Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss‐of‐function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen‐specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid‐mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short‐chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.     

Functional characterization of UDP‐rhamnose‐dependent rhamnosyltransferase involved in anthocyanin modification,a key enzyme determining blue coloration in Lobelia erinus

Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue‐flowered and accumulates delphinidin 3‐O‐p‐coumaroylrutinoside‐5‐O‐malonylglucoside‐3′5′‐O‐dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve‐flowered, and LC‐MS analyses showed that AL accumulated delphinidin 3‐O‐glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP‐rhamnose‐dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5‐bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.     

Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.     

昆虫杆状病毒的蜕皮甾体尿苷二磷酸葡糖转移酶

昆虫杆状病毒的蜕皮甾体尿苷二磷酸葡糖转移酶（ecdysteroid UDP-glucosyltransferase, EGT）能催化UDP-糖基连接到蜕皮甾体上,使之失活,从而阻止或推迟蜕皮,有利于病毒复制,所以缺失egt基因的病毒能改良杀虫效果,如加速死亡和减少摄食量等.最近几年的研究进展主要集中在基因和蛋白质的一级结构、酶催化反应、以及对宿主的生物学功能等几方面.     

祁连圆柏和圆柏色素含量及其花青苷合成酶活性的季节性变化

以祁连圆柏(Sabina przewalskii)和圆柏(S. chinensis)为材料, 测定2种植物花青苷、类黄酮、紫松果黄素、叶绿素和 类胡萝卜素的含量及花青苷合成过程中关键酶苯丙氨酸解氨酶(PAL)和类黄酮糖基转移酶(UFGT)的活性, 并分析了各值的季节性变化。结果表明, 祁连圆柏和圆柏叶片中PAL及UFGT的活性、花青苷、类黄酮﹑紫松果黄素以及类胡萝卜素的含量在低温季节均明显高于其它季节; 叶绿素含量在低温季节低于其它季节; 并且祁连圆柏中花青苷含量及其合成酶PAL和UFGT的活性以及类黄酮、紫松果黄素和叶绿素含量始终高于圆柏。结果说明花青苷是圆柏属植物中具有抗冻特性的重要次生代谢物,是抵御低温和辐射胁迫的一种重要保护物质; 紫松果黄素等色素对圆柏属植物抵抗低温诱导的光抑制起重要作用。     

Variations in chemical composition and in vitro and in situ ruminal degradation characteristics of dried distillers’ grains with solubles from European ethanol plants

The objective of this study was to characterise variations in the composition and nutritive value of dried distillers’ grains with solubles (DDGS) for ruminants, and to estimate the undegradable crude protein (UDP) in DDGS. Thirteen samples originating from wheat, corn, barley and blends of different substrates were studied. The rumen degradation of crude protein (CP) was determined using the nylon bag technique. Samples were incubated for 0, 1, 2, 4, 8, 16, 32 and 72 h, and in situ degradation kinetics were determined. The UDP was estimated using a passage rate of 8%/h. In vitro gas production was measured to estimate the metabolisable energy (ME), net energy for lactation (NE_L) and in vitro digestibility of organic matter (IVDOM). Chemical profiles varied among samples [in g/kg dry matter (DM) ± standard deviation]; the values were 310 ± 33 CP, 86 ± 37 ether extract, 89 ± 18 crude fibre, 408 ± 39 neutral detergent fibre, 151 ± 39 acid detergent fibre and 62 ± 31 acid detergent lignin, as well as in protein fractions according to the Cornell Net Carbohydrate and Protein System [in g/kg CP]; the values were for fractions A, 161 ± 82; B1, 24 ± 11; B2, 404 ± 105; B3, 242 ± 61; and C, 170 ± 87. The ME, NE_L [MJ/kg DM] and IVDOM [%] also varied among samples: 12.1 ± 0.59, 7.3 ± 0.39 and 72.5 ± 4.30, respectively. The in situ rapidly degradable CP fraction (a) varied from 10.2% to 30.6%, and the potentially degradable fraction (b) averaged to 66.8%. The UDP varied from 8.6% to 62.6% of CP. The present study suggests significant variations in composition and nutritive value among different sources of DDGS. The UDP could be predicted on the basis of analysed CP fractions, but the accuracy of UDP prediction improved upon the inclusion of neutral-detergent insoluble nitrogen, explaining 94% of the variation in the UDP values. We conclude that chemical protein fractions may be used to predict the UDP values of DDGS and that the variability in the protein fractions of DDGS should be considered when formulating diets for dairy cows.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

Mechanism of cell contact-dependent glycolipid synthesis: further studies with glycolipid-glass complex

An initiation factor from rabbit reticulocytes can overcome the block in initiation of protein synthesis occurring in reticulocyte lysates when exogenous hemin is not present, or when double-stranded RNA is added. This factor has been identified with IF-MP, an initiation factor capable of forming ternary complexes with GTP and methionyl-tRNA_F. Initiation factor IF-M3 by itself is unable to overcome the block in initiation, but appears to stimulate this activity of IF-MP. IF-MP binds to single-stranded R17 RNA as well as to double-stranded RNA, while IF-M3 only binds to double-stranded RNA. The protein synthetic activity of IF-MP is sensitive to N-ethylmaleimide, but its ability to bind RNA is resistant.