Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete 1-4. Here we report referencegrade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes 1,3 , these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement. Cotton represents the largest source of natural textile fibers in the world. Over 90% of annual fiber production comes from allotetraploid cotton (G. hirsutum and G. barbadense), which originated from an allopolyplodization event approximately 1-2 million year ago, followed by millennia of asymmetric subgenome selection 5,6. G. hirsutum is cultivated all over the world because of its high yield and G. barbadense is prized for its superior fiber quality. To cultivate G. hirsutum that produces longer, finer and stronger fibers, one approach is to introduce the superior fiber traits from G. barbadense into G. hirsutum. A genomics-enabled breeding strategy requires a detailed and robust understanding of genomic organization. Genomic feature G. hirsutum G. barbadense

Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. Dt, 0.56 × 10 -3 ) ( Fig. 1d and Supplementary Fig. 3). This shows that a large amount is associated with the development of the long fiber trait in cultivated cotton (Fig. 3b). 217Domestication has led to the transformation of cotton fiber from brown to white. 218To understand this phenomenon, we examined two homoeologous gene pairs only 219 subjected to domestication selection in the Dt, 4-COUMARATE:COA LIGASE (4CL) 220 and CHALCONE SYNTHASE (CHS), which encode enzymes involved in the 221 phenylpropanoid metabolic pathway ( Fig. 3c and Supplementary Fig. 6 Fig. 3c). These SNPs display reductions in nucleotide diversity that occurred 225 during domestication (Fig. 3c). Interestingly, we found that the two SNPs in the Fig. 8) 42 . We identified a total of 188,360 DNase I-hypersensitive 248 sites (DHSs) in cotton leaves and fibers, of which ca. 47% are common to both tissues 249 (Fig. 4a). DHSs were preferentially identified in chromosomal arms and 250 approximately half were detected in promoter and intergenic regions ( Fig. 4b and 251 Supplementary Fig. 9). We found DHSs are hypo-methylated, consistent with 252 previous studies 42 (Fig. 4c) H3K4me1 and inactive H3K9me2 (Fig. 4d). Intergenic DHSs were also found to 255 exhibit an enrichment of H3K4me3 and H3K27me3, but depletion of H3K9me2 and 256 no enrichment of H3K4me1 (Fig. 4e). As predicted, the patterns of chromatin 257 modification marks in cotton are different between genic and TE regions 258 ( Supplementary Fig. 10). In addition, genes with promoter DHSs are generally 259 expressed at a higher level in both tissues than those without promoter DHSs (Fig. 4f), 260 and tissue-specific promoter DHSs corresponded to higher levels of gene expression 261 ( Fig. 4g) Hi-C analysis was carried out using the TM-1 accession to characterize global 296 chromatin interactions. We generated 1.1 billion Hi-C paired-end reads, of which ca. possible Hi-C bias, HindIII fragments of less than 2 kb were merged to obtain 299 305,682 chromosomal anchor regions (Fig. 5a). On the basis of a high-quality 300 genome assembly of TM-1 (Supplementary Fig. 11), we used the Hi-C data to 301 characterize the cotton chromatin interactome (Supplementary Fig. 12) and ( Fig. 5b), but many topologically associated domain-like (TAD-like) regions were 305 identified (Fig. 5c, Supplementary Fig. 13 and Supplementary are less frequent at regions marked by H3K9me2 (Fig. 5d). (Fig. 5g). 320We...

Summary Root‐knot nematodes differentiate highly specialized feeding cells in roots (giant cells, GCs), through poorly characterized mechanisms that include extensive transcriptional changes. While global transcriptome analyses have used galls, which are complex root structures that include GCs and surrounding tissues, no global gene expression changes specific to GCs have been described. We report on the differential transcriptome of GCs versus root vascular cells, induced in Arabidopsis by Meloidogyne javanica at a very early stage of their development, 3 days after infection (d.p.i.). Laser microdissection was used to capture GCs and root vascular cells for microarray analysis, which was validated through qPCR and by a promoter‐GUS fusion study. Results show that by 3 d.p.i., GCs exhibit major gene repression. Although some genes showed similar regulation in both galls and GCs, the majority had different expression patterns, confirming the molecular distinctiveness of the GCs within the gall. Most of the differentially regulated genes in GCs have no previously assigned function. Comparisons with other transcriptome analyses revealed similarities between GCs and cell suspensions differentiating into xylem cells. This suggests a molecular link between GCs and developing vascular cells, which represent putative GC stem cells. Gene expression in GCs at 3 d.p.i. was also found to be similar to crown galls induced by Agrobacterium tumefaciens, a specialized root biotroph.

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a ⌬ 8-⌬ 7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function. INTRODUCTIONSterols are essential components of fungal, plant, and animal membranes. They regulate fluidity and interact with lipids and proteins within the membrane, and they are the precursors for the brassinosteroid (BR) hormones in plants (Hartmann, 1998). The sterol biosynthetic pathway in plants, therefore, can be viewed as comprising two parts: one branch produces the bulk membrane sterols (the principal sterols in Arabidopsis being stigmasterol, campesterol, and sitosterol), and the second part represents the BR synthesis branch. Sterol biosynthesis has been well characterized in yeast, supported by a powerful system of genetic analysis. In animals, and more recently in plants, sterol biosynthetic enzyme function has been confirmed via the functional complementation of yeast mutants (Gachotte et al., 1996). Functional analysis of sterol function in plants has involved a range of approaches, but recently, genetic studies have provided useful information on the requirement for particular enzymes in sterol and BR biosynthesis and, for BRs, perception and signal transduction (Clouse, 2000;Diener et al., 2000;Schaeffer et al., 2001).In animals, sterols appear to be important to maintain correct cell-signaling activities. For example, drugs such as the ligand SR31747A, which inhibits the activity of the receptor (emopamil binding protein [EBP], which has ⌬ 8-⌬ 7 sterol isomerase activity), cause defects in a diversity of cellular processes, including the inhibition of mammalian lymphocyte proliferation in response to mitogens (Derocq et al., 1995) and the inhibition of graft rejection in mouse via the modulation of gene expression (Carayon et al., 1995), and they may influence lipoprotein functions leading to immunosuppressive effects (Dussossoy et al., 1999). In plants, a lack of detailed pharmacological studies has precluded analogous investigations of the role of sterols in plant cell biology.However, mutational and transgenic studies have given new insight into the roles of sterols in plant development. sterol methyltransferase1 ( smt1 ) mutants accumulate cholesterol...

Genome editing (GE) has revolutionized biological research through the new ability to precisely edit the genomes of living organisms. In recent years, various GE tools have been explored for editing simple and complex genomes. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has widely been used in GE due to its high efficiency, ease of use, and accuracy. It can be used to add desirable and remove undesirable alleles simultaneously in a single event. Here, we discuss various applications of CRISPR/Cas9 in a range of important crops, compare it with other GE tools, and review its mechanism, limitations, and future possibilities. Various newly emerging CRISPR/Cas systems, including base editing (BE), xCas9, and Cas12a (Cpf1), are also considered. Genome EditingGenome editing (GE) (see Glossary) is a technique which introduces DNA mutations in the form of insertions and/or deletions (indels) or base substitutions in target sequences. GE comprises various techniques, such as the use of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the most recently developed clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system. ZFNs are targetable DNA cleavage proteins used to cut DNA sequences at any site. TALENs induce double-stranded breaks (DSBs) in target sequences, which trigger DNA damage response pathways, leading to genome modification [1]. Although ZFNs and TALENs have been widely used (since 2002 and 2011, respectively) for GE in human, animal, and plant cells, there are still some limitations which hinder their effective use. The specificity of ZFN is limited and it frequently introduces off-target mutations [2]. Vector construction for ZFNs and TALENs is time-and labor-consuming [3]. Therefore, since 2013, attention has been diverted towards the use of CRISPR/Cas9 and more recently towards several newly emerging CRISPR/Cas variants. CRISPR/Cas9 is an RNA-guided endonuclease that specifically targets DNA sequences via nucleotide base pairing (Box 1). Here, we review the applications of CRISPR/Cas9 in crop plants and its comparisons with other GE tools, such as ZFNs and TALENS. We focus on target efficiency and specificity, mechanism, and challenges and limitations, but also discuss prospects for the use of newly emerging GE tools, such as base editing (BE), xCas9, Cas12a (Cpf1), and Cas 13 in plants. Emerging CRISPR/Cas Systems for GETo overcome the limitations of the CRISPR/Cas9 system (Box 2), a variety of CRISPR systems have been generated for efficient GE. The Cas9 variant CjCas9, derived from Campylobacter jejuni, is composed of 984 amino acid residues (2.95 kbp) and has been used for efficient GE in vitro and in vivo. CjCas9 is highly specific and cuts only a limited number of sites in the genomes of mouse or human. Delivered through adeno-associated virus (AAV), it has been shown to induce targeted mutations at high frequencies in retinal pigment epithelium (RPE) cells or mouse m...

Summary Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture.We examine the effect of osmotic stress on abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth through effects on PIN proteins. We integrate experimental data to construct hormonal crosstalk networks to formulate a systems view of root growth regulation by multiple hormones.Experimental analysis shows: that ABA‐dependent and ABA‐independent stress responses increase under osmotic stress, but cytokinin responses are only slightly reduced; inhibition of root growth under osmotic stress does not require ethylene signalling, but auxin can rescue root growth and meristem size; osmotic stress modulates auxin transporter levels and localization, reducing root auxin concentrations; PIN1 levels are reduced under stress in an ABA‐dependent manner, overriding ethylene effects; and the interplay among ABA, ethylene, cytokinin and auxin is tissue‐specific, as evidenced by differential responses of PIN1 and PIN2 to osmotic stress.Combining experimental analysis with network construction reveals that ABA regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with b,g-methyleneadenosine 59-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

SummaryIt is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes.