Hee Jong Koh

SPL28 encodes a clathrin‐associated adaptor protein complex 1, medium subunit μ1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa)

To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3′-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T1 plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

ZEBRA-NECROSIS, a thylakoid-bound protein, is critical for the photoprotection of developing chloroplasts during early leaf development

The zebra-necrosis (zn) mutant of rice (Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness.

Protein Disulfide Isomerase-Like Protein 1-1 Controls Endosperm Development through Regulation of the Amount and Composition of Seed Proteins in Rice

Protein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species) scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

Expression of hpa1 Gene Encoding a Bacterial Harpin Protein in Xanthomonas oryzae pv. oryzae Enhances Disease Resistance to Both Fungal and Bacterial Pathogens in Rice and Arabidopsis

Xanthomonas oryzae pv. oryzae causing bacterial leaf blight disease in rice produces and secretes Hpa1 protein that belongs to harpin protein family. Previously it was reported that Hpa1 induced defense responses when it was produced in tobacco. In this study, we expressed hpa1 gene in rice and Arabidopsis to examine the effects of Hpa1 expression on disease resistance to both fungal and bacterial pathogens. Expression of hpa1 gene in rice enhanced disease resistance to both X. oryzae pv. oryzae and Magnaporthe grisea. Interestingly, individual transgenic rice plants could be divided into four groups, depending on responses to both pathogens. hpa1 expression in Arabidopsis also enhanced disease resistance to both Botrytis cineria and Xanthomonas campestris pv. campestris. To examine genes that are upregulated in the transgenic rice plants after inoculation with X. oryzae pv. oryzae, known defense-related genes were assessed, and also microarray analysis with the Rice 5 K DNA chip was performed. Interestingly, expression of OsACS1 gene, which was found as the gene that showed the highest induction, was induced earlier and stronger than that in the wild type plant. These results indicate that hpa1 expression in the diverse plant species, including monocot and dicot, can enhance disease resistance to both fungal and bacterial plant pathogens.