The auxin efflux carrier PIN1a regulates vascular patterning in cereal roots.

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.

Lipo-chitooligosaccharides promote lateral root formation and modify auxin homeostasis in Brachypodium distachyon.

Lipo-chitooligosaccharides (LCOs) are microbial symbiotic signals that also influence root growth. In Medicago truncatula, LCOs stimulate lateral root formation (LRF) synergistically with auxin. However, the molecular mechanisms of this phenomenon and whether it is restricted to legume plants are not known. We have addressed the capacity of the model monocot Brachypodium distachyon (Brachypodium) to respond to LCOs and auxin for LRF. For this, we used a combination of root phenotyping assays, live-imaging and auxin quantification, and analysed the regulation of auxin homeostasis genes. We show that LCOs and a low dose of the auxin precursor indole-3-butyric acid (IBA) stimulated LRF in Brachypodium, while a combination of LCOs and IBA led to different regulations. Both LCO and IBA treatments locally increased endogenous indole-3-acetic acid (IAA) content, whereas the combination of LCO and IBA locally increased the endogenous concentration of a conjugated form of IAA (IAA-Ala). LCOs, IBA and the combination differentially controlled expression of auxin homeostasis genes. These results demonstrate that LCOs are active on Brachypodium roots and stimulate LRF probably through regulation of auxin homeostasis. The interaction between LCO and auxin treatments observed in Brachypodium on root architecture opens interesting avenues regarding their possible combined effects during the arbuscular mycorrhizal symbiosis.

Live Confocal Imaging of Brachypodium Spikelet Meristems.

Live confocal imaging of fluorescent reporters and stains in plant meristems provides valuable measurements of gene expression, protein dynamics, cell polarity, cell division, and growth. The spikelet meristem in the grass Brachypodium distachyon (Brachypodium) is well suited to live imaging because of the ease of dissection, small meristem size, simple arrangement of organs, and because each plant provides abundant spikelet meristems. Brachypodium is also far easier to genetically transform than other grass species. Presented here is a protocol for the growth, staging, dissection, mounting, and imaging of Brachypodium spikelet meristems for live confocal imaging.

Cross-species functional diversity within the PIN auxin efflux protein family.

In Arabidopsis, development during flowering is coordinated by transport of the hormone auxin mediated by polar-localized PIN-FORMED1 (AtPIN1). However Arabidopsis has lost a PIN clade sister to AtPIN1, Sister-of-PIN1 (SoPIN1), which is conserved in flowering plants. We previously proposed that the AtPIN1 organ initiation and vein patterning functions are split between the SoPIN1 and PIN1 clades in grasses. Here we show that in the grass Brachypodium sopin1 mutants have organ initiation defects similar to Arabidopsis atpin1, while loss of PIN1 function in Brachypodium has little effect on organ initiation but alters stem growth. Heterologous expression of Brachypodium SoPIN1 and PIN1b in Arabidopsis provides further evidence of functional specificity. SoPIN1 but not PIN1b can mediate flower formation in null atpin1 mutants, although both can complement a missense allele. The behavior of SoPIN1 and PIN1b in Arabidopsis illustrates how membrane and tissue-level accumulation, transport activity, and interaction contribute to PIN functional specificity.

Miscibility Gap Closure, Interface Morphology, and Phase Microstructure of 3D Li(x)FePO4 Nanoparticles from Surface Wetting and Coherency Strain.

We study the mesoscopic effects which modify phase-segregation in LixFePO4 nanoparticles using a multiphysics phase-field model implement on a high performance cluster. We simulate 3D spherical particles of radii from 3 to 40 nm and examine the equilibrium microstructure and voltage profiles as they depend on size and overall lithiation. The model includes anisotropic, concentration-dependent elastic moduli, misfit strain, and facet dependent surface wetting within a Cahn-Hilliard formulation. We find that the miscibility gap vanishes for particles of radius ∼5 nm, and the solubility limits change with overall particle lithiation. Surface wetting stabilizes minority phases by aligning them with energetically beneficial facets. The equilibrium voltage profile is modified by these effects in magnitude, and the length and slope of the voltage plateau during two-phase coexistence.

Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana.

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

Plasma membrane-targeted PIN proteins drive shoot development in a moss.

BACKGROUND: Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. RESULTS: We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. CONCLUSIONS: Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development.

A division in PIN-mediated auxin patterning during organ initiation in grasses.

The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae.

The influence of acute resistance exercise on cyclooxygenase-1 and -2 activity and protein levels in human skeletal muscle.

This study evaluated the activity and content of cyclooxygenase (COX)-1 and -2 in response to acute resistance exercise (RE) in human skeletal muscle. Previous work suggests that COX-1, but not COX-2, is the primary COX isoform elevated with resistance exercise in human skeletal muscle. COX activity, however, has not been assessed after resistance exercise in humans. It was hypothesized that RE would increase COX-1 but not COX-2 activity. Muscle biopsies were taken from the vastus lateralis of nine young men (25 ± 1 yr) at baseline (preexercise), 4, and 24 h after a single bout of knee extensor RE (three sets of 10 repetitions at 70% of maximum). Tissue lysate was assayed for COX-1 and COX-2 activity. COX-1 and COX-2 protein levels were measured via Western blot analysis. COX-1 activity increased at 4 h (P < 0.05) compared with preexercise, but returned to baseline at 24 h (PRE: 60 ± 10, 4 h: 106 ± 22, 24 h: 72 ± 8 nmol PGH2·g total protein(-1)·min(-1)). COX-2 activity was elevated at 4 and 24 h after RE (P < 0.05, PRE: 51 ± 7, 4 h: 100 ± 19, 24 h: 98 ± 14 nmol PGH2·g total protein(-1)·min(-1)). The protein level of COX-1 was not altered (P > 0.05) with acute RE. In contrast, COX-2 protein levels were nearly 3-fold greater (P > 0.05) at 4 h and 5-fold greater (P = 0.06) at 24 h, compared with preexercise. In conclusion, COX-1 activity increases transiently with exercise independent of COX-1 protein levels. In contrast, both COX-2 activity and protein levels were elevated with exercise, and this elevation persisted to at least 24 h after RE.

Unraveling the KNOTTED1 regulatory network in maize meristems.

KNOTTED1 (KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. We defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci, including 643 genes that are modulated in one or multiple tissues. These KN1 direct targets are strongly enriched for transcription factors (including other homeobox genes) and genes participating in hormonal pathways, most significantly auxin, demonstrating that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function.

Estrogen receptor beta dependent attenuation of cytokine-induced cyclooxygenase-2 by androgens in human brain vascular smooth muscle cells and rat mesenteric arteries.

Androgens may provide protective effects in the vasculature under pathophysiological conditions. Our past studies have shown that dihydrotestosterone (DHT) decreases expression of cyclooxygenase-2 (COX-2) during cytokine, endotoxin, or hypoxic stimulation in human vascular smooth muscle cells, in an androgen receptor (AR)-independent fashion. Classically DHT is regarded as a pure AR agonist; however, it can be endogenously metabolized to 5α-androstane-3ß, 17ß-diol (3ß-diol), which has recently been shown to be a selective estrogen receptor (ERß) agonist. Therefore, we hypothesized that DHT's anti-inflammatory properties following cytokine stimulation are mediated through ERß. Using primary human brain vascular smooth muscle cells (HBVSMC), we tested whether DHT's effect on IL-1ß induced COX-2 expression was mediated via AR or ERß. The metabolism of DHT to 3ß-diol is a viable pathway in HBVSMC since mRNA for enzymes necessary for the synthesis and metabolism of 3ß-diol [3alpha-hydroxysteroid dehydrogenase (HSD), 3ß-HSD, 17ß-HSD, CYP7B1] was detected. In addition, the expression of AR, ERα, and ERß mRNA was detected. When applied to HBVSMC, DHT (10nM; 18 h) attenuated IL-1ß-induced increases in COX-2 protein expression. The AR antagonist bicalutamide did not block DHT's ability to reduce COX-2. Both the non-selective estrogen receptor antagonist ICI 182,780 (1 µM) and the selective ERß antagonist PHTPP (1 µM) inhibited the effect of DHT, suggesting that DHT actions are ERß-mediated. In HBVSMC and in rat mesenteric arteries, 3ß-diol, similar to DHT, reduced cytokine-induced COX-2 levels. In conclusion, DHT appears to be protective against the progression of vascular inflammation through metabolism to 3ß-diol and activation of ERß.

Small RNAs going the distance during plant development.

Small RNAs are 19-27 nucleotide long RNAs that negatively regulate gene expression and function as important regulators of diverse aspects of plant development. Current models for how they function continue to be modified as new research uncovers additional aspects of their biology. Unexpectedly, several previously characterized small RNAs appear to function non-cell autonomously, some moving a few cells away, others moving throughout the plant. This fact may reflect that small RNAs are an essential component of a larger signaling network that orchestrates plant development.