Ultrasound and dynamic functional imaging in vascular cognitive impairment and Alzheimer's disease.

BACKGROUND: The vascular contributions to neurodegeneration and neuroinflammation may be assessed by magnetic resonance imaging (MRI) and ultrasonography (US). This review summarises the methodology for these widely available, safe and relatively low cost tools and analyses recent work highlighting their potential utility as biomarkers for differentiating subtypes of cognitive impairment and dementia, tracking disease progression and evaluating response to treatment in various neurocognitive disorders. METHODS: At the 9th International Congress on Vascular Dementia (Ljubljana, Slovenia, October 2015) a writing group of experts was formed to review the evidence on the utility of US and arterial spin labelling (ASL) as neurophysiological markers of normal ageing, vascular cognitive impairment (VCI) and Alzheimer's disease (AD). Original articles, systematic literature reviews, guidelines and expert opinions published until September 2016 were critically analysed to summarise existing evidence, indicate gaps in current knowledge and, when appropriate, suggest standards of use for the most widely used US and ASL applications. RESULTS: Cerebral hypoperfusion has been linked to cognitive decline either as a risk or an aggravating factor. Hypoperfusion as a consequence of microangiopathy, macroangiopathy or cardiac dysfunction can promote or accelerate neurodegeneration, blood-brain barrier disruption and neuroinflammation. US can evaluate the cerebrovascular tree for pathological structure and functional changes contributing to cerebral hypoperfusion. Microvascular pathology and hypoperfusion at the level of capillaries and small arterioles can also be assessed by ASL, an MRI signal. Despite increasing evidence supporting the utility of these methods in detection of microvascular pathology, cerebral hypoperfusion, neurovascular unit dysfunction and, most importantly, disease progression, incomplete standardisation and missing validated cut-off values limit their use in daily routine. CONCLUSIONS: US and ASL are promising tools with excellent temporal resolution, which will have a significant impact on our understanding of the vascular contributions to VCI and AD and may also be relevant for assessing future prevention and therapeutic strategies for these conditions. Our work provides recommendations regarding the use of non-invasive imaging techniques to investigate the functional consequences of vascular burden in dementia.

Two major quantitative trait loci controlling the number of seminal roots in maize co-map with the root developmental genes rtcs and rum1.

The genetic dissection of root architecture and functions allows for a more effective and informed design of novel root ideotypes and paves the way to evaluate their effects on crop resilience to a number of abiotic stresses. In maize, limited attention has been devoted to the genetic analysis of root architecture diversity at the early stage. The difference in embryonic (including seminal and primary) root architecture between the maize reference line B73 (which mostly develops three seminal roots) and the landrace Gaspé Flint (with virtually no seminal roots) was genetically dissected using a collection of introgression lines grown in paper rolls and pots. Quantitative trait locus (QTL) analysis identified three QTLs controlling seminal root number (SRN) on chromosome bins 1.02, 3.07, and 8.04-8.05, which collectively explained 66% of the phenotypic variation. In all three cases, Gaspé Flint contributed the allele for lower SRN. Primary root dry weight was negatively correlated with SRN (r= -0.52), and QTLs for primary root size co-mapped with SRN QTLs, suggesting a pleiotropic effect of SRN QTLs on the primary root, most probably caused by competition for seed resources. Interestingly, two out of three SRN QTLs co-mapped with the only two known maize genes (rtcs and rum1) affecting the number of seminal roots. The strong additive effect of the three QTLs and the development of near isogenic lines for each QTL in the elite B73 background provide unique opportunities to characterize functionally the genes involved in root development and to evaluate how root architecture affects seedling establishment, early development, and eventually yield in maize.

Sterols and sphingolipids differentially function in trafficking of the Arabidopsis ABCB19 auxin transporter.

The Arabidopsis ATP-binding cassette B19 (ABCB19, P-glycoprotein19) transporter functions coordinately with ABCB1 and PIN1 to motivate long-distance transport of the phytohormone auxin from the shoot to root apex. ABCB19 exhibits a predominantly apolar plasma membrane (PM) localization and stabilizes PIN1 when the two proteins co-occur. Biochemical evidence associates ABCB19 and PIN1 with sterol- and sphingolipid-enriched PM fractions. Mutants deficient in structural sterols and sphingolipids exhibit similarity to abcb19 mutants. Sphingolipid-defective tsc10a mutants and, to a lesser extent, sterol-deficient cvp1 mutants phenocopy abcb19 mutants. Live imaging studies show that sterols function in trafficking of ABCB19 from the trans-Golgi network to the PM. Pharmacological or genetic sphingolipid depletion has an even greater impact on ABCB19 PM targeting and interferes with ABCB19 trafficking from the Golgi. Our results also show that sphingolipids function in trafficking associated with compartments marked by the VTI12 syntaxin, and that ABCB19 mediates PIN1 stability in sphingolipid-containing membranes. The TWD1/FKBP42 co-chaperone immunophilin is required for exit of ABCB19 from the ER, but ABCB19 interactions with sterols, sphingolipids and PIN1 are spatially distinct from FKBP42 activity at the ER. The accessibility of this system to direct live imaging and biochemical analysis makes it ideal for the modeling and analysis of sterol and sphingolipid regulation of ABCB/P-glycoprotein transporters.

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis.

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-ß-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.

Genetic dissection of maize phenology using an intraspecific introgression library.

BACKGROUND: Collections of nearly isogenic lines where each line carries a delimited portion of a donor source genome into a common recipient genetic background are known as introgression libraries and have already shown to be instrumental for the dissection of quantitative traits. By means of marker-assisted backcrossing, we have produced an introgression library using the extremely early-flowering maize (Zea mays L.) variety Gaspé Flint and the elite line B73 as donor and recipient genotypes, respectively, and utilized this collection to investigate the genetic basis of flowering time and related traits of adaptive and agronomic importance in maize. RESULTS: The collection includes 75 lines with an average Gaspé Flint introgression length of 43.1 cM. The collection was evaluated for flowering time, internode length, number of ears, number of nodes (phytomeres), number of nodes above the ear, number and proportion of nodes below the ear and plant height. Five QTLs for flowering time were mapped, all corresponding to major QTLs for number of nodes. Three additional QTLs for number of nodes were mapped. Besides flowering time, the QTLs for number of nodes drove phenotypic variation for plant height and number of nodes below and above the top ear, but not for internode length. A number of apparently Mendelian-inherited phenotypes were also observed. CONCLUSIONS: While the inheritance of flowering time was dominated by the well-known QTL Vgt1, a number of other important flowering time QTLs were identified and, thanks to the type of plant material here utilized, immediately isogenized and made available for fine mapping. At each flowering time QTL, early flowering correlated with fewer vegetative phytomeres, indicating the latter as a key developmental strategy to adapt the maize crop from the original tropical environment to the northern border of the temperate zone (southern Canada), where Gaspé Flint was originally cultivated. Because of the trait differences between the two parental genotypes, this collection will serve as a permanent source of nearly isogenic materials for multiple studies of QTL analysis and cloning.

Corrigendum: The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves.

[This corrects the article DOI: 10.3389/fpls.2019.00806.].

The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves.

The phytohormone auxin plays significant roles in regulating plant growth and development. In Arabidopsis, a subset of ATP-BINDING CASSETTE subfamily B (ABCB) transporters participate in polar movement of auxin by exclusion from and prevention of reuptake at the plasma membrane. A previous analysis identified ABCB21 as a conditional auxin uptake/efflux transporter that regulates cellular auxin levels, but clear physiological roles for ABCB21 in planta remain unknown. Here we show that ABCB21 maintains the acropetal auxin transport stream by regulating auxin levels in the pericycle. Loss of ABCB21 reduces rootward auxin transport and delays lateral root emergence. In seedling shoots, ABCB21 regulates mobilization of auxin from the photosynthetic cotyledons that is important for phototropic bending. In rosette leaves ABCB21 contributes to lateral auxin distribution. These results support a primary role for ABCB21 in regulating auxin distribution supplementary to the primary ABCB auxin transporters ABCB1 and 19.

Whole-Genome Sequence Accuracy Is Improved by Replication in a Population of Mutagenized Sorghum.

The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS)-treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive-induced variants in next-generation sequencing (NGS) data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7×) from 586 independently mutagenized individuals and detected 5,399,493 homozygous single nucleotide polymorphisms (SNPs). Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error-prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error-prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs, which, congruent with the mutagenic mechanism of EMS, were >98% G:C to A:T transitions. Through this analysis, we generated a collection of sequence indexed mutants of sorghum. This collection contains 4035 high-impact homozygous mutations in 3637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. This method for detecting false positive-induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory-induced mutations or somatic mutation detection in medicine.

MicroV Technology to Improve Transcranial Color Coded Doppler Examinations.

The purpose of this review is to provide an update on technology related to Transcranial Color Coded Doppler Examinations. Microvascularization (MicroV) is an emerging Power Doppler technology which can allow visualization of low and weak blood flows even at high depths, thus providing a suitable technique for transcranial ultrasound analysis. With MicroV, reconstruction of the vessel shape can be improved, without any overestimation. Furthermore, by analyzing the Doppler signal, MicroV allows a global image of the Circle of Willis. Transcranial Doppler was originally developed for the velocimetric analysis of intracranial vessels, in particular to detect stenoses and the assessment of collateral circulation. Doppler velocimetric analysis was then compared to other neuroimaging techniques, thus providing a cut-off threshold. Transcranial Color Coded Doppler sonography allowed the characterization of vessel morphology. In both Color Doppler and Power Doppler, the signal overestimated the shape of the intracranial vessels, mostly in the presence of thin vessels and high depths of study. In further neurosonology technology development efforts, attempts have been made to address morphology issues and overcome technical limitations. The use of contrast agents has helped in this regard by introducing harmonics and subtraction software, which allowed better morphological studies of vessels, due to their increased signal-to-noise ratio. Having no limitations in the learning curve, in time and contrast agent techniques, and due to its high signal-to-noise ratio, MicroV has shown great potential to obtain the best morphological definition.

MMP-9 microsatellite polymorphism and susceptibility to carotid arteries atherosclerosis.

OBJECTIVE: The aims of this study were to compare a microsatellite polymorphism (PM) of matrix metalloproteinase (MMP)-9 in patients with carotid atherosclerosis and control population, and to assess the relationship between this PM and plaque structure. METHODS AND RESULTS: One hundred fifty patients referring to vascular diagnostic centers for suspected carotid atherosclerosis (at ultrasound examination: 110 positive, 40 negative) and controls (n=110) have been genotyped for MMP-9 PM. After controlling for risk factors, allelic and genotype frequencies were significantly different among the groups, with significant prevalence of long microsatellites in patients with carotid atherosclerosis. Long microsatellites (settled as 22 to 27 repeats) were associated with carotid atherosclerosis (odds ratio [OR], 5.2; 95% confidence interval [CI], 2.9 to 9.2), compared with controls; an independent case control study on patients with coronary atherosclerosis confirmed such result. Binary logistic regression showed that hypertension, long microsatellites in MMP-9 PM and smoking habits were variables accounting for the difference between ultrasound-positive patients and controls. Long microsatellites were also associated to plaques with thin fibrous cap and echolucent core (OR, 13.1; 95% CI, 1.6 to 100). These alleles were slightly more represented in female patients (chi2 test=0.019; OR, 2.7; 95% CI, 1.2 to 6) but not associated with other risk factors. Plasma MMP-9 levels were related neither to MMP-9 PM nor to plaque type, and were related to gender and extension of atherosclerosis in carotid arteries. CONCLUSIONS: The number of repeats (> or =22 CA) in the microsatellite of MMP-9 promoter, but not MMP-9 plasma levels, is associated to carotid atherosclerosis and particularly to plaques with a thin fibrous cap.

Immunolocalization of PIN and ABCB Transporters in Plants.

PIN auxin efflux carriers and ABCB auxin transporters are important for polar auxin transport, organogenesis and long distance auxin transport. Along with the auxin influx symporter AUX1, they are essential for tropic responses such as gravitropism and phototropism where lateral redistribution of auxin is required for the tropic response to occur. Immunolocalization of plant membrane transporters is an effective technique to determine the transporters' subcellular localization patterns in the tissues of interest, especially when fluorescent protein fusions of the protein of interest are not available. Immunolocalization is also a valuable tool for validation of the localization of fluorescent protein fusions when the fusions are available. Here we describe the procedures to prepare plant tissue samples and fix them for whole mount or embedding and sectioning. We focus on immunolocalizations of PINs and ABCBs in Arabidopsis and maize tissues. In addition, we describe treatments of roots with inhibitors of cellular trafficking: brefeldin A (BFA), a fungal compound that blocks exocytosis; wortmannin, a fungal compound that inhibits phosphatidylinositol 3-kinase and induces fusion of pre-vacuolar compartments and multi-vascular bodies; and oryzalin, a fungal compound that depolymerizes microtubules. Inhibitor treatments are performed prior to fixation and affect the localization patterns of PINs and ABCBs, giving insight into cell type -specific trafficking mechanisms. The procedures described for Arabidopsis and maize can be easily adapted for other herbaceous plants.

MMP-9 microsatellite polymorphism: association with the progression of intima-media thickening and constrictive remodeling of carotid atherosclerotic plaques.

Intima-media thickening (IMT) of carotid arteries and constrictive remodeling (CR) of atherosclerotic plaques are vascular pathologic characteristics that precede the onset of vascular events. SMC migration and proliferation are linked both to IMT and CR and are matrix metalloproteinase 9 (MMP-9) dependent. A genetic polymorphism (PM) of MMP-9, a CA (13-27) microsatellite in the promoter region, which accounts for differential expression of MMP-9, could be linked to progression of IMT and CR. Progression of IMT and CR of plaques in carotid arteries were studied in 55 consecutive patients with a 12-18 months follow-up. All patients were genotyped for MMP-9 PM. A positive linear relationship between the number of repeats and the progression of IMT (P=0.028) as well as of CR (P=0.018) was found. The analogous relationship was obtained when only the allele with longer microsatellite was considered. Carriers of more than 20 repeats in one allele showed faster both IMT growth (P=0.045) and stenosis progressions of plaques (P=0.019). In multivariate analysis, age, dyslipidemia, and MMP-9 PM were determinants of IMT progression, while MMP-9 PM was the only one of CR. In conclusion, the high number of CA repeats in MMP-9 promoter is positively correlated with faster IMT and CR progression.

Peculiar aspects of reading and writing performances in patients with olivopontocerebellar atrophy.

Olivopontocerebellar atrophy (OPCA) is a still debated condition, of which motor disruption is the most common feature. A high incidence of associated mood disorders may exist, but there are few studies on concomitant cognitive impairment. Our aim was to assess whether there is reading and writing disruption in olivopontocerebellar atrophy (OPCA). 6 patients were administered different reading and writing tasks. Scores were then compared to those obtained by healthy volunteers. There was an evident impairment in reading and writing execution in our patients compared to those of the control group. On the contrary, no difference could be found in abstraction, problem-solving, and memory tasks. We discuss the results, debating the role of the cerebellum in the conscious process of cognition or in ocular movement control (necessary for reading and writing fluidity and effective execution) and in the dynamic activation of all the cerebral cortex mediated by the diffuse projection to the reticular system.

A novel phylogeny and morphological reconstruction of the PIN genes and first phylogeny of the ACC-oxidases (ACOs).

The PIN and ACO gene families present interesting questions about the evolution of plant physiology, including testing hypotheses about the ecological drivers of their diversification and whether unrelated genes have been recruited for similar functions. The PIN-formed proteins contribute to the polar transport of auxin, a hormone which regulates plant growth and development. PIN loci are categorized into groups according to their protein length and structure, as well as subcellular localization. An interesting question with PIN genes is the nature of the ancestral form and location. ACOs are members of a superfamily of oxygenases and oxidases that catalyze the last step of ethylene synthesis, which regulates many aspects of the plant life cycle. We used publicly available PIN and ACO sequences to conduct phylogenetic analyses. Third codon positions of these genes in monocots have a high GC content, which could be historical but is more likely due to a mutational bias. Thus, we developed methods to extract phylogenetic information from nucleotide sequences while avoiding this convergent feature. One method consisted in using only A-T transformations, and another used only the first and second codon positions for serine, which can only take A or T and G or C, respectively. We also conducted tree-searches for both gene families using unaligned amino acid sequences and dynamic homology. PIN genes appear to have diversified earlier than ACOs, with monocot and dicot copies more mixed in the phylogeny. However, gymnosperm PINs appear to be derived and not closely related to those from primitive plants. We find strong support for a long PIN gene ancestor with short forms subsequently evolving one or more times. ACO genes appear to have diversified mostly since the dicot-monocot split, as most genes cluster into a small number of monocot and dicot clades when the tree is rooted by genes from mosses. Gymnosperm ACOs were recovered as closely related and derived.

The role of auxin transporters in monocots development.

Auxin is a key regulator of plant growth and development, orchestrating cell division, elongation and differentiation, embryonic development, root and stem tropisms, apical dominance, and transition to flowering. Auxin levels are higher in undifferentiated cell populations and decrease following organ initiation and tissue differentiation. This differential auxin distribution is achieved by polar auxin transport (PAT) mediated by auxin transport proteins. There are four major families of auxin transporters in plants: PIN-FORMED (PIN), ATP-binding cassette family B (ABCB), AUXIN1/LIKE-AUX1s, and PIN-LIKES. These families include proteins located at the plasma membrane or at the endoplasmic reticulum (ER), which participate in auxin influx, efflux or both, from the apoplast into the cell or from the cytosol into the ER compartment. Auxin transporters have been largely studied in the dicotyledon model species Arabidopsis, but there is increasing evidence of their role in auxin regulated development in monocotyledon species. In monocots, families of auxin transporters are enlarged and often include duplicated genes and proteins with high sequence similarity. Some of these proteins underwent sub- and neo-functionalization with substantial modification to their structure and expression in organs such as adventitious roots, panicles, tassels, and ears. Most of the present information on monocot auxin transporters function derives from studies conducted in rice, maize, sorghum, and Brachypodium, using pharmacological applications (PAT inhibitors) or down-/up-regulation (over-expression and RNA interference) of candidate genes. Gene expression studies and comparison of predicted protein structures have also increased our knowledge of the role of PAT in monocots. However, knockout mutants and functional characterization of single genes are still scarce and the future availability of such resources will prove crucial to elucidate the role of auxin transporters in monocots development.

Optic nerve and its arterial-venous vascularization: an ultrasonologic study in multiple sclerosis patients and healthy controls.

BACKGROUND: Recent studies suggest that alterations in the cerebrospinal venous system may play a role in multiple sclerosis (MS) and that chronic cerebrospinal venous insufficiency correlates with clinical features of MS patients. OBJECTIVES: To evaluate the vascularization of optic nerve (ONr) and measure ONe thickness by color Doppler ultrasonography in MS patients with and without previous optic neuritis (ONe). SUBJECTS AND METHODS: We assessed flow variables in the ophthalmic artery, central retinal artery, and central retinal vein and measured the diameter of ONe in 46 relapsing-remitting MS patients and 37 healthy controls (HC). Twenty-two MS patients had previous ONe and 24 MS patients had not. Patients with acute ONe were not included. We examined and compared 63 unaffected and 29 affected eyes of MS patients with 74 control eyes. RESULTS: Regarding flow variables, we did not find any significant difference between HC, MS affected, and unaffected eyes. Comparing ONr diameters, we found a progressive significant thinning of the ONr from HC to MS patients without and with past ONe. CONCLUSIONS: We found no significant alteration in the arterial-venous vascularization of both affected and unaffected ONr compared with HC. We demonstrated the possibility to detect ONr atrophy in MS patients.

Protein immunolocalization in maize tissues.

The analysis of gene expression at transcript and at protein level is of outstanding importance in plant developmental biology. Proteins can be localized with subcellular resolution by immunolocalization using specific antibodies or generating reporter lines carrying the specific protein fused to a fluorescent protein. Immunolocalization is particularly suitable to confirm the expression pattern of transgenic reporter lines. It also represents a valid alternative, especially for plants such as maize, for which transformation is time consuming and still often unsuccessful, by-passing also some side-effects of fusion proteins. Indeed, the availability of specific antibodies for immunolocalizations and observations of maize tissues under a confocal microscope is a powerful tool for studying protein targeting to different cellular compartments.In the following chapter we describe the complete procedure for the localization of proteins in different maize tissues both at cellular and sub-cellular level, using polyclonal or monoclonal antibodies. Tissues can be embedded in different substrates, such as paraplast, PEG400 and agarose, depending on the tissue and the desired use: epifluorescence or confocal microscope observations. An additional protocol for the analysis of the nuclear distribution of modified histones in squashed maize root apexes is also presented.

Diversification and Expression of the PIN, AUX/LAX, and ABCB Families of Putative Auxin Transporters in Populus.

Intercellular transport of the plant hormone auxin is mediated by three families of membrane-bound protein carriers, with the PIN and ABCB families coding primarily for efflux proteins and the AUX/LAX family coding for influx proteins. In the last decade our understanding of gene and protein function for these transporters in Arabidopsis has expanded rapidly but very little is known about their role in woody plant development. Here we present a comprehensive account of all three families in the model woody species Populus, including chromosome distribution, protein structure, quantitative gene expression, and evolutionary relationships. The PIN and AUX/LAX gene families in Populus comprise 16 and 8 members respectively and show evidence for the retention of paralogs following a relatively recent whole genome duplication. There is also differential expression across tissues within many gene pairs. The ABCB family is previously undescribed in Populus and includes 20 members, showing a much deeper evolutionary history, including both tandem and whole genome duplication as well as probable gene loss. A striking number of these transporters are expressed in developing Populus stems and we suggest that evolutionary and structural relationships with known auxin transporters in Arabidopsis can point toward candidate genes for further study in Populus. This is especially important for the ABCBs, which is a large family and includes members in Arabidopsis that are able to transport other substrates in addition to auxin. Protein modeling, sequence alignment and expression data all point to ABCB1.1 as a likely auxin transport protein in Populus. Given that basipetal auxin flow through the cambial zone shapes the development of woody stems, it is important that we identify the full complement of genes involved in this process. This work should lay the foundation for studies targeting specific proteins for functional characterization and in situ localization.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis.

Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.

ZmPIN1a and ZmPIN1b encode two novel putative candidates for polar auxin transport and plant architecture determination of maize.

Shoot apical meristems produce organs in a highly stereotypic pattern that involves auxin. Auxin is supposed to be actively transported from cell to cell by influx (AUXIN/LIKE AUXIN proteins) and efflux (PIN-FORMED proteins) membrane carriers. Current hypotheses propose that, at the meristem surface, PIN proteins create patterns of auxin gradients that, in turn, create patterns of gene expression and morphogenesis. These hypotheses are entirely based on work in Arabidopsis (Arabidopsis thaliana). To verify whether these models also apply to other species, we studied the behavior of PIN proteins during maize (Zea mays) development. We identified two novel putative orthologs of AtPIN1 in maize and analyzed their expression pattern during development. The expression studies were complemented by immunolocalization studies using an anti-AtPIN1 antibody. Interestingly, the maize proteins visualized by this antibody are almost exclusively localized in subepidermal meristematic layers. Both tassel and ear were characterized by a compact group of cells, just below the surface, carrying PIN. In contrast to or to complement what was shown in Arabidopsis, these results point to the importance of internally localized cells in the patterning process. We chose the barren inflorescence2 (bif2) maize mutant to study the role of auxin polar fluxes in inflorescence development. In severe alleles of bif2, the tassel and the ear present altered ZmPIN1a and ZmPIN1b protein expression and localization patterns. In particular, the compact groups of cells in the tassel and ear of the mutant were missing. We conclude that BIF2 is important for PIN organization and could play a role in the establishment of polar auxin fluxes in maize inflorescence, indirectly modulating the process of axillary meristem formation and development.