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To more sustainably mitigate the impact of crop diseases on plant health and productivity, there is a need for broader spectrum, long-lasting resistance traits. Defense response (DR) genes, located throughout the genome, participate in cellular and system-wide defense mechanisms to stave off infection by diverse pathogens. This multigenic resistance avoids rapid evolution of a pathogen to overcome host resistance. DR genes reside within resistance-associated quantitative trait loci (QTL), and alleles of DR genes in resistant varieties are more active during pathogen attack relative to susceptible haplotypes. Differential expression of DR genes results from polymorphisms in their regulatory regions, that includes cis-regulatory elements such as transcription factor binding sites as well as features that influence epigenetic structural changes to modulate chromatin accessibility during infection. Many of these elements are found in clusters, known as cis-regulatory modules (CRMs), which are distributed throughout the host genome. Regulatory regions involved in plant–pathogen interactions may also contain pathogen effector binding elements that regulate DR gene expression, and that, when mutated, result in a change in the plants’ response. We posit that CRMs and the multiple regulatory elements that comprise them are potential targets for marker-assisted breeding for broad-spectrum, durable disease resistance.

Summary The cytokinins, which are N6‐substituted adenine derivatives, control key aspects of crop productivity. Cytokinin levels are controlled via biosynthesis by isopentenyl transferase (IPT), destruction by cytokinin oxidase/dehydrogenase (CKX), and inactivation via glucosylation by cytokinin glucosyl transferases (CGTs). While both yield components and tolerance to drought and related abiotic stressors have been positively addressed via manipulation of IPT and/or CKX expression, much less attention has been paid to the CGTs. As naming of the CGTs has been unclear, we suggest COGT, CNGT, CONGT and CNOGT to describe the O‐, N‐ and dual function CGTs. As specific CGT mutants of both rice and arabidopsis showed impacts on yield components, we interrogated the wheat genome database, IWGSC RefSeq v1.0 & v2.0, to investigate wheat CGTs. Besides providing unambiguous names for the 53 wheat CGTs, we show their expression patterns in 70 developmental tissues and their response characteristics to various stress conditions by reviewing more than 1000 RNA‐seq data sets. These revealed various patterns of responses and showed expression generally being more limited in reproductive tissues than in vegetative tissues. Multiple cis‐regulatory elements are present in the 3 kb upstream of the start codons of the 53 CGTs. Elements associated with abscisic acid, light and methyl jasmonate are particularly over‐represented, indicative of the responsiveness of CGTs to the environment. These data sets indicate that CGTs have potential value for wheat improvement and that these could be targeted in TILLING or gene editing wheat breeding programmes.

Summary LONELY GUY (LOG) was first identified in a screen of rice mutants with defects in meristem maintenance. In plants, LOG codes for cytokinin riboside 5′‐monophosphate phosphoribohydrolase, which converts inactive cytokinin nucleotides directly to the active free bases. Many enzymes with the PGGxGTxxE motif have been misannotated as lysine decarboxylases; conversely not all enzymes containing this motif are cytokinin‐specific LOGs. As LOG mutants clearly impact yield in rice, we investigated the LOG gene family in bread wheat. By interrogating the wheat (Triticum aestivum) genome database, we show that wheat has multiple LOGs. The close alignment of TaLOG1, TaLOG2 and TaLOG6 with the X‐ray structures of two functional Arabidopsis thaliana LOGs allows us to infer that the wheat LOGs 1‐11 are functional LOGs. Using RNA‐seq data sets, we assessed TaLOG expression across 70 tissue types, their responses to various stressors, the pattern of cis‐regulatory elements (CREs) and intron/exon patterns. TaLOG gene family members are expressed variously across tissue types. When the TaLOG CREs are compared with those of the cytokinin dehydrogenases (CKX) and glucosyltransferases (CGT), there is close alignment of CREs between TaLOGs and TaCKXs reflecting the key role of CKX in maintaining cytokinin homeostasis. However, we suggest that the main homeostatic mechanism controlling cytokinin levels in response to biotic and abiotic challenge resides in the CGTs, rather than LOG or CKX. However, LOG transgenics and identified mutants in rice variously impact yield, providing interesting avenues for investigation in wheat.

Summary Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic‐related and salt stress‐inducible synthetic promoters for use in hybrid poplar. De novo motif‐detecting algorithms yielded 30 water‐deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water‐deficit stress motifs (SD18, SD13 and SD9) found in 16 co‐expressed gene promoters, and we discovered a well‐conserved motif for salt response (SS16). We characterized several native poplar stress‐inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five‐to‐eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18‐1, SD9‐2, SS16‐1, SS16‐2 and SS16‐3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18‐1 and SD9‐2 were induced by water‐deficit stress, whereas SS16‐1, SS16‐2 and SS16‐3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

Transcription initiated at alternative sites can produce mRNA isoforms with different 5ʹUTRs, which are potentially subjected to differential translational regulation. However, the prevalence of such isoform‐specific translational control across mammalian genomes is currently unknown. By combining polysome profiling with high‐throughput mRNA 5ʹ end sequencing, we directly measured the translational status of mRNA isoforms with distinct start sites. Among 9,951 genes expressed in mouse fibroblasts, we identified 4,153 showed significant initiation at multiple sites, of which 745 genes exhibited significant isoform‐divergent translation. Systematic analyses of the isoform‐specific translation revealed that isoforms with longer 5ʹUTRs tended to translate less efficiently. Further investigation of cis‐ elements within 5ʹUTRs not only provided novel insights into the regulation by known sequence features, but also led to the discovery of novel regulatory sequence motifs. Quantitative models integrating all these features explained over half of the variance in the observed isoform‐divergent translation. Overall, our study demonstrated the extensive translational regulation by usage of alternative transcription start sites and offered comprehensive understanding of translational regulation by diverse sequence features embedded in 5ʹUTRs. Synopsis Polysome profiling combined with 5ʹ‐end sequencing in mouse fibroblasts shows pervasive isoform‐specific translational regulation via alternative transcription start sites (TSSs) and reveals 5′UTR sequence features linked to translational regulation. Isoform‐specific translational regulation is achieved through alternative TSS usage. Isoforms with longer 5ʹUTRs tend to have lower translational efficiency (TE). Systematic analyses of isoform‐specific translation offer new insights into the regulation by known sequence features and identifies novel regulatory sequence motifs. Quantitative models integrating all identified sequence features explain over half of the variance in the observed TE divergence between isoforms. Graphical Abstract Polysome profiling combined with 5ʹ‐end sequencing in mouse fibroblasts shows pervasive isoform‐specific translational regulation via alternative transcription start sites (TSSs) and reveals 5′UTR sequence features linked to translational regulation.

Epithelial cells at many sites in the body are affected by the inherited disorder cystic fibrosis (CF). The lung was the major focus of research until recently, when effective therapeutics became available for most people with CF. There is now renewed interest in CF aetiology in other locations in the body, where the regulatory mechanisms for the CF transmembrane conductance regulator (CFTR) gene are less well‐characterised. The definition of the genomic elements controlling CFTR expression and their associated transcription factors is important for the design of gene‐based therapies. Here we identify the cis‐regulatory elements (CREs) associated with the CFTR locus by open chromatin mapping in pancreatic adenocarcinoma cell lines, primary human pancreatic and bile duct (cholangiocyte) organoids and single cells from tissues, as well as sweat gland coil and duct epithelial cells. We show that broadly these cell types use a combination of CREs that were characterised previously either in airway or intestinal epithelial cells, though not occurring together in these two cell lineages. Moreover, the chromatin structure of the CFTR locus in pancreatic cell lines is consistent with earlier models. We also use bioinformatic tools to predict the transcription factor network in these rare cell lineages from open chromatin peaks genome‐wide.

• Plant volatile emissions can recruit predators of herbivores for indirect defense and attract pollinators to aid in pollination. Although volatiles involved in defense and pollinator attraction are primarily emitted from leaves and flowers, respectively, they will co-evolve if their underlying genetic basis is intrinsically linked, due either to pleiotropy or to genetic linkage. However, direct evidence of co-evolving defense and floral traits is scarce. • We characterized intraspecific variation of herbivory-induced plant volatiles (HIPVs), the key components of indirect defense against herbivores, and floral volatiles in wild tobacco Nicotiana attenuata. • We found that variation of (E)-β-ocimene and (E)-α-bergamotene contributed to the correlated changes in HIPVs and floral volatiles among N. attenuata natural accessions. Intraspecific variations of (E)-β-ocimene and (E)-α-bergamotene emissions resulted from allelic variation of two genetically co-localized terpene synthase genes, NaTPS25 and NaTPS38, respectively. Analyzing haplotypes of NaTPS25 and NaTPS38 revealed that allelic variations of NaTPS25 and NaTPS38 resulted in correlated changes of (E)-β-ocimene and (E)-α-bergamotene emission in HIPVs and floral volatiles in N. attenuata. • Together, these results provide evidence that pleiotropy and genetic linkage result in correlated changes in defenses and floral signals in natural populations, and the evolution of plant volatiles is probably under diffuse selection.

Background The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes ( ERF ) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. Results A set of 28 cDNA clones encoding ERFs in the tomato ( Solanum lycopersicon ) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis -acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters. Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. Conclusions The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

The maize (Zea mays) enzyme b-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of β-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5′-flanking region containing the 263 bp 5′-untranslated region (5′-UTR) including the first intron. We linked this to the β-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5′-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.

Abscisic acid (ABA)-responsive elements (ABREs) are the major cis-regulatory elements in ABA-induced gene expression. However, the impact of sequence variations on ABRE function is not yet well-understood. Here, we used synthetic STARR-seq to quantitatively assess the effects of single-nucleotide substitutions on ABRE activity. Our results revealed that the nucleotide substitutions in both the ACGT-core and ACGT-flank regions affected transcriptional strength. Alterations in the ACGT-core sequence had a more significant impact on ABRE activity than changes in the flanking region. Interestingly, we observed that the ACGT-flank variants with high activity exhibited a strong sequence preference in the downstream region, whereas the highly active core variants were diverse in sequence patterns. Our studies provide a quantitative map of ABRE activity at single-nucleotide resolution, which will facilitate the design of ABA-responsive promoters with desired activities in plants.