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Scientists accused of carrying out research that favours seed companies 0
The European Union's stringent laws on maximum residue levels could threaten agricultural exports from Kenya and Uganda. Maximum residue levels (MRLs) are the upper legal levels of pesticide residues in food or feed. MRLs are meant to ensure the lowest possible consumer exposure.
In 2010 the EU became jittery about MRLs in Kenya's fresh produce, although the country earned Ksh43.5 billion ($427 million) from horticulture exports to the EU in 2012.
Since 2000, Uganda's exports of organic produce have risen both in volume and in value. Between 2004 and 2012, earnings from organic agriculture exports rose from $6.2 million to $28.4 million.
With organic farming growing at nearly 40 per cent per year, and with about 200,000 certified organic agriculture farmers, Uganda risks losing its premium market, because of stringent MRL rules. An estimated 185,000 hectares in Uganda are under organic farming.
Canadian professor Matthew A Schnurr, in a study on GMOs in Uganda, showed how scientists from developing countries are being co-opted into studies to help seed multinationals make money and in the process the livelihoods of communities risk destruction.
Prof Schnurr's study quotes fears by activists that growing GMOs will undermine the livelihoods of smallholder farmers by supplanting their ecologically resilient seeds.
The study, titled Biotechnology and bio-hegemony in Uganda: Unravelling the social relations underpinning the promotion of genetically modified crops into new African markets, was based on over 70 interviews with research scientists, policy experts, lobbyists, and promotional organisations. It was conducted between 2009 and 2012.
Prof Schnurr details the promotion of GMO cotton. Traditionally, cotton in Uganda is a popular cash crop grown on mixed farms by smallholder farmers. At the time of Schnurr's research, Monsanto's Bt cotton was undergoing trials in the country.
Continue reading on The East African
by Dorothy Kweyu
Photo Credits TEA Graphic


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Welcome to Cleddens Early Learning and Childcare Centre, where we provide a safe, stimulating and happy care and learning environment for children up to the age of five years.
We offer children opportunities for learning through play in a new and vibrant environment, both indoors and outdoors.
There are areas for active play, creativity and learning and comfortable places for relaxation to ensure children have an appropriate pace throughout the day.
At Cleddens, we have developed a warm, friendly, comfortable environment for all our children and families.
We hope you find the information available on our website helpful. We have photo galleries displaying some of the learning taking place throughout the Centre and how it links to the Pre Birth to Three and Curriculum for Excellence documents. You will find news about current and future events, such as holiday closures and parent's evenings. You can view our latest standards and quality report and the Centre's handbook. We have set up a link for you to view our most recent Care Inspectorate report. Please use the links available to browse through the website.
Updates regularly!
Leona Stewart
Head of Centre

subset: commoncrawl


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Learn the tools to Balance Your Hormones, Replenish Your Body and Feel Your Best!
Get the support, information, tools, inspiration, and accountability you need to powerfully transform your health from deep down. Together we will use food, herbs, nutrients and lifestyle to help balance hormones, reduce inflammation, and nourish our entire body.
Don't miss this awesome opportunity to maximize your health and come away empowered to age your best as your own expert! Things we do today affect our health, hormones, brain function, mood, immune system, digestion, and energy decades down the line.
Let's do this, women!
These grass root changes will impact sleep, skin issues, libido, energy levels, headaches, immune problems, anxiety, depression, pms symptoms, moodiness, menopause symptoms, sore breasts, digestive problems, joint pain, and more!
Janel offers an incredibly unique and rich angle as a certified holistic nutritionist, a Functional Medicine Nutritionist, a Doctor of natural medicine, an Ayurvedic yoga therapist, Family Herbalist, and someone who has worked with women specifically for decades.
There is endless information out there and it can feel overwhelming to know what the best approach for YOU is. Let me help by teaching you how to listen to YOUR OWN BODY. We will learn how to listen to what our body is telling us, body literacy.
Join the Awesomeness and feel your best!!!
The Plan-
We will meet 4 times in person and 2 times online (with the option of joining online each week but it is way more fun to meet in person especially in my peaceful and zen office to share tea and chat).
Receive your materials on January 1st to have plenty of time to plan and organize. Meet each Monday evening at 7pm for 5 additional weeks online or in person starting on January 6th to learn what and why. Ask questions and learn about how our environment and food impacts how we function and how we age. Gain empowering ways to think into your own health so you can take charge for decades to come. Learn about Root Cause and Functional approach to wellness.
We will use herbs, supplements, food, and lifestyle to reboot and replenish our body. We will be learning a ton about hormones and how they work together and impact our life. Blood sugar, adrenals, thyroid, sex hormones, neurotransmitters, and more.
Be part of our Facebook group to stay connected, share ideas, struggles and, victories.
Learn how to get various optional labs of your choice to help see 'what's going on in there' including serum (blood) labs and hormone (salivary) labs.
Join us and invest in YOU!
My goal is to empower you to understand your body and to support you with all the information and tools you need. This is NOT a cookie cutter program but instead a unique opportunity to learn why and how, not just what to do. You also have unique access to Janel for personal questions and navigating.
All of this for $230 – Early bird price $199 (sign up before December 20th)
Space is limited so we can keep the group intimate
Schedule- Receive info online on January 1st to start getting organized, Meet on January 6th and 13th at my office at 7pm together, Jan 20th meet online together, Jan 27th and Feb 3rd are in my office. You can video call in if needed. Office is located in Truckee by the airport. Meetings will be 7pm-815/830pm.
Feel free to email Janel for more information firstname.lastname@example.com


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The Repository Services section on the Admin Console contains pages for setting the Activities feed, the clustering tool, setting which workflow engine is in use, enabling replication, enabling and setting the search service, and controlling the Subscription and Transformation services.
- Activities Feed Activities Feed emails are sent from Alfresco to all users, summarizing the activities they see in their My Activities dashlet. Users will not see these email unless the Activities Feed is enabled. Emails include activities in all sites they are a member of, and by people they are following. You can set the frequency with which these emails are sent, the maximum number of activities they contain, and the maximum age of the activities.
- Admin Console: Repository server clustering This topic describes how to manage members of a cluster using the Admin Console.
- Admin Console: Enabling workflow process engines Alfresco workflows run on an embedded Activiti workflow engine.
- Admin Console: Working with the replication service The Replication Service page in Admin Console displays the settings to enable or disable the replication service and to control permissions.
- Working with the Search Service The Search Service page in Admin Console enables you to manage and monitor the search service you want to use in Alfresco.
- Admin Console: Enabling the subscription service The Subscriptions page allows you to enable or disable the Follow feature for users to follow each other in Share. Alfresco users can keep track of other users activities by choosing to follow them. When users are being followed, the person(s) following them will receive activity notifications. The Subscription Service is the underlying service used to manage and generate activity notifications. You can use this page to enable/disable the Subscription Service and the follow feature on a system wide basis.
- Admin Console: Transformation services Transformation services let you convert content between different file formats. The JODConverter installation (part of OpenOffice.org) is used by Alfresco for document transformations, such as a text file to PDF. The ImageMagick and PDF2SWF installations (part of SWFTools) are used for image transformations and transformations to Flash respectively, and it is recommended that you do not change their settings.
Parent topic: Admin Console Quick Guide


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The Bachelorette is back tomorrow, and that means it's time for lots of wine, lots of drama, and lots of Chris Harrison! This season, we will watch Rachel search through 31 contestants to find her fiancé – yes, you read that right! She revealed that she DID find love and is currently engaged 🙂 let's see if it will last… But until then, have fun with this Bachelorette Bracket so you can compete with family and friends as she searches for love!!!
Click the link below to download the file.Rachel Bachelorette Bracket


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When I mentioned Air Buns, Monica said she hadn't ever heard about them before.
I had just made them a few months ago....So Monica, here is my handed down recipe:
2 TBLS Yeast or 2 pkgs:
Soak in 2 cups lukewarm water with 1 tsp. Sugar
3-1/2 cups Lukewarm water:
Dissolve 1/2 cup Sugar
1 tsp. salt
1/2 cup shortening
Add yeast mixture and 1 tbls. vinegar and add 10 cups flour
1. Let rise for 2 hours in buttered bowl.
2. Kneed down and rise for 1 more hour.
3. Kneed down and put in pans (I roll the dough in the palms of my hand to form a small bun) and let rise for 2 more hours.
Bake at 370-400 for 15 - 20 minutes.
Makes about 36 Buns.
These are so soft, they melt in your mouth.
xo Deb


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- American Society of Plant Biologists
Ethylene regulates multiple aspects of plant growth and development in dicotyledonous plants; however, its roles in monocotyledonous plants are poorly known. Here, we characterized a subfamily II ethylene receptor, ETHYLENE RESPONSE2 (ETR2), in rice (Oryza sativa). The ETR2 receptor with a diverged His kinase domain is a Ser/Thr kinase, but not a His kinase, and can phosphorylate its receiver domain. Mutation of the N box of the kinase domain abolished the kinase activity of ETR2. Overexpression of ETR2 in transgenic rice plants reduced ethylene sensitivity and delayed floral transition. Conversely, RNA interference (RNAi) plants exhibited early flowering and the ETR2 T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering. The effective panicles and seed-setting rate were reduced in the ETR2-overexpressing plants, while thousand-seed weight was substantially enhanced in both the ETR2-RNAi plants and the etr2 mutant compared with controls. Starch granules accumulated in the internodes of the ETR2-overexpressing plants, but not in the etr2 mutant. The GIGANTEA and TERMINAL FLOWER1/CENTRORADIALIS homolog (RCN1) that cause delayed flowering were upregulated in ETR2-overexpressing plants but downregulated in the etr2 mutant. Conversely, the α-amylase gene RAmy3D was suppressed in ETR2-overexpressing plants but enhanced in the etr2 mutant. Thus, ETR2 may delay flowering and cause starch accumulation in stems by regulating downstream genes.
The gaseous plant hormone ethylene plays important roles in multiple aspects of plant growth and development, including seed germination, hypocotyl elongation, root hair initiation, leaf and flower senescence, fruit ripening, and organ abscission (Abeles et al., 1992). It is also involved in plant responses to biotic and abiotic stresses, such as pathogen attack, wounding, hypoxia, and drought stress (Morgan and Drew, 1997). However, the role of ethylene in plants appears to be multifunctional in terms of growth stimulation and disease resistance (Pierik et al., 2006; van Loon et al., 2006). Through analysis of a series of ethylene response mutants of Arabidopsis thaliana, the backbone of the ethylene signaling pathway has been established and the major components have been identified (Bleecker and Kende, 2000; Wang et al., 2002; Guo and Ecker, 2004; Chen et al., 2005; De Paepe and Van Der Straeten, 2005). These components include the ethylene receptors, the protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), the membrane protein ETHYLENE-INSENSTIVE2 (EIN2), and the downstream transcription factors EIN3, EIN3-LIKE1 (EIL1), and ETHYLENE RESPONSE FACTOR1 (ERF1). EIN3 is quickly degraded in the absence of ethylene through an ubiquitin/proteasome pathway mediated by two F box proteins, EIN3 BINDING F-BOX1 (EBF1) and EBF2 (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). EBF1 and EBF2 mRNA levels can be regulated indirectly by exoribonuclease XRN4/EIN5 (Olmedo et al., 2006; Potuschak et al., 2006). EIN2 stability is also disrupted by two F box proteins, EIN2 TARGETING PROTEIN1 (ETP1) and ETP2 (Qiao et al., 2009). New components of the ethylene signaling pathway have been proposed, and these components may either fill the gaps in the established ethylene signaling pathway or represent new branches of this pathway (Alonso et al., 2003; Larsen and Cancel, 2003; Moshkov et al., 2003; Quaked et al., 2003; Hass et al., 2004; Yoo et al., 2008).
Ethylene receptors are the first component in ethylene signaling and can be classified into two subfamilies, subfamily I and subfamily II. In Arabidopsis, subfamily I has two members, whereas subfamily II has three members (Klee, 2004; Chen et al., 2005; Chen and Zhang, 2006; Wang et al., 2006; Hall et al., 2007). All of the Arabidopsis and tomato (Solanum lycopersicum) receptors can bind ethylene, and the important regions for ethylene binding of Arabidopsis ETR1 have been identified in the transmembrane domain (Schaller and Bleecker, 1995; Rodriguez et al., 1999; O'Malley et al., 2005). Ethylene binding activity is confined to land plants, Chara, and a group of cyanobacteria (Wang et al., 2006). Ethylene negatively regulates the functions of ethylene receptors (Hua and Meyerowitz, 1998). Ethylene receptors appear to be localized in membrane systems, such as the endoplasmic reticulum (Chen et al., 2002, 2007; Xie et al., 2003; Ma et al., 2006; Dong et al., 2008). ETR1, a subfamily I member, has histidine kinase activity, whereas tobacco (Nicotiana tabacum) subfamily II members NTHK1 (Nicotiana tabacum histidine kinase1) and NTHK2, and other Arabidopsis receptors, have Ser/Thr kinase activities (Xie et al., 2003; Moussatche and Klee, 2004; Zhang et al., 2004). The NTHK2 and ETHYLENE RESPONSE SENSOR1 (ERS1) receptors also have histidine kinase activities in the presence of Ca2+ and Mn2+, respectively (Moussatche and Klee, 2004; Zhang et al., 2004). The ETR1 kinase domain has substantial roles in the regulation of ethylene responses and seedling growth recovery. However, whereas His kinase activity has little or no role in response to ethylene, it has a significant role in growth recovery (Wang et al., 2003; Binder et al., 2004; Qu and Schaller, 2004). Signal transmission by the ETR1 N terminus is dependent on the other subfamily I members (Xie et al., 2006), and the ETR1 receptor function can be regulated by a membrane protein, REVERSION-TO-ETHYLENE SENSITIVITY1, and a copper transporter, RESPONSIVE-TO-ANTAGONIST1 Hirayama et al., 1999; Resnick et al., 2006, 2008; Zhou et al., 2007; Dong et al., 2008). Recently, two studies have shown that ethylene receptor degradation represents a mechanism for regulating ethylene responses (Chen et al., 2007; Kevany et al., 2007). Formation of ethylene receptor dimers and/or higher-order receptor complexes also regulates ethylene signal output (Gao et al., 2008).
Ethylene receptor genes can be induced by various abiotic stresses (Zhang et al., 1999a, 2001a, 2001b). Overexpression of the tobacco ethylene receptor NTHK1 results in large rosettes or seedlings, reduced ethylene sensitivity, and increased salt sensitivity in transgenic plants (Xie et al., 2002; Cao et al., 2006; Zhou et al., 2006a). NTHK1 affects salt stress responses by alternating the expression pattern of the gene encoding the NAC transcription factor or of other relevant genes (He et al., 2004, 2005; Cao et al., 2006, 2007; Zhou et al., 2006a). Ethylene receptors also regulate plant defense responses (Knoester et al., 1998; Ciardi et al., 2000; O'Donnell et al., 2003). In addition to stress responses, ethylene receptors are highly expressed in reproductive organs and may be involved in reproductive development (Hua et al., 1998; Sato-Nara et al., 1999; Tieman et al., 2000; Yamasaki et al., 2000; Zhang et al., 2001a, 2001b; Klee, 2002, 2004; Xie et al., 2002; Rieu et al., 2003; Zhou et al., 2006a).
In monocots, ethylene receptor genes have been identified in wheat (Triticum aestivum) and maize (Zea mays) (Ma and Wang, 2003; Gallie and Young, 2004). In rice (Oryza sativa), ethylene receptor genes, EIN2-like and EIN3-like, have also been cloned, and the functions of the EIN2-like and EIL1 genes have been analyzed (Cao et al., 2003; Cao, 2004; Jun et al., 2004; Watanabe et al., 2004; Yau et al., 2004; Mao et al., 2006). The rice ethylene receptor gene family has five members, including Protein Kinase1 (PK1)/ETR2/ETHYLENE RESPONSE2 like1 (ERL1), PK2/ETR3, PK3/ETR4, ERS1, and ERS2. ERS1 and ERS2 belong to subfamily I, whereas the other three belong to subfamily II. Expression of these genes can be affected by various stresses and hormone treatments (Cao et al., 2003; Cao, 2004; Watanabe et al., 2004; Yau et al., 2004). Alternative spliced forms of these receptors have also been reported after analysis of the whole rice genome (Pareek et al., 2006). Although rice ethylene receptor genes have been identified, the biochemical properties of the proteins are not known. The function of the receptors in plant growth and development remains unclear.
In this study, we further characterized the PK1/ETR2/ERL1 gene from rice (Cao, 2004; Watanabe et al., 2004; Yau et al., 2004). For simplicity, the name ETR2 was chosen to represent the three names of this ethylene receptor. The kinase activity of the ETR2 protein was investigated. The roles of this receptor protein in rice plant growth and reproductive transition were analyzed using overexpression and RNA interference (RNAi) approaches. The ETR2 T-DNA insertion mutant, etr2, was further identified and studied. Our results indicate that ETR2 has Ser/Thr kinase activity and can phosphorylate its receiver domain. In rice, ETR2 reduces ethylene sensitivity, delays the transition from the vegetative stage to the floral stage, and affects starch accumulation.
Structural Features of the ETR2 Ethylene Receptor in Rice
Based on the SMART analysis and comparison with ethylene receptors from Arabidopsis, tobacco, and maize, the putative domains in rice ETR2 were predicted. ETR2 contains four hydrophobic regions, a GAF domain (amino acid positions 190 to 349), a putative kinase domain (amino acid positions 375 to 615), and a receiver domain (amino acid positions 640 to 756) (Figure 1A ). The first hydrophobic region (amino acid positions 1 to 26) probably represents a signal peptide, whereas the other three hydrophobic regions (amino acid positions 58 to 78, 86 to 108, and 115 to 137) represent transmembrane domains. The putative kinase domain consists of two subdomains, an H-containing domain (HIS, amino acid positions 375 to 440) and an ATP binding domain (ATP, amino acid positions 487 to 615) (Figures 1A and 1B). HIS is a dimerization and phosphoacceptor subdomain in bacterial histidine kinases, whereas an ATP binding subdomain is found in several ATP binding proteins, such as histidine kinase, DNA gyrase B, topoisomerases, and heat shock protein HSP90 (Parkinson and Kofoid, 1992; Robinson et al., 2000). The receiver domain (RD; amino acid positions 640 to 756) contains a putative phosphoacceptor site (D692) that is normally phosphorylated by a histidine kinase in bacteria (Parkinson and Kofoid, 1992).
The rice ETR2 kinase domain was compared with those from other ethylene receptors or homologs (Figure 1B). In the H motif of the HIS domain, rice ETR2 had a D385, which was different from the phosphorylation site H353 in ETR1 of Arabidopsis (Gamble et al., 1998) or residues in other proteins in the corresponding region. However, maize ETR2 had a D400 residue at the corresponding position. Seven residues downstream of the D385 in rice ETR2, an H392 residue was identified, which was highly conserved at the corresponding position among all the proteins compared (Figure 1B). However, this residue was not required for the kinase activity observed in NTHK2, Arabidopsis ETR2, or EIN4 (Moussatche and Klee, 2004; Zhang et al., 2004). In the ATP subdomain of Arabidopsis ETR1 and other histidine kinases, four motifs, including the N, G1, F, and G2 boxes, have been identified (Robinson et al., 2000; Parkinson and Kofoid, 1992; Chang et al., 1993). However, in rice ETR2 and in the other subfamily II members compared, the G1, F, and G2 boxes were more divergent than those in Arabidopsis ETR1, whereas the N box and the nearby residues were relatively conserved (Figure 1B). The residues G487, D488, E489, R491, F493, M499, and G501 in rice ETR2 may play roles in the regulation of receptor activity.
Kinase Activity of the ETR2 Protein
Ethylene receptors in dicotyledonous plants have been found to possess kinase activities; Arabidopsis ETR1 has His kinase activity, whereas the other receptors tested mainly have Ser/Thr kinase activities. Arabidopsis ERS1 and tobacco NTHK2 also have His kinase activities in the presence of Mn2+ and Ca2+, respectively (Gamble et al., 1998; Xie et al., 2003; Moussatche and Klee, 2004; Zhang et al., 2004). Here, we investigated the kinase activity of ETR2 from monocotyledonous rice plants to establish if they have different biochemical properties from those found in dicotyledonous plants. Four truncated versions of ETR2 were expressed as glutathione S-transferase (GST) fusions in bacterial cells (Figures 2A and 2B ). The wild-type fusion contained the GAF domain, the HIS domain, and the ATP domain. The N protein had five mutations (G487A, E489Q, R491Q, F493A, and G501A) in the N box of the wild-type protein. The ΔATP fusion contained the GAF domain and the HIS domain. The RD fusion only contained the receiver domain. These purified proteins displayed apparent molecular masses consistent with their corresponding predicted molecular masses when analyzed by SDS-PAGE (Figure 2B). It should be noted that in the wild-type, N, and ΔATP proteins, a similar protein band of ∼50 kD was usually observed (Figure 2B). This band represented a degradation product of their original proteins.
The wild-type protein was subjected to a phosphorylation assay through incubation with [γ-32P]ATP. In the presence of Mn2+, 32P was incorporated into the wild-type fusion protein (Figure 2C). However, in the presence of Mg2+ and Ca2+, 32P incorporation was not found. This result indicates that the wild-type autophosphorylation requires Mn2+ as a cofactor. Other truncated ETR2 proteins were also compared for their phosphorylation ability in the presence of Mn2+. Only the wild type had the ability to autophosphorylate, whereas the ΔATP, RD, and GST did not have such an ability (Figure 2D). This result indicates that the ATP domain is required for the wild-type phosphorylation. To examine which classes of residues are phosphorylated, the phosphorylated wild-type protein was transferred onto polyvinylidene difluoride (PVDF) membrane and treated with acid or base to test its hydrolytic stability. The wild-type protein was stable under acidic treatment but sensitive to base treatment, and overnight treatment with NaOH abolished the phosphorylation level completely (Figure 2E). These results imply that the phosphorylated wild type contained phosphoserine, phosphothreonine, or phosphotyrosine, but not phosphohistidine. The phosphorylated wild-type protein was also subjected to phosphoamino acid analysis by two-dimensional thin-layer chromatograph electrophoresis, which revealed that the phosphorylated ETR2 protein contained phosphoserine and phosphotheronine (Figure 2F). The N box of the wild type was further mutated, and the mutant version N did not have any 32P incorporation, suggesting that the N box is important for the phosphorylation and that kinase activity is intrinsic to the wild type (Figure 2G).
The receiver domain of two-component proteins in bacteria and yeast usually accepts the phosphate group from histidine kinase (Parkinson and Kofoid, 1992; Wurgler-Murphy and Saito, 1997; Robinson et al., 2000). Thus, we investigated if the wild type can phosphorylate RD and the universal substrate myelin basic protein (MBP) in vitro. MBP has been used as a substrate to examine kinase activities of protein kinases and ethylene receptors (Cicirelli et al., 1988; Xie et al., 2003; Zhang et al., 2004). Our results indicate that the wild-type protein can phosphorylate both RD and MBP (Figure 2G). However, the mutant protein N did not phosphorylate the two proteins, suggesting that the N box is required for substrate phosphorylation. The wild type was also incubated with ΔATP in the presence of [γ-32P]ATP to test if the wild type could phosphorylate other domains. The wild type could not phosphorylate ΔATP, suggesting that the phosphorylated residues in the wild type are likely in the ATP subdomain and not in the GAF or HIS subdomains (Figure 2G). Neither the wild type nor N could phosphorylate GST, and GST itself did not have any 32P incorporation (Figure 2G).
Overexpression of ETR2 Reduces Ethylene Sensitivity in Transgenic Rice
To study the roles of ETR2 in rice growth and development, we made two constructs, pBIN438-OsETR2 and pZH01-OsETR2-RNAi, and transferred them into rice (TP309) via Agrobacterium tumefaciens–mediated transformation. The pBIN438-OsETR2 construct harbored the full-length genomic gene of ETR2 under the control of the double 35S promoter with an Ω enhancer. The pZH01-OsETR2-RNAi construct contained two inverted repeats of the partial ETR2 gene and was used for inhibiting ETR2 gene expression (Figure 3A ). Forty-one T0 overexpression lines and 20 T0 RNAi lines were obtained. From these, T2 or T3 homozygous lines (8-2, 12-1, 17-1, and 56-4, with one to two insertions) with higher expression of the ETR2 gene and two homozygous independent RNAi lines (41-4 and 66-1, with one and two insertions, respectively) were used for further analysis (Figure 3B). The two RNAi lines had reduced, but not eliminated, expression of ETR2. Generally, ETR2 was expressed in rice plants at a very low level (Figure 3B).
Since ETR2 is a homolog of ethylene receptors, we hypothesized that overexpression or reduction in ETR2 levels would alter ethylene sensitivity in the transgenic plants. We analyzed the response of the transgenic seedlings upon 1-aminocyclopropane-1-caroxylic acid (ACC; ethylene biosynthesis precursor) treatment. Root growth of etiolated wild-type rice seedlings (wild type) was inhibited in response to increasing concentrations of ACC, whereas shoot growth was not significantly affected (Figure 3C). In the four lines overexpressing ETR2, root lengths were only slightly reduced upon ACC treatment compared with the corresponding wild-type controls (Figure 3C, top and bottom left panels). In the two RNAi lines with reduced ETR2 expression, root lengths were similar to that of the wild-type plants in response to ACC treatment (Figure 3C, top and bottom right panels). Shoot lengths of the overexpression lines and the RNAi lines were not significantly different from those of the wild-type controls (Figure 3C, top panels). These results indicate that rice roots are more sensitive to ACC treatment than are rice shoots and that overexpression of ETR2 conferred reduced ethylene sensitivity in transgenic rice plants. Partial reduction of the ETR2 level had limited effects on ethylene sensitivity.
Ethylene can promote the growth of rice coleoptiles (Ku et al., 1970). We examined the sensitivity of the transgenic rice plants to ethylene by testing for the promotion of coleoptile growth. At higher ethylene concentrations (50 ppm), the transgenic plants overexpressing ETR2 exhibited significantly shorter coleoptiles than wild-type plants (Figure 3D), indicating that overexpression of ETR2 conferred reduced ethylene sensitivity in the regulation of coleoptile growth. The RNAi plants showed no significant changes in coleoptile length in comparison with the wild-type plants.
Phenotypes of Transgenic Rice Plants with Altered ETR2 Expression
We investigated the phenotypic changes in transgenic plants exhibiting increased or decreased ETR2 expression. Four lines overexpressing ETR2 had shorter seedlings than control plants after 5 d of germination (Figure 4A ) or 50 d of growth in the field (Figure 4B). However, seedlings from the two RNAi lines exhibited no significant difference in seedling length compared with control seedlings under both conditions. These results suggest that ETR2 has an inhibitory effect on rice seedling elongation.
Observations of growth of the transgenic rice plants in field conditions revealed that ETR2-overexpressing lines had delayed flowering. We thus examined panicle development in these transgenic plants more closely. At the stage of panicle development, the two ETR2-RNAi plants had more well-developed panicles than the wild-type plants. However, the four lines overexpressing ETR2 had only one to two young panicles developed (Figure 4C). Heading dates of field-grown plants were also recorded and three ETR2-overexpressing lines (8-2, 17-1, and 56-4) had a heading date about 1 week later than control plants, whereas line 12-1 showed a heading time nearly 2 weeks later than controls. Two RNAi lines, 41-4 and 66-1, exhibited a heading time 3 to 4 d earlier than controls (Figure 4D). These results indicate that ETR2 may delay plant transition from the vegetative stage to the reproductive stage.
The phenotype of field-grown plants at the mature stage was further observed. Control plants and the two RNAi lines all had downward panicles, whereas ETR2-overexpressing lines had erect panicles (Figure 4E). In overexpressing line 12-1, panicles were barely emerged, and leaves were still very green. These results suggest that ETR2 delays the development of plants to maturity. The length of each internode was also measured when plants stopped elongating after maturation. Two RNAi lines were not different from controls in the length of all four internodes (Figure 4F). However, four ETR2-overexpressing lines had shorter internodes compared with control plants (Figure 4F). The overall height of three ETR2-overexpressing lines (12-1, 17-1, and 56-4) was shorter than controls. The two RNAi lines were of similar heights as the control line (Figure 4G). These results suggest that ETR2 suppresses internode elongation and delays panicle development in rice.
Yield-Related Traits of the Transgenic Rice Plants with Altered ETR2 Expression
At maturity, the control and the two RNAi lines had yellow and drooping panicles, whereas four ETR2-expressing lines had yellow-green or green but upright panicles (Figure 5A ). After harvest, the ratio of effective tillers (tillers bearing panicles) was decreased in the ETR2-overexpressing line 12-1 (Figure 5B). However, this ratio was only slightly reduced or not significantly changed in other ETR2-overexpressing lines or in the two RNAi lines. The effective panicles (panicle having at least one full-sized grain) and the seed-setting rates were also evaluated. Both parameters were reduced in all four ETR2-overexpressing lines, whereas the two RNAi lines had no significant difference compared with the controls (Figure 5B). These results indicate that ETR2 significantly affects panicle development and seed set.
Since ETR2 delays floral transition of rice plants, this may cause the low seed-setting rate of ETR2-overexpressing lines. Alternatively, ETR2 may extend the maturation process and grain-filling process, leading to low seed-set in the normal harvest season. To distinguish between these two possibilities, control plants (WT2) were further grown 18 d later than normal control plants (WT1) and transgenic plants. We found that the late-grown WT2 had the same heading date as the ETR2-overexpressing line 12-1, which had a severe phenotype. However, the seed-setting rate of this late-grown WT2 remained similar to that of the normal WT1 (Figure 5C). These results indicate that late flowering of the late-grown WT2 did not significantly change its seed-setting rate, suggesting that the low seed-set in ETR2-overexpressing lines was not due to late heading, but likely due to the ETR2 roles in extension of the seed ripening process.
Seed phenotypes were compared, and seed coats from the two RNAi lines were yellow, whereas seed coats from control plants and the four ETR2-overexpressing lines had brown regions, suggesting that seeds from the two RNAi lines were riper than seeds from other lines (Figure 5D). The thousand-seed weights were substantially higher in the two RNAi lines 41-4 and 66-1 grown both in Beijing and Hainan compared with controls (Figure 5E). However, four ETR2-overexpressing lines had lower thousand-seed weight or no change in this parameter.
Starch Accumulation in the Internode of the ETR2-Overexpressing Rice Plants
Cross sections of the internodes of transgenic plants showed that starch granule accumulation was altered relative to the wild type. At the same growing period of 162 d, the ETR2-overexpressing line 12-1 had more starch granules in all four internodes compared with the controls (Figure 6A ). Consistently, the first internode of RNAi line 41-4 had fewer starch granules compared with the controls. However, other internodes of these RNAi plants showed no difference in numbers of starch granules. This result suggests that ETR2 promotes the accumulation of starch granules in internodes.
It is possible that the difference in starch accumulation between ETR2-overexpressing lines and the control plant was due to the difference in developmental stage. Thus, we measured numbers of starch granules in these plants that were at the same developmental stage of full heading, but with different growth time. The first internode of all four ETR2-overexpressing lines had higher average numbers of starch granules than control plants and the two RNAi lines (Figures 6B and 6C). However, in the other three internodes, no significant difference in number of starch granules was observed among the lines compared. Iodine staining further revealed that four ETR2-overexpressing lines had a high density of starch granules, whereas control plants and the two RNAi lines had a lower density of starch granules (Figure 6D). These results suggest that ETR2 may facilitate starch accumulation in the internodes of rice plants.
The ETR2 Receptor Regulates Gene Expression
To investigate if ETR2 affected rice growth and development by regulating downstream genes, we performed a microarray analysis using Affimetrix rice gene chips. Shoot apical meristems (SAMs) from field-grown rice plants, including control plants, the ETR2-overexpressing line 12-1, and the RNAi line 41-4, were used to isolate RNA for chip analysis. Compared with control plants, 57 genes were upregulated and 263 genes were downregulated in the ETR2-overexpressing line 12-1 (see Supplemental Data Sets 1 and 2 online). In the RNAi line 41-4, 157 genes were upregulated and 68 genes were downregulated compared with control plants (see Supplemental Data Sets 3 and 4 online). Through further comparison of genes that showed altered expression, we identified genes consistently regulated in the ETR2-overexpressing line 12-1 and the RNAi line 41-4 (see Supplemental Data Set 5 online). Among these, 63 genes were downregulated in the ETR2-overexpressing line but upregulated in the ETR2-RNAi line. By contrast, eight genes were upregulated in the overexpressing line but downregulated in the RNAi line. These analyses suggest that ETR2 mainly plays inhibitory roles on gene expression, and only 13% of the total regulated genes showed a reciprocal expression pattern in the overexpressing line and RNAi line. The microarray data were further analyzed using the GOEAST program (Zheng and Wang, 2008; http://omicslab.genetics.ac.cn/GOEAST/index.php). Flower development, and many other biological processes, cellular components, and molecular functions were found to be substantially affected in the ETR2-overexpressing line 12-1 and ETR2-RNAi line 41-4 (see Supplemental Figures 1 to 6 online).
Because ETR2 caused a late flowering phenotype in the overexpressing lines and its suppression in the RNAi lines resulted in early flowering, we chose several flowering-related genes (see Supplemental Data Set 6 online) from the microarray analysis, including OsGI, RCN1, MADS box genes, and ONAC300, and examined their expression in transgenic plants. OsGI and RCN1 genes have been reported to cause late flowering or late transition to the reproductive stage in rice plants (Nakagawa et al., 2002; Hayama et al., 2003). MADS box genes and NAC genes have been found to promote the differentiation of flower organs (Aida et al., 1997, 1999; Rijpkema et al., 2007). OsGI was enhanced in the shoot apical meristem (SAM) of the two ETR2-overexpressing lines 8-2 and 12-1, but slightly decreased in the SAM of the two RNAi lines 41-4 and 66-1 compared with controls (Figure 7A ). OsGI gene expression was further reduced in the panicles of control plants. RCN1 gene expression was enhanced in the two ETR2-overexpressing lines. However, its expression was not significantly changed in the two RNAi lines compared with controls. Expression of two MADS genes and ONAC300 was reduced in the two overexpressing lines, but not significantly altered in the two RNAi lines in comparison with control plants (Figure 7B). The ETR2 gene was highly expressed in the SAM of the two ETR2-overexpressing lines, and 12-1 had higher expression than the 8-2 line (Figure 7A). All of these results indicate that ETR2 may promote OsGI and RCN1 gene expression but inhibit MADS5, MADS7, and ONAC300 expression, leading to the late heading phenotype in ETR2-overexpressing rice plants.
Since starch accumulation was altered in the ETR2-overexpressing lines, we studied expression of the α-amylase gene RAmy3D. α-Amylase catalyzes the hydrolysis of α-1,4 glucan bonds of amylase and amylopectin (Beck and Ziegler, 1989). RAmy3D expression was inhibited in the SAM of the two overexpressing lines but not significantly affected in the two RNAi lines compared with control plants (Figure 7C). This result suggests that ETR2 may inhibit starch degradation, leading to starch accumulation in internodes of the ETR2-overexpressing lines. A gene encoding a putative monosaccharide transporter was also identified from microarray analysis, and this gene was reduced in SAM of the overexpressing lines but enhanced in the two RNAi lines (Figure 7C). This expression pattern indicates that ETR2 may also inhibit genes responsible for sugar translocation.
Expression patterns of the above genes were further investigated in different organs of rice plants. The two MADS box genes, RAmy3D, and the putative monosaccharide transporter gene were mainly expressed in SAM and panicles, whereas OsGI, RCN1, and ONAC300 were expressed in all organs examined (Figures 7A to 7C). These results suggest that the first four genes may function in SAM or the transition from the vegetative stage to the reproductive stage, whereas the last three genes may play roles in growth and development of multiple organs.
Previous studies have demonstrated functional compensation within the ethylene receptor family in tomato (Tieman et al., 2000). Thus, we examined whether alteration of ETR2 gene expression would affect expression of the other ethylene receptor genes. Figure 7D showed that although the ETR2 levels were elevated in the overexpressing lines and suppressed in the RNAi lines, no significant changes in expression of the other four ethylene receptor genes were observed in these rice seedlings (Figure 7D).
Identification and Phenotypic Comparison of T-DNA Insertion Mutants for ETR2 and Other Ethylene Receptor Genes
To further investigate ETR2 function, the T-DNA insertion mutant, etr2, for the ETR2 gene was requested from the Rice Mutant Database (http://rmd.ncpgr.cn) and analyzed. For comparison, the T-DNA insertion mutant etr3 and ers2 for rice ETR3 and ERS2, respectively, were also studied. These mutants were in the background of Zhong Hua 11 (ZH), a japonica variety. In etr2, the T-DNA was inserted at 0.6 kb upstream of the ATG start codon of ETR2 gene (Figure 8A ). In etr3, the insertion was found at 0.9 kb upstream of the ETR3 start codon. In ers2, the insertion was at 0.7 kb downstream of the ERS2 stop codon. Homozygous lines of these mutants were identified (Figure 8A). In etr2, ETR2 expression was barely detectable; however, in etr3 or ers2, ETR3 or ERS2 was still expressed at low levels (Figure 8B). Thus, etr2 appeared to be a near-knockout mutant, whereas etr3 and ers2 were knockdown mutants.
The three T-DNA insertion mutants were treated with ethylene, and their coleoptile growth response was examined. The etr2 mutant exhibited the longest coleoptile at all ethylene concentrations tested compared with controls (Figure 8C). The etr3 and ers2 mutants showed moderate length of coleoptiles with these treatments compared with controls (Figure 8C). These results indicate that the three mutants have an enhanced ethylene response, suggesting that reduction of ethylene receptor gene expression results in increased ethylene sensitivity.
The mutants were grown under field conditions to observe the effect of reducing expression of the receptor. We found that etr2 and etr3 developed panicles earlier than ZH control plants (Figure 9A ). During the heading period, etr2 and etr3 plants showed early heading panicles compared with ZH controls (Figure 9B). The heading time distribution of these mutants was also analyzed. The etr2 mutant showed a 3-d early heading, whereas the etr3 had a 7-d early heading compared with ZH control plants (Figure 9C). These results indicate that reduction of ethylene receptor gene expression leads to early flowering.
Since overexpression of ETR2 causes starch accumulation in stems (Figure 6), we examined whether reduction of ETR2 and ETR3 gene expression would affect starch accumulation. In both the etr2 and etr3 mutants, starch granules were almost absent in stems (Figure 9D). However, in the ZH control, starch granules were still abundant (Figure 9D). These results indicate that reduction of ethylene receptor gene expression causes disappearance of starch granules.
The seed phenotypes were compared, and the etr2 and etr3 mutant seeds appeared slightly larger than those of the ZH controls and ers2 (Figure 9E). The thousand-seed weight was also examined, and etr2 seeds had significantly higher thousand-seed weight than ZH control (Figure 9E). However, etr3 seeds and ers2 seeds had slightly higher and lower levels of the parameter, respectively, than the ZH control (Figure 9E). These analyses indicate that reduction of ETR2 expression leads to high thousand-seed weight. However, suppression of ETR3 and ERS2 results in more or less variation than ZH control in this parameter.
Since overexpression of ETR2 causes an increase of OsGI and RCN1 expression, but a decrease of RAmy3D and monosaccharide transporter gene expression (Figures 7A and 7C), we examined whether these genes were altered in the SAM of the three mutant rice plants. Both OsGI and RCN1 expression were reduced in the three mutants compared with the ZH controls (Figure 9F). By contrast, RAmy3D and monosaccharide transporter gene expression were upregulated in the three mutants compared with the ZH control (Figure 9F). The expression of the above genes in the etr2 mutant was consistent with that in the ETR2-overexpressing lines but not in the ETR2-RNAi lines (Figures 7A and 7C), probably because the ETR2-RNAi lines had a partial reduction but not an elimination of ETR2 expression. These results indicate that suppression of ethylene receptor gene expression in T-DNA insertion mutants leads to inhibition of OsGI and RCN1 but upregulation of RAmy3D and the monosaccharide transporter gene.
We have studied the ethylene receptor from the monocotyledonous rice plant and found that subfamily II ethylene receptor ETR2 is a Ser/Thr kinase that can phosphorylate its own receiver domain and an in vitro substrate MBP. Complete abolishment of ETR2 kinase activity by N box mutation suggests that the N box is the most important motif for ETR2 function (Figure 2). Overexpression of ETR2 leads to reduced ethylene sensitivity and late flowering, whereas T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering (Figures 3, 4, 8, and 9). Other yield-related traits, starch accumulation, and gene expressions were also affected by ETR2 overexpression or reduction (Figures 5 to 7⇑⇑). This study reveals conserved and diverged aspects in ethylene receptor signaling between the monocotyledous rice plant and dicotyledonous plant.
While Ser/Thr kinase activity has been found in subfamily II receptors from dicotyledonous plants (Xie et al., 2003; Moussatche and Klee, 2004; Zhang et al., 2004), we show here that ETR2, from a monocotyledonous plant rice, possesses Ser/Thr kinase activity. Mutations in the N box of NTHK2 and Arabidopsis receptors partially reduced their kinase activities (Moussatche and Klee, 2004; Zhang et al., 2004). However, the rice ethylene receptor ETR2 completely lost its kinase activity through mutation of the N box. This property demonstrates that the Ser/Thr kinase activity of ETR2 is intrinsic and not from other contaminating proteins, which might be questioned during analysis of yeast-expressed proteins. This feature may reflect the further divergence of the rice ethylene receptors in terms of structure and function, or it could have resulted from mutations of different amino acid residues. The phosphorylation sites in ETR2 are likely in the ATP subdomain, since the truncated form without the ATP subdomain fails to be phosphorylated by ETR2. In the tobacco ethylene receptor NTHK1, the phosphorylation sites are mainly located in the HIS and ATP subdomains (Zhou et al., 2006a). ETR2 can phosphorylate its RD. This property was not found for NTHK1, NTHK2, and Arabidopsis receptors (Xie et al., 2003; Moussatche and Klee, 2004; Zhang et al., 2004). However, whether and how the RD phosphorylation would mediate the receptor signal output needs to be further studied.
Although ethylene receptors have kinase activities, the physiological function of these activities is largely unknown and of uncertain significance. Histidine kinase activity of Arabidopsis ETR1 may have no role or might be involved in growth recovery or ethylene-independent growth regulation (Wang et al., 2003; Binder et al., 2004; Qu and Schaller, 2004; Cho and Yoo, 2007). The Ser/Thr kinase activity of tobacco NTHK1 appeared to be required for plant leaf growth and stress responses (Chen, 2008). However, the role of ETR2 Ser/Thr kinase activity in rice ethylene signaling needs to be investigated. It is possible that the Ser/Thr kinase activity of the ethylene receptor could phosphorylate its interacting proteins and then transfer the signal to downstream components. Identification of these interacting proteins should shed light on the mechanism of receptor signal transduction in the future.
Rice plants have at least five ethylene receptors. However, among these, no ETR1-like receptor (i.e., subfamily I receptor with a receiver domain) was identified after analysis of the whole rice genome. This is in contrast with the observation that dicotyledonous plants generally have ETR1-like proteins. Pareek et al. (2006) have analyzed the rice genome for two-component signaling machinery but did not identify an ETR1-like receptor. These results possibly imply that rice may not have an ETR1-like protein. However, rice does contain subfamily I receptors, ERS1 and ERS2, neither of which contains a receiver domain. Gallie and Young (2004) have analyzed ethylene perception machinery in maize and they failed to identify an ETR1-like gene, although they were able to isolate other ethylene receptor genes. The significance of the absence of an ETR1-like protein in monocotyledonous cereal plants remains to be studied.
Overexpression of the ETR2 gene conferred reduced ethylene sensitivity in rice plants, whereas the etr2 mutant showed enhanced ethylene sensitivity (Figures 3 and 8). The T-DNA insertion mutants etr3 and ers2, with reduced expression of ETR3 and ERS2, respectively, also had increased ethylene sensitivity (Figure 8). This observation was in line with the roles of the ethylene receptor NTHK1 in tobacco and Arabidopsis (Xie et al., 2002; Cao et al., 2007). Consistently, overexpression of the tomato ethylene receptor NR reduced ethylene sensitivity in seedlings and mature plants, whereas transgenic plants with reduced tomato ETR4 gene expression exhibited multiple symptoms of extreme ethylene sensitivity (Ciardi et al., 2000; Tieman et al., 2000). Although both the shoots and roots of rice are responsive to ethylene or ACC treatment, their responses are different: root growth was suppressed, whereas coleoptile growth was promoted. The etiolated rice seedlings did not have a typical triple response as Arabidopsis or other dicotyledonous plants do upon ethylene treatment. This phenomenon represented a special feature for rice seedlings due to difference in seedling structures from dicotyledonous plants. Dicotyledonous seedlings have two cotyledons and a long hypocotyl when grown in the dark, whereas etiolated rice seedlings have only very short mesocotyls (Maclean et al., 2002). Further studies should reveal how ethylene signaling could exert different effects on root and shoot growth.
ETR2 can delay floral transition, and this was supported by the results from overexpression, RNAi, and T-DNA insertion mutant analysis (Figures 4 and 9). An abundance of ETR2 transcripts in the SAM and panicle (Figure 7A) further supports this conclusion. Consistently, the T-DNA insertion mutant etr3 for another rice ethylene receptor gene ETR3 showed early flowering (Figure 8). The tobacco ethylene receptor NTHK1 also caused a late flowering phenotype in transgenic Arabidopsis (Cao et al., 2007). Similarly, the Arabidopsis gain-of-function mutant etr1-1 of ethylene receptor ETR1 displayed late flowering (Bleecker et al., 1988; Ogawara et al., 2003). An ethylene receptor gain-of-function mutant in tomato, NR (never ripe), originally identified by its lack of fruit ripening, exhibited delayed flower development (Lanahan et al., 1994). Since ethylene negatively regulates its receptors (Hua and Meyerowitz, 1998), ethylene should promote plant flowering through suppression of ethylene receptor functions. It has been reported that ethylene induced flowering in pineapple (Ananas comosus) and that silencing of the ACC synthase gene caused delayed flowering in pineapple (Abeles et al., 1992; Trusov and Botella, 2006). In addition to ethylene receptors, other components of ethylene signaling also affect flowering time. Ethylene-insensitive mutants ein2-1 and ein3-1 and the constitutive ethylene response mutant ctr1-1 showed a late flowering phenotype (Ogawara et al., 2003; Achard et al., 2007). Although ethylene can enhance floral development, it also inhibits flowering in some plant species (Abeles, 1967). Further studies should disclose the mechanism by which ethylene differentially regulates flowering in different plant species.
Microarray analysis of gene expression profiles in ETR2-overexpressing plants and ETR2-RNAi plants revealed that ETR2 affected flower development (see Supplemental Figures 1 and 4 online). Two flowering-related genes OsGI and RCN1 were consistently upregulated in ETR2-overexpressing plants but downregulated in the etr2 mutant. These two genes were also suppressed in etr3 and ers2 mutants (Figure 9). It has been reported that overexpression of OsGI caused late flowering in transgenic rice, and this function was likely due to the activation of the CONSTANS homolog Heading date1 (Hd1) and suppression of the FLOWERING LOCUS (FT) homolog Hd3a (Hayama et al., 2003). The Arabidopsis FT homolog TERMINAL FLOWER1/CENTRORADTALIS (TFL1/CEN) gene retards floral transition (Kobayashi et al., 1999), and RCN1, the rice homolog of TFL1/CEN, was also found to cause delayed transition from vegetative to reproductive development in the SAM of transgenic rice (Nakagawa et al., 2002). It is thus proposed that ETR2 may delay floral transition at least partially through activation of the OsGI-RCN1 pathway. However, it is not known whether the regulation of floral transition by ETR2 is through the CTR1-EIN2-EIN3–mediated ethylene response pathway or whether ETR2 has a role in flowering time that is distinct from its role in ethylene signaling. Further studies should test these possibilities.
ETR2 promoted starch accumulation in plant internodes (Figure 6), and this accumulation was possibly due to the reduced expression of the RAmy3D gene, whose product, α-amylase, hydrolyzes starch. A monosaccharide transporter gene was also suppressed in the ETR2 overexpression lines, and this suppression may prevent sugar translocation from the stem to grains, leading to reduced seed-setting rate and reduced thousand-seed weight (Figure 5). When expression of ETR2 was reduced in the etr2 mutant and/or RNAi lines, starch failed to accumulate, and sugar translocation was enhanced, resulting in higher thousand-seed weight (Figures 5, 6, and 9). Parameters from a different year also support this conclusion (see Supplemental Figure 7 online). Inhibition of RAmy3D by ETR2 in this study may be consistent with a report showing that ethylene induced RAmy3D gene expression in rice (Fukao et al., 2006). Under submergence in water, the intolerant rice cultivar M202 produces more ethylene than tolerant M202(Sub1) plants and has decreased starch content. This process of starch metabolism was also regulated by the ERF-type transcription factor Sub1C (Fukao et al., 2006). Ethylene, together with abscisic acid, also had an influence on grain filling (Yang et al., 2006). These studies imply that ethylene and receptor signaling can regulate carbohydrate metabolism, sugar translocation, and grain filling in rice. The mechanism reported here provides significant clues for improvement of yield-related traits in rice. It should be noted that although reduction of ETR2 expression could increase thousand-seed weight, suppression of ethylene receptor genes ETR3 and ERS2 in their corresponding mutants only slightly changed this parameter. However, the etr3 mutant seeds appeared larger than the other seeds evaluated (Figure 9E). These observations suggest that each ethylene receptor gene may regulate specific seed-related traits.
Arabidopsis GI affected both floral initiation and starch accumulation (Araki and Komeda, 1993; Eimert et al., 1995). We therefore speculate that the rice ethylene receptor ETR2 may regulate floral transition and starch accumulation partially by regulating the OsGI gene. Another cam1 mutant of Arabidopsis also had delayed floral initiation and altered starch metabolism. However, not all the late-flowering mutants had increased starch content, and the higher starch level is not the cause of the late floral transition (Eimert et al., 1995). It is possible that starch accumulation and floral transition share a common signaling pathway. Alternatively, plants may use the OsETR2-OsGI pathway to store energy and use it later for reproduction, once the ethylene signal is available for receptor inhibition at a proper time or appropriate developmental stage.
Plant Materials and Growth Conditions
Japonica rice (Oryza sativa) variety Taipei 309 (TP309) and Zhong Hua 11 (ZH) were used in this study. Rice seeds from control plants, OsETR2-overexpressing plants, OsETR2-RNAi transgenic plants, or T-DNA insertion mutants were immersed in water for 2 d and grown for ∼45 d in soil seed bed, and then the seedlings were transplanted to the field. The field was located in the Experimental Station of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (39°48'N, 116°28'E). The growing season is from the end of April to the end of October. During this period, the average lowest temperature is from ∼7 to ∼22°C, and the average highest temperature is from ∼19 to ∼31°C. The light period of the photoperiod is from ∼7.2 to ∼9.3 h. The plants were maintained following routine management.
Cloning of the ETR2 Genomic and cDNA Sequences
The rice ETR2 genomic sequence was amplified from genomic DNA using the primers 5′- GAAAGATCTATGCCACCGATCCCATCTCTG-3′ and 5′-CAAGGTACCTGTGATTGTATGGACCTAAC-3′. The amplified fragment was cloned into the T-easy vector and sequenced, and the position of its intron was determined by comparison with the cloned 3′-cDNA sequence spanning the intron region. The primers 5′-CATGGTAGGTACTCTAATAC-3′ and 5′-CAAGGTACCTGTGATTGTATGGACCTAAC-3′ were used to amplify the 3′-cDNA of ETR2. The full-length coding-region of ETR2 was also amplified from the first-strand cDNAs derived from the total RNAs of rice seedlings, cloned into a T-easy vector and used for further analysis.
Expression of the Truncated ETR2 Proteins
For expression of different truncated versions of ETR2 as GST fusions in Escherichia coli, the pGEX4T-1 vector was used. DNA fragments corresponding to the putative kinase domain (wild type, amino acids 180 to 619), the kinase domain without the ATP binding motif (ΔATP, amino acids 180 to 461), and the putative RD (amino acids 631 to 756) were amplified from the original rice ETR2 cDNA. For the wild type, the sense primer is OsETR2-KD-LEFT (5′-CGGGATCCATGCTCACGCACGAGATC-3′) and the antisense primer is OsETR2-KD-RIGHT (5′-ACGCGTCGACGAGTCACATGCTGCAGCT-3′). For ΔATP, the sense primer is the same as that for the wild type, and the antisense primer is OsETR2-KD(ΔATP)-RIGHT (5′-ACGCGTCGACCAACACTGATTGCCTCCT-3′). The primers for RD are OsETR2-RD-LEFT (5′-CGGGATCCGTCTCTGGAGCATCCTCG-3′) and OsETR2-RD-RIGHT (5′-ACGCGTCGACACAGTTCATCTCCCAGTG-3′). The multisite mutations in the N box (N, with mutations G487A, E489Q, R491Q, F493A, and G501A) were made through the overlapping PCR method by the Shanghai Sangon Company, and the mutation was confirmed by sequencing. Finally, the PCR products for the wild type, ΔATP, N, and RD were digested with BamHI/SalI, cloned into the E. coli expression vector pGEX4T-1, and confirmed by sequencing. The recombinant plasmids were transformed into E. coli strain BL21. The positive colony was grown in 5 mL 2× Yeast Extract Tryptone medium at 37°C for 12 h, and the cultured cells were transferred into 100 mL 2× Yeast Extract Tryptone medium and grown for 10 h at 37°C.The expression of recombinant proteins was induced by addition of isopropyl-β-D-thiogalactopyranoside to a concentration of 400 μM and grown at 30°C for 2.5 h. After induction, the E. coli cells were broken by sonication in 10 mL extraction buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, and 10% [v/v] glycerol), and the supernatant was loaded onto the GST affinity resin in the column. The resin was washed extensively with washing buffer (50 mM Tris-HCl, pH 7.7, 100 mM NaCl, 0.1% [v/v] Tween 20, and 10% [v/v] glycerol) and finally eluted for GST fusion proteins with elution buffer (10 mM reduced glutathione and 50 mM Tris-HCl, pH 8.0) (Xie et al., 2003; Zhang et al., 2004). The free glutathione was removed on a centriprep concentrator, and the buffer was changed to storage buffer (50 mM Tris-HCl, pH 7.6, 50 mM KCl, 2 mM DTT, and 10% [v/v] glycerol). The expression and purification of the GST fusion proteins were confirmed by protein gel blotting using a mouse anti-GST monoclonal antibody (Amersham). The GST protein itself was also expressed and used as a control.
Kinase Assay
The purified GST fusion proteins were examined for their kinase activity, and GST was used as a control. Phosphorylation was performed in 25 μL of assay buffer (50 mM Tris–HCl, pH 7.6, 50 mM KCl, 2 mM DTT, 10 μM ATP, and 10% [v/v] glycerol) containing 1 μg GST fusion proteins in the presence of 5 mM of MnCl2, MgCl2, or CaCl2. The phosphorylation was initiated by adding 25 μCi of [γ-32P] ATP (5000 Ci mmol−1) and incubated at 22°C for 45 min (Xie et al., 2003; Zhang et al., 2004). The phosphorylated proteins were subjected to 10% SDS-PAGE and transferred onto PVDF membranes (Amersham). The incorporated phosphate was visualized by autoradiography. Coomassie Brilliant Blue staining of the same membrane was also performed to verify the protein loading. The stability of the incorporated phosphate was determined by treating the membranes with water, 1 M HCl, or 3 M NaOH for 2 h to overnight at room temperature. The treated membranes were then subjected to autoradiography. For substrate phosphorylation, wild-type or N protein was incubated under phosphorylating conditions with no substrate or with MBP, ΔATP, RD, or GST. The phosphorylated proteins were resolved on an SDS-PAGE gel, transferred to PVDF membranes, and subjected to autoradiography or Coomassie Brilliant Blue staining.
Phosphoamino acid analysis followed previous descriptions (Xie et al., 2003).
Generation of Transgenic Rice Plants
The genomic DNA sequence of the ETR2 gene, which contains a 370-bp intron, was cloned into the BamHI/KpnI sites of the pBIN438 binary vector, between the cauliflower 35S promoter and nopaline synthase (nos) terminator, to generate the pBIN438-OsETR2 construct for overexpression analysis. The primers used to amplify this insert were 5′- GAAAGATCTATGCCACCGATCCCATCTCTG-3′ and 5′-CAAGGTACCTGTGATTGTATGGACCTAAC-3′. The tobacco mosaic virus Ω sequence was included downstream of the double 35S promoter to enhance the translation efficiency. For the RNAi construct, primers R1 (5′-CACTCTAGAGAGCTCAGAGCAAAGCATGAAACCACG-3′) and R2 (5′- CGCGTCGACGGTACCACAGCCTGCATTACACCTCTG-3′) were used for amplification of the 433-bp ETR2 fragment (from positions 1100 to 1533 bp). This fragment was inserted into the pZH01 binary vector in an inversely oriented manner, and with a β-glucuronidase fragment in between the two inserted fragments, to generate the RNAi construct pZH01-OsETR2-RNAi. The fused gene was under the control of the cauliflower 35S promoter. Transgenic rice plants were generated according to Agrobacterium tumefaciens–mediated transformation methods described by Zhou et al. (2006b). Briefly, the coat-stripped TP309 seeds were sterilized in 20% NaClO solution, washed with sterilized water three times, and plated on NB medium [N6 salts and B5 vitamins, supplemented with 30 g/l maltose, 500 mg/l proline, 300 mg/l casein hydrolate, 500 mg/l glutamine, and 2 mg/l 2,4-D (pH 5.8)]. The scutellum-driven embryonic calli were induced from TP309 seeds after 1 to 2 months. The calli were incubated with the A. tumefaciens strain AGL1, carrying the overexpression or RNAi plasmids, at room temperature for 20 to 30 min and then cocultured on NB medium containing 0.1 μM acetosyringone for 2 d. For selection of positive OsETR2-overexpressing transformants, the calli were transferred to NB medium containing 80 mg/L G-418 and 500 mg/L cefotaxime, maintained for 3 weeks at room temperature, and then transferred to NB medium containing 150 mg/L G-418 and 500 mg/L cefotaxime, and maintained for another 3 weeks. For selection of RNAi transformants, 50 mg/L hygromycin was used. After selection, the calli were transferred to Shoot Regeneration medium for shoot regeneration and the medium (half-strength Murashige and Skoog salts containing 0.5 mg/L napthalene acetic acid) for root development. The transgenic plants were then transferred to the field of the Experimental Station (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for normal growth and seed harvesting.
Measurement of Agronomic Traits and Material Collection
Seeds of control and T2 or T3 homozygous transgenic plants were soaked in water for 3 d at 37°C and then transferred to Petri dishes with wet cheesecloth at 28°C. Elongation of the shoot was measured at 5-d intervals. The seedling length was also measured for the 50-d-old seedling grown in the field. Flower development was observed at ∼100 d after germination and then the heading date of each transgenic line, controls (TP309 and Zhong Hua 11) or mutants were examined every day. Plant height and internode length were measured at the mature stage. The ratio of effective tillers, ratio of effective panicles, seed-setting rate, and thousand-seed weight were analyzed when the seeds were harvested. Effective tillers were defined as the tillers that have panicles, and the ratio of the effective tiller number to the total tiller number was regarded as the ratio of effective tillers. Effective panicles were defined as the panicles that have at least one full-sized grain, and the ratio of the effective panicle number to the total tiller number was regarded as the ratio of effective panicle. Seed-setting rate was defined as the ratio of full-size grain number to the total grain number. For each line, data from 30 individual plants were obtained and subjected to statistic analysis. For examination of gene expression, the roots and leaves were harvested from seedlings grown in water for 14 d. The SAM and panicles were collected from TP309 or transgenic rice plants grown in the field for 95 and 100 d after sowing, respectively. The materials were frozen in liquid nitrogen and stored at −80°C until extraction of DNA or RNA.
Isolation of Nucleic Acids and Hybridization Analysis
Total RNA isolation and RNA gel blot hybridization were performed following the description by Zhang et al. (1996, 1999b). Full-length rice ETR2 cDNA was used as the template for probe labeling. For detection of OsGI expression, a 1005-bp fragment (from 1720 to 2725 bp in the open reading frame) was amplified with the primers 5′-CCTGTGCTTCAATGGACCTT-3′ and 5′-GCCAGAGCAATGAGACAACA-3′. For the detection of RCN1, a 646-bp fragment (from 177 to 823 bp in the open reading frame) was amplified using the primers 5′-TGACCTGCGATCTTTCTTCA-3′ and 5′-CACCAGCATGAATTGGCTTA-3′. Genomic DNA from rice leaves was isolated by an extraction method with SDS (Chen et al., 1991). Genomic DNA (8 μg) was digested with DraI, resolved on a 0.8% (w/v) agarose gel, transferred to a nylon membrane, and hybridized using the labeled probes (Chen et al., 1991).
Determination of Ethylene Sensitivity
Rice seeds were immersed in water at 37°C for 2 d, transferred onto a plate containing wet cheesecloth, and grown at 28°C for another 3 d. The germinated seeds were then subjected to treatments with various concentrations of ACC (0, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) in the dark. After 1 week, the lengths of the shoots and roots were measured. The experiment was repeated independently three times, and the results were consistent. For ethylene treatment, germinating seeds were transferred into 4-cm Petri dishes (20 per dish) fitted with eight layers of moist gauze. The Petri dishes were placed in air-tight plastic boxes (41 × 31.5 × 15 cm), which was used for reducing the effect of endogenous ethylene. Ethylene gas was injected into the plastic boxes using a syringe. Seed transfer and ethylene treatment were performed under dim green light as described by Ku et al. (1970). Seedlings were grown in darkness at 25°C. Coleoptile length was measured 3 d after ethylene treatment.
Starch Analysis
Premature rice stems were collected and used for scanning electron microscopy analysis. For the same growth stage experiment, 162-d-old field-grown control TP309, overexpression line 12-1, and RNAi line 41-4 were used. For the same developmental stage experiment, the sampling days were varied for different lines. In detail, 123-d-old TP309, 120-d-old RNAi lines 41-4 and 66-1, 136-d-old overexpression lines 8-2, 17-1, and 56-4, and 150-d-old overexpression line 12-1 were used. The lower parts (adjacent to the nodes) of the first to fourth internodes were collected, and then the samples were cross-sectioned using a razor blade. The fixation, dehydration, and scanning electron microscopy observation followed the description by Cao et al. (2007). The starch numbers in 30 cells for each sample were calculated.
The cross sections were also subjected to iodine staining with 1% (w/v) KI solution to reveal the starch granules. The sections were washed with water and observed under a light microscope (Olympus U-25ND6).
Microarray Analysis
Two biological replicate samples of SAMs from Japonica control variety TP309 (95 d), the ETR2-overexpression line 12-1 (100 d), and the ETR2-RNAi line 41-4 (92 d) were collected for RNA extraction from field-grown rice plants. Each replicate contains 10 to 20 SAMs from ∼10 individual rice plants grown in the same field. These SAMs were at the same vegetative stage but were grown for different periods of time. Plants were grown under field conditions with regular management, including irrigation and routine pest control. The total RNAs were extracted from each replicate using the RNeasy Mini Kit (Qiagen) and used in target synthesis for the Rice Genome Array from Affymetrix. The cDNA synthesis and cRNA preparation were performed according to standard protocols (Affymetrix). Hybridization, washing, and staining were conducted in an Affymetrix Genechip Hybridization Oven 640 and Affymetrix Fluidics Station 450. The scanning and data collection and analysis were performed using a Gene array Scanner 3000 7G and GeneChip Operating Software (Affymetrix). The rice genome array contains probes to query 51,279 transcripts representing two rice cultivars, with ∼48,564 japonica transcripts and 1260 transcripts representing the indica cultivar. The entire analysis was performed at Shanghai Biochip Company and followed standard protocols from Affymetrix.
Examination of Gene Expression by RT-PCR Analysis
For RT-PCR analysis of the putative downstream genes, cDNA was reverse transcribed from the total RNAs of the SAM and used as templates for PCR amplification with specific primers for the selected genes. The PCR reactions were performed at 94°C for 50 s, 54 to 58°C for 60 s, and 72°C for 60 s for 30 cycles unless stated, and the products were visualized with ethidium bromide. For Actin, the primers are R-Actin A (5′-GGAACTGGTATGGTCAAGGC-3′) and R-Actin B (5′-AGTCTCATGGATAACCGCAG-3′); for ETR2, the primers are OsETR2-A (5′-CATGGTAGGTACTCTAATAC-3′) and OsETR2-B (5′-TGTGATTGTATGGACCTAAC-3′); for MADS5, the primers are 5′-GCGAGGGAAAGTAGAGCTGA-3′ and 5′-ACCCGCATGTAAAAGAGAGC-3′; for MADS7, the primers are 5′-TGGAGCTGAAGAGGATCGAG-3′ and 5′-AATTCATTGTCCCCGTCATC-3′; for RAmy3D, the primers are left primer (5′-CCCCTAAAATGGAACGGGTA-3′) and right primer (5′-ACTGCATCCTGAACCTGACA-3′); for ONAC300, the primers are 5′- AGAACGGCATCTGCTACACC-3′ and 5′-ATATACCTGCCCTGCCCTCT-3′; and for the putative monosaccharide transporter, the primers are 5′-GTCATCGTCATCGCCTTCTT-3′ and 5′-AGGACAAACACGGGATGGTA-3′.
The primers for RT-PCR analysis of other rice ethylene receptor genes were as follows. For ETR3, 5′-TGAAGTTTGTCATTGGTGTC-3′ and 5′-CAGGATTCACAGCTTTTACC-3′ were used. For ETR4, 5′-TCAGGGTGTCCATCTACGAC-3′ and 5′-TCCCATCTGAGAGGCTGGTAG-3′ were used. For ERS1, 5′-TGATAGCGCAAGAAATGGC-3′ and 5′-GTAAATACCTGAGGTAGATC-3′ were used. For ERS2, 5′-CGTGATCTACTAATGGAGCA-3′ and 5′-ACATCTACCATCCATACATC-3′ were used.
Identification and Analysis of Rice T-DNA Insertion Mutants
The T-DNA insertion mutants and their parental variety Zhong Hua 11 (ZH) were requested from the Rice Mutant Database (http://rmd.ncpgr.cn). The etr2, etr3, and ers2 mutant plants were identified using T-DNA primers p1 (5′-TGCAGGTTCTCTCCAAATGA-3′) and p2 (5′-AATCCAGATCCCCCGAATTA-3′) and specific primers for the ETR2, ETR3, and ERS2 genes as follows. For etr2, the 5′- and 3′-primers are 5′-ATCGTGCAAATCTGCTCACT-3′ and 5′-CTCCTCCACAATCTGGCTCT-3′, respectively. For etr3, the 5′- and 3′-primers are 5′-CAATGACACTCAACCCACAA-3′ and 5′-CAAAGTGAATACAGGACGAA-3′, respectively. For ers2, the 5′- and 3′-primers are 5′-GGGAAGGAAGTTCGAGAAGG-3′ and 5′-TCAAGCGTATGAGCAAGCACC-3′, respectively. The receptor gene expression in these mutants was examined by RT-PCR with the primers described in the above section.
Determination of ethylene sensitivity, growing of the plants in the Experimental Station, observation and analysis of rice plant heading date, thousand-seed weight measurement, and downstream gene examination were all based on procedures in the above sections. For starch granules, cross sections of the first internodes holding panicles, which are from field-grown rice plants (119 d) during grain filling, were observed under a scanning electron microscope.
Statistical Analysis
All the data were analyzed by one-way analysis of variance with the program of SPSS (version 10.0). For the microarray data, the interquantile range (IQR) of each probe among all the six array hybridization results was calculated using R packages. Genes with a IQR value greater than the median IQR of all genes on the array (see Supplemental Figure 8 online) and that had a P value of <0.05 and a fold change greater than 1.8 between the transgenic line (OsETR2-overexpressing line 12-1 or the RNAi line 41-4) and TP309 were considered as differentially expressed genes (see Supplemental Data Sets 1 to 4 online). Multiple testing corrections (Benjamini and Hochberg false discovery rate) were used to adjust the P value. For the genes consistently regulated in both the OsETR2-overexpressing line 12-1 and the RNAi line 41-4 compared with the control line (see Supplemental Data Set 5 online), a fold change of 1.5 was considered to be significant. The selected genes in the text were further examined for their expression using RNA gel blot analysis or RT-PCR analysis. The internal control probes on the Affymetrix rice microarray exhibited no obvious changes between the RNAi/overexpression lines and the control lines.
The microarray data were further analyzed using the GOEAST program (Zheng and Wang, 2008; http://omicslab.genetics.ac.cn/GOEAST/index.php). The enriched Gene Ontology (GO) terms and their hierarchical relationships in biological process, cellular component, or molecular function GO categories were identified and provided as Supplemental Figures 1 to 6 online. The P value of GOID enrichment was calculated as the hypergeometric probability and resulted in so many probes/probe sets/genes for a GO term, under the null hypothesis, that they were picked out randomly from the microarray/genome. GOEAST by default adjusts the raw P values into false discovery rate using the Benjamini-Yekutieli method. False discovery rate is used for control of the type I error rate.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: Os ETR2/Os PK1, AF420319; Os ETR3/Os PK2, AF420318; Os ETR4/Os PK3, AF497626; Os GI, AK067038; RCN1, AF159882; MADS5, OSU78890; MADS7, OSU78891; RAmy3D, AA753608; ONAC300, AK102794; putative monosaccharide transporter, AK072059; and Zm ETR2, AB040406.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Biological Processes Affected in ETR2-Overexpressing Rice Line 12-1 Compared with Control Line TP309.
Supplemental Figure 2. Cellular Components Affected in ETR2-Overexpressing Rice Line 12-1 Compared with Control Line TP309.
Supplemental Figure 3. Molecular Functions Affected in ETR2-Overexpressing Line 12-1 Compared with Control Line TP309.
Supplemental Figure 4. Biological Processes Altered in ETR2-RNAi Rice Line 41-4 Compared with Control Line TP309.
Supplemental Figure 5. Cellular Components Affected in ETR2-RNAi Rice Line 41-4 Compared with Control Line TP309.
Supplemental Figure 6. Molecular Functions Affected in ETR2-RNAi Rice Line 41-4 Compared with Control Line TP309.
Supplemental Figure 7. Comparison of Seed-Setting Rate and Thousand-Seed Weight among ETR2 Overexpression Lines, ETR2-RNAi Lines, and the Wild Type (TP309) Grown in a Year Different from Those in Figure 5.
Supplemental Figure 8. Plot of Gene Expression and IQR Values.
Supplemental Data Set 1. Genes Upregulated in ETR2-Overexpressing Rice Line 12-1 Compared with Control Line TP309.
Supplemental Data Set 2. Genes Downregulated in ETR2-Overexpressing Rice Line 12-1 Compared with Control Line TP309.
Supplemental Data Set 3. Genes Upregulated in ETR2-RNAi Rice Line 41-4 Compared with Control Line TP309.
Supplemental Data Set 4. Genes Downregulated in ETR2-RNAi Rice Line 41-4 Compared with Control Line TP309.
Supplemental Data Set 5. Genes Consistently Regulated in Both ETR2-Overexpressing Rice Line 12-1 and ETR2-RNAi Line 41-4 Compared with Control Line TP309.
Supplemental Data Set 6. Genes Selected for RT-PCR or RNA Gel Blot Analysis and Their Fold Changes in ETR2-Overexpressing Line 12-1 and ETR2-RNAi Line 41-4 Compared with Control Line TP309.
We thank David Clark (University of Florida) for critical reading of the manuscript and Cheng-Cai Chu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for helpful comments. We also thank Qi Zheng and Jun Liu for their helpful discussions on microarray analysis. We also thank the Rice Mutant Database (National Center of Plant Gene Research, Wuhan, at Huazhong Agricultural University) for providing rice mutant seeds. This work was supported by the National Natural Science Foundation of China (90717005), the National Basic Research Program of China (2006CB100102), the National Transgenic Research Project (2008ZX08009-003), and the High-Tech Program (2006AA10A101).
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.plantcell.org) is: Jin-Song Zhang (jszhang{at}genetics.ac.cn).
↵1 These authors contributed equally to this work.
↵2 Current address: Syngenta Biotechnology (China) Co., Ltd., Zhongguancun Life Science Park, Changping District, Beijing 102206, China.
↵[W] Online version contains Web-only data.
- Received December 24, 2008.
- Revised April 9, 2009.
- Accepted April 21, 2009.
- Published May 5, 2009.


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Learn how to use a simple paintbrush to create authentic looking hand-painted brush strokes. Von Glitschka does this with black acrylic paint.
- The term authentic means of undisputed origin or genuine. When it comes to artwork, authenticity simply means it resembles the real deal. When you create your own vector-based brushes in Illustrator, we're going for an authentic look and feel. To achieve this level of authenticity, we need to derive our brushes from a real world methodology. That means we're going to literally paint them with a paintbrush in order to capture true-to-life brush strokes we can then digitize.
A few years back I was approached by the local TEDx event and they asked me to brand it. The theme of the TEDx event was called Fearless. In the theme fearless is all about pursuing creativity without worrying about how or the possibility of failure. So I decided to approach that in a way that I normally never handle a logo design and that is to create it by hand. I want to share with you how that project inspired the ability to create brushes for Illustrator.
So I set out to hand paint all the letters I would need for the logo type I was designing for the local TEDx event called Fearless. This just shows a bunch of As that I painted out. I did a lot more iterations than this, but this just shows a page of As that I created. This is some of the Ss I created, and here's some of the Es I created. Now I painted out hundreds of these letters, but when it was all said and done, I then cherry picked the ones that I felt work best.
This shows how I pasted 'em up on one sheet, and I scanned this in. This became the final art for the final logo type of Fearless that the local TEDx event used. If you want to check out the digital art on this, you can go to the Exercise Files and you'll be able to zoom in and see how this was digitized. But now I want to walk you through the process of creating your own painted brushes. It's not complicated, it's very simple.
It first starts by having your own brushes. This first brush I want to show you, it's a nice brush but ironically it's too nice to paint with because it's not gonna create all the nice artifacts. So you don't want to use a nice brush like this. It's okay for other types of painting, but for this it's too nice, we won't use it. The only brush you really need is this one. This is just a one-inch simple paintbrush.
You can buy it at any paint store. I think this one cost me 89 cents. If you look at the bristles at the end, they're really kind of horrible, but horrible in a good way because, because they're so uneven, it's gonna create a lot of nice artifacting as you drag the pigment across the paper and that's what we want. So we're gonna be using this brush for sure. The other brushes I have here are just two smaller detailing type brushes, and these will form the line work brushes that we'll then use in Illustrator as well.
So I'm gonna go ahead and demonstrate how to paint out these brushes or dry brush with 'em to create the assets that we want to create. So that isn't complicated either. I like to use just simple white bond paper. You could use a textured paper if you wanted. That'd just give you more artifacts to work with, that's good too. But this is all we need. I like to just use a simple white paper plate to put my ink on. I'm using just simple black acrylic paint, and I'll just put some of this on this plate here.
If you're worried about getting paint all over your hands, you could wear surgical gloves as well. I usually do that at home so I don't get paint all over the place, but I'm kinda doing it without that today. But once you have paint loaded on your plate, I'm just gonna grab some water here. We're gonna mix it with some of the pigment. You don't want it to be too watery, just enough that it's gonna streak. So I think that's gonna work good.
You want to load the brushes. Kinda feel like Bob Ross here. Once you have that, you'll just drag it across and you can see how it makes those nice little artifacts and those streaks, and that's kind of what you want. It's not about painting nice, it's about capturing an idealistic looking paint stroke because that's what gonna work well. So I'll go back on some of these and just drag it a few times just to add some more artifacting to it like that.
I think that one looks pretty good, so we'll do another one. Now it doesn't always have to be so dense. You could make it really liney kinda like that. Sometimes I'll do smaller ones just like that. Those are always nice to have. So it's really up to you in terms of how you drag the brush, but don't press too hard. Just lightly let the bristles kind of form those nice lines and the negative spaces within the mark.
So I'll usually do a good 10 sheets of these. That one looks pretty good. And I like that. So there is no wrong way to do these. It's all about doing a lot of 'em and then you can go back and cherry pick the ones that look the best and that becomes your final brush set in Illustrator. So that's all I'll do for these. I want to show you how I'll paint some line work brushes, and we're just gonna use the small brush here to do this.
I'll do the same thing. Grab a little bit of water, a little bit of paint. What this is replicating and will turn into a brush in Illustrator is just to make an irregular kind of line brush that we can use on the perimeter of shapes. Once again, I'll just drag this just to create an imperfect line. This will wrap around the stroke of a shape and it'll make it look more organic. That's kinda the reason why I'm doing this. So these don't have to be perfect either.
I'll just experiment. I usually on these I'll push harder and thinner and harder so it's irregular in terms of its weight. So this is how I approach creating either a big paint brush or a line work brush like this. Once I've painted a lot of 'em out, this is what I end up with. I end up with sheets of these handpainted brushes and I'll actually tape 'em up. I'll cherry pick the ones that look the best, tape 'em up on a sheet.
So here's one sheet, here's another sheet. All of these appear in the course, by the way. They've all become assets I've used to create the artwork that I show in this course. This is some of the line work brushes I created. Now since you have your paint out, you don't have to limit yourself to just creating these brushes. Here's one where I just painted out the surface with the paintbrush before it dried out. This turned into a nice texture that I could also use. So it's up to you what kind of assets you want to create, but really isn't hard.
Now that we have all of our handcrafted brushstrokes painted out, we can now digitize them and turn 'em into useful vector assets for Illustrator. We'll do that later in this chapter.
Graphic designer Von Glitschka appreciates the endless brush stroke freedom that real-world brushes offer, and he knows how to replicate this offering in the digital world. In this course, he shows how to create your own custom, handmade brush strokes and import them into Adobe Illustrator. Von demonstrates how to compile and use custom brush strokes to achieve both a hand-painted aesthetic and a personal touch. Whether you're a painter, a designer, or just a fan of Illustrator, this course offers instructions on how to expand your use of Illustrator.
- Painting real brush strokes
- Transforming real brush strokes into digital brush strokes
- Importing brush strokes
- Creating bitmap surface textures
- Modifying vectors
- Compiling patterns, shapes, and strokes
- Working with layers and blend modes
- Using vector brushes


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The Kurdish-led Syrian Democratic Forces (SDF) has withdrawn from a 75-mile area between the border towns of Ras al-Ayn and Tal Abyad in compliance with a U.S.-brokered ceasefire, and will move back 19 miles from the Syria-Turkey border, a senior SDF official told the Associated Press on Monday.
SDF official Redur Khalil added however that while the Kurds were upholding their end of the ceasefire, the United States was failing to hold Turkey to its end, saying Turkey continues to conduct shellings and military operations.
Turkey halted its offensive in northern Syria on Thursday for five days under a deal agreed between Turkish President Tayyip Erdoğan and U.S. Vice President Mike Pence. The aim of the ceasefire is to give Kurdish forces time to withdraw from the border area Turkey is seeking to establish as a "safe zone."
While Erdoğan has said Turkey will resume its assault "with determination" when the deadline expires on Tuesday if all Kurdish forces, including the Kurdish People's Protection Units (YPG) militia, have not withdrawn, the U.K.-based Syrian Observatory for Human Rights reported that a Turkish drone hit a vehicle on Monday carrying four SDF fighters, some three kilometers from Ain Issa, south of Tal Abyad.
On Sunday, a Turkish soldier was reported killed and another wounded during an attack by the YPG militia in Tal Abyad. The Turkish Defense Ministry said the attack had targeted Turkish soldiers conducting a reconnaissance and surveillance mission, and that while Turkey had retaliated it remained committed to the ceasefire.
U.S. Defense Secretary Mark Esper said Saturday that all of the nearly 1,000 troops withdrawing from northern Syria will be stationed in western Iraq, where they will "help defend Iraq" and continue fighting the Islamic State.
There are currently more than 5,000 American troops based in Iraq.
Video circulated on social media showed residents of the city of Qamishli throwing potatoes at troops leaving the area, yelling "America liar" and "No America," expressing their sense of betrayal at the U.S. withdrawal.
Signs at protests included "Thanks for U.S. people but Trump betrayed us," and "To the U.S. army who are leaving northeast Syria: Tell your children that the children of the Kurds were killed by the Turks and we did nothing to protect them."
Some U.S. special forces soldiers that have fought alongside the Kurds in recent years have reportedly expressed remorse at the withdrawal.
According to reports, at least 114 civilians have been killed and 300,000 displaced since Turkey began its offensive on Oct. 9.
Erdoğan is scheduled to meet with Russian President Vladimir Putin in Sochi, Russia on Tuesday, coinciding with the end of the five-day ceasefire.
An SDF official called on Israel on Monday to take action against Turkey's military incursion into northern Syria, according to a report in the Times of Israel.
"The State of Israel must work to put an end to this war that is killing women and children and expelling civilians from their homes," the official, who was speaking on condition of anonymity, told the Times via text message.
"I am certain that the Jewish people understand the situation of the Kurdish people best because it lived these types of dangers throughout much of its history. I am sure that it will not stand by idly as our region faces Turkey's terror," said the official.


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• Single ownership and 31 miles from new. The Lancia Delta was first seen at the Frankfurt Motor Show in 1979. The Delta is best known for its domination of The World Rally Championship in the late 1980s and early 1990s. The first 'Evoluzione' cars were built at the end of 1991 and carried on through 1992. These 'Evoluzione' models were to be the final homologation cars built for the Lancia Rally Team which raced under the Martini Racing banner. In order to improve the handling, the Evoluzione had a wider track front and rear than earlier Deltas. So, in order to fit this track into the bodywork, the wide wheel arches were extended even further and in the process also became more rounded. The new Integrale retained the four wheel drive layout. It had an epicyclic center diff with torque splitter, plus a Ferguson viscous coupling and Torsen rear differential. The engine, although technically the same as the earlier 16V cars, was remapped to give 210bhp at 5750rpm in order to compensate for the slight increase in weight and increased frontal area. These improvements were aimed at improving the handling potential of the car and it worked, with the new Integrale being able to travel 5% faster over rally sections both asphalt and gravel. This example of one of the most iconic and sought after cars of its era. It is number 124 of the limited run of 400 cars built to celebrate Integrale's 5th consecutive victory in the World Championship for Manufacturer's. Finished in the distinctive white livery of the famous Martini drinks company, Lancia's sponsors at the time, the interior is trimmed in grey Alcantara. Despite the competition livery, this Integrale has only ever been used on the road once and that was when it was delivered by the supplying dealer to its only owner where it has remained in perfect storage conditions for the last 22 years. The car has covered a distance of only 50 kilometers from new and is supplied with its original service book, manual, invoice and tool kit. We are delighted to be able to offer this iconic car in what can only be described as 'brand new condition'.
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Using the most reliable components in the industry, DuSol has developed a whole new line of super efficient solar powered street lighting systems. We have named them Perpetual Sox (PS) . Keeping true to its name, the product offers virtually perpetual operation of the street lights without requiring any external power connection. With efficiency almost 3 times as that of normal street lights, Perpetual Sox make the best choice both in areas with no power supply as well as in areas with power availability. Output from the lamp is pale yellow colored making the lamp most desirable on road and streets as the human response time is know to be the best in this conditions. Moreover, reduced fatigue on the road is yet another advantage of PS street lights.
The system is shipped as a complete complete system (except for the pole) and can be mounted on poles at the best possible angle to capture the maximum sunlight. PS3514 is best suited for 4-6meter poles and PS5507 and PS 5514 is suited for higher poles of up to 10 meters. Our products has been tested in Dubai Airport and the reports have been very encouraging.


text:
Alicia Silverstone, who follows an organic vegan diet, sips this nutritious elixir, which personal chef and friend Lesa Carlson mixes for many of the star's private affairs. It's a tasty health potion: The mint, fresh peach juice, and honey deliver cancer-fighting antioxidants.
- Yield
- Makes 4 drinks
- 1 large bottle (16.9 oz) sparkling mineral water, chilled (or 1 bottle organic sweet or sparkling white wine, chilled)
- 2 tbsp honey (or more to taste)
- 4 fresh organic peaches, juiced in an electric juicer (or peel, slice and puree peaches in a blender and strain juice using a mesh strainer)
- 4 sprigs fresh mint
- Combine half the sparkling mineral water and honey in a blender. Pour about 1/4 mixture into a martini shaker; add 8 ice cubes and the rest of the sparkling water. Shake. Using the martini strainer, pour into a martini glass until almost full. Drizzle peach juice over top to fill. Garnish with mint, pushed into the drink, and serve.
Nutritional analysis per serving: 56 calories, 0 g fat (0 g saturated), 15.5 g carbohydrates, 0 g protein, 0 g fiber


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