A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.     

A graphical method for identifying the six types of non‐deep physiological dormancy in seeds

We present a new seed dormancy classification scheme for the non‐deep level of the class physiological dormancy (PD), which contains six types. Non‐deep PD is divided into two sublevels: one for seeds that exhibit a dormancy continuum (types 1, 2 and 3) and the other for those that do not exhibit a dormancy continuum (types 4, 5 and 6). Analysis of previous studies showed that different types of non‐deep PD also can be identified using a graphical method. Seeds with a dormancy (D) ? conditional dormancy (CD) ? non‐dormancy (ND) cycle have a low germination percentage in the early stages of CD, and during dormancy loss the germination capacity increases. However, seeds with a CD/ND (i.e. D→CD?ND) cycle germinate to a high percentage at a narrow range of temperatures in the early stages of CD. Cardinal temperatures for seeds with either a D/ND or a CD/ND cycle change during dormancy loss: the ceiling temperature increases in seeds with Type 1, the base temperature decreases in seeds with Type 2 and the base and ceiling temperatures decrease and increase, respectively, in seeds with Type 3. Criteria for distinguishing the six types of non‐deep PD and models of the temperature functions of seeds with types 1, 2 and 3 with both types of dormancy cycles are presented. The relevancy of our results to modelling the timing of weed seedling emergence is briefly discussed.     

Acquisition of physical dormancy and ontogeny of the micropyle--water-gap complex in developing seeds of Geranium carolinianum (Geraniaceae)

Background and Aims

The ‘hinged valve gap’ has been previously identified as the initial site of water entry (i.e. water gap) in physically dormant (PY) seeds of Geranium carolinianum (Geraniaceae). However, neither the ontogeny of the hinged valve gap nor acquisition of PY by seeds of Geraniaceae has been studied previously. The aims of the present study were to investigate the physiological events related to acquisition of PY and the ontogeny of the hinged valve gap and seed coat of G. carolinianum.

Methods

Seeds of G. carolinianum were studied from the ovule stage until dispersal. The developmental stages of acquisition of germinability, physiological maturity and PY were determined by seed measurement, germination and imbibition experiments using intact seeds and isolated embryos of both fresh and slow-dried seeds. Ontogeny of the seed coat and water gap was studied using light microscopy.

Key Results

Developing seeds achieved germinability, physiological maturity and PY on days 9, 14 and 20 after pollination (DAP), respectively. The critical moisture content of seeds on acquisition of PY was 11 %. Slow-drying caused the stage of acquisition of PY to shift from 20 to 13 DAP. Greater extent of cell division and differentiation at the micropyle, water gap and chalaza than at the rest of the seed coat resulted in particular anatomical features. Palisade and subpalisade cells of varying forms developed in these sites. A clear demarcation between the water gap and micropyle is not evident due to their close proximity.

Conclusions

Acquisition of PY in seeds of G. carolinianum occurs after physiological maturity and is triggered by maturation drying. The micropyle and water gap cannot be considered as two separate entities, and thus it is more appropriate to consider them together as a ‘micropyle–water-gap complex’.     

Deep simple epicotyl morphophysiological dormancy in seeds of two Viburnum species, with special reference to shoot growth and development inside the seed

Background and Aims

In seeds with deep simple epicotyl morphophysiological dormancy, warm and cold stratification are required to break dormancy of the radicle and shoot, respectively. Although the shoot remains inside the seed all winter, little is known about its growth and morphological development prior to emergence in spring. The aims of the present study were to determine the temperature requirements for radicle and shoot emergence in seeds of Viburnum betulifolium and V. parvifolium and to monitor growth of the epicotyl, plumule and cotyledons in root-emerged seeds.

Methods

Fresh and pre-treated seeds of V. betulifolium and V. parvifolium were incubated under various temperature regimes and monitored for radicle and shoot emergence. Growth of the epicotyl and cotyledons at different stages was observed with dissecting and scanning electron microscopes.

Key Results

The optimum temperature for radicle emergence of seeds of both species, either kept continuously at a single regime or exposed to a sequence of regimes, was 20/10 °C. GA₃ had no effect on radicle emergence. Cold stratification (5 °C) was required for shoot emergence. The shoot apical meristem in fresh seeds did not form a bulge until the embryo had grown to the critical length for radicle emergence. After radicle emergence, the epicotyl–plumule and cotyledons grew slowly at 5 and 20/10 °C, and the first pair of true leaves was initiated. However, the shoot emerged only from seeds that received cold stratification.

Conclusions

Seeds of V. betulifolium and V. parvifolium have deep simple epicotyl morphophysiological dormancy, C_1bB (root)–C₃ (epicotyl). Warm stratification was required to break the first part of physiological dormancy (PD), thereby allowing embryo growth and subsequently radicle emergence. Although cold stratification was not required for differentiation of the epicotyl–plumule, it was required to break the second part of PD, thereby allowing the shoot to emerge in spring.     

A novel approach for multi-domain and multi-gene family identification provides insights into evolutionary dynamics of disease resistance genes in core eudicot plants

Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.     

The Jak2 Small Molecule Inhibitor,G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo

Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.