Savithri Nambeesan

Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit-ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8-ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild-type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation-related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd-mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S-ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole-plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post-harvest senescence and decay.

The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution

The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives, including numerous extremophile species. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade and a sunflower-specific whole-genome duplication around 29 million years ago. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs.

Genetic Analysis of Floral Symmetry in Van Gogh's Sunflowers Reveals Independent Recruitment of CYCLOIDEA Genes in the Asteraceae

The genetic basis of floral symmetry is a topic of great interest because of its effect on pollinator behavior and, consequently, plant diversification. The Asteraceae, which is the largest family of flowering plants, is an ideal system in which to study this trait, as many species within the family exhibit a compound inflorescence containing both bilaterally symmetric (i.e., zygomorphic) and radially symmetric (i.e., actinomorphic) florets. In sunflower and related species, the inflorescence is composed of a single whorl of ray florets surrounding multiple whorls of disc florets. We show that in double-flowered (dbl) sunflower mutants (in which disc florets develop bilateral symmetry), such as those captured by Vincent van Gogh in his famous nineteenth-century sunflower paintings, an insertion into the promoter region of a CYCLOIDEA (CYC)-like gene (HaCYC2c) that is normally expressed specifically in WT rays is instead expressed throughout the inflorescence, presumably resulting in the observed loss of actinomorphy. This same gene is mutated in two independent tubular-rayed (tub) mutants, though these mutations involve apparently recent transposon insertions, resulting in little or no expression and radialization of the normally zygomorphic ray florets. Interestingly, a phylogenetic analysis of CYC-like genes from across the family suggests that different paralogs of this fascinating gene family have been independently recruited to specify zygomorphy in different species within the Asteraceae.

Polyamines Attenuate Ethylene-Mediated Defense Responses to Abrogate Resistance to Botrytis cinerea in Tomato

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense.

Association mapping and the genomic consequences of selection in sunflower.

The combination of large-scale population genomic analyses and trait-based mapping approaches has the potential to provide novel insights into the evolutionary history and genome organization of crop plants. Here, we describe the detailed genotypic and phenotypic analysis of a sunflower (Helianthus annuus L.) association mapping population that captures nearly 90% of the allelic diversity present within the cultivated sunflower germplasm collection. We used these data to characterize overall patterns of genomic diversity and to perform association analyses on plant architecture (i.e., branching) and flowering time, successfully identifying numerous associations underlying these agronomically and evolutionarily important traits. Overall, we found variable levels of linkage disequilibrium (LD) across the genome. In general, islands of elevated LD correspond to genomic regions underlying traits that are known to have been targeted by selection during the evolution of cultivated sunflower. In many cases, these regions also showed significantly elevated levels of differentiation between the two major sunflower breeding groups, consistent with the occurrence of divergence due to strong selection. One of these regions, which harbors a major branching locus, spans a surprisingly long genetic interval (ca. 25 cM), indicating the occurrence of an extended selective sweep in an otherwise recombinogenic interval.

The P Domain of the P0 Protein of Plasmodium falciparum Protects against Challenge with Malaria Parasites

ABSTRACT Monoclonal antibodies (MAbs) specific for the P domain of the Plasmodium falciparum P0 phosphoriboprotein (PfP0) blocked the invasion of RBCs by P. falciparum. Vaccination with this P-domain peptide protected mice upon malaria parasite challenge. The absolute specificity of the MAbs and the PfP0 P peptide makes them potential protective malaria reagents.

Development of an ultra-dense genetic map of the sunflower genome based on single-feature polymorphisms.

The development of ultra-dense genetic maps has the potential to facilitate detailed comparative genomic analyses and whole genome sequence assemblies. Here we describe the use of a custom Affymetrix GeneChip containing nearly 2.4 million features (25 bp sequences) targeting 86,023 unigenes from sunflower (Helianthus annuus L.) and related species to test for single-feature polymorphisms (SFPs) in a recombinant inbred line (RIL) mapping population derived from a cross between confectionery and oilseed sunflower lines (RHA280×RHA801). We then employed an existing genetic map derived from this same population to rigorously filter out low quality data and place 67,486 features corresponding to 22,481 unigenes on the sunflower genetic map. The resulting map contains a substantial fraction of all sunflower genes and will thus facilitate a number of downstream applications, including genome assembly and the identification of candidate genes underlying QTL or traits of interest.

Association mapping in sunflower (Helianthus annuus L.) reveals independent control of apical vs. basal branching

BackgroundShoot branching is an important determinant of plant architecture and influences various aspects of growth and development. Selection on branching has also played an important role in the domestication of crop plants, including sunflower (Helianthus annuus L.). Here, we describe an investigation of the genetic basis of variation in branching in sunflower via association mapping in a diverse collection of cultivated sunflower lines.ResultsDetailed phenotypic analyses revealed extensive variation in the extent and type of branching within the focal population. After correcting for population structure and kinship, association analyses were performed using a genome-wide collection of SNPs to identify genomic regions that influence a variety of branching-related traits. This work resulted in the identification of multiple previously unidentified genomic regions that contribute to variation in branching. Genomic regions that were associated with apical and mid-apical branching were generally distinct from those associated with basal and mid-basal branching. Homologs of known branching genes from other study systems (i.e., Arabidopsis, rice, pea, and petunia) were also identified from the draft assembly of the sunflower genome and their map positions were compared to those of associations identified herein. Numerous candidate branching genes were found to map in close proximity to significant branching associations.ConclusionsIn sunflower, variation in branching is genetically complex and overall branching patterns (i.e., apical vs. basal) were found to be influenced by distinct genomic regions. Moreover, numerous candidate branching genes mapped in close proximity to significant branching associations. Although the sunflower genome exhibits localized islands of elevated linkage disequilibrium (LD), these non-random associations are known to decay rapidly elsewhere. The subset of candidate genes that co-localized with significant associations in regions of low LD represents the most promising target for future functional analyses.

Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0.

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum

Molecular evolution of candidate genes for crop-related traits in sunflower (Helianthus annuus L.).

Evolutionary analyses aimed at detecting the molecular signature of selection during crop domestication and/or improvement can be used to identify genes or genomic regions of likely agronomic importance. Here, we describe the DNA sequence-based characterization of a pool of candidate genes for crop-related traits in sunflower. These genes, which were identified based on homology to genes of known effect in other study systems, were initially sequenced from a panel of improved lines. All genes that exhibited a paucity of sequence diversity, consistent with the possible effects of selection during the evolution of cultivated sunflower, were then sequenced from a panel of wild sunflower accessions an outgroup. These data enabled formal tests for the effects of selection in shaping sequence diversity at these loci. When selection was detected, we further sequenced these genes from a panel of primitive landraces, thereby allowing us to investigate the likely timing of selection (i.e., domestication vs. improvement). We ultimately identified seven genes that exhibited the signature of positive selection during either domestication or improvement. Genetic mapping of a subset of these genes revealed co-localization between candidates for genes involved in the determination of flowering time, seed germination, plant growth/development, and branching and QTL that were previously identified for these traits in cultivated × wild sunflower mapping populations.