NAC domain transcription factors VNI2 and ATAF2 form protein complexes and regulate leaf senescence.

The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND-INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two-hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.

A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis.

trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5'-capped and 3'-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5' terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5' and 3' fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3' fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5' fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process.

3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3.

trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)-mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1-RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3' cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3' cleavage fragment. When the 3' nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3' cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1-RISC via the double-stranded RNA formed by the 3'-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1-RISC molecular surface, (ii) SGS3 protects the 3' cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3' fragment of TAS2 RNA is key to tasiRNA production.

GLP-1 secretion is enhanced directly in the ileum but indirectly in the duodenum by a newly identified potent stimulator, zein hydrolysate, in rats.

Glucagon-like peptide-1 (GLP-1) is released from enteroendocrine cells (L cells) in response to food ingestion. The mechanism by which dietary peptides stimulate GLP-1 secretion in the gut is unknown. In the present study, we found that a hydrolysate prepared from zein, a major corn protein [zein hydrolysate (ZeinH)], strongly stimulates GLP-1 secretion in enteroendocrine GLUTag cells. Stimulatory mechanisms of GLP-1 secretion induced by ZeinH were investigated in the rat small intestine under anesthesia. Blood was collected through a portal catheter before and after ZeinH administration into different sites of the small intestine. The duodenal, jejunal, and ileal administration of ZeinH induced dose-dependent increases in portal GLP-1 concentration. GLP-1 secretion in response to the ileal administration of ZeinH was higher than that in the duodenal or jejunal administration. Capsaicin treatment on esophageal vagal trunks abolished the GLP-1 secretion induced by duodenal ZeinH but did not affect the secretion induced by jejunal or ileal ZeinH. These results suggest that ZeinH in the jejunum or ileum directly stimulates GLP-1 secretion but duodenal ZeinH indirectly stimulates GLP-1 secretion via the vagal afferent nerve. A direct blood sampling method from the duodenal vein and ileal mesenteric vein revealed that ZeinH administered into the ligated duodenal loop enhanced GLP-1 concentration in the ileal mesenteric vein but not in the duodenal vein. This confirmed that ZeinH in the duodenum induces GLP-1 secretion from L cells located in the ileum by an indirect mechanism. These results indicate that a potent GLP-1-releasing peptide, ZeinH, induces GLP-1 secretion by direct and indirect mechanisms in the rat intestine.

Flavonol glucuronides and C-glucosidic ellagitannins from Melaleuca squarrosa.

Two flavonoids and three ellagitannins, squarrosanins A, B, and C, were isolated from the leaves of Melaleuca squarrosa. The flavonoids were characterized structurally as kaempferol-3-O-(2''-O-galloyl)-glucuronide and herbacetin-3-O-glucuronide, while the ellagitannins were characterized as monomeric and dimeric C-glucosidic ellagitannins by application of spectroscopic and chemical methods. The antioxidant effect of the polyphenolic constituents of the M. squarrosa leaves was also examined in vitro, and C-glucosidic tannins including oligomers were shown to be more effective radical scavengers against 1,1-diphenyl-2-picrylhydrazyl (DPPH) than flavonoids and ordinary ellagitannins.

C-Glucosidic ellagitannin oligomers from Melaleuca squarrosa Donn ex Sm., Myrtaceae.

C-glucosidic ellagitannin dimers were classified as types A-C according to a putative biogenetic oligomerization mode. They were characterized by different positions of the C-C bond between the phenolic acyl unit in one monomer and the benzylic C-1 of the open-chain glucose core in the other monomer. In recent years, four C-glucosidic tannins, melasquanins A-D (18-21), have been found in the leaves of Melaleuca squarrosa Donn ex Sm. (Myrtaceae). These are characterized as a dimer (melasquanin A) of a dimerization mode (type D), and trimers (melasquanins B-D) based on spectroscopic analysis including various two-dimensional nuclear magnetic resonance (2D NMR) experiments. Melasquanins B (19) and D (21) are C-glucosidic tannin trimers with a structure containing, non-repeating condensation modes, which was hitherto unknown.

Cowaniin, a C-glucosidic ellagitannin dimer linked through catechin from Cowania mexicana.

A new complex tannin, cowaniin (1) was isolated from the leaves and stems of Cowania mexicana (Rosaceae), and its structure was characterized as novel C-glucosidic tannin dimer linked through (+)-catechin on the basis of spectral and chemical evidence. The inhibitory effect on activation of the Epstein-Barr virus early antigen was assessed for cowaniin. Six known polyphenols and related compounds, including a nitrile glucoside, purshianin, were also characterized.

Pork peptone stimulates cholecystokinin secretion from enteroendocrine cells and suppresses appetite in rats.

We found that soybean beta-conglycinin peptone (BconP) suppresses food intake through cholecystokinin (CCK) release from enteroendocrine cells in association with binding of the peptone to rat small intestinal brush border membrane (BBM). The aim of the present study was to find new appetite suppressing peptides. Peptones from chicken, pork, beef, beef liver, and egg white were examined for activities to bind with rat BBM, CCK-release from enteroendocrine cell line STC-1, and induce satiety in rats. Chicken and pork peptone (ChickP and PorkP) bound to BBM with highest ability as evaluated with a surface plasmon biosensor. PorkP and ChickP released CCK in higher amounts than BconP from STC-1 cells dose-dependently, with highest stimulation by PorkP. An orogastric preload of PorkP, but not ChickP, suppressed food intake similarly to BconP, dose-dependently. These results suggest that PorkP interacts directly with the small intestinal CCK cells to release CCK, and that it suppresses appetite in rats.

Efficient construction of cDNA microarrays utilizing normalized cDNA libraries of Arabidopsis thaliana.

We have demonstrated the utility of normalization for efficient cDNA microarray preparation using Arabidopsis as a model. Nonredundant cDNAs including 5722 species were efficiently collected from random 7914 expressed sequence tags (ESTs) in four normalized cDNA libraries. The prepared microarrays were successfully used to monitor gene expression. These methodologies should be applicable to the study of other species in plant biotechnology.