In dicots, the key developmental process by which immature plastids differentiate into photosynthetically competent chloroplasts commences in the shoot apical meristem (SAM), within the shoot apex. Using laser-capture microdissection and single-cell RNA sequencing methodology, we studied the changes in the transcriptome along the chloroplast developmental pathway in the shoot apex of tomato seedlings. The analysis revealed the presence of transcripts for different chloroplast functions already in the stem cell-containing region of the SAM. Thereafter, an en masse up-regulation of genes encoding for various proteins occurs, including chloroplast ribosomal proteins and proteins involved in photosynthesis, photoprotection and detoxification of reactive oxygen species. The results highlight transcriptional events that operate during chloroplast biogenesis, leading to the rapid establishment of photosynthetic competence.

Abstract Chickpea shows a distinct domestication trajectory vis-a-vis pod dehiscence and growth cycle mediated by vernalization insensitivity compared with its companion Near Eastern legumes. Our objectives were: (i) to map the quantitative trait loci (QTLs) associated with vernalization response and seed free tryptophan in domesticated × wild chickpea progeny and (ii) estimate the genetic correlation between vernalization response and free tryptophan content. A domesticated × wild chickpea cross was used to document phenotypic segregation in both traits and to construct a skeletal genetic map for QTL detection. A number of vernalization response and seed free tryptophan content QTLs were documented in both F2 and F3 generations. No significant genetic correlation between these two traits was observed. Epistatic relationship between two free tryptophan loci was documented. It is evident that selection for high seed tryptophan is easier to accomplish relative to selection for vernalization insensitivity. This suggests that the two traits were selected independently in antiquity, thereby corroborating earlier claims for conscious selection processes associated with chickpea domestication.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.

Fruit initiation following fertilization in angiosperms is strictly regulated by phytohormones. In tomato (), auxin and gibberellin (GA) play central roles in promoting fruit initiation. Without fertilization, elevated GA or auxin signaling can induce parthenocarpy (seedless fruit production). The GA-signaling repressor SlDELLA and auxin-signaling components SlIAA9 and SlARF7 repress parthenocarpy, but the underlying mechanism is unknown. Here, we show that SlDELLA and the SlARF7/SlIAA9 complex mediate crosstalk between GA and auxin pathways to regulate fruit initiation. Yeast-two-hybrid and coimmunoprecipitation assays showed that SlARF7 and additional activator SlARFs interact with SlDELLA and SlIAA9 through distinct domains. SlARF7/SlIAA9 and SlDELLA antagonistically modulate the expression of feedback-regulated genes involved in GA and auxin metabolism, whereas SlARF7/SlIAA9 and SlDELLA coregulate the expression of fruit growth-related genes. Analysis of (), (with downregulated expression of multiple activator ), and () single and double mutants indicated that these genes additively affect parthenocarpy, supporting the notion that the SlARFs/SlIAA9 and SlDELLA interaction plays an important role in regulating fruit initiation. Analysis of the GA-deficient mutant showed that active GA biosynthesis and signaling are required for auxin-induced fruit initiation. Our study reveals how direct crosstalk between auxin- and GA-signaling components is critical for tomato fruit initiation.

Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO -induced stomatal closing.

The genus Selaginella resides in an early branch of the land plant lineage that possesses a vasculature and roots. The majority of the Selaginella root system is shoot borne and emerges through a distinctive structure known as the rhizophore, the organ identity of which has been a long-debated question. The rhizophore of Selaginella moellendorffii - a model for the lycophytes - shows plasticity to develop into a root or shoot up until 8 d after angle meristem emergence, after which it is committed to root fate. We subsequently use morphology and plasticity to define the stage of rhizophore identity. Transcriptomic analysis of the rhizophore during its plastic stage reveals that, despite some resemblance to the root meristem, rhizophore gene expression patterns are largely distinct from both shoot and root meristems. Based on this transcriptomic analysis and on historical anatomical work, we conclude that the rhizophore is a distinct organ with unique features.

The domestication and subsequent genetic improvement of wheat led to the development of large-seeded cultivated wheat species relative to their smaller-seeded wild progenitors. While increased grain weight (GW) continues to be an important goal of many wheat breeding programs, few genes underlying this trait have been identified despite an abundance of studies reporting quantitative trait loci (QTL) for GW. Here we perform a QTL analysis for GW using a population of recombinant inbred lines (RILs) derived from the cross between wild emmer wheat accession 'Zavitan' and durum wheat variety 'Svevo'. Identified QTLs in this population were anchored to the recent Zavitan reference genome, along with previously published QTLs for GW in tetraploid wheat. This genome-based, meta-QTL analysis enabled the identification of a locus on chromosome 6A whose introgression from wild wheat positively affects GW. The locus was validated using an introgression line carrying the 6A GW QTL region from Zavitan in a Svevo background, resulting in >8% increase in GW compared to Svevo. Using the reference sequence for the 6A QTL region, we identified a wheat ortholog to OsGRF4, a rice gene previously associated with GW. The coding sequence of this gene (TtGRF4-A) contains four single nucleotide polymorphisms (SNPs) between Zavitan and Svevo, one of which reveals the Zavitan allele to be rare in a core collection of wild emmer and completely absent from the domesticated emmer genepool. Similarly, another wild emmer accession (G18-16) was found to carry a rare allele of TtGRF4-A that also positively affects GW and is characterized by a unique SNP absent from the entire core collection. These results exemplify the rich genetic diversity of wild wheat, posit TtGRF4-A as a candidate gene underlying the 6A GW QTL, and suggest that the natural Zavitan and G18-16 alleles of TtGRF4-A have potential to increase wheat yields in breeding programs.

BACKGROUND: The large persistent seed bank of invasive plants is a significant obstacle to restoration programs. Soil solarization was demonstrated to be an effective method for reducing the seed bank of Australian acacias. However, the use of this method in natural habitats might be limited due to the requirement to moisten the soil by irrigation. The present study examined the possibility of replacing irrigation by trapping the soil moisture caused by the last rainfall, i.e. rain-based soil solarization (RBS). RESULTS: Exposure of Acacia saligna seeds to 57 C at 20 % soil moisture for 68 h caused an almost complete loss of seed viability. Similarly, RBS treatment significantly reduced the viability of A. saligna seeds buried at a soil depth of 1-19 cm as well as the seed density in the natural seed bank, and almost completely eliminated seedling emergence from the natural seed banks of A. saligna and other environmental weeds. CONCLUSION: Our results indicate that RBS is an effective method for reducing the seed bank of invasive plants in natural habitats located in various climate regions characterized by different soil types. This is the first demonstration of a successful application of RBS for soil disinfestation. This article is protected by copyright. All rights reserved.

Background: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in and define organ-specific regulatory sites and binding motifs of TFs at these sites. Results: We coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in based on their accessibility to nuclease digestion. By applying this pipeline in roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements. Conclusions: By coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.

Photosynthesis is limited by three main factors: stomatal conductance (gs), mesophyll conductance (gm) and maximum capacity for Rubisco carboxylation (Vcmax). It is unclear how limiting factors vary under stress, particularly during long-term stress acclimation. In this work, we compared for the first time photosynthesis limitation resulting from long-term acclimation to three major abiotic stresses: drought, salinity and temperature. We used saplings of Ziziphus spina-christi, a thermophilic and drought-tolerant tree, which recently became more abundant in the Mediterranean, presumably due to increased winter temperatures. Stress acclimation was investigated by measuring growth, gas exchange, chlorophyll fluorescence and leaf structure. For each stress, photosynthesis-limiting factors were compared. We developed an integrative stress index that allowed us to precisely define stress level, enabling a comparison between stress types. Photosynthesis under all stresses was limited mostly by gs and gm (80-90%); whereas biochemistry (Vcmax) made a minor contribution (10-20%). The relative contribution of gs and gm on photosynthetic limitation was influenced by stress type. During acclimation to drought or salinity, photosynthesis was limited by a decline in gs, while intolerance to low temperatures was driven by decline in gm. In all the stresses, gm decreased only under progressive reduction in leaf physiological functionality and was associated with low turgor under drought, an increase in leaf Na+ under salinity and low leaf hydraulic conductance (Kleaf) at low temperatures. Mesophyll structure (mesophyll surface area exposed to the intercellular air spaces, leaf thickness, % intercellular air spaces) did not explain gm acclimation to stress. Current work gives methodology for stress studies, and defines the main factors underlying the plant response to climate change. The ability to minimize mesophyll-imposed limitations on photosynthesis was found as a strong indicator of progressive stress tolerance. Moreover, the results demonstrate how warming climate benefits the photosynthetic function in thermophilic species, such as Ziziphus spina-christi.

Silica cells are specialized epidermal cells found on both surfaces of grass leaves, with almost the entire lumen filled with solid silica. The mechanism precipitating silicic acid into silica is not known. Here we investigate this process in sorghum (Sorghum bicolor) leaves. Using fluorescent confocal microscopy, we followed silica cells' ontogeny, aiming to understand the fate of vacuoles and nuclei. Correlating the confocal and scanning electron microscopy, we timed the initiation of silica deposition in relation to cell's viability. Contrary to earlier reports, silica cells did not lose their nucleus before silica deposition. Vacuoles in silica cells did not concentrate silicic acid. Instead, postmaturation silicification initiated at the cell periphery in live cells. Less than 1% silica cells showed characteristics of programmed cell death in the cell maturation zone. In fully elongated mature leaves, 2.4% of silica cells were nonsilicified and 1.6% were partially silicified. Silica deposition occurs in the paramural space of live silica cells. The mineral does not kill the cells. Instead, silica cells are genetically programmed to undergo cell death independent of silicification. Fully silicified cells seem to have nonsilicified voids containing membrane remains after the completion of the cell death processes.

Seminal roots constitute the initial wheat root system and provide the main route for water absorption during early stages of development. Seminal root number (SRN) varies among species. However, the mechanisms through which SRN is controlled and in turn contribute to environmental adaptation are poorly understood. Here, we show that SRN increased upon wheat domestication from 3 to 5 due to the activation of 2 root primordia that are suppressed in wild wheat, a trait controlled by loci expressed in the germinating embryo. Suppression of root primordia did not limit water uptake, indicating that 3 seminal roots is adequate to maintain growth during seedling development. The persistence of roots at their primordial state promoted seedling recovery from water stress through reactivation of suppressed primordia upon rehydration. Our findings suggest that under well-watered conditions, SRN is not a limiting factor, and excessive number of roots may be costly and maladaptive. Following water stress, lack of substantial root system suppresses growth and rapid recovery of the root system is essential for seedling recovery. This study underscores SRN as key adaptive trait that was reshaped upon domestication. The maintenance of roots at their primordial state during seedling development may be regarded as seedling protective mechanism against water stress.

We report the complete nucleotide sequence of a new member of the Potyviridae family isolated from passion fruit plants grown in Israel, called Passiflora edulis symptomless virus (PeSV). The PeSV genome is 9,928 nucleotides long and encodes a 3,173 amino acids polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Our phylogenetic analysis shows that PeSV represents a new species, and is most closely related to rose yellow mosaic virus (RoYMV). According to currently accepted criteria for genus demarcation, both viruses should be assigned as representative isolates of new species in the recently approved genus, Roymovirus, in the Potyviridae family.

FtsZ proteins of the FtsZ1 and FtsZ2 families play important roles in the initiation and progression of plastid division in plants and green algae. Arabidopsis possesses a single FTSZ1 member and two FTSZ2 members, FTSZ2-1 and FTSZ2-2. The contribution of these to chloroplast division and partitioning has been mostly investigated in leaf mesophyll tissues. Here, we assessed the involvement of the three FtsZs in plastid division at earlier stages of chloroplast differentiation. To this end, we studied the effect of the absence of specific FtsZ proteins on plastids in the vegetative shoot apex, where the proplastid-to-chloroplast transition takes place. We found that the relative contribution of the two major leaf FtsZ isoforms, FtsZ1 and FtsZ2-1, to the division process varies with cell lineage and position within the shoot apex. While FtsZ2-1 dominates division in the L1 and L3 layers of the shoot apical meristem (SAM), in the L2 layer, FtsZ1 and FtsZ2-1 contribute equally toward the process. Depletion of the third isoform, FtsZ2-2, generally resulted in stronger effects in the shoot apex than those observed in mature leaves. The implications of these findings, along with additional observations made in this work, to our understanding of the mechanisms and regulation of plastid proliferation in the shoot apex are discussed.

BACKGROUND: Genomic analysis technologies can promote efficient fruit tree breeding. Genotyping by sequencing (GBS) enables generating efficient data for high-quality genetic map construction and QTL analysis in a relatively accessible way. Furthermore, High-resolution genetic map construction and accurate QTL detection can significantly narrow down the putative candidate genes associated with important plant traits. RESULTS: We genotyped 162 offspring in the F1 'Spadona' x 'Harrow Sweet' pear population using GBS. An additional 21 pear accessions, including the F1 population's parents, from our germplasm collection were subjected to GBS to examine diverse genetic backgrounds that are associated to agriculturally relevant traits and to enhance the power of SNP calling. A standard SNP calling pipeline identified 206,971 SNPs with Asian pear ('Suli') as the reference genome and 148,622 SNPs with the European genome ('Bartlett'). These results enabled constructing a genetic map, after further stringent SNP filtering, consisting of 2036 markers on 17 linkage groups with a length of 1433 cM and an average marker interval of 0.7 cM. We aligned 1030 scaffolds covering a total size of 165.5 Mbp (29%) of the European pear genome to the 17 linkage groups. For high-resolution QTL analysis covering the whole genome, we used phenotyping for vegetative budbreak time in the F1 population. New QTLs associated to vegetative budbreak time were detected on linkage groups 5, 13 and 15. A major QTL on linkage group 8 and an additional QTL on linkage group 9 were confirmed. Due to the significant genotype-by-environment (GxE) effect, we were able to identify novel interaction QTLs on linkage groups 5, 8, 9 and 17. Phenotype-genotype association analysis in the pear accessions for main genotype effect was conducted to support the QTLs detected in the F1 population. Significant markers were detected on every linkage group to which main genotype effect QTLs were mapped. CONCLUSIONS: This is the first vegetative budbreak study of European pear that makes use of high-resolution genetic mapping. These results provide tools for marker-assisted selection and accurate QTL analysis in pear, and specifically at vegetative budbreak, considering the significant GxE and phenotype-plasticity effects.

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. loss-of-function mutants arrest shortly after germination; therefore, the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing in the () expression domain in the mutant background resulted in a leafy-flower phenotype, and an intermediate allele enhanced the flower meristem identity phenotype of Transcriptional profiling of perturbation suggested that STM activity affects multiple floral fate genes, among them the F-box protein-encoding gene (). In agreement with this notion, enhanced the floral fate phenotype, and ectopic expression rescued the leafy flowers in genetic backgrounds with compromised and activities. This work suggests a genetic mechanism that underlies the activity of in the specification of flower meristem identity.

Degradation of the D1 protein of photosystem II (PSII) reaction center is a pre-requisite for the repair cycle from photoinhibition. Two types of thylakoid proteases, FtsH and Deg, have been demonstrated to participate in this process. However, the location of the proteolytic sites of the lumenal Deg1 protease within its internal sphere raised the question whether the lumenal-exposed regions of D1 are indeed long enough to reach these sites. Implanting these regions into the stable GFP rendered it sensitive to the presence of Deg1 in vitro, demonstrating that the flexible regions of D1 that protrude into the lumen can penetrate through the three side-openings of Deg1 and reach its internal proteolytic sites. This mode of action, facilitating cooperation between proteases on both sides of the thylakoid membranes, should be applicable to the degradation of other integral thylakoid membrane proteins as well.

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA-LIKE 2 (COBL2), a member of the COBRA-LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: 'unraveled' ray morphology, loss of primary cell wall 'pyramidal' organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage-modified 2 (mum2) suggest that COBL2 functions independently of the FEI-SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.

Multicellular organisms develop from a single cell that proliferates to form different cell types with specialized functions. Sixty years ago, Waddington suggested the 'epigenetic landscape' as a useful metaphor for the process. According to this view, cells move through a rugged identity space along genetically encoded trajectories, until arriving at one of the possible final fates. In plants in particular, these trajectories have strong spatial correlates, as cell identity is intimately linked to its relative position within the plant. During regeneration, however, positional signals are severely disrupted and differentiated cells are able to undergo rapid non-canonical identity changes. Moreover, while pluripotent properties have long been ascribed to plant cells, the introduction of induced pluripotent stem cells in animal studies suggests such plasticity may not be unique to plants. As a result, current concepts of differentiation as a gradual and hierarchical process are being reformulated across biological fields. Traditional studies of plant regeneration have placed strong emphasis on the emergence of patterns and tissue organization, and information regarding the events occurring at the level of individual cells is only now beginning to emerge. Here, I review the historical and current concepts of cell identity and identity transitions, and discuss how new views and tools may instruct the future understanding of differentiation and plant regeneration.