Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line.

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Early flowering phenotype of the Arabidopsis altered meristem program1 mutant is dependent on the FLOWERING LOCUS T-mediated pathway.

Controlling the flowering time is crucial for propagating plant species and crop production. ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis thaliana encodes a putative carboxypeptidase, and an AMP1 mutant (amp1) was found to cause highly pleiotropic phenotypes including a short plastochron, an enlarged shoot apical meristem, and reduced apical dominance. Although amp1 also shows an early flowering phenotype, its mechanism has not been investigated in detail. The most important floral integrator or florigen gene, FLOWERING LOCUS T (FT), has a close relative, TWIN SISTER OF FT (TSF). In this report, we generated a new allele of tsf using a genome-editing technique and produced ft tsf double and amp1 ft tsf triple mutants. The flowering time of amp1 ft tsf was equally as late as ft tsf under long-day conditions. In addition, the expression level of FT in amp1 was 2.4-fold higher than that in wild-type, even five days after germination under long-day conditions. These results suggest that the elevated expression level of FT is responsible for the early flowering phenotype of amp1. Furthermore, expression of FLOWERING LOCUS C (FLC), a negative regulator of FT expression, is severely repressed in amp1, raising the possibility that low expression levels of FLC contributes to upregulation of FT expression and the early flowering phenotype of amp1.

Analysis of Physiological Variations and Genetic Architecture for Photosynthetic Capacity of Japanese Soybean Germplasm.

The culmination of conventional yield improving parameters has widened the margin between food demand and crop yield, leaving the potential yield productivity to be bridged by the manipulation of photosynthetic processes in plants. Efficient strategies to assess photosynthetic capacity in crops need to be developed to identify suitable targets that have the potential to improve photosynthetic efficiencies. Here, we assessed the photosynthetic capacity of the Japanese soybean mini core collection (GmJMC) using a newly developed high-throughput photosynthesis measurement system "MIC-100" to analyze physiological mechanisms and genetic architecture underpinning photosynthesis. K-means clustering of light-saturated photosynthesis (Asat ) classified GmJMC accessions into four distinct clusters with Cluster2 comprised of highly photosynthesizing accessions. Genome-wide association analysis based on the variation of Asat revealed a significant association with a single nucleotide polymorphism (SNP) on chromosome 17. Among the candidate genes related to photosynthesis in the genomic region, variation in expression of a gene encoding G protein alpha subunit 1 (GPA1) showed a strong correlation (r = 0.72, p < 0.01) with that of Asat . Among GmJMC accessions, GmJMC47 was characterized by the highest Asat , stomatal conductance (gs ), stomatal density (SDensity ), electron transfer rate (ETR), and light use efficiency of photosystem II (Fv'/Fm') and the lowest non-photochemical quenching [NPQ(t)], indicating that GmJMC47 has greater CO2 supply and efficient light-harvesting systems. These results provide strong evidence that exploration of plant germplasm is a useful strategy to unlock the potential of resource use efficiencies for photosynthesis.

Genetic analysis of chlorophyll synthesis and degradation regulated by BALANCE of CHLOROPHYLL METABOLISM.

Chlorophyll (Chl) serves a number of essential functions, capturing and converting light energy as a component of photosystem supercomplexes. Chl degradation during leaf senescence is also required for adequate degeneration of chloroplasts and salvaging of nutrients from senescent leaves. In this study, we performed genetic analysis to determine the functions of BALANCE of CHLOROPHYLL METABOLISM1 (BCM1) and BCM2, which control Chl levels by regulating synthesis and degradation, and STAY-GREEN (SGR)1 (also known as NON-YELLOWING1 [NYE1]) and SGR2, which encode Mg-dechelatase and catalyze Chl a degradation in Arabidopsis (Arabidopsis thaliana). Analysis of bcm1 bcm2 revealed that both BCM1 and BCM2 are involved in the regulation of Chl levels in presenescent leaves and Chl degradation in senescing leaves. Analysis of bcm1 bcm2 nye1 nye2 suggested that BCMs repress Chl-degrading activity in both presenescent and senescing leaves by regulating SGR activity. Furthermore, transactivation analysis and chromatin immunoprecipitation (ChIP) assay revealed that GOLDEN2-LIKE1 (GLK1), a central transcription factor regulating the expression of genes encoding photosystem-related proteins, such as light-harvesting Chl a/b-binding proteins (LHCPs), directly regulates the transcription of BCM1. LHCPs are stabilized by Chl binding, suggesting that GLKs control the amount of LHCP through transcriptional and post-translational regulation via BCM-mediated Chl-level regulation. Meanwhile, we generated a mutant of the BCM ortholog in lettuce (Lactuca sativa) by genome editing and found that it showed an early yellowing phenotype, but only a slight reduction in Chl in presenescent leaves. Thus, this study revealed a conserved but slightly diversified regulation of Chl and LHCP levels via the GLK-BCM pathway in eudicots.

Highly pleiotropic functions of CYP78As and AMP1 are regulated in non-cell-autonomous/organ-specific manners.

The cytochrome P450 CYP78A5/KLUH in Arabidopsis thaliana is predicted to be involved in the synthesis of a mobile signal molecule that has a pleiotropic function that is distinct from classical phytohormones. CYP78A5 has five close relatives in Arabidopsis. We first investigated their functions, focusing on the plastochron, leaf size, and leaf senescence. Our analyses revealed that CYP78A5 and CYP78A7 are involved in the plastochron and leaf size, and CYP78A6 and CYP78A9 are involved in leaf senescence. Complementation analyses using heterologous promoters and expression analyses suggested that CYP78A isoforms have a common biochemical function and are functionally differentiated via organ-specific expression. The altered meristem program1 (amp1) carboxypeptidase mutant shows a phenotype very similar to that of the cyp78a5 mutant. Complementation analyses using boundary and organizing center-specific promoters suggested that both CYP78A5 and AMP1 act in a non-cell-autonomous manner. Analyses of multiple cyp78a mutants and crosses between cyp78a and amp1 mutants revealed that AMP1/LIKE AMP1 (LAMP1) and CYP78A isoforms regulate plastochron length and leaf senescence in the same genetic pathway, whereas leaf size is independently regulated. Furthermore, we detected feedback regulation between CYP78A6/CYP78A9 and AMP1 at the gene expression level. These observations raise the possibility that AMP1 and CYP78A isoforms are involved in the synthesis of the same mobile signal molecule, and suggest that AMP1 and CYP78A signaling pathways have a very close, albeit complex, functional relationship.

Identification and Characterization of an Early Leaf Senescence Gene ELS1 in Soybean.

Early leaf senescence phenotype in soybean could be helpful to shorten the maturation period and prevent green stem disorder. From a high-density mutation library, we identified two early leaf senescence soybean mutant lines, els1-1 (early leaf senescence 1) and els1-2. The chlorophyll contents of both els1-1 and els1-2 were low in pre-senescent leaves. They degraded rapidly in senescent leaves, revealing that ELS1 is involved in chlorophyll biosynthesis during leaf development and chlorophyll degradation during leaf senescence. The causal mutations in els1 were identified by next-generation sequencing-based bulked segregant analysis. ELS1 encodes the ortholog of the Arabidopsis CaaX-like protease BCM1, which is localized in chloroplasts. Soybean ELS1 was highly expressed in green tissue, especially in mature leaves. The accumulation of photosystem I core proteins and light-harvesting proteins in els1 was low even in pre-senescent leaves, and their degradation was accelerated during leaf senescence. These results suggest that soybean ELS1 is involved in both chlorophyll synthesis and degradation, consistent with the findings in Arabidopsis BCM1. The gene els1, characterized by early leaf senescence and subsequent early maturation, does not affect the flowering time. Hence, the early leaf senescence trait regulated by els1 helps shorten the harvesting period because of early maturation characteristics. The els1-1 allele with weakly impaired function of ELS1 has only a small effect on agricultural traits and could contribute to practical breeding.

Functional Divergence of G and Its Homologous Genes for Green Pigmentation in Soybean Seeds.

The degradation of chlorophyll in mature soybean seeds is closely related to the development of their yellow color. In this study, we examined G, its homologue G-like (GL), and their mutant alleles and investigated the relationship between these genes and chlorophyll accumulation in the seed coats of mature seeds. Transient expression of G and GL proteins fused with green fluorescent protein revealed that both were localized in plastids. Overexpression of G resulted in the accumulation of chlorophyll in the seed coats and cotyledons of mature seeds, indicating that high expression levels of G result in chlorophyll accumulation that exceeds its metabolism in the seeds of yellow soybean. Analysis of near isogenic lines at the G locus demonstrated a significant difference in the chlorophyll content of the seed coats and cotyledons of mature seeds when G and mutant g alleles were expressed in the d1d2 stay-green genetic background, indicating that the G protein might repress the SGR-independent degradation of chlorophyll. We examined the distribution of mutant alleles at the G and GL loci among cultivated and wild soybean germplasm. The g allele was widely distributed in cultivated soybean germplasm, except for green seed coat soybean lines, all of which contained the G allele. The gl alleles were much fewer in number than the g alleles and were mainly distributed in the genetic resources of cultivated soybean from Japan. None of the landraces and breeding lines investigated in this study were observed to contain both the g and gl alleles. Therefore, in conclusion, the mutation of the G locus alone is essential for establishing yellow soybeans, which are major current soybean breeding lines.

pCYOs: Binary vectors for simple visible selection of transformants using an albino-cotyledon mutant in Arabidopsis thaliana.

Several selection markers for the screening of transformants have been developed; however, simple and reliable methods are generally preferred. We have developed a novel visible selection system for the identification of transformants in Arabidopsis thaliana that does not require any special reagent and/or equipment except using the albino-cotyledon mutant cyo1. In this system, the pCYO vector carrying the CYO1 genomic fragment as a selection marker is introduced into the cyo1 mutant. Transformation is performed by the Agrobacterium-mediated floral dip method and resultant T1 seeds are sown in soil. Seedlings with green cotyledons, not albino, are expected to be 'complemented' transformants with the transgene of interest. This system provides a very simple selection method that can be performed without any special equipment, reagent, sterile conditions, or UV illumination. We have constructed three vectors, (1) pCYO1, an empty vector; (2) pCYO2, an overexpression vector carrying CaMV35S promoter; and (3) pCYO3, a vector for genome editing, carrying the CRISPR/Cas9 cassette. Example transformation experiments using these vectors, including genome editing, are shown.

Impairment of Lhca4, a subunit of LHCI, causes high accumulation of chlorophyll and the stay-green phenotype in rice.

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called 'stay-green' mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the 'senescent green leaves', suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll-protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

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.

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll - protein complexes during leaf senescence.

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll - protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4-1, a stay-green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4-1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation-related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity-dependent manner. Surprisingly, the Fv /Fm value remained high in nyc4-1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4-1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild-type and other non-functional stay-green mutants, including sgr-2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll - protein complexes during leaf senescence is discussed.

Intensity correlation measurement system by picosecond single shot soft x-ray laser.

We developed a new soft x-ray speckle intensity correlation spectroscopy system by use of a single shot high brilliant plasma soft x-ray laser. The plasma soft x-ray laser is characterized by several picoseconds in pulse width, more than 90% special coherence, and 10(11) soft x-ray photons within a single pulse. We developed a Michelson type delay pulse generator using a soft x-ray beam splitter to measure the intensity correlation of x-ray speckles from materials and succeeded in generating double coherent x-ray pulses with picosecond delay times. Moreover, we employed a high-speed soft x-ray streak camera for the picosecond time-resolved measurement of x-ray speckles caused by double coherent x-ray pulse illumination. We performed the x-ray speckle intensity correlation measurements for probing the relaxation phenomena of polarizations in polarization clusters in the paraelectric phase of the ferroelectric material BaTiO(3) near its Curie temperature and verified its performance.

Demonstration of a highly coherent 13.9 nm x-ray laser from a silver tape target.

A highly coherent 13.9 nm x-ray laser (XRL) is generated under an oscillator-amplifier configuration using a new tape target system and a driver laser system with a 0.1 Hz repetition rate. The output energy is comparable to the XRL generated with a silver-deposited slab target, and the pointing stability using the new tape target system is better than conventional slab targets.

Characterization of a high-brilliance soft x-ray laser at 13.9 nm by use of an oscillator-amplifier configuration.

We have improved a highly coherent x-ray laser at 13.9 nm using an oscillator-amplifier configuration. To improve a high-brilliance x-ray laser, we adopted traveling wave pumping for the amplifier target and rotated the amplifier target 3-4 mrad in the counterclockwise direction. Thereby, a seed x-ray laser can be amplified by medium plasma of the amplifier target with a high gain coefficient. The amplified x-ray laser has the output energy of approximately 1.3 microJ, corresponding to a large photon flux of 6.5 x 10(10) photons/pulse and a high peak brilliance of 5 x 10(26) photons/(s x mm(2) x mrad(2) x 0.01% bandwidth).