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Quality CSO Plans
Developing Lead Service Line Inventories Presented by the Association of State Drinking Water Administrators
Many state drinking water administrators are considering developing inventories of the materials used in service lines that are part of the distribution systems of community water systems (CWSs) they regulate. Some states have already conducted voluntary or mandatory surveys of CWSs whether on their own or in response to state legislation. Others are preparing to use the information in the next round of Drinking Water Infrastructure Needs Survey and Assessments (DWINSA) that the Environmental Protection Agency (EPA) is developing pursuant to Section 2015 of the America's Water Infrastructure Act of 2018. The 2020 DWINSA will include an estimate of the number of public and private lead service lines as well as an estimate of the costs to replace all lead service lines, which will be a significant undertaking for water systems to develop and states to collect information on. To assist states that are considering initiating a lead service line (LSL) inventory, the Association of State Drinking Water Administrators (ASDWA) has developed the following guidance based on the experience of the states that have already conducted or are preparing to develop a comprehensive inventory of service line materials.
Developing Lead Service Line Inventories Presented by the Association of State Drinking Water Administrators
Handout: Water Infrastructure Drives New Jersey's Economy
In order to lay the groundwork for wide-scale transformation of New Jersey's water infrastructure systems, members of the Jersey Water Works collaborative have prioritized educating the public and elected and appointed officials on the importance of taking care of water infrastructure. This booklet is a tool for doing so and Jersey Water Works encourages all to make this economic case to their local and state elected and appointed officials today.
Assessment of Eliminating Lead in Minnesota Drinking Water
The 2017 Minnesota Legislature directed the Minnesota Department of Health (MDH) to "conduct an analysis to determine the scope of the lead problem in Minnesota's water and the cost to eliminate lead exposure in drinking water." In this report, Assessment of Eliminating Lead in Minnesota Drinking Water, MDH assesses the scope of the lead problem by examining the extent of lead already in water systems as well as factors that allow lead to get in drinking water. However, because drinking water systems across the state are diverse and have varying requirements and resource needs, broad estimates are used to gauge costs.
Lead in Minnesota Water: Assessment of Eliminating Lead in Minnesota Drinking Water
Policy Options: Promoting Affordability of Public Water and Sewer Service for Low-Income Households in New Jersey
This discussion paper presents policy options for improving affordability of water and wastewater service for lower income residents in New Jersey—for the benefit not only of those customers, but of all New Jerseyans who depend on their local utilities for clean, safe, and reliable water and sewer service. Jersey Water Works aims to use it to spark discussion among state and local policy makers, utility managers, water and sewer customers, community organizations, and other public sector, private sector, and non-profit stakeholders, and to use it as a basis for further analysis and development of priority recommendations. The paper is based on a review of existing literature from around the country; research on existing New Jersey laws and programs; and interviews with leaders representing or associated with a wide range of stakeholders, including publicly and privately owned utilities, state regulators, affordable housing developers and advocates, consumer advocates, environmental justice advocates, business, and labor.
Promoting Affordability of Public Water and Sewer Service for Low-Income Households in New Jersey: Policy Options
"A major deterrent to proper investment in water infrastructure is affordability . . . Municipalities with a larger share of low-income residents find it difficult to raise rates to fund water infrastructure upgrades due to the detrimental effect higher rates will have on those residents. The result is underinvestment, which is a losing proposition . . . However, while increasing rates can adversely affect low-income households, as several witnesses noted, these effects are not inevitable and can be avoided . . ." —New Jersey Legislature, Joint Legislative Task Force on Drinking Water Infrastructure, Final Report (Jan. 2018)
Rates could fund lead pipe replacement in critical states: Laws in states with the most lead service lines support the practice
In Rates could fund lead pipe replacement in critical states, Emmett Environmental Law & Policy Clinic at Harvard Law School and Environmental Defense Fund reviewed state laws and policies in 13 states with the most lead service lines (LSLs), and found no explicit barriers to using rate funds to replace the lines on private property. These states have an estimated 4.2 million LSLs, more than two-thirds of the nation's total. In these states, publicly-owned utilities can act pursuant to existing state legislation by determining that the practice serves a public purpose—protecting public health. Investor-owned utilities can do the same, but typically need approval of the state's utility commission. While we have not reviewed the remaining states, we anticipate that the state laws and policies are similar to the ones we evaluated.
Rates could fund lead pipe replacement in critical states: Laws in states with the most lead service lines support the practice


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Want to know how to choose a water filter cartridge to remove sediment from the entire house? The advantages of the cartridge filter are low cost, easy installation, no electricity and no waste water.
The RO system removes dissolved salts, minerals and microorganisms, making the water taste suitable for drinking. Sometimes RO systems cannot remove dissolved impurities. This will affect the taste of the water.
Pay attention to how much water you drink every day is essential to maintain health. Most people drink when they are thirsty, which helps regulate daily water intake.
When selecting whole house water filters, some important factors concerning the system should be considered.
An outbreak of a respiratory disease called COVID-19 continues to spread around the world. During the crisis period, people have raised various questions about whether COVID-19 can be transferred through drinking water and waste water.
Inline water filters are a convenient way to ensure that all water you are consuming is completely safe drinking water. Many of the best inline water filters can remove 99.99% of the pollutants in the water. It includes chemical, physical, radioactive and

subset: commoncrawl


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AKA & Ricky Rick tease new collaboration & fans are excited
Are AKA and Riky Rick working on new music together?
Rikhado Makhado whose best known with his stage name, Rick have sparked suggestions that he is working with multi-award winning rapper AKA on new music.
The 32-year-old rapper, who previously collaborated on projects with Cassper Nyovest, teased a song with AKA featuring DJ Tira.
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Following the video, fans have been expressing their excitement on Twitter.
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— Kay (@Skay_Da_Rapper) August 12, 2019
Mega never dissapoints… it's gonna be a chilled song pic.twitter.com/kXxThS8F6z
— Msentiii (@Msentiii_) August 12, 2019


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The Zac Brown Band will kick off their new North American Welcome Home tour on May 12th in Atlanta. The 40-plus date will play in stadiums and amphitheaters around the country, including the Hollywood Bowl, Wrigley Field, and Coors Field in Denver.
The Zac Brown Band will release an album, also called Welcome Home, on May 12th. It's the group's fifth studio album, which Zac Brown told Rolling Stone would be a return "straight back to our roots, Foundation-style."
The band has blocked off Saturday May 20th and Sunday May 21st their annual Southern Ground Music & Food Festival taking place in Charleston, SC.


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Most Popular
Quarantined Italians Record Messages For Past Selves Warning Of Future Coronavirus Threat
Quarantined Italians have recorded messages for their younger selves, warning of what lies ahead as the coronavirus pandemic continues.
The country was put on national lockdown last week in a bid to restrict the spread of the disease.
And with some estimates putting the UK, US, and others just 10 days behind Italy in the progression of the pandemic in their countries, Italians who have been consigned to their homes have recorded videos with messages they wish they had heard 10 days ago, explaining the severity of the situation.
The videos have been put together and shared by a Milan-based creative group called 'A Thing By'.
Stitching together messages from multiple people across the country, the video describes what life is like for them and how they wish they had their time over again so they could take it seriously and treat the crisis with respect.
After introducing themselves, they say: "I'm speaking to you from the future. I know you're busy but wait a second.
"I wanted to update on Italy's latest: a huge mess is about to happen.
"I'm sure you've heard about coronavirus. And I'm also pretty sure you're underestimating it.
"I even used to make fun of people wearing masks. Stop making fun of mum for making you buy masks and go buy them... idiot."
It goes on: "Until 10 days ago, I used to go to meetings, I'd go to dinner with friends, to the gym, to the hairdresser. To usual boring things that never hurt anybody.
"But in 10 days, life won't be like this anymore."
Adding: "The worst case scenario? That is exactly what will happen. Yes, cause infections in Italy will continue to increase - proving that this wasn't bulls*** like you thought.
"Ten days ago we had 2,000 people infected, now we're at 18,000."
And with all the benefit of hindsight, they push home the point that it's vital those fortunate enough not to be in Italy should take the advice being given doctors, scientists, and healthcare experts.
One man says: "I know China is far away but this virus is faster than you think so maybe re-think your Saturday night plans.
"And who knows? Maybe had you stayed at home then I wouldn't have to be home now."
The video then ends with a simple but powerful message: "We underestimated this. You don't have to do the same. Stay at home."
It's okay to not panic. LADbible and UNILAD's aim with our Coronavirus campaign, Cutting Through, is to provide our community with facts and stories from the people who are either qualified to comment or have experienced first-hand the situation we're facing. For more information from the World Health Organisation on Coronavirus, click here.
Featured Image Credit: A Thing By
Topics: Science, World News, Interesting, coronavirus, Health


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International trade successes bolster Belarusian ruble
In June 2014, the international trade surplus totalled USD 240 million (the best result in 2014).
As a result, the pressure on the foreign exchange market has reduced, the international reserves have been used at a slower pace to pay out the outstanding public debt in foreign currency, foreign currency inflows have increased due to growing exports, and some measures, such as licensing, have proved their efficiency in curbing food and industrial imports. Given the expected negative impact of the sanctions on the Russian economy, Belarus' exports of engineering products may decrease further, but could be compensated with increased exports and higher prices on meat and dairy products.
Similar articles
Over the past year, military-political relations between Minsk and Kyiv have become complicated. Due to their high inertia and peculiarities, this downward trend would be extremely difficult to overcome.
The root cause of the crisis is the absence of a common political agenda in the Belarusian-Ukrainian relations. Minsk is looking for a market for Belarusian exports in Ukraine and offers its services as a negotiation platform for the settlement of the Russo-Ukrainian war, thereby hoping to avoid political issues in the dialogue with Kiev. Meanwhile, Ukraine is hoping for political support from Minsk in the confrontation with Moscow. In addition, Ukraine's integration with NATO presupposes her common position with the Alliance in relation to Belarus. The NATO leadership regards the Belarusian Armed Forces as an integral part of the Russian military machine in the western strategic front (the Baltic states and Poland). In addition, the ongoing military reform in Ukraine envisages a reduction in the number of generals and the domestic political struggle makes some Ukrainian top military leaders targets in politically motivated attacks.
Hence, the criticism of Belarus coming from Ukrainian military leadership is dictated primarily by internal and external political considerations, as well as by the need to protect the interests of generals, and only then by facts.
For instance, initially, the Ukrainian military leadership made statements about 100,000 Russian servicemen allegedly taking part in the Russo-Belarusian military drill West-2017. Then the exercises were labelled quazi-open and military observers from Ukraine refused to provide their assessment, which caused a negative reaction in Minsk. Further, without citing specific facts, it was stated that Russia was building up its military presence in Belarus.
Apparently, the Belarusian and Ukrainian Defence Ministries have entangled in a confrontational spiral (on the level of rhetoric). Moreover, only a small part of the overly hidden process has been disclosed. That said, third states are very likely to take advantage of the situation (or have already done so). This is not only about Russia.
The Belarusian Defence Ministry officials are restrained in assessing their Ukrainian counterparts. However, such a restraint is not enough. Current military-political relations between Belarus and Ukraine are unlikely to stabilise without the intervention of both presidents.


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Semi-detached house for sale in Wentworth Road, Birmingham B17
* Calls to this number will be recorded for quality, compliance and training purposes.
Property features
- No Chain
- Imposing Traditional Semi Detached
- Three Excellent Bedrooms
- Potential To Extend (stp)
- Two Spacious Reception Rooms
- Large Bathroom
- Double Garage
- Lovely Rear Garden
- Parking For Multiple Vehicles
- Sought After Location
Property description
**Large Family Home, No Chain**
A must see, this fantastic home offers spacious living and lots of potential. Ready to move into and offering no chain; do not miss the opportunity to secure it and reside in the sought after Harborne area, with it's easy access to Queen Elizabeth Hospital and highly sought after schools.
Comprising of: Two reception rooms, separate kitchen, utility area, outside WC, large bathroom, three excellent bedrooms, fabulous rear garden and a double garage. There is also off road parking for multiple vehicles.
Located on the corner of Wentworth Road and Woodville Road; this imposing semi-detached residence is perfectly located to be within easy reach of schools, transport links and Harborne High Street with its many amenities.
Having dwarf wall to front and side, hedging to side and lawn with a variety of shrubs. Paved drive and path leading to a storm porch with door into the entrance hall. Also having access to the double garage via an up and over door.
Entrance Hall
16'03" max x 6'05" max
Stained glass window and original door into, period feature coving and cornice to walls and ceiling. Having carpeted flooring, central heating radiator, stairs to first floor, doors to two reception rooms, storage cupboard under the stairs and kitchen.
Reception Room One
15'08" max x 13'01" max
Carpeted flooring, central heating radiator, double glazed bay window to front aspect, fire place with surround, shelving to the alcoves, picture rail to walls and coving to ceiling.
Reception Room Two
13'07" max x 10'03" max
Carpeted flooring, central heating radiator, double glazed window and door to rear access the rear garden. Having also a gas fire and surround, shelving to the alcoves, picture rail to walls, cornice and coving to ceiling.
9'10" max x 9'02" max
Having red brick flooring, central heating radiator, single glazed window to rear aspect, door into utility area and Potterton boiler.
Matching wall and base units with integrated sink and drainer with splash back tiles, gas hob and oven with extractor fan over, space for a dishwasher and low level fridge freezer.
Utility Area
9'03" max x 3'08" max
Vinyl flooring, single glazed window to side aspect, door to side accessing the garden and having plumbing for a washing machine.
First Floor
Carpeted stairs and landing, central heating radiator, loft hatch, doors to three bedrooms and a bathroom.
Bedroom One
14'11" max x 11'11" max
Carpeted flooring, central heating radiator, double glazed bay window to front aspect, wall mounted gas fire, picture rail to walls and coving to ceiling.
Bedroom Two
13'02" max x 11'11" max
Carpeted flooring, central heating radiator, single glazed window to rear aspect, original feature fire place, picture rail to walls, coving and cornice to ceiling.
Bedroom Three
8'01" max x 7'08" max
Carpeted flooring, central heating radiator, double glazed window to front aspect.
10'01" max x 7'05" max
Tiled floor, part tiled walls, obscure double glazed window to rear aspect, bath with shower over, low level WC, wash basin and bidet. Also having a store cupboard with the hot water tank within.
Rear Garden
Being fence enclosed and having a well proportioned concrete patio area, large lawn with a variety of shrubs and trees. Having also a door into the garage, door to an outside WC and double gates to the side giving access onto Woodville Road.
Outside W.C.
Low level WC and overhead lighting.
Double Garage
18'04" max x 14'04" max
Having and up and over door to the front, door to the rear, single glazed windows to rear aspect, electrical sockets and overhead lighting.
Property info
Floor Plan View original
For more information about this property, please contact
Purplebricks, Head Office, B90 on +44 24 7511 8874 * (local rate)
Property descriptions and related information displayed on this page, with the exclusion of Running Costs data, are marketing materials provided by Purplebricks, Head Office, and do not constitute property particulars. Please contact Purplebricks, Head Office for full details and further information. The Running Costs data displayed on this page are provided by PrimeLocation to give an indication of potential running costs based on various data sources. PrimeLocation does not warrant or accept any responsibility for the accuracy or completeness of the property descriptions, related information or Running Costs data provided here.


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Red Lips Cartoon Drawing Girl Tutorial Anime
|Include In Article
Cartoon Lips Drawing
|Published Date
|Wednesday , June 26th 2019
|Uploaded By
|Andi Zimmerman
Free Drawing. Wednesday , June 26th 2019.
Quote from Cartoon Lips Drawing :
Initially it could possibly be beneficial to use the grid technique to capture all the characteristics of the face. Detailed drawing techniques with descriptive captions make it possible for readers to produce their very own automotive designs. You might need to experiment with diverse numbers to find the line precisely where you desire.
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You should observe a butterfly. When the circle is made, you can alter the formatting in whatever way you demand.
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Another trailer to wet your appetites, this time for Pokémon Black & White 2, featuring a new rival, trainers, a
Category: Games
Are you looking for news, reviews, video game footage, trailers or general information about a particular Nintendo DS game? If so be sure to check out the QJ Nintendo DS games category for all of the latest news and reviews for the top Nintendo DS games.
New Pokemon Game Is A Collaboration Crossover
Can your brain take the change in routine? Ninty are going a new route with this Pokémon title.
Sega Games Land Official 3DS Release Dates
The former Nintendo rival confirms four new titles for the Nintendo 3DS and provides North American release dates to an
Professor Layton and the Last Specter's Hidden RPG
A new Professor Layton game hits shelves today, but did you know that The Last Specter contains a completely separate
Aliens: Infestation hits North America in October
Gearbox Software's Aliens: Colonial Marines is still a long way from release, but franchise fans who just really have to strap
This week's Nintendo downloads
Nintendo has once again updated its various download services with a few new releases for its gaming platforms.
Bakugan: Rise of the Resistance announced
Fans of the Bakugan anime and games will get a new handheld title to play with this holiday season.
This week's Nintendo USA downloads
This week's Nintendo USA downloads feature a mixed bag of platforming, hidden object mystery solving, and strategic maneuvering.
Nintendo: No Zelda collection for 25th anniversary
Nintendo has big things in store for Zelda fans, but a collection isn't one of them.
Devil Survivor sequel coming to DS in the West
There's more turn-based demon collecting in store for Nintendo DS gamers.


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Karrimor quarter Zip Short Sleeve T Shirt Ladies
Product Info
Karrimor 1/4 Zip Short Sleeved T Shirt Ladies
Conquer your next workout with the 1/4 Zip Short Sleeve T Shirt from Karrimor. Crafted with mesh panels and reflective details, this lightweight piece is perfect for training sessions.
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Standard delivery
Find out more
Using the standard service, your order should be with you within 4-5 working days.
Next day delivery
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Place your order before 7pm Sunday - Friday and receive your order within 1 to 2 days (excludes Sundays and Public holidays).
International delivery
Find out more
International delivery is available for this product. The cost and delivery time depend on the country.
If you need to return any item to us, please send it back to us within 14 days of receipt of the order (unless faulty).
It's important that any items returned to us, unless faulty, are returned in a re-saleable condition. We would like this to mean that you've kept all original packaging and labels, and that it's unused.
For more information, view our full Returns and Exchanges information.
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- Womens > Clothing
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Easy Query Builder 2.6 ~ crack/serial/keygen
Easy Query Builder is a Windows program made to simplify the users' job when it comes to putting together SQL queries, especially when they have minimum background in this area of IT. It lets them fiddle with some intuitive settings that automatically get translated into valid valid SQL statements, generate queries, and save data to CSV files.
The entire installation procedure is over in no time. As for the interface, Easy Query Builder opts for a professional-looking window with a neatly organized structure. You can either create a new database connection by filling out some some details like name, type, SQL dialect, open an existing one (NWind Access), or manage a collection of multiple connections.
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It is possible to add customer, order, product and employee columns, sort them, and add new query conditions by just tinkering with some options in the main frame. The corresponding SQL statements can be viewed in real time. When all settings are made, you can execute the query and check out results on the bottom part of the screen, as well as save the query and export the results to CSV format.
In addition, Easy Query Builder Serial lets you edit the data model in terms of tables, links, entities and operation, such as adding new data and virtual tables, as well as to edit connection properties.
The program does not put a strain on computer performance, since it runs on low CPU and RAM. It has a good response time and works smoothly. No errors were shown in our tests, and Easy Query Builder did not hang or crash. All in all, this software utility provides inexperienced users with a simple solution to building simple queries with standard SQL statements, and it can be handled by anyone.


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Water Damage Repair and Restoration
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Brainteaser 1510: Sum puzzle
From The Sunday Times, 18th August 1991 [link]
In this addition sum nine digits are each represented by a different letter and the remaining one digit it represented in various places by nine letters.
When the 47-figure number:
is added to the 46-digit number:
the answer is the 47-figure number:
What is the SOLUTION?
The text of this puzzle is taken from the book Brainteasers (2002, edited by Victor Bryant), so may differ from the puzzle originally published in the newspaper.


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Career Services provides career and job search services to Florida Tech students and alumni. Our mission is to help students in the development of career plans and job search skills. Career Services acts as a liaison among Florida Tech students, alumni, faculty, staff and employers.
福利视频(午夜),不卡福利午夜福利视频,国语92午夜福利2000Career Services is open 8 a.m. to 5 p.m., Monday through Friday (except holidays) and is located in Room 307 of the Harris Commons Building.
福利视频(午夜),不卡福利午夜福利视频,国语92午夜福利2000Our office provides a variety of services to our enrolled students. On our main campus we provide:
福利视频(午夜),不卡福利午夜福利视频,国语92午夜福利2000For online students we provide:
Click here to see which employers recruit at our school.


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Date to decommission INS Viraat not finalised: Dhowan
Navy Chief Admiral RK Dhowan on Wednesday said aircraft carrier INS Viraat was on its last legs, but the actual date of its decommissioning had not been decided yet.
"I must mention that INS Viraat has done excellent service to the Navy and the nation. I would like to congratulate the commanding officers and the entire ship company making sure that INS Viraat is maintained, operational at its peak efficiency. But now, it is on its last legs," Dhowan said, following the Investiture Ceremony at Naval dockyard onboard INS Viraat. "We have not yet decided on the date but we certainly are looking at the aspect when the ship would be decommissioned," he said.
About prospects of turning the country's flagship aircraft carrier into a museum after its decommissioning, he said, "Yes, we have proposed to the Ministry of Defence that it (INS Viraat) should be converted into a museum."
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- © 2013 American Society of Plant Biologists. All Rights Reserved.
Appropriate stimulus-response coupling requires that each signal induces a characteristic response, distinct from that induced by other signals, and that there is the potential for individual signals to initiate different downstream responses dependent on cell type. How such specificity is encoded in plant signaling is not known. One possibility is that information is encoded in signal transduction pathways to ensure stimulus- and cell type-specific responses. The calcium ion acts as a second messenger in response to mechanical stimulation, hydrogen peroxide, NaCl, and cold in plants and also in circadian timing. We use GAL4 transactivation of aequorin in enhancer trap lines of Arabidopsis (Arabidopsis thaliana) to test the hypothesis that stimulus- and cell-specific information can be encoded in the pattern of dynamic alterations in the concentration of intracellular free Ca2+ ([Ca2+]i). We demonstrate that mechanically induced increases in [Ca2+]i are largely restricted to the epidermal pavement cells of leaves, that NaCl induces oscillatory [Ca2+]i signals in spongy mesophyll and vascular bundle cells, but not other cell types, and detect circadian rhythms of [Ca2+]i only in the spongy mesophyll. We demonstrate stimulus-specific [Ca2+]i dynamics in response to touch, cold, and hydrogen peroxide, which in the case of the latter two signals are common to all cell types tested. GAL4 transactivation of aequorin in specific leaf cell types has allowed us to bypass the technical limitations associated with fluorescent Ca2+ reporter dyes in chlorophyll-containing tissues to identify the cell- and stimulus-specific complexity of [Ca2+]i dynamics in leaves of Arabidopsis and to determine from which tissues stress- and circadian-regulated [Ca2+]i signals arise.
Ca2+ is an important second messenger involved in a wide range of responses in plants (Dodd et al., 2010). Fluorescent reporters of Ca2+ have revolutionized the study of Ca2+ signaling, permitting measurement of the intracellular concentration of Ca2+ ([Ca2+]i) with high subcellular spatial and temporal resolution. In plants, the use of fluorescent reporters has identified stimulus-induced increases in [Ca2+]i that encode information in the temporal and subcellular distribution of the [Ca2+]i signal (Dodd et al., 2010). However, the utility of fluorescent Ca2+ indicators in plants has been usually restricted to a few specific cell types, including the guard cells, root hairs, and pollen tubes, because these tissues are low in chlorophyll. In other tissues, chlorophyll fluorescence reduces the signal-to-noise ratio to unacceptable levels. Furthermore, in situ measurements of cytosolic-free Ca2+ concentration ([Ca2+]cyt) signals in single cells in tissues below the plant surface using fluorescence or confocal microscopy has proved technically challenging, even in root tissues (Zhu et al., 2013), due to light scatter and fluorescence by the cell walls. To overcome some of these difficulties, Knight et al. (1991) introduced recombinant aequorin as a reporter of Ca2+ concentration in plant systems. Aequorin is a bioluminescent reporter and, therefore, has an intrinsically high signal-to-noise ratio, and it requires no damaging excitation light and, therefore, is suited to measurements in chlorophyll-containing tissue. The major limitation of the use of aequorin is low light emission, meaning that imaging even at the subtissue level has proved elusive (Zhu et al., 2013), and subcellular imaging in plants is not possible. Aequorin has found high utility and has identified roles for [Ca2+]cyt signaling in mechanical stimulation, cold, salinity, and pathogen stress and in the daily timing of plants (Campbell et al., 1996; Knight et al., 1996, 1997; Gong et al., 1998; Kawano et al., 1998; Baum et al., 1999; Love et al., 2004; Kosuta et al., 2008; Monshausen et al., 2009; Zhu et al., 2013). However, in leaves and shoots, it is not known from which tissues the stress- and circadian-regulated [Ca2+]i signals arise.
We describe targeting of the in vivo Ca2+ reporter aequorin to spongy mesophyll, trichome, vascular, and epidermal pavement cells using GAL4-mediated transactivation in enhancer trap lines (Gardner et al., 2009). In the GAL4 enhancer trap system as implemented in Arabidopsis (Arabidopsis thaliana), the yeast GAL4 transcriptional activator is inserted randomly into the genome under the control of a minimal promoter (Kiegle et al., 2000). When the insertion is located near an endogenous enhancer, GAL4 is expressed. GAL4 expression is visualized by combining on the same insertion cassette endoplasmic reticulum-targeted GFP downstream of a GAL4 upstream activation sequence (UAS). In some cases, cell-specific GFP expression can be detected in individual lines. These cell-specific driver lines can be used to deliver cell-specific expression of any transgene downstream from another GAL4 UAS. These GAL4 UAS-driven transgenes are introduced by supertransformation or crossing. Previously, GAL4 transactivation of aequorin has been described for root tissues (Kiegle et al., 2000) and guard cells (Dodd et al., 2006).
We test the hypothesis that there is cell and tissue specificity of the [Ca2+]i signaling networks in Arabidopsis. We use trichome, spongy mesophyll, epidermal pavement, vascular bundle, and guard cell-specific enhancer trap lines that we described previously (Dodd et al., 2006; Gardner et al., 2009) to investigate the responses of cell types to NaCl, cold, mechanical stimulation, and hydrogen peroxide (H2O2) because all cause a rapid increase in [Ca2+]i followed by dynamic alterations of [Ca2+]i when measured in seedlings in which aequorin has been targeted to the whole seedling (Lynch et al., 1989; Price et al., 1994; Knight et al., 1996, 1997; Monshausen et al., 2009). Additionally, we investigate from which cells diel oscillations of [Ca2+]i arise. Diel and circadian oscillations of [Ca2+]cyt were first detected in seedlings in which aequorin was constitutively expressed (Johnson et al., 1995) and later demonstrated to be occurring in the leaves (Love et al., 2004). The daily oscillations of [Ca2+]cyt peak at around 300 nm about 8 h after dawn (Love et al., 2004). The circadian clock and PHYTOCHROME A-, CRYPTOCHROME1-, and CRYPTOCHROME2-dependent signaling pathways drive daily rhythms of [Ca2+]cyt (Dalchau et al., 2010) with cyclic ADP ribose (cADPR) acting as an intermediary (Dodd et al., 2007). The core circadian clock gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is required for circadian oscillations of [Ca2+]cyt and cADPR, because cca1-1 loss of function abolishes the rhythms of both [Ca2+]cyt and cADPR, resulting in constitutively high [Ca2+]cyt, whereas overexpression of CCA1 results in constitutively low cADPR concentration and [Ca2+]cyt (Dodd et al., 2007; Xu et al., 2007).
Despite the central importance of [Ca2+]i signaling in the regulation of leaf biology, study of the dynamics of stimulus-induced and circadian-regulated [Ca2+]i signals in leaves has usually been limited to the stomatal guard cells, or data have only been gathered on the responses at the whole seedling level or from images of whole leaves, summing the responses of different cell types (Johnson et al., 1995; Wood et al., 2000, 2001; Love et al., 2004; Dodd et al., 2006, 2010; Zhu et al., 2013). In this study, we identify cell- and stimulus-specific [Ca2+]i signals in the leaf and other tissues of Arabidopsis, suggesting the presence of cell-specific signaling cassettes in plants.
GAL4 Transactivation of Aequorin in Cell-Specific GAL4-GFP Enhancer Trap Lines
Multiple independent lines transformed with pBINYFPAEQ were obtained for each of the enhancer trap lines used in this study. More than five independent transformants per GAL4 enhancer trap line were analyzed, and only those lines with the highest total aequorin activity were selected for further analysis. Consistent with previous reports of aequorin transformation under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter or using the GAL4 transactivation system (Dodd et al., 2006; Gardner et al., 2009), we observed no gross alterations in the visible phenotype of the plants when they were transformed with yellow fluorescent protein fused to apoaequorin (YFPAPOAEQUORIN). The tissue-specific localization of GFP in the selected enhancer trap lines and, therefore, the restriction of the GAL4 transactivation in all the cell-specific driver lines analyzed here was described previously (Gardner et al., 2009; see Fig. 6A). In this study, YFPAPOAEQUORIN was used to determine in which cells aequorin was expressed in each of the enhancer trap lines. The YFPAPOAEQUORIN fluorescence was detected only in the specific cells marked by GFP in the GAL4 driver lines. GFP/yellow fluorescent protein (YFP) fluorescence was not detected in other cells (Supplemental Figs. S1–S4). GFP and YFPAPOAEQUORIN were cotargeted to the living vascular cells, including the bundle sheath cells in KC274 (Supplemental Fig. S1; see also Fig. 6A in Gardner et al., 2009), the epidermal pavement cells of leaves in KC464 (Supplemental Fig. S2; see also Fig. 6A in Gardner et al., 2009), the leaf spongy mesophyll cells in JR11-2 (Supplemental Fig. S3; see also Fig. 6A in Gardner et al., 2009), and the trichomes in KC380 (Supplemental Fig. S4; see also Fig. 6A in Gardner et al., 2009). We also confirmed the localization of YFPAPOAEQUORIN specifically to mature stomatal guard cells in E1728, as described previously (Dodd et al., 2006). In all lines, the expression was restricted to the specific cells in the aerial tissues, with the exception of KC274, in which expression in the vascular tissues was detected in roots, shoots, and leaves.
YFPAPOAEQUORIN fluorescence was present in the cytosol and nucleus for all lines (Supplemental Figs. S1–S5), as reported previously by Dodd et al. (2006) for the E1728 GAL4-GFP enhancer trap line expressing YFPAPOAEQUORIN in guard cells. The GFP signal was excluded from the nucleoplasm due to an endoplasmic reticulum-targeting sequence in the mGFP5 variant used in the enhancer trap (Haseloff et al., 1997). The different subcellular localization allowed confirmation that the spectral separation of the GFP and YFP emission spectra was sufficient to conclude that there was colocalization of YFPAPOAEQUORIN and GFP to the same cell types, consistent with the enhancer trap expression pattern.
Localization of Nycthemeral and Circadian Oscillations of [Ca2+]i
In 12-h-light and 12-h-dark cycles (LD), rhythms of aequorin bioluminescence were detected from the spongy mesophyll of JR11-2 (Fig. 1A), the trichomes of KC380 (Fig. 1B), the vascular cells of KC274 (Fig. 1C), the epidermal cells of KC464 (Fig. 1D), and the guard cells of E1728 (Fig. 1E). However, only the nycthemeral [Ca2+]i oscillations reported from the spongy mesophyll cells had the dynamics of a peak in the middle to end of the photoperiod and anticipation of dawn in the night typical for nycthemeral rhythms of [Ca2+]cyt in LD reported by CaMV35S:AEQUORIN (Love et al., 2004). In constant light (LL), free-running circadian rhythms of [Ca2+]i with a period close to 24 h were detected in the JR11-2 enhancer trap line expressing YFPAPOAEQUORIN in spongy mesophyll cells in the presence of distilled water and mannitol (period in water, 22.1 ± 0.6 h, eight out of 12 plant clusters relative amplitude of error < 0.5, period in mannitol 22.6 ± 0.5 h, five out of six plant clusters relative amplitude of error < 0.5; Fig. 1A; Supplemental Fig. S6). The circadian oscillations of [Ca2+]i had the characteristics of circadian oscillations of [Ca2+]cyt detected previously in plants constitutively expressing CaMV35S::APOAEQUORIN, including peaking in the middle to end of the subjective day (Love et al., 2004), a declining amplitude (Dalchau et al., 2010), and being abolished by 3% (w/v) Suc (Johnson et al., 1995; no rhythm detected from six out of six plant clusters; Fig. 1A; Supplemental Fig. S6). No circadian oscillations of [Ca2+]i were detected in LL under any of the experimental conditions tested when GAL4-transactivated YFPAPOAEQUORIN was targeted specifically to the trichome, vascular, epidermal pavement, and guard cells (no rhythms detected from four out of four plant clusters tested for each line; Fig. 1, B–E). Due to the low signal-to-noise ratio of the enhancer trap lines, we were unable to calibrate the AEQUORIN signal during the long-time-course circadian analysis, but in previous studies, we estimated that the peak [Ca2+]i is around 300 nm (Love et al., 2004).
To determine whether the changes observed in AEQUORIN light emission over time were due to changes in [Ca2+]i or APOAEQUORIN expression, total AEQUORIN was quantified for all the lines at 7 to 9 h and 21 to 23 h after dawn in LD. This is the condition in which the amplitude of light emission was greatest and in which all the lines had cyclic light emission. There was no significant difference in total AEQUORIN pool size between the time points, representing the timing of the minima and maxima of the diel oscillations of light signal. The nonsignificant trend was for the AEQUORIN pool to be largest when the diel light emission was smallest. These data indicate that the oscillations in light emission we detected are a consequence of changes in [Ca2+]i rather than AEQUORIN pool size (Supplemental Table S1).
Analysis of Stimulus-Induced [Ca2+]i Increases in Spongy Mesophyll, Pavement Epidermis, Vasculature, Trichomes, and Guard Cells
Mechanical stimulation resulted in an immediate and rapid rise of [Ca2+]i levels in all plants tested of KC464 in which YFPAPOAEQUORIN was targeted to the epidermal pavement cells (n = 12; Supplemental Fig. S7), with a mean peak value of 646.6 ± 76.2 nm (Fig. 2A). The magnitude of the mechanical stimulation-induced increase in [Ca2+]i was smaller in all other tissues (approximately 250–370 nm; Fig. 2). For all tissues other than the epidermal pavement cells, not all the plants responded to the mechanical stimulation: seven out of 12, three out of 10, four out of 10, and seven out of 13 plants responded to mechanical stimulation in guard, mesophyll, trichome, and vascular cell enhancer trap lines, respectively (Supplemental Figs. S8–S11). In all tissues, when a response to mechanical stimulation was detected, it comprised an immediate transient elevation of [Ca2+]i that returned to resting within 20 s of stimulation (Fig. 2). Occasionally, following the transient, subsequent [Ca2+]i changes of low amplitude were detected in epidermal pavement cells (Supplemental Fig. S7).
In Arabidopsis seedlings expressing CaMV35S::APOAEQUORIN, NaCl results in a [Ca2+]cyt signature that consists of a rapid rise of [Ca2+]cyt levels immediately followed by a slower relaxation (Knight et al., 1997; Kiegle et al., 2000; Tracy et al., 2008). Here, we demonstrate that it is possible to partially deconvolute the NaCl-induced [Ca2+]i signatures based on the intensity and the pattern of the [Ca2+]i response in the cell types providing differential contributions to the whole-plant NaCl-induced [Ca2+]i signature. In epidermal cells, only one component of the NaCl-induced [Ca2+]i signature was discernible, a rapid rise of [Ca2+]i for approximately 20 to 30 s, peaking at a mean of 1,381.0 ± 66.6 nm (Fig. 3A). Subtracting the mean mechanical stimulation-induced increase derives a NaCl-specific [Ca2+]i transient with a mean peak of 735.0 ± 66.6 nm in epidermal pavement cells. A large transient NaCl-induced increase in [Ca2+]i was not detected in guard, trichome, and mesophyll cells but was detected from vascular cells (Fig. 3). The small NaCl-induced initial [Ca2+]i transients detected in guard, trichome, and mesophyll cells can be ascribed to mechanical stimulation (compare Fig. 2, B–D, and Fig. 3, B–D). In all cell types except the epidermal pavement cells, sustained increases in [Ca2+]i following NaCl stimulation were detected (Fig. 3). Vascular cells had a similar pattern of [Ca2+]i dynamics to previous reports from plants expressing CaMV35S::APOAEQUORIN (Knight et al., 1997; Kiegle et al., 2000; Tracy et al., 2008), a transient increase in [Ca2+]i that reached 778.0 ± 90.4 nm (527.8 ± 90.4 nm after correction for mechanical stimulation) followed by a gradual decrease over 150 s (Fig. 3E). The dynamics of the NaCl-induced [Ca2+]i signals from mesophyll and vasculature cells were more complex, with mesophyll cells reporting a sustained [Ca2+]i peaking 120 s after stimulation, whereas in the vasculature, there were prolonged relaxation kinetics following the initial transient (Fig. 3, C and E). However, for both mesophyll and vasculature, the mean average behavior obscures more complex dynamics reported by individual plants (Supplemental Figs. S12–S16). When the data from individual plants were analyzed, in both spongy mesophyll and vasculature cells, complex oscillatory [Ca2+]i signals were induced by NaCl (Supplemental Figs. S14 and S16).
H2O2 induced a characteristic biphasic increase in [Ca2+]i comprising a transient increase in [Ca2+]i ("spike") followed by a more prolonged elevation of [Ca2+]i peaking around 40 to 50 s after injection in the range of 200 nm for all tissues tested (Fig. 4; Supplemental Figs. S17–S21). However, the peak values and dynamics of the initial [Ca2+]i spike or transient (519.9 ± 58.0, 322.0 ± 77.9, 149.0 ± 31.5, 153.0 ± 4.4, and 200.8 ± 26.5 nm for epidermal, guard, mesophyll, trichome, and vasculature cells, respectively) were very similar to those obtained after mechanical stimulation. Therefore the H2O2-specific [Ca2+]i signal comprises a slow rise in [Ca2+]i followed by sustained elevation without cell-specific dynamics (Fig. 4).
All the plants tested of all the enhancer trap lines (10 out 10 for each specific cell type) reported cold shock-induced [Ca2+]i increases (Supplemental Figs. S22–S26), with a very large mean peak height compared with mechanical stimulation and NaCl, resulting in a peak reaching [Ca2+]i values of 1,242.3 ± 40.0, 1,168.3 ± 77.3, 688.3 ± 49.1, 709.7 ± 47.4, and 895.1 ± 42.8 nm for epidermal, guard, mesophyll, trichome, and vasculature cells, respectively (595.7 ± 40.0, 810.3 ± 77.3, 357.3 ± 49.1, 339.6 ± 47.4, and 644.9 ± 42.8 nm after mechanical stimulation correction; Fig. 5). There was no second component after cold stimulus in any of the cell lines analyzed.
Using GAL4 Transactivation of Aequorin to Measure [Ca2+]i in Specific Cell Types
We describe a suite of cell-specific aequorin reporter lines that permit the measurement of [Ca2+]i in leaves. The strong regulation by the GAL4 UAS drove sufficiently high aequorin expression to permit reproducible measurement of [Ca2+]i from relatively small populations of cells (e.g. trichomes or guard cells), cells that are rich in chlorophyll (spongy mesophyll), and tissues deep within the plant (vascular bundle). GAL4 is a yeast transcription factor with no orthologs in plants; therefore, GAL4 is unlikely to induce artifacts through the regulation of endogenous genes. This technical advance has permitted the measurement of cell- and stimulus-specific [Ca2+]i signals in Arabidopsis leaves.
Using GAL4 Transactivation of Aequorin to Measure Daily [Ca2+]i Rhythms in Specific Leaf Cell Types
A major motivation of this study was to determine the source of circadian oscillations of [Ca2+]i within the plant. It has been suggested that circadian oscillations of [Ca2+]cyt have the potential to be both an output and an input to the circadian oscillator (Johnson et al., 1995; Dodd et al., 2007). To understand the role of [Ca2+]cyt in the circadian network, it is necessary to determine specifically in which cells [Ca2+]cyt functions in the circadian clock. All plant cells are believed to have circadian rhythms that are cell autonomous (Dodd et al., 2004; Yakir et al., 2011). There is evidence of tissue-specific circadian behavior; for example, the vascular bundle circadian clock appears to have a different genetic architecture compared with other tissues (Para et al., 2007). We previously reported that in the timing of chlorophyll a/b binding protein expression1-1 mutant the circadian rhythms of [Ca2+]cyt become uncoupled from those of CHLOROPHYLL A/B BINDING PROTEIN2 expression, demonstrating the presence of at least two circadian oscillators running at different speeds (Xu et al., 2007). We speculated that these different circadian oscillators might be located in different cell types (Xu et al., 2007). Here, using GAL4 transactivation, we demonstrate that the predominant aequorin signal in the measurement of circadian oscillations of [Ca2+]i arises from the spongy mesophyll (Fig. 1A). This is consistent with our previous imaging of aequorin bioluminescence from plants constitutively expressing aequorin, which demonstrated that daily and circadian [Ca2+]cyt rhythms arise from leaf tissues (Love et al., 2004).
We were unable to detect circadian oscillations of [Ca2+]i in other leaf tissues. Our inability to detect circadian oscillations of [Ca2+]i from epidermal pavement cells, trichomes, guard cells, and the vascular bundle does not necessarily mean that circadian oscillations of [Ca2+]i are absent in those tissues. The dynamic range of circadian oscillations of [Ca2+]cyt is about 200 nm (Love et al., 2004); therefore, it is possible that we did not detect relatively low-amplitude changes in [Ca2+]i when aequorin was targeted to small populations of cells, but the signal-to-noise ratio was great enough when aequorin was targeted to the more populous spongy mesophyll cells. Nycthermeral oscillations of [Ca2+]i that occur in light and dark cycles have a higher amplitude of oscillations than the free-running circadian rhythm of [Ca2+]i that occurs in LL (Fig. 1A), and potentially nycthermeral [Ca2+]i oscillations were detected from aequorin targeted specifically to the trichomes, guard cells, epidermal pavement cells, and vascular bundle (Fig. 1, B–E). It appears, therefore, that daily oscillations of [Ca2+]i are common to all leaf cell types and that we were unable to detect low-amplitude circadian oscillations of [Ca2+]i from cell types other than the spongy mesophyll due to the low signal-to-noise ratio from aequorin when it was targeted to smaller populations of cells. A caveat to this conclusion that daily rhythms of [Ca2+]i can occur in all cell types is that only the spongy mesophyll cells reported a pattern of [Ca2+]i oscillations in light and dark cycles that are consistent with those reported by constitutively targeted aequorin (Love et al., 2004). It was previously suggested that the major bioluminescent signal from tobacco (Nicotiana plumbaginifolia) transformed with aequorin arises mostly from the epidermal layers (Wood et al., 2001); our data demonstrate that this is not the case for circadian rhythms of [Ca2+]i in Arabidopsis.
GAL4 Transactivation of Aequorin Identifies Cell- and Stimulus Type-Specific [Ca2+]i Signals
The role of [Ca2+]i in the response of Arabidopsis to stress signals, including mechanical stimulation, H2O2, NaCl, and cold, was first identified based on [Ca2+]cyt elevations detected in seedlings constitutively expressing aequorin under the control of the CaMV35S promoter (Knight et al., 1991, 1996, 1997). The canonical [Ca2+]cyt response measured in response to these stimuli in plants constitutively expressing aequorin in all tissues is a transient elevation immediately upon stimulation, to relatively high values (dependent on the strength and type of stimulus), followed by a rapid decline to intermediate values of [Ca2+]cyt and subsequently often a second slower elevation and decline of the [Ca2+]cyt signal, returning to resting values after a few minutes. The temporal dynamics of this "spike-shoulder" pattern of [Ca2+]cyt signal exhibits considerable variation dependent on the signal applied (McAinsh and Pittman, 2009). In root tissues, it is apparent that this whole-plant response masks more complex cell-specific behaviors (Kiegle et al., 2000), notably in the endodermis and pericycle of roots in response to NaCl. It is likely that summation of the behavior of many individual cells probably obscures the underlying oscillatory nature of NaCl-induced [Ca2+]cyt increases in root cells (Tracy et al., 2008).
GAL4 transactivation of aequorin to specific cell types has allowed partial deconvolution of the whole-plant [Ca2+]i signal to identify cell specificity and the dynamics of [Ca2+]i signals. We demonstrate that mechanical stimulation-induced [Ca2+]i signals are largely restricted to the epidermal pavement cells in the leaf (Fig. 2; Supplemental Figs. S7–S11). The epidermis being the location of the major mechanical stimulation-induced [Ca2+]i signals is intuitively appealing, since wind-, rain-, and animal-induced mechanical stimulation signals will first occur at the epidermis (Braam, 2005). In our study, mechanical stimulation was applied by an application of room-temperature water with minimal force; therefore, it is possible that mechanical stimulation-induced [Ca2+]i signals were not detected with the same intensity from tissues below the epidermis because the physical stimulus was attenuated during penetration through the leaf. Demonstration that epidermal pavement cells have a large mechanical stimulation-induced [Ca2+]i transient allowed post-data-capture correction of pavement epidermal cell [Ca2+]i signals to account for mechanical stimulation-induced signals when investigating the effects of other stimuli. For example, when the contribution to the [Ca2+]i dynamics made by mechanical stimulation is taken into account, all cells, including the epidermal pavement cells, had an essentially monophasic response to H2O2 (compare Figs. 2 and 4; Supplemental Figs. S17–S21).
The detection of H2O2-mediated [Ca2+]i increases in guard cells in E1728 (Fig. 4B) is consistent with the extensive literature linking plasma membrane NADPH oxidase-mediated H2O2 increases to abscisic acid-induced stomatal closure (Webb and Robertson, 2011). In guard cells, H2O2 increases are detected in the cytosol within 30 s of abscisic acid treatment and are thought to activate Ca2+ influx through plasma membrane hyperpolarization-activated Ca2+ channels (Pei et al., 2000). Chloroplast-derived H2O2 signals in response to high light, wounding, or infection with an incompatible hypersensitive response-inducing pathogen are localized specifically to the vascular bundle (Mullineaux et al., 2006). Similarly, the key high-light-induced antioxidant protein ASCORBATE PEROXIDASE2 is expressed specifically in the vascular bundle (Fryer et al., 2003). Further studies will be required to determine if the pathways that elevate [Ca2+]cyt in response to chloroplast-derived H2O2 signals in the vasculator are similar to or different from those that generate [Ca2+]cyt signals in the guard cell in response to plasma membrane-localized NADPH oxidase-derived H2O2 activity.
We previously suggested that the spike-shoulder pattern of [Ca2+]cyt increase often measured using constitutively targeted aequorin might not represent the underlying [Ca2+]cyt dynamics in single cells, due to the integration of potential cell-specific [Ca2+]cyt signals and/or out-of-phase oscillatory [Ca2+]cyt dynamics in single cells (Dodd et al., 2006). Here, we found evidence for stimulus- and cell type-specific [Ca2+]i dynamics using GAL4 transactivation of aequorin. For example, NaCl caused oscillatory [Ca2+]i signals specifically in spongy mesophyll and vascular cells (Supplemental Figs. S14 and S16). This is consistent with previous studies that found that NaCl caused oscillatory [Ca2+]cyt signals specifically in the endodermis and pericycle of roots (Kiegle et al., 2000) and that oscillatory [Ca2+]cyt dynamics are easier to resolve when measurements are made from smaller cell populations (Tracy et al., 2008). The NaCl-induced oscillatory [Ca2+]i signals in the vasculature and spongy mesophyll cells are indicative of a role in signal transduction and the regulation of NaCl-regulated downstream responses. By contrast, in the guard cell, NaCl appears to alter only the steady-state [Ca2+]i (Fig. 3B). The instantaneous nature of the change in steady-state [Ca2+]i in response to NaCl in the guard cell is suggestive of an alteration in plasma membrane potential as a consequence of the increased external NaCl concentration. Increasing NaCl concentration will cause Na+ influx through nonselective cation channels and could possibly result in blockage of inward rectifying K+ currents, which in turn will result in a more hyperpolarized plasma membrane potential due to reduced depolarizing influx of K+ (Véry et al., 1998). In other systems, the blockage of guard cell inward rectifying K+ currents by cytosolic Na+ has taken longer to develop than the nearly instantaneous effect on [Ca2+]i seen here (Véry et al., 1998); therefore, other mechanisms also might be at work.
For cold stimulation, there was consistency in the dynamics of response across cell types, similar to the dynamics described previously for whole plants, root-specific cell types, and guard cell populations (Knight et al., 1991, 1996; Kiegle et al., 2000; Dodd et al., 2006), suggesting that there are not cell-specific responses to cold in terms of Ca2+ signaling.
Here, we report the use of GAL4 transactivation of aequorin to analyze [Ca2+]i signaling in specific cell types, including those of the leaves. We identify a high degree of specialization in Ca2+ signaling networks within different cell types and also commonalities. Our data suggest commonality in the pathways by which cold, H2O2, and possibly daily timing signals are transduced within cells but identify cell-specific dynamics to [Ca2+]i signals induced by mechanical stimulation and NaCl. The library of GAL4-mediated aequorin transactivation lines with root- and now leaf/shoot cell-specific aequorin expression offers the opportunity to dissect in detail the role of Ca2+ signals in cell-specific stimulus-response coupling.
Plant Material and Growth Conditions
Seeds of the Arabidopsis (Arabidopsis thaliana) guard cell-specific GAL4 GFP enhancer trap line E1728 expressing YFPAPOAEQUORIN under the control of the GAL4 UAS promoter (Dodd et al., 2006) and vascular cell-specific KC274, trichome cell-specific KC380, epidermal cell-specific KC464, and spongy mesophyll cell-specific JR11-2 GAL4 GFP enhancer trap lines (Gardner et al., 2009) were used in this study. Seeds were surface sterilized by washing for 1 min in 100% (v/v) ethanol and then incubating them for 10 min in 50% (v/v) sodium hypochlorite followed by three washes with autoclaved distilled water. Seeds were sown on 0.5× Murashige and Skoog nutrient mixture dissolved in 0.8% agar (w/v), unless otherwise stated. Germination was synchronized by stratification at 4°C for 2 d in the dark. Seedlings were transferred to MLR30 growth chambers (Sanyo) and grown in LD at 19°C; the light intensity was 50 µmol m−2 s−1.
Generation and Selection of Lines Expressing Aequorin in Specific Leaf Cells
KC274, KC380, KC464, and JR11-2 were transformed with pBINYFPAEQ containing the YFPAPOEQUORIN fusion downstream from a GAL4 UAS (Kiegle et al., 2000) as described by Dodd et al. (2006). Homozygous transformants were selected by growth on medium containing 40 µg mL−1 hygromycin. Lines were selected for high total aequorin activity by destructive measurement of total aequorin activity in leaf samples of T1 and T2 seedlings by discharging all available aequorin in the presence of 1 m CaCl2 dissolved in 10% (v/v) ethanol (discharge solution) and measuring aequorin bioluminescence in a multifunctional microplate reader (FluoStar OPTIMA; BMG LabTech). Imaging of GFP and YFP to confirm the colocalization of GFP and YFPAPOAEQUORIN was performed as described by Kiegle et al. (2000), and GFP and YFP were collected using emission windows of 495 to 525 nm and 595 to 650 nm, respectively.
Measurement of [Ca2+]i and Statistical Analysis
Imaging of circadian oscillations of [Ca2+]i and the growth conditions, light regimes, and entrainment regime for circadian measurements were as described by Dodd et al. (2007). Circadian rhythms of aequorin luminescence, corresponding to circadian rhythms in [Ca2+]i, were analyzed using the software based on the fast Fourier transform-nonlinear least-squares method described by Plautz et al. (1997) using the Biological Rhythms Analysis Software System.
Luminometry of changes in [Ca2+]i in response to mechanical stimulation, cold, NaCl, and H2O2 and subsequent calibration of bioluminescence to estimate [Ca2+]i were measured as follows. In order to reconstitute aequorin, 14-d-old seedlings transformed with GAL4 UAS:YFPAPOAEQUORIN were incubated overnight in the dark at room temperature in 500 µL of 20 µm coelenterazine free base (Nanolight) dissolved in distilled water within a luminometer tube (51 mm long × 12 mm diameter; Sarstedt). Bioluminescence was measured using a photon-counting luminometer (photomultiplier tube 9899A) cooled to −20°C with a FACT50 housing (Electron Tubes). Injections were performed from a 1-mL light-tight syringe attached to a 75-mm needle inserted into a light-tight port in the luminometer sample housing at a distance of 40 mm approximately over the plants. Response to cold was determined by injecting 1 mL of 4°C distilled water onto the plants described above. Response to NaCl and H2O2 was determined by injecting 1 mL of the solutions onto the plants to reach final concentrations of 200 and 3.3 mm, respectively. The injection of 1 mL of room-temperature distilled water was used as a touch response control for all the treatments. All the stimuli injections were performed at 30 s over a 5-s interval followed after 300 s by the injection of 1 mL of discharge solution. Measurements were made until the detected luminescence reached 10% of the first peak after discharge injection. [Ca2+]i levels were determined according to Fricker et al. (1999).
Nycthemeral and circadian [Ca2+]i oscillations were determined in four independent experiments with at least four independent replicates in each; mean data for one of the experiments are reported. [Ca2+]i levels in response to stimulation are means of two independent experiments with at least 10 independent replicates per treatment and line.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Localization of GFP and YFPAPOAEQUORIN in the vascular cells of the KC274 enhancer trap line.
Supplemental Figure S2. Localization of GFP and YFPAPOAEQUORIN in the pavement epidermal cells of the KC464 enhancer trap line.
Supplemental Figure S3. Localization of GFP and YFPAPOAEQUORIN in the spongy mesophyll cells of the JR11-2 enhancer trap line.
Supplemental Figure S4. Localization of GFP and YFPAPOAEQUORIN in the trichome cells of the KC380 enhancer trap line.
Supplemental Figure S5. Different spatial localization of the GFP and YFPAPOAEQUORIN fluorescence signals in the cells.
Supplemental Figure S6. Analysis of the circadian [Ca2+]i oscillations in the spongy mesophyll cells of the JR11-2 enhancer trap line.
Supplemental Figure S7. [Ca2+]i response to mechanical stimulation in pavement epidermal cells.
Supplemental Figure S8. [Ca2+]i response to mechanical stimulation in guard cells.
Supplemental Figure S9. [Ca2+]i response to mechanical stimulation in spongy mesophyll cells.
Supplemental Figure S10. [Ca2+]i response to mechanical stimulation in trichome cells.
Supplemental Figure S11. [Ca2+]i response to mechanical stimulation in vascular cells.
Supplemental Figure S12. [Ca2+]i response to salt stress in pavement epidermal cells.
Supplemental Figure S13. [Ca2+]i response to salt stress in guard cells.
Supplemental Figure S14. [Ca2+]i response to salt stress in spongy mesophyll cells.
Supplemental Figure S15. [Ca2+]i response to salt stress in trichome cells.
Supplemental Figure S16. [Ca2+]i response to salt stress in vascular cells.
Supplemental Figure S17. [Ca2+]i response to H2O2 in pavement epidermal cells.
Supplemental Figure S18. [Ca2+]i response to H2O2 in guard cells.
Supplemental Figure S19. [Ca2+]i response to H2O2 in spongy mesophyll cells.
Supplemental Figure S20. [Ca2+]i response to H2O2 in trichome cells.
Supplemental Figure S21. [Ca2+]i response to H2O2 in vascular cells.
Supplemental Figure S22. [Ca2+]i response to cold stress in pavement epidermal cells.
Supplemental Figure S23. [Ca2+]i response to cold stress in guard cells.
Supplemental Figure S24. [Ca2+]i response to cold stress in spongy mesophyll cells.
Supplemental Figure S25. [Ca2+]i response to cold stress in trichome cells.
Supplemental Figure S26. [Ca2+]i response to cold stress in vascular cells.
Supplemental Table S1. Quantification of total AEQUORIN in light/dark cycles.
We are grateful to Dr. Fernán Federici for advice on confocal microscopy.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alex A.R. Webb (aarw2{at}cam.ac.uk).
↵1 This work was supported by the European Union, Marie Curie Intra European Fellowship (grant to M.C.M.).
↵[OPEN] Articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- [Ca2+]i
- intracellular concentration of Ca2+
- [Ca2+]cyt
- cytosolic-free Ca2+
- upstream activation sequence
- hydrogen peroxide
- cADPR
- cyclic ADP ribose
- yellow fluorescent protein fused to apoaequorin
- yellow fluorescent protein
- 12-h-light and 12-h-dark cycles
- constant light
- Received June 6, 2013.
- Accepted September 11, 2013.
- Published September 11, 2013.


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