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The widespread use of plant grafting enables eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years, and despite their economic and ecological relevance, we know little about how they graft. Here we developed a micrografting method in conifers using young tissues that allowed efficient grafting with closely related species and between distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell-wall-related genes. By comparing these genes to those induced during Arabidopsis thalianagraft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Norway spruce (Picea abies) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1(PAT1) acted as a core regulator of graft healing. This gene was strongly up-regulated during both spruce and Arabidopsisgrafting, and Arabidopsismutants lacking PATgenes failed to attach tissues or successfully graft. Complementing ArabidopsisPAT mutants with the spruce PAT1homolog rescued tissue attachment and enhanced callus formation. Together, our data show an ability for young tissues to graft with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration.

The plant DNA damage response (DDR) pathway safeguards genomic integrity by rapid recognition and repair of DNA lesions that, if unrepaired, may cause genome instability. Most frequently, DNA repair goes hand in hand with a transient cell cycle arrest, which allows cells to repair the DNA lesions before engaging in a mitotic event, but consequently also affects plant growth and yield. Through the identification of DDR proteins and cell cycle regulators that react to DNA double-strand breaks or replication defects, it has become clear that these proteins and regulators form highly interconnected networks. These networks operate at both the transcriptional and post-transcriptional levels and include liquid–liquid phase separation and epigenetic mechanisms. Strikingly, whereas the upstream DDR sensors and signalling components are well conserved across eukaryotes, some of the more downstream effectors are diverged in plants, probably to suit unique lifestyle features. Additionally, DDR components display functional diversity across ancient plant species, dicots and monocots. The observed resistance of DDR mutants towards aluminium toxicity, phosphate limitation and seed ageing indicates that gaining knowledge about the plant DDR may offer solutions to combat the effects of climate change and the associated risk for food security.

The leaf epidermis represents a multifunctional tissue consisting of trichomes, pavement cells and stomata, the specialized cellular pores of the leaf. Pavement cells and stomata both originate from regulated divisions of stomatal lineage ground cells (SLGCs), but whereas the ontogeny of the stomata is well characterized, the genetic pathways activating pavement cell differentiation remain relatively unexplored. Here, we reveal that the cell cycle inhibitor SIAMESE-RELATED1 (SMR1) is essential for timely differentiation of SLGCs into pavement cells by terminating SLGC self-renewal potency, which depends on CYCLIN A proteins and CYCLIN-DEPENDENT KINASE B1. By controlling SLGC-to-pavement cell differentiation, SMR1 determines the ratio of pavement cells to stomata and adjusts epidermal development to suit environmental conditions. We therefore propose SMR1 as an attractive target for engineering climate-resilient plants.

Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type - (MT-) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.

Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type - (MT-) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell-cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell-cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell-cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell-cycle regulators, e.g. CDKs, cyclins, or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G2/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G2/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G2/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G2/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G2/M progression in plants.

E2F/DP complexes play a pivotal role in the regulation of the G1/S transition in animals. Recently, plant E2F homologs have been cloned, but DP‐related sequences have not been identified so far. Here we report that Arabidopsis thalianacontains at least two different DP‐related genes, AtDPaand AtDPb. They exhibit an overall domain organization similar to that of their animal counterparts, although phylogenetic analysis demonstrated that they form a separate subgroup. AtDPs efficiently heterodimerize in vitro with the ArabidopsisE2F‐related proteins, AtE2Fa and AtE2Fb through their dimerization domains. AtDPaand AtE2Faare predominantly produced in actively dividing cells with highest transcript levels in early S phase cells.

A homologue of the human site-specific prolylcis/transisomerase PIN1 was identified inArabidopsis thaliana. The PIN1Atgene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1AtmRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolylcis/transisomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the α1 helix observed in PIN1 that could mediate the protein-protein interactions.

TheCKS1Atgene product, p10CKS1AtfromArabidopsis thaliana,is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At,a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to expressCKS1AtinEscherichia coliunder the control of the T7 promoter was not successful.E. coliBL21(DE3) cotransformed with theCKS1Atgene and theE. coli argUgene that encoded the arginine acceptor tRNAUCUproduced a sufficient amount of p10CKS1Atto start the structural study by NMR. Replacement of four rare codons in theCKS1Atgene sequence, including a tandem arginine, by highly used codons inE. coli,restored also a high expression of the recombinant protein. Double-isotopic enrichment by13C and15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1Atwas purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1Atprotein.

Abstract.: D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.

Abstract.: Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle.

In Arabidopsis, two cyclin‐dependent kinases (CDK), Cdc2aAt and Cdc2bAt, have been described. Here, we have used the yeast two‐hybrid system to identify Arabidopsisproteins interacting with Cdc2aAt. Three different clones were isolated, one of which encodes a Suc1/Cks1 homologue. The functionality of the ArabidopsisSuc1/Cks1 homologue, designed Cks1At, was demonstrated by its ability to rescue the temperature‐sensitive cdc2‐L7strain of fission yeast at low and intermediate expression levels. In contrast, high cks1Atexpression levels inhibited cell division in both mutant and wild‐type yeast strains. Cks1At binds both Cdc2aAt and Cdc2bAt in vivo and in vitro. Furthermore, we demonstrate that the fission yeast Suc1 binds Cdc2aAt but only weakly Cdc2bAt, whereas the human CksHs1 associated exclusively with Cdc2aAt.

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase SnRK2.2 and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth.

Regeneration of a tissue damaged by injury represents a physiological response for organ recovery1–3. Although this regeneration process is conserved across multicellular taxa, plants appear to display extremely high regenerative capacities, a feature widely used in tissue culture for clonal propagation and grafting4,5. Regenerated cells arise predominantly from pre-existing populations of division-competent cells6,7; however, the mechanisms by which these cells are triggered to divide in response to injury remain largely elusive8. Here, we demonstrate that the heterodimeric transcription factor complex ETHYLENE RESPONSE FACTOR115 (ERF115)–PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1) sustains meristem function by promoting cell renewal after stem cell loss. High-resolution time-lapse imaging revealed that cell death promotes ERF115 activity in cells that are in direct contact with damaged cells, triggering divisions that replenish the collapsed stem cells. Correspondingly, the ERF115–PAT1 complex plays an important role in full stem cell niche recovery upon root tip excision, whereas its ectopic expression triggers neoplastic growth, correlated with activation of the putative target gene WOUND INDUCED DEDIFFERENTIATION1 (WIND1)9. We conclude that the ERF115–PAT1 complex accounts for the high regenerative potential of plants, granting them the ability to efficiently replace damaged cells with new ones.

Plant somatic cells are generally acknowledged to retain totipotency, the potential to develop into any cell type within an organism. This astonishing plasticity may contribute to a high regenerative capacity on severe damage, but how plants control this potential during normal post-embryonic development remains largely unknown1,2. Here we show that POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a chromatin regulator that maintains gene repression through histone modification, prevents dedifferentiation of mature somatic cells in Arabidopsis thaliana roots. Loss-of-function mutants in PRC2 subunits initially develop unicellular root hairs indistinguishable from those in wild type but fail to retain the differentiated state, ultimately resulting in the generation of an unorganized cell mass and somatic embryos from a single root hair. Strikingly, mutant root hairs complete the normal endoreduplication programme, increasing their nuclear ploidy, but subsequently reinitiate mitotic division coupled with successive DNA replication. Our data show that the WOUND INDUCED DEDIFFERENTIATION3 (WIND3) and LEAFY COTYLEDON2 (LEC2) genes are among the PRC2 targets involved in this reprogramming, as their ectopic overexpression partly phenocopies the dedifferentiation phenotype of PRC2 mutants. These findings unveil the pivotal role of PRC2-mediated gene repression in preventing unscheduled reprogramming of fully differentiated plant cells.

Because of their sessile lifestyle, plants have developed extensive mechanisms to safeguard their genetic information from one generation to the next. The WEE1 kinase is one of the guardians of genome integrity, being important during S-phase progression under replication stress. Knock-out plants for WEE1(WEE1KO) show a hypersensitive response when grown on replication-inhibiting drugs. Recently, we reported the identification of a mutant in the RNase H2Agene that could partially complement this replication phenotype. Here, we present the identification of a second member of the RNase H2 complex, RNase H2B, being able to complement the root growth phenotype of WEE1KOplants. We additionally show that deletion of a conserved domain in RNase H2B leads to loss of interaction with the RNase H2C subunit, likely explaining the loss of activity of the RNase H2 complex.

Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.