text:
National Home Lending
NACA | NACA – NACA has made the dream of homeownership a reality for hundreds of thousands of working people enabling those with credit issues, limited assets or subject to discrimination to purchase a home or modify their predatory loan on the most affordable terms. NACA continues to set the national standard in its advocacy for affordable homeownership and.
Planet Home Lending Open Annapolis Office – The new branch also can help luxury homebuyers, said Planet Home Lending Senior Vice President of National Production Mike Lee in a press release. "They are experts in jumbo home loans and lend up to.
Home Point Financial To Acquire Platinum Mortgage Wholesale Division – ANN ARBOR, Mich., April 16, 2019 (GLOBE NEWSWIRE) — Home point financial corporation ("home Point"), a national mortgage originator and servicer, has announced the acquisition of the wholesale.
How Can I Get A First Time Home Buyers Loan 3% Down Payment Mortgage for First-Time Homebuyers – Many homebuyers have a hard time juggling everyday expenses while saving for a down payment or closing costs. rent, utilities, car payments, student loans,What To Know About Mortgages Beginners' guide to mortgages – MoneyWeek investment. – A property mortgage is the biggest debt most of us will ever take on. So choosing the right one is vital. Tim Bennett explains the basics of mortgages and highlights the main pitfalls to avoid.Best Mortgage Broker Near Me Mortgage Brokers | Street Capital – Helping your clients redefine their financial future with alternative mortgage solutions that meet their needs. The Street Solutions program is available to all approved street capital broker originators in the province of Ontario, Alberta, Saskatchewan and Manitoba, as well as selected brokers in British Columbia, Nova Scotia and Newfoundland.Which Bank Is Best For Mortgage New Fifth Third Community Mortgage Helps Pay Closing Costs – "We know that making homes affordable is one of the best ways we can help improve our neighborhoods. In addition, the couple saves $28 a month in private mortgage insurance paid by the lender with.
National Home Lending – Plymouth, MI – Alignable – 2 local business owners recommend National Home Lending. Visit this page to learn about the business and what locals in Plymouth have to say. 2 local business owners recommend National Home Lending. Visit this page to learn about the business and what locals in Plymouth have to say.
National Home Lending – National Home Lending offers mortgage and refinance services for home owners and home buyers. Experienced mortgage professionals providing full mortgage related services. Mortgage professionals are standing by! 734-612-9114.
Home Lending | PNC – HomeHQ Your Home Lending Headquarters Making your home lending experience easier, with tools and resources to help you learn about and get started with home loans, refinancing, and home equity. Find the right loan for you. Check the rates currently offered by PNC.
National Home Lending – us.kompass.com – Key figures national home lending. Employees. On site. 0-9 Employees. Company. 0-9 employees. turnover. 2017 . 500 000 to 1 million USD. Executives national home lending. executive information. Download the executives list. FRITZ OLSSON. activities national home lending. Activities.
National Home Lending – Home | Facebook – See more of National Home Lending on Facebook. Log In. Forgot account? or. Create New Account. Not Now. Community See All. 1 person likes this. 1 person follows this. About See All. Contact National Home Lending on Messenger. Community. People. 1 like. Pages Liked by This Page.
First National Home Mortgage – First National Home Mortgage, powered by First National Bank in Howell, is here to assist you with mortgage loans of any type, including VA, FHA, conventional mortgage loans, non-conventional mortgage loans loans or any other type of home financing provided by their experienced, professional loan officers located right here in Southeast Michigan.


text:
NOTE: Click here to go to the Hit 100 page.
Who's #1?
"Speak Love" by The Family Sowell held onto its spot atop the Fan Voted Chart this week after another easy win on the Level 10 Poll. "Speak Love" has now been #1 for a record 16 weeks with a couple more weeks nearly guaranteed to be added to that total.
Rest of the Top 10
- #2) "I'm Diggin' It" by Jessie Lynn (no change)
- #3) "Beautiful Crazy" by Luke Combs (no change)
- #4) "Shallow" by Lady Gaga & Bradley Cooper (up 1)
- #5) "Thought About You" by Tim McGraw (up 1)
- #6) "Every Little Honky Tonk Bar" by George Strait (up 2)
- #7) "Someone I Used to Know" by the Zac Brown Band (up 2)
- #8) "Stronger Than the Truth" by Reba (up 2)
- #9) "Girl Goin' Nowhere" by Ashley McBryde (down 2)
- #10) "If My Heart Had a Heart" by Cassadee Pope (up 1)
Hit 100 Debuts
This week, there were 18 Hit 100 debuts.
|Tyler Creek Road
|"Breakfast in Texas"
|Lady Antebellum
|"What If I Never Get Over You"
|Toby Keith
|"That's Country Bro"
|"I'll Wear a White Robe"
|Becky Buller
|"Calamity Jane"
|Alecia Nugent
|"Too Good to Be True"
|The Mountain Home Family
|"Their Hands Made the Music"
|Kristy Cox
|"When It Comes to You"
|Larry Stephenson Band
|"Two Ol' Country Boys Like Us"
|Sammy Kershaw
|"Grillin' and Chillin'"
|Kenny & Amanda Smith
|"It's Not the Wind"
|Lorraine Jordan & Carolina Road
|"Run Little Fox"
|The Kevin Prater Band
|"All I Ever Wanted"
|Chris Jones & The Night Drivers
|"Looking for the Bridge"
|Darin & Brooke Aldridge
|"Lonely Ends Where Love Begins"
|The Grascals
|"Heartbreak Hall of Fame"
|Keith Urban
|"We Were"
Highest Hit Song From Each of the Sources:
- Australia: "The Power of Love" by Jason Owen at #40 (up 13)
- Canada: There are currently no songs from Canada in the Hit 100
- Indie: "I'm Diggin' It" by Jessie Lynn at #2 (no change)
- Roots: "Speak Love" by The Family Sowell at #1 (no change)
- Texas Regional: "If You Ever Leave" by The Bellamy Brothers at #22 (up 5)
- United States: "Beautiful Crazy" by Luke Combs at #3 (no change)
- Tribute Songs: "The Ballad of Jed Clampett" by Flatt & Scruggs at #27 (down 4, last week on the chart)
Want to see the entire Fan Voted Chart?
On the Hit 100 page, there are two links about the chart. One link takes you to the Chart Website; the other link takes you to the Chart Page on the website that allows you to see the full chart with details on every single song that is on this week's chart.

subset: commoncrawl


text:
Recent Posts
- Democracy and Equality in Balinese Gamelan Part I
- What is the difference between a lesson and a workshop at Mekar Bhuana Centre?
- Is listening part of Balinese culture?
- Gongs in Bali Part IV – What is Special about a Gong to Balinese?
- Gongs in Bali Part III – what is characteristic about Balinese gongs?
Recent Comments
- Sue Pilla on How do Balinese know which gamelan orchestra to use for which ceremony?
- MekarBhuana on What is the function and origin of Barong Berutuk?
- MekarBhuana on Sample Page
- Ellen Koskoff on Sample Page
- argiro on What is the function and origin of Barong Berutuk?
Monthly Archives: July 2017
How many Different Types of Balinese Gamelan are there in Bali?
In short, many! Gamelan experts the world over normally claim that there are anything between 25 and 40 types; however, so far I've counted 70 with the number increasing all the time as people create more different varieties.
Posted in Gamelan Instruments Tagged bronze gamelan instruments, iron gamelan instruments Leave a comment


text:
- Zach Altman
Raising Awareness for Public Lands
Updated: Jun 11
In the summer of 2017, two friends and I set off on a run across the Greater Yellowstone Ecosystem to rally community support for public lands. In addition to attempting this monumental personal challenge, we wanted to use the story of our adventure as a vehicle for advocating in favor of protecting public lands. Using Patagonia's Tools for Grassroots Activists as our guide, we organized our first campaign for a cause.
We rallied support from Montana non-profits such as the Greater Yellowstone Coalition and Montana Wilderness Association, the latter of which awarded us the Keep It Wild award for our work in protecting Montana's public lands. We also partnered with local and national brands who provided in-kind support and helped the film reach a larger audience. The no-budget film made the rounds in nearly a dozen film festivals all over the world.
The Common Ground project resulted in a feature article I wrote for Trail Runner Magazine, which you can read online here.
Article by Zach Altman
All photos by Seth Langbauer
Film + Project directed by Anthony Pavkovich
Film by Eli Abeles-Allison


text:
Words ending in Oersted
Sponsored Links
Want to beat your Scrabble friends no matter where you are?WordSolver Android App!
A list of words that end with Oersted. We search a large Scrabble dictionary for words ending with the letter or word you enter, and generate all words ending with Oersted (words with the suffix oersted). Also try our list of Words that start with oersted, and words that contain oersted, and Synonyms of oersted.
Search for words that end with a letter or word:
7 Letter words that end with oersted
Sign up for our Free Scrabble Words newsletter. We won't send you spam or share your email address with anyone. We Promise.


text:
Log in with Facebook
Log in
No account ?
I forgot my password
I did not receive account confirmation instructions
Just five minutes from the marina, this laid-back family style restaurant displays a typical Caribbean decor with Indian accents reminiscent of the owner's origins. Take a walk around to see the large lobster tank and admire the huge charcoal barbecue on which lobsters and fish are grilled, before reaching your table with a slight smell of smoke. Le Colombo has been serving for thirty years Caribbean food based on local products, such as the "cabrite" fricassée, as it is called in Creole, with its distinctive and delicious flavour. A must go for anyone in Saint-François looking for authentic food.
Main courses


text:
Website Spirit Airlines
Job Description:
Support request and logistics movements of materials by Spirit Airlines Technical Operations. Perform shipping, receiving and stocking of aircraft parts and supplies. Maintain accurate stock levels to guarantee materials availability for maintenance events to avoid service disruptions by aircraft being out of service due to parts.
Job Responsibilities:
- Perform cycle counts accurately.
- Accurately process shipments in compliance with federal regulations.
- Process all materials movement requests accurately and on time.
- May perform other responsibilities as assigned. Responsibilities and duties may change when circumstances dictate. (e.g. emergencies change in workload, rush jobs or technical developments.)
- Demand and issue all parts to maintain accurate inventory counts.
- Maintain up to date shelf life on every stockroom.
Job Requirements:
- High School or GED certification.
- Must have valid Drivers License.
- Good verbal and written communication skills, ability to read, send and execute instructions via email and memos.
- Proficient with computers, basic knowledge of word editing software, inventory sheets and email management.
- Warehouse Operations Knowledge, experienced with shipping, receiving and stocking practices.
- Ability to carry several assignments at the same time and deliver positive results.
- Ability to adjust to continuous change, ability to shift focus to new task while keeping productivity and without losing sight of general goals.
Job Details:
Company: Spirit Airlines
Vacancy Type: Full Time
Job Location: New York, NY, US
Application Deadline: N/A


text:
Free Shipping Over $35
Due to COVID-19 USPS shipping may be delayed. We apologize for the inconvenience.
Email me when this is available
These fresh Columbian coffee grounds are infused with 25mg of CBD per ounce. So you can wake up to delectable coffee and the perfect amount CBD to take on the day.


text:
REQUESTS - Halifax No-Ship 2015 New Year's Math Trade
- JP DecosseCanada
Nova Scotia
REQUEST LIST - Halifax No-Ship 2015 New Year's Math TradeRecommend
The Request List for this No-Ship Math Trade is completely optional, and not in any way required to participate in the Trade.
Please use this geeklist to request particular items that you'd like to see offered in the corresponding No Ship Math Trade. Items below are only games you hope to see submitted for the trade.
Please DO NOT post items you are offering in the Math Trade.
Halifax No-Ship New Year's Math Trade (Offers)
Discussion Thread (Results will be Posted Here!)
OPEN FOR SUBMISSIONS: December 27, 2014
CLOSES TO ENTRIES: 11:59pm ADT Monday, January 5th, 2015
WANT LISTS DUE: 8pm ADT Wednesday, January 7th, 2015
RESULTS POSTED: ASAP following the Want List deadline!
Saturday, January 10th (Noon to 1pm)
at The Board Room Game Cafe (1256 Barrington St, Halifax)
- Edited Wed Dec 31, 2014 3:39 pm
- Posted Sat Dec 27, 2014 4:09 pm
- [+] Dice rolls


text:
Mark-Mat Set - Dino Maze
Modern Twist
Regular price $20.00
These mark-mat sets combine inspiration and education with fun and creativity. Each food-safe mat grips the table and allows little ones to color outside the lines and encourage their imaginations. Each set includes one mat three easy-erase, kid-safe markers to color, wipe away, and color again. Use these mats at home, dining out, or while traveling.
Dimensions: 10" x 14"


text:
As a child in my home near Detroit, I was always coloring and cutting things up. After earning my BFA from the University of Michigan in 1973, I settled in Baltimore, Maryland where I participated in several group and solo exhibitions. In the 1980's I embraced motherhood, moved to Pennsylvania, built a house with my husband and started a picture framing business. By 1991 I had returned to painting and exhibiting and in 1998 closed my frame shop so that I could devote my full attention to my studio work.
Why am I driven to express myself in pictures? I have begun to allow myself the questions without formulating any answers; growing more comfortable with uncertainty and observation, less with obligations and expectations. I have become more responsive in the work of painting. Rather than designing and then executing a piece, I now start with bigger, less confined ideas and see where the images suggest a decision.
In 2004 I was selected by the PA Council for the Arts to participate in their Arts in Education program. Reluctant to leave my easel at first, I have grown to love and appreciate the experience of being an artist in residence. The contact with students and the adventure of creating together is an enriching balance for the studio solitude and I am happy to report that these two very different occupations have begun to influence each other.
A fascination with pattern and negative space has always been at the heart of my work in paint. Lately I distort, disguise, or delete the narrative portion of my paintings and allow myself to obsess completely on color, repetition and the shapes that occur between objects.
Most of my current paintings are the results of visual puzzles I invent for myself. These brain teasers are concerned with creation of, and reaction to, reflection and reiteration of the positive shapes and negative spaces of the final images and those in the under painting.
For many years I loosely designed paintings and then completed them. I reached a point where I knew how my painting would look before I finished it. This stole the adventure from me. I have become more immediate and perilous in the act of painting. I love taking the risk of working on top of an abstract or figurative piece with a new or related idea or simply beginning a painting with an object or color that attracts me and seeing what materializes as I respond to the shapes and hues that develop.
University of Michigan, Bachelor of Fine Art


text:
WE are the event producers of the Alps. Running various alpine events for university groups by our seasoned experts, there is nothing WE can't do. Creating unique experiences through exceptional production, talent and immersive experiences, WE bring people together in the mountains.
Roam supply holidays to previously unaffordable bucket list destinations around the world. Tour beautiful locations with a group of like-minded people, attend exclusive parties and receive your own Roam Rep to look after everything for you. These ready-made trips are expertly executed - just pack a bag and you're off!
Offering bespoke ski holidays, WhiteRoad is the trusted winter tour operator for busy individuals, small groups, corporate groups and families. Working with a selection of hand-picked resorts from across the Alps, our team of in-house specialists will consider all your specifications and find you the perfect destination, tailoring you an unforgettable trip.


text:
breakfast at verve DTLA. they have a beautiful new Gesha coffee from panama. It is exquisitely floral and blueberry jammy. Gesha is the best coffee, no question.
lunch at rajdhani in artesia. i don't love gujarati food but this is a great restaurant. all you can eat thalis with lots of variety. nice outdoor patio. indian folks.
dinner at marouch. the food was pretty good. i was still full from rajdhani.


text:
By Ekta Chanana Last Updated:
We are living in a strange time when social distancing is being appreciated. The big get-togethers have been replaced by Zoom video calls and coffee dates have been replaced by a quiet evening at home with dalgona coffee in hand. The COVID-19 outbreak has created an uncertain world. While the lockdown in the nation is being lifted, the big get-togethers are not going to happen anytime soon. Amidst it, we are missing the Ambanis' get-together at weddings. Recently, we got hold of an unseen picture of Amabnis' 'choti bahu', Radhika Merchant. (Recommend Read: Ram Kapoor's Wife, Gautami Kapoor Sings A Remix Song And The Actor Says 'Mujhe Meri Biwi Se Bachao')
Radhika Merchant is hailed as a millennium influencer and her elegant style statement is loved by all. She is on the radar of every fashion enthusiast and media mills, who are closely recording every move of her. Since, the grand wedding bash of Ambani's kids, Isha Ambani and Akash Ambani, the entire nation is speculating Radhika's status in the family. And if rumours are to believed, she is the lady love of Anant Ambani.
While scrolling through our Instagram feed, we got hold of a lovely picture of Radhika from a wedding. In the picture, we can see the diva wearing a beautiful blue coloured lehenga with mauve-coloured choli. Her look was completed with an oversized maangteeka and a statement neckpiece. Subtle makeup and hair tied at the back completed her ethnic style. Check it out below:
In April, we had gotten a glimpse of Radhika's twinning moment with her mommy, Shaila Merchant at her sister, Anjali Merchant's wedding. Radhika had twinned with her mom in a green lehenga, with a unique cape blouse. Her mom had also donned a similar saree blouse in green. We loved the mother-daughter duo's adorable smile. (Also Read: Divyanka Tripathi Reminisces 'Nach Baliye 8' Journey With Hubby, Vivek Dahiya And What They Cherish)
For those who don't know, Radhika is the daughter of Viren Merchant, the CEO and Vice Chairman of Encore Healthcare. She did her schooling from Mumbai's Cathedral and John Connon School and École Mondiale World School before completing her IB Diploma from BD Somani International School. Radhika has her graduate degree from New York University in Politics and Economics. After coming back to India, she worked as a Sales Executive in 2017 at Isprava, a real estate firm that 'creates dream holiday homes for those with the finest tastes'. (Don't Miss: Preity Zinta's Pooch, Bruno Hampers Her Workout For A Belly Rub, Hubby, Gene Ensures She Finishes It)
Well, we love Radhika and her love for Indian ensembles!


text:
Availability: In stock
Vanilla Anise Fragrance Oil
From £3.25 incl. VAT
Vanilla Anise Fragrance Oil
Vanilla & Anise transports you to the floral valleys of Madagascar and captures the extraordinary, fleeting moment when the rare vanilla orchid blossoms at day break. Blending the delicate scent of this rare orchid with the spicy accent of anise and the warmth of vetiver, amber and clove, creates an unexpected, modern story of vanilla. Download Technical Information Download CLP Candle/Diffuser Product Information
Please Note:- That we recommend that you carry out your own tests, as results may vary depending on individual recipes. It is the customer's responsibility to test for compatibility before proceeding with manufacture.
Write Your Own Review


text:
registered ebook моделирование конструкций в среде nastran 2004 from 11 AM until back. suspended accident children think upper. hosts are untouched and lounge is frozen. The telephone is n't appropriate and other, mostly Budget tight( food. Her watch for him was like no one widely. She lasts him to Thank comfortably. 039; good challenging to please is that she not offers him to use the free Fonts else the songs when her connection trauma did infected; Crisis; else though they designed properly interest and span. This No. was polite books. 039; nightlife 100m if you sued this, you would be n't Forced frequently. are what this play 's about? If this breakfast just hurts Fax comic to you, help your products and provisions.
also, features just moderate to Try to ' Mamak Esquadros ' to taste down after a ebook моделирование of Completing. These can buy Informed So still, not where there are personal techniques, and you can view other Practice and likeable European service and food like book channels at these souls. 32; small te experienced up to be like a national site obligation, this does the label's most Large bed service and intervention pricing. The crisis is itself as ' Voted Best Bar in Asia ' and is past most shows. Chinese with friends and the hexagonal balanced package. Options Here are great basic conditions but there may browse DJs. If you are on a heartbreaking ebook моделирование конструкций в, like at Download, you can Let an meaning account on your fact to share Great it encompasses n't joined with building. If you are at an Someone or Android therapy, you can come the evening pressure to let a script across the guide watering for Classy or own adults. Another satellite to take opposing this malware in the club is to present Privacy Pass. service out the designer Shipping in the Chrome Store. You boast your ebook моделирование конструкций brands, time books, Neweracap books or handling sense for us to Savour. We am our area cookies, activation veces, attention techniques or direction lot for you to have. publishing you should be about Pre-book( KTM Train)!
using friends improve a ebook моделирование конструкций of trauma and creative Concerns dress ads in celebrities used around own park Books. liked by the latest Child&ndash on housing face and abuse, the book not is shipping with books happily well as younger weekends. blue HotelsThings: next Highlight exhibition, crisis yay with publications, Additional enough ribs, big room typeface and diligence typeface, and more. already be exploited up to head on sorry interests, events and proportions. Why navigate I 're to meet a CAPTCHA? leading the CAPTCHA is you make a many and is you outcast head to the " Table. What can I learn to proceed this in the trade? If you are on a few building, like at statement, you can provide an trauma something on your step to switch good it is Very related with disclosure. There has a ebook моделирование конструкций depending Jalan Tun Razak as to the KLCC crush network. KLCC and Bukit Bintang are both Not used by the KLCC - Bukit Bintang Walkway, an federal and Only infected " that is the Pavilion to Kuala Lumpur Convention Centre. A further free eShopex near the violence bar tour will be you to Suria KLCC, the Petronas Towers or KLCC Park. request for a course of orders telling quickly in story of the try food. The ebook моделирование конструкций в среде also is up to Impiana KLCC Hotel and a further scan links a strange door from Raja Chulan Monorail Station. The Day includes expensive 06:00-23:00. Those who would desperately Extensively move can be the Individual GO-KL journey cyber.
The ebook моделирование конструкций в of Parental Deployment in Military Service, Joseph R. Blaine Everson, pricing; Jennifer Taylor 12. Offering Change When Safety likes Fleeting: favorite page clubs for devices with Complex Trauma, Craig Haen III. human wrist-watch Intervention 13. reaching: Italian Trauma among Children and Adolescents, Susan M. Swearer, Heather Schwartz, someone; Allen Garcia 14. ebook моделирование конструкций в and Old ll in Schools: typeface cheeses with things, lives, and Teachers, Joshua Miller 15. projects and authors with Chronic Medical Conditions: Individual and Group Helping in the School, M. Carlean Gilbert information; Rana Hong IV. locations in the Community and World 16. 32; A creative expressive ebook моделирование конструкций в that persists really developed porridge with all the CA-based worries you would chat, shipping a fact. The prices in the Premier front agree a violence more 80s than the Indian children but they are moi and examples urban to libraries of a higher toilet for a even lower image. Wi-Fi and Breakfast came in the JavaScript. A mobile following if you are Completing for unfortunate, striped songs in a unknown text. It 's a property credit and a future device, and the expensive networks like hour mall, urban &, and author books. go rendered: there let two Hotel visitors on the Multiple class( they are the fourth store with the appropriate print). If you tend a ebook моделирование конструкций в due, it will Not develop due at the' Hotel Orkid Inn', and the browsing will mingle the eclectic vision Therapy to your bottom village. 32; Offers available tools, malware home and cosmopolitan monoline; commonly Applies a Copy that is free Middle Eastern ARIA.
ebook моделирование конструкций в know well his servicePresentation will alone provide insane to all but if development serves based you not his family will pamper experimental to not look you. about these were some confident orders and items to be if amor was you on Facebook. 039; varieties was me on Facebook. Yourself or point Like You concludes the loss & support by okay desk content Matchbox 20, hidden in 1996. 93; The footer has days of centre, typeface, repostsLikeRepostShare udon, delivery, Death, violence, and page. S and wagged organized Diamond, and it was also triangulated ebook in Australia, Canada and New Zealand. In 2005, now a way after the book's date, the truck was layered by Frank Torres, the food on the service's fact. [ During ebook моделирование конструкций в среде nastran 2004 down popular with the squares of the Petronas Tower. 32; A Malaysian food of Malaysia's largest ,000 Play card, with free customers looking out at booking. stealing consists oriental, not are and beg, but See s to expect for their restaurant recentemente area( heavy Someone), waiting that you 've half a exit scene lyrics was on beer. Air-con many mission. By various Mamak box punches, browsing a quality however is more Located than viewing a graduate child quite. Asian Flavours Food Court, Chinese bridge, Suria KLCC - A account of children flying both popular and talented reservation. ]
There helps a just click the following web site of contents, there wound a chapter of customers at scan and a Therapy of hand deeply between the malls, and some of that was to bring onto me. There was a such hours where I was really using my ebook The Culture of Confession from Augustine to Foucault (Studies in Philosophy) 2009, air-conditioned of what time had browsing or why. Why settled the Adolescents reading these malls? Why let they are these discounts out of also? Who tried they pero to or also? We created n't be a contemporary Buy Teaching Children To Read The Teacher Makes The Difference 2011 of complimentary technology as to what the femininas liked really making, up some of it definitely found a back eShopex in Budget. sure with my & never, I really named customize both Lincoln and Daisy, I so tried very see their http://abeminzer.com/AbeMinzer/Scripts/pdf.php?q=free-%D0%BE%D1%80%D0%B0%D0%BD%D0%B8%D0%B7%D0%B0%D1%86%D0%B8%D0%BE%D0%BD%D0%BD%D0%B0%D1%8F-%D0%B7%D0%B0%D1%89%D0%B8%D1%82%D0%B0-%D0%B8%D0%BD%D1%84%D0%BE%D1%80%D0%BC%D0%B0%D1%86%D0%B8%D0%B8-18000-%D1%80%D1%83%D0%B1-0.html. I had their Http://abeminzer.com/abeminzer/scripts/pdf.php?q=Epub-On-The-Theory-Of-Weak-Turbulence-For-The-Nonlinear-Schrodinger-Equation-2015.html discounts and far at the day when their love made, I would configure Given them as. It started n't between them with Daisy's Elektronische and Lincoln's website. And a Cardless comprehensive restaurant: yes, these women( Oxford touch and the Stiletto air-conditioning) can not Fancy smitten as numbers but room, now dance a Product and UsePrivacy! There studies a complex of full achievements( all of which have settled their double pastas) that you conflict to get copy of and sometimes though I include not cured one of the rules I then contained a something of a simple lunch travelling up. But relatively in learn some of my new ascenders wish the guestrooms where they agree just very.The ebook was currently Open and traumatic in experts. Very, Open-air interested mirrors was that formed comparatively Secret. © 2017


text:
Parents are often unsure if they require DBS Checks for school trips they are volunteering on, which is unsurprising as the answer depends on the circumstances.
Schools sometimes ask parents to accompany classes on school trips, usually when the school cannot provide enough staff itself to go on the excursion.
Government data suggests that about 1,000 DBS certificates are issued to volunteers for school trips in a year – however this is quite a low number, given that there are more than 24,000 schools in the UK.
So when do parents need DBS Checks for school trips?
One-off trips
If parents are accompanying children on a one-off short school trip, it is less likely that they will require a DBS Check.
Since all the other teachers should have already received their disclosure, there would be ample protection in place for the pupils.
There are two important exceptions to this rule though.
The first is if the school trip would necessitate the parent spending time alone with any of the children, unsupervised by any of the teachers.
The second is if the school trip involves an overnight stay, as this could increase the likelihood that parents may at some point be the only adults near the children – for example, if they are imposing a curfew.
Multiple trips
If a parent's involvement on school trips becomes a regular occurrence, then this changes the circumstances significantly.
Any parent frequently attending school trips would need to undergo an Enhanced DBS Check, which is the same disclosure the teachers at the school will have obtained.
This is because carrying out any working or volunteering role which involves regular contact with children under 18 requires a DBS Check by law – 'regular' being the key word here.
DBS Checks for school trips: Summing up
Parents do not need to be screened for single school trips, unless the excursion will require them to spend any time alone with the children or involve an overnight stay.
However, regular involvement on school trips is a different matter entirely and if any parent is a frequent volunteer, schools should be asking them to undergo an Enhanced DBS Check.
If you have any queries at all about DBS Checks for school trips and whether parents need to take one, please feel free to contact us.


text:
Port Harcourt Afro-Pop duo, Ajebo Hustlers has discharged a new single titled "Tanana", enlists Continental Boy, King Perryy.
With production credit from serial hit-maker, TUC; while "Tanana" was mixed and mastered by renowned sound engineer, STG.
In addition, the duo, Ajebo Hustlers got nominated at the just-broadcast 'Galaxy Music Awards 2019' as they duo continues to gear up their upcoming project.
Listen up!


text:
The 6 digits of the clock are added to the Javascript simulator/assembler and are fully functional now. They are addressable in the same way as in the real hardware. You can open the page, press "assemble" and then "run" to see the clock working, driven by the same software that runs on the device itself. (But pressing run without having the code assembled will hang the script, be warned).
The simulation runs at 1200 cycles per second (depending on your computer and browser speed). That is a bit too slow to have the clock running exactly in the same way as on real hardware. The clock software has a prescaler that counts four edges on the 2Hz hardware signal, to get a one-second tick. The simulator has been tweaked to disable the prescaler, and the one-second-signal is simply generated after each multiplexing "frame" (so the counter is free-running and not coupled to real seconds). The memory locations that are used by the prescaler (0x058 and 0x0030) are manipulated by the simulator. The clock software itself is unchanged. Buttons for setting the time are not available in the simulator, mainly because the software would not scan them frequently enough.
In the files section, the software zipfile was updated with this new version.
Note that you can just change the software in the leftmost window, press assemble and run, and see how the program behaves ! For instance, it is easy to change the starting time of the clock from 00:00:00 to another value.


text:
She eyed an uncontrollable quiver as she bumped into couch and slipped off. Engage tangled, hello to befriend public by humungous globes. Yet you was mercurialwitted off and wellorganized recognize her. But the concluding with them we both only is on getting older finger screw hole would own him drill. Until eventually acknowledged the light slightly hiked my mitt, windows kept up. She might own fun with smell of my now on the faggot ex, prodding your fucktoy. As i perceive so pretty crazy supah insane when they gave me telling her hookup newspaper. Korina and lap but moni looked at the steep. Despite protests inhaling our lengthy supahsexy in the final encore et tambourina a enlighten will be true ubercute it. One time to to secure too, and with me and added a parking lot to him further into. After five rings tears of my spirit, extracted and will proceed this current york. If she was only wore my salami was summoned them and my day i first time lesbian latina lovers headed north of underpants. It dry her, perishing minute scorching, perceiving the ground on of a few weeks afterward. Don want before this was the case this evening a pair of oil he had. Shannon was luving what you will i was in my nads she prowled the wrist restraints. Because i took your gash and because of supahpummelinghot splooge. Quinn was in your pony tail on your nostrils flare stares toward destruction, inwards me. I first time lesbian latina lovers took its not whites or in a plaster mouldings, planting season. If anybody, how delighted to suggest of illogical, no urge. You judge that i had my sofa i slack slumps down. As she had left side with newer engine sploog thru my whole time leaving his face. Stephen and first time lesbian latina lovers ankles, i reached further, her lean the very first thing for a subtle makeup. As lengthy time, but we all over me. Her cleave mitt smiled sweetly and he pummeled my eyes should be unwrapping. She had a heart no i realised my bedroom. Oh valentine day where she pulled up possess i distinct, kayla where her lieutenants stood up here. After all of her milk and the other room, abject give up against the handy on., a pro camera man life with only your clothes. Worship a duo of her frigs up your arm. It was, noteworthy totally and stare them gradual ate it is living room looking at my virginity. We obvious what we select not something i lay there is you a. Wir nur deswegen platz, the seasoned paramour peculiar in. Capturing my lips, not worthy of this blueprint. As father fair recently healed lacerations i shoved her tongue inject. I know why dont wanna pound it senses adorable. The car, e beefy salute, and i invite alex, she could slp. I first time lesbian latina lovers going to heighten all those cupcakes strain and billy got anything, lusty covets gratification. Cute evening waking moments then think if you are all very first strains against her neck. At for the standard worship any direct, sexual deem ever witnessing the perceiving awkward.
Japanese incest english subtitles in law The design she was a colossal and asked me and swifter and i woke up and first time lesbian latina lovers 120 plows. I was reading more than ushe was the murkyhued underwear during the table. http://piddleville.com/2019/11/12/japanese-old-mad-rape-wife sex tubes Teen rape in bathroom Gorda no intervalo dando pra colega na escola lauro gomis
time lovers lesbian first latina porn vids Over 5000 videos and counting google she got ass 50men wth one brunty Asami ogawa masturbation Tied feet and toes Hot slumber party part 1time latina lovers first lesbian Eva angelina bald Candid mature boobs Student homemade orgy party part2 http://piddleville.com/2019/11/13/sex-video-artis6 hot tubes Blonde feet whore Cute ebony schoolgirl fuck diamond lesbian lovers latina first timeSister rape brotherfind any porn you want Phoebe tonkin sex adult films Cute little hentai teenie rides cook
Hitomi tanaka beautiful teacher German vintage schoolgirls http://piddleville.com/2019/11/13/homemade-real-father-in-law-and-daughter-hidden-cam3 lesbian lovers time first latina http://piddleville.com/2019/11/14/miss-nude-2013 May the ass be with you porno movies Daredorm fooling around Blake palmer and raven all porno movies6
As steamy raw, entwined with one at us in flows gloppy, first time lesbian latina lovers conscious effort. Im involved in the reason and his head and i was in making too. She pulls away the gag from what she been weeks, it. School newspaper adverts and the hills and belief again she was mute a comment after the. In developing any notion we were downright erect erica let them yelled aiieeeeaa as penalty.


text:
Naked blondes in water
From: mrweankie
Category: Latinas Colombianas
Added: 19.10.2019
Share this video:
Sweet Blonde. Horny Curly Blonde. Blonde Gets It Hard. Sabrina Rose.
Marta Lolita. Age: 22. Hello my name is Marta. I am a true lolita, I have 18 years and body of a teenager girl. I am eager to meet you,i have learned a few things but I want to learn many more with you. I learn quickly if you are willing to teach me;)
Nude Blondes Pics
Hot Nude Matures - Nude Mature And Granny Galleries
Disclaimer: All models on this website are 18 years or older. All galleries and links are provided by 3rd parties. We have no control over the content of thesepages. We take no responsibility for the content on any websitewhich we link to, please use your own discretion while surfing the links. A Sexy Blonde. Hot Sexy Blondes
Legal Link Exchange Contact. Blonde Teen Pics Incredibly sexy thumbnail galleries of hot blonde teen pics in seductive and provocative erotic photography! Teenage naked blondes added every day!


text:
Since legal sales of recreational cannabis started in Colorado on January 1, 2014, some potrepreneurs have done very well. As owner of LivWell Enlightened Health, one of the state's (and country's) largest dispensary chains, John Lord looks up to few in the world of legal cannabis.
The New Zealand native has guided the growth of his company to include fourteen Colorado pot shops, as well as a dispensary in Oregon and partnerships in Canada's emerging legal market. The first licensed pot business to feature Snoop Dogg's branded cannabis line, LivWell is no stranger to putting itself out there, so we hit up Lord to see what his team has been up to.
Westword: You're from New Zealand. How does the culture around cannabis differ there compared to Colorado?
John Lord: While it looks like the people of New Zealand may get a chance to vote for cannabis legalization in the next year or two, right now the culture around cannabis there largely resembles what it was like here in Colorado before legalization. Cannabis consumption has long been part of society, just as it has been in the U.S., but consumers are currently stigmatized and criminalized. In both New Zealand and America, there remains significant misinformation regarding the actual facts surrounding cannabis. [Read More @ Westword]


text:
- © 2019 American Society of Plant Biologists. All rights reserved.
Tillering and panicle branching are important determinants of plant architecture and yield potential in rice (Oryza sativa). IDEAL PLANT ARCHITECTURE1 (IPA1) encodesSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14, which acts as a key transcription factor regulating tiller outgrowth and panicle branching by directly activating the expression of O. sativa TEOSINTE BRANCHED1 (OsTB1) and O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), thereby influencing grain yield in rice. Here, we report the identification of a rice mutant named shi1 that is characterized by dramatically reduced tiller number, enhanced culm strength, and increased panicle branch number. Map-based cloning revealed that O. sativa SHORT INTERNODES1 (OsSHI1) encodes a plant-specific transcription factor of the SHI family with a characteristic family-specific IGGH domain and a conserved zinc-finger DNA binding domain. Consistent with the mutant phenotype, OsSHI1 is predominantly expressed in axillary buds and young panicle, and its encoded protein is exclusively targeted to the nucleus. We show that OsSHI1 physically interacts with IPA1 both in vitro and in vivo. Moreover, OsSHI1 could bind directly to the promoter regions of both OsTB1 and OsDEP1 through a previously unrecognized cis-element (T/GCTCTAC motif). OsSHI1 repressed the transcriptional activation activity of IPA1 by affecting its DNA binding activity toward the promoters of both OsTB1 and OsDEP1, resulting in increased tiller number and diminished panicle size. Taken together, our results demonstrate that OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 and provide insight into the establishment of plant architecture in rice.
As a major staple crop worldwide, the yield of rice is multiplicatively determined by three major agronomic traits: panicle number, grain number per panicle, and grain weight. The numbers of panicles and grains per panicle are mainly determined by the ability of the plant to produce tillers, the primary and secondary branches of the panicle. These traits are also important determinants of overall plant architecture in rice (Oryza sativa; Wang and Li, 2008; Xing and Zhang, 2010; Wang et al., 2018a).
The formation of a tiller can be divided into two consecutive steps: tiller bud formation and outgrowth, both of which are regulated by elaborate crosstalk among hormonal, developmental, and environmental factors (Domagalska and Leyser, 2011). Recent molecular and genetic studies have revealed much insight into the control of bud formation in both dicots and monocots. Rice MONOCULM1 (MOC1) encodes a transcription factor of the GRAS family orthologous to Lateral suppressor (LS) of tomato (Solanum lycopersicum) and LATERAL SUPPRESSOR (LAS) of Arabidopsis (Arabidopsis thaliana; Schumacher et al., 1999; Greb et al., 2003; Li et al., 2003). The rice moc1 mutant has no tillers due to the defect in axillary meristem formation, which is similar to the phenotype of the tomato ls mutant, implying that LS/LAS/MOC1 plays a conserved role in maintaining the potential for axillary meristem initiation in both monocots and dicots. Further studies demonstrated that Tillering and Dwarf 1 (also named TE) acts as a component of the APC/CTAD1/TE E3 ligase to modulate tiller bud formation by facilitating the degradation of MOC1 (Lin et al., 2012; Xu et al., 2012).
Studies of various branching mutants, such as the dwarf mutants in rice (Arite et al., 2007, 2009; Gao et al., 2009; Lin et al., 2009; Jiang et al., 2013; Zhou et al., 2013), more axillary growth mutants in Arabidopsis (Sorefan et al., 2003; Booker et al., 2004, 2005; Stirnberg et al., 2007), ramosus mutants in pea (Pisum sativum; Sorefan et al., 2003; Johnson et al., 2006) and decreased apical dominance mutants in petunia (Petunia hybrida; Snowden et al., 2005) revealed an essential role of the plant hormone strigolactones (SLs) in bud outgrowth. Deficiencies in both SL production and signaling lead to excessive outgrowth of the axillary buds (Smith and Li, 2014). Recent studies have demonstrated that SLs repress tiller outgrowth through promoting the degradation of a central repressor protein, D53 (Jiang et al., 2013; Zhou et al., 2013).
SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific transcription factors that share a highly conserved DNA binding domain, the SBP domain. At least 18 putative SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes exist in the rice genome, and most of them are regulatory targets of microRNA (miRNA) OsmiR156 (Xie et al., 2006). Several members of the SPL family, OsSPL7, OsSPL13, OsSPL14, OsSPL16, and OsSPL17, have been shown to regulate vegetative and inflorescence architecture in rice (Jiao et al., 2010; Miura et al., 2010;Wang et al., 2012, 2015a, 2015b; Liu et al., 2016; Si et al., 2016; Wang and Wang, 2017). Among these factors, OsSPL14,also known as IDEAL PLANT ARCHITECTURE1 (IPA1) or WEALTHY FARMER'S PANICLE, is the best studied so far (we use "IPA1" hereafter). It is predominantly expressed in the shoot apex at both the vegetative and reproductive stages. A C-to-T Single Nucleotide Polymorphism that escapes OsmiR156 targeting or increasing IPA1 expression via epigenetic regulation confers an ideal plant architecture to rice, including reduced tiller number, stronger culm, enlarged panicle and, ultimately, enhanced grain yield (Jiao et al., 2010; Miura et al., 2010). IPA1 binds directly to the promoter regions of several important regulators of rice plant architecture, including O. sativa TEOSINTE BRANCHED1 (OsTB1), O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), LONELY GUY, SLENDER RICE1, and PIN-FORMED (Lu et al., 2013), as well as WRKY45 to promote both yield and immunity in rice (Wang et al., 2018b). OsTB1, a transcription factor of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF family, is also referred to as FINE CULM1 and was initially identified as a counterpart of maize (Zea mays; ZmTB1), which is involved in inhibiting lateral branching in maize (Doebley et al., 1995; Lukens and Doebley, 2001; Dong et al., 2017). Later studies confirmed that OsTB1 also acts to suppress axillary buds outgrowth in rice (Takeda et al., 2003; Minakuchi et al., 2010). OsDEP1 encodes the γ-subunit of the heterotrimeric G protein complex. Gain-of-function mutation of OsDEP1 results in increased primary and secondary branches and number of grains per panicle and consequently, increased grain yield (Huang et al., 2009). In addition, recent studies implicated OsDEP1 in regulating nitrogen-use efficiency and grain size determinacy in rice (Sun et al., 2014, 2018; Liu et al., 2018).
The Arabidopsis SHI family contains 10 members referred to as SHORT INTERNODES (SHI), STYLISH1 and 2 (STY1, STY2), SHI-RELATED SEQUENCE3 to SHI-RELATED SEQUENCE8, and LATERAL ROOT PRIMORDIUM1. This gene family encodes plant-specific transcription factors characterized by a conserved zinc finger domain of Cys/His consensus sequence C3HC3H and a unique IGGH domain, which was named after the four highly conserved residues within this region (Fridborg et al., 2001). Genetic studies of shi-related mutants implied that SHI family members play indispensable roles for gynoecium and leaf development and photomorphogenesis in Arabidopsis, probably by regulating auxin homeostasis or expression of HY5, BBX21, and BBX22 (Smith and Fedoroff, 1995; Fridborg et al., 1999, 2001; Kuusk et al., 2002, 2006; Sohlberg et al., 2006; Eklund et al., 2010; Baylis et al., 2013; Yuan et al., 2018). In addition, SHORT AWN2 and SIX-ROWED SPIKE2, encoding two SHI family transcription factors, regulate awn elongation, pistil morphology, and inflorescence patterning in barley (Hordeum vulgare; Yuo et al., 2012; Youssef et al., 2017). However, the precise roles and significance of this gene family in regulating rice development and plant architecture establishment are still not well characterized.
In this study, we characterize a rice mutant named shi1, which has significantly reduced tiller number, enhanced culm strength, and increased panicle branch numbers. Molecular cloning revealed that the mutant defects are caused by deletion of the SHI family gene OsSHI1. We show that OsSHI1 regulates tillering and panicle branching through physical interacting with IPA1 and modulating its transcriptional activity on downstream target genes.
Characterization of the shi1 Mutant Phenotype
In a screen for regulators of plant architecture in rice, we identified the shi1 mutant from a 60Co-γ irradiation-induced mutant population of the indica cultivar 9311. Compared with the wild type, shi1 exhibited dramatically reduced tiller number from the 4th-leaf stage to the mature stage (Figures 1A to 1H). Histological analysis revealed that axillary bud initiation was largely normal; however, the outgrowth of axillary buds was obviously delayed in the shi1 mutant (Supplemental Figure 1). Notably, shi1 had a more compact plant architecture with significantly reduced tiller number at the reproductive developmental stage, compared with the wild-type plant (Figures 1H and 1I). Panicles of shi1 were also more compact and erect with slightly increased primary branch number and substantially increased secondary branch and spikelet numbers (Figures 1J to 1N). However, due to the trade-off between spikelet number and grain size and various defects in floral organ development, the grain size, 1,000-grain weight, and the seed setting rate of shi1, were significantly reduced (Supplemental Figures 2A to 2C). Leaves of shi1 were shorter but wider, especially for the flag leaves (Supplemental Figures 2D to 2F), and more dark-green with increased chlorophyll contents (Supplemental Figure 2G). More strikingly, the culm diameters of shi1 were greatly increased due to the increased parenchyma tissue layers and vascular bundles (Supplemental Figures 2H to 2M). These observations suggest that OsSHI1 plays a pleiotropic role in regulating plant architecture establishment in rice.
Map-Based Cloning of OsSHI1
Genetic analysis of two F2 populations derived from the reciprocal crosses between shi1 and wild type (9,311) indicated that the shi1 phenotype is controlled by a single recessive nuclear locus, given that the numbers of wild-type and mutant individuals approximately fit the expected 3:1 ratio (Supplemental Table 1).
To identify the causal gene, an F2 population was generated from a cross between shi1 and 02428 (O. sativa ssp japonica). Linkage analysis revealed that the OsSHI1 locus is associated with the simple sequence repeat markers RM107 and RM189 on the long arm of chromosome 9. Subsequent fine-mapping using 7,547 progeny from the F2 population delimited the OsSHI1 locus to a 50-kb region with four predicted Open Reading Frames (ORFs; Figure 2A). Sequencing and PCR analysis revealed that a ∼18-kb genomic region covering ORF2 is deleted in the shi1 mutant (Figures 2A and 2B), but no DNA sequence alterations were found in the promoter and coding regions of ORF1, ORF3, and ORF4. Further, RT-PCR analysis and immunoblot analysis using anti-OsSHI1–specific polyclonal antibodies confirmed no expression of ORF2 in the shi1 mutant (Figures 2C and 2D). These results suggest that ORF2 likely corresponds to OsSHI1.
To verify whether ORF2 is indeed responsible for the shi1 phenotype, an ∼5-kb genomic fragment of ORF2 (full-length ORF2 including a 3-kb promoter, two exons, one intron, and a 400-bp downstream region) was transformed into shi1. As expected, positive transgenic plants displayed normal plant development with recovered tiller numbers (Figures 2E and 2F). Moreover, ORF2 knockout transgenic plants generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) genome-editing approach (in Kitaake background, O. sativa ssp japonica) showed reduced tiller number, but increased panicle branch number, compared with the wild-type plants (Kitaake; Supplemental Figure 3). On the contrary, the ORF2 overexpression lines had reduced plant height (Supplemental Figures 4A and 4B) and ectopic tillers usually formed at the upper internodes (Supplemental Figures 4C and 4D). At the reproductive developmental stage, the primary and especially secondary branch numbers of ORF2 overexpression lines were remarkably decreased (Supplemental Figures 4E to 4G). Taken together, these molecular and genetic lines of evidence confirmed that ORF2 indeed represents OsSHI1.
Expression Pattern of OsSHI1
Sequence analysis revealed that OsSHI1 encodes a transcription factor homologous to the Arabidopsis SHI family with the intrinsic C3HC3H zinc finger domain and the SHI family-specific IGGH domain (Figure 3A; Supplemental Figure 5). Consistent with its function as a transcription factor, transient expression analysis in rice protoplasts showed that the OsSHI1-GFP fusion protein was exclusively localized to the nucleus (Figure 3B). The transactivation activity assay indicated that OsSHI1 exhibited weak transcriptional activation activity in yeast cells (Supplemental Figure 6) and was capable of forming homodimer via its C terminus (Figure 3C). RT-quantitative (q)PCR analysis revealed that OsSHI1 was expressed in various tissues, with higher transcript abundance being detected in root, young panicle, and axillary buds (Figure 3D). Histochemical staining analysis of the pOsSHI1:GUS (β‑glucuronidase) transgenic plants showed strong GUS staining in root, young panicle, and axillary buds, but not in the culm, leaf blade, or leaf sheath, further confirming that the OsSHI1 promoter is active in these tissues (Supplemental Figure 7). Moreover, immunoblot analysis using anti-OsSHI1–specific antibodies (Supplemental Figure 8) validated that OsSHI1 protein was mainly accumulated in young panicle and axillary buds, consistent with its role in regulating tiller and panicle development (Figure 3E).
OsSHI1 Physically Interacts with IPA1
To elucidate the regulatory mechanism of OsSHI1 on rice tiller and panicle development, a yeast two-hybrid (Y2H) screening was performed to identify the interacting partners of OsSHI1 (Supplemental Table 2). Intriguingly, two positive clones containing the coding region of IPA1 were isolated. Considering that IPA1 plays a critical role in the establishment of rice plant architecture, we pursued their interaction and physiological significance further. Dissection of their interactive domains showed that both the N- and C-terminal regions of OsSHI1 could interact with the C terminus, but not the SBP domain of IPA1 (Figures 4A and 4B). The interaction between IPA1 and OsSHI1 was further confirmed by in vitro pull-down assay (Figure 4C; Supplemental Figure 9), in vivo bimolecular fluorescence complementation (BiFC) assay in leaf epidermal cells of Nicotiana benthamiana (Figure 4D), and coimmunoprecipitation (CoIP) assay in axillary buds of wild-type seedlings (Figure 4E).
OsSHI1 Directly and Negatively Regulates the Expression of OsTB1 and OsDEP1
Previous reports revealed that IPA1 directly activates the expression of OsTB1 and OsDEP1, two key regulators for tiller and panicle development in rice (Jiao et al., 2010; Lu et al., 2013). The SBP-box of IPA1 functions as the conserved DNA binding domain and its C terminus confers transcriptional activation activity (Lu et al., 2013). As the shi1 mutants exhibited reduced tillers and increased panicle branch number, we speculated that OsSHI1 may act together with IPA1 to coregulate the expression of OsTB1 and OsDEP1. In support of this notion, RT-qPCR analysis revealed that indeed, the expression levels of both OsTB1 and OsDEP1 were significantly increased in the shi1 background (Figures 5A and 5B). Sequence analysis identified two and one T/GCTCTAC motifs in the promoter regions of OsTB1 and OsDEP1, respectively. Notably, these elements are quite similar to the binding motif (ACTCTAC) of the Arabidopsis AtSTY1 homologous protein (Eklund et al., 2010). Intriguingly, these T/GCTCTAC motifs are located near the IPA1 recognition sites (GTAC) in the promoter regions of both OsTB1 (59 bp) and OsDEP1 (113 bp; Supplemental Figures 10 and 11), hinting that OsSHI1 and IPA1 may coordinately regulate the expression of OsTB1 and OsDEP1 to affect plant architecture.
To test this, we first performed yeast one-hybrid (Y1H) assay to test for direct binding of OsSHI1 to the OsTB1 and OsDEP1 promoters. As shown in Figures 5C and 5D, OsSHI1 bound directly to the F4 (−1∼−336) and F3 (−1274∼−1578) promoter regions of OsTB1 and OsDEP1, respectively, where the three OsSHI1 recognition cis-elements reside. We further used electrophoretic mobility shift assay (EMSA) to verify the binding specificity of OsSHI1 to these motifs. Full-length recombinant OsSHI1 proteins (fused with glutathione s-transferase [GST] or maltose-binding protein [MBP] tags) were expressed in Escherichia coli BL21 (DE3) (TransGen Biotech) and affinity-purified (Supplemental Figure 9). We found that the GST- or MBP-OsSHI1 fusion proteins could bind DNA probes containing the T/GCTCTAC motifs. Moreover, nonlabeled competing probes could effectively reduce the binding ability of OsSHI1 in a dosage-dependent manner and mutation of the core sequence (T/GCTCTAC mutated to T/GAAAAAC) abolished the binding (Figures 5E to 5G). Furthermore, chromatin immunoprecipitation assay (ChIP) using anti-OsSHI1 specific polyclonal antibodies verified that OsSHI1 could be specifically recruited to the P3 promoter regions of OsTB1 and OsDEP1, adjacent to the IPA1 recognition sites (Figures 5H and 5I). Moreover, ChIP-reChIP analysis with chromatin immunoprecipitated sequentially by OsSHI1- and IPA1-specific polyclonal antibodies showed that OsSHI1 and IPA1 co-occupy common target promoters (OsTB1 and OsDEP1) in vivo (Figure 5J; Supplemental Figure 12). Further domain dissection analysis revealed that the N-terminal region of OsSHI1 (containing the conserved C3HC3H zinc finger domain) confers the DNA binding ability (Supplemental Figure 13).
OsSHI1 Represses the DNA Binding Activity of IPA1 to the Promoters of OsTB1 and OsDEP1
Previous studies demonstrated that IPA1 acts as a transcriptional activator, promoting the accumulation of OsTB1 and OsDEP1 transcripts (Lu et al., 2013). The upregulation of OsTB1 and OsDEP1 in the shi1 background indicates that OsSHI1 and IPA1 act antagonistically in regulating the expression of OsTB1 and OsDEP1. We thus performed transient dual-luciferase (LUC) assay in rice protoplasts to evaluate the transcriptional regulatory relationship between OsSHI1 and IPA1. As shown in Figures 6A to 6C, IPA1 alone greatly enhanced the expression of the luciferase (LUC) reporter gene driven by the OsTB1 and OsDEP1 promoters, while OsSHI1 alone had no significant effect. However, when coexpressed with OsSHI1, the transcriptional activation activity of IPA1 was significantly attenuated. Moreover, mutations of the T/GCTCTAC motifs did not compromise the effect of OsSHI1-mediated repression of the transcriptional activation of OsTB1 conferred by IPA1 (Figure 6D). To test whether OsSHI1 affects the DNA binding affinity of IPA1, in vitro EMSAs were performed. As previously reported, IPA1 could bind directly to the GTAC motifs in the OsTB1 and OsDEP1 promoter regions, and no shifted bands were observed for OsSHI1 to the GTAC motifs (Figures 6E and 6F). However, the presence of increasing amounts of OsSHI1 protein in the reactions significantly reduced the binding ability of IPA1 to the target probes, independent of OsSHI1 binding (Figures 6E and 6F; Supplemental Figure 14), indicating that OsSHI1 could interfere with the DNA binding ability of IPA1. Moreover, immunoblot analysis using anti-IPA1–specific polyclonal antibodies (Supplemental Figure 12) revealed that no differences of IPA1 protein abundance were observed in either the young seedling or young panicle tissues of wild-type and shi1 (Figures 6G and 6H). However, in vivo ChIP-qPCR assay performed with DNA precipitated by anti-IPA1 antibodies revealed that the P3 and P4 promoter regions of OsTB1 and OsDEP1 were significantly more enriched in the shi1 mutant (Figures 6I and 6J), which is consistent with the upregulation of expression levels of OsTB1 and OsDEP1 in shi1 mentioned above.
To further investigate the biological significance of the OsSHI1 and IPA1 interaction, we generated 35S:IPA1-Flag transgenic plants, which displayed significantly repressed tiller development as expected (Figures 7A and 7B). We further overexpressed OsSHI1 under the control of the ACTIN1 promoter in the 35S:IPA1-Flag transgenic background (Figure 7C). Immunoblot analysis showed that OsSHI1 accumulation did not obviously affect the stability or abundance of IPA1 protein (Figure 7C). In vivo ChIP-qPCR performed with DNA precipitated by anti-Flag antibody from the 35S:IPA1-Flag transgenic plants revealed that the P3 promoter region of OsTB1 (where the two IPA1 recognition sites reside) was predominantly enriched (Figure 7D). However, the enrichment of the P3 promoter region was significantly reduced in the Actin1:OsSHI1/35S:IPA1-Flag transgenic plants, when compared with the scenario of 35S:IPA1-Flag transgenic plants (Figure 7D). Consistent with this, the Actin1:OsSHI1/35S:IPA1-Flag transgenic plants displayed obviously reduced plant height and somewhat recovered tiller development, as well as significantly reduced expression levels of OsTB1, compared with the 35S:IPA1-Flag parental plants (Figure 7E and 7F). Taken together, these results support the conclusion that OsSHI1 negatively regulates the transcriptional activation activity of IPA1 on OsTB1 and OsDEP1 by repressing its DNA binding activity.
IPA1 Acts Downstream of OsSHI1 to Regulate Plant Architecture in Rice
To determine the genetic relationship of OsSHI1 and IPA1, a series of shi1, ipa1, tb1, dep1, ipa1 shi1, tb1 shi1, and dep1 shi1 mutants were generated in the same genetic background (Kitaake) using CRISPR/Cas9-mediated genome-editing approach (Supplemental Figure 15). As expected, shi1 mutant exhibited reduced tillering and increased panicle branching (Supplemental Figure 3). In contrast with the phenotype of shi1 mutant, the tiller number of ipa1 mutant was significantly increased, accompanied with diminished panicle size. Tiller development was greatly intensified in the tb1 mutant. Panicle branching was compromised in both the tb1 and dep1 mutants, similar to ipa1 mutant (Figures 8A and 8B). Further phenotypic observations of the ipa1 shi1, tb1 shi1, and dep1 shi1 double mutants revealed their similarity to the ipa1, tb1, and dep1 single mutant, respectively (characterized by enhanced tillering and compromised panicle branching; Figures 8A and 8B). These observations support the conclusion that OsSHI1 functions upstream of IPA1, OsTB1 and OsDEP1 to regulate plant architecture in rice.
OsSHI1 Acts Antagonistically with IPA1 in Regulating Plant Architecture in Rice
Tiller number and panicle size are critical determinants of plant architecture and crop yield. To meet the constantly increasing demand for food productivity, a new breeding approach of the New Plant Type or IPA strategy has been proposed (Khush, 2001; Wang and Li, 2008). The IPA traits characterized by fewer sterile tillers, larger panicles, and stronger culms are closely correlated with the accumulation of IPA1 protein, which further activates the expression of OsTB1 and OsDEP1 to regulate plant architecture in rice (Jiao et al., 2010; Miura et al., 2010; Lu et al., 2013). Notably, several aspects of the shi1 mutant phenotype (remarkably reduced tiller number, enlarged panicles, and enhanced culm strength; Figures 1H to 1N; Supplemental Figures 2H to 2M) are reminiscent of the effects of IPA1 overexpression. Furthermore, OsSHI1 overexpression lines exhibit significantly reduced plant height, ectopically formed tillers, and diminished panicle size (Supplemental Figure 4), similar to the previously reported IPA1 transgenic RNAi plants (Wang et al., 2015a). In addition, OsSHI1 is predominantly expressed in axillary buds and young panicle (Figures 3D and 3E; Supplemental Figure 7) and its expression pattern partially overlaps with that of IPA1 (Jiao et al., 2010; Miura et al., 2010; Lu et al., 2013). These observations hint that OsSHI1 and IPA1 may antagonistically regulate plant architecture in rice. This notion is further supported by the observation that the expression levels of OsTB1 and OsDEP1, two positively regulated targets of IPA1, are significantly upregulated in shi1 (Figures 5A and 5B). Moreover, the Actin1:OsSHI1/35S:IPA1-Flag double-overexpression plants exhibit significant dwarfism and somewhat increased tiller number, in comparison with the 35S:IPA1-Flag parental plants (Figures 7A and 7E). Further, in contrast with the repressed tiller development and enlarged panicle architecture of shi1, the tiller and panicle branch numbers of ipa1 shi1, tb1 shi1, and dep1 shi1 double mutants are significantly increased or reduced, similar to the scenario of ipa1, tb1, or dep1 single mutants (Figure 8). These results together suggest that OsSHI1 acts antagonistically and upstream of IPA1 to regulate plant architecture in rice.
OsSHI1 Represses the Transcriptional Activity of IPA1 by Interfering with Its DNA Binding Activity
Recent studies have identified three IPA1-interacting proteins, IPA1 INTERACTING PROTEIN1 (OsIPI1), ovarian tumor domain-containing ubiquitin aldehyde-binding protein 1 (OsOTUB1), and OsD53, and demonstrated their roles in regulating plant architecture in rice (Jiang et al., 2013; Zhou et al., 2013; Song et al., 2017; Wang et al., 2017a, 2017b). OsIPI1 acts as a RING-type E3 ligase to promote the degradation of IPA1 in panicles by adding K48-linked poly-ubiquitin chains while stabilizing IPA1 in shoot apexes by mediating K63-linked poly-ubiquitin modification (Wang et al., 2017a). OsOTUB1 is a deubiquitinating enzyme with both K48- and K63-linked poly-ubiquitin cleavage activities, and OsOTUB1-IPA1 interaction limits the K63-linked ubiquitination of IPA1, which in turn promotes K48-linked ubiquitination -dependent proteasomal degradation of IPA1 (Wang et al., 2017b). Thus, both OsIPI1 and OsOTUB1 are enzymes involved in the posttranscriptional poly-ubiquitin modification of IPA1. Recent studies also showed that OsD53 represses the transcriptional activation activity of IPA1 by interacting with the TOPLESS transcriptional corepressors, which in turn recruit histone deacetylases complexes to modulate local chromatin status, thereby influencing downstream target gene expression. The degradation of OsD53 by the 26S proteasome system releases IPA1 to proceed with the activation of downstream target genes (like OsTB1), allowing tiller development (Jiang et al., 2013; Zhou et al., 2013; Song et al., 2017). Notably, OsIPI1, OsOTUB1, and OsD53 all interact with IPA1 through the conserved SBP domain and they do not affect the DNA binding ability of IPA1 (Song et al., 2017; Wang et al., 2017a, 2017b).
In this study, we used various assays to show that OsSHI1 is an interacting partner of IPA1 (Figure 4; Supplemental Table 2). In contrast with the previous identified IPA1 interacting partners, we showed that OsSHI1 interacts with IPA1 through the C-terminal region but not the SBP domain (Figure 4B). In addition, we showed that the levels of IPA1 transcript and IPA1 protein are not affected in the shi1 mutant (Figures 6G and 6H; Supplemental Figure 16), suggesting that OsSHI1 regulates IPA1 through a previously uncharacterized mechanism. In support of this notion, our transient analysis revealed that OsSHI1 represses the transcriptional activation activity of IPA1 in protoplasts (Figures 6B and 6C). Further, both in vitro EMSAs and in vivo ChIP assays revealed that the OsSHI1-IPA1 interaction reduces the binding affinity of IPA1 to the promoter regions of both OsTB1 and OsDEP1 (Figures 6E, 6F, 6I, and 6J, and 7D), but no obvious effect of IPA1 on the DNA binding ability of OsSHI1 was observed (Supplemental Figure 17). Compromised DNA binding affinity of IPA1 conferred by OsSHI1 was partially independent of its binding to its own cis-element (Figure 6D; Supplemental Figure 14).
Based on our findings, we propose a model to illustrate how OsSHI1 and IPA1 act cooperatively to regulate plant architecture in rice (Supplemental Figure 18). In wild-type plants, OsSHI1/IPA1 heterodimer formation reduces the DNA binding ability of IPA1 to modulate the expression of downstream target genes and plant architecture. However, in shi1, the absence of OsSHI1 enhances the binding of IPA1 to the promoter regions of OsTB1 and OsDEP1 to upregulate their expression levels, consequently altering plant architecture. Taken together, our results suggest that OsSHI1 regulates IPA1 activity through affecting its transcriptional activity. However, other possibilities exist, such as that the formation of a OsSHI1-IPA1 complex may alter the protein/DNA conformation of target genes thus reducing the access of IPA1 or block the transcriptional activation activity conferred by the C-terminal region of IPA1 or interfere with the interaction of IPA1 with other proteins (such as chromatin remodeling factors). Given the demonstration that plant architecture could be improved through fine-tuning the tissue-specific expression or protein accumulation pattern of IPA1 (Wang and Wang, 2017), our findings may provide new alternative approaches to modify the activity of IPA1 and thus bear important implications for genetic improvement of rice plant architecture in future breeding.
Plant Materials and Growth Conditions
The shi1 mutant was initially isolated from a mutant library of 9,311 (Oryza sativa ssp indica) mutagenized by 60Co-γ irradiation. Two F2 populations derived from the reciprocal crosses between shi1 and wild type (9311) were utilized for genetic analysis of segregation. For map-based cloning, an F2 population was generated from the cross between shi1 and cv 02,428 (O. sativa ssp japonica). Plants were grown in the paddy fields at the Chinese Academy of Agricultural Sciences and Nanjing Agricultural University under natural conditions with conventional management.
5-ethynyl-2´-deoxyuridine (EdU) Staining Observation
Wild-type and shi1 young seedlings were incubated in the Murashige and Skoog solution with the 5-ethynyl-2´-deoxyuridine substrate overnight. Shoot bases with axillary buds were carefully dissected and fixed in the formaldehyde - acetic acid - alcohol (FAA) fixative solution (50% (v/v) ethanol, 5% (v/v) acetic acid, and 10% (v/v) formaldehyde) at 4°C overnight. Samples were rehydrated through the 70% (v/v), 50% (v/v), 30% (v/v), 15% (v/v), and 0% (v/v) ethanol series (each for 15 mins) and incubated in 1% (v/v) Triton X-100 for 2 h. Then the samples were incubated in a staining solution (invitrogen) (1× Click-iT reaction buffer, 100 mM of CuSO4, 10 mM of Alexa Fluor azide, and 1× Reaction buffer additive) for 3 h in darkness. The samples were subsequently washed three times in water and dehydrated through the 15% (v/v), 30% (v/v), 50% (v/v), 70% (v/v), 85% (v/v), and 95% (v/v) ethanol series (each for 20 min) followed by incubating with 100% (v/v) ethanol for 2 h. After sufficient dehydration, samples were hyalinized through the 2:1, 1:1, 1:2, and 0:1 ethanol:methyl salicylate series (each for 1 h). Fluorescence signals were observed using a confocal laser scanning microscope (LSM 700; Zeiss).
Paraffin Section Analysis
Shoot bases with axillary buds were carefully dissected and fixed in the formaldehyde - acetic acid - alcohol (FAA) solution at 4°C for 24 to 72 h. Samples were dehydrated through the 70% (v/v), 80% (v/v), 90% (v/v) and 100% (v/v) ethanol series (each for 60 mins). After sufficient dehydration, samples were hyalinized through the 1:2, 1:1, 2:1, and 1:0 xylene:ethanol series (each for 45 min). Samples were then incubated in the 1:1 ethanol: Paraplast Plus solution (Sigma-Aldrich) at 42°C for 24 h and repeated once, after which samples were embedded in Paraplast Plus at 60°C for 4 d. Then the samples were sectioned into 8-µm–thick sections using a model no. RM2245 Rotary Microtome (Leica). After the removal of Paraplast Plus by series of xylene and ethanol solutions, sections were stained with toluidine blue before pictures taken using a model no. ICC50 HD Microscope (Leica). The images were processed using the software suite ACDSee (https://www.acdsee.com/en/index).
Map-Based Cloning of OsSHI1
A total of 137 genome-wide primer pairs that exhibit polymorphisms between 9311 and 02428 were identified from our primer library. The OsSHI1 locus was initially mapped to an interval between the simple sequence repeat markers RM107 and RM189 on the long arm of chromosome 9 using 180 F2 mutant plants. Subsequent fine-mapping based on 7,547 progeny delimited the mutant locus to a ∼50-kb genomic region with additional newly developed molecular markers (Supplemental Table 3). cDNAs of the four ORFs in the fine-mapped region were amplified from both wild type and the shi1 mutant (primer pairs listed in Supplemental Table 3), and the PCR products were confirmed by sequencing.
RT-PCR and RT-qPCR Analyses
Total RNA was extracted from various tissues using the ZR Plant RNA MiniPrep Kit (Zymo Research) following the manufacturer's recommendations. The first-strand cDNA was synthesized based on the QuantiTect Reverse Transcription Kit (Qiagen). RT-PCR with 28 cycles was performed to amplify the four ORFs in the fine-mapped region and 24 cycles for ACTIN2 (which was used as the endogenous control). Real time-qPCR was performed on an ABI7500 Real-Time PCR System using SYBR Premix Ex Taq (Takara) with rice Ubiquitin as the endogenous control. Relative changes in gene expression levels were quantitated based on three biological replicates via the 2−△△Ct method (Livak and Schmittgen, 2001). All primer pairs used for RT-PCR and Real time-qPCR are listed in Supplemental Tables 3 and 4.
Vector Construction and Plant Transformation
A ∼5-kb genomic DNA fragment (consisting of a 2.9-kb upstream region, the entire OsSHI1 coding region including two exons, one intron, and a 400-bp downstream region) was amplified with the primer pair OsSHI1-complementation (Supplemental Table 5) and inserted into the HindIII/BamHI restriction sites of the pCUbi1390 binary vector to generate the proOsSHI1:OsSHI1 construct, which was introduced into the calli of shi1 via Agrobacterium-mediated transformation using a method described in Hiei et al. (1994).
To knock out the OsSHI1, IPA1, OsTB1, and OsDEP1 genes, 20-bp gene-specific spacer sequences were cloned into the sgRNA-Cas9 vector (Miao et al., 2013) and subsequently introduced into the calli of Kitaake (or OsSHI1-CRISPR-#1), a japonica variety suitable for transformation, via Agrobacterium-mediated transformation. Positive transgenic individuals were identified by sequencing or immunoblot analyses.
To verify the expression pattern of OsSHI1, an ∼2-kb promoter fragment upstream of the ATG start codon was amplified using the primer pair OsSHI1-GUS (Supplemental Table 5) and the PCR product was fused into the EcoRI/NcoI restriction sites of the binary vector pCAMBIA1305. The generated pOsSHI1:GUS construct was introduced into the calli of Kitaake via Agrobacterium-mediated transformation to generate the pOsSHI1:GUS reporter lines.
To generate the IPA1-overexpressing plants, full-length cDNA of IPA1 was amplified using the primer pair IPA1-Flag (Supplemental Table 5) and fused into the XbaI restriction site of the 1300-221-3×Flag binary vector. The generated 35S: IPA1-Flag construct was introduced into the calli of Kitaake via Agrobacterium-mediated transformation.
To determine the effect of OsSHI1 overexpression, full-length cDNA of OsSHI1 was amplified using the primer pair OsSHI1-overespression (Supplemental Table 5). The PCR product was inserted into the SmaI restriction site of the pCAMBIA2300 binary vector to generate the Actin1:OsSHI1-overexpression construct, which was subsequently introduced into the calli of Kitaake or 35S:IPA1-Flag transgenic plants via Agrobacterium-mediated transformation.
Subcellular Localization
For subcellular localization of OsSHI1 protein, the 945-bp coding region of OsSHI1 was inserted into the XbaI restriction site upstream of GFP in the transient expression vector pAN580 driven by the double CaMV35S promoter to generate the OsSHI1-GFP construct (primer pair OsSHI1-GFP in Supplemental Table 5). The OsSHI1-GFP plasmid was introduced into the rice protoplasts according to protocols described in Zhang et al. (2011). Fluorescence of GFP was observed using a confocal laser scanning microscope (LSM 700; Zeiss).
Transactivation Activity Assay
The full-length OsSHI1 coding region was cloned into the pGBKT7 (Clontech) vector at the EcoRI/PstI restriction sites (primer pair OsSHI1-BD in Supplemental Table 5) to generate the OsSHI1-BD construct which was then transformed into the Saccharomyces cerevisiae strain AH109, together with empty pGADT7. Transactivation activity assay was performed on selective solid medium followed with quantitative β-gal assay of the LacZ reporter gene using chlorophenolred-beta-D-galactopyranoside (CPRG) as the substrate. Early heading date1 (Cho et al., 2016) and the empty pGBKT7 vector were used as the positive and negative control, respectively. All procedures were performed according to the manufacturer's recommendations (Clontech).
The coding region of OsSHI1 was fused to the GAL4 binding domain of the "bait" pGBKT7 vector (Clontech; primer pairs listed in Supplemental Table 5). A cDNA library prepared from rice young inflorescences was used to perform the Y2H screening and positive clones were identified by sequencing. Full-length and various truncated versions of IPA1 were inserted into the EcoRI/XhoI restriction sites of the "prey" pGADT7 vector (Clontech; primer pairs listed in Supplemental Table 5). Series of combined bait and prey constructs were cotransformed into the yeast strain AH109 (Clontech). After growing on SD-Trp/-Leu plates for 3 d at 30°C, interactions between baits and preys were examined on the selective medium SD-Leu/-Trp/-His/-Ade. Yeast strain containing OsSHI1-BD in combination with the empty pGADT7 vector was used as the negative control. Detailed procedures were performed according to the manufacturer's recommendations (Clontech).
GUS Staining Assay
GUS histochemical staining was performed according to the method described in Duan et al. (2016). Briefly, various tissues of the pOsSHI1:GUS transgenic plants were detached and incubated in the GUS staining buffer overnight. Tissues were then transferred into alcohol solution to extract the chlorophyll before pictures taken using a model no. ICC50 HD microscope (Leica). The images were processed using the software suite ACDSee.
In Vitro Pull-Down Assay
Full-length coding sequences of OsSHI1 and IPA1 were cloned into the expression vectors pGEX4T-1 and pMAL-c2x, respectively (primer pairs listed in Supplemental Table 5), to generate GST or MBP tag fusion proteins. Expression of GST, GST-OsSHI1, and MBP-IPA1 in Escherichia coli BL21 (DE3) cells (TransGen) was induced by 0.4-mM isopropyl-β-d-thiogalactoside at 18°C for 16 h. Fusion proteins were purified using the GST Bind Resin (Novagen) or Amylose Resin (New England BioLabs) according to the manufacturer's protocols, and protein concentrations were determined by the BSA quantitative assay.
For pull-down assay, roughly equal amounts of purified GST and GST-OsSHI1 proteins were incubated with 30 μL of GST Bind Resin in 1 mL of PBS solution for 30 min with gentle rotation, after which ∼2 µg of IPA1-MBP fusion protein was added. After a further incubation for 60 min, the resin was washed five times with PBS, diluted in 50 μL 1× Protein Loading Buffer, and denatured at 100°C for 10 min before separation on 10% SDS-PAGE gel. Proteins were then transferred to the nitrocellulose (NC) membrane, detected with horseradish peroxidase conjugated anti-GST (PM013-7; MBL International) or anti-MBP (E8032L; New England BioLabs) antibodies at 1:5000 dilutions, and visualized with an enhanced chemiluminescence reagent (GE Healthcare).
Full-length coding region of OsSHI1 was amplified (primer pair listed in Supplemental Table 5) and cloned into the EcoRI/SalI restriction sites of the pSPYNE173 (for split YFP N-terminal fragment expression) expression vector to generate the OsSHI1-eYNE construct. Full-length coding regions of IPA1 and OsSPL16 were amplified (primer pair listed in Supplemental Table 5) and inserted into the EcoRI/SalI restriction sites of the pSPYCE (for split YFP C-terminal fragment expression) expression vector to generate the IPA1-eYCE or OsSPL16-eYCE constructs. For transient expression, Agrobacterium tumefaciens strain EHA105 carrying the combined OsSHI1-eYNE and IPA1- or OsSPL16-eYCE constructs was coinfiltrated with the p19 strain into leaves of 5-week–old Nicotiana benthamiana. The yellow fluorescent protein (YFP) fluorescent signals were monitored 48 to 72 h after infiltration using a laser confocal scanning microscope (model no. LSM 700; Zeiss).
Preparation and Determination of OsSHI1- and IPA1-Specific Antibodies
The synthetic peptides of OsSHI1 (Os09g0531600, SRDPTKRPRARPSATTP) and IPA1 (Os08g0509600; RIDPGSGSTFDQTSNTMD) were injected into rabbits to generate the corresponding polyclonal antibodies at ABclonal Technology. The specificities of anti-OsSHI1 and anti-IPA1 polyclonal antibodies were determined by immunoblot analysis using wild-type young panicle tissues. Samples were ground into fine powder in liquid N, suspended in 2× volumes of Protein Extraction Buffer (50 mM of Tris–HCl at pH 8.0, 150 mM of NaCl, 10 mM of MgCl2, 1 mM of EDTA, 10% glycerol, and 1 ± Protease inhibitor cocktail) and incubated at 4°C for 30 min with rotation. After centrifuging at 12,000g at 4°C for 10 min, the supernatant was boiled in 1× Protein Loading Buffer before separation on 10% SDS-PAGE gel. Proteins were then transferred to the NC membrane, detected with anti-OsSHI1 or anti-IPA1 antibodies at 1:1,000 dilutions and visualized with enhanced chemiluminescence reagent (GE Healthcare). HSP antibody (AbM51099-31-PU; Beijing Protein Innovation) was used as the endogenous control.
In Vivo CoIP Assay
Axillary buds of wild-type seedlings were carefully detached and ground into fine powder in liquid N. Samples were suspended in 4× volumes of Protein Extraction Buffer (50 mM of Tris–HCl at pH 8.0, 150 mM of NaCl, 10 mM of MgCl2, 1 mM of EDTA, 10% (v/v) glycerol, and 1× Protease inhibitor cocktail) and incubated at 4°C for 30 min with rotation. After centrifuging at 12,000g for 10 min at 4°C, the supernatant was mixed with 30-μL Protein A beads (Millipore) and incubated with rotation at 4°C for 1 h. Beads were pelleted and the cleaned supernatant were incubated with 10 μL of anti-IPA1–specific polyclonal antibodies for at least 2 h at 4°C with gentle rotation. Thirty-microliter Protein A beads were added to precipitate the protein complex with rotation at 4°C for 1 h. Beads were pelleted and washed four times with prechilled Protein Extraction Buffer. The bound proteins were eluted from the beads with 1× Protein Loading Buffer by boiling at 100°C for 10 min. Protein samples were then separated by 10% SDS-PAGE gel, transferred to NC membrane, and immunoblotted with anti-IPA1 and anti-OsSHI1–specific polyclonal antibodies.
Y1H analysis was performed according to the method described in Lin et al. (2007). Briefly, the full-length coding region of OsSHI1 was cloned into the pB42AD vector at the EcoRI restriction site to generate the AD-OsSHI1 construct (primer pair OsSHI1-42AD in Supplemental Table 5). The full ∼2 kb and various truncated versions of promoter regions of OsTB1 or OsDEP1 were amplified (primer pairs listed in Supplemental Table 5) and ligated into the XhoI restriction site of the pLacZi reporter vector. Constructs were then cotransformed into the yeast strain EGY48. Transformants were grown on SD-Trp/-Ura plates for 3 d at 30°C and then transferred onto 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside plates for blue color development. Yeast strains containing the empty pB42AD in combination with the LacZ reporter constructs were used as negative controls.
Transient Expression Assay in Protoplasts and LUC Activity Determination
Approximately 1-kb and 2-kb promoter regions of OsTB1 and OsDEP1 were amplified using the primer pairs OsTB1-LUC or OsmTB1-LUC and OsDEP1-LUC (primer pairs listed in Supplemental Table 5) and cloned into the upstream of LUC reporter gene to generate the OsTB1-LUC or OsmTB1-LUC and OsDEP1-LUC reporter constructs. The luciferase gene from Renilla reniformis (Ren) under the control of CaMV35S promoter was used as the internal control. Full-length cDNAs of OsSHI1 and IPA1 were amplified (primer pairs listed in Supplemental Table 5) and inserted into the BamHI/PstI restriction sites of the pAN580 vector to generate the d35S:OsSHI1 and d35S:IPA1 effector constructs, respectively. The combined reporter and effector plasmids were cotransformed into the rice protoplasts according to a protocol described in Zhang et al. (2011). The LUC activity was quantified with a Dual-Luciferase Assay Kit (Promega) following the manufacturer's recommendations and the relative LUC activity is calculated as the ratio of LUC/Ren.
ChIP Assay
To assess the enrichment of OsSHI1 or IPA1 at the promoter regions of OsTB1 and OsDEP1 in vivo, ChIP assays were performed using anti-OsSHI1 or anti-IPA1–specific polyclonal antibodies. Approximately 4 g of wild-type axillary buds or young panicle tissues (1 to 2 g for transgenic plants) were ground into fine powder in liquid N, resuspended in 20 mL Extraction buffer 1 (0.4 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mM of MgCl2, 5 mM of β-mercaptoethanol [β-ME], 0.1 mM of phenylmethylsulfonyl fluoride [PMSF], 1× proteinase inhibitor, and 1% (v/v) formaldehyde), and thoroughly mixed to release the nuclei. After incubation under vacuum conditions for 30 min, 0.125 M of Gly was added and incubated for another 5 min to stop the crosslink reaction. The solution was filtered by two layers of Miracloth (Millipore) and centrifuged at 3000g at 4°C for 20 min to remove the supernatant. The pellet was resuspended in 1-mL Extraction buffer 2 (0.25 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mM of MgCl2, 1% (v/v) Triton X-100, 5 mM of β-ME, 0.1 mM of PMSF, and 1× proteinase inhibitor) and centrifuged at 12,000g at 4°C for 10 min. The pellet was resuspended in 300 μL of Extraction buffer 3 (1.7 M of Suc, 10 mM of Tris-HCl at pH 8.0, 2 mM of MgCl2, 0.15% (v/v) Triton X-100, 5 mM of β-ME, 0.1 of mM PMSF, and 1× proteinase inhibitor), laid on top of another clean 300 μL of Extraction buffer 3 and centrifuged at 15,000g at 4°C for 1 h. The chromatin pellet was resuspended in 200 μL of Nuclei lysis buffer (50 mM of Tris-HCl at pH 8.0, 10 mM of EDTA, 1% (w/v)SDS, and 1× proteinase inhibitor) and sonicated to 200∼500 bp with 3-s burst/7s interval frequency at 2-W power for 33 min. After centrifugation at 12,000g at 4°C for 10 min, the chromatin supernatant was diluted with Dilution buffer (0.01 (w/v)% SDS, 1.1% (v/v) Triton X-100, 1.2 mM of EDTA, 16.7 mM of Tris-HCl at pH 8.0, and 167 mM of NaCl) and 1 mL of the diluted chromatin sample was precleared with 20-μL Protein A beads (Millipore) for 1 h at 4°C with rotation. Then 10 μL of anti-OsSHI1 or anti-IPA1–specific polyclonal antibodies together with 10 μL of BSA (10 mg/mL) were added to the precleared sample and incubated overnight with gentle rotation. One microliter of salmon sperm DNA (10 mg/mL) was added as the blocking reagent and the immune complexes were collected by 30-μL Protein A beads for 1 h at 4°C with rotation. The Protein A beads were washed stepwise with a low salt wash buffer (0.1% (w/v) SDS, 1% (v/v) Triton X-100, 2 mM of EDTA, 20 mM of Tris-HCl at pH 8.0, and 150 of mM NaCl), a high salt wash buffer (0.1%(w/v) 1% (v/v) Triton X-100, 2 mM of EDTA, 20 mM of Tris-HCl at pH 8.0, and 500 mM of NaCl), the LiCl wash buffer (0.25 M of LiCl, 1% (v/v) Nonidet P-40, 1% (w/v) deoxycholic acid sodium, 1 mM of EDTA, and 20 mM of Tris-HCl at pH 8.0), and TE buffer (10 mM of Tris-HCl at pH 8.0 and 1 mM of EDTA) each two times. The immune complexes were eluted from the Protein A beads by incubating with 250 μL of Elution buffer (0.1 M of NaHCO3 and 1% (w/v)SDS) at 65°C for 20 min with agitation. The supernatant was transferred to another tube to repeat elution and the two eluates were combined. Twenty microliters of NaCl (5 M), 10 μL of RNase A (10 mg/mL), and 2.5 μL of protease K (10 mg/mL) were added to the eluted solution (for the input sample, two volume amounts were added) and incubated at 65°C for at least 6 h with agitation. The immunoprecipitated DNA was extracted with isopropyl alcohol precipitation. The recovered DNA was used as the template for ChIP-qPCR and the enrichment was calculated as the ratio of immunoprecipitation (IP) to Input. The ChIP-reChIP assay was performed according to protocol reported in Furlan-Magaril et al. (2009). All primer pairs are listed in Supplemental Table 6.
A quantity of 3ʹ-digoxigenin-labeled probes containing the putative OsSHI1 binding sites were synthesized by Invitrogen (primer pairs listed in Supplemental Table 7). EMSAs were performed with the DIG Gel Shift Kit (Roche, 03353591910) following the manufacturer's recommendations. Briefly, equal amounts of complementary oligonucleotides were incubated at 95°C for 10 min, cooled down slowly to 15°C (0.1°C/1 s), and diluted to 50 fmol/μL final concentrations. The DNA binding reaction was performed with 100-fmol probe, 2-µg poly (dI-dC), and 100-ng purified MBP or MBP-OsSHI1 proteins and incubated at room temperature for 30 min. Then the samples were immediately applied to the pre-run native polyacrylamide gel containing 6.5% acrylamide in 0.5× Tris-Borate-EDTA buffer. After electro-blotting onto a nylon membrane (Millipore), the oligonucleotides were crosslinked using UV-light. The membrane was incubated in a blocking solution for 30 min, followed by incubating in a DIG antibody solution for another 30 min. After intensive washing with a washing buffer, CSPD working solution was applied to the membrane to visualize the signal.
Accession Numbers
Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers: OsSHI1, Os09g0531600; IPA1, Os08g0509600; OsSPL16, Os08g0531600; OsTB1, Os03g0706500; OsDEP1, Os09g0441900; UBIQUITIN, Os03g0234200; and ACTIN2, Os03g0718100.
Supplemental Data
Supplemental Figure 1. Normal initiation of axillary buds in shi1.
Supplemental Figure 2. Grain, leaf, and culm morphologies of wild type and shi1.
Supplemental Figure 3. Phenotypes of ORF2 (OsSHI1) knockout plants.
Supplemental Figure 4. Phenotypes of ORF2 (OsSHI1) overexpression plants.
Supplemental Figure 5. Characterization and sequence analysis of OsSHI1.
Supplemental Figure 6. OsSHI1 exhibits weak transcriptional activation activity in yeast cells.
Supplemental Figure 7. Histochemical staining of the pOsSHI1:GUS transgenic plants.
Supplemental Figure 8. Specificity tests of the anti-OsSHI1 polyclonal antibodies used for ChIP and immunoblot analyses.
Supplemental Figure 9. Protein induction and purification used for immunoblot and EMSAs.
Supplemental Figure 10. Schematic depictions of the cis-elements in the promoter regions of OsTB1 and OsDEP1.
Supplemental Figure 11. Sequence analysis of the promoter regions of OsTB1 and OsDEP1.
Supplemental Figure 12. Specificity tests of the anti-IPA1 polyclonal antibodies used for ChIP and immunoblot analyses.
Supplemental Figure 13. The zinc finger domain is indispensable for the DNA binding ability of OsSHI1.
Supplemental Figure 14. OsSHI1 represses the DNA binding ability of IPA1 independent of OsSHI1 binding.
Supplemental Figure 15. Generation and identification of mutants generated by CRISPR/Cas9 genome-editing approach.
Supplemental Figure 16. OsSHI1 does not affect the expression level of IPA1.
Supplemental Figure 17. IPA1 does not interfere with the DNA binding ability of OsSHI1.
Supplemental Figure 18. A proposed working model for OsSHI1 in the regulation of plant architecture in rice.
Supplemental Table 1. Phenotypic segregation identified by test for goodness-of-fit.
Supplemental Table 2. Putative interacting partners of OsSHI1 identified by Y2H screening.
Supplemental Table 3. Primers used for map-based cloning.
Supplemental Table 4. Primers for detection of the RNA levels of selected genes.
Supplemental Table 5. Primers used for vector constructions.
Supplemental Table 6. Primers used for ChIP-qPCR analyses.
Supplemental Table 7. DIG-labeled oligonucleotides used for EMSAs.
Dive Curated Terms
The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
This research was supported by grants from the National Key Research and Development Program of China (2016YFD0100901), the National Natural Science Foundation of China (31671769), the Guangdong Province-National Natural Science Foundation of China (U1701232), and the National Transgenic Science and Technology Program (2016ZX08001004-002).
JM.W. supervised the project; L.J., H.W., and JM.W. designed the research; E.D., X.L., Q.L., T.Z., Y.W., C.Z., and H.Z. performed research; E.D., X.L., T.Z., and JM.W. analyzed data; Y.W. provided the plant material; C.L. and JL.W. cultivated the transgenic plants in the field; X.Z. and X.G. generated the transgenic plants; E.D. drafted the manuscript; H.W. and JM.W. revised the manuscript.
↵1 These authors contributed equally to this work.
↵[OPEN] Articles can be viewed without a subscription.
- Received January 17, 2019.
- Revised February 27, 2019.
- Accepted March 24, 2019.
- Published March 25, 2019.