Different acyl-CoA:diacylglycerol acyltransferases vary widely in function, and a targeted amino acid substitution enhances oil accumulation.

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content.

CO2 -responsive CCT protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes.

CO2 -responsive CCT protein (CRCT) is a positive regulator of starch synthesis-related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis-related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5'-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa; however, CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provides important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

Expression level of Rubisco activase negatively correlates with Rubisco content in transgenic rice.

The relationship between ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (Rca) levels was studied using transgenic rice overexpressing maize Rca (OX-mRca) and knockdown transgenic rice expressing antisense Rca (KD-Rca). The ratio of Rubisco to total soluble protein was lower in OX-mRca, whereas it was higher in KD-Rca than in WT, indicating that Rca expression was negatively correlated with Rubisco content. The expressions of other Calvin-Benson-Bassham cycle enzymes such as sedoheptulose-1,7-bisphosphatase and phosphoribulokinase analyzed by immunoblotting did not show such a negative correlation with Rca, suggesting that the effect of Rca on protein expression may be specific for Rubisco. Although Rubisco content was decreased in OX-mRca, the transcript levels of the Rubisco large subunit (OsRbcL) and the Rubisco small subunit mostly increased in OX-mRca as well as in KD-Rca. Additionally, polysome loading of OsRbcL was slightly higher in OX-mRca than it was in WT, suggesting that the OsRbcL translation activity was likely stimulated by overexpression of Rca. 35S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized Rubisco among genotypes. However, 35S-methionine-labeled Rubisco was marginally decreased in OX-mRca and increased in KD-Rca compared to the WT. These results suggest that Rca negatively affects the Rubisco content, possibly in the synthesis step.

Growth promotion and inhibition of the Amazonian wild rice species Oryza grandiglumis to survive flooding.

In Asian cultivated rice (Oryza sativa), distinct mechanisms to survive flooding are activated in two groups of varieties. Submergence-tolerant rice varieties possessing the SUBMERGENCE1A (SUB1A) gene display reduced growth during flash floods at the seedling stage and resume growth after the flood recedes, whereas deepwater rice varieties possessing the SNORKEL1 (SK1) and SNORKEL2 (SK2) genes display enhanced growth based on internodal elongation during prolonged submergence at the mature stage. In this study, we investigated the occurrence of these growth responses to submergence in the wild rice species Oryza grandiglumis, which is native to the Amazon floodplains. When subjected to gradual submergence, adult plants of O. grandiglumis accessions showed enhanced internodal elongation with rising water level and their growth response closely resembled that of deepwater varieties of O. sativa with high floating capacity. On the other hand, when subjected to complete submergence, seedlings of O. grandiglumis accessions displayed reduced shoot growth and resumed normal growth after desubmergence, similar to the response of submergence-tolerant varieties of O. sativa. Neither SUB1A nor the SK genes were detected in the O. grandiglumis accessions. These results indicate that the O. grandiglumis accessions are capable of adapting successfully to flooding by activating two contrasting mechanisms as the situation demands and that each mechanism of adaptation to flooding is not mediated by SUB1A or the SK genes.

The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell.

BACKGROUND: PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs. RESULTS: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway. CONCLUSIONS: These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.

Suppression and promotion of growth by ethylene in rice seedlings depends on ambient humidity.

We examined the effect of ethylene on the growth of rice seedlings (Oryza sativa L.) at various degrees of humidity. Ethylene significantly suppressed the growth of shoots when applied to seedlings grown under 30% relative humidity (RH), but promoted the growth of shoots when applied to seedlings grown under 100% RH. The application of gibberellic acid (GA(3)) promoted the elongation of shoots in seedlings grown under 30% and 100% RH. Ethylene inhibited the shoot elongation induced by GA(3) at 30% RH, but enhanced the elongation induced by GA(3) at 100% RH. These results indicate that ethylene can either promote or suppress the growth of rice shoots depending on ambient humidity, and that these actions of ethylene may be mediated through modulating the responsiveness of shoots to gibberellin.

The M3 Phosphorylation Site Is Required for Trafficking and Biological Roles of PIN-FORMED1, 2, and 7 in Arabidopsis.

Asymmetrically localized PIN-FORMED (PIN) auxin efflux carriers play key roles in regulating directional intercellular auxin movement, generating local auxin gradients, and diverse auxin-mediated growth and development. The polar localization of PINs is controlled by phosphorylation in the central hydrophilic loop (HL) of PINs. Although the M3 phosphorylation site, including phosphorylatable 5 Ser/Thr residues, is conserved among long HL-PINs, its native role has only been characterized in PIN3. In this study, we examined the role of M3 phosphorylation site of PIN1, PIN2, and PIN7 in intracellular trafficking, phosphorylation, and biological functions of those PINs in their native expressing tissues. Phosphorylation-defective mutations of the phosphorylatable residues in the M3 site of PIN1-HL led to alteration in subcellular polarity of PIN1 and caused defects in PIN1-mediated biological functions such as cotyledon development, phyllotaxy of vegetative leaves, and development of reproductive organs. The M3 mutations of PIN7 interfered with its polar recycling in the root columella cell in response to gravity stimulus and partially disrupted root gravitropism. On the other hand, the M3 site of PIN2 was shown to be necessary for its targeting to the plasma membrane. In vitro phosphorylation assay showed that the M3 phosphorylation residues of PIN1 are the partial targets by PINOID kinase. Our data suggest that the M3 phosphorylation site is functionally conserved among long HL-PINs by playing roles for their subcellular trafficking and auxin-mediated developmental processes.

Direct mono-insertion of isocyanides into terminal alkynes catalyzed by rare-earth silylamides.

Rare-earth silylamide complexes, Ln[N(SiMe3)2]3 (Ln = Y, La, Sm, Yb), effectively catalyzed the coupling reaction of isocyanides with both aliphatic and aromatic terminal alkynes under mild conditions.

Ethylene is not involved in adaptive responses to flooding in the Amazonian wild rice species Oryza grandiglumis.

The Amazonian wild rice Oryza grandiglumis has two contrasting adaptation mechanisms to flooding submergence: a quiescence response to complete submergence at the seedling stage and an escape response based on internodal elongation to partial submergence at the mature stage. We investigated possible factors that trigger these responses. In stem segments excised from mature O. grandiglumis plants, complete submergence only slightly promoted internodal elongation with increased ethylene levels in the internodes, while partial submergence substantially promoted internodal elongation without increased ethylene levels in the internodes. Incubation of non-submerged stem segments under a continuous flow of humidified ethylene-free air promoted internodal elongation to the same extent as that observed for partially submerged segments. Applied ethylene had little effect on the internodal elongation of non-submerged segments irrespective of humidity conditions. These results indicate that the enhanced internodal elongation of submerged O. grandiglumis plants is not triggered by ethylene accumulated during submergence but by the moist surroundings provided by submergence. The growth of shoots in O. grandiglumis seedlings was not promoted by ethylene or complete submergence, as is the case in O. sativa cultivars possessing the submergence-tolerant gene SUB1A. However, because the genome of O. grandiglumis lacks the SUB1A gene, the quiescence response of O. grandiglumis seedlings to complete submergence may be regulated by a mechanism distinct from that involved in the response of submergence-tolerant O. sativa cultivars.

Anoxia promotes gravitropic curvature in rice pulvini but inhibits it in wheat and oat pulvini.

Gravitropic curvature of pulvini of wheat and oat stem segments gradually declined with decreasing atmospheric O2 concentration and was almost completely blocked under anoxia, whereas that of rice stem segments was enhanced under hypoxia and anoxia. Anoxia substantially increased the ethanol content in pulvini of gravistimulated stem segments in rice, wheat and oat, but the ethanol content showed no marked difference between rice pulvini and wheat and oat pulvini. The concentrations of exogenous ethanol and acetaldehyde required to inhibit the gravitropic curvature of pulvini were significantly higher in rice segments than in wheat and oat segments. However, in all three species, the concentrations of ethanol and acetaldehyde required to completely inhibit curvature were several-fold higher than the endogenous levels that accumulated in pulvini gravistimulated in N2. The pulvini of rice segments gravistimulated in N2 did not contain much more ATP than those of wheat or oat segments gravistimulated in N2. When applied unilaterally to the pulvini of vertically oriented stem segments incubated in N2, indole-3-acetic acid induced bending in rice stem segments but not in wheat and oat stem segments. Transference of graviresponsive pulvini of rice, as well as those of wheat and oat, from aerobic conditions to anaerobic conditions led to cessation of gravitropic curvature within several minutes, but subsequently only gravitropic curvature of anoxic rice pulvini was completely recovered within 2 h. A large portion of this recovery was blocked by cordycepin, a transcription inhibitor. These results suggested that anoxia-induced expression of any gene or genes enables rice pulvini to respond to gravistimulation under anaerobic conditions, and that such a gene or genes might be unrelated to ethanol fermentation and ATP production in anaerobic conditions.

Regulation of the polarity of protein trafficking by phosphorylation.

The asymmetry of environmental stimuli and the execution of developmental programs at the organism level require a corresponding polarity at the cellular level, in both unicellular and multicellular organisms. In plants, cell polarity is important in major developmental processes such as cell division, cell enlargement, cell morphogenesis, embryogenesis, axis formation, organ development, and defense. One of the most important factors controlling cell polarity is the asymmetric distribution of polarity determinants. In particular, phosphorylation is implicated in the polar distribution of the determinant protein factors, a mechanism conserved in both prokaryotes and eukaryotes. In plants, formation of local gradients of auxin, the morphogenic hormone, is critical for plant developmental processes exhibiting polarity. The auxin efflux carriers PIN-FORMEDs (PINs) localize asymmetrically in the plasma membrane and cause the formation of local auxin gradients throughout the plant. The asymmetry of PIN distribution in the plasma membrane is determined by phosphorylationmediated polar trafficking of PIN proteins. This review discusses recent studies on the role of phosphorylation in polar PIN trafficking.

Involvement of cell wall-bound phenolic acids in decrease in cell wall susceptibility to expansins during the cessation of rapid growth in internodes of floating rice.

The cell walls in the elongating zone of submerged floating rice internodes show high susceptibility to expansins. When internode sections corresponding to such an elongation zone were incubated for 24h under osmotic stress conditions produced by treatment with 100mM polyethylene glycol 4000 (PEG), the cell wall susceptibility to expansins remained at its initial level, while the susceptibility of internode sections incubated under unstressed conditions decreased considerably during the same period. The contents of polysaccharides and phenolic acids as ferulic, diferulic and p-coumaric acids in the cell walls of internode sections increased substantially under unstressed conditions, but the increases were almost completely prevented by osmotic stress. Ferulic acid applied to internode sections under osmotic stress reduced the susceptibility of the cell walls to expansins and increased the levels of ferulic and diferulic acids in the cell walls, with little effect on the accumulation of polysaccharides. In contrast, applied p-coumaric acid increased the level of p-coumaric acid in the cell walls without a change in the levels of ferulic and diferulic acids but did not reduce the susceptibility to expansins. These results suggest that the deposition of ferulic and diferulic acids is a primary determinant in regulating the reduction of the susceptibility of cell walls to expansins in floating rice internodes.

Rare-earth silylamide-catalyzed monocoupling reaction of isocyanides with terminal alkynes.

[reaction: see text] Rare-earth silylamides, Ln[N(SiMe3)2]3 (Ln = Y, La, Sm, Yb), serve as good catalysts for monoinsertion of isocyanides into terminal alkynes in the presence of amine additives, leading to 1-aza-1,3-enyens in excellent yields. The reaction is applicable to a diverse set of terminal alkynes with various functionalities such as ethers, acetals, and amino groups. Larger metals (La and Sm) give a better performance than smaller ones (Y and Yb). Using less hindered primary amines and, in contrast, bulky isocyanides is crucial for the coupling reaction; otherwise, competitive oligomerization of the isocyanides occurs predominantly. In the mechanistic study, the rate-determining step of the reaction seems to be the first insertion of the isocyanides into rare-earth alkynides, which is followed by spontaneous protonation with the amine additives.