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The Washington Nationals replaced regular first baseman Ryan Zimmerman with Howie Kendrick, their outstanding utility man Tuesday night at Busch Stadium. After all, Kendrick was six for eight in his career against Cardinals starter Miles Mikolas.
Make that eight for 11 now.
Kendrick tripled and scored in the second. He hit a solo homer, his 16th home run of the season, in the fourth. After Mikolas left, Kendrick singled off reliever John Gant to open a two-run eighth inning and the beleaguered Nationals bullpen was spotless as Washington beat the Cardinals 6-2, handing them their third loss in five games on their next-to-last-home stand of the season which ends Wednesday afternoon.
Both Cardinals runs were unearned, the result of Washington errors. Mikolas fell to 9-14 as he allowed three runs in six innings, but the Cardinals' bullpen tripped up for the second time in three days as Gant, Ryan Helsley and Genesis Cabrera each allowed a run over the final two innings, stretching out what was a 3-2 Washington lead.
The Milwaukee Brewers won to cut the Cardinals' lead over them to two games in the National League Central Division race while the Chicago Cubs were losing to Cincinnati and remained two games behind.
The Cardinals' magic number for eliminating both is 10.
Cardinals relievers John Gant and Ryan Helsley, both of whom faltered Sunday when the Cardinals gave up a couple of leads to the Milwaukee Brewers, failed again Tuesday night.
Each allowed a run in the eighth inning as the Washington Nationals stretched their lead to 5-2.
Giovanny Gallegos was pulled with one out in the eighth after he had struck out three of the first four men to face him. But Gant, who walked three hitters in succession on Sunday, allowed Howie Kendrick's third hit and walked Asdrubal Cabrera.
Rookie Henlsey entered and threw a pitch off the backstop but the ball rebounded to catcher Yadier Molina, who threw out Kendrick at third for the second out, with Cabrera advancing to second. But Victor Robles drove in his second run with a single and Yan Gomes added a run-scoring double.
While Howie Kendrick was powering the Washington Nationals with a triple and home run off Cardinals starter Miles Mikolas, the Nats' defense was giving it right back.
After the Nationals had moved ahead of the Cardinals 3-1 in the top of the sixth inning Tuesday night at Busch Stadium, Washington handed the Cardinals a second unearned run in the home half. Catcher Yan Gomes, whose throwing has been off-line the entire series, gave up his fifth base in two nights as he fired the ball into center field, allowing Tommy Edman to score from third as Harrison Bader stole second to cut the lead to 3-2.
Edman walked with one out and sped to third on Bader's single off Patrick Corbin, who registered his 11th strikeout on pinch hitter Jose Martinez before his catcher winged the ball into the outfield.
The Washington Nationals changed first basemen from Ryan Zimmerman to valuable utility man Howie Kendrick for game two of their series with the Cardinals Tuesday night at Busch Stadium. For good reason.
Kendrick, who had been six for eight in his career against Cardinals starter Miles Mikolas, tripled off the wrist of leaping center fielder Harrison Bader and scored the first Washington run in the second.
The next time up, after the Cardinals had tied the score, Kendrick hit it where Bader, leaping again, had no chance. The drive, off an 0-2 pitch, went into the center-field greenery for Kendrick's 16th homer and a 2-1 Nationals lead. Lefthander Patrick Corbin struck out two more in the Cardinals' fourth, giving him nine already for the night.
Washington had a chance to extend that lead in the fifth when Yan Gomes doubled to right and Corbin crossed up a Cardinals' infield expecting a bunt by singling to left center. Gomes stopped at third _ and stayed there.
Trea Turner lined to jumping shortstop Paul DeJong. Adam Eaton flied to left, not deep enough to score catcher Gomes. And Anthony Rendon lined to deep right. It still was 2-1.
After two-out walks to Paul Goldschmidt and Marcell Ozuna in the Cardinals' fifth, DeJong grounded sharply to Washington shortstop Trea Turner.
Mikolas was touched for another run in the sixth, opened by a double from Juan Soto, who, the Cardinals thought came off the bag on his head-first slide into second. They challenged. They lost. Kendrick finally was retired on a fly to right _ he is eight for 11 against Mikolas _ and Asdrubal Cabrera bounced to the mound.
Mikolas took a look at second where Soto appeared to be trapped but took the safe out at first. Victor Robles then singled to score Soto and it was 3-1.
The Washington Nationals took a 1-0 lead over the Cardinals in the second inning Tuesday night. Cardinals center fielder Harrison Bader, who has made many spectacular plays this year, couldn't make a leaping catch of Howie Kendrick's drive to deep center, with the ball clanking off Bader's left wrist and Kendrick reaching third on a triple.
Asdrubal Carrerra's sacrifice fly to deep left against Miles Mikolas easily scored Kendrick.
Mikolas escaped trouble in the third when he induced dangerous Anthony Rendon to ground into an inning-ending double play and then the Cardinals' pitcher opened the Cardinals' third with a single to left off lefthander Patrick Corbin.
Dexter Fowler followed with another single to left and Klten Wong sacrificed the runners to second and third. Paul Goldschmidt walked on a 3-2 pitch to fill the bases.
Marcell Ozuna struck out, bringing up Paul DeJong, a .207 hitter with men in scoring position. DeJong worked the count to 3-0 and swinging on the next offering, grounded to the left of shortstop Trea Turner, who bobbled the ball as he attempted to get a forceout and Mikolas scored the tying run.
Yadier Molna fanned on a slider for the third out.
Tying a season high achieved first in late April, Cardinals manager Mike Shildt is employing the same eight-man position player lineup for the fifth consecutive game Tuesday night against Washington at Busch Stadium.
That means no Matt Carpenter at third base and it means Tommy Edman will hit seventh, where he has been six for 14 with two homers in the first four games of the stretch.
Righthander Miles Mikolas, who is 8-3 in 13 career starts against National League Eastern Division opponents, will seek his 10th overall win this season against lefthander Patrick Corbin. The latter is holding lefthanded batters to a major-league low .260 slugging percentage.
The Cardinals (84-66) begin the night two games ahead of Chicago and three ahead of Milwaukee in the Central Division race. Washington holds a one-half game lead over Chicago and 1½ over Milwaukee in the wild-card derby, from which two teams will qualify.
The Cardinals are hitting a modest .256 with runners in scoring position, compared to Washington's .281. But No. 5 hitter Paul DeJong of the Cardinals is only at .207 with just one of his 27 homers coming with a man in scoring position. Cleanup man Marcell Ozuna, for instance, has nine homers with a man in scoring position, including one in the first inning on Monday.
"Paulie's gotten some big hits for us," said Shildt. "He'll work through it. He's going to be a big-time run producer."
Switch-hitting backup catcher Matt Wieters, who hasn't played since Aug. 31 because of a left calf strain, has been cleared for full duty. That would include use as a lefthanded pinch-hitter, Shildt said.
1. Dexter Fowler rf
2. Kolten Wong 2b
3. Paul Goldschmidt 1b
4. Marcell Ozuna lf
5. Paul DeJong ss
6. Yadier Molina c
7. Tommy Edman 3b
8. Harrison Bader cf
9. Miles Mikolas p
1. Trea Turner ss
2. Adam Eaton rf
3. Anthony Rendon 3b
4. Juan Soto lf
5. Howie Kendrick 1b
6. Asdrubal Carrera 2b
7. Victor Robles cf
8. Yan Gomes c
9. Patrick Corbin p
Tickets for a potential wild-card game at Busch Stadium on Oct. 1 and for up to three division series games (more likely two) will go on sale Thursday at noon.
They will be available at cardinals.com or by calling 314 345-9000. All individual tickets will be delivered digitally by the MLB Ballpark app.
In advance of the public sale, fans also can receive ticket access to postseason games this year by placing a deposit on 2020 season tickets.


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GDS is totally different from a computer reservations system , which is a reservation system utilized by the service providers(often known as vendors). Note: I also think portable journey bar units are an important gift for any caregiver. Not solely have earlier entries received funding as a result of the Awards, they have also had the chance to satisfy and do business with travel trade friends. A journey journal (or travel blog) can turn out to be useful at this time, particularly when there are things you forgot about, or if you end up requested a question you aren't positive the best way to answer.
Indeed, such have been the fluctuations that some UK holidaymakers had been required to pay a surcharge after the referendum to cover the additional cost incurred by the journey supplier. From over 10 years of experience in the air journey industry, I can tell that 99% of the time your ticket may be changed and also you don't have to lose money and throw ticket away when you can't journey. Travel Guard is the industry's leading provider of journey insurance coverage for North American residents. I'm delighted to bless this outstanding travel information to Vancouver and its environs.
Travel tip primary can be to get good insurance coverage, I haven't been to Nepal however Tibet could be very comparable and health care is pretty basic by our requirements in loads of locations and the altitude sure takes some getting used to in case you are a coastal dweller :). Travel mild and be prepared to benefit from the people and the nation and you should have a ball particularly if you make an effort to meet the locals.
Any or all of those moveable cocktail bars would make great birthday, Mother's Day, Father's Day, Christmas or Chanukah presents for somebody who likes to journey and imbibe. Open a Credit Card with 0% overseas transaction charge: If you do not have one already, I counsel opening both a Bank of America Travel Rewards Credit Card or a Capital One Quiksilver Cash Rewards Cards. I want we had that form of dedication to public infrastructure within the US. On a brighter word, although, Amtrak is contemplating a plan where their frequent journey program (Amtrak Guest Rewards) will allow you to earn and redeem credit on European trains. More than 12,000 journey corporations by nearly each distribution channel in the journey industry promote Travel Guard.
Two metalhead slackers journey via time as a lot as 7000 years, starting from 2068, in a time machine formed like a phone sales space. When deciding what to pay for a used camper van, you'll must do your research and go to the assorted places people use to sell an RV. There are many labeled RV listing gross sales websites you can check each locally and on-line. Good Sam Club presents guarantee insurance that will pay the bills for newer models.

subset: commoncrawl


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Hello, hello! Two posts in one day, WHAT?! Before December begins and things get crazy around here with end of the year posts and holiday merriment and such, I wanted to send out a quick survey to get your feedback about this ole blog here! I'm really curious to hear what types of posts you all are enjoying and what you'd like to see more of as I start to dream about 2019…
If you wouldn't mind, could you take a few quick minutes to fill out the survey below and share your thoughts?! I'd be so grateful!
Thank you SO MUCH!
Your feedback is so appreciated!
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In How To Sell Your Crafts Online, I speak extensively about Facebook and Twitter. As well as a series of other Social Media options, including Tumblr, Posterous and, Squidoo and HubPages.
All of these platforms work. They all do the job you need – broadcasting your creativity, information and interests.
Often however, it seems that a lot of people tend to favor one Social Media site over another. Facebook has been "in favor" for a long time. And why not? Facebook can be fantastic, particularly if you build a decent Fan Page for your online store.
But give it a little time and a few privacy "changes" or unpopular updates and you may find that a lot more people jump ship elsewhere. To Tumblr for instance, whose popularity and community is soaring all the time…
Until Tumblr make potential unpopular changes and then people swarm back to Facebook or Twitter. Not to mention Google+, which is a wonderful platform (although unavaiable for Marketing Businesses at the time of writing).
Talking of Twitter, I just read an interesting Mashable article which reveals:
"Twitter has been on a roll in recent months. The company surpassed 200 million tweets per day in June but has since jumped to nearly 250 million daily tweets. The growth has been tremendous: Twitter had around 100 million tweets per day in January 2011."
Which is why you need to make sure your spending as much time on Twitter as you are on Facebook, as well as maintaining a presence on all of the popular Social Media platforms.
Maintain an anchor in the ebb and flow.
There are various ways of doing this and I'll show you a very simple one in a future blog post. And don't forget Social Media Marketing Magic which is the free book I created to give you a very simple Twitter Strategy.
With the rise in popularity of these Social Media Platforms comes changes and not all of them are seen as positive. But these changes are ofen in a state of flux, just like the Internet. Many of the larger Social Media Giants seem to listen to their users feedback, so jumping ship from one to the other can be self-defeating.
I find it best to maintain a presence on each of the big platforms and to make sure I'm not placing all my Social Media "Eggs" in one basket.
Using the techniques I outline in my book How To Sell Your Crafts Online makes this a simple process.
This is a truly amazing time…
Never before have we had the opportunity to "broadcast" ourselves across the world, from so many platforms.
And for free.


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Today , I'm trying to get back on track from all the yummy food I ate last weekend and yesterday for the fourth. I figured out that I do consume too much sugar and salt when I eat whatever I want. So, if I am going to make anymore progress, I really need to cut down on these things. Easier said than done!
I think I did pretty good today. I had a protein shake for breakfast. Then for lunch I had zucchini noodles. Dinner was a little harder because we had leftovers. I managed only two-four bites of the bad things. I'm happy to say I have all my calories added up and everything looks good.
I read somewhere that if you crave something, just take three bites. This is supposed to help you not over indulge. This will be my next thing to try. I can say eating in a certain amount of time is good if you don't go overboard. I kinda went overboard. That being said I need to add the few bite thing in with the no late night snacking.
Time is running out on me. Good thing I work well under pressure. It stresses me out but I seem to make it work.
I have in over 13,000 steps so far for today and 75 active minutes. Very little Zumba today but a lot of cardio. I did add in some arm weights for awhile and I did a mile on the elliptical.
How was your day?
Thanks so much for reading and God Bless!


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Norcros Wet Seal Tanking Kit
Norcros Wet Seal Waterproofing Kit for Showers & Wetrooms
Norcros Wet Seal is a white brush applied tanking membrane, specifically designed for use beneath ceramic, porcelain and natural stone tiles and mosaics on internal walls and floors in installations subject to frequent wetting, such as domestic and commercial showers, wet rooms and saunas. Norcros Wet Seal is suitable for use on all types of substrates for interior tile installations including moisture sensitive backgrounds such as timber, plaster and plasterboard. It is not however suitable for use externally or in areas of total immersions i.e. swimming pools
Norcros Wet Seal is sold in a kit, consisting of 5kg of liquid membrane, 500ml of Norcros Prime Bondprimer and 10m of 100mm wide waterproof polyester tape
Key Features
- Coverage (dependant on bed depth and surface regularity): one Norcros Wet Seal kit is sufficient to cover 5m2 at 1mm thickness of liquid (generally 2 coats)
- Drying time 24 hours
- Flexible, waterproof tanking membrane
- Sold in kit format; 5kg liquid membrane, 500ml Norcros Prime Bond primer and 10m of 100mm wide waterproof polyester tape
- Brush applied
- Suitable for use on all types of substrate including, timber, plaster and plasterboard
- Suitable for use on walls and floors
- Suitable for use with ceramic, porcelain, natural stone tiles and mosaics
- Ideal for tanking domestic and commercial showers, wet rooms and saunas
- NOT suitable for use in areas of total immersion i.e. swimming pools
- Suitable for internal use ONLY
Surface Preparation
- Ensure the surface is clean, dry and strong enough to support the combined weight of the tiles, adhesive and grout
- Check the substrate is flat and true and free from any gaps or cracks
– narrow gaps can be bridged using either Norcros Waterproof Tape or , DURAL Durabase CI De-Coupling and Waterproofing Membrane
– floors with space exceeding 3mm under a 2m straight edge should be leveled using Pro 50 Floor Leveller
Norcros Wet Seal is sold in a kit, consisting of 5kg of liquid membrane, 500ml of Norcros Prime Bondprimer and 10m of 100mm wide waterproof polyester tape
WALL Application
- Dilute the primer 1:2 with water and brush apply on to the area to be tanked
- Allow to dry
- Brush apply Norcros Wet Seal liquid membrane to internal and external corners, junctions between walls and floors, junctions between boards and/or different substrates, areas where pipes penetrate and any small cracks and gaps in the background
- Fix Norcros Waterproof Tape to the wet membrane, ensuring that there are no gaps between the lengths of tape
- Paint a second layer of Norcros Wet Seal liquid membrane over the tape
- Brush apply Norcros Wet Seal liquid membrane over the remaining area of the walls to a height no less than 1m
Note. in frequently used showers or wet rooms the full area of the walls may require tanking
FLOOR Application
- Dilute the primer 1:2 with water and brush apply on to the area to be tanked
- Allow to dry
- Brush apply Norcros Wet Seal liquid membrane over the whole floor area
Note. on timber floors, suspended floors and in areas which may be subject to heavy use, DURAL Durabase CI De-Coupling and Waterproofing Membrane should be laid into the wet membrane, taking care to overlap the matting by approx. 20mm, and a second layer of the Norcros Wet Seal liquid membrane painted over the matting
Tiling can commence as soon as the liquid membrane has dried , usually 24 hours
Delivery Times
Delivery times are Monday through Friday (excluding Bank Holidays) from 7:30am-5:30pm. Most deliveries leave on Express Delivery for 1-2 working day delivery service.
A 'working day' is Monday to Friday, excluding bank holidays. Weekends and bank holidays do not count as working days.
FREE 1-2 working day delivery service for all orders over £99 (excluding VAT). This excludes shower screens, Lustrolite and special order items. Conditions apply - see Delivery page for further details.
Saturday deliveries are available for Mainland UK only and are for orders over £99 (excluding VAT). There is an additional charge of £15 (excluding VAT) for a Saturday delivery.
Delivery Damages
It's important that you indicate the damages on the delivery note when you sign for the goods. Notify Tile Fix Direct by calling our advice line on 0333 320 04 24 if there are any damages or irregularities to your order within 24 hours of delivery.
Delivery Costs
For additional information on delivery costs, please visit the main Delivery page.
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Star Skin: Home
All i ever wanted was to come home. My first home was the solace of infinity, to feel space tug ineffably on my tiny heart so that I felt both me and YOU for the first time. All I ever wanted was to come home. To know that touching you, loving you, didn't divert or replace the love of this ALL. Loving you would just make this star skin we wear brighter. All I ever wanted was to come home. To see the fullest shape with no eyes (or sight), or ears (to hear), no mouth (to sing), to nose (to breathe), or hands (to touch), only one thing in this shape, only one thing there ever is. And I do know I am and have always been home and if love bore me to breathe, love will bear me to cease -- and that is all the same. But you my sweet, consider coming to a place with me where I may come to know the shape of all things true and you.


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#8: Nathan Koren | Founder, Podaris
How can digital collaboration tools help deliver major infrastructure projects in a more timely and less costly manner? What is the future of transport?
In the 40 years leading up to 1870, Great Britain managed to construct more rail network (13,500 miles) than exists in the country today (10,072 miles). Since then, transport infrastructure delivery has become slower and more expensive as projects require both more experts but also, in a more democratic society, more stakeholders.
Nathan Koren has designed a digital collaboration tool at Podaris which enables real-time collaboration & stake-holder engagement, which he believes will lead to faster, more accurate, more creative, and ultimately more inclusive, transport infrastructure planning. With real-time collaboration, increasing numbers of stakeholders & experts can be an advantage (additional creativity and thought), rather than a disadvantage (additional complexity, time, costs).
Nathan is an architect, transport planner, software engineer and entrepreneur. Having worked for many years as an architect specialising in sustainable transit-oriented developments, Nathan became interested in how new transportation technologies could positively influence urban design. This led him to work on planning and design stages of urban-scale Automated Transit Networks, before launching Podaris to tackle the issue of collaboration.
There are several tools aiming to act as the single source of truth in construction projects (for example, BIM level 4) but creating a single model that accommodates all the needs of planners, economists and engineers needs enormous complexity. At the heart of Nathan's ethos is ensuring that the Podaris design is sufficiently sophisticated to ensure that all key decisions can be made but simple enough so that it focuses on just the key shared information. It achieves this by acting a parametric model with key data in the cloud and APIs to stay synchronised with 3rd party software.
We touch on the issue of Big Data and it's worth taking a look at the data pyramid. The point is to remember that contextualised data is meaningless. We need to provide context to turn it into information and process the information to make knowledge. We can then implement that knowledge in the for of wisdom. It's worth bearing in mind next time you consider gathering data!
Nathan previously worked on the autonomous Heathrow POD system (designed by Ultra Global PRT). We discuss the benefits and why it has failed to take off elsewhere.
Given Nathan's background, it may be surprising that he predicts that the transport mode of the future will be feet. In the context of our conversations with Chris Choa, Andrew Waugh and Vanessa Butz about the importance of human connectivity, however, this seems more logical. As information becomes more democratic and widely available, Nathan sees a growing importance of human concepts of trust and creativity which require direct relationships to flourish and argues that it's in dense urban environments where this will continue to flourish.
For those, like me, looking forward to recapturing green urban spaces through the Autonomous Vehicle revolution, Nathan offers some pretty glum thoughts! While AVs may lead to less cars sitting idly (cars are parked for 95% of their average lives), the supply / demand nature of AVs is likely to mean that it will lead to more cars on the road. Moreover, they may not offer the panacea to traffic congestion which I'd assumed they would. Nathan argues that:
- The issue of slow acceleration from traffic lights will only be slightly improved. Cars will not be able to simultaneously accelerate at an equal rate. If AVs will need to retain safe stopping distances between one another, it follows that cars further back at the slights will need to accelerate at increasingly slower rates in order to achieve that stopping distance.
- Secondly, while the issue of standing wave congestion may be solved, given that there'll be more cars on the road (see above), AVs will accommodate more capacity by slowing down (the slower you go, the smaller the safe distance between cars and the more cars can be fitted onto any given road). The optimal speed under these circumstances will be just 13-16mph!
Nathan's favourite building is the Pantheon in Rome. If you're in any doubt as to whether or not you should visit, I'll leave to listen to his explanation as to why you should.
In terms of innovation, the use of CLT and computational manufacturing in buildings is what excites Nathan. His hope is that, as digitisation increases in the building manufacturing process, we may soon get to a point where we can achieve extraordinary detail at relatively low-cost. While detailed stone masonry of the past required teams of highly skilled masons (often economically unfeasible in the modern era), machine manufacturing combined with a skilled stone 3-D modeller, for example, could make this kind of work the norm.


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The successful applicant will be expected to serve customers from our vibrant kiosk in Westfield London, which includes the following:
SushiDog is forecasting rapid growth with three kiosks expected to be open by the end of 2019. As one of the first employees in a growing company there will be excellent opportunities to progress and take on a larger role as the company grows.
Are you the perfect candidate? Click the button 'Apply Now! and we'll contact you as soon as possible!
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ANAHEIM, Calif. — The Cubs took over Disneyland territory and made a Los Angeles Angels cast headlined by Mike Trout and Albert Pujols feel like The Other Team.
The Arizona Diamondbacks might not be an afterthought on Thursday night at Chase Field, but the Cubs have no other choice except to "Embrace The Target" for the next 160 games. Sweeping the Angels by a 15-1 aggregate score and hearing fans sing "Go Cubs Go" on their way out to the parking lots showed what could be ahead this season.
Before the Cubs became Revenants on a Sports Illustrated cover, the last time they played at Angel Stadium of Anaheim was June 4-5, 2013. That's a jumping-off point for how the stars aligned for what had been a star-crossed franchise.
• Anthony Rizzo: During the first offseason for the Theo Epstein administration in Chicago, the Cubs looked at Pujols and Prince Fielder and pictured the first basemen on a stock chart. The front-office projections had Rizzo's performance eventually rising to the point where he would pass Pujols and Fielder as they inevitably declined in their later years.
The Cubs still don't have the kind of TV contract that led the Angels to splurge on a 10-year, $254 million megadeal for Pujols at the 2011 winter meetings. One month later, Cubs executives who knew Rizzo from their time together with the Boston Red Sox and San Diego Padres looked beyond his struggles at Petco Park, giving up hard-throwing/injury-prone pitcher Andrew Cashner.
Rizzo may never come close to matching what Pujols did for the St. Louis Cardinals (two World Series rings, three MVP awards) or finding the level of consistency that allowed the future Hall of Famer to earn 10 All-Star selections and hit 40 homers in his age-35 season last year.
But Rizzo is clearly the player to build around now — at a fraction of the cost — while Pujols tries to stay healthy and transition into being a part-time designated hitter. Rizzo is a Gold Glove-level defender coming off a 31-homer, 101-RBI season, and the team has taken on his personality, striking the right balance between goofy and serious.
"We're really hungry," Rizzo said. "If anyone in this clubhouse is thinking about the World Series right now, we're in the wrong spot. We need to think about tomorrow, winning the game (and) dominating April."
[MORE: After all the hype, Jon Lester ready to roll with Cubs]
• Kris Bryant: Before Darwin Barney batted leadoff on June 5, 2013 in Orange County, the Cubs had already eliminated University of North Carolina third baseman Colin Moran from consideration for the No. 2 overall pick they couldn't whiff on, leaving two pitchers and a fast-rising hitter still in the conversation.
As the draft unfolded the next day, the Houston Astros selected Stanford University right-hander Mark Appel with the top pick, giving the Cubs their shot at the University of San Diego slugger who led the nation in homers, runs scored, walks and slugging percentage.
Jon Gray — the University of Oklahoma right-hander who dropped to No. 3 — put up a 5.53 ERA in nine starts for the Colorado Rockies last year. The Astros packaged Appel (5.12 ERA in the minors) in the Ken Giles trade with the Philadelphia Phillies this winter. Houston had already acquired Moran — the sixth overall pick who spent last year at the Double-A level — from the Miami Marlins in the Jarred Cosart trade at the 2014 deadline.
Meanwhile, all lines are open at Bryzzo Souvenir Co.
"This could be a very special thing that we have here," said Bryant, already an All-Star third baseman and a Rookie of the Year. "But we just really need to focus on this year and not really get too ahead of ourselves. There are so many things that can happen in this game. We're here focusing on today."
• Jake Arrieta: The pitching infrastructure that just helped shut down Trout and Pujols (0-for-15 with five strikeouts) had already been in place by June 4, 2013, when sign-and-flip guy Scott Feldman got a tough-luck no-decision in a one-run loss to the Angels.
Five weeks later, the Cubs sold high on Feldman (7-6, 3.46 ERA) and shipped him to the Baltimore Orioles in a trade for change-of-scenery pitchers Arrieta and Pedro Strop.
Arrieta's unconventional workout routine/nutrition regimen has been covered at length, and there are almost two full seasons left to place over-under bets ($200 million) on how much super-agent Scott Boras will get his client in free agency.
But in listening to a Cy Young Award winner explain how to attack the Angels, it's also clear how much Arrieta enjoys thinking about his craft and preparing for each start. Meaning there are intangible benefits to his presence and reasons to believe he will keep performing at a high level.
"Trout's a guy who is exceptional at hitting the ball down in the zone really well for power," Arrieta said, "both to the pull side and to right-center field. So you got to move the ball away from him and into him — to kind of keep him from getting extended — and elevate from time to time. Remaining unpredictable is really big against guys like him (and) Pujols (who) hit the fastball well.
"I expect to pitch this way every time I take the mound. Obviously, once the ball leaves your hand, you can't dictate the results. But I expect to execute at a pretty high percentage. If I execute — and pound the strike zone with my stuff and keep them guessing — I have a pretty good opportunity to have another good year."
[SHOP: Gear up for the new season, Cubs fans!]
• Joe Maddon: The Cubs lucked out when Andrew Friedman bolted from the Tampa Bay Rays after the 2014 season and took a president's job with the Los Angeles Dodgers, allowing the star manager to use the escape clause in his contract.
But Maddon still knows the groundskeeper by name in Anaheim and appreciates what he learned during his three-plus decades in the Angels organization. Places like Gene Autry Park and Idaho Falls — and the long climb to the top — shaped him into the perfect ringleader for this circus.
"I really trust our players," Maddon said. "When you talk about pressure and expectations, understand it's spread out among the whole group. It's not just on one guy. We have so many good players here. A lot of them have been through the baseball wars before where they've been very successful. We're going to have our rough moments. (But) we have the ability – mentally, physically – to fight through those moments.
"If you factor in everything, experience, talent level, motivation — because all of them want to be here to become part of the first team that wins the World Series for the Cubs in a long, long time — there are so many good things here to repel pressure and expectations."


text: What an adorable bundle of joy! His mom has been so exited to finally meet him and once he arrived I just melted watching them interact together. This session was a part of my Tiny Moments Package This package included a maternity and newborn session bundle. I love being able to get to know my clients before and after baby! What a blessing!


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The Charles O'Conor Collection
The Charles O'Conor Collection
Charles O'Conor, was a leading New York attorney with pronounced pro-Southern, pro-slavery views, who left the library $21,000, and the multi-volume bound sets of "My Own Cases" and "My Own Opinions," relating to the most notable cases of his long career. The volumes in this collection include the well-known Lemmon Slave Case of 1860 in which the "New York Court of Appeals made the strongest statement against slavery of the highest court of any state before the Civil War."* Interestingly, the judge presiding over this landmark case was Elijah Payne, Jr., former librarian at the New York Law Institute. Also included in these cases are the The New Almaden Mine case and the Vigilance Committee case.
Collection Items
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Today's Fxshnjunke Spotlight goes to 25 year old Monte Woodruff from Nashville , Tennessee. This lover of all things fashion who loves to dress up likes the idea of styling people and finding their perfect outfit to accommodate their style . "I love when my close friends ask me or send me pictures to my phone so they can get my opinion on what they should wear " . Read more below !
Q: What do you love most about fashion ?
Monte : What I love about fashion is that it gives you the power to express yourself through colors and clothes
Q: How would you describe your style ?
Monte : I would describe my style as the more hip hop urban streetwear
Q: Where are your favorite places to shop ?
Monte : Asos, h&&m, forever, pacsun urban outfitters and even the goodwill I just love to mix and match all there pieces
Q: What are your plans for 2016 ?
Monte : My plans is to meet people who are just like me networking and even help people who are also stylist designers in the making
Make sure to follow Monte @astronaut_Kidd_ for more stylish looks .
Thoughts on his article ? Comment below
Written by @dasiatheceo


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Confused: I created a viewshed using Azimuth (140) and 2 (220) to limit viewing angle. To add clarity I add a line in create features using direction and distance. I select the point feature to start the line and right mouse click to bring up drawing options. I select the Direction/Length option (140, 1500) which draws a line. I repeat the process for the second line (220,1500). They do not match the viewshed azimuth and when I add geometry to the line to add values in attribute table the direction (Azimuth) values do not match those imputed in create feature.
The line created with direction140 becomes 230
The line created with direction 220 becomes 310
How do I get all the Azimuth values to agree?
Image attached to show displacement.
Any and all help appreciated.


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201-1405 St. Paul St.
Kelowna BC, V1Y 2E4
I am passionate about exploring new technology to find better ways to solve problems and developing web applications that make the world a better place. The tools I use are PHP, Drupal, Laravel, Javascript, jQuery, AngularJS, CSS, HTML, and SASS. I implement automated testing frameworks, responsive design and use Linux, Apache, MySQL and Git.


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Let the wonder of watercoloring commence with this awesome All-in-One Watercolor Art Set. A bounty of painting supplies await, including 24 Watercolor Pencils, 5 Paint Brushes, 50 Pages of Watercolor Paper, and 24 Washable Watercolor Paints all conveniently kept in a Decorative Storage Box. Use the Watercolor Colored Pencils on the Watercolor Paper—designs can be left dry or wetted to create beautiful watercolor effects. Simply wet the Watercolor Paper or a Paint Brush and bring the fine details to life. The Watercolor Palette also gives little artists a colorful way to express themselves through paint. Great for kids and adults, this Watercolor Painting Set has everything needed for marvelous masterpieces.


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Nestled in a magical pocket of Gippsland, just 2.5 hours drive southeast of Melbourne, lies the spectacular Grand Strzelecki Track (GST), poised to become one of Victoria's premier hiking, bushwalking and camping adventure destinations.
Over 100 km of track includes both easy and challenging walks that will suit trekkers with varying levels of fitness and experience.
The Grand Strzelecki Track has something for everyone, whatever your level of experience or fitness. Overall it is Class 4 under the Australian Standard which means that some parts are narrow foot track with minimal infrastructure provided,
requiring self-sufficiency, navigation skills and responsibility for your own safety.
The Strzelecki Ranges are unique and provide an enormous wealth of biodiversity and scenic values. The Parks charm visitors with pristine rainforest and tall eucalypt forests second to none and a proliferation of plant and animal life such as the South
Gippsland Koala, which is the only genetically intact remnant of the original population remaining in Victoria.
The Grand Strzelecki Track opens up many natural treasures including endangered ecosystems, stunning views, waterfalls and much more. Wander among enchanting giant tree ferns and across pristine mountain streams in one of Victoria's last
remaining patches of rainforest.
These walks take in four catchments on both sides of the range, with a corresponding diversity of forest ecosystems and landscapes including the highest point in the range, Mt. Tassie at 720 metres, which offers dramatic views across the Latrobe Valley and the Great Dividing Range to the North and Wilsons Promontory (the southernmost part of the Australian mainland) and Corner Inlet to the south. A 26.4 km section links the National Parks, with a rest area half way providing a place to camp.
Morwell National Park (Junction Road car park), Balook (Tarra-Bulga Visitors Centre car park), Tarra Valley picnic area car park or Macks Creek Hall.
- Street: Grand Ridge Road
- City: Balook
- State: Victoria


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Doesn't God provide for us in amazing ways? It is encouraging to come to the comforting realization that when you confide your trust in Him, God is not only aware of your needs and desires but is faithful to meet those needs. He even blesses us from the most unlikely sources.
This was very evident to me when my great-grandmother, yes my great-grandmother, generously offered to bless me by giving her 2006 Honda Accord as a gift for the college days ahead that loom in my future. Since I'll be starting college in January, a vehicle was something that we were looking out for, but wow! Out of nowhere, here was a car —but not just a car. It very well could have been a battered up lemon, a toolbox on wheels, and that would have been fine, adequate enough for my needs as a college kid; but no, this was a beautiful, sleek, white Honda with leather seats, a sunroof, and all the fancy gadgets, complete with a spoiler on the back.
The cool thing about this story is my great-grandmother. She is an amazing woman and a godly member of our family who we love dearly. Now, she is getting up there in years and stopped driving. She told me, "Morgan, you know that I love you boys. I'm getting older now, and I've stopped driving, and I have this car here. It's a nice car you know, and I've taken good care of it. I just want you to know that God told me to give that little car to you and your brothers to use when you start school. That's what He told me." What an amazing statement!
In this situation, through the generosity of my great-grandma, God was so good to bless me by providing not only a functioning vehicle but a very nice one at that. I felt so privileged. We have witnessed God's provision for our family time and time again on the mission field overseas and here in America as well. But each time I get into that car from my great-grandma and buckle up, it is yet a constant reminder to me of God's faithfulness and provision. It solidifies the fact that when you trust in God, He is very able to generously supply all of our needs.
© 2019 Answers in Genesis


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Training a new dog can be a stressful endeavor. However, dog training is important to ensure that your property, your home, your pet and other people are safe. You need to always use positive reinforcement to train your pet. And that means also being patient and consistent in your training. Every day, you should devote about half an hour to training your pet as this is the optimal duration for a single session. Longer sessions than that result in boredom for the dog.
Make sure your teething puppy has a bunch of chew toys readily available to him, and keep other things he likes to chew on away from him. Replace the tasty item you do not want chewed with a designated chew toy. Teething can cause puppies significant pain. Give them relief by letting them chew on a washcloth that is wet and frozen.
It is dangerous to put more than one dog together in a close space. Doing so could cause injuries to the dogs if they become entangled in the chains and attempt to free themselves. If two dogs' chains become entangled, it is possible that one or both dogs could experience difficulty breathing or even strangulation.
If you intend to use a crate in puppy training, make sure the crate is an appropriate size for your dog. Remember, puppies grow up. Choose a crate which will be suitable for the puppy when he matures into an adult. The dog must have enough room to lie down and turn around without getting crowded.
It is very easy to teach a pooch to roll over as long as you have some tasty treats on hand. First, make your dog lay down. Then, put a treat close to the floor near the side of the dog's head, and gradually raise it over its head to the opposite side. Ideally, your dog will use his nose to follow the treat, rolling his whole body in the process. As he rolls, state the command "roll over" in a firm tone. Repeat the process until your dog associates the command with the action. Be patient while he masters this trick. He will be a star once he does!
If your dog is a jumper, squeeze his paws when he jumps on you to let him know he cannot do this any longer. Do something that makes them uncomfortable so that they can associate their action with something negative. It won't take much time for your dog to associate the unpleasant squeeze with jumping on a person, and he will quickly refrain from doing so.
To keep your dog from barking, teach them to obey to a simple order that will make them quiet. Use negative reinforcement to stop your dog from barking when it engages in this action. Then, you can treat them. When you repeat this action multiple times, your dog will eventually learn that the order means to be quiet.
By using these tactics you can effectively train your dog. As a result, your property at home and your pet will be safer. When you use the training techniques listed here, you'll surely have a better time in training your pet, especially when it comes to practicing patience and staying consistent.


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Good morning! Are you having a good weekend? The weather has been so nice! Not too hot, not too cool. Perfect for the last couple days of spring. I wanted to show you the card I made for my hubby for Father's Day. It's a very simple card. I let the image do all the talking on this one. No embellishments at all. Just used some patterned paper from Pink Petticoat matted with Bazzill cardstock. This fabulous image is from Kenny K and I think she's gorgeous!! I've got a really cute picture of myself that I got a couple years back when I went to Vegas with my two very favorite ladies in the entire world. We were celebrating turning 50 together. We came across one of those places that insert your face into a sexy pic. We had a blast with that one. Anyway, I had my face inserted into a Cowgirl pinup, so I couldn't pass up this image.
Of course I had to come up with my own sentiment. LOL! And I've got to tell you that he truly LOVED this card when he opened it. heehee Well....what guy wouldn't??!!
She's colored with Copic markers - The T series of grays and the R20's series for the reds. I used E11, E01, and E00 for her skin. For her hair, I used E97, 99. and 17. I didn't care for how it came out, but it was one of the last things I colored and I just didn't have the time to redo her again. She did take a while to color. And for those of you that know me, the first part of the sentiment is on the front, and here's the inside
And now I'd like to share some pics in memory of my wonderful dad....Happy Father's Day Dad!
My dad when he was about two years old
What a handsome guy!
He sure did love to fish!
And last but not least, a picture of my mom and dad
Have a wonderful day everyone!! Thanks so much for stopping by to see me today, and hope to see you back again soon! Hugs!


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NUPD to conduct 'violent incident' training exercise
July 29, 2018
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University Police will conduct a training exercise next week to raise awareness among the Northwestern community about what to do in the event of a violent incident on campus, according to a Friday email from Chief of Police Bruce Lewis.
The exercise will be filmed for a safety video, and parts of it will involve events that look like a real incident, such as police cars, crime scene tape and scenes with actors and unloaded weapons, the email said.
This training comes several months after a swatting incident at NU, in which police received a call reporting a man with a gun on campus and the community was instructed to find shelter. The incident was determined to be a hoax and the investigation concluded.
Each day of the exercise, UP will send reminders to the community detailing where the it will occur and signs will be posted at filming sites when the training is in progress. The training will take place July 30 through August 3 at various locations around campus, including Norris University Center, the Rock and the Lakefill. In the email, Lewis said they are in different locations "to emphasize that no matter where you are on campus, you will know what to do."
Northwestern is joining the Department of Homeland Security's efforts to train the public in the "Run, Hide, Fight" strategy in the event of an incident involving a gun, the email said. The UP safety video will educate the NU community about what to do during an incident specifically at the University.
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Twitter: @allymauch


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A Barbadian mother and son are facing four drug charges after trying to bring cannabis into the island.
Marita Eugene Odle, 42, and her 24-year-old son Rico Antonio Odle, both of 1st Avenue Weekes Land, Goodland, St. Michael, allegedly had 5.6 kilogrammes of cannabis in their suitcases when they came in from Canada yesterday.
They arrived on the same flight but were separately stopped and interviewed by police.
When customs searched their luggage, they found the illegal drugs in false bottoms of their suitcases.
They are charged with possession, possession with intent to supply, trafficking and importation.


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The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.
THE biogenesis of the lignified plant cell wall is a complex metabolic process in which the formation and modification of several cell wall polymers need to be coordinated. Lignin is synthesized via the oxidative coupling of several phenolic monomers. The traditional view holds that lignin is derived from the monolignols p-coumaryl, coniferyl, and sinapyl alcohol, which give rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) residues, respectively. Small amounts of other phenolic molecules can be incorporated as well, as a function of the species, genetic make-up, and tissue (reviewed by Ralph et al. 2004). The last step in monolignol biosynthesis is the reduction of cinnamyl aldehyde precursors (reviewed by Humphreys and Chapple 2002), catalyzed by the enzyme cinnamyl alcohol dehydrogenase (CAD; EC 184.108.40.206; Mansell et al. 1974). Two enzymes with cinnamyl alcohol dehydrogenase activity were identified in Eucalyptus gunnii, and named EgCAD1 and EgCAD2 (Goffner et al. 1992). The enzymes are structurally unrelated and share no sequence similarity. EgCAD1 is more similar to cinnamoyl-CoA reductase (EC 220.127.116.11), its active form is a monomer, and it is active toward a range of substrates, including coniferaldehyde and benzaldehyde derivatives, but not sinapaldehyde (Goffner et al. 1992, 1998; Hawkins and Boudet 1994). EgCAD2 is active as a homo- or heterodimer (Hawkins and Boudet 1994; Boudet et al. 2004) and can synthesize all three monolignols. EgCAD2 was considered the predominant CAD involved in lignification (Goffner et al. 1992), and was the basis for studies on the role of CAD in lignification in other species. Recently, however, downregulation of the tobacco ortholog of EgCAD1 was shown to have a subtle effect on the synthesis of coniferyl alcohol (Damiani et al. 2005). Unless stated differently, further mention of CAD refers to EgCAD2-type enzymes.
In gymnosperms, which contain lignin composed primarily of G residues, the CAD activity is strongly coniferaldehyde specific. Several CAD isoforms have been identified in gymnosperm species, although it is still unclear whether those isoforms represent alleles of a single locus or paralogs (Galliano et al. 1993; MacKay et al. 1997; Schubert et al. 1998). In angiosperm species CAD is encoded by a multigene family (Dixon et al. 2001; Li et al. 2001; Lynch et al. 2002; Sibout et al. 2003; Tobias and Chow 2005). The Arabidopsis and rice CAD gene families consist of 9 and 12 members, respectively (Tavares et al. 2000; Sibout et al. 2003; Tobias and Chow 2005). Angiosperm CAD proteins are multifunctional enzymes capable of catalyzing the reduction of p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols (Hawkins and Boudet 1994; Kim et al. 2004). The greater variation in lignin composition in angiosperms related to maturity and tissue type, together with the presence of multiple CAD genes, has led to the hypothesis that specialized CAD proteins with differential affinities for their aldehyde substrates play a role in regulating lignin composition during development (Li et al. 2001). The identification of a sinapyl alcohol dehydrogenase (SAD) in aspen (Populus tremuloides), which has greater affinity for sinapaldehyde than coniferaldehyde (Li et al. 2001), provides support for this hypothesis. However, no specific requirement for SAD activity for the synthesis of S units has been identified in Arabidopsis (Kim et al. 2004; Sibout et al. 2005).
Phylogenetic analyses of the Arabidopsis and rice CAD gene families reveal several subgroups (Tavares et al. 2000; Raes et al. 2003; Sibout et al. 2003; Tobias and Chow 2005), one of which displays strong similarity to the gymnosperm CAD genes. This group contains the genes that have been implicated in lignification of the vascular tissue by biochemical, expression, and mutant studies, including AtCAD4 and AtCAD5 (Sibout et al. 2003; Kim et al. 2004; Sibout et al. 2005), ZmCAD2 (Halpin et al. 1998; Guillaumie et al. 2007a), and OsCAD2 (Tobias and Chow 2005; Zhang et al. 2006). The enzymes encoded by the genes belonging to other groups have lower sequence similarity to these bona fide CADs and present wider substrate preferences and varying degrees of enzymatic activity and expression (for review see Raes et al. 2003; Kim et al. 2004; Guillaumie et al. 2007a). It is likely that some of the genes are involved in defense response (Kiedrowski et al. 1992; Brill et al. 1999) or metabolic processes not related to the lignification of the vascular tissue.
Although modifications in lignin content and composition have been observed in the vascular tissue of mutants and transgenics with reduced CAD activity (Halpin et al. 1994, 1998; Baucher et al. 1996, 1999; Chabannes et al. 2001; Sibout et al. 2005), it has been difficult to assign unique functions to individual CAD genes, underscoring the complementation capacity of the CAD multienzyme network. Without clear differences in substrate specificity, the spatiotemporal control of lignin biosynthesis in Arabidopsis and maize may therefore be the result of the regulated expression of tissue-specific CAD genes (Raes et al. 2003; Guillaumie et al. 2007a).
The maize brown midrib1 (bm1) mutant, which has reduced CAD activity (Halpin et al. 1998), reportedly has lower expression of several CAD genes (Guillaumie et al. 2007b) and several other monolignol biosynthetic genes. This led to the hypothesis that the Bm1 gene may encode a transcription factor. Regulatory genes implicated in the regulation of lignification were shown to be reduced in expression (Guillaumie et al. 2007b). In contrast, a phenotypically similar mutant in rice, the golden hull and internode2 (gh2) mutant, is caused by a mutation in the OsCAD2 gene, the ortholog of the maize ZmCAD2 gene identified by Halpin et al. (1998). Genetic complementation experiments demonstrated that expression of the wild-type OsCAD2 gene was sufficient to restore normal cell wall composition of gh2 plants (Zhang et al. 2006). The brown midrib (bmr) mutants of sorghum (Porter et al. 1978) are similar to the bm mutants of maize. The abbreviation bmr was adopted to distinguish it from bm, already in use for the sorghum bloomless mutants (Ayyangar and Ponnaiya 1941). There are at least four independent bmr loci (Bittinger et al. 1981; Saballos et al. 2008). One of the mutants, bmr6, displays decreased CAD activity (Bucholtz et al. 1980; Palmer et al. 2008) and contains cell walls with higher levels of cinnamaldehydes (Pillonel et al. 1991; Saballos et al. 2008). Aided by the release of the sorghum genome sequence (www.phytozome.net/sorghum), we report here the systematic analysis of the sorghum CAD gene family, its relationship with the gene families from other species, and the identification of the Bmr6 gene.
Genome analysis:
CAD-like genes were identified by performing a BLAST homology search (Altschul et al. 1990) of the sorghum genome sequence database (http://www.phytozome.net/sorghum) using as queries the DNA or deduced amino acid sequences of the rice OsCAD2 (AK105011; Tobias and Chow 2005), Arabidopsis AtCAD4 and AtCAD5 (At3g19450 and At4g34230, respectively; Sibout et al. 2003), and maize ZmCAD2 (AJ005702.1; Halpin et al. 1998; Guillaumie et al. 2007b). The deduced amino acid sequences were used to query the translation of the sorghum genome in all six frames. The deduced amino acid sequence of the best DNA sequence match was used as a new query in both the sorghum and Arabidopsis (www.arabidopsis.org) genomes. This process was repeated until no additional CAD-like sequences were identified. Gene structure was predicted using Fgenesh+ (Salamov and Solovyev 2000), and predicted intron/exon structures were compared with available EST sequences of rice and other grass species. Phylogenetic analysis included all rice and Arabidopsis CAD protein family members (Kim et al. 2004; Tobias and Chow 2005). Two sorghum CAD genes (genomic accession nos. Sb02g024200.1 and Sb10g006310.1) were excluded from the analysis, because their sequences did not appear to encode functional proteins. Multiple sequence alignments were generated using CLUSTAL W version 3.2 (Thompson et al. 1994) with default parameters (http://workbench.sdsc.edu). A Bayesian phylogenetic analysis was performed using MrBayes software (http://mrbayes.csit.fsu.edu/index.php; Huelsenbeck and Ronquist 2001) with the amino acid model set to pr = mixed, parameters lset rates = gamma, number of generations = 256,000 with sampling every 100 generations (Ronquist et al. 2005). The output file was visualized for analysis using TreeView (Page 1996).
Plant materials and nucleic acid extraction:
The brown midrib mutants bmr6-ref, bmr6-3, and bmr6-27, belonging to the bmr6 group (Saballos et al. 2008) and their normal isolines, were planted in 2004 and 2008 at the Purdue Agronomy Center for Research and Education (ACRE, West Lafayette, IN). DNA was extracted from leaf tissue by a modified CTAB protocol (Bernatzky and Tanksley 1986). Three bmr6-ref near-isogenic lines (NILs) in the inbred lines BTx623, RTx430, and Kansas Collier (Pedersen et al. 2006a,b) were planted in the greenhouse at the University of Florida (Gainesville, FL). Young leaf tissue was harvested and extracted using MoBio Power Plant DNA isolation kit (Carlsbad, CA).
A population of recombinant inbred lines (RILs) were created by crossing Brown County, a non-bmr sweet sorghum cultivar, with the bmr6-ref line. The F1 generation was selfed to produce an F2 population of 218 plants. Individual selfed F2 plants were advanced to the F6 generation using the single seed descent method. The phenotype (bmr vs. wild type) was recorded, and DNA of the individual F6 RILs was extracted from leaf tissue using a modified CTAB method (Bernatzky and Tanksley 1986).
For expression assays, BTx623 and BTx623-bmr6-ref lines were planted in the greenhouse at the University of Florida. Tissue was collected at developmental stage 2 (Vanderlip 1993). At this stage, the stalk is small and covered by the leaf sheaths; therefore, leaves and stacked internodes were harvested together to represent the shoot. In addition, the complete root ball was harvested. Tissue was also collected at stage 4 (growing point differentiation) from the apex of developing roots, the first internode (counting from the top of the stem), and the whorl. For both stages, each line was harvested in triplicate and assayed separately. The tissue was frozen in liquid nitrogen and stored at −80° until RNA extraction with the RNeasy Plant RNA extraction kit with in-column DNase digestion (QIAGEN, Carlsbad, CA). First-strand cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Tissue from the bmr6-3 mutant and the normal isoline N3, planted in the greenhouse (West Lafayette, IN), was harvested at stage 2 and processed as described above.
PCR and cloning:
A homolog to the maize ZmCAD2 gene was identified in the sorghum genome (accession no. Sb04g005950.1) and used to design primers for cloning and gene expression experiments. The identified genomic sequence corresponded to the cDNA sequence identified by Tsuruta et al. (2007) (GenBank accession no. AB288109). The SbCAD2 gene was cloned from bmr6 lines and their respective wild-type isolines. Primer pair U168 (5′ CGG CTC CGT CGT CGT GTT C 3′) and L3923 (Table 1) was used to amplify a 710-bp fragment from the 5′ end of the gene using cDNA as template, with an annealing temperature of 62°. cDNA was used as template due to the technical difficulties of amplifying and sequencing through the first intron of the gene. A 2516-bp 3′ fragment (with partial overlap to the 5′ fragment) was amplified from genomic DNA using primers U2237 (5′ TTT CAG CAC CTT TGG GAT CGA GT 3′) and L4753 (5′ GCA ACA TTT GGG CAT ACG TCT 3′) as above, except that the annealing temperature used was 61°.
PCR was performed with Jump Start RedAccuTaq (Sigma, St. Louis) according to the manufacturer's instructions, using a three-step program. The products were analyzed by gel electrophoresis, purified from the gel using the MinElute gel purification kit (QIAGEN), cloned in vector pT7-Blue3 (EMDBiosciences, Gibbstown, NJ), and introduced in Escherichia coli NovaBlue cells (EMDBiosciences). Plasmids with inserts were sequenced in both directions at the University of Florida's Interdisciplinary Center for Biotechnology Research on an Applied Biosystems 3700 capillary electrophoresis sequencer. The sequence from a minimum of three plasmids for each insert was compared to eliminate discrepancies in the sequences due to PCR and sequencing errors. Sequence analysis was performed using Biology Workbench 3.2 software.
CAPS analysis:
CAPS markers (Konieczny and Ausubel 1993) were based on amplification of the SbCAD2 gene with primers U2931 and L3923 (Table 1), and JumpStart AccuTaq ReadyMix PCR reaction mixes (Sigma), using a three-step PCR program with annealing temperature of 59°. The PCR products were purified using AccuPrep PCR purification kit (Bioneer, Seoul, South Korea) and digested with restriction endonucleases according to manufacturer's directions. The enzymes TaiI and TauI (Fermentas, Glen Burnie, MD) were used for the identification of the bmr6-ref and bmr6-3 alleles, respectively. The digested DNA was separated on a 2% agarose gel.
Structural modeling of SbCAD2 and its mutant versions:
The crystal structure of the AtCAD5 (PDB ID: 2CF6, Youn et al. 2006) was used to build a model for SbCAD2 and the bmr6-3 and bmr6-27 mutant versions. The substitutions, insertions, and deletions were superimposed on the coordinates of AtCAD5, followed by a quick energy minimization by CNS v1.1 (Brünger et al. 1998) using potential function parameters of CHARMM19 as described previously (Kim et al. 2004). Substrate position was generated through the solid docking module on QUANTA (BioSYM/Micron Separations), which is based on conformational space, followed by a quick energy minimization by CNS v1.1.
Expression studies of the CAD-like family of genes in normal, bmr6-ref and bmr6-3 mutants of sorghum:
Gene-specific primer pairs flanking introns were designed to differentiate amplification resulting from cDNA and residual genomic DNA (Table 1). cDNA from shoots and roots of three plants each of BTx623/BTx623 bmr6-ref NILs at stages 2 and 4, and from roots and shoots of N3/bmr6-3 NILs at stage 2 was tested for amplification of seven different sorghum CAD genes. Amplification of the Ubiquitin cDNA was used to control for cDNA loading. The Ubiquitin primers were based on the maize cDNA sequence (GenBank accession no. BT016881). The reactions were carried out using RedTaq Readymix PCR reaction mix (Sigma) in a three-step PCR program consisting of 28 cycles. This was empirically determined to be within the exponential phase of amplification. PCR products were visualized in a 2% agarose gel.
The CAD family in sorghum:
The availability of the sorghum genome sequence has made it relatively straightforward to identify the different members of the CAD gene family. An iterative search of the published sorghum genome sequence using as query the maize, rice, and Arabidopsis bona fide CAD sequences resulted in the identification of 14 CAD-like genes at seven different loci in the sorghum genome. Two of the loci (SbCAD4 and SbCAD8) contain clusters of genes (SbCAD4-1 to -5, and SbCAD8-1 to -4). The DNA sequence of two members of these groups, SbCAD4-1 and SbCAD8-3, are interspersed with retroelements, appear to not encode full-length proteins, and are likely pseudogenes. Sorghum genes in cluster SbCAD8 are very similar to the genes in the rice cluster OsCAD8, but they differ in the gene arrangement. The comparison of the sorghum and rice CAD8 gene clusters is presented in Figure 1. The chromosomal location and EST information of the entire CAD family is presented in Table 2.
Phylogenetic analysis:
To understand the structure of the CAD gene family and the possible functional divergence of the individual members, a phylogenetic analysis was performed using deduced amino acid sequences. The nomenclature of sorghum CAD genes was based on similarity with the corresponding rice orthologs (Tobias and Chow 2005). In general, good correspondence between the rice and sorghum family structure was observed, with the exception of OsCAD1, OsCAD3, and OsCAD9, for which no sorghum orthologs were found, and SbCAD10, for which no unambiguous rice ortholog was identified. The predicted length of the proteins varied between 342 and 383 residues and had a maximum and a minimum identity to maize CAD2 of 95% (SbCAD2) and 40% (SbCAD4-2), respectively.
All of the sorghum CADs contain the conserved GHE(X)2G(X)5G(X)2V motif involved in the binding of the catalytic Zn2+, the GD(X)10C(X)2C(X)2(X)7C structural Zn2+ binding domain, and the conserved glycine-rich repeat G(X)GGV(L)G involved in NADP(H) cofactor binding. On the basis of these motifs, these proteins appear to be zinc-dependent alcohol dehydrogenases and members of the medium-chain dehydrogenase/reductase (MDR) superfamily (Persson et al. 1994; Riveros-Rosas et al. 2003). Youn et al. (2006) identified 12 amino acids that constitute the proposed substrate binding site of the bona fide CADs in a number of angiosperms and gymnosperms. Of the 14 identified CAD-like proteins, SbCAD2 contains 10 of these 12 conserved amino acids (Figure 2). All other family members contained a varying number of amino acid substitutions in these residues, as is the case for other CAD family members in Arabidopsis, poplar, and rice (Li et al. 2001; Raes et al. 2003; Bomati and Noel 2005; Tobias and Chow 2005).
An unrooted phylogenetic tree (Figure 3) constructed from the deduced amino acid sequences shows the relationship between the sorghum CAD family members and previously reported CADs from other species. Sorghum sequence Sb07g025220.1, the closest sequence outside the CAD family, was included in the analysis for comparison. The CAD proteins included in this analysis cluster in five groups.
Group I corresponds to class I of Raes et al. (2003), and contains the sequences identified as the bona fide CAD enzymes involved in the reduction of coniferyl and sinapyl aldehydes to their corresponding alcohols for the formation of lignin in lycophytes, gymnosperms, and monocot and dicot angiosperms. SbCAD2 belongs to this group and it is closely related to the maize ZmCAD2 and rice OsCAD2 proteins. Group II contains AtCAD2, AtCAD3, AtCAD9 [following the nomenclature of Kim et al. (2004)], designated by Raes et al. (2003) as class III. Group III encompasses enzymes of diverse substrate specificity, including the proposed poplar SAD (Li et al. 2001) and Arabidopsis enzymes belonging to class II of Raes et al. (2003), involved in pathogen defense (Somssich et al. 1996). Group IV contains the SbCAD6 and OsCAD6 sequences (Tobias and Chow 2005). Both the rice and the sorghum sequences contain the SKL peroxisome targeting sequence at their C terminus. Group V contains monocot and dicot CADs. Even though there appear to be groups specific to monocots (group IV) and dicots (group II), sequence resources are probably still too limited for definitive conclusions.
The bmr6 mutation affects the SbCAD2 gene:
The increased levels of cinnamaldehyde endgroups in the lignin of the bmr6-ref mutant are consistent with reduced CAD activity (Bucholtz et al. 1980; Pillonel et al. 1991; Palmer et al. 2008; Saballos et al. 2008). On the basis of the clustering of SbCAD2 in group I with the bona fide CADs, we hypothesized that the SbCAD2 gene is the main CAD gene in sorghum and that a mutation in this gene would affect lignification.
The SbCAD2 alleles of three different inbred lines in which the bmr6-ref allele had been introgressed revealed a C-to-T transition at position 2800 of the SbCAD2 genomic sequence. This particular nucleotide change creates a premature stop codon. The resulting predicted protein contains only 131 amino acids and lacks the nucleotide binding site and most of the residues that form the substrate binding site. Such a truncated protein is likely to be nonfunctional even if produced. The mutation creates a novel restriction site for endonuclease TaiI (ACGC → ACGT). Restriction digest of a fragment of the gene (nucleotides 2931–3923 of the genomic DNA) resulted in three fragments (585, 407, and 24 bp) from the wild-type alleles, whereas the bmr6-ref allele produced four fragments (508, 363, 44, and 24 bp). This CAPS marker is therefore diagnostic for the presence of the bmr6-ref allele. The mutation was confirmed with the CAPS marker using independently isolated DNA from N6 and bmr6-ref (Figure 4). Furthermore, this CAPS marker was used in a cosegregation analysis of an F6 RIL population derived from a cross between Brown County × bmr6, and indicated complete linkage between the mutated SbCAD2 gene (bmr6-ref allele) and the bmr phenotype.
In the absence of a facile transformation protocol for sorghum, proving beyond reasonable doubt that the SbCAD2 gene is the Bmr6 gene requires the identification of additional independent mutations in this gene in mutants that are allelic to bmr6-ref. Indeed, sequencing of the bmr6-3 allele revealed a G-to-A transition at position 3875 of the genomic sequence. This mutation results in an amino acid substitution from Gly (G) to Ser (S) at position 191 of the protein sequence. The glycine-rich motif 188G(X)GGV(L)G193 is highly conserved among studied MDR proteins. The motif is located at the first β-α-β-unit of the cofactor binding site and is presumed to participate in the binding of the pyrophosphate group of the NADP+-cofactor (Youn et al. 2006). Although the mutated allele would encode a full-length protein, its catalytic activity may be compromised. A rice mutant with a single mutation in this same region, 185 G → D, results in complete loss of enzymatic activity, and is the causal mutation of the gh2 phenotype (Zhang et al. 2006).
The base substitution in the bmr6-3 allele eliminates a restriction site for the enzyme TauI. A restriction digest of the amplified gene fragment 2931–3923 with TauI resulted in four fragments in the bmr6-3 allele (796, 138, 54, and 5 bp) with a diagnostic fragment of 138 bp. This product was cleaved into two fragments (92 and 46 bp) in the wild-type allele. The mutation was confirmed with this marker using independently isolated DNA from N3 and bmr6-3 (Figure 4).
The bmr6-27 mutation is a nucleotide insertion after position 4288 (+G), which causes a frameshift that results in the formation of a protein with nonconservative substitutions in the 31 C-terminal residues. The mutation also created a premature stop codon that renders the protein four residues shorter than the wild-type protein. In addition to the mutation responsible for the phenotype, the bmr6-27 allele also contains a deletion of base 3077 (−T), but this is a silent mutation because of its location in the third intron of the gene. This mutation does not cause splicing abnormalities, as sequencing of the CAD2 cDNA from wild type and bmr6-27 showed no sequence variation in this region.
Structural comparison of SbCAD2 and its mutant versions with AtCAD5:
The amino acid sequence comparison between SbCAD2 and AtCAD5 (Figure 2) revealed 93 residue changes in total. Among those, 48 are conservative changes and 45 are nonconservative changes. A close inspection of the 3D structure of AtCAD5 (PDB ID: 2CF6) revealed that 52 of these changes are surface residues, whereas 39 occur in the secondary structure. In general, the 3D model of SbCAD2 remains very similar to that of AtCAD5, with many of the changes not having a significant impact on the overall structure (Figure 5). All the secondary structural elements observed in AtCAD5 are maintained in SbCAD2. The functional unit of AtCAD5 is a dimer tightly associated through two twofold-related β-strands. SbCAD2 very likely maintains the dimer character on the basis of the intact nature of βD, βE, and βF. There is a minor perturbation caused by L271H in βE, which is due to a delocalization of the imidazole ring of residue 271 by the neighboring D250 and H256 residues, but the magnitude of the impact is not enough to disrupt the dimer formation at the βF contact points.
Two of the amino acid changes between AtCAD5 and SbCAD2 are in positions that may alter the enzymatic activity (Figure 6). The first of those is residue 60 M → A. This residue resides near the surface of SbCAD2 and may possibly affect the entry of substrate. The methionine residue can act as a hydrophobic gating mechanism in concert with surrounding side chains. Thus, by replacing it with alanine, the specificity of this potential gate becomes less stringent. The second change, residue 95 C → V, occurs in close proximity of the substrate-binding pocket. The amino acid change generates a more hydrophobic character to this pocket, which is filled with water molecules in the apo-form AtCAD5. Consequently, there might be fewer water molecules in the substrate-binding pocket of SbCAD2, resulting in a significant reduction of the entropic contribution to the free energy associated with substrate binding. Therefore, it is very likely that SbCAD2 has not only reduced efficiency of substrate binding, but also altered substrate specificity compared to AtCAD5.
SbCAD2 in the bmr6-3 and bmr6-27 mutants:
The only amino acid change observed in the bmr6-3 mutant is 191 G → S, which is part of the highly conserved 188GXGGV(L)G193 motif. A serine residue at this site is a very rare alteration. Among the Zn-dependent medium-chain dehydrogenases in the database, this site is highly conserved with only hydrophobic residues. The unique serine substitution at position 191 in the bmr6-3 mutant will change the characteristics of this motif and consequently affect the binding affinity for the NADP+ cofactor (Figure 7).
In the bmr6-27 mutant, there is a total of 27 amino acid changes, all within the C terminus, spanning residues 331–361. Since the mutation affects a relatively small portion of the total sequence, and does not directly affect the active site of the enzyme or its dimerization domain, it is important to determine whether the changes produced structural alterations that would affect its activity. Among those 27 mutations, only 24 can be modeled in because the remaining C-terminal three residues are disordered in the AtCAD5 structure. Those remaining mutations are distributed through the secondary structures β12 (346–349) and α6 (330–338). In AtCAD5, those secondary structural elements, together with β11 (323–326) in the C terminus, maintain their interface through hydrophobic interactions. Those interfaces among β11 and β12 and α6 appear to be significantly affected by several arginine substitutions, which include 333 A → R, 348 V → R, and 350 V → R. Specifically, 333 A → R disrupts the hydrophobic area that maintains β11, 348 V → R disrupts the hydrophobic integrity of α6, while 350 V → R perturbs a hydrophobic pocket generated by V40, P67, G134, and F135. In addition, the 346 F → P substitution disrupts the pocket, which in turn affects the integrity of β12.
The secondary structural prediction algorithms show that both α6 (330–338) and β12 (346–349) are no longer maintained. However, the amino acid changes are unlikely to affect the ability to dimerize due to their distal locations from βD, βE, and βF (Figure 5). Another significant substitution is 342 V → R, which approaches up to 5 Å toward the Sγ-atom of 47C, and could thus alter the coordination of the catalytic Zn2+. The various amino acid substitutions present in bmr6-27 could result in either serious disruption of the catalytic domain or expansion of the intersecondary distances among β11, β12, and α6 (Figure 8). Even though we performed modeling for the bmr6-27 variant of SbCAD2, the number of amino acid substitutions and the consequent potential disruption due to the change in chemical properties is beyond the capabilities of the energy minimization-modeling algorithm. Additional experiments, such as circular dichroism spectroscopy or X-ray crystallography, are required to validate the correct model for this mutant.
Semiquantitative RT–PCR analysis of SbCAD family members in normal and bmr plants:
Comparison of the transcript levels of SbCAD2 in the near-isogenic pair BTx623/BTx623-bmr6-ref showed that in the latter the transcript levels of SbCAD2 were downregulated (Figure 9B). Such downregulation of transcripts with premature stop codons is known to occur as a result of nonsense-mediated RNA decay (Bout and Vermerris 2003; Conti and Izaurralte 2005). It is likely that the bmr6-ref allele is a null mutation. In contrast, missense mutations are unlikely to trigger mRNA degradation. Indeed, expression analysis showed that the transcript levels of SbCAD2 were not severely reduced in the bmr6-3 mutant compared to its wild-type isoline N3 (Figure 9A).
The expression of the CAD family genes varied according to tissue type and stage of development of the plant. Table 3 summarizes the expression differences among the growth stages and tissues tested. The presence of the bmr6-ref or bmr6-3 allele of the SbCAD2 gene in homozygous form did not alter the pattern of expression of the other CAD family members, with the exception of SbCAD5. This gene was upregulated in the shoot of all the bmr6-ref plants, and two of the bmr6-3 plants at stage 2. However, one of the wild-type plants also showed upregulation of this gene. Additional expression experiments with field-grown N6 and bmr6-ref plants confirmed strong plant-to-plant variation in the level of expression of the SbCAD5 gene (data not shown), but in general, this gene was more frequently upregulated in bmr6-ref plants than in wild-type plants.
As previously shown in other plants species, CAD is encoded by a small gene family in sorghum. Several genes with high similarity to the bona fide CAD genes are present in the sorghum genome. Of the 14 CAD genes identified with homology searches, there is evidence that at least 10 genes are expressed, on the basis of the presence of corresponding ESTs and expression analysis. Two loci in the genome contain closely related groups of CAD genes. The SbCAD8 1-4 cluster is orthologous with the rice OsCAD8 cluster described by Tobias and Chow (2005). Although the rice and sorghum genes within this cluster are highly similar, the gene arrangement differs between the species, raising the question whether the duplication event occurred prior to the divergence of the species or whether the clusters are the result of independent duplication events. The cluster is divided in two main branches, each of which contains sorghum and rice sequences. This suggests two duplication events, one before and one after speciation. This may explain the different orientation of the rice and sorghum genes. A homology search of the recently published maize draft genome sequence (http://maizesequence.org) with the sorghum SbCAD8 genomic sequences revealed a single ortholog (AC197840.3_FG008). This maize gene is located on chromosome 7, which is syntenic with sorghum chromosome 2 (Wilson et al. 1999) on which the SbCAD8 cluster resides. The presence of a single gene in maize, a species more closely related to sorghum than rice, is not consistent with the hypothesis that a duplication event occurred prior to the divergence of these species unless additional paralogs of AC197840.3_FG008 or remnants thereof are identified as the maize genome sequence reaches completion. The second gene cluster in sorghum (SbCAD4 1-5) has no reported orthologs in other species for which the genome sequence is available at this time. The closest rice sequence to the members of this group is a single gene, OsCAD4.
On the basis of the phylogenetic analysis, the sorghum CAD genes cluster into four groups. SbCAD2 groups together with the bona fide CADs from other species. The high sequence similarity and conservation of important substrate binding pocket residues within this group point to a conservation of the gene function. Indeed, mutations or downregulation of the expression of genes belonging to this group have significant impact in lignin content and composition in tobacco, loblolly pine, poplar, and Arabidopsis (Halpin et al. 1994; MacKay et al. 1997; Ralph et al. 1997; Baucher et al. 1999; Lapierre et al. 1999; Pilate et al. 2002; Sibout et al. 2003). Modeling SbCAD2 over the crystal structure of AtCAD5 revealed that even though SbCAD2 contains a large number of residue changes, it remains similar in overall structure, with secondary structure preserved throughout. The close relationship of SbCAD2 with the other members of this group supports the idea that it is the main sorghum CAD involved in lignification. Consistent with this hypothesis, we observed that SbCAD2 is the predominantly expressed CAD gene in sorghum. In addition, a mutation in this gene is linked to the bmr phenotype in three independently introgressed isolines and it cosegregates with the bmr phenotype in a RIL population. Furthermore, sequencing of three independent bmr6 alleles all revealed nonconservative mutations in the SbCAD2 gene. Therefore, the evidence strongly suggests that mutations in this gene are responsible for the phenotype of the bmr6 mutants.
Members of the other groups present much more diverse substitutions in the substrate binding pocket. These substitutions would allow for a wider variety of substrate specificity by changing the size, conformation, and hydrophobicity of the binding pocket. The role of these enzymes in lignification has not yet been elucidated. Only the Populus SAD has been implicated directly in lignification, catalyzing the reduction of sinapyl aldehyde to sinapyl alcohol (Li et al. 2001; Bomati and Noel 2005). Wound-inducible genes in parsley (Petroselinum crispum) and Arabidopsis cluster together in group III (Schmelzer et al. 1989; Trezzini et al. 1992; Somssich et al. 1996), suggesting that the genes in this group may be involved in the production of defense-related compounds (Hawkins and Boudet 2003). Kinetic analysis of the Arabidopsis genes in groups II, III, and V have shown that the activity against phenylpropanoid aldehydes is much lower than the Arabidopsis CADs belonging to group I. AtCAD7 has been shown to behave as an aromatic alcohol oxidoreductase of wide substrate affinity (Somssich et al. 1996). Consequently, these CAD-like genes are currently annotated as putative mannitol or benzyl alcohol dehydrogenases. However, it is evident that there is some redundancy in the CAD metabolic network. Compositional studies of Arabidopsis, rice, and sorghum mutants in which the bona fide CAD genes are downregulated have shown that their lignin still contains a certain amount of the normal G and S residues (Sibout et al. 2003, 2005; Zhang et al. 2006; Saballos et al. 2008). Reporter gene studies of the Arabidopsis CAD family showed that AtCAD7 and AtCAD8 are expressed in lignifying tissues (Kim et al. 2007). In the Arabidopsis double mutant Atcad5/Atcad4, which lacks activity of the two bona fide CAD proteins, the AtCAD1 gene is highly upregulated (Sibout et al. 2005), suggesting it may act to compensate for the lack of the CAD activity from AtCAD5 and AtCAD4. Transgenic expression of the AtCAD9 gene under control of the AtCAD5 promoter was able to partially rescue the phenotype of the double mutant (Eudes et al. 2006). In our study, we did not find consistent upregulation of any of the SbCAD genes of groups III, IV, or V in the bmr6 lines, suggesting that the steady-state level of the genes in the CAD family are sufficient to produce the observed levels of G and S units in the mutant. The SbCAD8-1 gene is highly expressed in the aerial part of the plant. In the seedling (stage 2) it is the most highly expressed CAD gene. Guillaumie et al. (2007a) analyzed the expression of the CAD genes in maize and its relation with tissue-specific lignification patterns. The EST sequence 3071507.2.1II is the likely ortholog of SbCAD8. The spatiotemporal expression of this gene agrees with our results, but it differs in that it is not the main CAD expressed in the aerial portion of the plant at the seedling stage. Of the identified CAD-like genes, the genes in the SbCAD8 cluster are the most similar to SbCAD2 (52–48% identity, except SbCAD8-3). To our knowledge, the role of the SbCAD8 genes in sorghum has not been investigated. On the basis of the high level of expression of these genes, we hypothesize that the SbCAD8 enzymes may be responsible for the production of S and G residues observed in the bmr6 mutants. Studies of a Sbcad2/Sbcad8 double mutant could help determine the role of SbCAD8 in lignification.
At this moment, it is difficult to attribute specific functions to the other sorghum CAD-like genes from groups III and V on the basis of phylogenetic relationships. We observed that the expression of the CAD-like genes in sorghum varies with the stage of development and tissue type, in agreement with what was observed in maize (Guillaumie et al. 2007a). Given the evolutionary relationship and the general agreement of gene expression patterns between maize and sorghum, it is likely that their functions are conserved. One exception may be SbCAD5. The maize ortholog, EST 4424417.2.1III, is the predominantly expressed CAD in leaves and stems of maize seedlings. In contrast, SbCAD5 showed variable expression levels in the leaves and stems of individual plants, both in seedlings and preflowering (stage 4) plants. More bmr6-ref and bmr6-3 plants showed upregulation of SbCAD5 than their wild-type isolines. It would be tempting to speculate that the gene is upregulated to compensate for the loss of SbCAD2 activity, but the lack of consistent upregulation in all bmr plants does not support this conclusion. Another possible explanation is that SbCAD5 gene is upregulated as part of a pathogen- or other stress-response mechanism, as it clusters in the same group as the Arabidopsis pathogen-inducible defense genes AtCAD7 and AtCAD8. The bmr phenotype is associated with decreased lignin content and results in a fitness penalty in the plant (Oliver et al. 2005a,b; Pedersen et al. 2005), and it is possible that the bmr plants are under increased stress compared to wild-type plants. It is interesting that while in the aerial parts of both maize and sorghum seedlings the bona fide CAD is not the most highly expressed CAD gene, the predominant CAD genes in those tissues represent nonorthologous paralogs in the two species.
To our knowledge, no orthologs of SbCAD6 and OsCAD6 (group IV) have been described in the literature. SbCAD6 is mainly expressed in the roots of stage 4 plants, although at low levels. Both SbCAD6 and OsCAD6 (Tobias and Chow 2005) contain a predicted peroxisomal targeting signal at their C terminus. Dehydrogenase activities in the peroxisome are involved in the metabolism of alcohol, branched amino acids, fatty acids, and plant hormones (Reumann et al. 2004). Detailed biochemical and expression studies are needed to assign a physiological function to this gene.
We have described three different mutant alleles of SbCAD2: bmr6-ref, a likely null allele, bmr6-3, which produces a full-length protein with a single disruption in the cofactor binding site, and bmr6-27, encoding an enzyme with a disrupted secondary structure outside the active site. Although all three mutants produce the visual brown midrib phenotype, it is likely that the mutations have different effects on enzyme activity. There is evidence that the bmr6-3 allele has a stronger effect on enzymatic saccharification of stover than the bmr6-ref allele, wheras the latter allele results in higher coniferaldehyde levels in the midrib tissue than either the bmr6-3 or bmr6-27 allele (Saballos et al. 2008). Now that the molecular basis of the mutations is known, variation in penetrance can be investigated. Tailoring of the effect of the activity of the enzyme may be achieved on the basis of the choice of allele introgressed in a particular sorghum line. The development of molecular markers specific for each of those variants will greatly facilitate the use of the alleles in genetic studies and breeding programs. There are at least six additional known bmr6 alleles available (Saballos et al. 2008), which opens the possibility of finding more useful variants.
The authors thank Jeff Pedersen (U.S. Department of Agriculture (USDA)-Agricultural Research Service, Lincoln NE) for providing seeds of the bmr6-ref mutants in different inbred lines. We thank Terry Lemming for excellent management of the field plots. This research was supported by the Office of Science (BER), U.S. Department of Energy, grant DE-FG02-07ER64458 (W.V. and G.E.), and National Research Initiative of the USDA grant 2006-35318-17454 (C.H.K.), with additional funding from Purdue University Agricultural Research Programs, Purdue Research Foundation, and the University of Florida.
- Received November 21, 2008.
- Accepted December 10, 2008.
- Copyright © 2009 by the Genetics Society of America


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