A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana.

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

GhSBI1, a CUP-SHAPED COTYLEDON 2 homologue, modulates branch internode elongation in cotton.

Branch length is an important plant architecture trait in cotton (Gossypium) breeding. Development of cultivars with short branch has been proposed as a main object to enhance cotton yield potential, because they are suitable for high planting density. Here, we report the molecular cloning and characterization of a semi-dominant quantitative trait locus, Short Branch Internode 1(GhSBI1), which encodes a NAC transcription factor homologous to CUP-SHAPED COTYLEDON 2 (CUC2) and is regulated by microRNA ghr-miR164. We demonstrate that a point mutation found in sbi1 mutants perturbs ghr-miR164-directed regulation of GhSBI1, resulting in an increased expression level of GhSBI1. The sbi1 mutant was sensitive to exogenous gibberellic acid (GA) treatments. Overexpression of GhSBI1 inhibited branch internode elongation and led to the decreased levels of bioactive GAs. In addition, gene knockout analysis showed that GhSBI1 is required for the maintenance of the boundaries of multiple tissues in cotton. Transcriptome analysis revealed that overexpression of GhSBI1 affects the expression of plant hormone signalling-, axillary meristems initiation-, and abiotic stress response-related genes. GhSBI1 interacted with GAIs, the DELLA repressors of GA signalling. GhSBI1 represses expression of GA signalling- and cell elongation-related genes by directly targeting their promoters. Our work thus provides new insights into the molecular mechanisms for branch length and paves the way for the development of elite cultivars with suitable plant architecture in cotton.

Two adjacent NAC transcription factors regulate fruit maturity date and flavor in peach.

Although maturity date (MD) is an essential factor affecting fresh fruit marketing and has a pleiotropic effect on fruit taste qualities, the underlying mechanisms remain largely unclear. In this study, we functionally characterized two adjacent NAM-ATAF1/2-CUC2 (NAC) transcription factors (TFs), PpNAC1 and PpNAC5, both of which were associated with fruit MD in peach. PpNAC1 and PpNAC5 were found capable of activating transcription of genes associated with cell elongation, cell wall degradation and ethylene biosynthesis, suggesting their regulatory roles in fruit enlargement and ripening. Furthermore, PpNAC1 and PpNAC5 had pleiotropic effects on fruit taste due to their ability to activate transcription of genes for sugar accumulation and organic acid degradation. Interestingly, both PpNAC1 and PpNAC5 orthologues were found in fruit-producing angiosperms and adjacently arranged in all 91 tested dicots but absent in fruitless gymnosperms, suggesting their important roles in fruit development. Our results provide insight into the regulatory roles of NAC TFs in MD and fruit taste.

WOX1 controls leaf serration development via temporally restricting BZR1 and CUC3 expression in Arabidopsis.

Leaves evolve shape diversity ranging from simple leaves with smooth margin to complicated shape with toothed/serrated, lobed and dissected leaves with leaflets. In the model plant Arabidopsis thaliana with simple leaves producing serrated margin, boundary regulatory factors CUP SHAPED COTYLEDON 2 (CUC2) and CUC3 play important roles in promoting leaf serration initiation and maintenance. Stem cell related WUSCHEL-RELATED HOMEOBOX1 (WOX1) and PRESSED FLOWER/WOX3 are also essential for leaf margin morphogenesis, but the role of WOX1 and PRS as well as the relationships between CUCs and WOXs on tooth development was unclear. In this study, we found that WOX1, but not PRS, prevents overproduction of tooth number and excessive tooth size by limiting CUC3 expression to a moderate level in a temporally regulated manner. We also revealed that BRASSINAZOLE RESISTANT 1 (BZR1), a known regulator for plant development including boundary regions, is involved in WOX1 negative regulation of tooth development by repressing CUC3 expression during the initiation/early stage of tooth development. WOX1 parallelly limits BZR1 and CUC3 expression from the late stage of the first 2 teeth, while restricts CUC3 activity in a BZR1 dependent manner from the initiation/early stage of subsequently developed teeth. This study uncovers a new mechanism for WOX1 in fine-tuning the leaf margin geometry.

An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development.

The sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promoted the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene-insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.

Genome-Wide Identification of NAC Gene Family Members of Tree Peony (Paeonia suffruticosa Andrews) and Their Expression under Heat and Waterlogging Stress.

An important family of transcription factors (TFs) in plants known as NAC (NAM, ATAF1/2, and CUC2) is crucial for the responses of plants to environmental stressors. In this study, we mined the NAC TF family members of tree peony (Paeonia suffruticosa Andrews) from genome-wide data and analyzed their response to heat and waterlogging stresses in conjunction with transcriptome data. Based on tree peony's genomic information, a total of 48 PsNAC genes were discovered. Based on how similar their protein sequences were, these PsNAC genes were divided into 14 branches. While the gene structures and conserved protein motifs of the PsNAC genes within each branch were largely the same, the cis-acting elements in the promoter region varied significantly. Transcriptome data revealed the presence of five PsNAC genes (PsNAC06, PsNAC23, PsNAC38, PsNAC41, PsNAC47) and one PsNAC gene (PsNAC37) in response to heat and waterlogging stresses, respectively. qRT-PCR analysis reconfirmed the response of these five PsNAC genes to heat stress and one PsNAC gene to waterlogging stress. This study lays a foundation for the study of the functions and regulatory mechanisms of NAC TFs in tree peony. Meanwhile, the NAC TFs of tree peony in response to heat and waterlogging stress were excavated, which is of great significance for the selection and breeding of new tree peony varieties with strong heat and waterlogging tolerance.

Gibberellins regulate ovule number through a DELLA-CUC2 complex in Arabidopsis.

Ovule development is a key process for plant reproduction, helping to ensure correct seed production. Several molecular factors and plant hormones such as gibberellins are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been shown to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA acts in ovule primordia initiation remained unknown. In this study we report that DELLA proteins participate in ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, through the direct GAI-CUC2 interaction in placental cells that would determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and co-localization in the placenta supports this hypothesis. Moreover, molecular analysis identified a subset of the loci for which the GAI protein may act as a transcriptional co-regulator in a CUC2-dependent manner. The DELLA-CUC2 complex is a component of the gene regulatory network controlling ovule primordia initiation in Arabidopsis.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Glyoxalase I-4 functions downstream of NAC72 to modulate downy mildew resistance in grapevine.

Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α-ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI-4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain-containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI-4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI-4 via the CACGTG element, leading to inhibition of VvGLYI-4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI-4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI-4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG-associated ROS homeostasis through a salicylic acid-mediated defense pathway.

Ethylene regulates miRNA-mediated lignin biosynthesis and leaf serration in Arabidopsis thaliana.

microRNAs (miRNAs) regulate target gene expression by pairing to target mRNAs, leading to mRNA degradation or translation inhibition. Out of several miRNAs in Arabidopsis, miR397b and miR857 regulate secondary growth by modulating lignin polymerization and deposition in secondary xylem cells by targeting laccases. Interestingly, the phytohormone ethylene is also suggested to have a role in lignin biosynthesis in tension wood formation. Despite this information, it is not known whether ethylene has any role in controlling secondary growth via miRNAs-mediated pathways. In this study, we elucidate that ethylene acts upstream to the miR397b/miR857-laccases module and negatively regulates lignin biosynthesis by directly activating the expression of both the miRNAs. The binding of EIN3 to the promoter of miR397b is further validated by yeast one-hybrid assay. In addition to its role in lignification, ethylene also regulates leaf serration by directly regulating the expression of NAC transcription factors, like CUP-SHAPED COTYLEDON2 (CUC2) and CUC3. Together, our study suggests a novel mechanism involving ethylene and miRNAs in lignin biosynthesis and leaf serration in Arabidopsis thaliana.

Interspecific complementation-restoration of phenotype in Arabidopsis cuc2cuc3 mutant by sugarcane CUC2 gene.

BACKGROUND: In plants, a critical balance between differentiation and proliferation of stem cells at the shoot apical meristem zone is essential for proper growth. The spatiotemporal regulation of some crucial genes dictates the formation of a boundary within and around budding organs. The boundary plays a pivotal role in distinguishing one tissue type from another and provides a defined shape to the organs at their developed stage. NAM/CUC subfamily of the NAC transcription factors control the boundary formation during meristematic development. RESULTS: Here, we have identified the CUP-SHAPED COTYLEDON (CUC) genes in sugarcane and named SsCUC2 (for the orthologous gene of CUC1 and CUC2) and SsCUC3. The phylogenetic reconstruction showed that SsCUCs occupy the CUC2 and CUC3 clade together with monocots, whereas eudicot CUC2 and CUC3 settled separately in the different clade. The structural analysis of CUC genes showed that most of the CUC3 genes were accompanied by an intron gain during eudicot divergence. Besides, the study of SsCUCs expression in the RNA-seq obtained during different stages of ovule development revealed that SsCUCs express in developing young tissues, and the expression of SsCUC2 is regulated by miR164. We also demonstrate that SsCUC2 (a monocot) could complement the cuc2cuc3 mutant phenotype of Arabidopsis (eudicot). CONCLUSIONS: This study further supports that CUC2 has diverged in CUC1 and CUC2 during the evolution of monocots and eudicots from ancestral plants. The functional analysis of CUC expression patterns during sugarcane ovule development and ectopic expression of SsCUC2 in Arabidopsis showed that SsCUC2 has a conserved role in boundary formation. Overall, these findings improve our understanding of the functions of sugarcane CUC genes. Our results reveal the crucial functional role of CUC genes in sugarcane.

What's unique? The unfolded protein response in plants.

The investigation of a phenomenon called the unfolded protein response (UPR) started approximately three decades ago, and we now know that the UPR is involved in a number of cellular events among metazoans, higher plants, and algae. The relevance of the UPR in human diseases featuring protein folding defects, such as Alzheimer's and Huntington's diseases, has drawn much attention to the response in medical research to date. While metazoans and plants share similar molecular mechanisms of the UPR, recent studies shed light on the uniqueness of the plant UPR, with plant-specific protein families appearing to play pivotal roles. Given the considerable emphasis on the original discoveries of key factors in metazoans, this review highlights the uniqueness of the plant UPR based on current knowledge.

Coordination of differentiation rate and local patterning in compound-leaf development.

The variability in leaf form in nature is immense. Leaf patterning occurs by differential growth, taking place during a limited window of morphogenetic activity at the leaf marginal meristem. While many regulators have been implicated in the designation of the morphogenetic window and in leaf patterning, how these effectors interact to generate a particular form is still not well understood. We investigated the interaction among different effectors of tomato (Solanum lycopersicum) compound-leaf development, using genetic and molecular analyses. Mutations in the tomato auxin response factor SlARF5/SlMP, which normally promotes leaflet formation, suppressed the increased leaf complexity of mutants with extended morphogenetic window. Impaired activity of the NAC/CUC transcription factor GOBLET (GOB), which specifies leaflet boundaries, also reduced leaf complexity in these backgrounds. Analysis of genetic interactions showed that the patterning factors SlMP, GOB and the MYB transcription factor LYRATE (LYR) coordinately regulate leaf patterning by modulating in parallel different aspects of leaflet formation and shaping. This work places an array of developmental regulators in a morphogenetic context. It reveals how organ-level differentiation rate and local growth are coordinated to sculpture an organ. These concepts are applicable to the coordination of pattering and differentiation in other species and developmental processes.

Serum HDL cholesterol uptake capacity in subjects from the MASHAD cohort study: Its value in determining the risk of cardiovascular endpoints.

BACKGROUND: The efficiency of high-density lipoprotein (HDL) to efflux cholesterol contributes to the reverse cholesterol transport (RCT) pathway as one of HDL's proposed functions and depends on the ability of HDL to uptake cholesterol. We aimed to investigate cholesterol uptake capacity (CUC) by a newly developed assay in samples from the MASHAD (Mashhad Stroke and Heart Atherosclerotic Disorders) cohort study. METHOD: The study population comprised 153 individuals developed CVD diagnosed by a specialist cardiologist, over 6 years of follow-up, and 350 subjects without CVD. We used a modified CUC method to evaluate the functionality of HDL in serum samples. RESULT: The CUC assay was highly reproducible with values for inter- and intra-assay variation of 13.07 and 6.65, respectively. The mean serum CUC was significantly lower in the CVD group compared to control (p = 0.01). Although, there were no significant differences in serum HDL-C between the groups and there was no significantly association with risk of progressive CVD. Multivariate logistic regression analysis showed that there was a significantly negative association between CUC and risk of CVD after adjustment for confounding parameters (OR = 0.57, 95% CI = 0.38-0.87, p = 0.009). The CUC was also inversely and independently associated with the risk of CVD event using Cox proportional hazards models analysis (HR = 0.62; 95% CI = 0.41-0.94, p = 0.02). We determined the optimum cutoff value of 1.7 a.u for CUC in the population. Furthermore, the CUC value was important in determining the CVD risk stratification derived from data mining analysis. CONCLUSIONS: Reduced HDL functionality, as measured by CUC, appears to predict CVD in population sample from north-eastern Iran.

Revisiting floral fusion: the evolution and molecular basis of a developmental innovation.

Throughout the evolution of the angiosperm flower, developmental innovations have enabled the modification or elaboration of novel floral organs enabling subsequent diversification and expansion into new niches, for example the formation of novel pollinator relationships. One such developmental innovation is the fusion of various floral organs to form complex structures. Multiple types of floral fusion exist; each type may be the result of different developmental processes and is likely to have evolved multiple times independently across the angiosperm tree of life. The development of fused organs is thought to be mediated by the NAM/CUC3 subfamily of NAC transcription factors, which mediate boundary formation during meristematic development. The goal of this review is to (i) introduce the development of fused floral organs as a key 'developmental innovation', facilitated by a change in the expression of NAM/CUC3 transcription factors; (ii) provide a comprehensive overview of floral fusion phenotypes amongst the angiosperms, defining well-known fusion phenotypes and applying them to a systematic context; and (iii) summarize the current molecular knowledge of this phenomenon, highlighting the evolution of the NAM/CUC3 subfamily of transcription factors implicated in the development of fused organs. The need for a network-based analysis of fusion is discussed, and a gene regulatory network responsible for directing fusion is proposed to guide future research in this area.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.

Conserved and novel roles of miR164-CUC2 regulatory module in specifying leaf and floral organ morphology in strawberry.

MicroRNAs (miRNAs) are a kind of short noncoding RNA (20-24 nt), playing versatile roles in plant growth and development. Strawberry generates leaves and flowers with unique features. However, few miRNAs have been functionally characterised in strawberry, especially for their developmental regulation. Here, we identified one ethyl methanesulfonate (EMS) mutant, deeply serrated (des), in the woodland strawberry Fragaria vesca that has wrinkled leaves with deeper serrations, serrated petals and deformed carpels. The causative mutation occurs in the 19th nucleotide of the FvemiR164a mature sequence. Overexpressing FveMIR164A rescued the phenotypes of des/fvemir164a except the petal serrations. Furthermore, we identified two allelic mutants of FveCUC2a, one target of FvemiR164a, which developed leaves with smooth margins and fused leaflets. Phenotypes of the double mutant fvemir164a fvecuc2a indicated that the two genes act linearly in leaf and carpel development, but synergistically in the development of other floral organs and inflorescence architecture. This work demonstrates the conserved and novel roles of the miR164-CUC2 module in leaf and flower development in different plant species, and reveals that the 19th nucleotide of FvemiR164a is important for its processing.

Expression pattern of CUC3 ortholog in Zeylanidium tailichenoides (Podostemaceae) infers organization of a unique distichous shoot in Podostemoideae.

Shoots of the aquatic eudicot family, Podostemaceae, exhibit unusual organogenesis with mixed leaf and stem identities. New shoots arise at the base of the older shoot with shoot apical meristem (SAM) identity but the entire SAM differentiates into a "leaf" as it develops in the Podostemoideae subfamily. The "leaves" are tightly arranged in a zigzag manner to form an apparent distichous shoot as a whole. Although previous studies have suggested that Podostemoideae shoots have evolved by modifying the ancestral sympodial branching system in the basal Tristichoideae subfamily, this evolutionary scenario requires elucidation at the molecular level. To confirm that the shoots arise as axillary shoots, in the present study, we examined gene expression patterns in plumular shoots of Zeylanidium tailichenoides using CUP-SHAPED COTYLEDON 3 (CUC3) and SHOOT MERISTEMLESS (STM) orthologs, which are involved in the determination of axils and meristem formation in model plants. Expression of the CUC3 ortholog was detected at the adaxial base of cotyledons and parental shoots where the new shoots are initiated, while STM ortholog was expressed at the initiation site and in the young shoot primordia throughout early shoot development. The results demonstrate that each Z. tailichenoides shoot arises as an axillary bud in a manner similar to axillary meristem formation in model plants involving CUC3 and STM genes. Considering that each of the two cotyledons produces an axillary bud that in turn continues to form its own axillary bud independently, the apparent distichous shoot in Z.tailichenoides is not a single shoot, but a composite of two sympodially branched shoots.