A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin.

Septins are a family of filament-forming GTP-binding proteins that regulate fundamental cellular activities, such as cytokinesis and cell polarity. In general, septin filaments function as barriers and scaffolds on the cell cortex. However, little is known about the mechanism that governs the recruitment and localization of the septin complex to the cell cortex. Here, we identified the Cdc42 GTPase-activating protein Rga6 as a key protein involved in promoting the localization of the septin complex to the cell cortex in the fission yeast Schizosaccharomyces pombe. Rga6 interacts with the septin complex and partially colocalizes with the septin complex on the cell cortex. Live-cell microscopy analysis further showed septin enrichment at the cortical regions adjacent to the growing cell tip. The septin enrichment likely plays a crucial role in confining active Cdc42 to the growing cell tip. Hence, our findings support a model whereby Rga6 regulates polarized cell growth partly through promoting targeted localization of the septin complex on the cell cortex. This article has an associated First Person interview with the first author of the paper.

RGA1 regulates grain size, rice quality and seed germination in the small and round grain mutant srg5.

BACKGROUND: Generating elite rice varieties with high yield and superior quality is the main goal of rice breeding programs. Key agronomic traits, including grain size and seed germination characteristics, affect the final yield and quality of rice. The RGA1 gene, which encodes the α-subunit of rice G-protein, plays an important role in regulating rice architecture, seed size and abiotic stress responses. However, whether RGA1 is involved in the regulation of rice quality and seed germination traits is still unclear. RESULTS: In this study, a rice mutant small and round grain 5 (srg5), was identified in an EMS-induced rice mutant library. Systematic analysis of its major agronomic traits revealed that the srg5 mutant exhibited a semi-dwarf plant height with small and round grain and reduced panicle length. Analysis of the physicochemical properties of rice showed that the difference in rice eating and cooking quality (ECQ) between the srg5 mutant and its wild-type control was small, but the appearance quality was significantly improved. Interestingly, a significant suppression of rice seed germination and shoot growth was observed in the srg5 mutant, which was mainly related to the regulation of ABA metabolism. RGA1 was identified as the candidate gene for the srg5 mutant by BSA analysis. A SNP at the splice site of the first intron disrupted the normal splicing of the RGA1 transcript precursor, resulting in a premature stop codon. Additional linkage analysis confirmed that the target gene causing the srg5 mutant phenotype was RGA1. Finally, the introduction of the RGA1 mutant allele into two indica rice varieties also resulted in small and round rice grains with less chalkiness. CONCLUSIONS: These results indicate that RGA1 is not only involved in the control of rice architecture and grain size, but also in the regulation of rice quality and seed germination. This study sheds new light on the biological functions of RGA1, thereby providing valuable information for future systematic analysis of the G-protein pathway and its potential application in rice breeding programs.

Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Pan-genome of Citrullus genus highlights the extent of presence/absence variation during domestication and selection.

The rich genetic diversity in Citrullus lanatus and the other six species in the Citrullus genus provides important sources in watermelon breeding. Here, we present the Citrullus genus pan-genome based on the 400 Citrullus genus resequencing data, showing that 477 Mb contigs and 6249 protein-coding genes were absent in the Citrullus lanatus reference genome. In the Citrullus genus pan-genome, there are a total of 8795 (30.5%) genes that exhibit presence/absence variations (PAVs). Presence/absence variation (PAV) analysis showed that a lot of gene PAV were selected during the domestication and improvement, such as 53 favorable genes and 40 unfavorable genes were identified during the C. mucosospermus to C. lanatus landrace domestication. We also identified 661 resistance gene analogs (RGAs) in the Citrullus genus pan-genome, which contains 90 RGAs (89 variable and 1 core gene) located on the pangenome additional contigs. By gene PAV-based GWAS, 8 gene presence/absence variations were found associated with flesh color. Finally, based on the results of gene PAV selection analysis between watermelon populations with different fruit colors, we identified four non-reference candidate genes associated with carotenoid accumulation, which had a significantly higher frequency in the white flesh. These results will provide an important source for watermelon breeding.

Cdc42 reactivation at growth sites is regulated by local cell-cycle-dependent loss of its GTPase-activating protein Rga4 in fission yeast.

In fission yeast, polarized cell growth stops during division and resumes after cytokinesis completes and cells separate. It is unclear how growth reactivation is timed to occur immediately after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth during septation, displaying a polar elongation simultaneous with septation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, during septation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. Although the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that, for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs).

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.

Rapid Identification by Resequencing-Based QTL Mapping of a Novel Allele RGA1-FH Decreasing Grain Length in a Rice Restorer Line 'Fuhui212'.

Grain size is one of the most frequently selected traits during domestication and modern breeding. The continued discovery and characterization of new genes and alleles in controlling grain size are important in safeguarding the food supply for the world's growing population. Previously, a small grain size was observed in a rice restorer line 'Fuhui212', while the underlying genetic factors controlling this trait were unknown. In this study, by combining QTL mapping, variant effect prediction, and complementation experiments, we recovered a novel allele RGA1-FH that explains most of the phenotypic changes. The RGA1-FH allele contains an A-to-T splicing site variant that disrupts the normal function of RGA1. While population analysis suggests extremely strong artificial selection in maintaining a functional allele of RGA1, our study is the first, to the best of our knowledge, to prove that a dysfunctional RGA1 allele can also be beneficial in real agricultural production. Future breeding programs would benefit from paying more attention to the rational utilization of those overlooked 'unfavored' alleles.

GA-Mediated Disruption of RGA/BZR1 Complex Requires HSP90 to Promote Hypocotyl Elongation.

Circuitries of signaling pathways integrate distinct hormonal and environmental signals, and influence development in plants. While a crosstalk between brassinosteroid (BR) and gibberellin (GA) signaling pathways has recently been established, little is known about other components engaged in the integration of the two pathways. Here, we provide supporting evidence for the role of HSP90 (HEAT SHOCK PROTEIN 90) in regulating the interplay of the GA and BR signaling pathways to control hypocotyl elongation of etiolated seedlings in Arabidopsis. Both pharmacological and genetic depletion of HSP90 alter the expression of GA biosynthesis and catabolism genes. Major components of the GA pathway, like RGA (REPRESSOR of ga1-3) and GAI (GA-INSENSITIVE) DELLA proteins, have been identified as physically interacting with HSP90. Interestingly, GA-promoted DELLA degradation depends on the ATPase activity of HSP90, and inhibition of HSP90 function stabilizes the DELLA/BZR1 (BRASSINAZOLE-RESISTANT 1) complex, modifying the expression of downstream transcriptional targets. Our results collectively reveal that HSP90, through physical interactions with DELLA proteins and BZR1, modulates DELLA abundance and regulates the expression of BZR1-dependent transcriptional targets to promote plant growth.

Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family.

BACKGROUND: Proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine (Pinus flexilis) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus (Cr4) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown. RESULTS: To identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4-segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4. The tight linkage of NLRs with Cr4-controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR. CONCLUSION: This study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.

A novel Arabidopsis gene RGAT1 is required for GA-mediated tapetum and pollen development.

The phytohormone gibberellin (GA) is critical for anther development. RGA, a member of the DELLA family of proteins that are central GA signalling repressors, is a key regulator of male fertility in plants. However, the downstream genes in GA-RGA-mediated anther development remain to be characterised. We identified RGA Target 1 (RGAT1), a novel Arabidopsis gene, that functions as an important RGA-regulated target in pollen development. RGAT1 is predominantly expressed in the tapetum and microspores during anther stages 8-11, and can be directly activated by RGA and suppressed by GA in inflorescence apices. Both loss of function and gain of function of RGAT1 led to abnormal tapetum development, resulting in abortive pollen and short siliques. In RGAT1-knockdown and overexpression lines, pollen abortion occurred at stage 10. Loss of RGAT1 function induced the premature degeneration of tapetal cells with defective ER-derived tapetosomes, while RGAT1 overexpression delayed tapetum degeneration. TUNEL assay confirmed that RGAT1 participates in timely tapetal programmed cell death. Moreover, reducing RGAT1 expression partially rescued the tapetal developmental defects in GA-deficient ga1-3 mutant. Our findings revealed that RGAT1 is a direct target of RGA and plays an essential role in GA-mediated tapetum and pollen development.

Intricate genetic variation networks control the adventitious root growth angle in apple.

BACKGROUND: The root growth angle (RGA) typically determines plant rooting depth, which is significant for plant anchorage and abiotic stress tolerance. Several quantitative trait loci (QTLs) for RGA have been identified in crops. However, the underlying mechanisms of the RGA remain poorly understood, especially in apple rootstocks. The objective of this study was to identify QTLs, validate genetic variation networks, and develop molecular markers for the RGA in apple rootstock. RESULTS: Bulked segregant analysis by sequencing (BSA-seq) identified 25 QTLs for RGA using 1955 hybrids of the apple rootstock cultivars 'Baleng Crab' (Malus robusta Rehd., large RGA) and 'M9' (M. pumila Mill., small RGA). With RNA sequencing (RNA-seq) and parental resequencing, six major functional genes were identified and constituted two genetic variation networks for the RGA. Two single nucleotide polymorphisms (SNPs) of the MdLAZY1 promoter damaged the binding sites of MdDREB2A and MdHSFB3, while one SNP of MdDREB2A and MdIAA1 affected the interactions of MdDREB2A/MdHSFB3 and MdIAA1/MdLAZY1, respectively. A SNP within the MdNPR5 promoter damaged the interaction between MdNPR5 and MdLBD41, while one SNP of MdLBD41 interrupted the MdLBD41/MdbHLH48 interaction that affected the binding ability of MdLBD41 on the MdNPR5 promoter. Twenty six SNP markers were designed on candidate genes in each QTL interval, and the marker effects varied from 0.22°-26.11°. CONCLUSIONS: Six diagnostic markers, SNP592, G122, b13, Z312, S1272, and S1288, were used to identify two intricate genetic variation networks that control the RGA and may provide new insights into the accuracy of the molecular markers. The QTLs and SNP markers can potentially be used to select deep-rooted apple rootstocks.

miR319a/TCP module and DELLA protein regulate trichome initiation synergistically and improve insect defenses in Populus tomentosa.

Trichomes are specialized epidermal cells that contribute to plant resistance against herbivores. Their formation is controlled precisely by multiple genetic and environmental signals. Previous studies have shown that microRNA319 (miR319) and gibberellin (GA) signaling are involved in trichome development in Arabidopsis, but little is known about their interaction between these factors. Here we reported that the miR319a/TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) module participates in trichome initiation synergistically with GA signaling in Populus tomentosa. We demonstrated that overexpression of miR319a decreased transcription levels of its targeted TCPs and significantly elevated leaf trichome density in transgenic poplar, resulting in decreasing insect herbivory. Conversely, repressing miR319a by short tandem target mimics (STTM) elevated TCP expression levels and decreased trichome density in transgenic plants. The trichome phenotype of 35S:miR319a plants could be abolished by introducing a miR319a-resistant form of TCP19. Furthermore, the miR319a-targeted TCP19 interacted directly with REPRESSOR OF ga1-3 (RGA), a downstream repressor of GA signaling. TCP19 and RGA synergistically inhibited the GLABROUS1 (GL1)-induced expression of trichome marker gene GLABRA2 (GL2), thereby repressing leaf trichome initiation. Our results provide an insight into the molecular mechanism by which miR319/TCP19 module and GA signaling coordinated regulating trichome initiation in P. tomentosa.

Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay.

Interferons (IFNs) are important glycoproteins which can stimulate or inhibit up to three hundred different genes encoding proteins involved in antiviral defense mechanisms, inflammation, adaptive immunity, angiogenesis and among other processes. Nevertheless, different genetic alterations may lead to interferon alpha (IFN-α) overproduction in human autoimmune diseases like systemic lupus erythematosus. As a consequence, IFN-α is a central molecule whose activity must be regulated to block their harmful effect on those disorders where the endogenous cytokine production constitutes the etiology of the illnesses. In this work, we evaluate the biological activity of eighty-eight compounds, from our own chemo-library, to find potential IFN-α inhibitors by using a reporter gene assay (RGA) WISH-Mx2/EGFP. We identified some compounds able to modulate negatively the IFN-α activity. The most active IFN-α inhibitors were further studied achieving promising results. In addition, some combinations of the most active compounds were analyzed accomplishing a stronger effect to decrease the IFN-α activity than each compound alone. Furthermore, the complete inhibition of the cytokine activity was reached with some combinations of compounds.

Frontiers in Dissecting and Managing Brassica Diseases: From Reference-Based RGA Candidate Identification to Building Pan-RGAomes.

The Brassica genus contains abundant economically important vegetable and oilseed crops, which are under threat of diseases caused by fungal, bacterial and viral pathogens. Resistance gene analogues (RGAs) are associated with quantitative and qualitative disease resistance and the identification of candidate RGAs associated with disease resistance is crucial for understanding the mechanism and management of diseases through breeding. The availability of Brassica genome assemblies has greatly facilitated reference-based quantitative trait loci (QTL) mapping for disease resistance. In addition, pangenomes, which characterise both core and variable genes, have been constructed for B. rapa, B. oleracea and B. napus. Genome-wide characterisation of RGAs using conserved domains and motifs in reference genomes and pangenomes reveals their clustered arrangements and presence of structural variations. Here, we comprehensively review RGA identification in important Brassica genome and pangenome assemblies. Comparison of the RGAs in QTL between resistant and susceptible individuals allows for efficient identification of candidate disease resistance genes. However, the reference-based QTL mapping and RGA candidate identification approach is restricted by the under-represented RGA diversity characterised in the limited number of Brassica assemblies. The species-wide repertoire of RGAs make up the pan-resistance gene analogue genome (pan-RGAome). Building a pan-RGAome, through either whole genome resequencing or resistance gene enrichment sequencing, would effectively capture RGA diversity, greatly expanding breeding resources that can be utilised for crop improvement.

Proteomic Analysis of a Rice Mutant sd58 Possessing a Novel d1 Allele of Heterotrimeric G Protein Alpha Subunit (RGA1) in Salt Stress with a Focus on ROS Scavenging.

High salinity severely restrains plant growth and results in decrease of crop yield in agricultural production. Thus, it is of great significance to discover the crucial regulators involved in plant salt resistance. Here, we report a novel mutant, sd58, which displays enhanced salt tolerance and dwarf phenotype, by screening from ethyl methane sulfonate (EMS) mutagenized rice mutant library. Genetic analysis showed that sd58 was caused by a single recessive locus. Map-based cloning and allelic test revealed that the phenotypes of sd58 were due to the mutation of RGA1, encoding the alpha subunit of heterotrimeric G protein (Gα). A point mutation (G to A) was identified at the splicing site (GT-AG) of the first intron in RGA1, which gives rise to the generation of abnormal mRNA splicing forms. Furthermore, 332 differentially abundant proteins (DAPs) were identified by using an Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)-based proteomic technique from seedlings of sd58 and Kitaake in response to salt treatment. Gene Ontology (GO) and KEGG pathway enrichment analysis revealed these proteins were mainly involved in regulation of the processes such as metabolic pathways, photosynthesis and reactive oxygen species (ROS) homeostasis. Under salt stress, sd58 displayed lower ROS accumulation than Kitaake, which is consistent with the higher enzyme activities involved in ROS scavenging. Taken together, we propose that RGA1 is one of the regulators in salt response partially through ROS scavenging, which might be helpful in elucidating salt tolerant mechanisms of heterotrimeric G protein in rice.

Illuminating the role of the Gα heterotrimeric G protein subunit, RGA1, in regulating photoprotection and photoavoidance in rice.

We studied physiological mechanisms of photoavoidance and photoprotection of a dwarf rice mutant with erect leaves, d1, in which the RGA1 gene, which encodes the Gα subunit of the heterotrimeric G protein, is non-functional. Leaves of d1 exhibit lower leaf temperature and higher photochemical reflectance index relative to wild type (WT), indicative of increased photoavoidance and more efficient light harvesting. RNA sequencing analysis of flag leaves revealed that messenger RNA levels of genes encoding heat shock proteins, enzymes associated with chlorophyll breakdown, and ROS scavengers were down-regulated in d1. By contrast, genes encoding proteins associated with light harvesting, Photosystem II, cyclic electron transport, Photosystem I, and chlorophyll biosynthesis were up-regulated in d1. Consistent with these observations, when WT and d1 plants were experimentally subjected to the same light intensity, d1 plants exhibited a greater capacity to dissipate excess irradiance (increased nonphotochemical quenching) relative to WT. The increased capacity in d1 for both photoavoidance and photoprotection reduced sustained photoinhibitory damage, as revealed by a higher Fv /Fm . We therefore propose RGA1 as a regulator of photoavoidance and photoprotection mechanisms in rice and highlight the prospect of exploiting modulation of heterotrimeric G protein signalling to increase these characteristics and improve the yield of cereals in the event of abiotic stress.

In-Situ Molecular Vapor Composition Measurements During Lyophilization.

PURPOSE: Monitoring process conditions during lyophilization is essential to ensuring product quality for lyophilized pharmaceutical products. Residual gas analysis has been applied previously in lyophilization applications for leak detection, determination of endpoint in primary and secondary drying, monitoring sterilization processes, and measuring complex solvents. The purpose of this study is to investigate the temporal evolution of the process gas for various formulations during lyophilization to better understand the relative extraction rates of various molecular compounds over the course of primary drying. METHODS: In this study, residual gas analysis is used to monitor molecular composition of gases in the product chamber during lyophilization of aqueous formulations typical for pharmaceuticals. Residual gas analysis is also used in the determination of the primary drying endpoint and compared to the results obtained using the comparative pressure measurement technique. RESULTS: The dynamics of solvent vapors, those species dissolved therein, and the ballast gas (the gas supplied to maintain a set-point pressure in the product chamber) are observed throughout the course of lyophilization. In addition to water vapor and nitrogen, the two most abundant gases for all considered aqueous formulations are oxygen and carbon dioxide. In particular, it is observed that the relative concentrations of carbon dioxide and oxygen vary depending on the formulation, an observation which stems from the varying solubility of these species. This result has implications on product shelf life and stability during the lyophilization process. CONCLUSIONS: Chamber process gas composition during lyophilization is quantified for several representative formulations using residual gas analysis. The advantages of the technique lie in its ability to measure the relative concentration of various species during the lyophilization process. This feature gives residual gas analysis utility in a host of applications from endpoint determination to quality assurance. In contrast to other methods, residual gas analysis is able to determine oxygen and water vapor content in the process gas. These compounds have been shown to directly influence product shelf life. With these results, residual gas analysis technique presents a potential new method for real-time lyophilization process control and improved understanding of formulation and processing effects for lyophilized pharmaceutical products.

Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16.

BACKGROUND: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS). RESULTS: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel. CONCLUSIONS: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

Microdissection of the Ah01 chromosome in upland cotton and microcloning of resistance gene anologs from the single chromosome.

BACKGROUND: Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. RESULTS: Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. CONCLUSIONS: Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.