Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

Field transcriptome analysis reveals a molecular mechanism for cassava-flowering in a mountainous environment in Southeast Asia.

KEY MESSAGE: The field survey in this article showed in 'KU50', a popular variety and late-branching type of cassava in Southeast Asia, that flowering rarely occurs in normal-field conditions in Southeast Asia but is strongly induced in the dry season in the mountainous region. Flowering time is correlated with the expression patterns of MeFT1 and homologs of Arabidopsis GI, PHYA, and NF-Ys. Cassava (Manihot esculenta Crantz) is a tropical crop that is propagated vegetatively rather than sexually by seed. Flowering rarely occurs in the erect-type variety grown in Southeast Asia, but it is known that cassava produces flowers every year in mountainous regions. Data pertaining to the effect of environmental factors on flowering time and gene expression in cassava, however, is limited. The aim of the present study was to determine the kinds of environmental conditions that regulate flowering time in cassava and the underlying molecular mechanisms. The flowering status of KU50, a popular variety in Southeast Asia and late-branching type of cassava, was monitored in six fields in Vietnam and Cambodia. At non-flowering and flowering field locations in North Vietnam, the two FLOWERING LOCUS T (FT)-like genes, MeFT1 and MeFT2, were characterized by qPCR, and the pattern of expression of flowering-related genes and genes responsive to environmental signals were analyzed by using RNA sequencing data from time-series samples. Results indicate that cassava flowering was induced in the dry season in the mountain region, and that flowering time was correlated with the expression of MeFT1, and homologs of Arabidopsis GI, PHYA, and NF-Ys. Based upon these data, we hypothesize that floral induction in cassava is triggered by some conditions present in the mountain regions during the dry season.

Field-transcriptome analyses reveal developmental transitions during flowering in cassava (Manihot esculenta Crantz).

KEY MESSAGE: We characterized genes that function in the photoperiodic flowering pathway in cassava. Transcriptome analysis of field-grown plants revealed characteristic expression patterns of these genes, demonstrating that field-grown cassava experiences two distinct developmental transitions. Cassava is an important crop for both edible and industrial purposes. Cassava develops storage roots that accumulate starch, providing an important source of staple food in tropical regions. To facilitate cassava breeding, it is important to elucidate how flowering is controlled. Several important genes that control flowering time have been identified in model plants; however, comprehensive characterization of these genes in cassava is still lacking. In this study, we identified genes encoding central flowering time regulators and examined these sequences for the presence or absence of conserved motifs. We found that cassava shares conserved genes for the photoperiodic flowering pathway, including florigen, anti-florigen and its associated transcription factor (GIGANTEA, CONSTANS, FLOWERING LOCUS T, CENTRORADIALIS/TERMINAL FLOWER1 and FD) and florigen downstream genes (SUPRESSOR OF OVEREXPRESSION OF CONSTANS1 and APETALA1/FRUITFUL). We conducted RNA-seq analysis of field-grown cassava plants and characterized the expression of flowering control genes. Finally, from the transcriptome analysis we identified two distinct developmental transitions that occur in field-grown cassava.

DNA methylation is reconfigured at the onset of reproduction in rice shoot apical meristem.

DNA methylation is an epigenetic modification that specifies the basic state of pluripotent stem cells and regulates the developmental transition from stem cells to various cell types. In flowering plants, the shoot apical meristem (SAM) contains a pluripotent stem cell population which generates the aerial part of plants including the germ cells. Under appropriate conditions, the SAM undergoes a developmental transition from a leaf-forming vegetative SAM to an inflorescence- and flower-forming reproductive SAM. While SAM characteristics are largely altered in this transition, the complete picture of DNA methylation remains elusive. Here, by analyzing whole-genome DNA methylation of isolated rice SAMs in the vegetative and reproductive stages, we show that methylation at CHH sites is kept high, particularly at transposable elements (TEs), in the vegetative SAM relative to the differentiated leaf, and increases in the reproductive SAM via the RNA-dependent DNA methylation pathway. We also show that half of the TEs that were highly methylated in gametes had already undergone CHH hypermethylation in the SAM. Our results indicate that changes in DNA methylation begin in the SAM long before germ cell differentiation to protect the genome from harmful TEs.

Building new insights in plant gametogenesis from an evolutionary perspective.

Extant bryophytes are thought to preserve characteristics of ancestral land plants, with a life cycle dominated by the haploid gametophyte. The gametophyte produces gametes in specialized organs that differentiate after an extensive phase of vegetative development. During land plant evolution, these organs became extremely reduced. As a result, in flowers of angiosperms the haploid phase of the life cycle is reduced to few-celled gametophytes, namely the embryo sac (female) and pollen (male). Although many factors contributing to gametogenesis have been identified in flowering plants, the extreme reduction of the gametophytes has prevented a clear molecular dissection of key processes of gametogenesis. Recent studies in the model bryophyte Marchantia polymorpha have identified conserved transcription factors regulating the equivalent steps in the sexual reproduction of land plants. These include FEMALE GAMETOPHYTE MYB for female gametophyte development, BONOBO for gamete progenitor cell specification, DUO POLLEN1 for sperm differentiation and members of the RWP-RK domain family for female gamete formation. These studies demonstrate that M. polymorpha is a powerful model to untangle the core processes of gametogenesis in land plants. We anticipate that a deeper understanding of gametogenesis in bryophytes will circumscribe the origin of plant germ cells and define the differentiation programmes of sperm and eggs.

Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage.

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Transcriptional Framework of Male Gametogenesis in the Liverwort Marchantia polymorpha L.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.

BRANCHED1 interacts with FLOWERING LOCUS T to repress the floral transition of the axillary meristems in Arabidopsis.

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.