Phototropin-and photosynthesis-dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells^FA

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic in-teractions. We previously reported that, in leaf palisade cel s of Arabidopsis thaliana, mitochondria exhibit blue-light-dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue-light receptor phototropins. In this study, precise motility analyses by fluorescent micro-scopy revealed that the individual mitochondria in palisade cel s, labeled with green fluorescent protein, exhibit typical stop-and-go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mi-tochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma-membrane ghost assay suggested that the stopped mi-tochondria are firmly adhered to chloroplasts. These re-sults indicate that the physical interaction between mi-tochondria and chloroplasts is cooperatively mediated by phototropin2- and photosynthesis-dependent signals. The present study might add novel regulatory mechanism for light-dependent plant organel e interactions.

Appadtion of the NAC Transcription Factors Predates the Emergence of Land Plants

Dear Editor The NAC （for NO APICAL MERISTEM [NAM], Arabidopsis TRANSCRII^TION ACTIVATOR FACTOR [ATAF] and CUP- SHAPED COTYLEDON [CUC]） proteins are one of the largest families of plant-specific transcription factors （Nagata et aL, 2015）. They are involved in many different biological processes including development and the response to biotic and abiotic stresses. To date, NAC transcription factors have only been identified in land plants （Zhu et al., 2012; Nagata et al., 2015）; and some of them control land-plant-specific processes, such as the formation of specialized water-conducting systems （Xu et al., 2014）. Therefore, it has been suggested that their appearance accompanied the emergence of land plants. Here, we exploited the transcriptomic data generated within the 1000 plants project （www.onekp.com, Wickett et al., 2014） to revisit the origin of NAC proteins. Our careful analysis shows that NAC transcription factors appeared before the ernergence of land plants as we detected their homologs in several groups of streptophyte green algae. The possible significance of this finding is discussed.

The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato

Precision genome editing is highly desired for crop improvement.The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering.A prime editing(PE)approach combining a reverse transcriptase(RT)with a Cas9 nickase and a“priming”extended guide RNA(gRNA)has shown a high frequency for precise genome modification in mammalian cells and several plant species.Nevertheless,the applications of the PE approach in dicot plants are still limited and inefficient.We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation.Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems.Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs(pegRNAs)negatively affected the cleaving activity of the Cas9 nuclease.The self-complementarity between the primer binding sequences(PBSs)and spacer sequence might pose risks to the activity of the Cas9 complex.However,modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein(SlMBP21),alcobaca(SlALC),and acetolactate synthase 1(SlALS1)loci.Our data show challenges of the PE approach in tomato,indicating that a further improvement of the PE system for successful applications is demanded,such as the use of improved expression systems for enriching active PE complexes.