Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (Vitis vinifera L. ssp. vinifera). In contrast, all extant wild Vitis species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species V. cinerea and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the Vitis genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.

Stomata, the gas exchange structures of plants, are formed by the division and differentiation of stem cells, or meristemoids. Although diverse patterns of meristemoid behavior have been observed among different lineages of land plants, the ecological significance and diversification processes of these different patterns are not well understood. Here we describe an intrageneric diversity in the patterns of meristemoid division within the ecologically diverse genus Callitriche (Plantaginaceae). Meristemoids underwent a series of divisions before differentiating into stomata in the terrestrial species of Callitriche, but these divisions did not occur in amphibious species, which can grow in both air and water, in which meristemoids differentiated directly into stomata. These findings imply the adaptive significance of diversity in meristemoid division. Molecular genetic analyses showed that the different expression times of the stomatal key transcription factors SPEECHLESS and MUTE, which maintain and terminate the meristemoid division, respectively, underlie the different division patterns of meristemoids. Unlike terrestrial species, amphibious species prematurely expressed MUTE immediately after expressing SPEECHLESS, which corresponded to their early termination of stomatal division. By linking morphological, ecological, and genetic elements of stomatal development, this study provides significant insight that should aid ecological evolutionary developmental biology investigations of stomata.

Phyllotaxis, the distribution of organs such as leaves and flowers on their support, is a key attribute of plant architecture. The geometric regularity of phyllotaxis has attracted multidisciplinary interest for centuries, resulting in an understanding of the patterns in the model plants Arabidopsis and tomato down to the molecular level. Nevertheless, the iconic example of phyllotaxis, the arrangement of individual florets into spirals in the heads of the daisy family of plants (Asteraceae), has not been fully explained. We integrate experimental data and computational models to explain phyllotaxis in Gerbera hybrida. We show that phyllotactic patterning in gerbera is governed by changes in the size of the morphogenetically active zone coordinated with the growth of the head. The dynamics of these changes divides the patterning process into three phases: the development of an approximately circular pattern with a Fibonacci number of primordia near the head rim, its gradual transition to a zigzag pattern, and the development of a spiral pattern that fills the head on the template of this zigzag pattern. Fibonacci spiral numbers arise due to the intercalary insertion and lateral displacement of incipient primordia in the first phase. Our results demonstrate the essential role of the growth and active zone dynamics in the patterning of flower heads.

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV’s 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3′ ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr. Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

The plant ultraviolet-B (UV-B) photoreceptor UVR8 plays an important role in UV-B acclimation and survival. UV-B absorption by homodimeric UVR8 induces its monomerization and interaction with the E3 ubiquitin ligase COP1, leading ultimately to gene expression changes. UVR8 is inactivated through redimerization, facilitated by RUP1 and RUP2. Here, we describe a semidominant, hyperactive allele, namely uvr8-17D, that harbors a glycine-101 to serine mutation. UVR8^G101S overexpression led to weak constitutive photomorphogenesis and extreme UV-B responsiveness. UVR8^G101S was observed to be predominantly monomeric in vivo and, once activated by UV-B, was not efficiently inactivated. Analysis of a UVR8 crystal structure containing the G101S mutation revealed the distortion of a loop region normally involved in stabilization of the UVR8 homodimer. Plants expressing a UVR8 variant combining G101S with the previously described W285A mutation exhibited robust constitutive photomorphogenesis. This work provides further insight into UVR8 activation and inactivation mechanisms and describes a genetic tool for the manipulation of photomorphogenic responses.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that “fine regulation of the activities of these enzymes is crucial for efficient photosynthesis.” However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

An important question is what genes govern the differentiation of plant embryos into suspensor and embryo proper regions following fertilization and division of the zygote. We compared embryo proper and suspensor transcriptomes of four plants that vary in embryo morphology within the suspensor region. We determined that genes encoding enzymes in several metabolic pathways leading to the formation of hormones, such as gibberellic acid, and other metabolites are up-regulated in giant scarlet runner bean and common bean suspensors. Genes involved in transport and Golgi body organization are up-regulated within the suspensors of these plants as well, strengthening the view that giant specialized suspensors serve as a hormone factory and a conduit for transferring substances to the developing embryo proper. By contrast, genes controlling transcriptional regulation, development, and cell division are up-regulated primarily within the embryo proper. Transcriptomes from less specialized soybean and Arabidopsis suspensors demonstrated that fewer genes encoding metabolic enzymes and hormones are up-regulated. Genes active in the embryo proper, however, are functionally similar to those active in scarlet runner bean and common bean embryo proper regions. We uncovered a set of suspensor- and embryo proper–specific transcription factors (TFs) that are shared by all embryos irrespective of morphology, suggesting that they are involved in early differentiation processes common to all plants. Chromatin immunoprecipitation sequencing (ChIP-Seq) experiments with scarlet runner bean and soybean WOX9, an up-regulated suspensor TF, gained entry into a regulatory network important for suspensor development irrespective of morphology.

Inflorescence architecture dictates the number of flowers and, ultimately, seeds. The architectural discrepancies between two related cereals, barley and wheat, are controlled by differences in determinacy of inflorescence and spikelet meristems. Here, we characterize two allelic series of mutations named intermedium-m (int-m) and double seed1 (dub1) that convert barley indeterminate inflorescences into wheat-like determinate inflorescences bearing a multifloreted terminal spikelet and spikelets with additional florets. INT-M/DUB1 encodes an APETALA2-like transcription factor (HvAP2L-H5) that suppresses ectopic and precocious spikelet initiation signals and maintains meristem activity. HvAP2L-H5 inhibits the identity shift of an inflorescence meristem (IM) to a terminal spikelet meristem (TSM) in barley. Null mutations in AP2L-5 lead to fewer spikelets per inflorescence but extra florets per spikelet. In wheat, prolonged and elevated AP2L-A5 activity in rAP2L-A5 mutants delays but does not suppress the IM−TSM transition. We hypothesize that the regulation of AP2L-5 orthologs and downstream genes contributes to the different inflorescence determinacy in barley and wheat. We show that AP2L-5 proteins are evolutionarily conserved in grasses, promote IM activity, and restrict floret number per spikelet. This study provides insights into the regulation of spikelet and floret number, and hence grain yield in barley and wheat.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ–meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

Plant cystatins are cysteine proteinase inhibitors that play key roles in defense responses. In this work, we describe an unexpected role for the cystatin-like protein DEFORMED FLORAL BUD1 (CsDFB1) as a transcriptional regulator of local auxin distribution in cucumber (Cucumis sativus L.). CsDFB1 was strongly expressed in the floral meristems, floral primordia, and vasculature. RNA interference (RNAi)-mediated silencing of CsDFB1 led to a significantly increased number of floral organs and vascular bundles, together with a pronounced accumulation of auxin. Conversely, accompanied by a decrease of auxin, overexpression of CsDFB1 resulted in a dramatic reduction in floral organ number and an obvious defect in vascular patterning, as well as organ fusion. CsDFB1 physically interacted with the cucumber ortholog of PHABULOSA (CsPHB), an HD-ZIP III transcription factor whose transcripts exhibit the same pattern as CsDFB1. Overexpression of CsPHB increased auxin accumulation in shoot tips and induced a floral phenotype similar to that of CsDFB1-RNAi lines. Furthermore, genetic and biochemical analyses revealed that CsDFB1 impairs CsPHB-mediated transcriptional regulation of the auxin biosynthetic gene YUCCA2 and the auxin efflux carrier PIN-FORMED1, and thus plays a pivotal role in auxin distribution. In summary, we propose that the CsDFB1-CsPHB module represents a regulatory pathway for local auxin distribution that governs floral organogenesis and vascular differentiation in cucumber.

Plant meristems are self-renewing groups of pluripotent stem cells that produce lateral organs in a stereotypical pattern. Of interest is how the radially symmetrical meristem produces laminar lateral organs. Both the male and female inflorescence meristems of the dominant Fascicled ear (Fas1) mutant fail to grow as a single point and instead show deep branching. Positional cloning of two independent Fas1 alleles identified an ∼160 kb region containing two floral genes, the MADS-box gene, zmm8, and the YABBY gene, drooping leaf2 (drl2). Both genes are duplicated within the Fas1 locus and spatiotemporally misexpressed in the mutant inflorescence meristems. Increased zmm8 expression alone does not affect inflorescence development; however, combined misexpression of zmm8, drl2, and their syntenic paralogs zmm14 and drl1, perturbs meristem organization. We hypothesize that misexpression of the floral genes in the inflorescence and their potential interaction cause ectopic activation of a laminar program, thereby disrupting signaling necessary for maintenance of radially symmetrical inflorescence meristems. Consistent with this hypothesis, RNA sequencing and in situ analysis reveal altered expression patterns of genes that define distinct zones of the meristem and developing leaf. Our findings highlight the importance of strict spatiotemporal patterns of expression for both zmm8 and drl2 and provide an example of phenotypes arising from tandem gene duplications.

Plant cell walls are complex structures subject to dynamic remodeling in response to developmental and environmental cues and play essential functions in disease resistance responses. We tested the specific contribution of plant cell walls to immunity by determining the susceptibility of a set of Arabidopsis cell wall mutants (cwm) to pathogens with different parasitic styles: a vascular bacterium, a necrotrophic fungus, and a biotrophic oomycete. Remarkably, most cwm mutants tested (29/34; 85.3%) showed alterations in their resistance responses to at least one of these pathogens in comparison to wild-type plants, illustrating the relevance of wall composition in determining disease-resistance phenotypes. We found that the enhanced resistance of cwm plants to the necrotrophic and vascular pathogens negatively impacted cwm fitness traits, such as biomass and seed yield. Enhanced resistance of cwm plants is not only mediated by canonical immune pathways, like those modulated by phytohormones or microbe-associated molecular patterns, which are not deregulated in the cwm tested. Pectin-enriched wall fractions isolated from cwm plants triggered immune responses in wild-type plants, suggesting that wall-mediated defensive pathways might contribute to cwm resistance. Cell walls of cwm plants show a high diversity of composition alterations as revealed by glycome profiling that detect specific wall carbohydrate moieties. Mathematical analysis of glycome profiling data identified correlations between the amounts of specific wall carbohydrate moieties and disease resistance phenotypes of cwm plants. These data support the relevant and specific function of plant wall composition in plant immune response modulation and in balancing disease resistance/development trade-offs.

Plant secondary cell-wall (SCW) deposition and lignification are affected by both seasonal factors and abiotic stress, and these responses may involve the hormone abscisic acid (ABA). However, the mechanisms involved are not clear. Here we show that mutations that limit ABA synthesis or signaling reduce the extent of SCW thickness and lignification in Arabidopsis thaliana through the core ABA-signaling pathway involving SnRK2 kinases. SnRK2.2. 3 and 6 physically interact with the SCW regulator NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), a NAC family transcription factor that orchestrates the transcriptional activation of a suite of downstream SCW biosynthesis genes, some of which are involved in the biosynthesis of cellulose and lignin. This interaction leads to phosphorylation of NST1 at Ser316, a residue that is highly conserved among NST1 proteins from dicots, but not monocots, and is required for transcriptional activation of downstream SCW-related gene promoters. Loss of function of NST1 in the snd1 mutant background results in lack of SCWs in the interfascicular fiber region of the stem, and the Ser316Ala mutant of NST1 fails to complement this phenotype and ABA-induced lignin pathway gene expression. The discovery of NST1 as a key substrate for phosphorylation by SnRK2 suggests that the ABA-mediated core-signaling cascade provided land plants with a hormone-modulated, competitive desiccation-tolerance strategy allowing them to differentiate water-conducting and supporting tissues built of cells with thicker cell walls.

DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.

Phycobilisomes are the major pigment–protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin β-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.