The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6^DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6^DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6^DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6^DN npr1 and rop6^DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6^DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6^DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6^DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.Rho of Plants (ROPs), also known as RACs (for clarity, the ROP nomenclature will be used throughout this article), comprise a plant-specific group of Rho family small G proteins. Like other members of the Ras superfamily of small G proteins, ROPs function as molecular switches, existing in a GTP-bound “on” state and a GDP-bound “off” state. In the GTP-bound state, ROPs interact with specific effectors that transduce downstream signaling or function as scaffolds for interaction with additional effector molecules (Berken and Wittinghofer, 2008). Conserved point mutations in the G1 (P loop) Gly-15 or the G3 (switch II) Gln-64, which abolish GTP hydrolysis, or the G1 Thr-20 or G4 Asp-121 that compromise GDP/GTP exchange, can form either constitutively active or dominant negative mutants, respectively (Feig, 1999; Berken et al., 2005; Berken and Wittinghofer, 2008; Sorek et al., 2010). Primarily based on studies with neomorphic mutants, ROPs have been implicated in the regulation of cytoskeleton organization and dynamics, vesicle trafficking, auxin transport and response, abscisic acid (ABA) response, and response to pathogens (Nibau et al., 2006; Yalovsky et al., 2008; Yang, 2008; Lorek et al., 2010; Wu et al., 2011; and refs. therein).In Arabidopsis (Arabidopsis thaliana), there are 11 ROP proteins (Winge et al., 1997). Assigning specific functions to individual members of this family is difficult, however, because ROPs are functionally redundant. A ROP10 loss-of-function mutant was reported to be ABA hypersensitive (Zheng et al., 2002), displaying enhanced expression of tens of genes in response to ABA treatments (Xin et al., 2005). However, in the absence of exogenous ABA, gene expression in the rop10 mutant was similar to that in wild-type plants (Xin et al., 2005). Loss of leaf epidermis pavement cell polarity was reported for rop4 rop2-RNAi (for RNA interference) double mutant plants (Fu et al., 2005). Mild changes in pavement and hypocotyl cell structure and microtubule (MT) organization were reported for a rop6 loss-of-function mutant (Fu et al., 2009).The involvement of ROPs in auxin-regulated development has been addressed in several studies (Wu et al., 2011). Ectopic expression of a dominant negative ROP2 (rop2^DN) mutant under regulation of the 35S promoter resulted in a loss of apical dominance and a reduction in the number of lateral roots. In contrast, ectopic expression of constitutively active ROP2 (rop2^CA) caused an increase in the number of lateral roots and an enhanced decrease in primary root length in response to auxin. Consistent with these findings, the expression of a constitutively active NtRAC1 in tobacco (Nicotiana tabacum) protoplasts induced the expression of auxin-regulated genes in the absence of auxin and promoted the formation of protein nuclear bodies containing components of the proteasome and COP9 signalosome (Tao et al., 2002, 2005; Wu et al., 2011). The ROP effector ICR1 (for interactor of constitutively active ROP1) regulates polarized secretion and is required for polar auxin transport (Lavy et al., 2007; Bloch et al., 2008; Hazak et al., 2010; Hazak and Yalovsky, 2010). In the root, local auxin gradients induce the accumulation of ROPs in trichoblasts at the site of future root hair formation (Fischer et al., 2006). Recently, it was shown that interdigitation of leaf epidermis pavement cells depends on Auxin-Binding Protein1 (ABP1)-mediated ROP activation (Xu et al., 2010). Taken together, these data indicate that ROPs are involved in both mediating the auxin response and facilitating directional auxin transport. It is still unclear, however, which ROPs function in these processes.ROP function was linked to plant defense responses in several studies. In rice (Oryza sativa), OsRAC1 is a positive regulator of the hypersensitive response, possibly through interactions with the NADPH oxidase RbohB, Required for Mla12 Resistance, and Heat Shock Protein90 (Ono et al., 2001; Thao et al., 2007; Wong et al., 2007). Interestingly, other members of the rice ROP family, namely RAC4 and RAC5, are negative regulators of resistance to the rice blast pathogen Magnaporthe grisea (Chen et al., 2010). Similar to rice, when expressed in tobacco, dominant negative OsRAC1 suppressed the hypersensitive response (Moeder et al., 2005). In barley (Hordeum vulgare), several constitutively active ROP/RAC mutants and a MT-associated ROPGAP1 loss-of-function mutant enhanced susceptibility to the powdery mildew Blumeria graminis f. sp. hordei (Bgh). The activated ROP-enhanced susceptibility to Bgh was attributed to disorganization of the actin cytoskeleton and was shown to depend on Mildew Resistance Locus O (MLO; Schultheiss et al., 2002, 2003; Opalski et al., 2005; Hoefle et al., 2011). In barley, three ROP proteins, HvRACB, HvRAC1, and HvRAC3, were linked to both development and pathogen response (Schultheiss et al., 2005; Pathuri et al., 2008; Hoefle et al., 2011).We have analyzed the function of the Arabidopsis AtROP6 (ROP6) by characterizing its expression pattern and its regulation by auxin and the phenotype of plants that express rop6^DN under the regulation of its endogenous promoter. The utilization of the dominant negative mutant overcame functional redundancy, while expression under the regulation of the endogenous promoter enabled the analysis of ROP6 function in a developmental context. Phenotypic and gene expression analyses indicate that ROP6 functions in developmental, salicylic acid (SA)-dependent, and SA-independent defense response pathways.     

Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

Short-term calorie restriction enhances skeletal muscle stem cell function

Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.     

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction  

Interleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4⁺ and TCRγδ⁺ T cells was evaluated systemically as well as at the site of inflammation.     

KFERQ sequence in ribonuclease A-mediated cytotoxicity.

Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.