Nam Chon Paek

Rice Virescent3 and Stripe1 Encoding the Large and Small Subunits of Ribonucleotide Reductase Are Required for Chloroplast Biogenesis during Early Leaf Development

The virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20°C or 30°C but almost green leaves under diurnal 30°C/20°C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise α2β2 heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 > RNRL1:rnrs1(st1) > rnrl1(v3):RNRS1 > rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1(st1) attenuate the first αβ dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth.

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.

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.

Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing

Summary Under mild abiotic-stress conditions, Arabidopsis atg mutants showed a functional stay-green phenotype which is probably caused by the lack of chloroplastic autophagy and the retrograde regulation of senescence-associated gene expression.

EFFECTS OF NITROGEN SOURCE AND TIMING OF SULFUR DEFICIENCY ON SEED YIELD AND EXPRESSION OF 11S AND 7S SEED STORAGE PROTEINS OF SOYBEAN

Abstract The nutritional value of soybean (Glycine max [L.] Merr.) seed as a protein source for nonruminants is limited by the amount of the S-containing amino acids, methionine and cysteine, they contain. The nutritional quality of S soybean is known to be influenced by both S and N nutrition. The capacity of the plant for S assimilation during late seed filling is an additional factor that might limit protein quality even when S availability is more than adequate. This paper describes experiments conducted to examine the effects of S and reduced N availability on protein quality, and the capacity of the plant for assimilation of sulfate-S during seed filling. Availability of sulfate-S was varied in the hydroponic culture during various vegetative and reproductive stages and N was supplied as either urea (reduced form) or as nitrate (oxidized form). The ratio of 11S/7S seed storage proteins was used as an indicator of protein quality. The 11S protein is of greater nutritional value than is the 7S protein; therefore, the greater the 11S/7S ratio the better the nutritional quality of the seed protein. Provision of reduced N caused seed yield to increase across S treatments and the 11S/7S ratio to decline among plants that were S-sufficient but not among those that were S-deficient. Seed yield was very sensitive to S-deficiency occurring during vegetative growth, but not to S-deficiency occurring during reproductive growth. The 11S/7S ratio was strongly influenced by S-deficiency occurring during reproductive growth, but was relatively insensitive to S availability during vegetative growth. Provision of S near the middle of the seed filling period to previously S-deficient plants caused 11S/7S ratio to increase threefold over plants that were maintained S-deficient throughout seed filling. We conclude that the plant maintains a substantial capacity for S assimilation late into seed filling, and that mobilization of vegetative S is not a large source of S for developing seeds.

Rapid upregulation ofDehyrin3 andDehydrin4 in response to dehydration is a characteristic of drought-tolerant genotypes in barley

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.

Differential accumulation of Soybean seed storage protein subunits in response to sulfur and nitrogen nutritional sources

Abstract Soybean (Glycine max [L.] Merr.) seed storage proteins consist of subunits that differ in amino acid profile, the β-subunit of 7S protein being essentially devoid of the S-containing amino acids, methionine and cysteine. Our objective was to examine the interaction of N and S nutrition on the relative abundance of these storage protein subunits in soybean seed. ‘Kenwood’ soybean was grown in hydroponic culture, and during vegetative growth (V2–R4.5) N was provided as 5 mM KNO3 to plants grown under sulfur-deficient (0.004 raM Na2SO4) or sulfur-sufficient (0.4 mM Na2S04) conditions. During seed fill (R4.5–R7) N was supplied as 5 mM KNO3 or 2.5 mM urea. Each N group was given S treatments of 1) no sulfur, 2) 0.4mM Na2SO4, 3) 0.2 mM L-cystine, or 4) 0.4 mM L-methionine. Effects on seed protein quality of S deficiency during vegetative growth were essentially overcome by supplying sulfate as late as R4.5. Total protein and seed storage protein were increased with urea as N source, but urea also increased the β-subunit. Provision of reduced S as methionine essentially suppressed β-subunit production, but cystine did not, suggesting that cystine did not influence methionine level in the seed. We also report the accumulation of two as yet unreported proteins which occur at extremes of S nutrition : (1) a putative β-subunit of 7S protein occurring in the embryonic axis under S-deficiency ; and (2) a ca. 14kD protein in cotyledon tissue under provision of L-methionine. Though S and N did interact to a limited extent to influence seed protein composition, major effects were from S or N acting individually.