Cassava flowering with emphasis on flowering pattern, morphology and phenology; pollen biology on viability and dimorphism, and histology on male and female gametophyte development are demonstrated. Reduced pollen viability at anthesis and the existence of pollen tri-morphism are the key findings.
Background and aims
Cassava (Manihot esculenta), a major food staple in the tropics and subtropics, thrives even in environments undergoing threatening climate change. To satisfy the increasing demand for crop improvement and overcome the limitations of conventional breeding, the introduction of inbreeding techniques such as the production of doubled haploid lines via androgenesis or gynogenesis offers advantages. However, comprehensive studies on cassava flower bud biology or structural development are lacking and precise structural and biological information is a prerequisite to enhance the efficiency of these techniques.
The floral biology of three selected cassava lines was studied, focusing on morphology, phenology and pollen biology (quantity, viability and dimorphism). Histological studies were also conducted on microsporogenesis/microgametogenesis and megasporogenesis/megagameto-genesis to generate precise developmental data for these lines.
Male and female cyathia have distinct developmental phases. Pollen viability was high during immature stages of plant development; however, pollen mortality was common at later stages. Pollen trimorphism in male gametophytes towards the larger or smaller pollen size, as compared with normal size, was observed. Ten characteristic events were identified in male gametogenesis and six in female gametogenesis that were correlated with flower bud diameter. Male gametophyte diameter at different developmental stages was also determined.
Results indicate that the three lines did not differ significantly, except regarding a few morphological aspects such as plant height, flower colour and number of male cyathia. Pollen grains were initially viable, but viability decreased drastically at later stages of growth. Abnormal meiosis or mitosis triggered pollen trimorphism. The demonstrated sequential events of reproductive development generated valuable information at the cellular level, which will help close the current information gap for cassava improvement via breeding programmes and doubled haploid plant production.
Free auxin and expression of auxin-related genes were measured at ripening in MADS8 suppressed apple fruit. It was found that the delayed ripening in these fruit was associated with high auxin and changes in auxin homeostasis and response genes.
Background and aims
Fruit ripening is an important developmental trait in fleshy fruits, making the fruit palatable for seed dispersers. In some fruit species, there is a strong association between auxin concentrations and fruit ripening. We investigated the relationship between auxin concentrations and the onset of ethylene-related ripening in Malus × domestica (apples) at both the hormone and transcriptome levels.
Transgenic apples suppressed for the SEPALLATA1/2 (SEP1/2) class of gene (MADS8/9) that showed severely reduced ripening were compared with untransformed control apples. In each apple type, free indole-3-acetic acid (IAA) concentrations were measured during early ripening. The changes observed in auxin were assessed in light of global changes in gene expression.
It was found that mature MADS8/9-suppressed apples had a higher concentration of free IAA. This was associated with increased expression of the auxin biosynthetic genes in the indole-3-acetamide pathway. Additionally, in the MADS8/9-suppressed apples, there was less expression of the GH3 auxin-conjugating enzymes. A number of genes involved in the auxin-regulated transcription (AUX/IAA and ARF classes of genes) were also observed to change in expression, suggesting a mechanism for signal transduction at the start of ripening.
The delay in ripening observed in MADS8/9-suppressed apples may be partly due to high auxin concentrations. We propose that, to achieve low auxin associated with fruit maturation, the auxin homeostasis is controlled in a two-pronged manner: (i) by the reduction in biosynthesis and (ii) by an increase in auxin conjugation. This is associated with the change in expression of auxin-signalling genes and the up-regulation of ripening-related genes.
In russeting of apple and pear fruit, a stiff cuticle is replaced by a more plastic periderm. Furthermore, the cell layers underlying the cuticle and the periderm represent the load-bearing structure in the fruit skin in both apple and pear.
Background and aims
Russeting in apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) is a disorder of the fruit skin that results from microscopic cracks in the cuticle and the subsequent formation of a periderm. To better understand russeting, rheological properties of cuticular membranes (CM) and periderm membranes (PM) were studied from the russet-sensitive apple ‘Karmijn de Sonnaville’ and from ‘Conference’ pear.
The CM and PM were isolated enzymatically, investigated by microscopy and subjected to tensile tests, creep/relaxation tests and to stepwise creep tests using a material testing machine.
The isolated CM formed a continuous polymer, whereas the PM represented a cellular structure of stacked cork cells. Tensile tests revealed higher plasticity of the hydrated PM compared with the CM, as indicated by a higher strain at the maximum force (ɛmax) and a lower modulus of elasticity (E). In apple, the maximum force (Fmax) was higher in the CM than in the PM but in pear the higher Fmax value was found for the PM. In specimens obtained from the CM : PM transition zone, the weak point in apple was found to be at the CM : PM borderline but in pear it was within the CM. In both apple and pear, creep/relaxation tests revealed elastic strain, creep strain, viscoelastic strain and viscous strain components in both the PM and CM. For any particular force, strains were always greater in the PM than in the CM and were also greater in pear than in apple. The ɛmax and Fmax values of the CM and PM were lower than those of non-russeted and russeted whole-fruit skin segments, which included adhering tissue.
In russeting, stiff CM are replaced by more plastic PM. Further, the cell layers underlying the CM and PM represent the load-bearing structure in the fruit skin in apple and pear.
cuticular membrane; fracture; fruit skin; mechanical properties; rheology; russet; strain
Nitric oxide (NO) is a plant signal contributing to plant stress responses and development. We here review some of the key advances in this field but also highlight certain key aspects of plant NO biology that require further attention.
Background and aims
After a series of seminal works during the last decade of the 20th century, nitric oxide (NO) is now firmly placed in the pantheon of plant signals. Nitric oxide acts in plant–microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes. By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention.
Scope and conclusions
The following questions will be considered. While cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms, including a poorly defined arginine oxidizing activity, need to be characterized at the molecular level. Other oxidative pathways utilizing polyamine and hydroxylamine also need further attention. Nitric oxide action is dependent on its concentration and spatial generation patterns. However, no single technology currently available is able to provide accurate in planta measurements of spatio-temporal patterns of NO production. It is also the case that pharmaceutical NO donors are used in studies, sometimes with little consideration of the kinetics of NO production. We here include in planta assessments of NO production from diethylamine nitric oxide, S-nitrosoglutathione and sodium nitroprusside following infiltration of tobacco leaves, which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather NO appears to act as a modifier of other signalling pathways. Thus, early reports that NO signalling involves cGMP—as in animal systems—require revisiting. Finally, as plants are exposed to NO from a number of external sources, investigations into the control of NO scavenging by such as non-symbiotic haemoglobins and other sinks for NO should feature more highly. By crystallizing these questions the authors encourage their resolution through the concerted efforts of the plant NO community.
Using sensitive and real-time detection of volatiles from plants with state-of-the-art laser based- and mass spectrometry-based methods many, hypotheses can be tested, revealing the role of the key elements in signalling and action mechanisms in plants.
Trace gas monitoring plays an important role in many areas of life sciences ranging from agrotechnology, microbiology, molecular biology, physiology, and phytopathology. In plants, many processes can be followed by their low-concentration gas emission, for compounds such as ethylene, nitric oxide, ethanol or other volatile organic compounds (VOCs). For this, numerous gas-sensing devices are currently available based on various methods. Among them are the online trace gas detection methods; these have attracted much interest in recent years. Laser-based infrared spectroscopy and proton transfer reaction mass spectrometry are the two most widely used methods, thanks to their high sensitivity at the single part per billion level and their response time of seconds. This paper starts with a short description of each method and presents performances within a wide variety of biological applications. Using these methods, the dynamics of trace gases for ethylene, nitric oxide and other VOCs released by plants under different conditions are recorded and analysed under natural conditions. In this way many hypotheses can be tested, revealing the role of the key elements in signalling and action mechanisms in plants.
Ethylene; laser-based detection; nitric oxide; plant volatiles; proton transfer reaction mass spectrometry; real-time emission; trace gas detection; volatile organic compounds
In this point of view paper, we argue that the monocot genus Hemerocallis (daylily) satisfies multiple criteria for selection as a ‘new model organism’ for intensive biological investigation. We discuss its important and interesting attributes at the biological, horticultural and medicinal levels. These include an intriguing self-incompatibility system, a sophisticated mechanism for flower bud opening and programmed floral death, and a long history of use by man as a vegetable, ornamental and medicinal plant. We examine the potential for modern technical developments to transform Hemerocallis into a valuable model plant.
Genetic model organisms have revolutionized science, and today, with the rapid advances in technology, there is significant potential to launch many more plant species towards model status. However, these new model organisms will have to be carefully selected. Here, we argue that Hemerocallis (daylily) satisfies multiple criteria for selection and deserves serious consideration as a subject of intensive biological investigation. Several attributes of the genus are of great biological interest. These include the strict control of flower opening and, within a short period, the precisely regulated floral death by a programmed cell death system. The self-incompatibility system in Hemerocallis is also noteworthy and deserves more attention. Importantly, the genus is widely cultivated for food, medicinal value and ornamental interest. Hemerocallis has considerable potential as a ‘nutraceutical’ food plant and the source of new compounds with biomedical activity. The genus has also been embraced by ornamental plant breeders and the extraordinary morphological diversity of hybrid cultivars, produced within a relatively short time by amateur enthusiasts, is an exceptional resource for botanical and genetic studies. We explore these points in detail, explaining the reasons why this genus has considerable value—both academic and socio-economic—and deserves new resources devoted to its exploration as a model. Its impact as a future model will be enhanced by its amenability to cultivation in laboratory and field conditions. In addition, established methods for various tissue and cell culture systems as well as transformation will permit maximum exploitation of this genus by science.
Asparagales; daylily; flower opening; medicinal plant; model organism; programmed cell death; self-incompatibility.
Here we discuss opportunities for system-wide analysis of plant volatiles provided by the implementation of non-supervised data processing. We illustrate the value of such approaches by presenting recent findings on wild tobacco volatile emissions using two-dimensional gas chromatography.
Plant volatile organic compound (VOC) production requires a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, remains only partly understood with regard to its physiological and genetic regulation. Additionally, although analytical procedures for plant VOC measurement have become increasingly faster and more sensitive in recent years, pinpointing relevant shifts in VOC production from the thousands of molecular fragments that are generated by modern mass spectrometer instruments remains challenging. Here we discuss novel opportunities for system-wide analysis provided by the implementation of non-targeted data processing and multivariate statistics in VOC analysis. We illustrate the value of implementing non-targeted data processing with examples of recent findings from our group on the interactive control exerted by salivary components of a lepidopteran herbivore, Manduca sexta, on herbivory-induced VOC emissions in the wild tobacco Nicotiana attenuata. Finally, we briefly discuss the use of multi-platform data integration for probing the nature of metabolic and regulatory systems underlying VOC emissions.
Herbivory; metabolomics; Nicotiana attenuata; plant volatiles; two-dimensional gas chromatography; untargeted analysis.
During pollination and pollen tube growth, DNA damage is induced in the male gametes. However, detailed data on the DNA damage response is not available. Our results indicate that the generative cells of plants recognize and manage genomic lesions during pollen tube growth. In particular, we have shown that cell cycle progression of pollen mitosis II is strictly regulated by the spindle assembly checkpoint in response to genomic lesions.
Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AX (γH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired.
DNA double-strand break; DNA repair; generative cell; heavy ion beam; pollen; spindle assembly checkpoint; sperm cell
Little is known about how herbivore-induced plant volatiles affect omnivorous predators. Here we show that the key predator Anthocoris nemorum is differentially attracted to three Salix clones when these are damaged by the detrimental blue willow beetle (Phratora vulgatissima). At least two volatile plant compounds were induced by the herbivore, and these were antennal active in the predator. The results elucidate how plants may recruit omnivorous predators when damaged. These findings could be utilized in crop breeding for increased resistance against herbivores.
While carnivores are known to be attracted to herbivore-induced plant volatiles, little is known about how such volatiles may affect the behaviour of omnivorous predators that may use both plants and herbivores as food. Here, we examine how systemically produced plant volatiles, in response to local herbivore damage, differentially attract a key omnivorous predator, Anthocoris nemorum (Heteroptera: Anthocoridae), to single clones of three species of Salix: S. viminalis, S. dasyclados and S. cinerea. The profiles of the plant volatiles produced were found to vary among Salix clones and between herbivore-damaged and intact plants. Anthocoris nemorum was attracted to the volatiles released from undamaged plants of all three species, but most strongly to a native S. cinerea clone. Plants damaged by the herbivorous leaf beetle Phratora vulgatissima (Coleoptera: Chrysomelidae) were generally more attractive than undamaged plants, with A. nemorum responding to systemic changes in the damaged plants where the experimental design specifically excluded volatiles released from the actual site of damage. When comparing damaged plants, the S. dasyclados clone was more attractive to A. nemorum than the S. viminalis clone—a somewhat surprising result since this Salix clone is considered relatively resistant to P. vulgatissima, and hence offers a limited amount of prey. Our experiments highlight that both constitutive and induced plant volatiles play a role in omnivore attraction, and this emphasizes the importance of considering odours of released volatiles when cropping and breeding Salix for increased resistance to herbivores.
Biocontrol; biological control; blue willow beetle; common flowerbug; E-4,8-dimethyl-1,3,7-nonatriene; GC electro-antennogram; Z-3-hexenyl acetate; short rotation coppice
We developed microsatellites for Manilkara multifida for future conservation genetics studies. M. multifida is a tropical tree that is endemic to Brazil which is currently restricted to fragmented landscapes. Our analysis indicated that all eight microsatellites are promising for assessing population genetics questions in this species.
Manilkara multifida is a tropical tree that is endemic to the Atlantic forests of southern Bahia, Brazil. Currently, populations of this species are restricted to fragmented landscapes that are susceptible to anthropogenic disturbances. Considering this issue, and that there is no genetic information available for this endangered species, we developed microsatellite markers for M. multifida to provide resources for future conservation genetics studies. Using an enriched genomic library, we isolated eight polymorphic microsatellite loci and optimized the amplification conditions for M. multifida. For each locus, we estimated the number of alleles, HE and HO, paternity exclusion Q, individual identity I and fixation index F, and examined the presence of null alleles. The mean number of alleles was 11.9, and the heterozygosity was high at all loci (average HE = 0.809 and HO = 0.777). The combined values for both paternity exclusion and individual identity were Q = 0.9959 and I = 5.45 × 10–11, respectively. No evidence of null alleles was detected. The results of our analysis indicated that all eight microsatellites are promising for assessing questions involving inbreeding, gene flow, co-ancestry and mating patterns in M. multifida.
Conservation; fragmentation; molecular ecology; molecular marker; PCR; population genetics; SSR; tropical rainforest.
While the physiological basis of cassava drought tolerance has been characterized, evaluation of the molecular responses to drought stress remains largely unexplored. This study provides an initial characterization of the molecular response of cassava to drought stress resembling field conditions. The candidate drought tolerance genes in cassava identified in this study can be used as expression-based markers of drought tolerance in cassava or be tested in the context of breeding and engineering drought tolerance in transgenics.
Cassava is an important root crop to resource-poor farmers in marginal areas, where its production faces drought stress constraints. Given the difficulties associated with cassava breeding, a molecular understanding of drought tolerance in cassava will help in the identification of markers for use in marker-assisted selection and genes for transgenic improvement of drought tolerance. This study was carried out to identify candidate drought-tolerance genes and expression-based markers of drought stress in cassava. One drought-tolerant (improved variety) and one drought-susceptible (farmer-preferred) cassava landrace were grown in the glasshouse under well-watered and water-stressed conditions. Their morphological, physiological and molecular responses to drought were characterized. Morphological and physiological measurements indicate that the tolerance of the improved variety is based on drought avoidance, through reduction of water loss via partial stomatal closure. Ten genes that have previously been biologically validated as conferring or being associated with drought tolerance in other plant species were confirmed as being drought responsive in cassava. Four genes (MeALDH, MeZFP, MeMSD and MeRD28) were identified as candidate cassava drought-tolerance genes, as they were exclusively up-regulated in the drought-tolerant genotype to comparable levels known to confer drought tolerance in other species. Based on these genes, we hypothesize that the basis of the tolerance at the cellular level is probably through mitigation of the oxidative burst and osmotic adjustment. This study provides an initial characterization of the molecular response of cassava to drought stress resembling field conditions. The drought-responsive genes can now be used as expression-based markers of drought stress tolerance in cassava, and the candidate tolerance genes tested in the context of breeding (as possible quantitative trait loci) and engineering drought tolerance in transgenics.
Cassava; drought avoidance; drought tolerance; gene expression; osmotic adjustment; oxidative stress; real-time PCR
The order Zingiberales comprises ∼2500 species of tropical to subtropical plants, including agriculturally (e.g. banana, ginger) and horticulturally (e.g. cannas, heliconias, bird-of-paradise) important plants. Throughout the evolution of this order, the stamens have been modified from the ancestral filamentous structures that produce pollen (seen in Banana flowers) to petal-like structures that no longer bear pollen sacs (seen in Canna flowers). This results in a reduction of pollen, but an effective increase in the overall size of the floral display and perhaps in the efficacy of specialized pollinators by converting stamens into ‘petals’. This study investigates the genetic mechanisms that are involved in making petal-like structures in place of pollen-producing stamens.
Flowers of the order Zingiberales demonstrate a remarkable trend of reduction in the number of fertile stamens; from five or six fertile, filamentous stamens bearing two thecae each in Musaceae and Strelitziaceae to just a single petaloid stamen bearing a single theca in Cannaceae and Marantaceae. As one progresses from ancestral to derived floral forms, 5–6 fertile stamens are replaced by 4–5 petaloid staminodes. In Cannaceae and Costaceae, all members of the androecial whorls exhibit petaloidy, including the fertile stamen. In Costaceae, a single fertile stamen develops two thecae embedded on a broad petaloid appendage, while in Cannaceae the single fertile stamen is further reduced to a single theca with a prominent, expanded petaloid appendage. Whether petaloidy of the fertile stamen is a synapomorphy of the entire ginger clade (including Cannaceae, Costaceae, Zingiberaceae and Marantaceae), or the result of independent convergent evolution in Cannaceae, Costaceae, and some Zingiberaceae, is unclear. We combine a developmental series of the formation of the petaloid fertile stamen in Canna indica with data on the expression of B- and C-class floral organ identity genes to elucidate the organogenetic identity of the petaloid stamen and staminodes. Our data indicate that the single fertile theca in C. indica and its petaloid appendage are derived from one-half of the primordium of a single stamen, with no contribution from the remaining part of the stamen (i.e. the second theca primordium) which aborts early in development. The petaloid appendage expands later, and develops from the position of the filament/connective of the developing theca. Floral identity gene expression shows that petal identity genes (i.e. B-class genes) are expressed in all floral organs studied while C-class gene AG-1 is expressed in an increasing gradient from sepals to gynoecium, and AG-2 is expressed in all floral organs except the petals. The canonical model for molecular specification of floral organ identity is not sufficient to explain petaloidy in the androecial whorl in Canna sp. Further studies understanding the regulation of gene networks are required.
Canna; evo-devo; floral development; MADS-box genes; petaloid stamens; petaloidy; Zingiberales
The plant hormone ethylene regulates growth and development as well as stress responses. This review focuses on recent discoveries that support a model for ethylene signal transduction that involves overlapping and non-overlapping roles for members of the ethylene receptor family. The roles of ethylene receptors in regulating plant growth, pathogen responses, and development are discussed. Mechanisms are proposed by which receptors can modulate downstream responses together and independently.
The plant hormone ethylene regulates growth and development as well as responses to biotic and abiotic stresses. Over the last few decades, key elements involved in ethylene signal transduction have been identified through genetic approaches, these elements defining a pathway that extends from initial ethylene perception at the endoplasmic reticulum to changes in transcriptional regulation within the nucleus. Here, we present our current understanding of ethylene signal transduction, focusing on recent developments that support a model with overlapping and non-overlapping roles for members of the ethylene receptor family. We consider the evidence supporting this model for sub-functionalization within the receptor family, and then discuss mechanisms by which such a sub-functionalization may occur. To this end, we consider the importance of receptor interactions in modulating their signal output and how such interactions vary in the receptor family. In addition, we consider evidence indicating that ethylene signal output by the receptors involves both phosphorylation-dependent and phosphorylation-independent mechanisms. We conclude with a current model for signalling by the ethylene receptors placed within the overall context of ethylene signal transduction.
Arabidopsis; ethylene; ethylene receptors; histidine kinase; hormone signalling; sub-functionalization; two-component system
Photosynthetic characteristics of Euphorbia milii are reported for the first time. The occurrence of CAM-cycling is shown to serve as a mechanism of water conservation. This is a detailed and novel report of such CAM mode in the genus, which is abundant in constitutive CAM and C4 species. Anatomical evidence for the possible operation of a CO2-concentrating mechanism around the vascular bundle, the C2 route, is provided. Findings are important for our understanding of evolution of CAM in the genus.
Crassulacean acid metabolism (CAM) occurs in many Euphorbiaceae, particularly Euphorbia, a genus with C3 and C4 species as well. With the aim of contributing to our knowledge of the evolution of CAM in this genus, this study examined the possible occurrence of CAM in Euphorbia milii, a species with leaf succulence and drought tolerance suggestive of this carbon fixation pathway. Leaf anatomy consisted of a palisade parenchyma, a spongy parenchyma and a bundle sheath with chloroplasts, which indicates the possible functioning of C2 photosynthesis. No evidence of nocturnal CO2 fixation was found in plants of E. milii either watered or under drought; watered plants had a low nocturnal respiration rate (R). After 12 days without watering, the photosynthetic rate (PN) decreased 85 % and nocturnal R was nearly zero. Nocturnal H+ accumulation (ΔH+) in watered plants was 18 ± 2 (corresponding to malate) and 18 ± 4 (citrate) μmol H+ (g fresh mass)−1. Respiratory CO2 recycling through acid synthesis contributed to a night-time water saving of 2 and 86 % in watered plants and plants under drought, respectively. Carbon isotopic composition (δ13C) was −25.2 ± 0.7 ‰ in leaves and −24.7 ± 0.1 ‰ in stems. Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH+, no nocturnal CO2 uptake and values of δ13C intermediate between C3 and constitutive CAM plants; ΔH+ was apparently attributable to both malate and citrate. The results suggest that daily malate accumulation results from recycling of part of the nocturnal respiratory CO2, which helps explain the occurrence of an intermediate value of leaf δ13C. Euphorbia milii can be considered as a CAM-cycling species. The significance of the operation of CAM-cycling in E. milii lies in water conservation, rather than carbon acquisition. The possible occurrence of C2 photosynthesis merits research.
CAM-cycling; citrate; transpiration; water saving; water-use efficiency
Apple is a unique horticultural crop that is available to consumers year round, though harvested just once annually. A year-long supply is reliant on current postharvest practices such as refrigeration, controlled atmosphere and chemical treatment. However, disorders can develop during storage leading to loss of the crop at great cost to orchardists and storage facilities. The goal of this work is to develop predictive biomarkers the apple industry can use to market apples susceptible to disorders early, consequently reducing postharvest losses. This article outlines the genomics based approach we are taking to develop such tools, and presents our first list of putative predictive biomarkers.
Several apple cultivars are susceptible to CO2 injury, a physiological disorder that can be expressed either externally or internally, and which can cause major losses of fruit during controlled atmosphere (CA) storage. Disorder development can also be enhanced using SmartFresh™ technology, based on the inhibition of ethylene perception by 1-methylcyclopropene (1-MCP). Injury development is associated with less mature fruit with lower ethylene production, but the aetiology of the disorder is poorly understood. Here we report on the progress made using mRNAseq approaches to explore the transcriptome during the development of external CO2 injury. Next-generation sequencing was used to mine the apple transcriptome for gene expression changes that are associated with the development of external CO2 injury. ‘Empire’ apples from a single orchard were treated with either 1 µL L−1 1-MCP or 1 g L−1 diphenylamine or left untreated, and then stored in a CA of 5 kPa CO2 and 2 kPa O2. In addition, susceptibility to the disorder in the ‘Empire’ apples from five different orchards was investigated and the methylation state of the ACS1 promoter investigated using McrBC endonuclease digestion and real-time quantitative polymerase chain reaction. Expression of over 30 000 genes, aligned to the apple genome, was monitored, with clear divergence of expression among treatments after 1 day of CA storage. Symptom development, internal ethylene concentrations (IECs) and methylation state of the ACS1 promoter were different for each of five orchards. With transcriptomic changes affected by treatment, this dataset will be useful in discovering biomarkers that assess disorder susceptibility. An inverse correlation between the frequency of this disorder and the IEC was detected in a multiple orchard trial. Differential methylation state of the ACS1 promoter correlated with both IEC and injury occurrence, indicating epigenetic regulation of ethylene biosynthesis and possibly events leading to disorder development.
Apple; biomarker; controlled atmosphere; DNA methylation; ethylene; external carbon dioxide injury; transcriptome
Napier grass is an important forage for smallholder dairy farms. However, there has been a comparatively low effort to improve Napier grass. It is necessary to strengthen forage breeding programs for development of cultivars with superior traits like. With a high rich gene pool; correct identification of Napier grass accessions is a prerequisite because the existing germplasm information is scanty and cannot be relied upon for crop improvement. Thus the genetic assessment of various Napier grass accessions from the Eastern Africa region is important for correct cultivar identification in order to fully exploit them in crop improvement strategies.
Napier grass is an important forage crop for dairy production in the tropics; as such, its existing genetic diversity needs to be assessed for conservation. The current study assessed the genetic variation of Napier grass collections from selected regions in Eastern Africa and the International Livestock Research Institute Forage Germplasm-Ethiopia. The diversity of 281 cultivars was investigated using five selective amplified fragment length polymorphism (AFLP) markers and classical population genetic parameters analysed using various software. The number of bands generated was 216 with fragments per primer set ranging from 50 to 115. Mean percentage polymorphic loci was 63.40. Genetic diversity coefficients based on Nei's genetic diversity ranged from 0.0783 to 0.2142 and Shannon's information index ranged from 0.1293 to 0.3445. The Fst value obtained was moderately significant (Fst = 0.1688). Neighbour-joining analysis gave two distinct clusters which did not reflect geographical locations. Analysis of molecular variance showed all variance components to be highly significant (P < 0.001), indicating more variation within (91 %) than between populations (9 %). Results suggested moderate genetic differentiation among Napier grass populations sampled, which could imply a high germplasm exchange within the region. The AFLP markers used in this study efficiently discriminate among cultivars and could be useful in identification and germplasm conservation.
AFLP; conservation; cultivars; genetic diversity; germplasm; Napier grass
Using DNA markers and sequencing we investigated patterns of genetic variability in purple saxifrage, Saxifraga oppositifolia in the isolated Arctic Svalbard archipelago. Purple saxifrage is a circumpolar, ecologically and morphologically variable species with a wide range of habitats. Population genetic structures showed that both genetic variation and differentiation levels are modest, outcrossing is the main mating system, and dispersal and gene flow are important, likely accountable to strong winds and human and animal vectors. Different growth habits (compact, trailing and intermediate) did not possess distinct genetic composition.
We investigated patterns of genetic variability in Saxifraga oppositifolia in the isolated Arctic Svalbard archipelago. The genetic analysis included genotyping using nine polymorphic microsatellite markers and sequencing of the nuclear internal transcribed spacer region. Among populations, mean allele numbers per microsatellite locus ranged from 2.0 to 2.6, and 9 % of alleles were unique. Observed (HO) and expected (HE) heterozygosities averaged 0.522 and 0.445, respectively. Typically negative but non-significant FIS values (mean −0.173) were found in S. oppositifolia populations. FST values were relatively low (mean 0.123). The Bayesian structure analysis provided additional information on population genetic structures. Seven out of 11 studied populations, including populations located both near each other and far apart (distances 5–210 km), showed relatively homogeneous clustering patterns, while one population located on a slope in the main settlement of Longyearbyen possessed a unique genetic structure. The Mantel test proved that there is no significant correlation between genetic and geographical distances. Different growth habits (compact, trailing and intermediate) did not possess distinct genetic compositions based on microsatellite variation. Internal transcribed spacer sequencing revealed 12 polymorphic sites. Among 24 sequenced Svalbard samples, eight haplotypes were detected, none shared by the mainland samples. Population genetic structures of S. oppositifolia in Svalbard show that both genetic variation and differentiation levels are modest, outcrossing is the main mating system, and dispersal and gene flow are important, probably attributable to strong winds and human and animal vectors.
Arctic; ITS sequencing; microsatellites; population genetic structure; Saxifraga oppositifolia
There is a great breach between research made at universities and applications of these “academic results” to commercial purposes. This research is a successful example of this interaction. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of the astaxanthin-producing microalga, H. pluvialis and that improved carotenogenic capacity attained is maintained when the volume of the cultures is scaled up to a commercial size. This research allowed to the company dispose of an improved strain accumulating 30% more astaxanthin that the wild type strain (per dry weight basis) and a 72% more (per culture volume basis).
Astaxanthin is a red ketocarotenoid, widely used as a natural red colourant in marine fish aquaculture and poultry and, recently, as an antioxidant supplement for humans and animals. The green microalga Haematococcus pluvialis is one of the richest natural sources of this pigment. However, its slow growth rate and complex life cycle make mass culture difficult for commercial purposes. The aims of this research were (i) to standardize and apply a genetic improvement programme to a Chilean strain of H. pluvialis in order to improve its carotenogenic capacity and (ii) to evaluate the performance of a selected mutant strain in commercial-sized (125 000 L) open ponds in the north of Chile. Haematococcus pluvialis strain 114 was mutated by ethyl methanesulfonate. The level of mutagen dose (exposure time and concentration) was one that induced at least 90 % mortality. Surviving colonies were screened for resistance to the carotenoid biosynthesis inhibitor diphenylamine (25 µM). Resistant mutants were grown in a 30-mL volume for 30 days, after which the total carotenoid content was determined by spectrophotometry. Tens of mutants with improved carotenogenic capacity compared with the wild-type strain were isolated by the application of these standardized protocols. Some mutants exhibited curious morphological features such as spontaneous release of astaxanthin and loss of flagella. One of the mutants was grown outdoors in commercial-sized open ponds of 125 000 L in the north of Chile. Grown under similar conditions, the mutant strain accumulated 30 % more astaxanthin than the wild-type strain on a per dry weight basis and 72 % more on a per culture volume basis. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of H. pluvialis and that improved carotenogenic capacity is maintained when the volume of the cultures is scaled up to a commercial size.
Astaxanthin; commercial-sized open ponds; Haematococcus pluvialis mutant; North Chile; random mutagenesis.
Misidentification of Aframomum melegueta, an important medicinal plant has huge adverse health implications. Thus, the scientific description and characterization of the accessions in Ghana was imperative. The study found the plant to bear creepy stolons which bear terminal bud for production of tillers instead of rhizomes which had earlier been reported by other investigators. All the accessions had a distinctive non storage bulbous structure at the base of the pseudostem. The UPGMA dendrogram clustered the accession into Ashanti and Eastern accessions based on ecological location. The Eastern accessions were sub-clustered in two groups based on fruit colour.
In spite of the huge economic importance of Aframomum melegueta in the herbal and pharmaceutical industries, its production is limited by lack of planting materials (propagules). The plant also lacks scientific descriptors, which has often led to misidentification with adverse health implications. We therefore aimed at developing a descriptor list to facilitate the identification of A. melegueta using 34 morphometric traits comprising 18 quantitative and 16 qualitative characters. The morphological traits showed that A. melegueta has a characteristic stolon that produces tillers instead of rhizomes. The unweighted pair group method with arithmetic mean using both the nearest-neighbour and complete-linkage methods based on the 34 morphometric traits clustered the eight accessions into two main groups based on ecological location. The accessions from the Eastern and Ashanti regions were separated at similarity coefficients of 0.822 and 0.644, respectively, with a highly significant discriminant function. The Eastern accessions were further clustered into red or yellow fruits at similarity indexes of 0.936 and 0.865 using the nearest-neighbour and complete-linkage methods, respectively. The present study has shown that morphometric traits of A. melegueta are greatly influenced by its ecological habitat. It is envisaged that the descriptor list developed coupled with a morphometric description would enhance its identification and utilization.
Characterization; cluster analysis; descriptors; morphometric traits; phenetic cluster; rhizome; stolon.
ACC oxidase (Malus. domestica ACCO1) catalyzes the final step in the biosynthesis of the plant hormone ethylene. ACCO converts 1-aminocyclopropane-1-carboxylic acid(ACC) to ethylene, cyanide, carbon dioxide and water in the presence of ferrous ion, oxygen, ascorbic acid and bicarbonate. Cyanide, a product of the reaction, activates ACCO. Site-directed mutagenesis investigations revealed binding sites for ACC, bicarbonate and ascorbic acid to include; Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction through post-translational modifications. ACCO is subject to auto-phosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.
1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.
ACC oxidase; ascorbate free radical; ascorbic acid; bicarbonate; cyanide; cysteine protease; phosphorylation; post-translational activities; reaction mechanism; site-directed mutagenesis.
Crop domestication is a remarkable example of evolution of wildly growing plants into cultivable forms through human selection. Following the domestication of rice almost 10,000 years ago, ancient farmers selected many rice lineages for diverse agronomic and cultural traits, like grain size, shape and colour; awn length; pest resistance; and aroma etc. In this study, examining phenotypic traits of a large collection of Indian rice landraces (all accessed from Vrihi, rice seed bank, www.cintdis.org/vrihi) we characterize the huge phenotypic diversity, and find that a few grain, panicle and leaf traits are major drivers of this diversity. We also demonstrate the existence of short grain aromatic landraces perhaps with independently evolved aroma trait; unlike introgression from japonica into indica group, as evidenced in Basmati-type long grain varieties. The independent origin of aroma in indica rice is fascinating as it explores lesser known aspects of indica rice domestication and diversification.
Rice landraces are lineages developed by farmers through artificial selection during the long-term domestication process. Despite huge potential for crop improvement, they are largely understudied in India. Here, we analyse a suite of phenotypic characters from large numbers of Indian landraces comprised of both aromatic and non-aromatic varieties. Our primary aim was to investigate the major determinants of diversity, the strength of segregation among aromatic and non-aromatic landraces as well as that within aromatic landraces. Using principal component analysis, we found that grain length, width and weight, panicle weight and leaf length have the most substantial contribution. Discriminant analysis can effectively distinguish the majority of aromatic from non-aromatic landraces. More interestingly, within aromatic landraces long-grain traditional Basmati and short-grain non-Basmati aromatics remain morphologically well differentiated. The present research emphasizes the general patterns of phenotypic diversity and finds out the most important characters. It also confirms the existence of very unique short-grain aromatic landraces, perhaps carrying signatures of independent origin of an additional aroma quantitative trait locus in the indica group, unlike introgression of specific alleles of the BADH2 gene from the japonica group as in Basmati. We presume that this parallel origin and evolution of aroma in short-grain indica landraces are linked to the long history of rice domestication that involved inheritance of several traits from Oryza nivara, in addition to O. rufipogon. We conclude with a note that the insights from the phenotypic analysis essentially comprise the first part, which will likely be validated with subsequent molecular analysis.
Aromatic; Basmati; domestication; grain length; landraces; phenotypic diversity; rice
Grapevine roots can be exposed to a range of temperatures at any particular moment because the root system can explore large volumes of soil over great depths and distances. A split-pot experiment was designed to assess how vegetative and reproductive development respond to partial and whole root-zone warming following winter dormancy. Simultaneous cooling and warming of parts of the root system slowed shoot elongation, leaf expansion and berry development compared to plants with a fully warmed root-zone, but not to the same extent as those with a fully cooled root-zone.
Heterogeneity in root-zone temperature both vertically and horizontally may contribute to the uneven vegetative and reproductive growth often observed across vineyards. An experiment was designed to assess whether the warmed half of a grapevine root zone could compensate for the cooled half in terms of vegetative growth and reproductive development. We divided the root system of potted Shiraz grapevines bilaterally and applied either a cool or a warm treatment to each half from budburst to fruit set. Shoot growth and inflorescence development were monitored over the season. Simultaneous cooling and warming of parts of the root system decreased shoot elongation, leaf emergence and leaf expansion below that of plants with a fully warmed root zone, but not to the same extent as those with a fully cooled root zone. Inflorescence rachis length, flower number and berry number after fertilization were smaller only in those vines exposed to fully cooled root zones. After terminating the treatments, berry enlargement and the onset of veraison were slowed in those vines that had been exposed to complete or partial root-zone cooling. Grapevines exposed to partial root-zone cooling were thus delayed in vegetative and reproductive development, but the inhibition was greater in those plants whose entire root system had been cooled.
Berry composition; grapevine; Shiraz; shoot growth; soil temperature; split pot; Vitis vinifera.
FISH-mapping to meiotic chromosomes at pachytene is an important tool in plant cytogenetic research as it provides good resolution measurements of physical distances. This publication brings a new and more efficient protocol for the application of FISH technique for the first time in meiotic pachytene chromosomes of coffee.This new protocol involves some procedures for obtain suitable pachytene chromosomes that allows the making of a higher-resolution cytogenetic mapping on coffee chromosomes than that mapping on mitotic chromosomes. The use of this method expands the possibilities for high definition physical mapping of coffee chromosomes.
Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes.
Coffea; FISH; meiotic chromosomes; molecular cytogenetics; physical mapping; rDNA.
In the study of geographic range boundary development, the focus has been on leading rather than on trailing edge dynamics. This is an important caveat as trailing edge dynamics will be critical for an understanding population level persistence. Our study begins to fill this knowledge gap and extends the conceptual framework of the field by focusing on trans-generational environmental effects. We found that while these effects may overcome some constraints on stress tolerance evolution and range expansion, other constraints may be created to limit range.
Areas just across species range boundaries are often stressful, but even with ample genetic variation within and among range-margin populations, adaptation towards stress tolerance across range boundaries often does not occur. Adaptive trans-generational plasticity should allow organisms to circumvent these problems for temporary range expansion; however, range boundaries often persist. To investigate this dilemma, we drought stressed a parent generation of Boechera stricta (A.Gray) A. Löve & D. Löve, a perennial wild relative of Arabidopsis, representing genetic variation within and among several low-elevation range margin populations. Boechera stricta is restricted to higher, moister elevations in temperate regions where generalist herbivores are often less common. Previous reports indicate a negative genetic correlation (genetic tradeoff) between chemical defence allocation and abiotic stress tolerance that may prevent the simultaneous evolution of defence and drought tolerance that would be needed for range expansion. In growth chamber experiments, the genetic tradeoff became undetectable among offspring sib-families whose parents had been drought treated, suggesting that the stress-induced trans-generational plasticity may circumvent the genetic tradeoff and thus enable range expansion. However, the trans-generational effects also included a conflict between plastic responses (environmental tradeoff); offspring whose parents were drought treated were more drought tolerant, but had lower levels of glucosinolate toxins that function in defence against generalist herbivores. We suggest that either the genetic or environmental tradeoff between defence allocation and stress tolerance has the potential to contribute to range limit development in upland mustards.
Boechera stricta; chemical defence; drought tolerance; epigenetic; glucosinolate; range limit; tradeoff
Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.
Background and aims
Dendrobium hookerianum is a rare and threatened epiphytic orchid of northeast India. Prospects for conservation would be strengthened by developing an in vitro method for mass propagation. Seeds are minute and difficult to use directly in the field for this purpose, being non-endospermous with a low nutrient content and dependent on a specific fungus for germination and early seedling development. Although produced in large numbers (2–3 million per capsule), <5 % germinate naturally in the wild. Our objective was to develop a rapid and successful method for in vitro propagation based on an initial in vitro asymbiotic seed germination step that achieved high percentages.
Effects of four different media, i.e. (i) Murashige and Skoog (MS), (ii) Mitra et al., (iii) Knudson (KC) and (iv) Gamborg et al. (B5), were evaluated for large-scale multiplication by asymbiotic seed germination. Seedling leaf number, shoot number, shoot length, root number and root length were scored. After 7–8 months, large numbers of well-rooted plantlets were transferred to a glasshouse in thermocol pots containing compost. Six different composts based on broken brick and charcoal were compared for their ability to support further development over 90 days of hardening.
The fastest and highest percentage seed germination was achieved using MS medium. Seeds on MS medium germinated in 3–4 weeks compared with 7–8 weeks on B5 medium. Seedling development was also superior on MS medium. The inclusion of plant growth regulators was unnecessary. Compost comprising broken brick and charcoal with an upper layer of moss was found to be the most suitable for the survival of transferred plantlets. Ninety per cent survival of plantlets was achieved 90 days after transfer to a glasshouse.
The use of MS culture medium is well suited for the mass multiplication of D. hookerianum plants intended for re-introducing this threatened orchid into the wild.