A chromosome-level genome sequence of Chrysanthemum seticuspe, a model species for hexaploid cultivated chrysanthemum.

Chrysanthemums are one of the most industrially important cut flowers worldwide. However, their segmental allopolyploidy and self-incompatibility have prevented the application of genetic analysis and modern breeding strategies. We thus developed a model strain, Gojo-0 (Chrysanthemum seticuspe), which is a diploid and self-compatible pure line. Here, we present the 3.05 Gb chromosome-level reference genome sequence, which covered 97% of the C. seticuspe genome. The genome contained more than 80% interspersed repeats, of which retrotransposons accounted for 72%. We identified recent segmental duplication and retrotransposon expansion in C. seticuspe, contributing to arelatively large genome size. Furthermore, we identified a retrotransposon family, SbdRT, which was enriched in gene-dense genome regions and had experienced a very recent transposition burst. We also demonstrated that the chromosome-level genome sequence facilitates positional cloning in C. seticuspe. The genome sequence obtained here can greatly contribute as a reference for chrysanthemum in front-line breeding including genome editing.

Genetic Interaction Among Phytochrome, Ethylene and Abscisic Acid Signaling During Dark-Induced Senescence in Arabidopsis thaliana.

Leaf senescence is induced by various internal and external stimuli. Dark-induced senescence has been extensively investigated, but the detailed mechanism underlying it is not well understood. The red light/far-red light receptor phytochrome B and its downstream transcription factors, PYHTOCHROME INTERACTING FACTORs (PIFs) 4 and 5, are known to play an important role in dark-induced senescence. Furthermore, the senescence-inducing phytohormones, ethylene and abscisic acid (ABA) are reported to be involved in dark-induced senescence. In this study, we analyzed the relationship between ethylene, ABA and PIFs in dark-induced leaf senescence. A triple mutant of the core ABA signaling components; SNF1-related protein kinases 2D (SRK2D), SRK2E, and SRK2I, displayed an ABA insensitive phenotype in ABA-induced senescence, whilst the ethylene insensitive mutant ein2 demonstrated low sensitivity to ABA, suggesting that ethylene signaling is involved in ABA-induced senescence. However, the pif4 pif5 mutant did not display low sensitivity to ABA, suggesting that PIF4 and PIF5 act upstream of ABA signaling. Although PIF4 and PIF5 reportedly regulate ethylene production, the triple mutant ein2 pif4 pif5 showed a stronger delayed senescence phenotype than ein2 or pif4 pif5, suggesting that EIN2 and PIF4/PIF5 partially regulate leaf senescence independently of each other. While direct target genes for PIF4 and PIF5, such as LONG HYPOCOTYL IN FAR-RED1 (HFR1) and PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1), showed transient upregulation under dark conditions (as is seen in the shade avoidance response), expression of STAY GREEN1 (SGR1), ORESARA1 (ORE1) and other direct target genes of PIF5, continued to increase during dark incubation. It is possible that transcription factors other than PIF4 and PIF5 are involved in the upregulation of SGR1 and ORE1 at a later stage of dark-induced senescence. Possible candidates are senescence-induced senescence regulators (SIRs), which include the NAC transcription factors ORE1 and AtNAP. In fact, ORE1 is known to bind to the SGR1 promoter and promotes its expression. It is therefore inferred that the phytochrome-PIF pathway regulates initial activation of senescence and subsequently, induced SIRs reinforce leaf senescence during dark-induced senescence.

Regulation of Sugar and Storage Oil Metabolism by Phytochrome during De-etiolation.

Exposure of dark-grown (etiolated) seedlings to light induces the heterotrophic-to-photoautotrophic transition (de-etiolation) processes, including the formation of photosynthetic machinery in the chloroplast and cotyledon expansion. Phytochrome is a red (R)/far-red (FR) light photoreceptor that is involved in the various aspects of de-etiolation. However, how phytochrome regulates metabolic dynamics in response to light stimulus has remained largely unknown. In this study, to elucidate the involvement of phytochrome in the metabolic response during de-etiolation, we performed widely targeted metabolomics in Arabidopsis (Arabidopsis thaliana) wild-type and phytochrome A and B double mutant seedlings de-etiolated under R or FR light. The results revealed that phytochrome had strong impacts on the primary and secondary metabolism during the first 24 h of de-etiolation. Among those metabolites, sugar levels decreased during de-etiolation in a phytochrome-dependent manner. At the same time, phytochrome upregulated processes requiring sugars. Triacylglycerols are stored in the oil bodies as a source of sugars in Arabidopsis seedlings. Sugars are provided from triacylglycerols through fatty acid ß-oxidation and the glyoxylate cycle in glyoxysomes. We examined if and how phytochrome regulates sugar production from oil bodies. Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle-deficient mutants not only failed to mobilize oil bodies but also failed to develop thylakoid membranes and expand cotyledon cells upon exposure to light. Hence, phytochrome plays a key role in the regulation of metabolism during de-etiolation.

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis.

Leaf senescence is not a passive degenerative process; it represents a process of nutrient relocation, in which materials are salvaged for growth at a later stage or to produce the next generation. Leaf senescence is regulated by various factors, such as darkness, stress, aging, and phytohormones. Strigolactone is a recently identified phytohormone, and it has multiple functions in plant development, including repression of branching. Although strigolactone is implicated in the regulation of leaf senescence, little is known about its molecular mechanism of action. In this study, strigolactone biosynthesis mutant strains of Arabidopsis (Arabidopsis thaliana) showed a delayed senescence phenotype during dark incubation. The strigolactone biosynthesis genes MORE AXIALLY GROWTH3 (MAX3) and MAX4 were drastically induced during dark incubation and treatment with the senescence-promoting phytohormone ethylene, suggesting that strigolactone is synthesized in the leaf during leaf senescence. This hypothesis was confirmed by a grafting experiment using max4 as the stock and Columbia-0 as the scion, in which the leaves from the Columbia-0 scion senesced earlier than max4 stock leaves. Dark incubation induced the synthesis of ethylene independent of strigolactone. Strigolactone biosynthesis mutants showed a delayed senescence phenotype during ethylene treatment in the light. Furthermore, leaf senescence was strongly accelerated by the application of strigolactone in the presence of ethylene and not by strigolactone alone. These observations suggest that strigolactone promotes leaf senescence by enhancing the action of ethylene. Thus, dark-induced senescence is regulated by a two-step mechanism: induction of ethylene synthesis and consequent induction of strigolactone synthesis in the leaf.