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A Talk With Mike Cohen and Dixon's Violin
in Music
On this week's radio show, my guests are Mike Cohen and Dixon's Violin. Michael H. Cohen is an internationally renowned Kirtan artist, teacher, healer and leader who guides Kirtan participants into heights of joy, energy and awareness through his unforgettable style of Kirtan music. Dixon's life mission is to inspire people. He has done so across North America captivating audiences at intimate shows and wowing crowds of thousands, including Burning Man and giving two TED talks/performances.
Black Violin drops by the Conversation
in Self Help
On "the Conversation" tonight!!! We're featuring 3 great guests who have done some amazing accomplishments and we're excited to hear HOW?
Black Violin: To most people, jazz, hip-hop, funk, and classical are musical genres. But to revolutionary music group Black Violin, they're nothing but ingredients.
Combining a daunting array of musical styles and influences to produce a signature sound that is not quite maestro, not quite emcee, this group of two classically trained violinists and their DJ is redefining the music world-one string at a time.
LaTosha Brown: We've discovered a fabulous singer who's making her mark in the indie industry! Sit back and let us delve into her comings and goings AND let's listen to her melodic voice….
Lola Jenkins: We're sitting down w/ Lola Jenkins and her Quilt Artistry!!! If you love folk art and design you'll love her amazing work!
Tune in and enjoy!!! The nextwork you can't LIVE Without!!!
Pablo Casals
"Forgotten music on 78 records" by Curt Hahn
Pablo Casals
Pablo Casals – Le Cygne (The Swan)
Moment Musical
Link to podcast to listen and enjoy this music:
For information on the background of the songs, Requests or contacting Curt Hahn, write to www.Facebook.com/s3productions2 or Forgotten Music on 78 records or email@example.com
All on Shellac 10" 78 rpm and released in the US
Pablo Casals – Le Cygne (The Swan) / Moment Musical
Le Cygne (The Swan) / Moment Musical (Shellac, 10")
Victrola 1143
A: Le Cygne (The Swan) Violin cello solo
The Carnival of the Animals (Le carnaval des animaux) is a musical suite of fourteen movements by the French Romantic composer Camille Saint-Saëns. The work was written for private performance by an ad hoc ensemble of two pianos and other instruments, and lasts around 25 minutes.
Composer Saint-Saens piano Nicolai Medikoff
B: Moment Musical Violin cello solo
Composer Shubert
Piano Nicolai Medikoff
Pau Casals i Defilló (Catalan: December 29, 1876 – October 22, 1973), known during his professional career as Pablo Casals, was a Spanish Catalan cellist and conductor. He is generally regarded as the pre-eminent cellist of the first half of the 20th century, and one of the greatest cellists of all time. He made many recordings throughout his career, of solo, chamber, and orchestral music, also as conductor, but he is perhaps best remembered for the recordings of the Bach Cello Suites he made from 1936 to 1939
A Theory of Parenting - Philharmonic Concert in the Park
June 19, 2015
WFMT Radio Network Broadcast
downloads at: nyphil.org/watchlisten
welcome to A theory of Parenting on blogtalkradio.com
my name is tammi joyner
i am your host for the show
today we are recapping the New York Philharmonic Concerts in the Park - presented by Didi and Oscar Schafer
Thursday June 18, 2015 at 8:00pm
location: The Great Lawn Central Park, Manhattan NY
Concert: 15,926 in the park
Conductor: Charles Dutoit
Violin: Renaud Capucon
This program is supported in part by public fund from the New York city dept. of cultural affairs in partnership with the City Council.
The concert in the part are presented in cooperation with the city of new york dept of parks and recreation bill de blasio mayor the borough president and city council of new york
Time length of the concert: one and three quarters hours one intermission
-Roman Carnival Overture
-Violin Concerto
-La Valse
NyAirWaves Radio Show! Live Interview With Rock Band Stereo Love!
in Music
Tune in and get into the NOW .... From the People's Perspective. Get the HOTTEST gossip, advice, interviews, prizes , and MUCH MORE!!!
** Tonight's Guest**
Jewel Steele picked up the guitar when she was 9 years old and has been playing non-stop ever since. Jewel began performing in public the Summer of 2010 on her local street corner. Jewel Steele is endorsed by Daisy Rock Girl Guitars and she also rocks the violin and drums too. Lula Steele picked up the bass guitar when she was 10 years old. Influenced by her jazz musician Grandfather, Lula also plays Upright Bass and was the only female bass player in her middle school orchestra. Lula is endorsed by Daisy Rock Girl Guitars and loves to thump da bass everytime she gets a chance. Judd Steele brings years of playing drums to the table. Judd has toured for the troops, playing in Afghanistan and other destinations around the world. Looking for a fun family activity to do together, Judd being a rock and roll dad, decided to start a band with his two daughters and wala... a 3-piece made in heaven! Judd plays Premier, English made drums, like his father, Judd Sr., did. STEREO LOVE recently won 'Hollywood Rocks' at the Las Vegas Rocks Music Awards 2013. The band played some 200+ shows since 2011... including festivals, clubs, bars and schools all the way from the west coast to Las Vegas. STEREO LOVE's motto is "good things will come to those, if you work like hell while waiting".
Website: www.StereoLove.us
Tony DeBlois: Journey of the Heart
in Motivation
Our Host:
Svetlana Kim, Speaker, Author of White Pearl and I: A Memoir of a Political Refugee, Speaker, Consultant, and Community Advocate. www.svetlanakim.com
Our Guest:
Tony DeBlois was born weighing 1 lb., ¾ oz. But that was only the first obstacle that he had to overcome. Tony is also blind and autistic, with Savant Syndrome.
This 41-year-old pianist from Randolph, MA, who has been playing piano since age two, was the subject of a movie inspired by actual events in Tony's life. "Journey of the Heart," with Cybil Shepard, was a 1997 CBS made-for-TV movie of the week. Additionally, he has appeared on 2 Catholic Global Showcase Specials (2001) and The Learning Channel's "Uncommon Genius," Strange Science "Unusual People," and "Understanding the Mysteries of Memory."
Tony graduated magna cum laude from Berklee College of Music. In addition to piano and organ, he plays harmonica, guitar, harpsichord, English hand bells, violin, banjo, drums, saxophone, clarinet, ukulele, mandolin, flute and trumpet. His extensive repertoire includes jazz, classical, pop, rock and show tunes. Tony's favorite phrase seems to be, "I haven't learned that yet." He is on Facebook and has a website, www.tonydeblois.com.
Kristy Cambron and June Foster
in Books
Kristy Cambron fancies life as a vintage-inspired storyteller. Her debut historical novel, THE BUTTERFLY AND THE VIOLIN (HarperCollins Christian Publishing, 2014), was named to Library Journal Reviews' Best Books of 2014 and Family Fiction's Top Ten Novels of 2014. Her debut also received a nomination for RT Reviewers' Choice Awards Best Inspirational Novel of 2014 and a nomination for the 2015 INSPY Awards for Best Debut Novel. Her second novel, A SPARROW IN TEREZIN (HarperCollins Christian Publishing, April 2015), was named Library Journal Reviews' Pick of the Month (Christian Fiction) for February 2015 and a Top Pick for RT Book Reviews.
An award-winning author, June Foster is also a retired teacher with a BA in Education and a MA in counseling. She is the mother of two and grandmother of ten. June began writing Christian romance in 2010. She penned her first novel on her Toshiba laptop as she and her husband traveled the US in their RV. Her adventures provided a rich source of information for her novels. In 2013, June's book Give Us This Day was a finalist in EPIC's eBook awards and in 2014 a finalist in the National Readers Choice Awards for best first book. Ryan's Father won The Clash of the Titles book of the month for January 2014 and was one of three finalists in the published contemporary fiction category of the 2014 Oregon Christian Writers Cascade Writing Contest and Awards. Deliver Us was a finalist in COTT's 2014 Laurel Awards.
June enjoys writing stories about characters who overcome the circumstances in their lives by the power of God and His Word. June uses her training in counseling and her Christian beliefs in creating characters who find freedom to live godly lives.
The Importance And Value In Hiring Creative And Professional Entertainment
in Lifestyle
Wednesday May 27th on Y-CLAD CHAT, we have special guest Gigi from Music By Gigi. She will play something special live on our show, just for our listeners.
Gigi is one of the most in-demand violinists in New York City. A native of St. Petersburg, Russia, she is a graduate of the St. Petersburg Conservatory, where she studied violin performance. Gigi is also a Juilliard educated violinist. She has been dazzling audiences all over the world with her vibrant and luxurious sound. She has shared the stage with stars like Steven Tyler and Josh Groban and has appeared on The Today Show, The View, Good Morning America and so much more.
She has been recognized by and performed for today's most successful lifestyle gurus and event planners such as Colin Cowie, Marcy Blum, Bryan Ranelli and Martha Stewart.
Gigi founded Music By Gigi so that anyone planning an event will have access to the best in entertainment. Because Music By Gigi was founded by a professional musician, the entertainers on staff are the most professional, highly trained, sophisticated, and FUN entertainers for hire.
Music By Gigi offers a full spectrum of event entertainment from musicians to performers, and can bring entertainment to you anywhere in the country. Best of all, they can work within most budgets to make your event spectacular.
Designer Feature: Chain & Spikes Necklace by Adia Kibur Collection.
Chain & Spikes Necklace is a silver and Hematite chunky 4-tier necklace with one tier of spikes. Each tier has different widths and sizes to add drama. It's definitely a conversation piece and as part of Y-Clad's more affordable line we're offering the look without the hefty price tag.
Souvenir De Moscow, William Tell Overture
Playing Forgotten Music on 78 RPM Records by Curt Hahn
Victor Record Souvenir De Moscow
Performed Mischa Elman
William Tell overture / Arthur Pryor's Band
Podcast Link:
All credit due to the artist, musicians and Record Companies
No profit or income derived from this podcast.
Have a historic 78 RPM and story you want to share with my listeners? Contact me at:
www.facebook.com/s3productions2 or firstname.lastname@example.com
Souvenir de Moscow (Primary title)
Mischa Elman (instrumentalist: violin)
Percy B. Kahn (instrumentalist: piano)
Takes 1-3.
Josef Bonime (instrumentalist: piano)
Takes 4-11.
Master Size: 12-in.
Discography of American Historical Recordings, s.v. "Victor matrix C-8814. Souvenir de Moscow / Mischa Elman," accessed May 21, 2015, http://adp.library.ucsb.edu/index.php/matrix/detail/200008913/C-8814-Souvenir_de_Moscow.
Victor Record 35120 (Black label (popular) 12-in. double-faced)
Victor C-1275 9/23/1909
William Tell overture / Arthur Pryor's Band
Discography of American Historical Recordings, s.v. "Victor 35120 (Black label (popular) 12-in. double-faced)," accessed May 21, 2015, http://adp.library.ucsb.edu/index.php/object/detail/86756/Victor_35120
Struck by Affiniti Irish Trio Kickstarter on Tamara Leigh's Trend On
Everybody loves it when the luck of the #Irish goes their way, but this classically trained Celtic trio is looking for a little love & support back to their kickstarter campaign. Artists Emer Barry (soprano vocals), Aisling Ennis (harp), & Mary McCague (violin) join Tamara Leigh's Trend On - Engaging Your Audience to Trend On You! to share their current kickstart campaign LIVE Wed., 5/13/15 at 10amCT.
tamaraleighllc.com; @tamaraleighllc;
http://www.affinitimusic.com/ http://kck.st/1JNVFZj
Kickstarter: https://www.kickstarter.com/projects/726507088/struck-by-affiniti-help-us-create-a-new-album-and
Inside Raleigh with Featured Guest Alice Osborn
in Business
Alice Osborn's (http://aliceosborn.com/) past educational and work experience is unusually varied, and it now feeds her work as a poet, as well as an editor-for-hire, writing coach, Reiki Master practitioner, dynamic speaker/facilitator. After the Steaming Stops is her most recent collection of poetry; previous collections are Right Lane Ends and Unfinished Projects. Alice is also the editor of the anthologies Tattoos and Creatures of Habitat, both from Main Street Rag Publishing Company.
She's currently at work on her upcoming collection, Heroes without Capes. A Pushcart Prize nominee, her work has appeared in the News and Observer, The Broad River Review, The Pedestal Magazine, Soundings Review and in numerous journals and anthologies.She serves on the North Carolina Writers' Network Board of Trustees. When she's not editing or writing, Alice is a competitive Irish dancer, as well as an aspiring musician on guitar and violin. She lives in Raleigh with her husband, two children and four messy birds.
Education: MA English, Rhetoric/Composition, North Carolina State University
Master's Thesis, "A Conversation in the Composition Classroom: Finding Our Voice Through Creative Nonfiction" Advisor: Chris M. Anson, Phd
Whirl With Merle
in Travel
Jenny Undercofler is a leading force in contemporary music education, having served as the music director for the Kaufman Center's Special Music School since 2004. The Special Music School is the only public school in New York City that integrates pre-conservatory music education with academic studies, providing an unparalleled training environment for musically gifted children.
Ms. Undercofler also co-founded and leads the youth ensemble Face the Music. In residence at the Kaufman Center, Face the Music breaks the boundaries of classical music education and performance, featuring today's music presented by the emerging artistic voices of tomorrow. The group burst onto the scene in 2005, performing works by such new-music mainstays as Michael Gordon, Phil Kline and John Adams.
Her daughter,Amelia a 4th grader, plays the violin and viola as part of this progrsm.
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StartUpHire is the place to find Manager of Benefits jobs. Review the list of Manager of Benefits career opportunities to the left. These Manager of Benefits jobs represent some of the most rewarding careers available.
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At Tender Rose, we work one-on-one exclusively with people who have dementia, maximizing quality of life and specializing in challenging cases. We're looking for a dynamic, proactive, disciplined salesperson in San Francisco to join our growing agency.7/23/2015 San Francisco, CA Save This Job
Our clients include some well-known names in film, tv and online video. You will set up and deploy measurable video, programmatic, mobile and social campaigns in advanced platforms. You will be a day-to-day campaign manager and optimize campaigns which pi7/6/2015 New York, NY Save This Job
Location Cleveland, OH 44115 Other Location Description This role involves managing the day to day activities and employees within your team. Our Customer Success Manager should work to ensure consistency with customer interactions, internal documentation, and cross department7/29/2015 Cleveland, OH Save This Job
New York, NY Advertising Sales Job Description Complex is the go to informational source for the millennial male consumer through videos, digital content, and events. Our hyper growth over the last several years has led us to create this new and important role in the Sales6/29/2015 New York, NY Save This Job
Location: Mountain View, CA Department: Product Management Elasticsearch is on a mission to make real-time data exploration easy and available to anyone. We spend our days engineering the world?s most advanced open source search and analytics engine, which is rapidly becoming the6/21/2015 Mountain View, CA Save This Job
MaxPoint is seeking a highly motivated Manger of Software Engineering (Infrastructure) to lead a team of software engineers responsible for developing software solutions that drive the real-time bidding and delivery of web-based advertising campaigns. The ideal candidate is6/7/2015 Morrisville, NC Save This Job
NO PHONE CALLS OR AGENCIES PLEASE. In this newly created position, the Program Manager is responsible for the management of all daily program activities for one or more of Company's clinical stage programs. He/she will work closely with functional representatives for assigned6/13/2015 Watertown, MA Save This Job
Full time 2015-07-17 Nashville, TN The Project Manager is responsible for the successful deployment of ENA products and services to individual customers. Provides end-to-end project leadership, coordinating internal and external resources in the completion of project7/24/2015 Nashville, TN Save This Job
Makerâs Row is redefining âMade in Americaâ for the 21st Century. We are looking for a Senior Digital Product Manager who is obsessed with digital innovation, and will drive our product strategy and process to the next level.7/9/2015 Brooklyn, NY Save This Job
We are seeking a talented Product Manager to coordinate the conceptual and technical development of new online and mobile products and experiences for consumers and partners.6/29/2015 Washington, DC Save This Job
Main Street Hub is the largest âdo-it-for-youâ marketing platform for great local businesses. Our mission is to create thriving local economies. Come join the #Hublife!7/1/2015 Austin, TX Save This Job
Startup Saas marketing analytics platform seeking Data savvy Junior, Mid and Senior Data Analyst/Client Success Managers to play a key role in driving North American expansion. Equity and make an impact!6/30/2015 NY Save This Job
The Glooko Healthcare Clinical Program Manager develops and executes on strategies and plans to clinically measure outcomes from Glooko usage through Glooko sponsored studies and partner driven studies.7/13/2015 Palo Alto, CA Save This Job
We are looking for a Product Manager to join our rapidly growing team and develop next-generation, mobile social gaming experiences for our users. Many of our games have been in the App Store's top 10 grossing list, and some of our better knownSan Francisco, CA Save This Job
In order to support our rapid growth and reach the next level of scale, we are currently seeking exceptionally talented consulting professionals who can drive successful customer deployments and continuously enhance our services portfolio and engagement b7/18/2015 San Francisco, CA Save This Job
In order to support our rapid growth and reach the next level of scale, we are currently seeking exceptionally talented consulting professionals who can drive successful customer deployments and continuously enhance our services portfolio and engagement b7/18/2015 New York, NY Save This Job
As Recruiting Manager, you will find and attract top talent for Via, introducing candidates to our ground-breaking company and guiding them through the hiring process.7/7/2015 New York City, NY Save This Job
SIM Partners is looking for a Product Marketing Manager to transform ah-ha moments into remarkable products. Reporting to the CMO, and working closely with the product and marketing teams, the Product Marketing Manager will lead and execute product market7/16/2015 Chicago, IL Save This Job
Engagement Managers own a book of clients and are responsible for helping those clients unlock the full potential of their business on the AppNexus platform. The EM builds trusted advisor relationships with senior executives, seize opportunities to unlock6/7/2015 New York, NY Save This Job
The Manager, Global Compensation and Rewards will leverage the large data resource behind Quantcast's greatest asset, our talented employees, to ensure that we continually foster a high performance, open culture. We are evolving our compensation pro6/29/2015 San Francisco, CA Save This Job

subset: commoncrawl


text:
While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening.
PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit.
These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.
Keywords:Apple (Malus x domestica); Fruit softening; Intercellular adhesion; Polygalacturonase; Texture; Pectin
Fruit softening is a complex developmental programme that involves the disassembly of various pectin and hemicellulose components of the primary cell wall, as well as alterations to cell turgor and fruit water status [1-4]. Pectin components of the cell wall are diverse, consisting of domains of contiguous 1,4-α-linked GalA residues known as homogalacturonan, and two different domains of rhamnogalacturonan [5]. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the cell wall where two adjacent cells abut, and is believed to play a major role in intercellular adhesion [6,7]. During fruit ripening, homogalacturonan can be depolymerised by endo-polygalacturonases (PGs), cell wall-localised enzymes that cleave stretches of unesterified GalA residues and weaken the middle lamella [8]. In ripe fruit, calcium cross-links between stretches of de-methylesterified GalA residues of homogalacturonan provide the main bonding between adjacent cells [9,10].
In many species, softening during early ripening does not appear to involve pectin depolymerisation, but a correlation between PG gene expression and softening rate, together with QTL analysis, has consistently implied some involvement of PG in the softening and textural change of fleshy fruit, principally during late ripening [4,11-21]. Despite extensive study, the specific contribution of endo-PGs to fruit softening remains an open question - particularly since studies where PG mRNA abundance and activity were directly manipulated in transgenic tomato and strawberry fruit have produced quite contrasting results.
In tomato (Solanum lycopersicum), down-regulation of the ripening-specific SlPG gene only slightly reduced the softening of the fruit [22,23]; and over-expression of SlPG in the ripening-inhibited rin background partially restored PG activity but did not restore fruit softening [24]. Cell wall analysis subsequently showed that pectin depolymerisation during ripening was not prevented and was only slightly reduced in SlPG-suppressed fruit [25], presumably due to the incomplete gene silencing that left ~1% of PG activity remaining [22]. Nevertheless, these experiments were interpreted as indicating that PG-mediated pectin depolymerisation was neither necessary nor sufficient for tomato fruit softening. However, in strawberry (Fragaria X ananassa), down-regulation of the ripening-related FaPG gene led to significantly firmer fruit [26], suggesting that PG plays a central role in strawberry fruit softening.
Ripening in both tomato and strawberry is associated with a rapid loss of firmness producing a soft fruit. In contrast, fruit of other species such as water melon (Citrullus lanatus), Asian pear (Pyrus pyrifolia) and apple (Malus domestica) don't soften to the same extent and are still crisp when ripe [27]. The role of PGs in determining crisp fruit texture has not been directly assessed. Apple possesses a PG gene (PG1) that increases in expression during fruit ripening and after exposure of apple fruit to ethylene and the cold temperature [28-31]. Over-expression of PG1 in apple trees led to increased intercellular separation in leaves [32], demonstrating a role for PG in the loss of intercellular adhesion. This was also suggested by Ben-Arie et al.[33], who showed that application of tomato PG to apple fruit discs caused similar ultrastructural changes to those occurring in over-ripe fruit, including dissolution of the pectin-rich middle lamella. Two genetic studies have also pointed to PG as being an important determinant of apple fruit softening: a major fruit firmness-related QTL has been mapped to the PG1 locus on chromosome 10 [15,34] and reduced levels of PG1 expression have been correlated with firmer fruit [35,36].
In this paper we investigate the role of PG1 in determining apple fruit texture by generating antisense apple lines with the fruit-specific PG1 gene suppressed. The fruit were phenotyped for a range of chemical, ultrastructural and biophysical features to assess changes in the cell wall, cell packing and the mode of tissue failure. The present study provides new evidence for the role of PG1 in apple fruit, and presents new opportunities for understanding physiological processes not previously linked with PG.
Suppression of PG1 leads to firmer apple fruit and reduced pectin depolymerisation
To directly assess the role of PG1 in apple fruit ripening, a binary vector containing a construct with the cauliflower mosaic virus 35S promoter controlling the transcription of a PG1 transgene in the antisense orientation was assembled. Ten independent PG1 antisense (PG1as) transgenic apple lines were generated in the cultivar 'Royal Gala' using Agrobacterium-mediated transformation [37]. These plants were grown alongside untransformed 'Royal Gala' control lines in a containment greenhouse.
In the first two seasons of fruiting, only some of the PG1as lines produced small numbers of fruit (n = 4–10). Fruit were harvested when mature and assessed when ripe using a destructive puncture (penetrometric) test in the fruit cortex. One transgenic line, PG1as-41, was identified as having fruit that were consistently firmer when compared with those obtained from untransformed 'Royal Gala' controls and the other transgenic lines (Figure 1A).
Figure 1. Cortical firmness of transgenic PG1-suppressed and 'Royal Gala' control fruit harvested from three seasons.A Cortical firmness of wild-type 'Royal Gala' (RG) apple fruit and transgenic PG1-suppressed (PG1as) lines in fruiting seasons 1 and 2. Fruit firmness was measured after ripening for 32 weeks at 5°C (n = 4–10 fruit ± Standard Error). B Mean loss of cortical firmness of transgenic PG1-suppressed and 'Royal Gala' fruit in season 3 after ripening for 16 weeks at 5°C. (n = 6–10 fruit ± Standard Error). Lines PG1as-41 and PG1as-275 highlighted in black showed the least change in firmness.
In season 3, all ten independent PG1as lines produced fruit. Relative to controls, no differences in final fruit size or shape were observed (data not shown). Two transgenic lines (PG1as-41 and PG1as-275) were identified that were firmer after ripening than 'Royal Gala' controls (Figure 1B). In these two lines, softening was only ~20-30 N compared with ~40-50 N in the control and the other PG1as lines.
For each of the transgenic lines, proteins were extracted from mature fruit at harvest and from fruit following ripening. The extracts were then screened for the presence of immunodetectable PG1 protein using protein gel blot analysis with a polyclonal PG1 antibody. At harvest, low levels of PG1 protein were detected in control fruit and one PG1as line, PG1as-290 (Figure 2A). After ripening, PG1 protein was observed at higher abundance than at harvest in the control and PG1as-290 lines. PG1 protein was also detected in seven other PG1as lines, but was undetectable in fruit of lines PG1as-41 and PG1as-275 (Figure 2B). These two independent transgenic lines with low PG1 protein abundance were also the lines with the firmest fruit after ripening (Figure 1B) and were therefore chosen for further detailed study.
Figure 2. Molecular characterisation of transgenic PG1as apple lines. Western analysis of protein extracts from fruit of transgenic PG1as lines and 'Royal Gala' (RG) control after SDS-PAGE (8 μg protein per lane) at harvest (A), and after ripening for 16 weeks at 5°C (B), using a polyclonal antibody raised to recombinant apple PG1. mRNA abundance of PG1(C), and a ripening-related apple β-GALACTOSIDASE gene (D)[38], at harvest (H) and after ripening for 16 weeks at 5°C (16 w). mRNA abundance of the PG1as-41 sample at harvest was set as 1 (n = 4 ± Standard Error).
In untransformed 'Royal Gala' controls, PG1 mRNA abundance was strongly induced during ripening (Figure 2C). In the transgenic lines, the suppression of PG1 protein accumulation was reflected in the abundance of the PG1 mRNA, with PG1as-275 and PG1as-41 fruit showing a 75% and 92% reduction in PG1 mRNA accumulation during ripening, respectively, compared with the control (Figure 2C). As an internal ripening control, the expression of a ripening-related apple β-GALACTOSIDASE gene [38] was shown to be induced to a similar level in PG1as-41, PG1as-275 and control fruit (Figure 2D). This result suggested that other aspects of cell wall metabolism related to ripening were proceeding as normal.
In vitro, PGs hydrolyse the homogalacturonan backbone of pectin polymers with a low degree of methylesterification, and hence may play a role in pectin solubilisation and depolymerisation during fruit ripening. To determine whether there was a difference in the pectin components of the cell walls of the two PG1-suppressed lines compared with 'Royal Gala' controls, cell walls were prepared from fruit after ripening then extracted to solubilise pectins. Sequential extractions solubilised water-soluble pectin, which is essentially unattached to the cell wall, followed by CDTA-soluble pectin, which is held in the wall by calcium bonds and is attributed to pectin of the middle lamellae. Relative to controls, suppression of the PG1 gene substantially reduced the amount of water-soluble pectin and increased the amount of CDTA-soluble pectin (Figure 3A). The polyuronide content in the water-soluble extract from the PG1as-41 line was less than half that of 'Royal Gala' control fruit, with PG1as-275 being intermediate, but also substantially reduced relative to the control. A corresponding increase in the amount of CDTA-soluble polyuronides was found in the two PG1-suppressed lines, which possessed a polyuronide content at least 50% greater than the control. These data indicate that in the PG1-suppressed lines pectin is less soluble than in controls, and a greater proportion is retained in the wall by calcium bonds.
Figure 3. Polyuronide content and size distribution of water-soluble and CDTA-soluble polyuronides from the cell walls of transgenic PG1as and 'Royal Gala' (RG) control apple fruit after ripening for 16 weeks at 5°C.A) Polyuronide content, expressed as mg of uronic acid per g fresh weight of tissue, of water-soluble extracts (white bars) and CDTA-soluble extracts (grey bars) (n = 3 ± Standard Deviation). B) Size distribution of water-soluble polyuronides, and (C) CDTA-soluble polyuronides after elution on Sepharose CL-2B (profiles are typical examples from two extractions). Arrows indicate elution of dextran molecular weight markers (kDa).
Analysis of the size distribution of water-soluble polyuronides showed that the relative average molecular mass was similar between PG1as lines and 'Royal Gala' control, with the polyuronides of the two transgenic lines on average being only slightly larger than the control (Figure 3B). The CDTA-soluble polyuronides from both PG1as-41 and PG1as-275 were considerably larger than in the 'Royal Gala' control, seen as a substantial shift in the peak average molecular weight to higher molecular mass (Figure 3C).
Suppression of PG1 reduces water loss from ripe fruit and changes the structure of hypodermal cell layers
The rate of water loss in 'Royal Gala' control, PG1as-41 and PG1as-275 fruit was assessed at 20°C for 9 weeks following low temperature ripening. After 9 weeks, the PG1as-41 and PG1as-275 fruit showed less shrivelling compared with control fruit (Figure 4A). Whereas control fruit exhibited a relatively large mean decrease in fresh weight of 0.36% weight loss per day, the two independent PG1-suppressed lines showed only 0.21% (PG1as-41) and 0.23% (PG1as-275) weight loss per day (Figure 4B). This unexpected observation indicated that silencing the PG1 gene reduced water loss from the fruit.
Figure 4. Transpirational water loss in transgenic PG1as and 'Royal Gala' control fruit. Fruit were initially ripened at 5°C for 16 weeks then transferred to 20°C for 9 weeks. A) Representative photograph of PG1as-41, PG1as-275 and 'Royal Gala' (RG) control fruit after being held for 9 weeks at 20°C. Note how the PG1as-41 and PG1as-275 fruit were substantially less wrinkled than the control. B) Weight loss (representing mainly water loss) was measured every 1–2 weeks for the 9 weeks fruit were held at 20°C. Weight loss is presented as a percentage of starting weight after transfer to 20°C (n = 4–6 ± Standard Error and T-test statistics between apples containing the transgene and untransformed controls are shown).
To assess whether this difference in water loss was due to changes in cellular structure of the exocarp (i.e. the skin including cuticle, epidermis and hypodermis), light microscopy was conducted on fruit at harvest and following ripening. In the 'Royal Gala' control (Figure 5A-D), toluidine blue staining of sections of the exocarp and outer cortex showed a hypodermal layer of small cells with thickened cell walls 4–5 cells deep that extended to a depth of ~50–70 μm into the fruit (Figure 5A, D). The walls of these cells stained densely at harvest. Following ripening, the cells of the hypodermal layers in all control samples appeared to be larger and more rounded, and the cell walls (particularly in the corners) were swollen, increasing the depth of this zone to ~100 μm (Figure 5C, D). In PG1as-41 fruit at harvest, the hypodermal layer was similar to the 'Royal Gala' control (Figure 5E, F). However, following ripening the hypodermal layer in all samples of PG1as-41 fruit did not appear to undergo the same level of cell swelling, maintaining a more 'at-harvest' appearance than the ripened 'Royal Gala' control (Figure 5G, H).
Figure 5. Structural changes occurring in the exocarp and cortex of transgenic PG1as and 'Royal Gala' control fruit. Toluidine blue-stained sections from: 'Royal Gala' (RG) at harvest (A, B) and following ripening for 16 weeks at 5°C - stored (C, D); PG1as-41 at harvest (E, F) and following ripening for 16 weeks at 5°C - stored (G, H). Coloured lines show the approximate extent of the epidermis/cuticle (blue), hypodermal cell layers (red) and cortex (green). Note the thickening of the hypodermal layers observed in the control (C, D) does not occur in the suppressed PG1as-41 line (G, H). Scale bars = 100 μm (A, C, E, G) and 50 μm (B, D, F, H).
Suppression of PG1 in the fruit cortex changes intercellular adhesion and alters fracture properties
Underneath the hypodermal layers, apple cortical cells are larger and rounder than near the epidermis and there are more visible intercellular spaces (Figure 5A). At harvest, the cell walls in this region stained similarly in both the 'Royal Gala' control and PG1as-41 fruit. Cell walls typically stained evenly, except at the junctions between three cells, or between two cells and an intercellular space where staining was more intense. There was no sign of cell-to-cell separation in either controls or in PG1-suppressed fruit (Figure 6A, B). Following ripening, cells in the 'Royal Gala' control showed separation at the corners of attachment between three cells and along the middle lamella (Figure 6C). Once the corners had separated, two or three remnants of wall material protruded into the intercellular space. In the PG1as-41 fruit, neither separation of the corners nor protruding cell wall remnants were observed (Figure 6D).
Figure 6. Intercellular adhesion in the cortex tissue of 'Royal Gala' (RG) control (A, C, E) and transgenic PG1as-41 (B, D, F) fruit. Toluidine blue-stained sections of cell wall boundaries at harvest (A, B); and following ripening for 16 weeks at 5°C - stored (C, D). Immunolocalisation of a pectin epitope with a low degree of esterification in the cell walls of apple fruit after ripening for 16 weeks at 5°C visualised using the antibody JIM5 (E, F). Arrows indicate materials filling the space where two cells join (A, B, D, F). In (C) and (E), solid arrowheads indicate remnants of cell wall material protruding into the intercellular space. In (C) the open arrowhead indicates a region where material has been lost between adjacent cells. Bar = 10 μm.
Immunolabelling of ripened 'Royal Gala' cortex tissue with the JIM5 antibody (recognising homogalacturonan with a low degree of methylesterification) showed labelling along the cell wall, and strong labelling of the cell wall protrusions (Figure 6E). In ripened tissue from the PG1as-41 line, labelling was evenly spread over the cell wall and no cell wall protrusions were observed (Figure 6F). These changes in cell separation and immunolabelling suggested that there might be a change in intercellular adhesion properties that could be measured using a tensile test (force needed to pull tissue apart) rather than a puncture test (mixture of compression and shear forces).
To test this hypothesis, in season 4, 'Royal Gala' control and PG1as-41 fruit were re-assessed using puncture and tensile tests at harvest and following a shorter ripening treatment (10 weeks, 0.5°C). At harvest, there were no significant differences in maximum puncture firmness (Figure 7A) or maximum tensile force between the 'Royal Gala' control and PG1as-41 apples (Figure 7B). In contrast, in ripened fruit, maximum puncture firmness and maximum tensile force were both significantly higher in PG1as-41 apples compared to the 'Royal Gala' control (Figure 7A, B). Force deformation analysis showed that the ripened PGas-41 apples had a higher Young's modulus (gradient of stress–strain curve) and higher work (area under stress–strain curve) for both puncture and tensile tests when compared to 'Royal Gala' control apples (Figure 7C-F). However these two differences in mechanical properties correlated with fruit firmness, with apples of similar firmness showing the same mechanical properties regardless of the presence of the transgene. Different probe sizes were also used to assess the relative contributions of shear (perimeter) and compression (area) forces during the puncture test. This approach showed that the compression forces were dominant and closely reflected the maximum firmness values (Figure 7G). In contrast, while a more minor component, the shear forces were higher for the PG1as-41 apples both at harvest and after ripening (Figure 7H). This was the only mechanical feature which was higher in the PG1as-41 apples at harvest.
Figure 7. Differences in puncture maximum force (A), tensile maximum force (B), puncture gradient calculated from stress–strain curve (C), tensile gradient calculated from stress–strain curve (D), puncture work calculated from area of stress–strain curve (E), tensile work calculated from area of stress–strain curve (F), compression force coefficient (G), and shear force coefficient (H), between transgenic PG1as-41 and 'Royal Gala' (RG) control fruit at harvest and following ripening at 0.5°C for 10 weeks (n = 15 ± Standard Deviation). Statistics for A-F were performed using a T-test. The coefficients and standard errors in (G) and (H) were determined using non-linear regression. Scanning electron microscope views are of a 'Royal Gala' section (I) and a PG1as-41 section (J) taken after tensile tests from fruit following ripening. White arrow heads show the presence of ruptured cells. Scale bar represents 500 μm.
Sections of ripened fruit after the tensile strength tests were snap frozen in liquid nitrogen and examined on a cold stage scanning electron microscope (SEM). The fracture surfaces of the pulled-apart sections of 'Royal Gala' showed areas of undamaged, un-ruptured rounded cells and low juice (Figure 7I, Additional file 1: Table S1), whereas sections from PG1as-41 showed a relatively large number of damaged (deflated) cells and sheet-like layers of juice obscuring cortex cells, indicating more cellular fractures and ruptures, and more free fluid in the cortex tissue (Figure 7J, Additional file 1: Table S1).
Additional file 1. Table S1. Assessment of cell integrity and juice in pulled-apart sections.
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Historically, apple has been reported to lack detectable depolymerisation of pectin during ripening [39]. Here we show that suppression of apple PG1 results in a clear increase in the average molecular mass of pectin, particularly in a CDTA-soluble fraction. Since PG1-suppressed fruit were significantly firmer than control fruit, it appears that the molecular weight distribution of pectin influences the firmness of apple fruit, presumably through increased integrity of the middle lamella. This result is consistent with data concerning the down-regulation of FaPG and pectate lyase in transgenic strawberry lines [26,40], but not with those regarding down-regulation of SlPG in transgenic tomato lines. The reason for this difference may relate partly to the degree of PG down-regulation achieved in tomato, and partly to the system studied. There are large differences in the extent to which depolymerisation naturally occurs in these fruit; pectin depolymerisation in both apple and strawberry is slight, whilst in tomato it is more extensive [2]. Tomato possesses unusually high levels of PG activity, and the ~1% of activity remaining in SlPG-suppressed fruit was apparently sufficient to cause almost wild-type levels of pectin depolymerisation [25]. Based on the transgenic work in strawberry and that presented here, it thus seems likely that PG-mediated pectin depolymerisation provides a component of the softening seen in many ripening fruit.
Sensory studies have shown that upon biting and chewing, the primary walls of apple fruit rupture and the cellular juices are released, resulting in juiciness [41]. The link between sensory juiciness and free fluid in pulled-apart sections in apple has been correlated with differences in pectin composition of which the most notable is a reduced galacturonic acid content in the water-soluble pectin fraction [42]. In this study, assessments of tensile properties and shear forces during puncture testing in conjunction with scanning electron microscopy showed that ripening in apple is normally accompanied by decreasing strength of the middle lamellae, resulting in reduced intercellular adhesion and cell separation under stress. In PG1-suppressed tissue, middle lamella strength was retained and the point of failure under stress became the primary cell walls, resulting in cellular rupture and release of free fluid. These higher levels of free fluid and increased tensile strength and shear forces indicate potential for a fruit with a juicier, crisper texture.
An unexpected consequence of PG1 suppression that has emerged from this work is a change in hypodermal anatomy that may influence fruit firmness by regulating water status. Declining cell turgor is a normal part of fruit softening [1,43-45] and is brought about by a combination of the movement of osmotic solutes and water to the apoplast [46-48] and by transpirational water loss from the fruit [3]. In tomato, the lack of softening in the DFD mutant was attributed to very low water loss and increased cellular turgor [3]. The data of Figures 4 and 5 show not only that an increase in transpirational water loss from the fruit is a part of ripening in apple, but that this is at least in part controlled by developmental changes in the hypodermal layers brought about by the action of PG. In control fruit, a swelling of the hypodermal cell layers occurred during ripening, and was correlated with increasing whole-fruit water loss. In the PG1-suppressed lines, cells in the hypodermal layers of the fruit below the cuticle remained densely packed, which was correlated with slower water loss from the fruit and reduced shrivelling. The cuticle and surface cell layers of fruit are known to change both in wax composition and in thickness and structure during ripening [49,50]. This can include a reduction in the amount of cell wall polysaccharide that is associated with the cuticle [51]. In apple, although the surface conductance declines from an initial high level in developing fruit, the rate of water loss per fruit declines only slightly by the time fruit are ripe [52]. The data presented here suggest that the action of PG may indirectly promote fruit water loss during ripening, in part by reducing intercellular adhesion and allowing a swelling of the hypodermal cell layers (Figure 4C).
A relationship between pectin fine structure and tissue anatomy has been shown both in fruit and in other organs. In potato tubers, fragmentation of rhamnogalacturonan I by a fungal rhamnogalaturononan lyase induced radial swelling of the periderm cells and the development of cortical intercellular air spaces [53]. In tomato fruit, down-regulation of the ethylene-regulated auxin response factor DR12 reduced pectin solubilisation and altered the distribution of methyl ester side groups, which produced a higher proportion of small cells in the outer pericarp and a thinner cuticle, and fruit firmness was increased [54]. Interestingly, over-expression of a fungal PG in tobacco altered cellular arrangement and enhanced fresh weight loss from detached leaves [55], suggesting that PG may play a wider role in altering intercellular adhesion that can affect cell shape and packing, which in some tissue results in increased water loss.
By comparing transgenic apple lines that differ only in the expression of a single gene, we show for the first time the effects of PG1 action on apple cell wall metabolism during ripening. Our data confirm PG1's role in apple fruit softening and textural change and suggest that this is achieved in part by cell wall disassembly and loss of intercellular adhesion. The unexpected effects of PG1 suppression on fruit anatomy and transpirational water loss show that PG1's function in fruit ripening is more complex than previously reported.
Generation of transgenic plants and plant growth
A cDNA derived from the fruit-specific apple polygalacturonase gene MdPG1[32] was ligated in the antisense orientation into pART7 to produce a 35S:PG1-as:nos cassette, and then the cassette was ligated into the Not1 site in the pART27 binary vector [56]. pART27-PG1as was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation, and used to generate 10 independent transgenic apple (Malus domestica 'Royal Gala') plants [37]. Transgenic plants were grown in a containment greenhouse alongside untransformed 'Royal Gala' controls. Plants were grown under natural daylight and temperature conditions in 50 L planter pots. Plants received 8 weeks winter chilling at 7°C each year to promote and synchronise flowering. Flowers were hand pollinated each spring using compatible 'Granny Smith' pollen. Wild-type and PG1as knockdown trees were managed in the same way by thinning fruit to one/two fruit per cluster, with trees having a similar TCA (trunk cross-sectional area). Trees typically carried between 10–30 fruit per plant.
Force deformation analysis during ripening
Fruit were harvested when mature based on background skin colour and starch pattern index [57]. All misshapen, marked or damaged fruit were discarded. Pedicels were removed from all fruit which were then placed in single layer plastic trays under polyethylene plastic liners in commercial cardboard cartons. Fruit were subjected to low temperature ripening in a constant temperature room at 5°C for 16 weeks and a relative humidity of 80-85%, unless stated otherwise. A cold treatment was used, since expression of the apple PG1 gene increases strongly in wild-type fruit after exposure to cold [28] thereby accentuating the physiological effects of altering PG levels in control vs. transgenic fruit. Furthermore, the exposure of apples to cold can be important for ensuring the ethylene climacteric and ripening proceeds fully [58]. The requirement for cold exposure in the present study is also accentuated by the apples being grown in a greenhouse and having less cold night conditioning before harvest than would normally occur in the field.
Mechanical properties during tissue failure were assessed using puncture [57] and tensile tests ( [59]. A puncture test was performed by driving cylindrical probes (2, 5, 8 and 11 mm diameter) into the flesh at a constant speed of 4 mm·s-1 to a depth of 9 mm using a TA.XTPlus Texture Analyser (Stable Microsystems, United Kingdom). All assessments were performed following the removal of the skin (~1-2 mm deep - including cuticle, epidermis and hypodermis) from two opposing locations on the fruit equator. Multiple probe sizes were used to minimise complications associated with performing puncture tests on small fruit and to allow estimates of puncture and shear coefficients [60]. Tensile properties were measured using an excised block of fruit tissue (10 mm × 4 mm), with notches cut at each side through the middle to provide a weakened zone and allow the tissue to be pulled apart with metal claws attached to the TA.XTPlus Texture Analyser. The claws moved apart at a constant rate of 10 mm·min-1 until tissue failure. For both tensile and puncture tests, full force deformation analysis was done to determine Young's modulus (slope before failure), maximum force, distance at maximum force, and work (area under curve) [61].
Antibody generation, protein extraction and western analysis
The mature ORF of apple PG1 was amplified using primers RA136 5'- ACG
To determine the presence of PG protein in apple fruit, frozen powdered tissue (500 mg) was mixed with 50 mg PVPP and extracted with 2 volumes of ice cold low salt buffer (0.2 M Na acetate, 10 mM DTT, 50 mM NaCl, pH 4.7) and allowed to thaw on ice for 10 min before centrifugation for 10 min, 4°C 16000 × g. The extraction was repeated again using 1 volume of ice cold low salt buffer. Two volumes of high salt buffer (0.3 M MES, 10 mM DTT, 0.6 M NaCl, pH 6.0) was mixed with the pellet and incubated on ice for 15 min before centrifugation was repeated. The supernatant was retained and the pellet extracted again with 1 volume of high salt buffer. The two high salt protein extractions were combined and protein concentrations in each sample were measured using a Protein Assay Kit (Bio-Rad Laboratories, Auckland New Zealand) using bovine serum albumin as a standard. Concentrations were verified on gels by Coomassie staining.
Proteins were separated on 12% (w/v) polyacrylamide SDS-Tris-Tricine gels using a Mini-PROTEAN3 electrophoresis system (Bio-Rad Laboratories, Auckland New Zealand), electroblotted onto polyvinyldifluoride membrane, and blocked as described by Atkinson et al.[32]. Proteins were immunodetected using the antiserum raised to apple PG1, which was diluted (1:1000, v/v) in TBS buffer containing 5% non-fat dried milk. Membranes were incubated with an anti-rabbit secondary antibody conjugated to alkaline phosphatase (Sigma-Aldrich), and binding to PG1 was visualised using 1-Step NBT/BCIP (Pierce).
Real-time qPCR
Total RNA was extracted from apple tissue of the 'Royal Gala' control and PG1as-41 and PG1as-275 lines as described in Chang et al.[63]. cDNA synthesis and qPCR were performed as described in Tacken et al.[28]. qPCR primers used for assessment of mRNA abundance of PG1 and the actin reference gene were as described by Tacken et al.,[28]. Primers for the ripening-related apple β-galactosidase gene [38] were MdBGALF 5'-GCCATCAAGT CATGCGAGTA-3' and MdBGALR 5'-CGTGTCATAG TTTGCGAGGA-3'.
Cell wall analyses
Cell walls were prepared and extracted as described in Melton and Smith [64]. Apple cortex was snap frozen in liquid N2, ground to a fine powder and buffer-saturated phenol (UltraPure™, Invitrogen) added. The slurry was homogenised using a Polytron, centrifuged, the supernatants recovered, and the residue washed twice in H2O to collect water soluble polysaccharides. Supernatants were combined, dialysed and freeze-dried to give the water soluble fraction. The pellet was extracted twice with DMSO to remove starch, and the supernatants recovered, dialysed and freeze-dried to give the DMSO-soluble extract. The pellet after DMSO extraction was dialysed and freeze-dried to give the cell wall material (CWM). An aliquot was extracted with CDTA (trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetic acid) in sodium acetate, pH 6.0, and the recovered supernatants were dialysed against ammonium acetate buffer (pH 5.2) for five days, followed by H2O, and freeze-dried to give the CDTA-soluble extract.
Extracts were analysed for uronic acid content after Blumenkrantz and Asboe-Hansen [65] using D-galacturonic acid (Sigma) as a standard. Water- and CDTA-soluble fractions were separated by size exclusion chromatography using a column of Sepharose CL-2B (1.6 × 90 cm; Pharmacia Biotech) eluted with 0.2 M ammonium acetate, pH 5.0, at a flow rate of ~6 ml h-1 and a fraction size of 20 min. The polyuronide content of the column fractions was determined as above. The column was calibrated with dextran molecular weight standards of 2000, 500 and 40 kDa (Pharmacia, Uppsala, Sweden).
Structural analyses
Multiple apples (n ≥5) that were matched for size and ripeness were selected for structural analyses. Pieces of apple fruit tissue 5 mm3 were fixed in either 2% (v/v) formaldehyde with 0.1% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, or 2% (v/v) formaldehyde with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, under vacuum for 1 h, washed in buffer, dehydrated with an alcohol series, and embedded in LR White resin [66]. Embedded material was sectioned at a thickness of 1 μm and stained with 0.05% (w/v) toluidine blue in borate buffer (pH 4.4) for gross cytological examination and at a thickness of 200 nm for immunolabelling [67]. The thin sections were antibody-labelled as described in Sutherland et al.[68]. Light microscopy was carried out on an Olympus Vanox AHT3 compound microscope (Olympus, Tokyo) with image recording by a CoolSnap digital camera (Roper Scientific, Tucson, AZ). Multiple sections (n ≥20) from multiple tissue blocks (n ≥2) were examined to obtain representative views.
Fracture surfaces from fruit after tensile testing were observed using Cryo-Scanning Electron Microscopy [41]. Blocks of tissue 4–5 mm high were cut from the fracture face to fit into 8 mm diameter shallow copper dish holders and secured with tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) for adhesion. The fracture face protruded above the surface of the holder. The assembly was rapidly frozen in liquid N2 and stored in containers under liquid N2. Prior to observation, the holder was transferred under N2 to a copper specimen mount of a Sputter Cryo system (model SP 2000, Emscope, Ashford, UK) and then moved in a dry argon atmosphere to the specimen chamber of the Sputter Cryo. The sample was sputter-coated with gold at a temperature less than −130°C. The sample was transferred under vacuum to a cryo-stage (temperature −150°C or lower) in a PSEM 505 scanning electron microscope (Philips, Eindhoven, The Netherlands), where it was observed using an accelerating voltage of 12 kV. Digital images were collected using an ADDAII digital interface and Scandium software (Olympus Soft Imaging Solutions, Münster, Germany).
PG1: ENDO-POLYGALACTURONASE1; CWM: Cell Wall Material; cDNA: Complementary DNA; qPCR: Quantitative Polymerase Chain Reaction.
Competing interest
The authors declare that they have no competing interests.
Authors' contributions
RGA conceived of the project, DM made the transgenic plants, PWS and ICH undertook the microscopy, SLJ and RS undertook cell wall composition assessments, KG did fruit assessments and molecular and protein characterisation, RGA, RS, JWJ, and RJS designed the experiments and were involved in the data analysis, RGA, RS, DAB, ICH, JWJ and RJS wrote the paper. All authors read and approved the final manuscript.
We thank Paul Pidakala and Miriam Farrell for assistance with fruit physiological measurements; Jia-Long Yao and Jun Hong Ma for supervising the production and screening of transgenic apple plants, Julie Nicholls, Gnanaseela Wadasinghe and Dan Cohen for care of transgenic plants; Neelam Sharma for generation of the PG antibody and Ian Ferguson for comments on the manuscript. This work was supported by the Foundation for Research, Science and Technology of New Zealand (C06X0705 and C06X0403).
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Wakasa Y, Kudo H, Ishikawa R, Akada S, Senda M, Niizeki M, Harada T: Low expression of an endopolygalacturonase gene in apple fruit with long-term storage potential.
Postharvest Biol Technol 2006, 39:193-198. Publisher Full Text
Mann HS, Alton JJ, Kim SH, Tong CBS: Differential expression of cell-wall-modifying genes and novel cDNAs in apple fruit during storage.
Yao JL, Cohen D, Atkinson R, Richardson K, Morris B: Regeneration of transgenic plants from the commercial apple cultivar Royal Gala.
Ross GS, Wegrzyn T, MacRae EA, Redgwell RJ: Apple β-galactosidase - Activity against cell-wall polysaccharides and characterization of a related cDNA clone.
Plant Physiol 1994, 106:521-528. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Yoshioka H, Aoba K, Kashimura Y: Molecular weight and degree of methoxylation in cell wall polyuronide during softening in pear and apple fruit.
Santiago-Domenech N, Jimenez-Bemudez S, Matas AJ, Rose JKC, Munoz-Blanco J, Mercado JA, Quesada MA: Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.
J Exp Bot 2008, 59:2769-2779. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Harker FR, Hallett IC: Physiological changes associated with development of mealiness of apple fruit during cool storage.
Billy L, Mehinagic E, Royer G, Renard CMGC, Arvisenet G, Prost C, Jourjon F: Relationship between texture and pectin composition of two apple cultivars during storage.
Postharvest Biol Technol 2008, 47:315-324. Publisher Full Text
Harker FR, Sutherland P: Physiological chnages associated with fruit ripening and the development of mealy texture during storage of nectarines.
Postharvest Biol Technol 1993, 2:269-277. Publisher Full Text
Thomas TR, Shackel KA, Matthews MA: Mesocarp cell turgor in Vitis vinifera L. berries throughout development and its relation to firmness, growth, and the onset of of ripening.
Planta 2008, 228:1067-1076. PubMed Abstract | Publisher Full Text
Thomas TR, Matthews MA, Shackel KA: Direct in situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water stress.
Plant Cell Environ 2006, 29:993-1001. PubMed Abstract | Publisher Full Text
MacDougall AJ, Parker R, Selvendran RR: Nonaqueous fractionation to assess the ionic composition of the apolast during fruit ripening.
Plant Physiol 1995, 108:1679-1689. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Almeida DPF, Huber DJ: Apoplastic pH and inorganic ion levels in tomato fruit: a potential means for regulation of cell wall metabolism during ripening.
Physiol Plant 1999, 105:506-512. Publisher Full Text
Wada H, Shackel KA, Matthews MA: Fruit ripening in Vitis vinifera: apoplastic solute accumulation accounts for pre-veraison turgor loss in berries.
Planta 2008, 227:1351-1361. PubMed Abstract | Publisher Full Text
Bargel H, Neinhuis C: Tomato (Lycopersicon esculentumMill.) fruit growth and ripening as related to the biomechanical properties of fruit skin and isolated cuticle.
Leide J, Hildebrandt U, Reussing K, Riederer M, Vogg G: The developmental pattern of tomato fruit wax accumulation and its impacts on cuticular transpiration barrier properties: effects of a deficiency in a β-ketoacyl-coenzyme A synthase (LeCER6).
Plant Physiol 2007, 144:1667-1679. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Domínguez E, López-Casado G, Cuartero J, Heredia A: Development of fruit cuticle in cherry tomato (Solanum lycopersicum).
Funct Plant Biol 2008, 35:403-411. Publisher Full Text
Jones HG, Higgs KH: Surface conductance and water balance of developing apple (Malus pumila Mill.) fruits.
J Exp Bot 1982, 33:67-77. Publisher Full Text
Oomen RJFJ, Doeswijk-Voragen CHL, Bush MS, Vincken JP, Borkhardt B, van den Broek LAM, Corsar J, Ulvskov P, Voragen AGJ, McCann MC, et al.: In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side-chains and abnormal periderm development.
Plant J 2002, 30:403-413. PubMed Abstract | Publisher Full Text
Guillon F, Philippe S, Bouchet B, Devaux MF, Frasse P, Jones B, Bouzayen M, Lahaye M: Down-regulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture.
J Exp Bot 2008, 59:273-288. PubMed Abstract | Publisher Full Text
Capodicasa C, Vairo D, Zabotina O, McCartney L, Caprari C, Mattei B, Manfredini C, Aracri B, Benen J, Knox JP, et al.: Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth.
Plant Physiol 2004, 135:1294-1304. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gleave AP: A versatile binary vector system with a T-DNA organizational-structure conducive to efficient integration of cloned DNA into the plant genome.
Plant Mol Biol 1992, 20:1203-1207. PubMed Abstract | Publisher Full Text
Johnston JW, Gunaseelan K, Pidakala P, Wang M, Schaffer RJ: Co-ordination of early and late ripening events in apples is regulated through differential sensitivities to ethylene.
J Exp Bot 2009, 60:2689-2699. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Larrigaudiere C, Vendrell M: Cold-induced activation of 1-aminocyclopropane-1-carboxylic acid metabolism in rewarmed Granny Smith apples - consequences on ripening.
Harker FR, Stec MGH, Hallett IC, Bennett CL: Texture of parenchymatous plant tissue: A comparison between tensile and other instrumental and sensory measurements of tissue strength and juiciness.
Postharvest Biol Technol 1997, 11:63-72. Publisher Full Text
Volz RK, Harker FR, Lang S: Firmness decline in 'Gala' apple during fruit development.
Camps C, Guillermin P, Mauget JC, Bertrand D: Data analysis of penetrometric force/displacement curves for the characterization of whole apple fruits.
Journal of Texture Studies 2005, 36(4):387-401. Publisher Full Text
Schröder R, Atkinson RG, Langenkämper G, Redgwell RJ: Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit.
Planta 1998, 204:242-251. PubMed Abstract | Publisher Full Text
Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees.
Plant Mol Biol Rep 1993, 11:113-116. Publisher Full Text
Melton L, Smith B: Isolation of plant cell walls and fractionation of cell wall polysaccharides. In Current Protocols in Food Analytical Chemistry. Edited by Wrolstad R, Acree T, An H, Decker E, Penner M, Reid D, Schwartz S, Shoemaker C, Sporns P. Wiley and Sons, vol. Unit E3.1. New York; 2001:1-23.
Blumenkrantz N, Asboe-Hansen G: New method for quantitative determination of uronic acids.
Anal Biochem 1973, 54:484-489. PubMed Abstract | Publisher Full Text
Hallett IC, MacRae EA, Wegrzyn TF: Changes in kiwifruit cell wall ultrastructure and cell packing during postharvest ripening.
Int J Plant Sci 1992, 153:49-60. Publisher Full Text
Sutherland P, Hallett I, Jones M: Probing cell wall structure and development by the use of antibodies: A personal perspective.
New Zeal J For Sci 2009, 39:197-205. Publisher Full Text
Sutherland P, Hallett I, Redgwell R, Benhamou N, MacRae E: Localization of cell wall polysaccharides during kiwifruit (Actinidia deliciosa) ripening.
Int J Plant Sci 1999, 160:1099-1109. PubMed Abstract | Publisher Full Text


text:
Indonesia Topics
Democracy Now! stories, posts and pages that relate to Indonesia
- Six years ago today — on November 12, 1991 — Indonesian troops armed with American-made M16 rifles fired on a crowd of several thousand unarmed East Timorese civilians gathered at the Santa Cruz cemetery in Dili, East Timor. At least 271 people were killed that day and more later as they sought medical treatment in local hospitals. [includes rush transcript]November 12, 1997 | Story
- To mark the sixth anniversary of the Santa Cruz massacre, where Indonesian soldiers with U.S. equipment opened fire on a funeral procession, killing more than 250 East Timorese people, activists around the country will be protesting the close ties between Washington and Indonesian dictator Suharto. [includes rush transcript]November 12, 1997 | Story
- Sportswear giant Nike is facing trouble again. Already reeling from charges that it exploits children in its factories in Indonesia and Vietnam, women's groups are now slamming the multinational company over its new advertising campaign.October 28, 1997 | Story
- One of the big scandals of the campaign-finance saga has been the role of a giant Indonesian conglomerate that allegedly funneled huge amounts of money into the Democratic Party. The Lippo Group, headed by James and Mochtar Riady, is closely tied to Indonesian dictator Suharto and has vast business interests throughout East Asia.July 09, 1997 | Story
- Amy is joined by two guests, who do not reveal their real names, discusses the trials and sentences of pro-democracy activists in Indonesia, the role of journalists in Indonesia as well as the upcoming elections. Indonesia will hold elections soon, although they are considered a sham and the current government is expected to win 70% of the vote.May 05, 1997 | Story
- Amy Goodman and Allan Nairn visited East Timor for the first time in the summer of 1990, 16 years after the invasion. They interviewed people, and excerpts of their stories are broadcast in this segment. They traveled to East Timor in order to be present for a historic event: a special delegation from United Nations and Portugal was due to visit East Timor. Despite threats and a dramatic increase in the number of disappearances, torture and...December 10, 1996 | Story
- In the face of the massacre story, even Suharto's longtime allies came under public pressure and cut aid to Indonesia. There were even open protests inside Indonesia where student demonstrators were beaten and arrested. But the U.S. Bush administration increased U.S. military training aid to Indonesia, which is called IMET (International Military, Education and Training). [includes rush transcript]December 10, 1996 | Story
- There have been important changes in the U.S. policy toward East Timor, but they are all results of grassroots pressure. But put simply, the U.S. continues to arm and back the Suharto regime, despite sometimes tough rhetoric. Excerpts of statements by President Clinton, Vice President Al Gore are broadcast. While the Riady money may have bought him access to the White House and some favors, the U.S. policy toward Indonesia has been far more...December 10, 1996 | Story
- As we've been reporting over the last week, Indonesia has been making headlines ever since two East Timorese independence and human rights activists were named winners of this year's Nobel Peace Prize. In the past week, the Los Angeles Times and other media have revealed in dozens of articles and news reports that the Clinton campaign has accepted hundreds of thousands of dollars from billionaire Indonesian family tied to the...October 18, 1996 | Story
- A Roman catholic bishop and an exiled activist have won the noble peace prize today for their work in East Timor. Jose Ramos Horta & Bishop Carlos Filipe Ximenes Belo will share the 1.1 million dollars. The men are being sited for their work towards a just and peaceful solution to the conflict in East Timor which was invaded by Indonesia 21 year ago and since that time they have killed more than 200 hundred thousand people.October 11, 1996 | Story
Bishop Carlos...
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text:
The Manian Language Test
Their result for The Manian Language Test ...
76 % Manian fluency!
You know enough about men to know when to turn on the charm, and when to get down to business. You could be dangerous if you use your powers for evil rather than good.
You are amusing in a good way, and men are very comfortable around you. Just be careful that they don't get too comfortable, or you could find yourself being the "FWB" more often than you like. Because people in this range are usually down to earth, we (and I say we because without male assistance, I'd have never been able to create this test) are the friend guys want to shag, but not the one most want to take home to show mom and dad. They desire us because we're not so mainstream, but this is the same reason they disqualify us as long term partners.
So even though you're a Geisha, guard your heart with the determination and decisiveness of a Samurai. Do not put the sword in the sheath until it has tasted blood.
Their Analysis (Vertical line = Average)
They scored 76% on fluency, higher than 46% of your peers.
All possible test results
You better be careful out there. Though there are many liars, cheats, and manipulators out there, most men are pretty straightforward. Rather than extrapolating hidden meanings behind the words, try... Read more
Foreign Resident
You've got an okay understanding of Manian, and can find your way around the airport. You could probably have a pretty decent relationship with a man who's sensitive, and will take the time to learn ... Read more
You got it going on, and probably will well into your older years. You know men quite well, and understand their needs, even if you don't always cater to their whims. You know enough about men to ... Read more
You know men almost too well. None you set your eyes on in earnest can resist you forever. You are the Mistress (or Master), and likely have no problems with men that don't boil down to your own pic... Read more
Recent Results
elvenlogicx25 / f / Straight
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text:
NEWS: The Penn State men's basketball team will take on Colorado in the Continental Tire Las Vegas Classic at 10:30 p.m. ET Tuesday, Dec. 22.
FEATURE: Jordan Dickerson, Devin Foster, Donovon Jack and Brandon Taylor will not be looking around for a veteran leader come October when the Nittany Lion basketball team begins official practice. The group of 2015-16 seniors has already laid the groundwork in taking...
NEWS: A pair of Penn State men's basketball alums participated in the NBA Summer Leagues over the last two weeks.
NEWS: The Penn State men's basketball program finished the 2014-15 season with the fifth-highest increase in attendance among all Division I teams as illustrated in the official basketball attendance figures released this week by the NCAA.
NEWS: Terrence Samuel has signed with the Penn State men's basketball team, Patrick Chambers, head coach of the Nittany Lions, announced Wednesday.
NEWS: Penn State is among eight teams in the 2015 Continental Tire Las Vegas Classic to be held December 22 and 23 at the Orleans Arena, tournament organizers announced Thursday.


text:
Statoil has awarded a contract to Dolphin AS and a letter of intent to Seadrill for two rigs which will operate on the Norwegian continental shelf (NCS). The Bideford Dolphin rig has won a three-year contract with start-up from 27 January 2011. The contract is worth about $421m. Statoil has an option to extend the contract from three to four years by 1 November 2010. The value of the contract will then increase to about $553m. Dolphin AS is a wholly-owned subsidiary of Fred Olsen Energy ASA.
Seadrill's West Epsilon rig has been awarded a letter of intent for a four-year contract to take effect on 29 December 2010. This contract is worth about $394m. The agreement is conditional on partner approval. Statoil can extend the contract period to five or six years before 1 October 2010. There will also be an option to extend the contract for an additional two years.
Both rigs are working for Statoil at present.
Before the global economic problems took full effect Statoil found that the rig rates had increased too much, and they did not decrease correspondingly when the market circumstances changed. Anders Opedal, senior vice president for procurement in Projects & Procurement, says that the rig rates are now at a more sustainable level.
U.S. energy firms added 5 oil rigs this week after putting 21 rigs into service last week, the most in over a year, despite a collapse in U.S. crude prices from recent highs in June,
DryShips Inc. (NASDAQ: DRYS), a global provider of marine transportation services for drybulk and petroleum cargoes, and through its subsidiary, Ocean Rig UDW Inc.
MHI gives Notice Regarding Conclusion of Absorption-type Split Agreements Relating to Transfer of Ship Construction and Hull Block Manufacture Businesses to Two
One of the largest Italian tug owners, Fratelli Neri S.p.A, ordered its first Damen tug, an ASD 3212, on 21 July 2015.
The vessel will be the first ASD tug
NOIA President Randall Luthi issued the following statement on the Senate Energy and Natural Resources Committee's passage of the OPENS Act, which will open new
Shell Oil's icebreaker MSV Fennica weaved through nine remaining protesters hanging from the St. Johns Bridge and made its way toward the Pacific Ocean.


text:
Tom Day
- BiographyOver his 1 year long porn career, Tom Day has appeared in 1 video scene and 1 picture set, debuting in Curious for Cock around May '14. Other actors co-starring with Tom are, most notably, Robin Ward. His most often performed activities include Solo Masturbation, Guy On Guy, Blowjobs, Handjobs, and Cum on Stomach. The content containing this actor has been requested 1,434 times in total, with more than 0.1 Gigabytes of video having been downloaded.
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text:
Ivan Reitman: 'There Is Only One New 'Ghostbusters' Movie'
There has been much dispute lately regarding the matter of who, in the event of a ghost-related emergency, ya gonna call.
There has been much dispute lately regarding the matter of who, in the event of a ghost-related emergency, ya gonna call.
The Dixie Chicks thrilled European fans recently by announcing they'd be reuniting and going on tour across the pond, but is it possible the band will take a tour stateside? According to Natalie Maines' Twitter page, it might be in the cards.
The world is still steamed about the devastating death of Cecil the lion, so why not get a look at some other lions that will put a smile on your face?
The extreme summer heat got you down? Why not take a load off by watching the best TV news bloopers from July?
This fly is pretty fly.
What would you do if you were quietly working in your office and all of a sudden you experienced a "Sharknado."
Now that President Obama has come and gone, Jon Stewart's final round of Daily Show outings will bring out a few more big guns to help send him off. Among the sure to be extensive list, we already have top comedians (and near successors) Amy Schumer, Louis C.K. and even Denis Leary booking slots on Jon Stewart's final run.
This blind dog has gotten some help that should revolutionize how it gets around.
Two days after the owner of a Danbury Pet Store was arrested on animal cruelty charges, one of the managers has now also been arrested.
What's beer than ice cold beer on beautiful warm day? We can't think of anything else either. The beer event of the year is coming to Connecticut on Saturday, Sept. 12 at Ives Concert Park in Danbury.


text:
Nonstop Fun
"Pablo Gigante" champions the SA Forums obsession with the act of partying hard:
"NoneMoreNegative" sure has strange tastes in games!
"FOOKOOF" sure knows a thing or two about awesome game making. Yep, a thing or two.
This "Anarchon" game isn't as revolutionary as it sounds, as EverQuest and Ultima Online have already pioneered the feeling of virtual rape.
"StreetPreacher" just keeps spinning, and spinning...
"juankerr" took a break from water balloon fights and tree climbing to make this:
This Halloween, the most terrifying house for policemen is opening its doors. If you're a cop, get ready to have nightmares!
Ariana Grande? No way! Here's a podcast that looks squarely at Ariana Grande and says, 'No way!'
Blood spills, dwarves cavort and intrigue abounds as this epic tale reaches its dramatic conclusion!
League of Legends has 600 characters and 11,000 skills. These are the only ones you need.
The water got bigger? my sand castle was destroyed and we had to move. Who did this?
Photoshop Phriday showcases the tremendous image manipulation talents of the Something Awful Forum Goons. Each week they tackle a new theme, parodying movies, video games, comics, history, and anything else you can think of. If you want in on the action, join us on the Something Awful Forums!
Copyright ©2015 Rich "Lowtax" Kyanka & Something Awful LLC.


text:
Melinda Taub and Adam Sacks imagine what it might look when the Hunger Games inevitably become real, and Coca-Cola inevitably becomes their sponsor.
Painting Characters Into Thrift Store Paintings Makes Them ...
Here's Why You Never Ask the Internet for Help... Ever
Poly Bridge Proves That Most of Us Shouldn't Be Civil Engineers
Happens All The Time
Here's What Women in Video Games Would Look Like if They ...
Links of the Day: Satan Statues, Baby Owls and What You Missed ...
It Makes No Sense
Professor Oak Was a Master Troll
Do You Have Photos of Your Husband?
Pick Your Site Name
Tell us more about it


text:
Male Perverts Granted Access to High School Girls' Locker Rooms
The triumph of moonbattery makes the Age of Hope & Change a great time to be a pervert. From Los Angeles:
One of the largest public charter high schools in the nation allows a boy who identifies as female to use the girls locker room and bathrooms — despite protests from a female student who says she feels uncomfortable and vulnerable by his presence.
What's more, the female student — who just finished her sophomore year at the Los Angeles-based Granada Hills Charter High School — claims the boy in question harassed her, touched her hair, stared strangely at her, and also peeked at girls over the stalls while they used the toilet.
"It's amazing to me that a public school who is responsible to protect the rights of all students will blatantly ignore the Constitutional right to privacy of young female students within the locker rooms and restrooms of the school," Robert Tyler, the girl's attorney, told The College Fix.
That's what they get for being normal. Political correctness dictates that normal people don't deserve rights, because they are oppressors of the sacred castes, prominent among which are perverts.
It will get worse before it gets better in the Land of Fruits and Nuts.
The California Assembly (same as House of Representatives in most states) passed Assembly Bill 1266 by a 46-25 vote. …
AB 1266 states that public schools cannot discriminate in any way concerning the sex of the student.
Sounds nice, since we all know that to "discriminate" is the ultimate sin. But if you swallow that, you have to swallow this:
Section 221.5 of the Education Code is amended to read: …
A pupil shall be permitted to participate in sex-segregated school programs and activities, including athletic teams and competitions, and use facilities consistent with his or her gender identity, irrespective of the gender listed on the pupil's records.
This means that horny adolescent boys no longer need to drill holes in the wall to the girls' shower, as in the movie Porky's. They can just proclaim themselves to be female at heart and march right in, boners a'bobbing.
The liberal campaign against decency will continue its blitzkrieg until it encounters meaningful resistance — which is no longer possible in California, as the demographic transformation of the state by massive Third World immigration has ensured an unbreakable Democrat stranglehold. All 46 votes for this depraved bill were by Democrats.
As goes California, so goes the nation, just as soon as Chuck Schumer and his RINO collaborators can ram amnesty home.
On tips from Mike B and Henry. Cross-posted at Moonbattery.
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