Ray flower initiation in the Helianthus radula inflorescence is influenced by a functional allele of the HrCYC2c gene.

The radiate pseudanthium, with actinomorphic disk flowers surrounded by showy marginal zygomorphic ray flowers, is the most common inflorescence in the Helianthus genus. In Helianthus radula, ray flower primordia are normally absent at the dorsal domain of the inner phyllaries (discoid heads) while the occurrence of radiate inflorescences is uncommon. In Helianthus spp., flower symmetry and inflorescence architecture are mainly controlled by CYCLOIDEA (CYC)-like genes but the putative role of these genes in the development of discoid inflorescences has not been investigate. Three CYC genes of H. radula with a role in ray flower identity (HrCYC2c, HrCYC2d, and HrCYC2e) were isolated. The phylogenetic analysis placed these genes within the CYC2 subclade. We identified two different alleles for the HrCYC2c gene. A mutant allele, designed HrCYC2c-m, shows a thymine to adenine transversion, which generates a TGA stop codon after a translation of 14 amino acids. We established homozygous dominant (HrCYC2c/HrCYC2c) and recessive (HrCYC2c-m/HrCYC2c-m) plants for this nonsense mutation. Inflorescences of both HrCYC2c/HrCYC2c and HrCYC2c/HrCYC2c-m plants initiated ray flowers, despite at low frequency. By contrast, plants homozygous for the mutant allele (HrCYC2c-m/HrCYC2c-m) failed at all to develop ray flowers. The results support, for the first time, a role of the HrCYC2c gene on the initiation of ray flower primordia. However, also in the two dominant phenotypes, discoid heads are the prevalent architecture suggesting that this gene is required but not sufficient to initiate ray flowers in pseudanthia. Other unknown major genes are most likely required in the shift from discoid to radiate inflorescence.

Terahertz probing of sunflower leaf multilayer organization.

We analyze the multilayer structure of sunflower leaves from Terahertz data measured in the time-domain at a ps scale. Thin film reverse engineering techniques are applied to the Fourier amplitude of the reflected and transmitted signals in the frequency range f < 1.5 Terahertz (THz). Validation is first performed with success on etalon samples. The optimal structure of the leaf is found to be a 8-layer stack, in good agreement with microscopy investigations. Results may open the door to a complementary classification of leaves.

Massive haplotypes underlie ecotypic differentiation in sunflowers.

Species often include multiple ecotypes that are adapted to different environments1. However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are maintained despite hybridization with non-adapted populations2-4. Here, by resequencing 1,506 wild sunflowers from 3 species (Helianthus annuus, Helianthus petiolaris and Helianthus argophyllus), we identify 37 large (1-100 Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes of the silverleaf sunflower H. argophyllus (probably through deletion of a homologue of FLOWERING LOCUS T (FT)), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other-possibly now-extinct-congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation.

Genetic and phenotypic analyses indicate that resistance to flooding stress is uncoupled from performance in cultivated sunflower.

Given the rising risk of extreme weather caused by climate change, enhancement of abiotic stress resistance in crops is increasingly urgent. But will the development of stress-resistant cultivars come at the cost of yield under ideal conditions? We hypothesize that this need not be inevitable, because resistance alleles with minimal pleiotropic costs may evade artificial selection and be retained in crop germplasm. Genome-wide association (GWA) analyses for variation in plant performance and flooding response were conducted in cultivated sunflower, a globally important oilseed. We observed broad variation in flooding responses among genotypes. Flooding resistance was not strongly correlated with performance in control conditions, suggesting no inherent trade-offs. Consistent with this finding, we identified a subset of loci conferring flooding resistance, but lacking antagonistic effects on growth. Genetic diversity loss at candidate genes underlying these loci was significantly less than for other resistance genes during cultivated sunflower evolution. Despite bottlenecks associated with domestication and improvement, low-cost resistance alleles remain within the cultivated sunflower gene pool. Thus, development of cultivars that are both flooding-tolerant and highly productive should be straightforward. Results further indicate that estimates of pleiotropic costs from GWA analyses explain, in part, patterns of diversity loss in crop genomes.

Deciphering the nitric oxide, cyanide and iron-mediated actions of sodium nitroprusside in cotyledons of salt stressed sunflower seedlings.

Nitric oxide (NO) is an endogenous signaling molecule in plants. Sodium nitroprusside (SNP), an established NO donor used in plant science research, simultaneously releases NO, cyanide (CN-) and iron (Fe) in solution. Since cyanide and iron mask NO effect of SNP, its use in NO research is debatable. Deciphering the action of SNP through NO, CN- or Fe has been undertaken in the present work. Cotyledons from salt stressed sunflower seedlings grown in the presence of NO donors were subjected to spectrofluorometric analysis of NO, CN- and Fe contents, and proteome and biochemical analyses. Diethylenetriamine NONOate (DETA) proved to be a better NO source since SNP enhanced ROS accumulation in the tissue. Abundance of 127 proteins is modulated by salt stress. SNP and exhausted SNP (exSNP) alter the abundance of 117 and 129 proteins, respectively. These proteins belong to primary metabolism, stress-response, transport, translation, proteolysis, chaperone, regulatory, and storage. Salt-responsive proteins, such as, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and isocitrate dehydrogenase are negatively modulated. DETA and SNP lower the activities of GAPDH and S-adenosylmethionine synthase (SAMS). Abundance of heat shock 70 kDa protein and actin are sensitive to both NaCl and NO. SNP affects plant growth by modulating proteome though iron, cyanide and NO. Its use only as an NO donor is thus debatable. exSNP control also releases substantial amount of cyanide and iron, thus questioning its use as control in NO research.

Characterization of lingering hope, a new brachytic mutant in sunflower (Helianthus annuus L.) with altered salicylic acid metabolism.

Dwarf mutants are useful to elucidate regulatory mechanisms of plant growth and development. A brachytic mutant, named lingering hope (linho), was recently isolated from sunflower (Helianthus annuus). The aim of this report is the characterization of the mutant through genetic, morphometric, physiological and gene expression analyses. The brachytic trait is controlled by a recessive gene. The reduction of plant height depends on shorter apical internodes. The mutant shows an altered ratio length/width of the leaf blade, chlorosis and defects in inflorescence development. The brachytic trait is not associated to a specific hormonal deficiency, but an increased level of several gibberellins is detected in leaves. Notably, the endogenous salicylic acid (SA) content in young leaves of the mutant is very high despite a low level of SA 2-O-ß-d-glucoside (SAG). The CO2 assimilation rate significantly decreases in the second pair of leaves of linho, due to effects of both stomatal and non-stomatal constraints. In addition, the reduction of both actual and potential photochemical efficiency of photosystem II is associated with a reduced content of chlorophylls and carotenoids, a lower chlorophyll a to chlorophyll b ratio and a higher SA content. In comparison to wild type, linho shows a different pattern of gene expression with respect two pathogenesis-related genes and two genes involved in SA biosynthesis and SA metabolism. linho is the first mutant described in sunflower with altered SA metabolism and this genotype could be useful to improve information about the effects of high endogenous content of SA on plant development, reproductive growth and photosynthesis, in a major crop.

Multiple genomic regions influence root morphology and seedling growth in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited conditions.

With climate change and an ever-increasing human population threatening food security, developing a better understanding of the genetic basis of crop performance under stressful conditions has become increasingly important. Here, we used genome-wide association studies to genetically dissect variation in seedling growth traits in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited (i.e., osmotic stress) conditions, with a particular focus on root morphology. Water limitation reduced seedling size and produced a shift toward deeper rooting. These effects varied across genotypes, and we identified 13 genomic regions that were associated with traits of interest across the two environments. These regions varied in size from a single marker to 186.2 Mbp and harbored numerous genes, some of which are known to be involved in the plant growth/development as well as the response to osmotic stress. In many cases, these associations corresponded to growth traits where the common allele outperformed the rare variant, suggesting that selection for increased vigor during the evolution of cultivated sunflower might be responsible for the relatively high frequency of these alleles. We also found evidence of pleiotropy across multiple traits, as well as numerous environmentally independent genetic effects. Overall, our results indicate the existence of genetic variation in root morphology and allocation and further suggest that the majority of alleles associated with these traits have consistent effects across environments.

Identification of SSR and retrotransposon-based molecular markers linked to morphological characters in oily sunfl ower (Helianthus annuus L.) under natural and water-limited states.

Sunflower is an important source of edible oil. Drought is known as an important factor limiting the growth and productivity of field crops in most parts of the world. Agricultural biotechnology mainly aims at developing crops with higher tolerance to the challenging environmental conditions, such as drought. This study examined a number of morphological characters, along with relative water content (RWC) in 100 inbred sunflower lines. A 10 × 10 simple lattice design with two replications was employed to measure the mentioned parameters under natural and water-limited states during two successive years. In molecular trial, 30 simple sequence repeat (SSR) primer pairs, as well as 14 inter-retrotransposon amplified polymorphism (IRAP) and 14 retrotransposon-microsatellite amplified polymorphism (REMAP) primer combinations were used for DNA fingerprinting of the lines. Most of the examined characters had lower average values under water-limited than natural states. Maximum and minimum reductions were observed in the cases of yield and oil percentage, respectively. The broad-sense heritabilities for all the examined characters were 0.20-0.73 and 0.10-0.34 under natural and water-limited states, respectively. In the studied samples, 8.97% of the 435 possible locus pairs of the SSRs represented significant linkage disequilibrium (LD) levels. In the association analysis using SSR markers, 22 and 21 markers were identified (P ≤ 0.05) for the studied characters under natural and water-limited states, respectively. The corresponding values were 50 and 37 using retrotransposon-based molecular markers. Some detected markers were communal between the characters under water-limited and natural states. This was in line with the phenotypic correlations detected between the characters. Communal markers facilitate the simultaneous selection of several characters and can thus improve the efficacy of selection based on markers in the plant-breeding activities.

Source-sink relations of sunflower plants as affected by a parasite modifies carbon allocations and leaf traits.

Sunflower broomrape (Orobanche cumana) is a root holoparasitic plant causing major damage to sunflower (Helianthus annuus L.). Parasite infection initiates source-sink relations between the parasite (sink) and the host (source), allocating carbohydrates, water and nutrients to the parasite. The primary aim of the current study was to explore responses of sunflower to broomrape parasitism, specifically to examine alternations in leaf area, leaf mass per area (LMA), mesophyll structure and root hydraulic conductivity. Leaf changes revealed modifications similar to described previously in shade adapted plants, causing larger and thinner leaves. These traits were accompanied with significantly higher root hydraulics. These changes were caused by carbohydrate depletion due to source-sink relationships between the host and parasite. An Imazapic herbicide (ALS inhibitor) was used for controlling broomrape attachments and by to investigate the plasticity of the traits found. Broomrape infected plants which were treated with Imazapic had leaves similar to non-infected plants, including mesophyll structure and carbon assimilation rates. These results demonstrated source-sink effects of broomrape which cause a low-light-like acclimation behavior which is reversible.

A new method for the rapid characterization of root growth and distribution using digital image correlation.

Rapidly determining root growth patterns is biologically important and technically challenging. Current methods focus on direct observation of roots and require destructive excavations or time-consuming root tracing. We developed a novel methodology based on analyzing soil particle displacement, rather than direct observation of roots. This inferred root growth method uses digital image correlation (DIC) analysis, an established and high-throughput method used in many engineering and science disciplines. By applying DIC analyses to repeated images of plants grown in clear window boxes, we produced visually intuitive and quantifiable strain maps, indicating the magnitude and direction of soil movement. From this, we could infer root growth and rapidly quantify root system metrics. Strain measures were closely associated with the spatial distribution of roots and correlated with root length measured using conventional approaches. The method also allowed for the detection of root proliferation in nutrient-enriched soil patches, indicating its suitability for quantifying biological patterns. This novel application of DIC in root biology is effective, scalable, low cost, flexible and complementary to existing technologies. This method offers a new tool for answering questions in plant biology and will be particularly useful in studies involving temporal dynamics of root processes.

Intraspecific variation in embolism resistance and stem anatomy across four sunflower (Helianthus annuus L.) accessions.

Drought-induced xylem embolism is a key process closely related to plant mortality during extreme drought events. However, this process has been poorly investigated in crop species to date, despite the observed decline of crop productivity under extreme drought conditions. Interspecific variation in hydraulic traits has frequently been reported, but less is known about intraspecific variation in crops. We assessed the intraspecific variability of embolism resistance in four sunflower (Helianthus annuus L.) accessions grown in well-watered conditions. Vulnerability to embolism was determined by the in situ flow-centrifuge method (cavitron), and possible trade-offs between xylem safety, xylem efficiency and growth were assessed. The relationship between stem anatomy and hydraulic traits was also investigated. Mean P50 was -3 MPa, but significant variation was observed between accessions, with values ranging between -2.67 and -3.22 MPa. Embolism resistance was negatively related to growth and positively related to xylem-specific hydraulic conductivity. There is, therefore, a trade-off between hydraulic safety and growth but not between hydraulic safety and efficiency. Finally, we found that a few anatomical traits, such as vessel density and the area of the vessel lumen relative to that of the secondary xylem, were related to embolism resistance, whereas stem tissue lignification was not. Further investigations are now required to investigate the link between the observed variability of embolism resistance and yield, to facilitate the identification of breeding strategies to improve yields in an increasingly arid world.

Growing larger with domestication: a matter of physiology, morphology or allocation?

Domestication might affect plant size. We investigated whether herbaceous crops are larger than their wild progenitors, and the traits that influence size variation. We grew six crop plants and their wild progenitors under common garden conditions. We measured the aboveground biomass gain by individual plants during the vegetative stage. We then tested whether photosynthesis rate, biomass allocation to leaves, leaf size and specific leaf area (SLA) accounted for variations in whole-plant photosynthesis, and ultimately in aboveground biomass. Despite variations among crops, domestication generally increased the aboveground biomass (average effect +1.38, Cohen's d effect size). Domesticated plants invested less in leaves and more in stems than their wild progenitors. Photosynthesis rates remained similar after domestication. Variations in whole-plant C gains could not be explained by changes in leaf photosynthesis. Leaves were larger after domestication, which provided the main contribution to increases in leaf area per plant and plant-level C gain, and ultimately to larger aboveground biomass. In general, cultivated plants have become larger since domestication. In our six crops, this occurred despite lower investment in leaves, comparable leaf-level photosynthesis and similar biomass costs of leaf area (i.e. SLA) than their wild progenitors. Increased leaf size was the main driver of increases in aboveground size. Thus, we suggest that large seeds, which are also typical of crops, might produce individuals with larger organs (i.e. leaves) via cascading effects throughout ontogeny. Larger leaves would then scale into larger whole plants, which might partly explain the increases in size that accompanied domestication.

Evolution of the leaf economics spectrum in herbs: Evidence from environmental divergences in leaf physiology across Helianthus (Asteraceae).

The leaf economics spectrum (LES) describes a major axis of plant functional trait variation worldwide, defining suites of leaf traits aligned with resource-acquisitive to resource-conservative ecological strategies. The LES has been interpreted to arise from leaf-level trade-offs among ecophysiological traits common to all plants. However, it has been suggested that the defining leaf-level trade-offs of the LES may not hold within specific functional groups (e.g., herbs) nor within many groups of closely related species, which challenges the usefulness of the LES paradigm across evolutionary scales. Here, we examine the evolution of the LES across 28 species of the diverse herbaceous genus Helianthus (the sunflowers), which occupies a wide range of habitats and climate variation across North America. Using a phylogenetic comparative approach, we find repeated evolution of more resource-acquisitive LES strategies in cooler, drier, and more fertile environments. We also find macroevolutionary correlations among LES traits that recapitulate aspects of the global LES, but with one major difference: leaf mass per area is uncorrelated with leaf lifespan. This indicates that whole-plant processes likely drive variation in leaf lifespan across Helianthus, rather than leaf-level trade-offs. These results suggest that LES patterns do not reflect universal physiological trade-offs at small evolutionary scales.

Antimony (SbIII) reduces growth, declines photosynthesis, and modifies leaf tissue anatomy in sunflower (Helianthus annuus L.).

The role of antimony (Sb)--a non-essential trace metalloid--in physiological processes running in crops is still poorly understood. Present paper describes the effect of Sb tartrate (SbIII) on growth, Sb uptake, photosynthesis, photosynthetic pigments, and leaf tissue organization in young sunflower plants grown in hydroponics. We found that growth of below- and aboveground part was reduced with increasing concentration of Sb in the medium. Although Sb was mostly taken up by sunflower roots and only small part (1-2%) was translocated to the shoots, decline in photosynthesis, transpiration, and decreased content of photosynthetic pigments were observed. This indicates that despite relatively low mobility of Sb in root-shoot system, Sb in shoot noticeably modifies physiological status and reduced plant growth. Additionally, leaf anatomical changes indicated that Sb reduced the size of intercellular spaces and made leaf tissue more compact.

Ectopic expression of the HAM59 gene causes homeotic transformations of reproductive organs in sunflower (Helianthus annuus L.).

The function of the HAM59 MADS-box gene in sunflower (Helianthus annuus L.) was studied to clarify homeotic C activity in the Asteraceae plant family. For the first time, transgenic sunflower plants with a modified pattern of HAM59 expression were obtained. It was shown that the HAM59 MADS-box transcription factor did mediate C activity in sunflower. In particular, it participated in termination of the floral meristem, repression of the cadastral function of A-activity, and together with other C-type sunflower protein HAM45-in the specification of the identity of stamens and pistils.

Pericarp anatomy and hormone profiles of cypselas in dormant and non-dormant inbred sunflower lines.

The pericarp anatomy and the effects of storage after harvest, storage temperature and early cypsela imbibition on phytohormone profiles were studied in inbred sunflower lines B123 and B91. On day 0, germination of B123 cypselas was near 0%, indicating dormancy, whereas that of B91 cypselas was near 100%, indicating non-dormancy. The germination of B123 and B91 on day 33 at room temperature (25 °C) storage was similar. Cell wall thickness and sclerification of the pericarp were higher in B123 than B91, suggesting that structural characteristics may contribute to physical dormancy in B123. Jasmonates (JAs), salicylic acid (SA) and abscisic acid (ABA) were measured in dry and imbibed pericarps. SA content of dry pericarp was higher on day 33 than day 0. SA content during imbibition on day 33 was similar for room and low (-20 °C) storage temperatures. ABA content after 12 h imbibition was similar on days 0 and 33 at low temperature, but it increased on day 33 at room temperature for B123. 12-Oxo-phytodienoic acid (OPDA) was maximal on day 0 for B123, but peaked at day 33 at low temperature for B91. JA was higher on days 0 and 33 at room temperature as compared with low temperature. Our findings indicate that pericarp hormone profiles are affected in the two lines with different dormancy degree depending on storage conditions and imbibition processes.

RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne).

Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

Ontogeny strongly and differentially alters leaf economic and other key traits in three diverse Helianthus species.

The leaf economics spectrum (LES) describes large cross-species variation in suites of leaf functional traits ranging from resource-acquisitive to resource-conservative strategies. Such strategies have been integral in explaining plant adaptation to diverse environments, and have been linked to numerous ecosystem processes. The LES has previously been found to be significantly modulated by climate, soil fertility, biogeography, growth form, and life history. One largely unexplored aspect of LES variation, whole-plant ontogeny, is investigated here using multiple populations of three very different species of sunflower: Helianthus annuus, Helianthus mollis, and Helianthus radula. Plants were grown under environmentally controlled conditions and assessed for LES and related traits at four key developmental stages, using recently matured leaves to standardize for leaf age. Nearly every trait exhibited a significant ontogenetic shift in one or more species, with trait patterns differing among populations and species. Photosynthetic rate, leaf nitrogen concentration, and leaf mass per area exhibited surprisingly large changes, spanning over two-thirds of the original cross-species LES variation and shifting from resource-acquisitive to resource-conservative strategies as the plants matured. Other traits being investigated in relation to the LES, such as leaf water content, pH, and vein density, also showed large changes. The finding that ontogenetic variation in LES strategy can be substantial leads to a recommendation of standardization by developmental stage when assessing 'species values' of labile traits for comparative approaches. Additionally, the substantial ontogenetic trait shifts seen within single individuals provide an opportunity to uncover the contribution of gene regulatory changes to variation in LES traits.

Morphological variation of mutant sunflowers (Helianthus annuus) induced by space flight and their genetic background detection by SSR primers.

After sunflower seeds were exposed to space conditions, various mutant plants were screened from the descendent plants. The morphological characters of plants changed in flower color from golden to yellow, light yellow, or even to yellowish green. The ligulate petals of the unisexual floret broadened, or became thin, while the short tubular petals of bisexual floret elongated to some extent, or even turned into semi-ligulate petals or ligulate petals, making the phenotype of the whole inflorescence like a chrysanthemum. The shape and thickness of leaves varied in some of these plants. Absolute sterile plants in mutant plants were found to possess neither normal bisexual florets nor unisexual florets, but the "pseudo-floret" only consisted of pieces of shield-like bracts on protuberant floral disc. Thirty-five pairs of simple sequence of repeat primers were used to detect the genetic variation of the plants, and the results showed that only a variation was tested in the mutant plants from 4 primers. The different PCR products obtained were extracted for sequencing and alignment analysis, and the aligned results showed that the DNA sequence changed by deletion, insertion and replacement that occurred at some sites. The results proved the high mutagenic efficacy of space flight, and ways of DNA transformation due to space conditions.

Systematic variations in divergence angle.

Practical methods for quantitative analysis of radial and angular coordinates of leafy organs of vascular plants are presented and applied to published phyllotactic patterns of various real systems from young leaves on a shoot tip to florets on a flower head. The constancy of divergence angle is borne out with accuracy of less than a degree. It is shown that apparent fluctuations in divergence angle are in large part systematic variations caused by the invalid assumption of a fixed center and/or by secondary deformations, while random fluctuations are of minor importance.