Elizabeth Harrison

High-Resolution Temporal Profiling of Transcripts during Arabidopsis Leaf Senescence Reveals a Distinct Chronology of Processes and Regulation

This work presents a high-resolution time-course analysis of gene expression during development of a leaf from expansion through senescence. Enrichment in ontologies, sequence motifs, and transcription factor families within genes showing altered expression over time identified both metabolic pathways and potential regulators active at different stages of leaf development and senescence. Leaf senescence is an essential developmental process that impacts dramatically on crop yields and involves altered regulation of thousands of genes and many metabolic and signaling pathways, resulting in major changes in the leaf. The regulation of senescence is complex, and although senescence regulatory genes have been characterized, there is little information on how these function in the global control of the process. We used microarray analysis to obtain a high-resolution time-course profile of gene expression during development of a single leaf over a 3-week period to senescence. A complex experimental design approach and a combination of methods were used to extract high-quality replicated data and to identify differentially expressed genes. The multiple time points enable the use of highly informative clustering to reveal distinct time points at which signaling and metabolic pathways change. Analysis of motif enrichment, as well as comparison of transcription factor (TF) families showing altered expression over the time course, identify clear groups of TFs active at different stages of leaf development and senescence. These data enable connection of metabolic processes, signaling pathways, and specific TF activity, which will underpin the development of network models to elucidate the process of senescence.

Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation

Abstract. Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO2 response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation.

Identification of the tomato ABA-deficient mutant sitiens as a member of the ABA-aldehyde oxidase gene family using genetic and genomic analysis

The sitiens (sit) wilty mutant of tomato (Solanum lycopersicum L.) is deficient in functional enzyme activity at the final step in abscisic acid (ABA) biosynthesis. The biochemical lesion is believed to be an impaired aldehyde oxidase (AO). Molecular mapping using various interspecies crosses has previously shown sit to co-map with a cluster of unresolved RFLP markers on the short arm of chromosome 1. Here, the utilisation of bridging lines to produce interspecies mapping populations involving a self-compatible S. peruvianum accession (LA2157) allowed the fine mapping of sit within this cluster. Identification of a novel AO gene, within the region now known to contain the sit locus, was confirmed by analysis of the tomato whole genome shotgun sequence assembly. This novel AO protein shares 76-78% identity at the amino acid level with the previously characterised tomato AO proteins. The DNA sequence of this putative sit gene was characterised in wild type and in two allelic sit mutants (sit and sitw): changes in DNA sequence were identified in these mutant alleles that cause a truncation of exon 2 and the deletion of exon 7, respectively. These results establish the identity of the tomato sit gene and are consistent with its proposed function of encoding the ABA aldehyde oxidase apoenzyme.

A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns

Petal development and senescence entails a normally irreversible process. It starts with petal expansion and pigment production, and ends with nutrient remobilization and ultimately cell death. In many species this is accompanied by petal abscission. Post-harvest stress is an important factor in limiting petal longevity in cut flowers and accelerates some of the processes of senescence such as petal wilting and abscission. However, some of the effects of moderate stress in young flowers are reversible with appropriate treatments. Transcriptomic studies have shown that distinct gene sets are expressed during petal development and senescence. Despite this, the overlap in gene expression between developmental and stress-induced senescence in petals has not been fully investigated in any species. Here a custom-made cDNA microarray from Alstroemeria petals was used to investigate the overlap in gene expression between developmental changes (bud to first sign of senescence) and typical post-harvest stress treatments. Young flowers were stressed by cold or ambient temperatures without water followed by a recovery and rehydration period. Stressed flowers were still at the bud stage after stress treatments. Microarray analysis showed that ambient dehydration stress accelerates many of the changes in gene expression patterns that would normally occur during developmental senescence. However, a higher proportion of gene expression changes in response to cold stress were specific to this stimulus and not senescence related. The expression of 21 transcription factors was characterized, showing that overlapping sets of regulatory genes are activated during developmental senescence and by different stresses.

Transcriptional regulation of plant senescence: from functional genomics to systems biology.

Leaf senescence is an active process that involves the increased expression of many hundreds of genes. Many putative transcription factors show enhanced transcription during leaf senescence in Arabidopsis and functional analysis of these should help to indicate their role in controlling gene expression during leaf senescence. In this paper, we describe the analysis of knockout insertion mutants in two different senescence-enhanced genes, one encodes a heat shock transcription factor and the other a zinc finger protein. Plants mutated in these genes show accelerated leaf senescence and reduced tolerance to drought stress, indicating that expression of these genes during senescence has a protective role to maintain viability during this essential developmental process. Analysis of gene expression changes in both mutants compared to the wild-type plants indicates an increased rate of senescence but does not show clearly the pathway that is dependent on these genes for expression. The complexities of signalling networks in plant stress and the plasticity of plant responses mean that the direct consequences of mutation are very difficult to define. The usefulness of this type of approach to address the burning question of how senescence is regulated is discussed, and an alternative approach aimed at a more global analysis of gene regulation using systems biology methods is described.

G287 Child maltreatment fatalities: A study of English serious case reviews, 2011–14

Aims To describe the features of all child maltreatment fatalities resulting in a Serious Case Review (SCR) in England in relation to the type of maltreatment fatality. Methods Preliminary data were obtained from the Department for Education (DfE) on all SCRs between April 2011 and December 2013. SCR overview reports were obtained and scrutinised for case characteristics. The deaths were categorised according to a previously developed framework. Case characteristics were compared between different categories of death using comparative statistics. Results A total of 194 child maltreatment fatalities were notified to DfE, of these, SCRs were obtained on 126 (65%). In 5 cases the death was not related to maltreatment; 6 were perpetrated by persons outside the family; and 3 cases could not be classified. A total of 194 child maltreatment fatalities were notified to DfE, of these, SCRs were obtained on 126 (65%). In 5 cases the death was not related to maltreatment; 6 were perpetrated by persons outside the family; and 3 cases could not be classified. Of the 112 remaining cases, 59 were directly caused by maltreatment, and 53 were related to but not directly caused by maltreatment (Table – serious case reviews), including sudden unexpected deaths in infancy with concerns about parenting or other evidence of abuse, and suicides where there were indications of child maltreatment in the background history.Abstract G287 Table 1 The different categories of death differed in relation to key characteristics, including the age of the child, whether or not they were known to social care, the relationship of the suspected perpetrator to the child, and background parental factors including the presence of domestic violence, mental health problems, and drug or alcohol abuse. 55% SCRs commented that the child’s death could not have been predicted or prevented. However, qualitative analysis of the overview reports identified potentially modifiable factors in a majority of cases examined. Conclusions Preventing child maltreatment fatalities does not depend on being able to accurately predict risk of death, but rather to understand the many and varied contexts within which children may be at risk.