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Purpose As an initial step in determining whether a spelling error analysis might be useful in measuring children's linguistic knowledge, the relation between the frequency of types of scores from a spelling error analysis and children's performance on measures of phonological and orthographic pattern awareness was examined. Method The spellings of first-grade children with typical spoken language skills were scored using the Spelling Sensitivity System (Masterson & Apel, 2010a); words were parsed into elements based on phonemes and then assigned a score based on the linguistic skills represented in the spelling. The children also completed more traditional measures of phonological and orthographic knowledge: an elision task and an orthographic pattern awareness task. Results There was a moderate negative correlation between number of elements omitted in the children's spellings (e.g., the child did not represent a phoneme with a letter[s] and performance on the phonological awareness task). There also was a moderate negative correlation between frequency of orthographically based spelling errors (e.g., spelling an element with a letter[s] that could never represent that sound in English) and performance on the orthographic pattern and phonological awareness measures. Conclusions These findings suggest that specific types of scores on the spelling error analysis provided information about the children's phonological awareness and orthographic knowledge. They also support continued investigations on the use of a systematic spelling error analysis for measuring phonological awareness and orthographic knowledge and highlight the potential utility of the analysis procedure in the educational setting.

Cholangiocarcinoma (CCA) is an aggressive malignancy with a 5-year-survival rate of <10%, mainly due to diagnosis in advanced stages and limited therapeutic options in case of progressive disease. Recently, evidence has indicated that alterations in the SWI/SNF-complex (SWI/SNF) may have an important role in the tumorigenesis of CCA. SWI/SNF-related chromatin remodeling has been reported to be crucial for differentiation and tumor suppression, and loss-of-function mutations of SWI/SNF are present in 20% of human malignancies; however, at present, little is known about its relevance in CCA. In the present study, a cohort of 52 patients with the diagnosis of primary CCA was retrospectively collected. All patients underwent surgery with curative intent. Tissue microarray analysis was performed on each tumor for immunohistochemical loss-of-protein analysis of the SWI/SNF core subunits ARID1A, INI-1, BRG1, PBRM-1 and BRM, corresponding to the following CCA subtypes: Extrahepatic CCA (ECCA), small duct or large duct intrahepatic CCA (ICCA). Kaplan-Meier analysis was used to determine survival distribution and survival differences were evaluated by log-rank test. In total, 14 of 52 patients (~35%) exhibited protein-loss of any tested SWI/SNF core subunit. Notably, 17% of patients exhibited a loss of ARID1a; this was the protein loss with the highest frequency. Patients with small and large duct ICCA with protein-loss of any tested SWI/SNF subunit exhibited significantly worse survival compared with the wild-type cohort with proficient protein expression (P=0.013 and P=0.002), whereas no significant survival difference was detected for patients with ECCA. SWI/SNF and its core subunits may be considered promising predictive and therapeutic targets, and require further investigation in patients with CCA., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Wagner et al.)

Introduction and Aims: The Self-Evaluation of Food Intake (SEFI®) is a simple tool to assess food intake that correlates well with the diagnosis of malnutrition in the hospital setting., Aims: to evaluate the validity of SEFI® for the diagnosis of malnutrition among adults in the primary care setting (primary aim); to assess the prevalence of malnutrition, the feasibility of the SEFI® and the variables associated with malnutrition (secondary aims)., Methods: A non-interventional prospective study on consecutive patients at three primary care practices. Primary endpoint: confrontation of a SEFI® visual analogue scale score <7/10 with the diagnosis of malnutrition as defined by the Global Leadership Initiative on Malnutrition criteria. Secondary endpoints: the proportion of patients for whom a SEFI® score was collected. Multivariate analysis: threshold α = 0.20 in univariate analyses, step-by-step logistic regression., Results: Among 747 eligible patients, 505 were included: mean age (±SD) 56 ± 19 yrs, 61% female, 49% presenting with acute medical problems, 15.8% (n = 80) with SEFI® score <7/10, and 4.2% (n = 21) with malnutrition. The predictive performance of the SEFI® score <7 for the diagnosis of malnutrition was good (AUC = 0.82 [95% confidence interval (CI), 0.72-0.92]): sensitivity 76.2% (n = 16/21, [58.0-94.4]), specificity 86.8% (n = 420/484, [83.8-89.8]), positive predictive value 20.0% (n = 16/80, [11.2-28.8]), and negative predictive value 98.8% (n = 420/425, [97.8-99.8]). The feasibility of the SEFI® 10-point visual analogue scale was 100% (505/505). The variables independently associated with malnutrition were: female gender (odds ratio 4.9 [95% CI, 1.7-14.2], P = 0.003), cancer (4.8 [1.4-15.9], P = 0.011) and chronic alcohol consumption (7.4 [1.3-41.4], P = 0.023)., Conclusions: The prevalence of malnutrition was 4.2% in this primary care setting. The SEFI® visual analogue scale for food intake is feasible and could be helpful for the diagnosis of malnutrition in this setting., Competing Interests: Conflicts of interest Ronan Thibault designed and received royalties for the Simple Evaluation of Food Intake® (SEFI®) (Knoë, le Kremlin Bicêtre, France). The other authors declare no conflict of interest related to this article., (Copyright © 2020. Published by Elsevier Ltd.)

Purpose Morphological awareness is the ability to consciously manipulate the smallest units of meaning in language. Morphological awareness contributes to success with literacy skills for children with typical language and those with language impairment. However, little research has focused on the morphological awareness skills of children with speech sound disorders (SSD), who may be at risk for literacy impairments. No researcher has examined the morphological awareness skills of children with SSD and compared their skills to children with typical speech using tasks representing a comprehensive definition of morphological awareness, which was the main purpose of this study. Method Thirty second- and third-grade students with SSD and 30 with typical speech skills, matched on age and receptive vocabulary, completed four morphological awareness tasks and measures of receptive vocabulary, real-word reading, pseudoword reading, and word-level spelling. Results Results indicated there was no difference between the morphological awareness skills of students with and without SSD. Although morphological awareness was moderately to strongly related to the students' literacy skills, performance on the morphological awareness tasks contributed little to no additional variance to the children's real-word reading and spelling skills beyond what was accounted for by pseudoword reading. Conclusions Findings suggest that early elementary-age students with SSD may not present with concomitant morphological awareness difficulties and that the morphological awareness skills of these students may not play a unique role in their word-level literacy skills. Limitations and suggestions for future research on the morphological awareness skills of children with SSD are discussed.

Purpose Spelling is a skill that relies on an individual's linguistic awareness, the ability to overtly manipulate language. The ability to accurately spell is important for academic and career success into adulthood. The spelling skills of adults have received some attention in the literature, but there is limited information regarding which approach for analyzing adults' spelling is optimal for guiding instruction or intervention for those who struggle. Thus, we aimed to examine the concurrent validity of four different scoring methods for measuring adults' spellings (a dichotomous scoring method and three continuous methods) and to determine whether adults' linguistic awareness skills differentially predict spelling outcomes based on the scoring method employed. Method Sixty undergraduate college students who were determined to be average readers as measured by a word reading and contextual word reading task were administered a spelling task as well as morphological, orthographic, phonemic, and syntactic awareness tasks. Results All four scoring methods were highly correlated suggesting high concurrent validity among the measures. Two linguistic awareness skills, morphological awareness and syntactic awareness, predicted spelling performance on both the dichotomous and continuous scoring methods. Contrastively, phonemic awareness and orthographic awareness predicted spelling performance only when spelling was scored using a continuous measure error analysis. Conclusions The results of this study confirm that multiple linguistic awareness skills are important for spelling in adults who are average readers. The results also highlight the need for using continuous measures of spelling when planning intervention or instruction, particularly in the areas of orthographic and phonemic awareness.

Singlet oxygen ( 1 O 2 ), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent ( flu ) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu -like phenotype. In flu ex1 safe1 , all 1 O 2 -induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1 O 2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1 O 2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu -produced 1 O 2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs., Competing Interests: The authors declare no competing interest.

Childhood asthma and allergies are particularly prevalent diseases. Our objective is to identify respiratory and allergic phenotypes from birth to 6 years of age, and to explore their environmental determinants, especially those related to the home environment. Data on respiratory and allergic health outcomes and domestic environmental exposure were collected for 935 mother-infant pairs from a longitudinal mother-child cohort based on mothers, included before 19 weeks of gestation in Brittany between 2002 and 2006. Information was obtained by self-administered questionnaires completed by parents at inclusion, delivery, and when the child was 2 and 6 years old. Kml3D clustering was used to describe profiles of children who shared similar trajectories of symptoms as phenotypes. Association with environmental determinants was estimated by polytomous logistic regression. Five phenotypes were identified: a reference group characterized by low symptom levels (31.1%), a transient cough phenotype (36.5%), an eczema/cough phenotype (12.3%), a wheeze/cough phenotype (11.8%), and finally a mixed phenotype (8.0%). The wheeze/cough profile was associated with postnatal exposure to glues used in renovation activities (aOR 2.3 [1.2-4.7]), and the mixed phenotype with postnatal exposure to paint (aOR 2.1 [1-4.5]). The phenotypes observed showed some consistencies with those seen in previous studies. Some exposures associated with respiratory/allergic phenotypes observed in this study are avoidable. If confirmed by further research including interventional trials, home-based environmental counseling could be a possible prevention target for primary care professionals.

In plants, highly reactive singlet oxygen (1O2) is known to inhibit photosynthesis and to damage the cell as a cytotoxin. However, more recent studies have also proposed 1O2 as a signal. In plants under stress, not only 1O2 but also other reactive oxygen species (ROS) are generated simultaneously, thus making it difficult to link a particular response to the release of 1O2 and establish a signaling role for this ROS. This obstacle has been overcome by the identification of conditional mutants of Arabidopsis thaliana that selectively generate 1O2 and trigger various 1O2-mediated responses. In chloroplasts of these mutants, chlorophyll or its biosynthetic intermediates may act as a photosensitizer and generate 1O2. These 1O2-mediated responses are not only dependent on the dosage of 1O2 but also are determined by the timing and suborganellar localization of its production. This spatial- and temporal-dependent variability of 1O2-mediated responses emphasizes the importance of 1O2 as a highly versatile and short-lived signal that acts throughout the life cycle of a plant.

Purpose: The purpose of this study was to examine the orthographic fast-mapping abilities of 5- and 6-year-old children across time to determine (a) growth in the ability to quickly acquire mental images of written words, (b) the effect of words' statistical regularities on the learning of written word images across time, (c) whether the statistical regularities of words impact children's eye movements during an orthographic fast-mapping task, and (d) the relation among written word learning and future literacy skills., Method: Twenty-eight 5- and 6-year-old children viewed and listened to 12 short stories while their eye movements were recorded across 2 time points (approximately 3 months apart). At each time point, objects in the stories represented 12 novel pseudowords differing in their phonotactic and orthotactic probabilities. After viewing each story, the children were asked to spell and identify the target pseudowords; they also completed a battery of literacy measures., Results: The children were able to quickly acquire mental orthographic representations of the novel written pseudowords as evidenced by their ability to identify and spell the target pseudowords after viewing the stories. This ability was related to future literacy performance and significantly improved over time. Performance on the orthographic fast-mapping tasks and the children's eye movements at Time 2 were influenced by the words' linguistic properties., Conclusions: This study adds to accumulating evidence that orthographic fast-mapping is largely influenced by the orthotactic probabilities of words. These findings, taken together with those from previous investigations, provide a rich amount of evidence indicating that children are statistical learners when developing their orthographic knowledge.

Formation of singlet oxygen ((1)O2) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, (1)O2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of (1)O2 was first documented in the fluorescent (flu) mutant of Arabidopsis It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by (1)O2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of (1)O2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of (1)O2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and (1)O2-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.

Purpose: We examined grade-level differences in 1st- through 3rd-grade students' performance on an experimenter-developed affix meaning task (AMT) and determined whether AMT performance explained unique variance in word-level reading and reading comprehension, beyond other known contributors to reading development., Method: Forty students at each grade level completed an assessment battery that included measures of phonological awareness, receptive vocabulary, word-level reading, reading comprehension, and affix meaning knowledge., Results: On the AMT, 1st-grade students were significantly less accurate than 2nd- and 3rd-grade students; there was no significant difference in performance between the 2nd- and 3rd-grade students. Regression analyses revealed that the AMT accounted for 8% unique variance of students' performance on word-level reading measures and 6% unique variance of students' performance on the reading comprehension measure, after age, phonological awareness, and receptive vocabulary were explained., Conclusion: These results provide initial information on the development of affix meaning knowledge via an explicit measure in 1st- through 3rd-grade students and demonstrate that affix meaning knowledge uniquely contributes to the development of reading abilities above other known literacy predictors. These findings provide empirical support for how students might use morphological problem solving to read unknown multimorphemic words successfully.

Purpose: As children develop skills in writing across academic contexts, clinicians and educators need to have a fundamental understanding of typical writing development as well as valid and reliable assessment methods. The purpose of this study was to examine the progression of linguistic elements in school-age children's narrative and expository writing development., Method: Narrative and expository writing samples produced by 89 children in Grades 2 through 4 were analyzed at the microstructure and macrostructure levels. Measures of receptive vocabulary, word-level reading, and reading comprehension were obtained., Results: Exploratory factor analyses revealed 4 microstructure factors (e.g., productivity, grammatical complexity, grammatical accuracy, and lexical density) and 1 macrostructure factor (e.g., a combination of organization, text structure, and cohesion). Multivariate analyses of covariance with reading comprehension as a covariate showed that productivity and macrostructure were sensitive to grade-level and genre differences and that expository grammatical complexity was sensitive to grade-level differences., Conclusions: Findings are discussed in light of grade-level standards for narrative and expository writing and current practices in writing assessment. Multiple suggestions are offered for clinical and educational implications, and specific directions are provided for future research.

Necrotrophic fungal pathogens produce toxic compounds that induce cell death in infected plants. Often, the primary targets of these toxins and the way a plant responds to them are not known. In the present work, the effect of tenuazonic acid (TeA), a non-host-specific toxin of Alternaria alternata, on Arabidopsis thaliana has been analysed. TeA blocks the QB -binding site at the acceptor side of photosystem II (PSII). As a result, charge recombination at the reaction centre (RC) of PSII is expected to enhance the formation of the excited triplet state of the RC chlorophyll that promotes generation of singlet oxygen ((1)O₂). (1)O₂ activates a signalling pathway that depends on the two EXECUTER (EX) proteins EX1 and EX2 and triggers a programmed cell death response. In seedlings treated with TeA at half-inhibition concentration (1)O₂-mediated and EX-dependent signalling is activated as indicated by the rapid and transient up-regulation of (1)O₂-responsive genes in wild type, and its suppression in ex1/ex2 mutants. Lesion formation occurs when seedlings are exposed to higher concentrations of TeA for a longer period of time. Under these conditions, the programmed cell death response triggered by (1)O₂-mediated and EX-dependent signalling is superimposed by other events that also contribute to lesion formation., (© 2014 John Wiley & Sons Ltd.)

Purpose: Written English is a morphophonemic language. Researchers have documented that a conscious awareness of the morphological structure of English morphology is predictive of students' written language skills and that morphological awareness instruction leads to improvements in morphological awareness and in other written language skills. The purpose of this tutorial is to provide specific information to clinical scientists and other educators for integrating morphological awareness instruction into their written language instruction., Method: The authors first define morphological awareness and provide an overview of the research on the effects of morphological awareness intervention on improving morphological awareness and written language skills. Measures used to assess morphological awareness ability are then discussed, followed by suggestions for how clinical scientists and other educators can provide morphological awareness instruction to improve the written language skills of the students they serve., Conclusion: By integrating morphological awareness instruction into the services they provide, clinical scientists and other educators will be providing their students with a strong tool to aid written language skills.

This paper describes the theoretical framework, as well as the development and testing of the intervention, Comprehension Tools for Teachers (CTT), which is composed of eight component interventions targeting malleable language and reading comprehension skills that emerging research indicates contribute to proficient reading for understanding for prekindergarteners through fourth graders. Component interventions target processes considered largely automatic as well as more reflective processes, with interacting and reciprocal effects. Specifically, we present component interventions targeting cognitive, linguistic, and text-specific processes, including morphological awareness, syntax, mental-state verbs, comprehension monitoring, narrative and expository text structure, enacted comprehension, academic knowledge, and reading to learn from informational text. Our aim was to develop a tool set composed of intensive meaningful individualized small group interventions. We improved feasibility in regular classrooms through the use of design-based iterative research methods including careful lesson planning, targeted scripting, pre- and postintervention proximal assessments, and technology. In addition to the overall framework, we discuss seven of the component interventions and general results of design and efficacy studies.

Purpose: To examine the utility of different dialect indices that have been used to characterize the Non-Mainstream American English (NMAE) dialect of African American children. The relationships among 4 popular dialect indices were examined and compared with the results of a standardized tool used to classify the language variation of child speakers at 3 different grade levels., Method: The authors used listener judgment ratings, 2 dialect density measures obtained from a narrative sample, a standardized tool (Part 1 of the Diagnostic Evaluation of Language Variation-Screener Test [DELV-ST; Seymour, Roper, & deVilliers, 2003]), and dialect variation scores (DVAR) obtained from the DELV-ST to characterize 113 African American children's spoken production of NMAE., Results: Grade-level effects on NMAE varied depending on the index used to measure dialect production. All of the dialect indices under investigation were related to one another. DELV-ST classification group effects were present on all but 1 of the indices used to capture NMAE., Conclusions: Newer measures of NMAE, such as the DELV-ST and DVAR scores, are comparable to older measures such as dialect density measures and listener judgment ratings. Like listener judgment ratings, the DELV-ST and DVAR scores offer clinicians and researchers alike a quicker alternative to dialect density measures for confirming and quantifying the spoken production of NMAE dialect. The present findings confirm that, depending on the type of data collected and questions posed, researchers and clinicians alike are able to choose from multiple, valid, and reliable measures of non-mainstream dialect use.

Purpose: In this article, the author comments on aspects of Kamhi's (2014) article, which caused the author to think more deeply about definitions of language, theories of learning, and how these two core components of intervention prepare clinical scientists as they search the literature for new knowledge. Interprofessional collaborative practice as a model for team-based intervention in school settings is also discussed., Method: The article addresses aspects of Kamhi's article that aligned or did not align with the author's definition of language and theory of learning and explains why considering these alignments is important for clinical scientists. Two challenges to staying current with the research base are also described, and suggestions are offered for addressing these challenges. Finally, the article provides an overview of interprofessional collaborative practice., Conclusion: Clinical scientists should continue to strive to use their knowledge of what language is and how it is learned, combined with the best available scientific information, to best serve their clients. Combining their thoughts and actions, they undoubtedly will continuously improve their clinical practices.

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant's resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen ((1)O2), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates (1)O2-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether (1)O2- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate (1)O2- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.

We investigated the efficacy of a morphological awareness intervention on the morphological awareness and reading skills of students from low-socioeconomic-status homes; we also examined whether the intervention was similarly effective for intervention students who differed in their initial morphological awareness abilities. The 8-week intervention was designed to increase awareness of affixes and the relations between base words and their inflected and derived forms for kindergarteners (n = 27) and first- (n = 22) and second-grade (n = 26) students. Students randomly assigned to the small group intervention were provided instruction four times a week, 25 min a day, whereas students assigned to the control group received "business as usual." Kindergarten and first- and second-grade students receiving the intervention showed statistically significant gains in morphological awareness with large effect sizes on most measures. Students in all three grades who received the intervention demonstrated nonsignificant gains in literacy abilities with null to small effect sizes. Further, students with low morphological awareness abilities at the onset of the study demonstrated similar gains from the intervention as their peers with typical morphological awareness abilities. Our results suggest that explicit morphological awareness instruction may produce gains of practical importance to young elementary students at risk for future literacy difficulties.

Singlet oxygen ((1)O2)-mediated signaling has been established in the conditional fluorescent (flu) mutant of Arabidopsis. In the dark, the flu mutant accumulates free protochlorophyllide (Pchlide), a photosensitizer that in the light generates (1)O2. The release of (1)O2 leads to growth inhibition of mature plants and bleaching of seedlings. These (1)O2-mediated responses depend on two plastid proteins, EXECUTER (EX) 1 and 2. An ex1/ex2/flu mutant accumulates in the dark Pchlide and upon illumination generates similar amounts of (1)O2 as flu, but (1)O2-mediated responses are abrogated in the triple mutant. The (1)O2- and EX-dependent signaling pathway operates also in wild type placed under light stress. However, it does not act alone as in flu, but interacts with other signaling pathways that modulate (1)O2-mediated responses. Depending on how severe the light stress is, (1)O2- and EX-dependent signaling may be superimposed by (1)O2-mediated signaling that does not depend on EX and is associated with photo-oxidative damage. Because of its high reactivity and short half-life, (1)O2 is unlikely to be a signal that is translocated across the chloroplast envelope, but is likely to interact with other plastid components close to its site of production and to generate more stable signaling molecules during this interaction. Depending on the site of (1)O2 production and the severity of stress, different signaling molecules may be expected that give rise to different (1)O2-mediated responses.

Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis thaliana) mutant snowy cotyledon4 (sco4) revealed a mutation in a chloroplast-targeted protein that shares limited homology with CaaX-type endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photooxidative stress. The sco4 mutation impairs the acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in photosystem I function, decreased linear electron transfer, yet increased nonphotochemical quenching. SCO4 is localized to the chloroplasts, which suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveal that SCO4 is a member of an unknown group of higher plant-specific proteinases quite distinct from the well-described CaaX-type endopeptidases RAS Converting Enzyme1 (RCE1) and zinc metallopeptidase STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light.

Purpose: The relations of phonological, orthographic, and morphological awareness and vocabulary to word reading and spelling were examined for 304 first-grade children who were receiving differentiated instruction in a Response to Intervention (RtI) model of instruction., Method: First-grade children were assessed on their phonological, orthographic, and morphological awareness; expressive vocabulary; word reading; and spelling. Year-end word reading and spelling were outcome variables, and phonological, orthographic, and morphological awareness; expressive vocabulary; and RtI status (Tiers 1, 2, & 3) were predictor variables., Results: The 3 linguistic awareness skills were unique predictors of word reading, and phonological and orthographic awareness were unique predictors of spelling. The contributions that these linguistic awareness skills and vocabulary made to word reading and spelling did not differ by children's RtI tier status., Conclusion: These results, in conjunction with previous studies, suggest that even beginning readers and spellers draw on multiple linguistic awareness skills for their word reading and spelling regardless of their level of literacy abilities. Educational implications are discussed.

Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded proteins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon (NF), an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species (ROS) were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among others, (1)O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.

Purpose: The effect of a morphological awareness intervention on the morphological awareness and literacy skills of students from low socioeconomic status homes was investigated., Method: A 9-week intervention designed to increase awareness of affixes and the relations between base words and their inflected and derived forms was conducted with students in kindergarten (n = 19), 1st grade (n = 21), and 2nd grade (n = 21). Groups of 4-5 students were provided with instruction 4 times a week for 25 min a day., Results: Results showed medium to very large clinically significant gains in morphological awareness and literacy abilities (ds = 0.29-2.96) across all participants., Conclusion: The results of this feasibility study suggest that morphological awareness instruction that requires students to analyze, recognize, orally produce, and determine the spelling patterns of multimorphemic words leads to therapeutic effects within a population of young students who are at risk for future reading difficulties. Initial clinical implications, limitations of the study, and research suggestions are discussed.

Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen ((1)O(2)) in chloroplasts of the flu mutant of Arabidopsis, reprogramming of nuclear gene expression reveals a rapid transfer of signals from the plastid to the nucleus. We have identified extraplastidic signaling constituents involved in (1)O(2)-initiated plastid-to-nucleus signaling and nuclear gene activation after mutagenizing a flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second-site mutations that lead to a constitutive up-regulation of the reporter gene or abrogate its (1)O(2)-dependent up-regulation. One of these mutants, caa39, turned out to be a weak mutant allele of the Topoisomerase VI (Topo VI) A-subunit gene with a single amino acid substitution. Transcript profile analysis of flu and flu caa39 mutants revealed that Topo VI is necessary for the full activation of AAA-ATPase and a set of (1)O(2)-responsive transcripts in response to (1)O(2). Topo VI binds to the promoter of the AAA-ATPase and other (1)O(2)-responsive genes, and hence could directly regulate their expression. Under photoinhibitory stress conditions, which enhance the production of (1)O(2) and H(2)O(2), Topo VI regulates (1)O(2)-responsive and H(2)O(2)-responsive genes in a distinct manner. These results suggest that Topo VI acts as an integrator of multiple signals generated by reactive oxygen species formed in plants under adverse environmental conditions.

Recently the porA-1 null mutant of Arabidopsis thaliana has been identified, which contains an insertion of the Dissociation (Ds) element in the PORA gene (Paddock et al. in Plant Mol Biol 78:447-460, 2012). Light-grown porA-1 seedlings suffer from a drastically reduced chlorophyll content and a developmental arrest beyond the cotyledon stage, suggesting that PORA is not only transiently involved in initiating chlorophyll synthesis during illumination of etiolated seedlings but is also essential for normal growth and plant development. Here we report the presence of a second Ds element in this porA-1 mutant line that inactivates the Speechless gene required for stomata formation. Similar to porA-1, speechless seedlings are severely impaired in their development. Our results suggest that the lack of stomata in porA-1 may contribute to the dwarfed phenotype of the mutant and thus emphasizes the need to re-address the proposed role of PORA during plant development by studying a porA mutant that retains its stomata formation.

Enhanced levels of singlet oxygen ((1)O(2)) in chloroplasts trigger programmed cell death. The impact of (1)O(2) production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates (1)O(2) upon a dark/light shift. The onset of (1)O(2) production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than (1)O(2) directly. In flu seedlings, (1)O(2)-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. (1)O(2)-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of (1)O(2) in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Retrograde plastid-to-nucleus signaling tightly controls and coordinates the nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activity. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens target plastid-specific but not extraplastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the constitutive activator of AAA-ATPase (caa33) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division, and thereby perturbing plastid homeostasis. This results in preconditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the capacity of the plant to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the susceptibility of the plant to stress, and emphasizes the need to consider the possible impact of preconditioning on retrograde plastid-to-nucleus signaling., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The purpose of this study was to examine the relation between the ability to quickly acquire initial mental graphemic representations (MGRs) in kindergarten and fourth grade literacy skills in children with typical language (TL) and children with language impairment (LI). The study is a longitudinal extension of a study conducted by Wolter and Apel in which kindergarten children with LI and TL were administered early literacy measures as well as a novel written pseudoword task of MGR learning (spelling and identification of target pseudowords). In the current study (4 years later), the authors administered reading and spelling measures to 37 of the original 45 children (18 children with LI, 19 children with TL). The children with LI performed significantly lower than their peers with TL on all fourth grade literacy measures. For both groups, kindergarten initial MGR acquisition ability significantly related to fourth grade real-word reading and spelling. For the children with LI, kindergarten initial MGR acquisition ability also related to fourth grade pseudoword decoding and reading comprehension. Collectively, the findings suggest that initial MGR learning in kindergarten is an essential skill that may uniquely relate to later literacy abilities.

Purpose: Orthographic knowledge refers to the information that is stored in memory that tells us how to represent spoken language in written form. Unfortunately, terms used to talk about orthographic knowledge and the two individual components that contribute to it have varied widely in the literature. Thus, consensus on the term, its meaning, and the tasks used to assess orthographic knowledge is needed., Method: In this tutorial, the problems and inconsistencies in terms and tasks used to define and measure orthographic knowledge are reviewed, and suggestions for using specific terms are offered. Additionally, orthographic knowledge development, considerations of its role in literacy development, and its independence from other factors that contribute to literacy development are discussed. Finally, ideas for overcoming current obstacles in discussions of orthographic knowledge and suggestions for future research endeavors are offered., Conclusion: With consensus on what orthographic knowledge is and how it should be measured, practitioners should be better prepared for assessing orthographic knowledge and implementing instructional practices accordingly.

Purpose: In this study, the authors compared the morphological awareness abilities of children with speech sound disorder (SSD) and children with typical speech skills and examined how morphological awareness ability predicted word-level reading and spelling performance above other known contributors to literacy development., Method: Eighty-eight first-grade students--44 students with SSD and no known history of language deficiencies, and 44 students with typical speech and language skills--completed an assessment battery designed to measure speech sound production, morphological awareness, phonemic awareness, letter-name knowledge, receptive vocabulary, word-level reading, and spelling abilities., Results: The children with SSD scored significantly lower than did their counterparts on the morphological awareness measures as well as on phonemic awareness, word-level reading, and spelling tasks. Regression analyses suggested that morphological awareness predicted significant unique variance on the spelling measure for both groups and on the word-level reading measure for the children with typical skills., Conclusion: These results suggest that children with SSD may present with a general linguistic awareness insufficiency, which puts them at risk for difficulties with literacy and literacy-related tasks.

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Purpose: I provide alternative views to Kamhi's (2011) assertion that clinical practice cannot be scientific. I also discuss how the field of communication sciences and disorders might encourage scientific thinking about clinical practices in researchers and clinicians., Method: Kamhi's three main points for why clinical practice cannot be scientific are discussed, and alternate perspectives are offered., Results: Clinicians and clinical practice can be scientific and can contribute to changes in scientific orthodoxy., Conclusion: Science is an attitude that needs to be instilled in future researchers and clinicians during their educational programs. Model clinical scientists should be used as role models when educating these individuals.

Here, we describe the snowy cotyledon3 (sco3-1) mutation, which impairs chloroplast and etioplast development in Arabidopsis thaliana seedlings. SCO3 is a member of a largely uncharacterized protein family unique to the plant kingdom. The sco3-1 mutation alters chloroplast morphology and development, reduces chlorophyll accumulation, impairs thylakoid formation and photosynthesis in seedlings, and results in photoinhibition under extreme CO(2) concentrations in mature leaves. There are no readily apparent changes to chloroplast biology, such as transcription or assembly that explain the disruption to chloroplast biogenesis. Indeed, SCO3 is actually targeted to another organelle, specifically to the periphery of peroxisomes. However, impaired chloroplast development cannot be attributed to perturbed peroxisomal metabolic processes involving germination, fatty acid β-oxidation or photorespiration, though there are so far undescribed changes in low and high CO(2) sensitivity in seedlings and young true leaves. Many of the chloroplasts are bilobed, and some have persistent membranous extensions that encircle other cellular components. Significantly, there are changes to the cytoskeleton in sco3-1, and microtubule inhibitors have similar effects on chloroplast biogenesis as sco3-1 does. The localization of SCO3 to the periphery of the peroxisomes was shown to be dependent on a functional microtubule cytoskeleton. Therefore, the microtubule and peroxisome-associated SCO3 protein is required for chloroplast development, and sco3-1, along with microtubule inhibitors, demonstrates an unexpected role for the cytoskeleton and peroxisomes in chloroplast biogenesis.

Purpose: The authors examined initial acquisition of mental graphemic representations (MGRs) for a set of pseudowords in children with language impairment (LI). They also determined whether the linguistic properties of the words (i.e., phonotactic and orthotactic probabilities) influenced MGR learning and whether the ability to acquire initial MGRs was related to performance on reading and spelling measures. The authors compared these children's initial acquisition of MGRs to a group of children with typical language (TL) skills., Method: A written fast mapping procedure was administered to 25 kindergarten children with LI and 31 with TL. During the procedure, the children were exposed to novel pseudowords varying in phonotactic and orthotactic probabilities in a computer-based, storybook reading task and then were asked to generate and identify those pseudoword spellings. Additionally, reading and spelling tasks were administered., Results: Children with LI acquired some initial MGRs as evidenced by their ability to identify some pseudowords; however, their ability to identify and generate pseudowords was significantly poorer than that of the comparison group. Similar to their TL peers, the ability to identify pseudoword spellings by children with LI was affected by the linguistic properties of words, and written fast mapping performance was related to spelling performance. However, unlike the comparison group, written fast mapping performance of children with LI was not related to performance on a reading composite measure., Conclusions: The authors propose that children with LI are less robust at developing initial MGRs than are children with TL and that this poor ability to acquire initial MGRs likely places them at risk for later literacy deficits.

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ((1)O(2)) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate (1)O(2)-mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of (1)O(2)-responsive genes after a dark-to-light shift they do not show a (1)O(2)-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent (1)O(2)-mediated cell death response without suppressing (1)O(2)-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as 'plastid signaling'.

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in (1)O(2)-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that (1)O(2)-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other (1)O(2)-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting (1)O(2)-mediated retrograde signaling with other signaling pathways.

Purpose: We examined the morphological awareness skills of fourth-grade African American children and the association between degree of African American English (AAE) use and performance on written measures of morphological awareness. Additional purposes were to determine whether performance on the morphological awareness tasks (a) was affected by the transparency of morphologically related words and the type of task administered, (b) was associated with other literacy and literacy-related skills, and (c) explained unique variance on these latter abilities., Method: Thirty fourth-grade African American children from low-income backgrounds were administered 2 morphological awareness tasks and completed norm-referenced measures of word-level reading, reading comprehension, spelling, phonemic awareness, and receptive vocabulary., Results: The degree of AAE use was not associated with students' performance on the morphological awareness tasks. On these tasks, significantly higher scores were obtained on items that represented a transparent relationship between a base word and its derived form. The students' performance on the morphological awareness tasks was significantly and moderately related to their performance on the word-level reading, spelling, and receptive vocabulary measures. Morphological awareness scores explained significant unique variance on measures of word-level reading and spelling, above that predicted by performance on measures of phonemic awareness and vocabulary., Conclusion: As shown in previous investigations of Caucasian children's morphological awareness skills, fourth-grade African American students' morphological awareness abilities are associated with select language and literacy skills. Professionals should capitalize on students' intact capabilities in morphological awareness during literacy instruction in an effort to maximize language and literacy performance for African American students.

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a (1)O(2)-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by (1)O(2) but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether (1)O(2) was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the (1)O(2)-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that (1)O(2)-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development.

Plastid development in seedlings of Arabidopsis thaliana is affected by the transfer of (1)O(2)-mediated retrograde signals from the plastid to the nucleus and changes in nuclear gene expression during late embryogenesis. The potential impact of these mechanisms on plastid differentiation is maintained throughout seed dormancy and becomes effective only after seed germination. Inactivation of the 2 nuclear-encoded plastid proteins EXECUTER1 and EXECUTER2 blocks (1)O(2)-mediated retrograde signaling before the onset of dormancy and impairs normal plastid formation in germinating seeds. This long-term effect of (1)O(2) retrograde signaling depends on the recruitment of abscisic acid (ABA) during seedling development. Unexpectedly, ABA acts as a positive regulator of plastid formation in etiolated and light-grown seedlings.

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

The conditional flu mutant of Arabidopsis accumulates excess amounts of protochlorophyllide within plastid membranes in the dark and generates singlet oxygen upon light exposure. By varying the length of the dark period, the level of the photosensitizer protochlorophyllide may be modulated, and conditions have been established that either endorse the cytotoxicity of (1)O(2) or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of (1)O(2): the impact of the EXECUTER1 mutation and the prevalence of either non-enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings, toxic effects of (1)O(2) prevail and non-enzymatic lipid peroxidation proceeds rapidly. In contrast, in light-grown flu plants that were subjected to an 8 h dark/light shift, lipid peroxidation occurred almost exclusively enzymatically. The resulting oxidation product, 13-hydroperoxy octadecatrienoic acid (13-HPOT), serves as a substrate for synthesis of 12-oxo phytodienoic acid (OPDA) and jasmonic acid (JA), both of which are known to control various metabolic and developmental processes in plants. Inactivation of the EXECUTER1 protein abrogates not only (1)O(2)-mediated cell death and growth inhibition of flu plants, but also enzymatic lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect (1)O(2)-mediated growth inhibition and cell death. Hence, JA and OPDA do not act as second messengers during (1)O(2) signaling, but form an integral part of a stress-related signaling cascade activated by (1)O(2) that encompasses several signaling pathways known to be activated by abiotic and biotic stressors.

In cotyledons of etiolated seedlings light-dependent transformation of etioplasts to chloroplasts marks the transition from heterotrophic to autotrophic growth. Genetic factors required for this developmental step were identified by isolating mutants of Arabidopsis thaliana that were impaired in chloroplast development in cotyledons but not in true leaves. Several mutants with chlorophyll-deficient cotyledons were isolated and dubbed snowy cotyledon (sco). Here we describe the identification and detailed characterization of the snowy cotyledon 2 mutant. The mutated SCO2 gene was identified using a map-based cloning strategy. SCO2 was shown to encode a novel protein which contains a single DnaJ-like zinc finger domain. The SCO2 protein fused to GFP was shown to be present in chloroplasts. Inactivation of SCO2 has almost no detectable impact on the levels of transcripts encoding plastid-specific proteins but leads to a significant reduction of plastid protein levels. Even though transcripts of SCO2 have been found ubiquitously in green tissues as well as in roots phenotypic changes due to SCO2 inactivation are confined to cotyledons. The cotyledons in embryos of sco2 are unaffected in their chloroplast biogenesis. Upon precocious germination seedlings of sco2 and wild type are indistinguishable. The SCO2 mutation affects chloroplast biogenesis only at the end of dormancy during seed germination. The transition from heterotrophic to autotrophic growth is dramatically impaired in sco2 when seedlings were kept in the dark for more than 5 days prior to light exposure.