Kaoru T. Yoshida

Scaling IP Routing with the Core Router-Integrated Overlay

IP routing scalability is based on hierarchical routing, which requires that the IP address hierarchy be aligned with the physical topology. Both site multi-homing and switching ISPs without renumbering break this alignment, resulting in large routing tables. This paper presents CRIO: a new approach to IP scalability for both global and VPN routing. Using tunneling and virtual prefixes, CRIO decouples address hierarchy and physical topology, effectively giving ISPs the ability to trade-off routing table size for path length. Though CRIO is a new routing architecture, it works with existing data-plane router mechanisms. Through static simulation on a Rocketfuel-measured Internet topology and traffic data from a Tier 1 ISP, we show that CRIO can shrink the BGP RIB by nearly two orders of magnitude, the global FIB by one order of magnitude, and the VPN FIB by ten to twenty times, all with very little increase in overall path length.

Dynamic changes in the distribution of minerals in relation to phytic-acid accumulation during rice seed development

Phytic acid (inositol hexakisphosphate [InsP6]) is the storage compound of phosphorus in seeds. As phytic acid binds strongly to metallic cations, it also acts as a storage compound of metals. To understand the mechanisms underlying metal accumulation and localization in relation to phytic acid storage, we applied synchrotron-based x-ray microfluorescence imaging analysis to characterize the simultaneous subcellular distribution of some mineral elements (phosphorus, calcium, potassium, iron, zinc, and copper) in immature and mature rice (Oryza sativa) seeds. This fine-imaging method can reveal whether these elements colocalize. We also determined their accumulation patterns and the changes in phosphate and InsP6 contents during seed development. While the InsP6 content in the outer parts of seeds rapidly increased during seed development, the phosphate contents of both the outer and inner parts of seeds remained low. Phosphorus, calcium, potassium, and iron were most abundant in the aleurone layer, and they colocalized throughout seed development. Zinc was broadly distributed from the aleurone layer to the inner endosperm. Copper localized outside the aleurone layer and did not colocalize with phosphorus. From these results, we suggest that phosphorus translocated from source organs was immediately converted to InsP6 and accumulated in aleurone layer cells and that calcium, potassium, and iron accumulated as phytic acid salt (phytate) in the aleurone layer, whereas zinc bound loosely to InsP6 and accumulated not only in phytate but also in another storage form. Copper accumulated in the endosperm and may exhibit a storage form other than phytate.

Molecular breeding for transgenic rice with low-phytic-acid phenotype through manipulating myo-inositol 3-phosphate synthase gene

In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6; phytic acid). Reducing the phytic acid content of seeds is a major breeding target, both to increase the availability of mineral nutrients and to decrease the environmental load of phosphorus. The first step in phytic acid biosynthesis and inositol metabolism is catalyzed by 1d-myo-inositol 3-phosphate (Ins(3)P1) synthase. In this study, we aimed to reduce phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)P1 synthase gene RINO1 using transgenic methods. RINO1 cDNA was transformed into rice plants in the antisense orientation under the control of the rice major storage protein glutelin GluB-1 promoter. The T4 generation of a stable transgenic line that contained four copies of the transgene showed little morphological differences compared to non-transgenic rice. In the T5 seeds of this line, severe reductions in RINO1 protein levels were observed during the late maturing stages of ripening. Most of the T5 seeds contained higher amounts of inorganic phosphates (Pi), without a reduction in total phosphorus levels, compared to non-transgenic seeds. Ion chromatography analysis suggested that the increase in available Pi is accompanied by a molar-equivalent decrease in phytic acid P. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerase chain-reaction (RT-PCR). Potential strategies for further molecular breeding to reduce phytic acid levels in seeds are discussed.

Reducing phosphorus accumulation in rice grains with an impaired transporter in the node

Phosphorus is an important nutrient for crop productivity. More than 60% of the total phosphorus in cereal crops is finally allocated into the grains and is therefore removed at harvest. This removal accounts for 85% of the phosphorus fertilizers applied to the field each year. However, because humans and non-ruminants such as poultry, swine and fish cannot digest phytate, the major form of phosphorus in the grains, the excreted phosphorus causes eutrophication of waterways. A reduction in phosphorus accumulation in the grain would contribute to sustainable and environmentally friendly agriculture. Here we describe a rice transporter, SULTR-like phosphorus distribution transporter (SPDT), that controls the allocation of phosphorus to the grain. SPDT is expressed in the xylem region of both enlarged- and diffuse-vascular bundles of the nodes, and encodes a plasma-membrane-localized transporter for phosphorus. Knockout of this gene in rice (Oryza sativa) altered the distribution of phosphorus, with decreased phosphorus in the grains but increased levels in the leaves. Total phosphorus and phytate in the brown de-husked rice were 20–30% lower in the knockout lines, whereas yield, seed germination and seedling vigour were not affected. These results indicate that SPDT functions in the rice node as a switch to allocate phosphorus preferentially to the grains. This finding provides a potential strategy to reduce the removal of phosphorus from the field and lower the risk of eutrophication of waterways.

Inferring POP-Level ISP Topology through End-to-End Delay Measurement

In this paper, we propose a new topology inference technique that aims to reveals how ISPs deploy their layer two and three networks at the POP level, without relying on ISP core network information such as router hops and domain names. This is because, even though most of previous works in this field leverage core network information to infer ISP topologies, some of our measured ISPs filter ICMP packets and do not allow us to access core network information through traceroute. And, several researchers point out that such information is not always reliable. So, to infer ISP core network topology without relying on ISP releasing information, we deploy systems to measure end-to-end communication delay between residential users, and map the collected delay and corresponding POP-by-POP paths. In our inference process, we introduce assumptions about how ISPs tend to deploy their layer one and two networks. To validate our methodology, we measure end-to-end communication delay of four nationwide ISPs between thirteen different cities in Japan and infer their POP-level topologies.

cDNA microarray analysis of gene expression changes during pollination, pollen-tube elongation, fertilization, and early embryogenesis in rice pistils

During the processes of pollen recognition, pollen-tube guidance, fertilization, and early embryogenesis, a complicated gene network is highly regulated in plants. To visualize the profile of gene expression during these processes, we performed cDNA microarray analysis with RNA isolated from rice pistils at several stages. We identified 32 cDNA clones, in 21 non-redundant groups, with pistil-specific expression patterns. These genes could be clustered into five groups based on expression patterns, and included pathogenesis-related genes, transcription factors, and genes related to pollen germination, pollen tube elongation, and starch degradation. The microarray analysis identified several previously undescribed genes that are expressed in a pistil-specific manner. These results provide new clues to the molecular mechanisms that underlie plant reproduction.

Molecular cloning and characterization of plasma membrane- and vacuolar-type Na + /H + antiporters of an alkaline-salt-tolerant monocot, Puccinellia tenuiflora

A better understanding of salt tolerance in plants might lead to the genetic engineering of crops that can grow in saline soils. Here we cloned and characterized plasma membrane and vacuolar Na+/H+ antiporters of a monocotyledonous alkaline-tolerant halophyte, Puccinellia tenuiflora. The predicted amino acid sequence of the transporters were very similar to those of orthologs in monocotyledonous crops. Expression analysis showed that (1) NHA was more strongly induced by NaCl in the roots of P. tenuiflora while in rice it was rather induced in the shoots, suggesting that the role of NHA in salt excretion from the roots partly accounts for the difference in the tolerance of the two species, and that (2) NHXs were specifically induced by NaHCO3 but not by NaCl in the roots of both species, suggesting that vacuolar-type Na+/H+ antiporters play roles in pH regulation under alkaline salt conditions. Overexpression of the antiporters resulted in increased tolerance of shoots to NaCl and roots to NaHCO3. Overexpression lines exhibited a lower Na+ content and a higher K+ content in shoots under NaCl treatments, leading to a higher K+/Na+ ratio.

Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility.

Protease activities with specificity toward synthetic substrates, Suc‐Gly‐Pro‐Leu‐Gly‐Pro‐MCA for prolyl endopeptidase or collagenase‐like peptidase, and Suc‐Ala‐Ala‐Pro‐Phe‐MCA for chymotrypsin were identified in the detergent‐soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm‐activating protein (HSAP). Among them a prolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity from herring testis. The molecular mass of the enzyme was 79 kDa and the properties of the enzyme were quite similar to prolyl endopeptidase from other tissues or cells. Both the enzyme activation and the sperm motility activation by HSAP were inhibited by benzyloxycarbonyl‐L‐thioproline‐thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility activation by HSAP was inhibited by substrates of the prolyl endopeptidase. Western blotting with mouse anti‐prolyl endopeptidase serum revealed the presence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm. These results suggest that prolyl endopeptidase exists on the surface of the sperm tail and interacts with the HSAP.

A novel endosperm transfer cell-containing region-specific gene and its promoter in rice

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT–PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.

A subtilisin-like serine protease specifically expressed in reproductive organs in rice

Abstract To identify genes specifically expressed in flowering pistils and that are related to reproductive phenomena, simplified differential display was performed with cDNA obtained from pistils and ovaries at several stages. One clone preferentially expressed in pistils at flowering and 1 day before flowering was identified as a rice subtilisin-like serine protease (RSP1). Sequence comparisons revealed that RSP1 has several characteristics in common with preproproteins. RT-PCR, northern blot, and in situ hybridization analysis revealed that RSP1 mRNA accumulates in pistils and in the filaments of stamens, whereas mRNA was undetectable in tissue from leaves, roots, panicles, and embryos. The mRNA levels in pistils increased slightly at flowering and decreased afterwards. Possible roles of the subtilisin-like serine protease in plant reproduction are discussed.