Multiscale digital Arabidopsis predicts individual organ and whole-organism growth.

Understanding how dynamic molecular networks affect whole-organism physiology, analogous to mapping genotype to phenotype, remains a key challenge in biology. Quantitative models that represent processes at multiple scales and link understanding from several research domains can help to tackle this problem. Such integrated models are more common in crop science and ecophysiology than in the research communities that elucidate molecular networks. Several laboratories have modeled particular aspects of growth in Arabidopsis thaliana, but it was unclear whether these existing models could productively be combined. We test this approach by constructing a multiscale model of Arabidopsis rosette growth. Four existing models were integrated with minimal parameter modification (leaf water content and one flowering parameter used measured data). The resulting framework model links genetic regulation and biochemical dynamics to events at the organ and whole-plant levels, helping to understand the combined effects of endogenous and environmental regulators on Arabidopsis growth. The framework model was validated and tested with metabolic, physiological, and biomass data from two laboratories, for five photoperiods, three accessions, and a transgenic line, highlighting the plasticity of plant growth strategies. The model was extended to include stochastic development. Model simulations gave insight into the developmental control of leaf production and provided a quantitative explanation for the pleiotropic developmental phenotype caused by overexpression of miR156, which was an open question. Modular, multiscale models, assembling knowledge from systems biology to ecophysiology, will help to understand and to engineer plant behavior from the genome to the field.

The stay-green trait.

Stay-green (sometimes staygreen) refers to the heritable delayed foliar senescence character in model and crop plant species. In a cosmetic stay-green, a lesion interferes with an early step in chlorophyll catabolism. The possible contribution of synthesis to chlorophyll turnover in cosmetic stay-greens is considered. In functional stay-greens, the transition from the carbon capture period to the nitrogen mobilization (senescence) phase of canopy development is delayed, and/or the senescence syndrome proceeds slowly. Yield and composition in high-carbon (C) crops such as cereals, and in high-nitrogen (N) species such as legumes, reflect the source-sink relationship with canopy C capture and N remobilization. Quantitative trait loci studies show that functional stay-green is a valuable trait for improving crop stress tolerance, and is associated with the domestication syndrome in cereals. Stay-green variants reveal how autumnal senescence and dormancy are coordinated in trees. The stay-green phenotype can be the result of alterations in hormone metabolism and signalling, particularly affecting networks involving cytokinins and ethylene. Members of the WRKY and NAC families, and an ever-expanding cast of additional senescence-associated transcription factors, are identifiable by mutations that result in stay-green. Empirical selection for functional stay-green has contributed to increasing crop yields, particularly where it is part of a strategy that also targets other traits such as sink capacity and environmental sensitivity and is associated with appropriate crop management methodology. The onset and progress of senescence are phenological metrics that show climate change sensitivity, indicating that understanding stay-green can contribute to the design of appropriate crop types for future environments.

Bioinformatics in the orphan crops.

Orphan crops are those which are grown as food, animal feed or other crops of some importance in agriculture, but which have not yet received the investment of research effort or funding required to develop significant public bioinformatics resources. Where an orphan crop is related to a well-characterised model plant species, comparative genomics and bioinformatics can often, though not always, be exploited to assist research and crop improvement. This review addresses some challenges and opportunities presented by bioinformatics in the orphan crops, using three examples: forage grasses from the genera Lolium and Festuca, forage legumes and the second generation energy crop Miscanthus.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis.

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6 h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6 hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6 hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6 hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

Evolution of plant senescence.

BACKGROUND: Senescence is integral to the flowering plant life-cycle. Senescence-like processes occur also in non-angiosperm land plants, algae and photosynthetic prokaryotes. Increasing numbers of genes have been assigned functions in the regulation and execution of angiosperm senescence. At the same time there has been a large expansion in the number and taxonomic spread of plant sequences in the genome databases. The present paper uses these resources to make a study of the evolutionary origins of angiosperm senescence based on a survey of the distribution, across plant and microbial taxa, and expression of senescence-related genes. RESULTS: Phylogeny analyses were carried out on protein sequences corresponding to genes with demonstrated functions in angiosperm senescence. They include proteins involved in chlorophyll catabolism and its control, homeoprotein transcription factors, metabolite transporters, enzymes and regulators of carotenoid metabolism and of anthocyanin biosynthesis. Evolutionary timelines for the origins and functions of particular genes were inferred from the taxonomic distribution of sequences homologous to those of angiosperm senescence-related proteins. Turnover of the light energy transduction apparatus is the most ancient element in the senescence syndrome. By contrast, the association of phenylpropanoid metabolism with senescence, and integration of senescence with development and adaptation mediated by transcription factors, are relatively recent innovations of land plants. An extended range of senescence-related genes of Arabidopsis was profiled for coexpression patterns and developmental relationships and revealed a clear carotenoid metabolism grouping, coordinated expression of genes for anthocyanin and flavonoid enzymes and regulators and a cluster pattern of genes for chlorophyll catabolism consistent with functional and evolutionary features of the pathway. CONCLUSION: The expression and phylogenetic characteristics of senescence-related genes allow a framework to be constructed of decisive events in the evolution of the senescence syndrome of modern land-plants. Combining phylogenetic, comparative sequence, gene expression and morphogenetic information leads to the conclusion that biochemical, cellular, integrative and adaptive systems were progressively added to the ancient primary core process of senescence as the evolving plant encountered new environmental and developmental contexts.

Markers and mapping revisited: finding your gene.

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of this highly dynamic arena of plant science.

Nondestructive analysis of senescence in mesophyll cells by spectral resolution of protein synthesis-dependent pigment metabolism.

* Over 6 d of dark-induced senescence, leaf segments of wild-type Lolium temulentum lost > 96% chlorophyll a + b; leaves from plants containing a staygreen mutation introgressed from Festuca pratensis, which has a lesion in the senescence-associated fragmentation of pigment-proteolipid complexes, retained over 43% of total chlorophyll over the same period. * Mutant segments preferentially retained thylakoid membrane proteins (exemplified by LHCP II) but lost other cellular proteins at the same rate as wild-type tissue. The protein synthesis inhibitor D-MDMP inhibited chlorophyll degradation and partially prevented protein loss in both genotypes, but tissues treated with the ineffective L-stereoisomer were indistinguishable from water controls. * Principal-components analysis of leaf reflectance spectra distinguished between genotypes, time points and D-MDMP treatments, showing the disruption of pigment metabolism during senescence brought about by the staygreen mutation, by inhibition of protein synthesis and by combinations of the two factors. * The build-up of oxidized, dephytylated and phaeo-derivatives of chl a during senescence of staygreen tissue was prevented by D-MDMP and associated with characteristic difference spectra when senescent mutant tissue was compared with wild-type or inhibitor-treated samples. The suitability of senescence as a subject for systems biology approaches is discussed.

The hypersensitive response; the centenary is upon us but how much do we know?

With the centenary of the first descriptions of 'hypersensitiveness' following pathogenic challenge upon us, it is appropriate to assess our current understanding of the hypersensitive response (HR) form of cell death. In recent decades our understanding of the initiation, associated signalling, and some important proteolytic events linked to the HR has dramatically increased. Genetic approaches are increasingly elucidating the function of the HR initiating resistance genes and there have been extensive analyses of death-associated signals, calcium, reactive oxygen species (ROS), nitric oxide, salicylic acid, and now sphingolipids. At the same time, attempts to draw parallels between mammalian apoptosis and the HR have been largely unsuccessful and it may be better to consider the HR to be a distinctive form of plant cell death. We will consider if the HR form of cell death may occur through metabolic dysfunction in which malfunctioning organelles may play a major role. This review will highlight that although our knowledge of parts of the HR is excellent, a comprehensive molecular model is still to be attained.

Deep sequence analysis reveals the ovine rumen as a reservoir of antibiotic resistance genes.

Antibiotic resistance is an increasingly important environmental pollutant with direct consequences for human health. Identification of environmental sources of antibiotic resistance genes (ARGs) makes it possible to follow their evolution and prevent their entry into the clinical setting. ARGs have been found in environmental sources exogenous to the original source and previous studies have shown that these genes are capable of being transferred from livestock to humans. Due to the nature of farming and the slaughter of ruminants for food, humans interact with these animals in close proximity, and for this reason it is important to consider the risks to human health. In this study, we characterised the ARG populations in the ovine rumen, termed the resistome. This was done using the Comprehensive Antibiotic Resistance Database (CARD) to identify the presence of genes conferring resistance to antibiotics within the rumen. Genes were successfully mapped to those that confer resistance to a total of 30 different antibiotics. Daptomycin was identified as the most common antibiotic for which resistance is present, suggesting that ruminants may be a source of daptomycin ARGs. Colistin resistance, conferred by the gene pmrE, was also found to be present within all samples, with an average abundance of 800 counts. Due to the high abundance of some ARGs (against daptomycin) and the presence of rare ARGs (against colistin), we suggest further study and monitoring of the rumen resistome as a possible source of clinically relevant ARGs.

Rice pseudomolecule-anchored cross-species DNA sequence alignments indicate regional genomic variation in expressed sequence conservation.

BACKGROUND: Various methods have been developed to explore inter-genomic relationships among plant species. Here, we present a sequence similarity analysis based upon comparison of transcript-assembly and methylation-filtered databases from five plant species and physically anchored rice coding sequences. RESULTS: A comparison of the frequency of sequence alignments, determined by MegaBLAST, between rice coding sequences in TIGR pseudomolecules and annotations vs 4.0 and comprehensive transcript-assembly and methylation-filtered databases from Lolium perenne (ryegrass), Zea mays (maize), Hordeum vulgare (barley), Glycine max (soybean) and Arabidopsis thaliana (thale cress) was undertaken. Each rice pseudomolecule was divided into 10 segments, each containing 10% of the functionally annotated, expressed genes. This indicated a correlation between relative segment position in the rice genome and numbers of alignments with all the queried monocot and dicot plant databases. Colour-coded moving windows of 100 functionally annotated, expressed genes along each pseudomolecule were used to generate 'heat-maps'. These revealed consistent intra- and inter-pseudomolecule variation in the relative concentrations of significant alignments with the tested plant databases. Analysis of the annotations and derived putative expression patterns of rice genes from 'hot-spots' and 'cold-spots' within the heat maps indicated possible functional differences. A similar comparison relating to ancestral duplications of the rice genome indicated that duplications were often associated with 'hot-spots'. CONCLUSION: Physical positions of expressed genes in the rice genome are correlated with the degree of conservation of similar sequences in the transcriptomes of other plant species. This relative conservation is associated with the distribution of different sized gene families and segmentally duplicated loci and may have functional and evolutionary implications.

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence.

Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6-benzylaminopurine (BAP 10(-4)M) or water (control) during 6d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of chloroplasts was maintained in BAP-treated leaves for up to 6d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H(2)O(2) in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark.

Gene expression under temperature stress.

In this review, changes in plant gene expression in response to environmental stresses are discussed using the examples of high and low temperature treatments. While some changes may contribute to acclimatory processes which improve plant survival or performance under stress, others may be 'shock' responses indicative of sensitivity. The heat-shock response, which is almost ubiquitous among eukaryotic organisms, is characterized by repression of normal cellular protein synthesis mediated at both the transcriptional and the translational level, and induction of heat-shock protein (HSP) synthesis. There is a correlation between HSP synthesis and induced thermotolerance in plants, but the evidence for a causal relationship is not conclusive. The possible biochemical functions of some of the HSPs are now becoming apparent; they are believed to play an important role in preventing accumulation of damaged proteins in the cell during heat shock. Although no other environmental stress elicits the full heat-shock response, certain treatments do induce synthesis of subsets of the HSPs, and the reasons for this are considered. Alterations in gene expression in response to low temperatures are more diverse and usually less dramatic than the heat-shock response, with which they share little, if any, homology. Biochemical adjustments during cold treatment are discussed, with particular reference to those which contribute to acclimation. Several genes whose expression is induced by cold have been cloned and characterized, and in some cases it is possible to attribute in vivo functions to them; they include enzymes of lipid, carbohydrate and protein metabolism, structural proteins and putative cryoprotectants. The use of transgenic plants is further facilitating an investigation of the biochemical factors which are important in cold acclimation. Drought, osmotic stress and abscisic acid induce expression of many of the same genes as does cold treatment; it seems likely that some of the products of these genes contribute to increased freezing tolerance by protecting against intracellular dehydration. Contents Summary 1 I. Introduction 1 II. High temperature stress 3 III. Low temperature stress 10 IV. Concluding remarks 20 Acknowledgements 21 References 21.

The effect of the slow-to-green mutation on cell division during leaf initiation and early leaf growth in Lolium temulentum.

Cell division was examined during leaf initiation in the slow-to-green mutant of Lolium temulentum L. to test the hypothesis that the cell cycle in the leaf primordium is a key regulator of the well characterized reduction in final leaf length in the mutant compared with that of the wild type. The cell doubling time (cdt, by colchicine method) was substantially longer in the youngest leaf primordium (YLP) of the mutant (107 h) than in the wild type (43 h) although the duration of the most rapid cell cycle (cc, by percentage labelled mitoses method) was between 18-20 h in each. As a consequence, the proportion of rapidly proliferating cells was only 20 % in the mutant compared with 47 % in the wild type. The size of the shoot apical meristem and the plastochron were similar between genotypes which indicates that the shoot meristem was largely buffered from the effects of the mutation. Mitotic cell area was also similar in the YLP of both genotypes. However, as the leaf elongated, mitotic cell area and interphase cell size were significantly larger in the mutant compared with the wild type. This change was coupled with a reduced number of cells per unit length of leaf in the mutant. The data are consistent in showing that the proportion of rapidly proliferating cells in the YLP (but not the rate of cell division) is a key parameter which influences growth of the leaf.

Leaf development in Lolium temulentum L.: characterization of a slow-to-green mutant.

A nuclear-gene mutation of the C3 grass Lolium temulentum L., which arose following cell suspension culture and plant regeneration, is manifested as delayed and incomplete greening, which occurs from the leaf tip downwards. Many plastids in the mutant exhibit abnormal morphology when examined by transmission electron microscopy; the plastid outer envelope lacks integrity and thylakoids, while still stacked, are spread over a wide area surrounded by diffuse stromal contents. These aberrant plastids can coexist with apparently normal chloroplasts in the same cell of mutant plants. Levels of chlorophyll a and b, and carotenoids, are all lower in the mutants than in normal Lolium temulentum. Leaf length, absolute growth rate, and number of cells per unit length at the leaf base, are greatly reduced (20-30% the normal values) in slow-to-green plants, but relative growth rate, duration of leaf growth, length of cell division zone and proportion of cells dividing are little affected. This novel mutant is a potentially valuable resource for studying interrelationships between photosynthetic function and leaf extension growth in grasses.

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum.

Pigments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 13(2) OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.

Unravelling the evolution of autumn colours: an interdisciplinary approach.

Leaf colour change is commonly observed in temperate deciduous forests in autumn. This is not simply a side effect of leaf senescence, and, in the past decade, several hypotheses have emerged to explain the evolution of autumn colours. Yet a lack of crosstalk between plant physiologists and evolutionary ecologists has resulted in slow progress, and so the adaptive value of this colour change remains a mystery. Here we provide an interdisciplinary summary of the current body of knowledge on autumn colours, and discuss unresolved issues and future avenues of research that might help reveal the evolutionary meaning of this spectacle of nature.

Modification of nitrogen remobilization, grain fill and leaf senescence in maize (Zea mays) by transposon insertional mutagenesis in a protease gene.

A maize (Zea mays) senescence-associated legumain gene, See2beta, was characterized at the physiological and molecular levels to determine its role in senescence and resource allocation. A reverse-genetics screen of a maize Mutator (Mu) population identified a Mu insertion in See2beta. Maize plants homozygous for the insertion were produced. These See2 mutant and sibling wild-type plants were grown under high or low quantities of nitrogen (N). The early development of both genotypes was similar; however, tassel tip and collar emergence occurred earlier in the mutant. Senescence of the mutant leaves followed a similar pattern to that of wild-type leaves, but at later sampling points mutant plants contained more chlorophyll than wild-type plants and showed a small extension in photosynthetic activity. Total plant weight was higher in the wild-type than in the mutant, and there was a genotype x N interaction. Mutant plants under low N maintained cob weight, in contrast to wild-type plants under the same treatment. It is concluded, on the basis of transposon mutagenesis, that See2beta has an important role in N-use and resource allocation under N-limited conditions, and a minor but significant function in the later stages of senescence.