Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll （Chl） degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 （SGR1） interacts with Chl catabolic enzymes （CCEs） and light-harvesting complex II （LHCII） at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 （At4g22920）, the Arabidopsis thaliana genome contains an additional homolog, SGR2 （At4g11910）, whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulat- ing Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.     

The Role of Endogenous Abscisic Acid in the Correlation Between the Cotyledon and Its Axillary Bud in PeaPisum sativum L

On cutting off one cotyledon from decapitated pea seedlings cultivated in the dark, the apical dominance is restored, as is well-known, by the growth of the bud of the removed cotyledon. As early as 12 h following cotyledon amputation(i.e. at the time when buds of both cotyledons-remaining and removed-are not yet differentiated in size), a decrease in the level of endogenous abscisic acid can be demonstrated in the bud of the removed cotyledon.     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

Phosphatidylinositol 3-Kinase Is a Negative Regulator of Cellular Differentiation

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor–induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.     

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

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Highlights? Loss of AMPKα1 cooperates with the Myc oncogene to accelerate lymphomagenesis ? AMPKα dysfunction enhances aerobic glycolysis (Warburg effect) ? Inhibiting HIF-1α reverses the metabolic effects of AMPKα loss ? HIF-1α mediates the growth advantage of tumors with reduced AMPK signaling     

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23^−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.     

Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

Sec22b Is a Negative Regulator of Phagocytosis in Macrophages

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.     

Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth But Not of Photosynthesis in Arabidopsis

Mitogen-activated protein kinase （MAPK） pathways regulate signal transduction from different cellular com- partments and from the extracellular environment to the nucleus in all eukaryotes. One of the best-characterized MAPKs in Arabidopsis thaliana is MPK4, which was shown to be a negative regulator of systemic-acquired resistance. The mpk4 mutant accumulates salicylic acid （SA）, possesses constitutive expression of pathogenesis-related （PR） genes, and has an extremely dwarf phenotype. We show that suppression of SA and phylloquinone synthesis in chloroplasts by knocking down the IC51 gene （by crossing it with the icsl mutant） in the mpk4 mutant background did not revert mpk4-impaired growth. However, it did cause changes in the photosynthetic apparatus and severely impaired the quantum yield of pho- tosystem Ih Transmission microscopy analysis revealed that the chloroplasts＇ structure was strongly altered in the mpk4 and mpk4/icsl double mutant. Analysis of reactive oxygen species （ROS）-scavenging enzymes expression showed that suppression of SA and phylloquinone synthesis in the chloroplasts of the mpk4 mutant caused imbalances in ROS homeo- stasis which were more pronounced in mpk4/icsl than in mpk4. Taken together, the presented results strongly suggest that MPK4 is an ROS/hormonal rheostat hub that negatively, in an SA-dependent manner, regulates immune defenses, but at the same time positively regulates photosynthesis, ROS metabolism, and growth. Therefore, we concluded that MPK4 is a complex regulator of chloroplastic retrograde signaling for photosynthesis, growth, and immune defenses in Arabidopsis.     

Abscisic Acid Represses Growth of the Arabidopsis Embryonic Axis after Germination by Enhancing Auxin Signaling

Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA–auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.     

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.