Enhancing Upland cotton for drought resilience, productivity, and fiber quality: comparative evaluation and genetic dissection.

To provision the world sustainably, modern society must increase overall crop production, while conserving and preserving natural resources. Producing more with diminishing water resources is an especially daunting endeavor. Toward the goal of genetically improving drought resilience of cultivated Upland cotton (Gossypium hirsutum L.), this study addresses the genetics of differential yield components referred to as productivity and fiber quality traits under regular-water versus low-water (LW) field conditions. We used ten traits to assess water stress deficit, which included six productivity and four fiber quality traits on two recombinant inbred line (RIL) populations from reciprocally crossed cultivars, Phytogen 72 and Stoneville 474. To facilitate genetic inferences, we genotyped RILs with the CottonSNP63K array, assembled high-density linkage maps of over 7000 SNPs and then analyzed quantitative trait variations. Analysis of variance revealed significant differences for all traits (p < 0.05) in these RIL populations. Although the LW irrigation regime significantly reduced all traits, except lint percent, the RILs exhibited a broad phenotypic spectrum of heritable differences across the water regimes. Transgressive segregation occurred among the RILs, suggesting the possibility of genetic gain through phenotypic selection for drought resilience and perhaps through marker-based selection. Analyses revealed more than 150 quantitative trait loci (QTLs) associated with productivity and fiber quality traits (p < 0.005) on different genomic regions of the cotton genome. The multiple-QTL models analysis with LOD > 3.0 detected 21 QTLs associated with productivity and 22 QTLs associated with fiber quality. For fiber traits, strong clustering and QTL associations occurred in c08 and its homolog c24 as well as c10, c14, and c21. Using contemporary genome sequence assemblies and bioinformatically related information, the identification of genomic regions associated with responses to plant stress/drought elevates the possibility of using marker-assisted and omics-based selection to enhance breeding for drought resilient cultivars and identifying candidate genes and networks. RILs with different responses to drought indicated that it is possible to maintain high fiber quality under LW conditions or reduce the of LW impact on quality. The heritable variation among elite bi-parental RILs for productivity and quality under field drought conditions, and their association of QTLs, and thus specific genomic regions, indicate opportunities for breeding-based gains in water resource conservation, i.e., enhancing cotton's agricultural sustainability.

Chlorophyll fluorescence analysis revealed essential roles of FtsH11 protease in regulation of the adaptive responses of photosynthetic systems to high temperature.

BACKGROUND: Photosynthetic systems are known to be sensitive to high temperature stress. To maintain a relatively "normal" level of photosynthetic activities, plants employ a variety of adaptive mechanisms in response to environmental temperature fluctuations. Previously, we reported that the chloroplast-targeted AtFtsH11 protease played an essential role for Arabidopsis plants to survive at high temperatures and to maintain normal photosynthetic efficiency at moderately elevated temperature. To investigate the factors contributing to the photosynthetic changes in FtsH11 mutant, we performed detailed chlorophyll fluorescence analyses of dark-adapted mutant plants and compared them to Col-0 WT plants under normal, two moderate high temperatures, and a high light conditions. RESULTS: We found that mutation of FtsH11 gene caused significant decreases in photosynthetic efficiency of photosystems when environmental temperature raised above optimal. Under moderately high temperatures, the FtsH11 mutant showed significant 1) decreases in electron transfer rates of photosystem II (PSII) and photosystem I (PSI), 2) decreases in photosynthetic capabilities of PSII and PSI, 3) increases in non-photochemical quenching, and a host of other chlorophyll fluorescence parameter changes. We also found that the degrees of these negative changes for utilizing the absorbed light energy for photosynthesis in FtsH11 mutant were correlated with the level and duration of the heat treatments. For plants grown under normal temperature and subjected to the high light treatment, no significant difference in chlorophyll fluorescence parameters was found between the FtsH11 mutant and Col-0 WT plants. CONCLUSIONS: The results of this study show that AtFtsH11 is essential for normal photosynthetic function under moderately elevated temperatures. The results also suggest that the network mediated by AtFtsH11 protease plays critical roles for maintaining the thermostability and possibly structural integrity of both photosystems under elevated temperatures. Elucidating the underlying mechanisms of FtsH11 protease in photosystems may lead to improvement of photosynthetic efficiency under heat stress conditions, hence, plant productivity.

Diversity analysis of cotton (Gossypium hirsutum L.) germplasm using the CottonSNP63K Array.

BACKGROUND: Cotton germplasm resources contain beneficial alleles that can be exploited to develop germplasm adapted to emerging environmental and climate conditions. Accessions and lines have traditionally been characterized based on phenotypes, but phenotypic profiles are limited by the cost, time, and space required to make visual observations and measurements. With advances in molecular genetic methods, genotypic profiles are increasingly able to identify differences among accessions due to the larger number of genetic markers that can be measured. A combination of both methods would greatly enhance our ability to characterize germplasm resources. Recent efforts have culminated in the identification of sufficient SNP markers to establish high-throughput genotyping systems, such as the CottonSNP63K array, which enables a researcher to efficiently analyze large numbers of SNP markers and obtain highly repeatable results. In the current investigation, we have utilized the SNP array for analyzing genetic diversity primarily among cotton cultivars, making comparisons to SSR-based phylogenetic analyses, and identifying loci associated with seed nutritional traits. RESULTS: The SNP markers distinctly separated G. hirsutum from other Gossypium species and distinguished the wild from cultivated types of G. hirsutum. The markers also efficiently discerned differences among cultivars, which was the primary goal when designing the CottonSNP63K array. Population structure within the genus compared favorably with previous results obtained using SSR markers, and an association study identified loci linked to factors that affect cottonseed protein content. CONCLUSIONS: Our results provide a large genome-wide variation data set for primarily cultivated cotton. Thousands of SNPs in representative cotton genotypes provide an opportunity to finely discriminate among cultivated cotton from around the world. The SNPs will be relevant as dense markers of genome variation for association mapping approaches aimed at correlating molecular polymorphisms with variation in phenotypic traits, as well as for molecular breeding approaches in cotton.

Genome-wide association analysis of seedling traits in diverse Sorghum germplasm under thermal stress.

BACKGROUND: Climate variability due to fluctuation in temperature is a worldwide concern that imperils crop production. The need to understand how the germplasm variation in major crops can be utilized to aid in discovering and developing breeding lines that can withstand and adapt to temperature fluctuations is more necessary than ever. Here, we analyzed the genetic variation associated with responses to thermal stresses in a sorghum association panel (SAP) representing major races and working groups to identify single nucleotide polymorphisms (SNPs) that are associated with resilience to temperature stress in a major cereal crop. RESULTS: The SAP exhibited extensive variation for seedling traits under cold and heat stress. Genome-wide analyses identified 30 SNPs that were strongly associated with traits measured at seedling stage under cold stress and tagged genes that act as regulators of anthocyanin expression and soluble carbohydrate metabolism. Meanwhile, 12 SNPs were significantly associated with seedling traits under heat stress and these SNPs tagged genes that function in sugar metabolism, and ion transport pathways. Evaluation of co-expression networks for genes near the significantly associated SNPs indicated complex gene interactions for cold and heat stresses in sorghum. We focused and validated the expression of four genes in the network of Sb06g025040, a basic-helix-loop-helix (bHLH) transcription factor that was proposed to be involved in purple color pigmentation of leaf, and observed that genes in this network were upregulated during cold stress in a moderately tolerant line as compared to the more sensitive line. CONCLUSION: This study facilitated the tagging of genome regions associated with variation in seedling traits of sorghum under cold and heat stress. These findings show the potential of genotype information for development of temperature resilient sorghum cultivars and further characterization of genes and their networks responsible for adaptation to thermal stresses. Knowledge on the gene networks from this research can be extended to the other cereal crops to better understand the genetic basis of resilience to temperature fluctuations during plant developmental stages.

Insights Into Upland Cotton (Gossypium hirsutum L.) Genetic Recombination Based on 3 High-Density Single-Nucleotide Polymorphism and a Consensus Map Developed Independently With Common Parents.

High-density linkage maps are vital to supporting the correct placement of scaffolds and gene sequences on chromosomes and fundamental to contemporary organismal research and scientific approaches to genetic improvement, especially in paleopolyploids with exceptionally complex genomes, eg, upland cotton (Gossypium hirsutum L., "2n = 52"). Three independently developed intraspecific upland mapping populations were analyzed to generate 3 high-density genetic linkage single-nucleotide polymorphism (SNP) maps and a consensus map using the CottonSNP63K array. The populations consisted of a previously reported F2, a recombinant inbred line (RIL), and reciprocal RIL population, from "Phytogen 72" and "Stoneville 474" cultivars. The cluster file provided 7417 genotyped SNP markers, resulting in 26 linkage groups corresponding to the 26 chromosomes (c) of the allotetraploid upland cotton (AD)1 arisen from the merging of 2 genomes ("A" Old World and "D" New World). Patterns of chromosome-specific recombination were largely consistent across mapping populations. The high-density genetic consensus map included 7244 SNP markers that spanned 3538 cM and comprised 3824 SNP bins, of which 1783 and 2041 were in the At and Dt subgenomes with 1825 and 1713 cM map lengths, respectively. Subgenome average distances were nearly identical, indicating that subgenomic differences in bin number arose due to the high numbers of SNPs on the Dt subgenome. Examination of expected recombination frequency or crossovers (COs) on the chromosomes within each population of the 2 subgenomes revealed that COs were also not affected by the SNPs or SNP bin number in these subgenomes. Comparative alignment analyses identified historical ancestral At-subgenomic translocations of c02 and c03, as well as of c04 and c05. The consensus map SNP sequences aligned with high congruency to the NBI assembly of Gossypium hirsutum. However, the genomic comparisons revealed evidence of additional unconfirmed possible duplications, inversions and translocations, and unbalance SNP sequence homology or SNP sequence/loci genomic dominance, or homeolog loci bias of the upland tetraploid At and Dt subgenomes. The alignments indicated that 364 SNP-associated previously unintegrated scaffolds can be placed in pseudochromosomes of the NBI G hirsutum assembly. This is the first intraspecific SNP genetic linkage consensus map assembled in G hirsutum with a core of reproducible mendelian SNP markers assayed on different populations and it provides further knowledge of chromosome arrangement of genic and nongenic SNPs. Together, the consensus map and RIL populations provide a synergistically useful platform for localizing and identifying agronomically important loci for improvement of the cotton crop.

Comparative Transcriptome and Lipidome Analyses Reveal Molecular Chilling Responses in Chilling-Tolerant Sorghums.

Chilling temperatures (0 to 15°C) are a major constraint for temperate cultivation of tropical-origin crops, including the cereal crop sorghum ( [L.] Moench). Northern Chinese sorghums have adapted to early-season chilling, but molecular mechanisms of chilling tolerance are unknown. We used RNA sequencing of seedlings to compare the chilling-responsive transcriptomes of a chilling-tolerant Chinese accession with a chilling-sensitive US reference line, and mass spectrometry to compare chilling-responsive lipidomes of four chilling-tolerant Chinese accessions with two US reference lines. Comparative transcriptomics revealed chilling-induced up-regulation of cold-response regulator C-repeat binding factor (CBF) transcription factor and genes involved in reactive oxygen detoxification, jasmonic acid (JA) biosynthesis, and lipid remodeling phospholipase Dα1 (α) gene in the chilling-tolerant Chinese line. Lipidomics revealed conserved chilling-induced increases in lipid unsaturation, as well as lipid remodeling of photosynthetic membranes that is specific to chilling-tolerant Chinese accessions. Our results point to CBF-mediated transcriptional regulation, galactolipid and phospholipid remodeling, and JA as potential molecular mechanisms underlying chilling adaptation in Chinese sorghums. These molecular systems underlying chilling response could be targeted in molecular breeding for chilling tolerance.

Diversion and Illicit Sale of Extended Release Tapentadol in the United States.

OBJECTIVE: Prescription opioid analgesics are commonly prescribed for moderate to severe pain. An unintended consequence of prescribing opioid analgesics is the abuse and diversion of these medications. Tapentadol ER is a recently approved centrally acting analgesic with synergistic mechanisms of action: µ-opioid receptor agonism and inhibition of norepinephrine reuptake. We assessed the amount of diversion and related cost of obtaining tapentadol IR (Nucynta®) and tapentadol ER (Nucynta ER®) as well as other Schedule II opioid medications in street transactions in the United States using the Researched Abuse, Diversion and Addiction-Related Surveillance (RADARS®) System. METHODS: The Drug Diversion Program measures the number of cases opened by 260 drug diversion investigators in 49 states. StreetRx(TM) uses a crowd-sourcing Website to collect the prices paid for licit or illicit drugs. RESULTS: The population-based rates of diversion were 0.003 (tapentadol IR), 0.001 (tapentadol ER), and 1.495 (other Schedule II opioid tablets) reports per 100,000 population. The tapentadol ER rate was lower than the other Schedule II opioid tablets (P < 0.001) and tapentadol IR (P= 0.004). Diversion rates based on drug availability were 0.03 (tapentadol IR), 0.016 (tapentadol ER), and 0.172 (other Schedule II opioid tablets) per 1,000 prescriptions dispensed. The median street price per milligram was $0.18 (tapentadol IR), $0.10 (tapentadol ER), and $1.00 (other Schedule II opioid tablets). DISCUSSION: Our results indicate that tapentadol ER is rarely sold illicitly in the United States. When sold illicitly, tapentadol ER costs less than other Schedule II opioid products.

AtRAV1 and AtRAV2 overexpression in cotton increases fiber length differentially under drought stress and delays flowering.

There is a longstanding problem of an inverse relationship between cotton fiber qualities versus high yields. To better understand drought stress signaling and adaptation in cotton (Gossypium hirsutum) fiber development, we expressed the Arabidopsis transcription factors RELATED_TO_ABA-INSENSITIVE3/VIVIPAROUS1/(RAV1) and AtRAV2, which encode APETALA2-Basic3 domain proteins shown to repress transcription of FLOWERING_LOCUS_T (FT) and to promote stomatal opening cell-autonomously. In three years of field trials, we show that AtRAV1 and AtRAV2-overexpressing cotton had ∼5% significantly longer fibers with only marginal decreases in yields under well-watered or drought stress conditions that resulted in 40-60% yield penalties and 3-7% fiber length penalties in control plants. The longer transgenic fibers from drought-stressed transgenics could be spun into yarn which was measurably stronger and more uniform than that from well-watered control fibers. The transgenic AtRAV1 and AtRAV2 lines flowered later and retained bolls at higher nodes, which correlated with repression of endogenous GhFT-Like (FTL) transcript accumulation. Elevated expression early in development of ovules was observed for GhRAV2L, GhMYB25-Like (MYB25L) involved in fiber initiation, and GhMYB2 and GhMYB25 involved in fiber elongation. Altered expression of RAVs controlling critical nodes in developmental and environmental signaling hierarchies has the potential for phenotypic modification of crops.

Developing fiber specific promoter-reporter transgenic lines to study the effect of abiotic stresses on fiber development in cotton.

Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Enhancement of reproductive heat tolerance in plants.

Comparison of average crop yields with reported record yields has shown that major crops exhibit annual average yields three- to seven-fold lower than record yields because of unfavorable environments. The current study investigated the enhancement of pollen heat tolerance through expressing an Arabidopsis thaliana heat shock protein 101 (AtHSP101) that is not normally expressed in pollen but reported to play a crucial role in vegetative thermotolerance. The AtHSP101 construct under the control of the constitutive ocs/mas 'superpromoter' was transformed into cotton Coker 312 and tobacco SRI lines via Agrobacterium mediated transformation. Thermotolerance of pollen was evaluated by in vitro pollen germination studies. Comparing with those of wild type and transgenic null lines, pollen from AtHSP101 transgenic tobacco and cotton lines exhibited significantly higher germination rate and much greater pollen tube elongation under elevated temperatures or after a heat exposure. In addition, significant increases in boll set and seed numbers were also observed in transgenic cotton lines exposed to elevated day and night temperatures in both greenhouse and field studies. The results of this study suggest that enhancing heat tolerance of reproductive tissues in plant holds promise in the development of crops with improved yield production and yield sustainability in unfavorable environments.

Natural Variation in Synthesis and Catabolism Genes Influences Dhurrin Content in Sorghum.

Cyanogenic glucosides are natural compounds found in more than 1000 species of angiosperms that produce HCN and are deemed undesirable for agricultural use. However, these compounds are important components of the primary defensive mechanisms of many plant species. One of the best-studied cyanogenic glucosides is dhurrin [(S)-p-hydroxymandelonitrile-ß-D-glucopyranoside], which is produced primarily in sorghum [Sorghum bicolor (L.) Moench]. The biochemical basis for dhurrin metabolism is well established; however, little information is available on its genetic control. Here, we dissect the genetic control of leaf dhurrin content through a genome-wide association study (GWAS) using a panel of 700 diverse converted sorghum lines (conversion panel) previously subjected to pre-breeding and selected for short stature (∼1 m in height) and photoperiod insensitivity. The conversion panel was grown for 2 yr in three environments. Wide variation for leaf dhurrin content was found in the sorghum conversion panel, with the Caudatum group exhibiting the highest dhurrin content and the Guinea group showing the lowest dhurrin content. A GWAS using a mixed linear model revealed significant associations (a false discovery rate [FDR] < 0.05) close to both UGT 185B1 in the canonical biosynthetic gene cluster on chromosome 1 and close to the catabolic dhurrinase loci on chromosome 8. Dhurrin content was associated consistently with biosynthetic genes in the two N-fertilized environments, while dhurrin content was associated with catabolic loci in the environment without supplemental N. These results suggest that genes for both biosynthesis and catabolism are important in determining natural variation for leaf dhurrin in sorghum in different environments.

Overexpression of MIC-3 indicates a direct role for the MIC gene family in mediating Upland cotton (Gossypium hirsutum) resistance to root-knot nematode (Meloidogyne incognita).

KEY MESSAGE: Transgene-based analysis of the MIC-3 gene provides the first report of a cotton gene having a direct role in mediating cotton resistance to root-knot nematode. Major quantitative trait loci have been mapped to Upland cotton (Gossypium hirsutum L.) chromosomes 11 and 14 that govern the highly resistant phenotype in response to infection by root-knot nematode (RKN; Meloidogyne incognita); however, nearly nothing is known regarding the underlying molecular determinants of this RKN-resistant phenotype. Multiple lines of circumstantial evidence have strongly suggested that the MIC (Meloidogyne Induced Cotton) gene family plays an integral role in mediating cotton resistance to RKN. In this report, we demonstrate that overexpression of MIC-3 in the RKN-susceptible genetic background Coker 312 reduces RKN egg production by ca. 60-75 % compared to non-transgenic controls and transgene-null sibling lines. MIC-3 transcript and protein overexpression were confirmed in root tissues of multiple independent transgenic lines with each line showing a similar level of increased resistance to RKN. In contrast to RKN fecundity, transgenic lines showed RKN-induced root galling similar to the susceptible controls. In addition, we determined that this effect of MIC-3 overexpression was specific to RKN as no effect was observed on reniform nematode (Rotylenchulus reniformis) reproduction. Transgenic lines did not show obvious alterations in growth, morphology, flowering, or fiber quality traits. Gene expression analyses showed that MIC-3 transcript levels in uninfected transgenic roots exceeded levels observed in RKN-infected roots of naturally resistant plants and that overexpression did not alter the regulation of native MIC genes in the genome. These results are the first report describing a direct role for a specific gene family in mediating cotton resistance to a plant-parasitic nematode.

Mapping genomic loci for cotton plant architecture, yield components, and fiber properties in an interspecific (Gossypium hirsutum L. × G. barbadense L.) RIL population.

A quantitative trait locus (QTL) mapping was conducted to better understand the genetic control of plant architecture (PA), yield components (YC), and fiber properties (FP) in the two cultivated tetraploid species of cotton (Gossypium hirsutum L. and G. barbadense L.). One hundred and fifty-nine genomic regions were identified on a saturated genetic map of more than 2,500 SSR and SNP markers, constructed with an interspecific recombinant inbred line (RIL) population derived from the genetic standards of the respective cotton species (G. hirsutum acc. TM-1 × G. barbadense acc. 3-79). Using the single nonparametric and MQM QTL model mapping procedures, we detected 428 putative loci in the 159 genomic regions that confer 24 cotton traits in three diverse production environments [College Station F&B Road (FB), TX; Brazos Bottom (BB), TX; and Shafter (SH), CA]. These putative QTL loci included 25 loci for PA, 60 for YC, and 343 for FP, of which 3, 12, and 60, respectively, were strongly associated with the traits (LOD score ≥ 3.0). Approximately 17.7 % of the PA putative QTL, 32.9 % of the YC QTL, and 48.3 % of the FP QTL had trait associations under multiple environments. The At subgenome (chromosomes 1-13) contributed 72.7 % of loci for PA, 46.2 % for YC, and 50.4 % for FP while the Dt subgenome (chromosomes 14-26) contributed 27.3 % of loci for PA, 53.8 % for YC, and 49.6 % for FP. The data obtained from this study augment prior evidence of QTL clusters or gene islands for specific traits or biological functions existing in several non-homoeologous cotton chromosomes. DNA markers identified in the 159 genomic regions will facilitate further dissection of genetic factors underlying these important traits and marker-assisted selection in cotton.

Related to ABA-Insensitive3(ABI3)/Viviparous1 and AtABI5 transcription factor coexpression in cotton enhances drought stress adaptation.

Drought tolerance is an important trait being pursued by the agbiotech industry. Abscisic acid (ABA) is a stress hormone that mediates a multitude of processes in growth and development, water use efficiency (WUE) and gene expression during seed development and in response to environmental stresses. Arabidopsis B3-domain transcription factor Related to ABA-Insensitive3 (ABI3)/Viviparous1 (namely AtRAV2) and basic leucine zipper (bZIPs) AtABI5 or AtABF3 transactivated ABA-inducible promoter:GUS reporter expression in a maize mesophyll protoplast transient assay and showed synergies in reporter transactivation when coexpressed. Transgenic cotton (Gossypium hirsutum) expressing AtRAV1/2 and/or AtABI5 showed resistance to imposed drought stress under field and greenhouse conditions and exhibited improved photosynthesis and WUEs associated with absorption through larger root system and greater leaf area. We observed synergy for root biomass accumulation in the greenhouse, intrinsic WUE in the field and drought tolerance in stacked AtRAV and AtABI5 double-transgenic cotton. We assessed AtABI5 and AtRAV1/2 involvement in drought stress adaptations through reactive oxygen species scavenging and osmotic adjustment by marker gene expression in cotton. Deficit irrigation-grown AtRAV1/2 and AtABI5 transgenics had 'less-stressed' molecular and physiological phenotypes under drought, likely due to improved photoassimilation and root and shoot sink strengths and enhanced expression of endogenous GhRAV and genes for antioxidant and osmolyte biosynthesis. Overexpression of bZIP and RAV TFs could impact sustainable cotton agriculture and potentially other crops under limited irrigation conditions.

Genetic analysis of recombinant inbred lines for Sorghum bicolor × Sorghum propinquum.

We describe a recombinant inbred line (RIL) population of 161 F5 genotypes for the widest euploid cross that can be made to cultivated sorghum (Sorghum bicolor) using conventional techniques, S. bicolor × Sorghum propinquum, that segregates for many traits related to plant architecture, growth and development, reproduction, and life history. The genetic map of the S. bicolor × S. propinquum RILs contains 141 loci on 10 linkage groups collectively spanning 773.1 cM. Although the genetic map has DNA marker density well-suited to quantitative trait loci mapping and samples most of the genome, our previous observations that sorghum pericentromeric heterochromatin is recalcitrant to recombination is highlighted by the finding that the vast majority of recombination in sorghum is concentrated in small regions of euchromatin that are distal to most chromosomes. The advancement of the RIL population in an environment to which the S. bicolor parent was well adapted (indeed bred for) but the S. propinquum parent was not largely eliminated an allele for short-day flowering that confounded many other traits, for example, permitting us to map new quantitative trait loci for flowering that previously eluded detection. Additional recombination that has accrued in the development of this RIL population also may have improved resolution of apices of heterozygote excess, accounting for their greater abundance in the F5 than the F2 generation. The S. bicolor × S. propinquum RIL population offers advantages over early-generation populations that will shed new light on genetic, environmental, and physiological/biochemical factors that regulate plant growth and development.

Prescription drugs purchased through the internet: who are the end users?

Although prescription drugs are readily available on the Internet, little is known about the prevalence of Internet use for the purchase of medications without a legitimate prescription, and the characteristics of those that obtain non-prescribed drugs through online sources. The scientific literature on this topic is limited to anecdotal reports or studies plagued by small sample sizes. Within this context, the focus of this paper is an examination of five national data sets from the U.S. with the purpose of estimating: (1) how common obtaining prescription medications from the Internet actually is, (2) who are the typical populations of "end users" of these non-prescribed medications, and (3) which drugs are being purchased without a prescription. Three of the data sets are drawn from the RADARS (Researched Abuse Diversion and Addiction-Related Surveillance) System, a comprehensive series of studies designed to collect timely and geographically specific data on the abuse and diversion of a number of prescription stimulants and opioid analgesics. The remaining data sets include the National Survey on Drug Use and Health (NSDUH) and the Monitoring the Future (MTF) survey. Our analysis yielded uniformly low rates of prescription drug acquisition from online sources across all five data systems we examined. The consistency of this finding across very diverse populations suggests that the Internet is a relatively minor source for illicit purchases of prescription medications by the individual end-users of these drugs.

Evaluation of source leaf responses to water-deficit stresses in cotton using a novel stress bioassay.

Water-deficit stresses preferentially reduce shoot growth, thereby disrupting the flow of carbohydrates from source leaves to the developing sinks. Here, we use a novel stress bioassay to dissect responses of field and greenhouse-grown cotton (Gossypium hirsutum) source leaves to water-deficit stresses. Fifth main stem leaf samples were harvested at sunrise and subjected to a prolonged elevated respiratory demand in the dark. Sucrose levels are lower in nonstressed cotton at sunrise compared to water-deficit stressed cotton, potentially predisposing the nonstressed tissue to succumb more rapidly. Tissue death was determined initially using the cell viability stain 2,3,5-triphenyltetrazolium chloride, but was determined in subsequent experiments by monitoring the decline in chlorophyll fluorescence yield. Fluorescence yield measurements were obtained within minutes of harvesting and individual samples were monitored over the time course of the treatment. Analyses of the time course and magnitude of chlorophyll fluorescence yield decline in samples from irrigated and dryland plots permitted the detection of stress responses within 24 h of the cessation of irrigation. The rate of fluorescence yield decline during the elevated respiratory demand treatment slowed as the water-deficit stress increased. Upon irrigation, the source leaves of the water-stressed plants recovered to prestress values within 4 d. Well-watered cotton overexpressing heat shock protein 101 had identical rates of fluorescence yield decline as nontransgenic cotton. These results suggest that the delayed decline in fluorescence yield of water-stressed tissue exposed to prolonged elevated respiratory demand can be used as a sensitive indicator of water-deficit stress responses.

Plant lipidomics: discerning biological function by profiling plant complex lipids using mass spectrometry.

Since 2002, plant biologists have begun to apply mass spectrometry to the comprehensive analysis of complex lipids. Such lipidomic analyses have been used to uncover roles for lipids in plant response to stresses and to identify in vivo functions of genes involved in lipid metabolism.

Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance.

Plants are constantly challenged with various abiotic stresses in their natural environment. Elevated temperatures have a detrimental impact on overall plant growth and productivity. Many plants increase their tolerance to high temperatures through an adaptation response known as acquired thermotolerance. To identify the various mechanisms that plants have evolved to cope with high temperature stress, we have isolated a series of Arabidopsis mutants that are defective in the acquisition of thermotolerance after an exposure to 38 degrees C, a treatment that induces acquired thermotolerance in wild-type plants. One of these mutants, atts02, was not only defective in acquiring thermotolerance after the treatment, but also displayed a reduced level of basal thermotolerance in a 30 degrees C growth assay. The affected gene in atts02 was identified by positional cloning and encodes digalactosyldiacylglycerol synthase 1 (DGD1) (the atts02 mutant was, at that point, renamed dgd1-2). An additional dgd1 allele, dgd1-3, was identified in two other mutant lines displaying altered acquired thermotolerance, atts100 and atts104. Expression patterns of several heat shock proteins (HSPs) in heat-treated dgd1-2 homozygous plants were similar to those from identically treated wild-type plants, suggesting that the thermosensitivity in the dgd1-2 mutant was not caused by a defect in HSP induction. Lipid analysis of wild-type and mutant plants indicated a close correlation between the ability to acquire thermotolerance and the increases in digalactosyldiacylglycerol (DGDG) level and in the ratio of DGDG to monogalactosyldiacylglycerol (MGDG). Thermosensitivity in dgd1-2 and dgd1-3 was associated with (1) a decreased DGDG level and (2) an inability to increase the ratio of DGDG to MGDG upon exposure to a 38 degrees C sublethal temperature treatment. Our results suggest that the DGDG level and/or the ratio of DGDG to MGDG may play an important role in basal as well as acquired thermotolerance in Arabidopsis.