text: Oct 03, 2022 · Stacker compiled a list of dogs available for adoption in Lincoln, Nebraska on Petfinder, ranging in age, breed composition, temperament, and needs. It's important to note that pet adoption is a huge responsibility and requires available savings for emergency and routine veterinary care, a day-to-day routine and game plan for coverage if the ... Oct 25, 2022 · The RuFF Rescue has them ready for those eager to bring them home. They also welcome other small dogs they found around the area of Lincoln. Their main tasks consist of rescuing, rehabilitating, and rehoming the "highlander kids", as they call these dogs. Heartland German Shepherd Rescue (HUGS) is a Nebraska-licensed, all-volunteer, 501c3 non-profit dog rescue organization founded May 7th, 2015. pretty brown dancers Adoptable Dogs. This page is updated in real-time based on every dog's current status.Each of our centres has a large and secure field which you and your pooch can hire - perfect to practise your dog's recall and enjoy stress free walkies and playtime together. 78p In every £1 donated is spent directly on caring for our rescue dogs 551 Dogs rehomed in 2019 47 The average amount of days a dog stays in rescue Happy tails Alfie the marc apartments for rent Find Rescue Dogs in Lincoln on Petlist Page 2, search 1000's of Dogs in your local area. Choose from hundreds of different Dog breeds including: Beagles, Bichon Frise, Border Terriers, Boxers, Cavalier King Charles Spaniels, Chihuahuas, Cocker Spaniels, Doberman Pinschers, English Springer Spaniels, Labrador Retrievers, Lhasa Apso, Pugs, Staffordshire Bull Terriers, Yorkshire Terriers expedite load board promo code One More Dog Rescue PO Box 1292, Lincoln, CA 95648 Contact Marcy Lopez Email [email protected] Phone (916) 960-8561 Website http://www.onemoredogrescue.com cats & dogs at One More Dog Rescue Pet Type Any Age Any Sex Any Mocha Winnie Wallace Local Chesapeake Bay Retriever Female, 9 mos Warwick, RI Daisy Taylor Chihuahua/Mixed Breed (Small)How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE.Aprox. 32.4 mi from Lincoln. Tags: Mutt Dogs for adoption in Gretna, NE, USA. German Shepherd Dog-Unknown Mix Dog for Adoption near Nebraska, Gretna, USA. ADN-1033912. Rose (nka Ruby Begonia) - Spring Litter - German Shepherd Dog / Mixed Dog For Adoption.Oct 25, 2022 · How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE. st charles lab locationsOct 25, 2022 · How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE. Brave Animal Rescue Lincoln, NE view our pets [email protected] Our Mission Brave Animal Rescue is dedicated to protecting vulnerable and abandoned animals through rescue, compassionate care, and advocacy. Adoption Policy The first step is completing the adoption application. pink bodycon dress midi WebWebRevolution Rescue is a volunteer & foster-based company out of Lincoln, NE, and is a registered 501(c)3 non-profit group. Mission Statement It is Revolution Rescue's sole drive and mission to help prevent cruelty to animals by providing exceptional foster care and re-homing services for homeless dogs and cats, concentrating on those who have ... WebSearch for dogs for adoption at shelters near Lincoln, CA. Find and adopt a pet on Petfinder today."Animal rescue organizations are responsible for providing the basic needs for housing, nutrition, and health of all animals in their care. The quality of life of the animals must be a priority,"... cummins 111 fault code Abraham Lincoln, the 16th president of the United States, was born in Hodgenville, Kentucky, on Feb. 12, 1809. Following a career in politics, which saw him help preserve the union during the Civil WaEstablished in 1985, we serve Lincoln County and the city of Fayetteville in Tennessee by ensuring proper care and humane treatment of all animals.Animal Control is located at 3131 O Street which is in the Lincoln Lancaster County ... What constitutes a "potentially dangerous" dog and a "dangerous dog? sefton street liverpool car dealers Find Rescue Dogs in Lincoln on Petlist Page 2, search 1000's of Dogs in your local area. Choose from hundreds of different Dog breeds including: Beagles, Bichon Frise, Border Terriers, Boxers, Cavalier King Charles Spaniels, Chihuahuas, Cocker Spaniels, Doberman Pinschers, English Springer Spaniels, Labrador Retrievers, Lhasa Apso, Pugs, Staffordshire Bull Terriers, Yorkshire Terriers capnproto vs flatbuffers benchmark ... the Humane Society of Lincoln County has worked to protect and care for the abandoned pets in our community. In 2020 we have moved into our new shelter ...5 Dolly's Legacy Animal Rescue is a 501c3 non-profit, foster home based rescue in Lincoln, NE. Our mission is to save orphaned and abandoned pets from overcrowded shelters and find them loving, forever homes. Adoptable Pets We have many deserving pets waiting for forever homes! Find a New Best Friend. Foster Fosters are essential to saving lives.Find Rescue Dogs in Lincolnshire on Petlist, search 1000's of Dogs in your local area. Choose from hundreds of different Dog breeds including: Beagles, Bichon Frise, Border Terriers, Boxers, Cavalier King Charles Spaniels, Chihuahuas, Cocker Spaniels, Doberman Pinschers, English Springer Spaniels, Labrador Retrievers, Lhasa Apso, Pugs, Staffordshire Bull Terriers, Yorkshire Terriers derek degrate Stacker compiled a list of dogs available for adoption in Lincoln, Nebraska on Petfinder, ranging in age, breed composition, temperament, and needs. It's important to note that pet adoption is a huge responsibility and requires available savings for emergency and routine veterinary care, a day-to-day routine and game plan for coverage if the ...... quality shelter, sanctuary, and adoptions, and by promoting animal welfare issues. Our efforts have resulted in thousands of dog and cat adoptions since ...Provide foster homes when a pet needs a safe and loving place to stay until they find their forever home. Focus on helping owners keep their own dogs by offering education and training to resolve behavioral issues. Offer assistance when owners post their pet (s) on social media sites. rogers ottawa WebOct 03, 2022 · Stacker compiled a list of dogs available for adoption in Lincoln, Nebraska on Petfinder, ranging in age, breed composition, temperament, and needs. It's important to note that pet adoption is a huge responsibility and requires available savings for emergency and routine veterinary care, a day-to-day routine and game plan for coverage if the ... There is an ongoing need for protection of vulnerable animals and education to society in general. Shelter Address Lincoln, NE Phone Contact Name Faith Jones Contact eMail [email protected] Website https://sites.google.com/site/ruffrescueinc/ Pet types Dogs Location served Adoption ProcessAbraham Lincoln's parents had two other children. Sarah was Abraham's older sister and Thomas was his younger brother, but he died in infancy. Abraham's parents were Thomas Lincoln and Nancy Hanks Lin desmos boxplot and histogram This week is the best opportunity ever to search for dog adoption near you in Lincoln, Maine. The Alpha Paw dog rescue center provides both nearby animal shelters close to Lincoln, Maine. All of our dogs for adoption near. We provide all puppies & dogs via our Pet Finder as well as pets SPCA and dogs that were brought into the Humane Society. Web stock option news Lincoln County Animal Shelter 510 NE Harney St. Newport, OR 97365. phone: 541-265-0720 fax: 541-574-0448 [email protected] Pet licensing: DocuPet The Lincoln County Humane Society is dedicated to the prevention of cruelty and abuse to animals, birds and wildlife. Whether you're bringing in a lost pet, ... molina healthcare nevada Webis a foster home based rescue in Lincoln, NE. Our mission is to save orphaned and abandoned pets from overcrowded shelters and find them loving, forever homes. Dolly's Legacy Animal Rescue's mission is to save orphaned and abandoned pets from overcrowded shelters where they are out of time & out of hope to make it out alive. Below are all of the organizations in Nebraska from which you can adopt a dog. Please continue on to each individual organization's website by clicking on their name to see dogs they have available for adoption or to learn more about the organization and their mission. Adopting is a great way of bringing home a dog in need of a loving home. seher dilovan Boarding for Cats & Dogs. ... range of roles . Read More. Head Office. Margaret Green Animal Rescue Church Knowle, Wareham, Dorset BH20 5NQ [email protected] Registered Charity Number: 1167990. Licence holder: Margaret Green Animal Rescue Church Knowle, Cat Boarding Licence issued by Dorset Council: licence number: 936. Lincoln Farm ...The Lincoln County Humane Society is dedicated to the prevention of cruelty and abuse to animals, birds and wildlife. Whether you're bringing in a lost pet, ...Web goldman sachs compliance associate salary Lincoln County Animal Shelter 510 NE Harney St. Newport, OR 97365. phone: 541-265-0720 fax: 541-574-0448 [email protected] Pet licensing: DocuPetAdoption fees vary depending on the type and age of the animal. Note: an administration fee of $5 or 5% (whichever is greater) will apply to all adoption fees upon checkout. Adult Cat – $99 Kitten (6 months or younger) – $150 Adult Dog (greater than 25 lbs) – $450 Puppy (less than 1 year) – $550 Small Adult Dog (25 lbs or under) – $550 Bunny – $35Abraham Lincoln had several professions over his lifetime, but most notably, he was the 16th president of the United States of America. Elected in 1860, Lincoln led the country through the American Ci damage contribution poke genie Adoption - Adoption Agencies If you are interested in adopting a pet, ... Family Addition Dog Rescue of Charlotte ... Lincoln County Humane Society ... uk drill stabbings video Bert is a male Lhasa Apso, weighs 25 lbs and was born approximately 12-17-14. He is neutered, vaccinated, microchipped, given flea/tick preventative, dewormed, tested negative for heartworm disease & started on preventative. Adoption fee is $50 to help offset the many expenses we have for vetting costs. Apr 26, 2021 ... Come meet adoptable dogs at the Lincolnton Petsmart, 10:30am –[...] Nov. 12. Sat · 10:30 am ...Revolution Rescue is a volunteer & foster-based company out of Lincoln, NE, and is a registered 501(c)3 non-profit group. Mission Statement It is Revolution Rescue's sole drive and mission to help prevent cruelty to animals by providing exceptional foster care and re-homing services for homeless dogs and cats, concentrating on those who have ... wordle hints reddit Search for a pet, rescue group or article Go. E.g. enter a pet's id into the search. E.g. enter a pet's name into the search. E.g. enter a rescue groups's name. E.g. enter in a keyword to find an article. ... RSPCA Port Lincoln Shelter hasn't added a link to donate online. Please contact RSPCA Port Lincoln Shelter directly to make a donation.WebHow to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE.1935 Q St Ste 1. Lincoln, NE 68503. 6. American Society Prevention of Cruelty to Animals - ASPCA. Social Service Organizations Community Organizations Humane Societies. (1) Website. linear interpolation How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE.In fact, in most cases, the adoption fee falls short of the actual medical expenses spent on our dogs and cats. The average amount we spend on a healthy rescued animal is $200-$250, but it could easily cost Revolution Rescue as much as $2,000 (or more) depending on any medical issues. Revolution Rescue depends solely on public donations. ** topps metazoo Sometimes your dog just can't go with you. At our North Lincolnshire boarding kennels your dog will experience the highest levels of care day and night.Animal Control is located at 3131 O Street which is in the Lincoln Lancaster County ... What constitutes a "potentially dangerous" dog and a "dangerous dog? 1 bedroom flat dss accepted birmingham WebR.u.F.F. Rescue, Inc. emerged from the desire of several community individuals that saw the need in Lincoln and its surrounding communities to provide a rescue for the "Highlander Kids" (meaning Westies, Cairns, and Scotties) plus other small dogs within the Lincoln area. There is an ongoing need for protection of vulnerable animals and ... Dolly's Legacy Animal Rescue is a non-profit, foster home based rescue in Lincoln, NE. We save orphaned & abandoned pets & find loving, forever homes. CONTACT: [email protected] Dolly's Legacy Animal Rescue P.O. Box 23122 Lincoln, NE 68542 Dolly's Legacy Animal Rescue's adopted pets See Adopted Pets list could not connect to smtp host smtp office365 com port 587Oct 03, 2022 · Dogs available for adoption in Lincoln When looking at the makeup of American families today, it's rare to see a home where a pet isn't part of the family. According to the most recent data from the APPA National Pet Owners Survey, 67% of households, or around 85 million homes, own a pet. Bert is a male Lhasa Apso, weighs 25 lbs and was born approximately 12-17-14. He is neutered, vaccinated, microchipped, given flea/tick preventative, dewormed, tested negative for heartworm disease & started on preventative. Adoption fee is $50 to help offset the many expenses we have for vetting costs. 2 bed property for sale fife Search for dogs for adoption at shelters near Lincoln, CA. Find and adopt a pet on Petfinder today. Oct 03, 2022 · Dogs available for adoption in Lincoln When looking at the makeup of American families today, it's rare to see a home where a pet isn't part of the family. According to the most recent data from the APPA National Pet Owners Survey, 67% of households, or around 85 million homes, own a pet. Lincoln County Animal Shelter 510 NE Harney St. Newport, OR 97365. phone: 541-265-0720 fax: 541-574-0448 [email protected] Pet licensing: DocuPet Lamb Chop is a Mixed Breed rescue dog for adoption in Lincoln, California. She is housed at Grubby Paws Rescue. regrow teeth and gums Find a Puppy · AKC Rescue Network · PuppyVisor Search Counselor ... Barn Hunt · Disc Dog · Diving Dogs · Flyball · Search & Rescue ... Limerick; Lincoln ...Pictures of dogs and cats who need a home. Search by breed, age, size and color. Adopt a dog, Adopt a cat. ... Dolly's Legacy Animal Rescue. P.O. Box 23122, Lincoln ...Web omni mae acrylic enamel Help An Animal ... Rescuing, Rehabilitating and Rehoming animals is not cheap…but it's the right thing to do. Help us fund lifesaving treatments and programs.How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE.Web the holmes building floor plans ... the Humane Society of Lincoln County has worked to protect and care for the abandoned pets in our community. In 2020 we have moved into our new shelter ...Midcoast Humane has committed to helping Bandit-O, Dieg-O, and Jackie-O, dogs who were brought to the Sumpter Humane Society in Georgia, a shelter that we know ...By submitting this form, you are consenting to receive marketing emails from: Boxer Rescue LA, 17514 Ventura Blvd, Encino, CA, 91316-3837, http://www.boxer-rescue-la.com. yamaha flush adapter Stacker compiled a list of dogs available for adoption in Lincoln, Nebraska on Petfinder, ranging in age, breed composition, temperament, and needs.Web the cat bandit Bert is a male Lhasa Apso, weighs 25 lbs and was born approximately 12-17-14. He is neutered, vaccinated, microchipped, given flea/tick preventative, dewormed, tested negative for heartworm disease & started on preventative. Adoption fee is $50 to help offset the many expenses we have for vetting costs.Complete our Adoption Agreement (Onsite or Printable Form Below) Adoption fee of $85.00 for dogs, and $20.00 for cats - unless otherwise indicated by one of our special adoption events. (Payments can be made with cash, local checks or credit/debit card. We accept Discover, Visa, Mastercard, Diners Club International or JCB)R.u.F.F. Rescue, Inc. emerged from the desire of several community individuals that saw the need in Lincoln and its surrounding communities to provide a rescue for the "Highlander Kids" (meaning Westies, Cairns, and Scotties) plus other small dogs within the Lincoln area. There is an ongoing need for protection of vulnerable animals and ... AT THE LICKING COUNTY HUMANE SOCIETY WE SAVE LIVES ; 49150456. Dog Female/Spayed Boxer/Terrier, American Pit Bull 9 years 11 months. Sponsor Bell ; 50532429. DogSearch for dogs for adoption at shelters near Lincoln, CA. Find and adopt a pet on Petfinder today. part time job in kuwait WebLincolnshire: Grimsby rehoming service ... Our Home Direct Scheme is for dogs, cats, rabbits and other small pets whose owners can sadly no longer keep them ... 1934 ford steel body for sale Find Rescue Dogs in Lincoln on Petlist, search 1000's of Dogs in your local area. Choose from hundreds of different Dog breeds including: Beagles, Bichon Frise, Border Terriers, Boxers, Cavalier King Charles Spaniels, Chihuahuas, Cocker Spaniels, Doberman Pinschers, English Springer Spaniels, Labrador Retrievers, Lhasa Apso, Pugs, Staffordshire Bull Terriers, Yorkshire Terriers Help find my dog. To check if we have your dog at our centre, please contact us directly on 01443 226659 or email [email protected] You can also check on our Facebook page. A Huge Puppy Farm Intake. What we do. Since 2005 Hope Rescue has been saving the lives of stray, abandoned and unwanted dogs that, through no fault of their own, need a ...Lamb Chop is a Mixed Breed rescue dog for adoption in Lincoln, California. She is housed at Grubby Paws Rescue. antique armchairs gumtree The Lincoln County Humane Society is dedicated to the prevention of cruelty and abuse to animals, birds and wildlife. Whether you're bringing in a lost pet, ...These companies are located in Lincoln CA, Princeton NJ, and Ypsilanti MI. SMALL DOG RESCUE, INC. CALIFORNIA NON-PROFIT CORPORATION - CA - PUBLIC BENEFIT. WRITE REVIEW. Address: 1146 Barnswood Loop. Lincoln, CA 95648. Registered Agent:WebSometimes your dog just can't go with you. At our North Lincolnshire boarding kennels your dog will experience the highest levels of care day and night. waterford armed robbery How to contact them: Website: Dolly's Legacy Animal Rescue. Mail Address: P.O. Box 23122, Lincoln, NE 68542. Phone: 402-499-2747. Email: [email protected] You may also reach out through their social media: Facebook and Twitter. 2. Brave Animal Rescue. Brave Animal Rescue is another volunteer-run, foster-based rescue in Lincoln, NE.Our adoption fee is $400-450 for puppies 7 months and up and dogs. The fee covers the cost of all medical care including all legally required inoculations, a fecal test and deworming (if required), spaying or neutering, and a microchip. Lifetime training is also included.Thomas Lincoln, Abraham's father, elected to leave the dog, but Abraham jumped into the icy river, waded to shore, and rescued the forgotten dog. When berated, he told his father: "The dog feels better, and except for cold feet, so do I." This theme of saving and protecting animals would remain constant throughout Lincoln's life.Dec 16, 2016 ... Broken Tail Foster & Rescue operates inside a grooming and pet supply store called Oh My Dog in Lincoln. If they don't pay off their $63,000 ... acc 2022 heart failure guidelines One More Dog Rescue. PO Box 1292, Lincoln, CA 95648 Contact Marcy Lopez. Email [email protected] Phone (916) 960-8561. ... One More Dog Rescue's adopted pets. See Adopted Pets list. Search and see photos of adoptable pets in Lincoln, CA area. Find a pet to adopt. Zip/Postal or City, State ... haunted ohio preble county WebThe Alpha Paw dog rescue center provides both nearby animal shelters close to Lincoln County, North Carolina. All of our dogs for adoption near. We provide all new born puppies and dogs via our Pet Finder as well as pets SPCA and dogs that were brought into the Humane Society. Nothing is greater than to give a new dog a loving home and when you ... how much does tradealgo cost New Leash On Life Rescue is a 501 (c)(3) non-profit that works to reduce pet overpopulation, keep animals out of shelters, and improve quality of life for pets and pet owners in need. We assist residents of Lincoln County, MO when facing financial hardship to help cover the cost of their pet's medical care and basic needs.Stacker compiled a list of dogs available for adoption in Lincoln, Nebraska on Petfinder, ranging in age, breed composition, temperament, and needs.WebWeb mt103 vs mt202


text:
import React from "react"
import FalcorController from "lib/falcor/FalcorController"

// create a curried createElement that injects a
// falcor model instance into each of the falcon controllers
// also passes a flag that lets the FalcorController know
// if its a server element or not
export function injectModelCreateElement(model) {
  return (Component, props) => {
    if (Component.prototype instanceof FalcorController) {
      return <Component model={model} {...props} />
    } else {
      return <Component {...props} />
    }
  }
}

// Turns to true on the client after the first render is entirely
// completed. Should be set only by the toplevel appController
// after it has mounted
let appReady = false

export function isAppReady() {
  return appReady
}

export function setAppReady() {
  console.log("APP SET AS READY")
  appReady = true
}

redpajama_set_name: RedPajamaGithub


King Mohammed VI, accompanied by Crown Prince Moulay El Hassan, was at the front row of the world leaders who attended the ceremony held Sunday in Paris to mark the centenary of the Armistice that ended World War I.
The King and the heir apparent sat next to US President Donald Trump & the First Lady Melania, German Chancellor Angela Merkel, French President Emmanuel Macron & his wife Brigitte and President Vladimir Putin of Russia.
According to some analysts, this eloquent image shows Morocco's status on international scene as one of the major global peace-makers and contributors, playing a key role for the world stability.
The participation of the Moroccan Sovereign in this historical event reflects the strong friendship ties existing Morocco and France. It pays tribute to the thousands of fallen Moroccan soldiers who fought for freedom and peace during World War I.
It also sends a strong message to the international community that Morocco is deeply committed to world peace & stability, multilateralism and universal values of democracy, freedom and equality.
The commemoration of Armistice Day honors and pays respect to all the fallen soldiers who participated in this bloody war regardless of their country of origin, color, or faith for the triumph of these universal ideals.
The event features speeches, concerts, parades, a luncheon for world leaders and the opening of the Paris Peace Forum, a global gathering of leaders and civil society dedicated to international cooperation and multilateralism.
Organized by the French Ministry of Foreign Affairs and several think tanks, the three-day forum will look into several issues related to international cooperation and world peace.
Posted by Hafid El Fassy on November 11, 2018. Filed under Columns, News, Zoom. You can follow any responses to this entry through the RSS 2.0. Both comments and pings are currently closed.


Hi I'm Gary Hart a professional driving instructor based in Westhoughton, Bolton covering the Leigh area. I am CRB checked, Driving Standards Agency (DSA) approved and a highly experienced driver. No two learner drivers are alike. Whether you are an ambitious 17 year old or an apprehensive 50 year old, my training methods are tailored to your individual requirements. I will guide you through all the stages of learning to drive from the theory test right through to your actual driving test. My aim is to teach you all the required knowledge and information to give you the best possible chance of passing your driving test first time. As a former police advanced driver you can be assured that I have the necessary skills and expertise to help you successfully pass your driving test. Call 01942 859260 for more information or to book driving lessons.


text:
A Humát al-Hima (حماة الحمى, "A menedékhely védelmezői") Tunézia nemzeti himnusza. A szöveget Musztafa Szádik er-Ráfii (1880–1937) és Abu l-Kászem es-Sábbi (1909–1934) írta, a dallamot pedig Mohammed Abd el-Vahháb (1915–1991) szerezte. A himnusz 1987-től lett hivatalos.

Szövege

Jegyzetek

Tunézia
Nemzeti himnuszok

redpajama_set_name: RedPajamaWikipedia


With all due respect to OSHA, they never actually said how far from a structure to place the generator. Being a Volunteer firefighter for 22 years and having responded to 911 calls for this exact type of incident I recommend making sure the generator and it's enclosure/cover/canopy are placed at least 20-30 feet from any structure that is is being run around. This allows for wind direction to dissipate the exhaust before actually reaching the structure. Any closer and you might be taking a risk of CO poisoning.


text:
Cast announced for Jose Rivera's MARISOL from Promethean Theatre Ensemble
Promethean Theatre Ensemble's twelfth season will open in October with Jose Rivera's MARISOL – an absurdist comedy in which a young New York City woman of Puerto Rican descent finds herself immersed in a battle led by angels to save the universe from a senile and ineffectual God. Marisol Perez is a copy editor for a Manhattan publisher. Though she is a white-collar worker, she still chooses to live alone in the tough section of the Bronx where she grew up. The play opens with Marisol barely escaping a vicious attack on her subway trip back to the Bronx. Later that evening, Marisol's guardian angel pays a visit to tell her she can no longer serve as Marisol's protector because she has been called to join the revolution already in progress against an old and senile God who is dying and "taking the rest of the universe with him." The angel warns Marisol that she will face many threats over the coming days and advises her to be prepared to defend herself. Soon, Marisol is caught up in the Armageddon being fought on the streets of New York City.
Director Juan Castañeda, former artistic director of Chicago's UrbanTheater Company, announced his cast for the production today. Leading the cast will be Rosie Ramos, playing Marisol. She'll be joined by Jazzma Pryor as the Angel, Promethean artistic associate Megan DeLay (twins Isabelle and Sabine in Promethean's THE LIAR) as June, Mike Cherry (Jerry in City Lit's AT HOME AT THE ZOO) as Lenny, and Promethean Ensemble Member Jamie Bragg (Oscar Wilde in Promethean's GROSS INDECENCY) as Woman in Furs. Amanda Lopez will understudy the role of Marisol.
The production team will include ensemble member Jeremiah Barr (scenic design), Uriel Gomez (costume design), John Kelly (lighting design), Sarah Espinoza (sound design), Arielle Valene (properties design), ensemble member Brendan Hutt (violence design), Hazel Marie Flowers-McCabe will be stage manager and Danielle Malenock will be assistant director.
Marisol premiered at the Humana Festival of New Plays in 1992. It was produced in New York City off-Broadway by the New York Public Theatre the following year in a staging directed by Michael Greif (RENT, GREY GARDENS, WAR PAINT) that won the 1993 Obie award for playwriting. Rivera has since written some thirty plays, including REFERENCES TO SALVADOR DALI MAKE ME HOT and BOLEROS FOR THE DISENCHANTED.
Director Juan Castañeda says, "I'm a huge fan of Rivera's work. I've directed or assistant directed six of his plays and I'm thrilled to be directing MARISOL in its 25th Anniversary year. The play reflects the anxiety of the time in which it was written, but it's equally resonant for us today in these divisive times. I think Rivera is telling us that while we have to push back against oppression, we should seek to achieve unity with the "other side" and not simply try to achieve victory over those with whom we disagree. We also have to work on self-healing in this shattered world. Rivera's magical realism helps me understand the world today better than a realistic 'kitchen sink' drama would."
Promethean's MARISOL will be performed on Raven Theatre's intimate West Stage, and will open on Saturday, October 28 at 8 pm, following previews from Friday, October 20 – Friday, October 27. More information is available at www.prometheantheatre.org. For box office inquiries, contact boxoffice@prometheantheatre.org
Juan Castañeda (director) has directed plays for Chicago Fusion Theatre at the DCA storefront, co-directed at the Steppenwolf Garage Rep with Urban Theater Company, Theatre on the Lake with UrbanTheater Company, Clockwise Theatre in Waukegan, Colectivo El Pozo and Halcyon Theatre. He has been a company member, literary manager and artistic director for UrbanTheatre Company. He is a 2006 graduate of The Theatre School at DePaul University.
Jose Rivera (playwright) is the Obie Award winning author of MARISOL, CLOUD TECTONICS, REFERENCES TO SALVADOR DALI MAKE ME HOT, and BOLEROS FOR THE DISENCHANTED, among many others. His screenplay for THE MOTORCYCLE DIARIES received an Academy Award nomination for Best Adapted Screenplay. More recently, he wrote the screenplays for LETTERS TO JULIET, ON THE ROAD and THE 33.
by José Rivera
directed by Juan Castañeda
Raven Theatre West Stage
6157 N. Clark St., Chicago
October 20-November 26, 2017
Performances Thursdays-Saturdays at 8 pm, Sundays at 3:30 pm
Previews Friday, October 20 – Friday, October 27
Press opening Saturday, October 28 at 8 pm
Regular run October 29 through November 26, 2017
No show on Thanksgiving
Tickets: General $25.00, Seniors (65+) $20.00, Students/Children $15.00
For box office inquiries, contact boxoffice@prometheantheatre.org

redpajama_set_name: RedPajamaCommonCrawl


The 3rd Annual 1K, 5K and 10K West Van Run 2014 was held on Sunday 2 March 2014 at the beautiful Dundarave Park West Vancouver.
This was my second year as the official Event Photographer for West Van Run.
Theme for this year's run was 80's style. Leg warmers and headbands not only looked the part but also kept warmth too.
Hi Kim, Thank you for getting in touch with me.
I just emailed your guys's pics 😉 Thank you for the great tip that you were dressed 80s totally helped me to track you guys down in no time.
I absolutely love the photos you sent me! I can't wait to share them with my daughter; family and friends!
Keep up the amazing work! I will definitely be (highly) recommending your work.
You captured a important event in my daughters life showing her support to cancer research. I'm thankful that you capture our moment! Thank for your speedy response.
OMG!!! The pictures are incredible!! Thank you so very much!
We loved all the shots! Great memories to have!!
I will certainly provide referrals in the future!
Your finish time at West Van Run was Epic !
Thanks for coming out and took photos of the event, Jennifer. They are absolutely awesome!
Such amazing photos from the West Van Run! These are by far the best race photos I've ever had. Thank you so much!
Thanks Jennifer. The clarity of the pictures is amazing. Truly professional. I will definitely pass this along.
Thanks. It's a beautiful picture from me, at the finish. My first Run, I will remember it.
Thank you Jennifer for such quality photos of my daughter and I coming across the finish line at the West Van 5K! Her first ever event has been captured with your amazing photos and we are grateful.


University of Social Sciences is the largest private educational institution in Poland, with more than 20 years of experience. We provide high-quality education to students from all over the world. The University offers a wide range of Bachelor's and Master's programs. Our offer also includes MBA and PhD studies and a unique American Master's degree program in cooperation with Clark University.
Why is the University of Social Sciences the best choice?
Modern educational infrastructure in major cities in Poland (Warsaw, Lodz and Cracow) and in London.


July Family fun activities for the whole family to enjoy.
July family Fun Continues! The weather here in the northern hemisphere is getting pretty warm. Maybe you spend a lot of time at the pool, or in air-conditioning but….there are still many fun things you can do.
Find calendar activities for the beginning of the month here!
July 15. National Tapioca Day.
Tapioca is a starch that is extracted from the root of the cassava plant. It is from the northwest regions of Brazil. As it lacks nutritional value and food energy, it is used as a thickening agent in manufactured foods.
I love tapioca pudding and want to share a couple of recipes with you: Old fashioned tapioca pudding and a super simple slow-cooker recipe. Many people like it served cold but try some warm—it is definitely a comfort food.
For the younger and adventurous ones in your group, try the following-Bubble Tea and clear slime. Both will add some fun to your summer days.
JUNE 16. National Ice Cream Day.
How many licks does it take to eat a scoop of ice cream? What are the three most popular flavors of ice cream – in order? Do you want the recipe for Thomas Jefferson's loved ice cream? The answers and more here!
Make your ice cream, or buy ice cream of your favorite flavors and make an ice cream sundae bar. Make your ice cream without an ice cream maker….
June 17 is a day for mathematicians.
Hampshire College has a day full of activities for the summer high school students.
To celebrate today, make or decorate a cake and cut it into 17 pieces and share and join those around the country in college — Brush up on your mathematical skills!
Find tips on finding a great location, food, entertainment and presents here!
June 19 is a great time to try new hot dog recipes. How about The Tiki Dog, The Frito Pie Dog, The French Onion Dog, The Monte Cristo Dog, the Nacho Dog, and more. Several fast food chains and convenience stores across America are offering free or discounted hot dogs to their customers on this day. In the past Sonic, 7-11, Quick-Trip and Kangaroo Express have offered discounted Hot Dogs. Check with your local establishments to see if they are offering discounts today.
June 20. There may be two Fortune Cookie Days.
One of them is today, and the other is September 13. Celebrate both! The Fortune Cookie is most popular in the United States and may have originated in Japan, not China as many assume. So, even they are not from China, try making them at home. Homemade is almost always better.
All you need to play Tug-of-War are some friends and rope. The longer the rope is, the more fun you will have. Divide the participants evenly and stand on either side of the rope. Find a spot on the ground to mark as the middle. Each team grabs the ropes. A bystander yells "GO, " and the pulling begins. The objective is for one team to pull the other team over the mark on the ground. When the whole team has been pulled across the mark, the team pulling them across is the winner. This is a real sport in some parts of the world as seen here.
1. Live each day with honesty and courage.
2. Take pride in your work. Always do your best.
3. Stay curious. Study hard and learn all you can.
4. Do what has to be done and finish what you start.
7. Be clean in thought, word, deed, and dress.
8. Practice tolerance and understanding of others.
9. Be willing to stand up for what's right.
10. Be an excellent steward of the land and its animals.
On National Parents' Day, we honor all parents do every day to raise up their children in a world that is constantly changing and challenging. President Clinton established National Parents Day in 1994. It was created to support the role of parents in raising children. So today, spend time with your parents and call them if they are not close. If they are close, spend some time together doing family together activities. Start a memory book, make a meal together, create a new family tradition, plan a family activity calendar, or just read together. Sometimes, it's just the time spent together to create stronger bonds. Today is also Vanilla Ice Cream Day. Get everyone together and have ice cream sundaes with all the fun fixings: Choose from the 74 fixings listed here or add Best Ice Cream Toppings or create your own.
JULY 24. National Tell an Old Joke Day.
There are knock-knock jokes, wordplay jokes, and just silly fun jokes. Frugal Fun for Boys and Baby Center have some great jokes for kids. The kids can pick their favorites and rehearse their delivery and have a family joke night. This is also Cousins Day. If you have cousins, give them a call if they are too far away to see in person. Have a relaxed, fun day with your family. You could even have a family stand-up comedy night with everyone sharing their new jokes.
Hot Fudge Sundaes are my very favorite dessert in the world. When I have individual time with my adult children, it is often spent talking while eating a hot fudge sundae. When we talk about getting together, it usually means "Let's have Hot Fudge" meaning a Hot Fudge Sundae. It is a simple thing but still very special to us. This recipe is like the one my mom has used, (but she never wrote it down!) So I will share this from Amber of Crazy Little Things. Once the sauce is made, you can use her recipe for Hot Fudge Sundae Cupcakes for something special!
July 26. Aunt and Uncles Day.
A family support system is a treasure to all families. If we are fortunate enough to have family nearby, life can be so much easier. Need a babysitter? Family emergencies are often easier to handle with people to lend a hand and emotional support. Aunts and Uncles can be invaluable, that someone close that your child can go to for advice, aid, support, and fun. July 26 is a day to recognize those great aunts and uncles and the contribution they can make for those they love and love them back.


A through road is for fast traffic, travelling longer distances, in large volumes. Motorways, trunk roads, bypasses, and so on. They are roads that human beings (either on foot, or cycling) should not 'engage with', by design - parallel routes should be distant, and crossings for these kinds of roads should be grade-separated.
The Dutch system of 'Sustainable Safety' means that all Dutch roads and streets have to be classified as either an access road, through road, or distributor road, under the principle of monofunctionality.


These days, the internet isn't just something you access from your computer and use to connect to other computers out there in the wider world. Today, home networks encompass not just other computers but a variety of devices, from smartphones and tablets to refrigerators and automated window coverings – the Internet of Things – and more and more homes are becoming fully connected this way. But, as has been true since the infancy of the internet, there are a plethora of malicious threats out there, from phishers attempting to steal your credit card information to infectious viruses that can take control of your machine. Most routers these days come with security systems that fend off simpler threats, but what if that's not enough?
One issue with the 'smart home' is that your personal data is spread around more devices than any before. Savvy attackers will take advantage of the often weak security around these devices – and the security is weak: research by HP has shown that the 10 most popular smart devices each have an average of 25 vulnerabilities. Basically, they're terrifyingly easy to crack if you know what you're doing, and the results could be catastrophic. It's bad enough to have your personal information stolen, but someone gaining control of your appliances could be really worrisome. Perhaps your thermostat is affected, or your motorized solar shades get rolled up at the wrong time, killing your houseplants. It sounds like a minor annoyance, but… maybe you have a baby monitor on the network and it gets shut off. It's worth thinking about.
The first line of defence is to make sure that your router has an appropriate firewall, so that the most straightforward attacks are filtered out. Unfortunately, this is only the beginning. Sophisticated malicious threats are becoming more and more common, and are often specifically crafted so as not to trigger even the most modern router security systems. Various pieces of software that can analyze and identify such threats are available, but they are often difficult to configure and likely too complex for the average consumer. But what if you could buy a device that sat in front of your network and stopped attacks from reaching it in the first place?
It's like giving your Doberman guard dog a bulletproof vest: protecting the protector. Corporate security systems have been doing this for a long time; with sensitive customer information and financial data on the line, it's crucial that just anyone can't waltz in and take whatever they want. Network intrusion detection systems are a core feature of corporate security. In the same way, a burglar alarm on top of locking the door of your home makes it that much less likely for people to physically enter. So why not put one on your network? There are a couple of products on the market, or close to it, that tackle this problem. They use the same kind of software that the consumer would find tricky to set up, but packaged up in an easy-to-use box that you just have to plug into your network to beef up its security.
Two similar products that perform these functions are the iGuardian (also known as Shield) from Itus Networks, which successfully completed a $125,000 Kickstarter campaign, and has to date raised $175,000; and BOX by BitDefender, which is already on the market. Both devices cost between $100–$200 plus a $100 or so yearly subscription, which enables the company to keep its response to threats current. It might be worth it for the peace of mind you'll get in your fully networked home, not to mention the money you'll save if your fridge is turned off and all the food you've bought for Thanksgiving spoils.
We're living in the future, but no matter how technologically advanced we get, if there's one thing that doesn't change with time, it's human behaviour. Protecting yourself is a must no matter what the arena – and the stakes just got a lot higher!


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Research ArticleENVIRONMENTAL STRESS AND ADAPTATION TO STRESS
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Leaf Senescence Is Accompanied by an Early Disruption of the Microtubule Network in Arabidopsis
Olivier Keech, Edouard Pesquet, Laurent Gutierrez, Abdul Ahad, Catherine Bellini, Steven M. Smith, Per Gardeström
Olivier Keech
For correspondence: okeech@cyllene.uwa.edu.au
Edouard Pesquet
Laurent Gutierrez
Abdul Ahad
Catherine Bellini
Per Gardeström
Published December 2010. DOI: https://doi.org/10.1104/pp.110.163402
© 2010 American Society of Plant Biologists
The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca2+-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.
Leaf senescence can be the result of natural aging but can also be initiated in response to various stresses such as ozone, shading, or pathogen infection (Woolhouse, 1967; Smart, 1994). In response to these developmental or environmental cues, the leaf will undergo three distinct phases sequentially: (1) an initiation period, where metabolic changes lead to a decrease in photosynthetic activity and a transition from source to sink; (2) a degenerative phase, mainly characterized by the disassembly of cellular components and their degradation; and (3) a terminal phase, where cell integrity is lost prior to cell death and death of the whole organ (Nooden et al., 1997; Yoshida, 2003). As leaf senescence is also induced by abiotic and biotic stresses, understanding its mechanisms is not only important for answering fundamental scientific questions but also as part of a challenge to increase crop yields.
In a recent study with Arabidopsis (Arabidopsis thaliana; Keech et al., 2007), we showed that regulation of metabolism differed significantly between an individually darkened leaf (idl) attached to a whole plant and an equivalent leaf from an entirely darkened plant. After 6 d of darkness, photosynthetic capacity was maintained in leaves from a whole darkened plant, whereas the capacity for mitochondrial respiration decreased. In contrast, idls showed an accelerated senescence with a rapid decline in photosynthetic capacity, while mitochondrial capacity remained high. Additionally, we observed drastic ultrastructural alterations, including aggregation of both chloroplasts and mitochondria during the accelerated senescence occurring in mesophyll cells of idls. We consequently hypothesized that the cytoskeleton, and in particular the cortical microtubules (MTs), could undergo modifications contributing to the pronounced rearrangements of the cellular content in the mesophyll cells during leaf senescence.
In plant cells, there are two main cytoskeleton components: (1) MTs, which are heterodimeric polymers of the globular proteins α- and β-tubulin; and (2) microfilaments, which are composed of actin monomers (Lloyd and Hussey, 2001; Boutté et al., 2007). MTs are assembled in a head-to-tail fashion to form protofilaments, and they are constantly undergoing polymerization and depolymerization at both the plus and minus ends (Hashimoto, 2003). They are mainly involved in cell elongation and cell division (Dixit and Cyr, 2004; Boutté et al., 2007), in the development of the plant cell wall, notably by facilitating or guiding cellulose deposition (Wasteneys, 2004; Lloyd, 2006; Wightman and Turner, 2008; Pesquet et al., 2010), and in organelle positioning (Van Gestel et al., 2002; Wada and Suetsugu, 2004; Romagnoli et al., 2007). Nevertheless, their function is intimately linked to their organizational state, which directly depends on the developmental and/or environmental conditions that the cells are facing (Dixit and Cyr, 2004). Modifications of these conditions trigger signals that, in turn, can be transduced through several types of proteins, generally referred to as microtubule-associated proteins (MAPs). MAPs, by their stabilizing, bundling, or severing activities, can then regulate the MT dynamics and can actively participate in the reorganization of the MT array (Lloyd and Hussey, 2001; Hashimoto and Kato, 2006; Hamada, 2007).
Although the dynamic assembly and disassembly of MTs is essential for cell stability and survival, the state of the MT cytoskeleton during senescence in plants remains largely unknown. Considering this in light of our previous data suggesting a remodeling of the cytoskeleton in leaves experiencing dark-induced senescence, we investigated changes in the cortical MT network and potential regulatory mechanisms during both natural and dark-induced leaf senescence in the model plant Arabidopsis.
Two Systems to Study Leaf Senescence
We compared attached leaves subjected to dark-induced senescence with Arabidopsis leaves naturally aging in order to assess whether the cytological modifications observed in our previous study (Keech et al., 2007) were dark induced or senescence specific. idls, previously described in detail by Weaver and Amasino (2001) and by Keech et al. (2007), offer a more synchronous and a better controlled process of senescence than age-induced senescence. Using this method in this work, we followed the progression of leaf senescence for 6 to 8 d, after which the leaves were yellow (Fig. 1A). Time zero idl (T0idl) represented leaves from 8-week-old plants with a chlorophyll content of 1.27 ± 0.04 mg mL−1 g−1 fresh weight. In parallel, from 11- to 13-week-old plants, we selected leaves at different stages of natural senescence (T0ns, T50ns, and T85ns). T0ns represented leaves with a chlorophyll content of 1.85 ± 0.02 mg mL−1 g−1 fresh weight, which was 30% more than in the T0idl. T50ns and T85ns were leaves undergoing natural senescence with approximately 50% and more than 85% reduction of their chlorophyll content, respectively. These three stages were consequently representative of the beginning, the middle, and the end of the process of natural senescence in Arabidopsis leaves (Fig. 1A). In addition, as we noticed that leaves undergoing natural senescence generally exhibit a heterogeneous pattern of chlorosis (Supplemental Fig. S1), we proposed that T50ns could thus represent a combination of the three more distinct and more synchronous stages T2idl, T4idl, and T6idl, as suggested in Figure 1A.
A, Representative time course of a mature rosette leaf of Arabidopsis plant undergoing either dark-induced senescence or natural senescence. For dark-induced senescence, leaves were individually darkened (idl) for 2 to 8 d. For natural senescence (ns), T50ns and T85ns represent 50% and at least 85% degradation of their chlorophyll content, respectively, in comparison with T0ns. Bars = 1 cm. B, Immunoblot analysis of total extracts from natural senescence (ns) and from idls (idl). Rubisco large subunit (Rbc L), Gln synthetase cytosolic (GS1) and chloroplastic (GS2) isoforms, cytochrome c oxidase subunit II (COX), α-tubulin (Tub-α), and F-actin (Actin) were tested. In each lane, 10 μg of protein was loaded.
To compare the similarities of the developmental stages between our two systems at the protein level, we ran immunoblot analyses with several relevant biological markers. In line with the progressive degradation of the chloroplasts during leaf senescence, the large subunit of Rubisco and the chloroplastic isoform of Gln synthetase (GS2) were progressively degraded during the two time courses of natural and dark-induced senescence (Fig. 1B). As expected, the relative content of the cytosolic isoform of Gln synthetase (GS1) increased during both natural and dark-induced senescence as part of the nitrogen remobilization and nutrient recycling pathway, in accordance with previous reports (Kawakami and Watanabe, 1988; Masclaux et al., 2000). It is worthwhile to note that the ratio GS1/GS2 was higher in T0ns leaves than in the T0idl leaves (Fig. 1B), reflecting an older stage of development of T0ns when compared with T0idl (see also quantitative real-time [qRT]-PCR analysis in Fig. 5C below). Mitochondrial degradation was tested with antibodies raised against cytochrome c oxidase subunit 2 (COX-II), the subunit of complex IV in the electron transport chain. We previously reported that the number of mitochondria per volume of leaf decreased during dark-induced senescence while their size increased (Keech et al., 2007). Here, we show that the amount of COX-II remained fairly stable during the 6 d of dark-induced and natural senescence, demonstrating that the ratio of mitochondrial proteins to total proteins remained almost unchanged during leaf senescence. This confirms that the degradation of mitochondria is slower than the degradation of chloroplasts and strengthens the prominent role of mitochondria during late events of leaf senescence, most likely to supply energy and the carbon skeleton to the cell for efficient nutrient remobilization. Finally, two cytoskeleton markers, α-tubulin for the MTs and actin for the actin filaments, decreased unexpectedly early during both dark-induced and natural leaf senescence.
In Vivo Visualization of the MT Cytoskeleton during Leaf Senescence
To investigate the consequences of such an early decrease in abundance of α-tubulin during both dark-induced (Fig. 2) and natural (Fig. 3) leaf senescence, the organization of the MT cytoskeleton was visualized in vivo by confocal laser microscopy using Arabidopsis plants constitutively expressing GFP-β-TUBULIN6 (TUB6; Nakamura et al., 2004). The GFP fusion to the N terminus of the Arabidopsis TUB6 under the control of the cauliflower mosaic virus 35S promoter was demonstrated to be a reliable reporter to fluorescently label MTs in aerial tissues (Abe and Hashimoto, 2005). In addition, the authors reported the incorporation of labeled β-tubulin subunits to be 20% to 30% of the endogenous tubulin level. Thus, it is likely that the in vivo visualization of the polymerized MTs was unbiased, despite the construct being driven by a strong promoter. MTs were observed in three different cell types from the abaxial side of leaf sections. Already 2 d after the start of dark-induced senescence, a reduction of the MT network was observed in both the epidermis and the adjacent spongy mesophyll cells (Fig. 2, B and G), and this reduction was accentuated at 4 and 6 d in the two cell types. However, no apparent modification of the MT cytoskeleton was observed in guard cells of stomata (Fig. 2, K–O). We also noticed a progressive reduction in size and in number of chloroplasts in mesophyll cells (Fig. 2, F–I), which led to their complete degradation after 8 d (Fig. 2J). In line with our previous observations (Keech et al., 2007), chloroplasts tended to aggregate after 6 d. Finally, after 8 d, the MT network was completely disrupted, with the exception of the stomatal guard cells, which retained both their MTs and their chloroplasts (Fig. 2, E, J, and O).
In folio imaging of the progressive disruption of MTs in epidermis (A–E), spongy mesophyll (F–J), and guard cells of stomata (K–O) during dark-induced senescence. Bars = 50 μm (A–J) and 10 μm (K–O).
In folio imaging of the progressive disruption of MTs in epidermis (A–C), spongy mesophyll (E–G), and guard cells of stomata (H–J) during natural senescence. The late longevity of stomatal guard cells is evidenced by the conserved MT lattice (H–J). Bars = 50 μm.
The situation was very similar in leaves undergoing natural senescence. The MT cytoskeleton from the epidermis and the mesophyll cells of T0ns leaves exhibited the same pattern as T0idl. Later, we observed a drastic reduction of the density of the MT cytoskeleton in epidermis and mesophyll cells after 50% degradation of the chlorophyll (T50ns; Fig. 3, B and F). After 85% degradation of the chlorophyll (T85ns), the depolymerization of the MTs was apparently complete in these two cell types (Fig. 3, C and G). We also noticed chloroplasts of a smaller size in mesophyll cells at T50ns (Fig. 3F) and their complete degradation at T85ns (Fig. 3G). Finally, as in dark-induced senescence, guard cells of stomata retained their MT network and their chloroplast until the very end of the leaf's life (Fig. 3, H–J).
Expression of Tubulin-Related Genes
In order to explain the progressive, tissue-dependent, disruption of the MTs during both dark-induced and natural senescence, we investigated the regulation of several molecular players that could be involved in the bundling/stabilization and the destabilization of the MT network. In Arabidopsis, there are six genes coding for α-tubulin subunits (Kopczak et al., 1992), nine for β-tubulin (Snustad et al., 1992), and two for γ-tubulin (Liu et al., 1994). From publicly available microarrays (van der Graaff et al., 2006) performed with leaves undergoing natural and dark-induced senescence in similar conditions to ours, we extracted relative expression profiles for all genes encoding tubulin subunits (Supplemental Table S3) and expressed the data as the log2 of the ratio between T25 T50, or T75 and the expression value of the gene at T0 (T0 being a 6-week-old leaf and T25, T50, and T75 being leaves with a loss of 25%, 50%, and 75% of their chlorophyll content, respectively). As shown in Figure 4, the expression pattern for the genes encoding α-, β-, and γ-subunits was fairly similar between natural (Fig. 4A) and dark-induced (Fig. 4B) senescence and showed a progressive repression for most of the genes. However, only a few of the genes showed a significant down-regulation. We subsequently used qRT-PCR to confirm that expression of TUB6 matches that of van der Graaff et al. (2006) in our systems (Fig. 5).
Gene expression analysis of α-, β-, and γ-tubulins during natural senescence (A) and dark-induced senescence (B). Data were primarily extracted from publicly available microarrays (van der Graaff et al., 2006). The normalized expression values were converted into the log2 of the ratio between the samples (25%, 50%, and 75% degradation of chlorophyll) and controls (leaves from 6-week-old plants). Asterisks indicate significant differences (P < 0.05) in comparison with the respective T0 determined by Student's t test. Samples are as follows: α1 (At1g64740), α2 (At1g50010), α3 (At5g19770), α4 (At1g04820), α5 (At5g19780), α6 (At4g14960), β1 (At1g75780), β2 (At5g62690), β3 (At5g62700), β4 (At5g44340), β5 (At1g20010), β6 (At5g12250), β7 (At2g29550), β8 (At5g23860), β9 (At4g20890), γ1 (At3g61650), γ2 (At5g05620).
A and B, qRT-PCR analysis of MT-related genes during natural senescence (A) and during dark-induced senescence (B). SAG12 was also included as a molecular marker of leaf senescence. Results are expressed on a log10 scale and represent the ratio between T50ns and T0ns (A) and between T2-idl, T4-idl, or T6-idl and T0-idl (B). C, The ratio between the level of expression of the genes at T0ns and T0idl indicates that senescence has already begun in T0ns. Samples are as follows: TUB6 (At5g12250), MAP18 (At5g44610), MAP65-1 (At5g55230), MAP65-2 (At4g26760), MAP65-3 (At5g51600), MAP65-4 (At3g60840), MAP65-5 (At2g38720), MAP65-6 (At2g01910), MAP65-7 (At1g14690), MAP65-8 (At1g27920), MAP65-9 (At5g62250), MAP70-1 (At1g68060), SAG12 (At5g45890), p60 (At1g80350), p80 (At5g23430), p80 (At1g03110), p80 (At4g34280), p80 (At4g04940).
While these results (van der Graaff et al., 2006) show reduced expression of the genes encoding tubulin subunits, we also hypothesized that other regulators could be involved in the disruption of the cortical MT array. To assess this possibility, we examined by qRT-PCR the level of transcripts of several MT regulators: the nine genes representing the MT-bundling MAP65 family (Smertenko et al., 2008), the MT-severing p60 subunit of katanin (Burk et al., 2001) and four of its putative p80 subunits containing WD40 repeats (Bouquin et al., 2003; Roll-Mecak and McNally, 2010; annotations in TAIR9), the MT-stabilizing MAP70-1 (Korolev et al., 2005), and finally the MT-destabilizing MAP18 (Wang et al., 2007). Results were expressed as the log10 of the ratio between the expression of the genes at T50ns and the expression of the genes at T0ns, for which the expression value was set to 1 (Fig. 5A). Similarly, Figure 5B represents the log10 of the ratio between the gene expression levels at T2idl, T4idl, or T6idl and the expression of the genes at T0idl (for which the value was set to 1). The low abundance of transcripts due to their degradation occurring at T85ns and T8idl did not allow qRT-PCR quantifications.
Often referred to as the main molecular marker of leaf senescence, the level of expression of SAG12 (for senescence-associated gene 12, encoding a papain-like Cys proteinase) was used as a control and shown to be up-regulated several thousand-fold between T0ns and T50ns and also gradually up-regulated during dark-induced senescence, strengthening the similarities between the two systems. Since the GFP-TUB6 construct was used for the visualization of the MTs by confocal laser microscopy (Nakamura et al., 2004), we also examined expression of the TUB6 gene. Reinforcing the validity of the arrays of van der Graaff et al. (2006), we found TUB6 to be progressively repressed during natural senescence and dark-induced senescence (Fig. 5, A and B).
Due to their central role in plant growth and development, MTs undergo constant reorganization during the different phases of cell expansion and actively participate in intracellular transport and signal transduction in response to hormone signals and/or environmental perturbations. As a direct consequence of their multiple functions, MTs are subjected to extremely complex regulation orchestrated by a plethora of proteins notably involved in building, remodeling, and interconnecting the MT network (Sedbrook, 2004). Among the increasing number of proteins discovered to interact with MTs, we primarily focused on the MAP65 family, as numerous studies have reported MAP65 proteins to be MT-bundling proteins. The Arabidopsis genome contains nine MAP65 genes sharing 25% to 78% amino acid sequence identity, suggesting an important variability in MAP65 activities (Smertenko et al., 2008). In an effort to unravel the localization and the biochemical roles of the different members of the MAP65 family in Arabidopsis, Smertenko et al. (2008) provided strong evidence for divergent dynamic association between the MAP65s and the MTs but also highlighted the difficulties of determining in planta the role of each MAP65, due to their tissue and developmental expression specificity. Nonetheless, most of the MAP65 proteins seem to promote MT polymerization at some stage. In this study, we show the expression of all nine MAP65 genes to be significantly repressed during natural and dark-induced senescence (Fig. 5, A and B). In addition, we also report another MAP gene involved in the stabilization of MTs, MAP70-1 (Korolev et al., 2005), to be down-regulated during the two processes (Fig. 5, A and B).
Interestingly, MAP18, whose protein has been demonstrated to have a depolymerizing activity on MTs in vitro (Wang et al., 2007), was found to be highly up-regulated during natural senescence, around 14-fold when compared with T0ns, and also during dark-induced senescence, where its regulation peaked at a 104-fold increase, suggesting a major role for MAP18 during leaf senescence. Strong promoter activity of MAP18 during leaf senescence was confirmed using a promMAP18::GUS reporter gene in transgenic plants (Wang et al., 2007). GUS staining was barely visible in either T0idl or T0ns, but surprisingly, we saw a pronounced staining in structures likely to be the hydathodes of the leaves. Progressively, the GUS staining appeared concomitantly with the progress of the senescence and had a peak of intensity at T4idl and at T50ns (Fig. 6). Although the expression of promMAP18::GUS was still detectable in T6idl and T85ns, the staining was not as strong as suggested by the results of the qRT-PCR analysis (Figs. 5 and 6). This discrepancy between the two results may be explained by the fact that the GUS staining reveals only the activity of the promoter of MAP18, whereas qRT-PCR reveals the steady-state level of MAP18 mRNA. Indeed, while the promoter activity is essential to produce MAP18 mRNAs, the quantity of steady MAP18 transcripts strongly depends on MAP18 mRNA stability. From a recently published study on mRNA decay rates in Arabidopsis (Narsai et al., 2007), we discovered that MAP18 had one of the most stable transcripts in Arabidopsis, with a half-life estimated at 46 h. The group of the most stable transcripts comprises transcripts with a half-life of at least 24 h and represents less than 1% of the 13,000 mRNAs studied (Narsai et al., 2007). The decrease of the promMAP18 activity in T6idl and the concomitantly high level of MAP18 mRNAs during senescence are consistent with a particularly high stability of the MAP18 mRNA in leaves.
GUS expression analysis of promMAP18 during dark-induced senescence and natural senescence. Experiments were performed with six independent biological replicates.
Finally, we also investigated the level of expression of katanin subunits. Katanin is a heterodimeric protein that mediates ATP-dependent destabilization of MTs in animals. The small subunit (p60) of katanin has been identified in Arabidopsis and presents a MT-severing activity in vitro, whereas a regulatory subunit of 80 kD (p80) is involved in the targeting of katanin (Stoppin-Mellet et al., 2007). In our work, the p60 subunit of katanin and four of its putative p80 partners were differentially regulated between natural and dark-induced senescence. Whereas the genes coding for the characterized p60 and the four putative p80 subunits were all down-regulated during natural senescence, p60 and two of the four putative p80 subunits appeared to be significantly up-regulated during dark-induced senescence. This discrepancy between natural and dark-induced senescence suggests katanin to be somehow regulated by light.
Whole Darkened Plants as a Control
In an attempt to dissociate the effects of darkness from the process of cell death occurring in leaves subjected to dark-induced senescence, we examined the expression level of the same set of genes in leaves harvested after 2, 4, and 6 d from entirely darkened plants. We previously showed (Keech et al., 2007) that after 6 d of darkness, leaves from entirely darkened plants were in a metabolic "standby" mode, where most of the cellular functions were reduced but still kept functional in order to optimize the recovery of the plant when a source of light became available. While idls are inexorably committed to senesce, this fundamentally different metabolic strategy confers to the entirely darkened plant a crucial survival ability. The qRT-PCR analysis revealed an intermediate transcript profile in the leaves from the entirely darkened plants. As in idls, MAP18, p60-At1g80350, p80-At4g34280, and p80-At5g23430 were progressively up-regulated during the 6 d of darkness. However, idls and whole darkened plants differed, as TUB6 was significantly up-regulated and all genes encoding MAP65 and MAP70-1 were stably expressed (Fig. 7A). Interestingly, only a minor reduction of the density of the MT network was observed in leaves from 6-d-darkened plants (Fig. 7B).
A, qRT-PCR analysis of MT-related genes in leaves from entirely darkened plants. Results are expressed on a log10 scale and represent the ratio between 2 d, 4 d, or 6 d and T0. All locus identifiers are available in the legend of Figure 5. B, In folio imaging of the MT array during 0, 2, 4, and 6 d in darkness. Bars = 20 μm.
Both Natural and Dark-Induced Senescence Are Accompanied by an Early, Cell-Specific Degradation of the Cortical MT Array
Although leaf senescence is a thoroughly studied developmental stage, the role of the cytoskeleton remains unknown. Consequently, our objective in this study was to characterize the cortical MT array during leaf senescence and to investigate its potential regulation. Natural senescence is the obvious physiological reference for studies about leaf senescence. However, working with different experimental systems can sometimes be advantageous because the comparison of the results unravels common or specific pathways and also helps to establish more accurately the interconnections between pathways leading to leaf senescence. In this study, despite T0ns being 4 weeks older than T0idl, no visible sign of senescence was apparent. Indeed, no degradation of chlorophyll was evident (the chlorophyll content was even 30% higher in T0ns than in T0-idl), the immunoblot analysis of Rubisco and of α-tubulin revealed a high amount of protein in the two T0s, and finally, the comparison of the MT cytoskeleton between the two did not show apparent differences. However, for a few genes of interest, the ratios between the expression levels in T0ns and in T0idl demonstrate that the T0ns leaves may have already entered the process of senescence (Fig. 5C). For instance, the high expression of SAG12 clearly indicates that the genetically controlled process of degradation has already begun in T0ns (Fig. 5C). In addition, it has been shown that in leaves under normal conditions, the chloroplastic isoform (GS2) represents the main fraction of the Gln synthetase pool, while the cytosolic isoform (GS1) is more abundant during leaf senescence (Kawakami and Watanabe, 1988; Brugière et al., 2000; Masclaux et al., 2000). The higher protein ratio of GS1 to GS2 in T0ns leaves when compared with T0idl indicates that these leaves have already modified their metabolism to enter their nutrient recycling process and in particular the salvage of nitrogen. In addition, as shown by Supplemental Figure S1, leaves undergoing natural senescence exhibit a more heterogeneous degradation pattern than leaves subjected to a dark-induced senescence. Taken together, these results show the difficulties in determining the starting point of natural senescence without using destructive techniques. For this reason, the use of other systems such as dark-induced senescence, which allows a fast, controlled, and more synchronous induction of leaf senescence, appears to be a powerful tool to study leaf senescence.
Here, we show that the degradation of the cortical MT array is an early event and takes place in the epidermis cells and the adjacent layer of mesophyll cells of Arabidopsis leaves undergoing either natural or dark-induced senescence (Figs. 2 and 3). Only a minor aggregation of the chloroplasts in mesophyll cells from both natural and dark-induced leaf senescence is observed, whereas such aggregation was more apparent in a previous study (Keech et al., 2007). However as infiltrations of calcofluor white were used to visualize the cell wall in our previous study (Keech et al., 2007), this might have accentuated the aggregation of the organelles, since they could not be properly anchored to the cytoskeleton due to its destabilization. In contrast, guard cells of stomata conserve their chloroplasts and their MT network, consistent with earlier studies reporting the extended longevity of chloroplasts from guard cells of yellowing leaves from both perennial and annual plants (Zeiger and Schwartz, 1982). It has been proposed that guard cells of stomata retain their functionalities to facilitate gas exchanges until the very end of the senescence process (Thimann and Satler, 1979). However, the heterogeneity in the metabolism of the different cell types during leaf senescence raises fundamental questions about cell-specific regulation and potential signaling between the cells undergoing senescence.
Molecular Mechanisms of the Degradation of the MT Arrays during Leaf Senescence
Smertenko et al. (2003) have reported disorganization of the MTs during embryonic programmed cell death in the gymnosperm Picea abies, and degradation of the MTs was also observed by Zottini et al. (2006) in cytokinin-treated cells of Medicago truncatula induced to enter programmed cell death. However, the molecular players and the regulatory mechanisms responsible for this destabilization remain unknown.
We assume that although MT assembly is energy dependent, the lack of ATP/GTP is not likely to be responsible for the disorganization of the MT, as we earlier showed mitochondria to provide sufficient ATP to maintain cellular functions until the end of the process of senescence (Keskitalo et al., 2005; Keech et al., 2007). So, in an attempt to elucidate the molecular mechanisms responsible for the degradation of the MT lattice during leaf senescence, we investigated the regulation of genes coding for tubulin subunits and for several MAPs in whole leaves. Expression of genes encoding α-, β-, and γ-tubulins is mainly repressed during leaf senescence, as shown by published microarray data (van der Graaff et al., 2006). In addition, we show that the MAP65 gene family and MAP70-1 are significantly repressed during both natural and dark-induced senescence but not in leaves from whole plants experiencing prolonged darkness. This consequently suggests that MT polymerization and stabilization mechanisms are specifically down-regulated during leaf senescence.
However, not all MAPs are involved in the stabilization of MTs. Recently identified as a new MAP (Wang et al., 2007), MAP18 was shown to bind to MTs and to inhibit tubulin polymerization in vitro. MAP18 was reported to be expressed mostly in expanding cells and localized in root, flower, cotyledon, hypocotyl, and trichome but surprisingly not in the root tip or mature leaf (Wang et al., 2007). Using the GUS reporter gene fused to the promoter of MAP18, we observed a specific staining of structures believed to be hydathodes of mature leaves under normal conditions (T0ns and T0idl). However, both qRT-PCR and GUS staining showed an increased expression of MAP18 during leaf senescence, strongly supporting the idea that MAP18 could play a physiological role in the senescence-specific degradation of MTs. However, our analysis showed that MAP18 was also induced in leaves from whole darkened plants, whereas only a minor destabilization of the MT network was observed. Furthermore, in a recent study, Kato et al. (2010) investigated the biochemical properties of MAP18 and concluded that this protein was a plasma membrane-associated Ca2+-binding protein (consequently renamed PCaP2 by the authors) and was very likely to be involved in the regulation of cellular functions, including stress response and cytoskeletal reorganization. In vivo, MAP18/PCaP2 appeared to be associated with the plasma membrane due to its N-myristoylation and strongly interacted with several phosphatidylinositol phosphates in a Ca2+-calmodulin-dependent manner (Kato et al., 2010). We consequently conclude that increased expression of MAP18 is not sufficient to destabilize the MT network in vivo but could instead be part of a more complex Ca2+-dependent signaling mechanism potentially leading to the perturbation of the MT cytoskeleton.
We also questioned whether katanin could be involved in the degradation of MTs. In a recent study, Stoppin-Mellet et al. (2006) examined in Arabidopsis the overexpression of the p60 subunit (also called AtKSS or AtKTN1) fused to the reporter monomeric Red Fluorescent Protein. Under the control of an ethanol-inducible promoter, overexpression of the katanin p60 subunit triggered MTs to organize into numerous and thick bundles, which ultimately depolymerized. In this work, our findings showed that all katanin subunit genes (i.e. the p60 and putative p80 genes) were all repressed during natural senescence, whereas the genes encoding p60 and two of the putative p80 genes were significantly up-regulated in idls (Fig. 5, A and B). Despite this up-regulation during dark-induced senescence, the formation of short and thick bundles was not apparent. Consequently, katanin does not seem to be required for the depolymerization of MTs during leaf senescence. However, the discrepancy between the data from natural senescence and from dark-induced senescence suggests that the severing activity of katanin could be involved in the rearrangement of MT in a light-dependent manner. In fact, this idea is further supported by the two following points. First, qRT-PCR performed on leaves from entirely darkened plants revealed the same p60 and p80 transcripts to be significantly up-regulated during 6 d of darkness (Fig. 7A), although only a minor degradation of the cortical MT array was noticed (Fig. 7B). Second, when we examined the ChIP-chip data set from Lee et al. (2007), we observed that AtKSS (p60-At1g80350) was among the in vivo binding targets of the transcription factor HY5 (for long hypocotyl 5). The bZIP protein HY5 is an extensively studied transcription factor involved in promoting photomorphogenesis. Mutations in HY5 cause a defect in the inhibition of hypocotyl elongation in all light conditions, suggesting that HY5 acts downstream of PHYA, PHYB, cryptochromes, and UV-B light (Lee et al., 2007). Taken together, these results strongly suggest that the severing activity of katanin on the remodeling of the cortical MT array can be light dependent, since darkness on the one hand enhances katanin expression and on the other hand represses the expression of HY5. However, much work remains to elucidate the regulation of the cortical MTs in response to developmental and environmental stimuli.
To conclude, in view of these new findings, we propose a tentative model (Fig. 8) for the early degradation of the cortical MT lattice during both natural and dark-induced senescence. Katanin p60 and p80 subunits may not be required during natural senescence; however, they could contribute to the reorganization and the severing of the MTs during prolonged darkness, including dark-induced senescence, potentially via the repression of the transcription factor HY5. In contrast, the corepression of the tubulin subunits and the bundling/stabilizing MAP65s/MAP70-1 could contribute to the destabilization of the MT network during leaf senescence; the minor disruption of the MTs observed in leaves from entirely darkened plants suggests that MAP18 expression alone is not sufficient for the disruption of the MTs. Altogether, this work highlights a novel trait of leaf senescence by demonstrating the surprising earliness of the degradation of the cortical MT array. This also leads to further questions about how vesicle shuttling, organelle mobility, and reorganization of metabolism are achieved in cells having a drastically reduced MT network.
Proposed model for the destabilization of the cortical MT array in epidermis and mesophyll cells during leaf senescence. [See online article for color version of this figure.]
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotype Columbia and Columbia transformed with GFP-TUB6 (Nakamura et al., 2004; Abe and Hashimoto, 2005) were grown in a controlled-environment growth chamber with a short-day photoperiod (8 h of light/16 h of dark), an irradiance of 250 μmol quanta m−2 s−1, a relative humidity of 75%, and a temperature cycle of 22°C day/17°C night.
Induction of Senescence
Leaves undergoing natural senescence were collected from rosettes (leaves 6–11) from plants aged 11 to 13 weeks. The three stages were labeled T0ns, T50ns, and T85ns. T0ns represented leaves with a chlorophyll content of 2.2 ± 0.4 mg mL−1 g−1 fresh weight. T50ns and T85ns were, respectively, the leaves undergoing natural senescence with 50% and more than 85% degradation of their chlorophyll content.
Dark-induced senescence was carried out as described by Keech et al. (2007). Briefly, with Arabidopsis plants aged 8 weeks, rosette leaves 6 to 11 were covered by mittens for 2, 4, 6, and 8 d while the rest of the plant remained in the light. These leaves are referred to as idls. In parallel, whole plants were placed in darkness for 2, 4, and 6 d.
Protein Extraction and Immunoblot Analysis
For each treatment, two leaf discs (diameter 15 mm) were ground in 500 μL of extraction buffer (50 mm Tris-HCl, pH 7.5, 5 mm EGTA, 5 mm MgCl2, 100 mm NaCl, 10% [v/v] glycerol, 0.1% [v/v] Triton-X-100, 0.5% [w/v] SDS, and 2 mm dithiothreitol). After centrifugation (4°C, 15 min, 20,000g), supernatants were collected and protein amount was quantified with the DC Protein Assay Kit from Bio-Rad.
Immunoblot analysis was done on nitrocellulose, and primary antibodies were used at the following concentrations: anti-large subunit of Rubisco (from Agrisera), 1:5,000; anti-COX-II (from Agrisera), 1:1,000; anti-GS1 and anti-GS2 (Sakakibara et al., 1992), 1:1,000; anti-TUBα (from Sigma; T5168), 1:2,000; anti-Actin (from Sigma; A0480), 1:1,000. Western blots were visualized by enhanced chemiluminescence (GE Healthcare).
Confocal Laser Imaging: MT Network Visualization
Imaging was performed with an SP2 inverted microscope (Leica) under a 63× water-immersion objective (numerical aperture 1.20). The MT cytoskeleton was visualized in the epidermis and the adjacent layer of spongy mesophyll cells from the abaxial side of the leaf sections. GFP was excited using a 488-nm silver laser, and emitted light was collected in the 503- to 562-nm window. Chloroplasts revealed by chlorophyll fluorescence were excited using a 633-nm helium-neon laser, and emitted light was collected in the 662- to 721-nm window. Spectral measurements were performed to confirm emission spectra. Three-dimensional images of leaves were made by sequential scans. For each time point, at least four biological replicates were processed. Images were mounted with the ImageJ software (version1.38x).
Transcript Quantification by qRT-PCR
RNA Isolation and cDNA Synthesis
Ten leaves at similar stages of senescence (i.e. T0ns and T50ns for natural senescence and T0, 2 d, 4 d, and 6 d for idl and whole darkened plants [see above for description]) were dissected from five different plants, pooled, frozen in liquid nitrogen, and ground into powder. Two sets of samples were prepared from two independent biological replicates. Total RNA was extracted using the hot phenol protocol described by Gutierrez et al. (2008). The resulting RNA preparations were treated with DNaseI using the DNAfree Kit (Ambion), and cDNA was synthesized by reverse transcribing 10 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The reaction was stopped by incubation at 70°C for 10 min, and the reaction mixture was then treated with RNaseH (Invitrogen) according to the manufacturer's instructions and diluted by adding 700 μL of deionized water. All cDNA samples were tested by PCR using specific primers flanking an intron sequence to confirm the absence of genomic DNA contamination.
Transcript levels were assessed by qRT-PCR in assays with triplicate reaction mixtures (final volume, 20 μL) containing 5 μL of cDNA, 0.5 μm of both forward and reverse primers, and 1× FastStart SYBR Green Master Mix (Roche) using an iCycler iQ Real-Time PCR Detection System (Bio-Rad), and threshold cycle (CT) values for each sample were acquired using iCycler iQ software 3.0 (Bio-Rad). The following standard protocol was applied for each amplification: 10 min at 95°C, followed by 40 cycles of 10 s at 95°C, 15 s at 60°C, and 15 s at 72°C. The sequences of primers used for all target genes are presented in Supplemental Table S1.
Reference genes were selected and validated as follows. Ten genes (for primer sequences, see Supplemental Table S2) were chosen for their putative stability of expression according to Czechowski et al. (2005) and Gutierrez et al. (2008). Their expression in our experimental material (i.e. leaves at different stages of natural or artificial senescence grown under our experimental conditions) was then assessed, and they were ranked according to their stability of expression using the geNorm software (Vandesompele et al., 2002). APT1 and TIP41 were the most stably expressed genes among the 10 tested (Supplemental Fig. S2) and thus were used to normalize the qRT-PCR data. The normalized expression patterns obtained using both reference genes were similar, so only the data normalized with APT1 are shown in Figures 4, 5, and 7. Relative standard curves describing the PCR efficiencies for each primer pair were generated for each amplicon, and the resulting values were used to calculate expression using the formula ET(CTT0 − CTX)/ER(CTT0 − CTX), where T is the target gene and R is the reference gene, CT is the crossing threshold value (i.e. the number of PCR cycles required for the accumulated fluorescence signal to cross a threshold above the background), X is related to cDNA from senescing leaves (i.e. T2idl, T4idl, T6idl, and T50ns), and T0 indicates the related T0 control (i.e. T0ns and T0idl). A melting curve analysis was added to each PCR program, and the size of PCR products was assessed by electrophoresis on agarose gels to check that the fluorescence signal was derived from the single intended amplicon.
promMAP18::GUS Analysis
Stable transformants for promMAP18::GUS (in the Columbia background) were kindly obtained from Prof. Ming Yuan. GUS staining was performed according to Wang et al. (2007).
The following materials are available in the online version of this article.
Supplemental Figure S1. Example of heterogeneity in the degradation of the MT network during natural leaf senescence.
Supplemental Figure S2. Validation of the reference genes (geNorm) for the qRT-PCR analysis.
Supplemental Table S1. Sequences of primers used for target genes.
Supplemental Table S2. Sequences of primers used for reference genes.
Supplemental Table S3. Relative expression values for natural and dark-induced senescence time courses.
We thank Dr. Hitoshi Sakakibara for GS1 and GS2 antibodies, Dr. Akashi Hashimoto for the transgenic Arabidopsis line GFP-TUB6, and Prof. Ming Yuan for the promMAP18::GUS line. Dr. Andreas Sjödin is acknowledged for his comments at the early stages of this work.
↵1 This work was supported by the Australian Research Council (award nos. FF0457721 and CE0561495), by the Centres of Excellence program of the Government of Western Australia, and by the Swedish Foundation for International Cooperation in Research and Higher Education.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Olivier Keech (okeech{at}cyllene.uwa.edu.au).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.110.163402
Received July 25, 2010.
Published October 21, 2010.
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