Endopolyploidy levels in barley vary in different root types and significantly decrease under phosphorus deficiency.

Increased endopolyploidy is important for plant growth and development as well as for adaptation to environmental stresses. However, little is known about the role of reduced endopolyploidy, especially in root systems. In this report, endopolyploidy variations were examined in different types of barley (Hordeum vulgare L.) roots, and the effects of phosphorus (P) deficiency and salinity (NaCl) stress on root endopolyploidy were also studied. The results showed that the endopolyploidy levels were lower in lateral roots than in either primary or nodal roots. The lower endopolyploidy in lateral roots was attributed to cortical cells. P deficiency reduced the endopolyploidy levels in lateral roots and mature zone of primary roots. By contrast, salinity had no effects on the endopolyploidy levels in either lateral or primary roots, but had a minor effect on nodal roots. Transcript analysis of cell cycle-related genes showed that multiple cell cycle-related genes were more highly expressed in lateral roots than in primary roots, suggesting their roles in lowering endopolyploidy. P deficiency reduced HvCCS52A1 transcripts in the mature zone of primary roots, but had little effect on the transcripts of 12 cell cycle-related genes in lateral roots, suggesting that endopolyploidy regulation differs between lateral roots and primary roots. Our results revealed that endopolyploidy reduction in root systems could be an integrated part of endopolyploidy plasticity in barley growth and development as well as in adaptation to a low P environment.

Right miniparasternotomy may be a good minimally invasive alternative to full sternotomy for cardiac valve operations: a propensity-adjusted analysis.

BACKGROUND: Limited real-world data existed for mini-parasternotomy approach with good sample size in Asian cohorts and most previous studies were eclipsed by case heterogeneity. The goal of this study was to compare safety and quality outcomes of cardiac non-coronary valve operations by mini-parasternotomy and full sternotomy approaches on risk-adjusted basis. METHODS From our hospital database, we retrieved the cases of non-coronary valve operations from 1 January 2005 to 31 December 2012, including re-do, emergent, and combined procedures. Estimated EuroScore-II and propensity score for choosing mini-parasternotomy were adjusted for in the regression models on hospital mortality, complications (pneumonia, stroke, sepsis, etc.), and quality parameters (length of stay, ICU time, ventilator time, etc.). Non-complicated cases, defined as survival to discharge, ventilator use not over one week, and intensive care unit stay not over two weeks, were used for quality parameters. RESULTS: There were 283 mini-parasternotomy and 177 full sternotomy cases. EuroScore-II differed significantly (medians 2.1 vs. 4.7, P<0.001). Propensity scores for choosing mini-parasternotomy were higher with lower EuroScore-II (OR=0.91 per 1%, P<0.001), aortic regurgitation (OR=2.3, P=0.005), and aortic non-mitral valve disease (OR=3.9, P<0.001). Adjusted for propensity score and EuroScore-II, mini-parasternotomy group had less pneumonia (OR=0.32, P=0.043), less sepsis (OR=0.31, P=0.045), and shorter non-complicated length of stay (coefficient=-7.2 (day), P<0.001) than full sternotomy group, whereas Kaplan-Meier survival, non-complicated ICU time, non-complicated ventilator time, and 30-day mortality did not differ significantly. CONCLUSION: The propensity-adjusted analysis demonstrated encouraging safety and quality outcomes for mini-parasternotomy valve operation in carefully selected patients.

Increased expression of six ZIP family genes by zinc (Zn) deficiency is associated with enhanced uptake and root-to-shoot translocation of Zn in barley (Hordeum vulgare).

Low zinc (Zn) in soils reduces yield and grain Zn content. Regulation of ZRT/IRT-like protein (ZIP) family genes is a major mechanism in plant adaptation to low and fluctuating Zn in soil. Although several Zn deficiency-inducible ZIP genes are identified in cereals, there has been no systematic study on the association of Zn deficiency-induced uptake and root-to-shoot translocation with expression of ZIP family genes. We measured Zn deficiency-induced uptake and root-to-shoot translocation of Zn in barley (Hordeum vulgare) plants by resupplying 0.5 µM Zn, and quantified the transcripts of thirteen HvZIP genes. Subcellular localization and tissue-specific expression were also determined for Zn deficiency-inducible HvZIP genes. Zn deficiency enhanced the capacity of uptake and root-to-shoot translocation of Zn, and sustained the enhanced capacity for 6 d after Zn resupply. Six HvZIP genes were highly induced in roots of Zn-deficient plants, and their proteins were localized in the plasma membrane. Tissue-specific expression in roots supports their roles in uptake and root-to-shoot translocation of Zn under low Zn conditions. Our results provide a comprehensive view on the physiological roles of ZIP genes in plant adaptation to low and fluctuating Zn in soil, and pave the way for development of new strategies to improve Zn-deficiency tolerance and biofortification in cereals.

HvZIP7 mediates zinc accumulation in barley (Hordeum vulgare) at moderately high zinc supply.

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

A DNA-based method for studying root responses to drought in field-grown wheat genotypes.

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Phosphate utilization efficiency correlates with expression of low-affinity phosphate transporters and noncoding RNA, IPS1, in barley.

Genetic variation in phosphorus (P) efficiency exists among wheat (Triticum aestivum) and barley (Hordeum vulgare) genotypes, but the underlying mechanisms for the variation remain elusive. High- and low-affinity phosphate (Pi) PHT1 transporters play an indispensable role in P acquisition and remobilization. However, little is known about genetic variation in PHT1 gene expression and association with P acquisition efficiency (PAE) and P utilization efficiency (PUE). Here, we present quantitative analyses of transcript levels of high- and low-affinity PHT1 Pi transporters in four barley genotypes differing in PAE. The results showed that there was no clear pattern in the expression of four paralogs of the high-affinity Pi transporter HvPHT1;1 among the four barley genotypes, but the expression of a low-affinity Pi transporter, HvPHT1;6, and its close homolog HvHPT1;3 was correlated with the genotypes differing in PUE. Interestingly, the expression of HvPHT1;6 and HvPHT1;3 was correlated with the expression of HvIPS1 (for P starvation inducible; noncoding RNA) but not with HvIPS2, suggesting that HvIPS1 plays a distinct role in the regulation of the low-affinity Pi transporters. In addition, high PUE was found to be associated with high root-shoot ratios in low-P conditions, indicating that high carbohydrate partitioning into roots occurs simultaneously with high PUE. However, high PUE accompanying high carbon partitioning into roots could result in low PAE. Therefore, the optimization of PUE through the modification of low-affinity Pi transporter expression may assist further improvement of PAE for low-input agriculture systems.

Proton-coupled high-affinity phosphate transport revealed from heterologous characterization in Xenopus of barley-root plasma membrane transporter, HvPHT1;1.

High-affinity phosphate transporters mediate uptake of inorganic phosphate (P(i) ) from soil solution under low P(i) conditions. The electrophysiological properties of any plant high-affinity P(i) transporter have not been described yet. Here, we report the detailed characterization of electrophysiological properties of the barley P(i) transporter, HvPHT1;1 in Xenopus laevis oocytes. A very low K(m) value (1.9 µm) for phosphate transport was observed in HvPHT1;1, which falls within the concentration range observed for barley roots. Inward currents at negative membrane potentials were identified as nH+ :P(i)⁻ (n > 1) co-transport based on simultaneous P(i) radiotracer uptake, oocyte voltage clamping and pH dependence. HvPHT1;1 showed preferential selectivity for P(i) and arsenate, but no transport of the other oxyanions SO4²â» and NO3⁻. In addition, HvPHT1;1 locates to the plasma membrane when expressed in onion (Allium cepa L.) epidermal cells, and is highly expressed in root segments with dense hairs. The electrophysiological properties, plasma membrane localization and cell-specific expression pattern of HvPHT1;1 support its role in the uptake of P(i) under low P(i) conditions.

Channel-like characteristics of the low-affinity barley phosphate transporter PHT1;6 when expressed in Xenopus oocytes.

Remobilization of inorganic phosphate (P(i)) within a plant is critical for sustaining growth and seed production under external P(i) fluctuation. The barley (Hordeum vulgare) transporter HvPHT1;6 has been implicated in P(i) remobilization. In this report, we expressed HvPHT1;6 in Xenopus laevis oocytes, allowing detailed characterization of voltage-dependent fluxes and currents induced by HvPHT1;6. HvPHT1;6 increased efflux of P(i) near oocyte resting membrane potentials, dependent on external P(i) concentration. Time-dependent inward currents were observed when membrane potentials were more negative than -160 mV, which was consistent with nH(+):HPO(4)(2-) (n > 2) cotransport, based on simultaneous radiotracer and oocyte voltage clamping, dependent upon P(i) concentration gradient and pH. Time- and voltage-dependent inward currents through HvPHT1;6 were also observed for SO(4)(2-)and to a lesser degree for NO(3)(-)Cl(-)but not for malate. Inward and outward currents showed linear dependence on the concentration of external HPO(4)(2-)similar to low-affinity P(i) transport in plant studies. The electrophysiological properties of HvPHT1;6, which locates to the plasma membrane when expressed in onion (Allium cepa) epidermal cells, are consistent with its suggested role in the remobilization of P(i) in barley plants.

Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in phosphate-deficient barley plants (Hordeum vulgare L.).

Plants modify metabolic processes for adaptation to low phosphate (P) conditions. Whilst transcriptomic analyses show that P deficiency changes hundreds of genes related to various metabolic processes, there is limited information available for global metabolite changes of P-deficient plants, especially for cereals. As changes in metabolites are the ultimate 'readout' of changes in gene expression, we profiled polar metabolites from both shoots and roots of P-deficient barley (Hordeum vulgare) using gas chromatography-mass spectrometry (GC-MS). The results showed that mildly P-deficient plants accumulated di- and trisaccharides (sucrose, maltose, raffinose and 6-kestose), especially in shoots. Severe P deficiency increased the levels of metabolites related to ammonium metabolism in addition to di- and trisaccharides, but reduced the levels of phosphorylated intermediates (glucose-6-P, fructose-6-P, inositol-1-P and glycerol-3-P) and organic acids (alpha-ketoglutarate, succinate, fumarate and malate). The results revealed that P-deficient plants modify carbohydrate metabolism initially to reduce P consumption, and salvage P from small P-containing metabolites when P deficiency is severe, which consequently reduced levels of organic acids in the tricarboxylic acid (TCA) cycle. The extent of the effect of severe P deficiency on ammonium metabolism was also revealed by liquid chromatography-mass spectrometry (LC-MS) quantitative analysis of free amino acids. A sharp increase in the concentrations of glutamine and asparagine was observed in both shoots and roots of severely P-deficient plants. Based on these data, a strategy for improving the ability of cereals to adapt to low P environments is proposed that involves alteration in partitioning of carbohydrates into organic acids and amino acids to enable more efficient utilization of carbon in P-deficient plants.

Mutational decay and age of chloroplast and mitochondrial genomes transferred recently to angiosperm nuclear chromosomes.

Transfers of organelle DNA to the nucleus established several thousand functional genes in eukaryotic chromosomes over evolutionary time. Recent transfers have also contributed nonfunctional plastid (pt)- and mitochondrion (mt)-derived DNA (termed nupts and numts, respectively) to plant nuclear genomes. The two largest transferred organelle genome copies are 131-kb nuptDNA in rice (Oryza sativa) and 262-kb numtDNA in Arabidopsis (Arabidopsis thaliana). These transferred copies were compared in detail with their bona fide organelle counterparts, to which they are 99.77% and 99.91% identical, respectively. No evidence for purifying selection was found in either nuclear integrant, indicating that they are nonfunctional. Mutations attributable to 5-methylcytosine hypermutation have occurred at a 6- to 10-fold higher rate than other point mutations in Arabidopsis numtDNA and rice nuptDNA, respectively, revealing this as a major mechanism of mutational decay for these transferred organelle sequences. Short indels occurred preferentially within homopolymeric stretches but were less frequent than point mutations. The 131-kb nuptDNA is absent in the O. sativa subsp. indica or Oryza rufipogon nuclear genome, suggesting that it was transferred within the O. sativa subsp. japonica lineage and, as revealed by sequence comparisons, after its divergence from the indica chloroplast lineage. The time of the transfer for the rice nupt was estimated as 148,000 (74,000--296,000) years ago and that for the Arabidopsis numtDNA as 88,000 (44,000--176,000) years ago. The results reveal transfer and integration of entire organelle genomes into the nucleus as an ongoing evolutionary process and uncover mutational mechanisms affecting organelle genomes recently transferred into a new mutational environment.

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco.

Transfer of organelle DNA into the nuclear genome has been significant in eukaryotic evolution, because it appears to be the origin of many nuclear genes. Most studies on organelle DNA transfer have been restricted to evolutionary events but experimental systems recently became available to monitor the process in real time. We designed an experimental screen to detect plastid DNA (ptDNA) transfers to the nucleus in whole plants grown under natural conditions. The resultant genotypes facilitated investigation of the evolutionary mechanisms underlying ptDNA transfer and nuclear integration. Here we report the characterization of nuclear loci formed by integration of newly transferred ptDNA. Large, often multiple, fragments of ptDNA between 6.0 and 22.3 kb in size are incorporated into chromosomes at single Mendelian loci. The lack of chloroplast transcripts of comparable size to the ptDNA integrants suggests that DNA molecules are directly involved in the transfer process. Microhomology (2-5 bp) and rearrangements of ptDNA and nuclear DNA were frequently found near integration sites, suggesting that nonhomologous recombination plays a major role in integration. The mechanisms of ptDNA integration appear similar to those of biolistic transformation of plant cells, but no sequence preference was identified near junctions. This article provides substantial molecular analysis of real-time ptDNA transfer and integration that has resulted from natural processes with no involvement of cell injury, infection, and tissue culture. We highlight the impact of cytoplasmic organellar genome mobility on nuclear genome evolution.

Direct measurement of the transfer rate of chloroplast DNA into the nucleus.

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.