tolerance to salt, osmotic, and heat stresses. Furthermore, acs7 seeds were hypersensitive to both ABA and glucose during germination. Transcript analyses revealed that acs7 had elevated transcript levels of the stress-responsive genes involved in the ABA-dependent pathway under salt stress. The ABA level was also higher in acs7 following salt treatment. Our data suggest that ACS7 acts as a negative regulator of ABA sensitivity and accumulation under stress and appears as a node in the cross-talk between ethylene and ABA.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Liasas/genética , Mutación , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Etilenos , Regulación de la Expresión Génica de las Plantas , Liasas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Estrés FisiológicoRESUMEN
The endosymbiont-derived Sec-dependent protein sorting pathway is essential for protein import into the thylakoid lumen and is important for the proper functioning of the chloroplast. Two loss-of-function mutants of cpSecA, the ATPase subunit of the chloroplast Sec translocation machinery, were analysed in Arabidopsis. The homozygous mutants were albino and seedling lethal under autotrophic conditions and remained dwarf and infertile with an exogenous carbon supply. They were subject to oxidative stress and accumulated superoxide under normal lighting conditions. Electron microscopy revealed that the chloroplast of the mutants had underdeveloped thylakoid structures. Histochemical GUS assay of the AtcpSecA::GUS transgenic plants confirmed that AtcpSecA was expressed in green organs in a light-inducible way. Real-time RT-PCR and microarray analysis revealed repressed transcription of nucleus- and chloroplast- encoded subunits of photosynthetic complexes, and induced transcription of chloroplast protein translocation machinery and mitochondrion-encoded respiratory complexes in the mutants. It is inferred that AtcpSecA plays an essential role in chloroplast biogenesis, the absence of which triggered a retrograde signal, eventually leading to a reprogramming of chloroplast and mitochondrial gene expression.