For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.

Background: Aspartic protease (APs) plays important roles in plant growth, development and biotic and abiotic stresses. We previously reported that the expression of a rice AP gene (OsAP77, Os10g0537800) was induced by probenazole (PBZ), a chemical inducer of disease resistance. In this study we examined some characteristics of this gene in response to fungal, bacterial and viral pathogens. Results: To elucidate the spatial and temporal expression of OsAP77, the chimeric gene was constructed carrying the structural gene encoding β-glucuronidase (GUS) driven by the OsAP77 promoter. This construct was introduced into rice and the transgenic lines were tested to analyze gene expression by fungal, bacterial and viral infections. Inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae revealed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. Moreover, GUS activity also increased after inoculation with Cucumber mosaic virus (CMV). Transgenic plants immersed in a solution containing salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide (H₂O₂) or abscisic acid (ABA) showed an increased level of GUS activity exclusively in vascular tissues. RT-PCR analysis showed that OsAP77 was induced not only by infection with these pathogens, but also after treatment with SA, INA, H₂O₂ or ABA. A knockout mutant line of OsAP77 by the insertion of Tos17 after inoculation with M. oryzae, X. oryzae pv. oryzae or CMV showed an enhanced susceptibility compared to wild type. Conclusion: These results suggest that the expression of OsAP77 is induced by pathogen infection and defense related signaling molecules in a vascular tissue specific manner and that this gene has a positive role of defense response against fungal, bacterial and viral infections.