Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

OsMKKK70 regulates grain size and leaf angle in rice through the OsMKK4-OsMAPK6-OsWRKY53 signaling pathway

Grain size and leaf angle are key agronomic traits that determine final yields in rice. However, the underlying molecular mechanisms are not well understood. Here we demonstrate that the Oryza sativa Mitogen Activated Protein Kinase Kinase Kinase OsMKKK70 regulates grain size and leaf angle in rice. Overexpressing OsMKKK70 caused plants to produce longer seeds. The osmkkk62/70 double mutant and the osmkkk55/62/70 triple mutant displayed significantly smaller seeds and a more erect leaf angle compared to the wild type, indicating that OsMKKK70 functions redundantly with its homologs OsMKKK62 and OsMKKK55. Biochemical analysis demonstrated that OsMKKK70 is an active kinase and that OsMKKK70 interacts with OsMKK4 and promotes OsMAPK6 phosphorylation. In addition, the osmkkk62/70 double mutant showed reduced sensitivity to Brassinosteroids (BRs). Finally, overexpressing constitutively active OsMKK4, OsMAPK6, and OsWRKY53 can partially complement the smaller seed size, erect leaf, and BR hyposensitivity of the osmkkk62/70 double mutant. Taken together, these findings suggest that OsMKKK70 might regulate grain size and leaf angle in rice by activating OsMAPK6 and that OsMKKK70, OsMKK4, OsMAPK6, and OsWRKY53 function in a common signaling pathway that controls grain shape and leaf angle.     

Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance

Significant achievements have been made in breeding programs for the heavy-panicle-type (HPT) rice (Oryza sativa) in Southwest China. The HPT varieties now exhibit excellent lodging resistance, allowing them to overcome the greater pressures caused by heavy panicles. However, the genetic mechanism of this lodging resistance remains elusive. Here, we isolated a major quantitative trait locus, Panicle Neck Diameter 1 (PND1), and identified the causal gene as GRAIN NUMBER 1A/CYTOKININ OXIDASE 2 (Gn1A/OsCKX2). The null gn1a allele from rice line R498 (gn1a^R498) improved lodging resistance through increasing the culm diameter and promoting crown root development. Loss-of-function of Gn1a/OsCKX2 led to cytokinin accumulation in the crown root tip and accelerated the development of adventitious roots. Gene pyramiding between the null gn1a^R498 allele with two gain-of-function alleles, STRONG CULM 2 (SCM2) and SCM3, further improved lodging resistance. Moreover, Gn1a/OsCKX2 had minimal influence on overall rice quality. Our research thus highlights the distinct genetic components of lodging resistance of HPT varieties and provides a strategy for tailor-made crop improvement of both yield and lodging resistance in rice.     

Application of an Effective Statistical Technique for an Accurate and Powerful Mining of Quantitative Trait Loci for Rice Aroma Trait

When a phenotype of interest is associated with an external/internal covariate, covariate inclusion in quantitative trait loci (QTL) analyses can diminish residual variation and subsequently enhance the ability of QTL detection. In the in vitro synthesis of 2-acetyl-1-pyrroline (2AP), the main fragrance compound in rice, the thermal processing during the Maillard-type reaction between proline and carbohydrate reduction produces a roasted, popcorn-like aroma. Hence, for the first time, we included the proline amino acid, an important precursor of 2AP, as a covariate in our QTL mapping analyses to precisely explore the genetic factors affecting natural variation for rice scent. Consequently, two QTLs were traced on chromosomes 4 and 8. They explained from 20% to 49% of the total aroma phenotypic variance. Additionally, by saturating the interval harboring the major QTL using gene-based primers, a putative allele of fgr (major genetic determinant of fragrance) was mapped in the QTL on the 8th chromosome in the interval RM223-SCU015RM (1.63 cM). These loci supported previous studies of different accessions. Such QTLs can be widely used by breeders in crop improvement programs and for further fine mapping. Moreover, no previous studies and findings were found on simultaneous assessment of the relationship among 2AP, proline and fragrance QTLs. Therefore, our findings can help further our understanding of the metabolomic and genetic basis of 2AP biosynthesis in aromatic rice.     

磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

斑马鱼tbx2b调控心脏房室间隔发育的功能研究*

tbx2是早期心脏发育的关键基因。为进一步探究其对房室间隔(AVC)发育的影响,研究利用CRISPR/Cas9介导的基因敲除技术,成功构建了斑马鱼tbx2b突变体。通过T7E1酶切对其F₀进行敲除效率检测,结果显示平均敲除效率约为57.5%。F₁进一步筛选获得tbx2b杂合突变体,测序结果显示突变类型为11 bp碱基缺失的移码突变。tbx2b杂合子内交获得纯合子,tbx2b纯合突变体在5 dpf死亡并出现早期心脏环化异常表型。斑马鱼整胚原位杂交实验显示在3 dpf tbx2b纯合突变体中, 心脏腔室分化特异性标志基因nppa、nppb表达上调并异位表达在AVC,而AVC发育关键基因has2的表达消失。高效构建tbx2b突变体并初探其对下游基因的影响,为后续深入研究tbx2b对心脏AVC发育的作用奠定了基础,同时加深了人们对早期心脏调控网络的认识。     

RID1 sets rice heading date by balancing its binding with SLR1 and SDG722

Rice (Oryza sativa) is a major crop that feeds billions of people, and its yield is strongly influenced by flowering time (heading date). Loss of RICE INDETERMINATE1 (RID1) function causes plants not to flower; thus, RID1 is considered a master switch among flowering-related genes. However, it remains unclear whether other proteins function together with RID1 to regulate rice floral transition. Here, we revealed that the chromatin accessibility and H3K9ac, H3K4me3, and H3K36me3 levels at Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) loci were significantly reduced in rid1 mutants. Notably, RID1 interacted with SET DOMAIN GROUP PROTEIN 722 (SDG722), a methyltransferase. We determined that SDG722 affects the global level of H3K4me2/3 and H3K36me2/3, and promotes flowering primarily through the Early heading date1-Hd3a/RFT1 pathway. We further established that rice DELLA protein SLENDER RICE1 (SLR1) interacted with RID1 to inhibit its transactivation activity, that SLR1 suppresses rice flowering, and that messenger RNA and protein levels of SLR1 gradually decrease with plant growth. Furthermore, SLR1 competed with SDG722 for interaction with RID1. Overall, our results establish that interplay between RID1, SLR1, and SDG722 feeds into rice flowering-time control.     

Phylogeny, expression and enzyme activity of zebrafish cyp19 (P450 aromatase) genes

The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.     

水稻矮化基因WLD1的图位克隆

水稻(Oryza sativa)矮化是与光合效率及产量等密切相关的重要农艺性状。发掘更多的水稻矮秆资源,不仅能够进一步加深对水稻株高分子遗传机制的认识,而且还能为水稻新品种培育提供新的种质资源。在水稻T-DNA插入突变体库中筛选到1个矮化、宽叶小粒突变体(wld1)。经图位克隆将WLD1基因定位在第5号染色体长臂,位于分子标记In Del37与InDel48之间,基因编号为LOC_Os05g32270,属于AP2转录因子家族。该基因第6外显子处胸腺嘧啶缺失,造成转录提前终止。石蜡切片观察结果显示,茎部第2节间横向细胞数目增加,而纵向细胞数目未变。RT-PCR检测结果表明,LOC_Os05g32270在突变体wld1中不表达,造成功能缺失。该基因与已报道的水稻OsSMOS1(SMALL ORGAN SIZE1)为等位基因。水稻突变体wld1的矮秆遗传效应可直接应用于育种中。该研究结果进一步明确了突变体wld1的表型特征与遗传基础,为解析其参与的信号途径提供参考。     

斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*

hox基因编码一类高度保守的转录因子家族,人类HOXA1的突变会导致阿萨巴斯卡发育不良综合征 (Athabascan brainstem dysgenesis syndrome, ABDS),使人出现因颅骨异常导致的面部畸形和面部麻痹等症状。利用模式生物斑马鱼研究其同源基因hoxa1a的功能机制。首先利用CRISPR/Cas9技术对斑马鱼hoxa1a进行基因编辑,获得了hoxa1a基因突变,T7E1酶切结果显示F₀酶切效率平均为70%。F₁进一步筛选到两种突变类型,分别是插入了8 bp和删除了7 bp的杂合突变体。杂合子自交得到hoxa1a F₂纯合突变体,并且测序验证hoxa1a基因突变成功。5 dpf时,hoxa1a纯合突变体出现颅面发育异常。阿尔新蓝软骨染色和茜素红硬骨染色结果表明,hoxa1a突变体中颅骨发育异常、筛骨板断裂,鳃弓发育出现缺损。成功在斑马鱼中构建ABDS疾病模型,表明hoxa1a突变会造成斑马鱼颅面骨骼发育异常,为其功能机制研究奠定了基础,为人类ABDS疾病的致病机制研究提供了新的思路。     

Biotransformation of podophyllotoxin by Hordeum vulgare cell suspension cultures

Hordeum vulgare cell suspension cultures were used to modify podophyllotoxin (1) One major product (1a) and one minor product (1b) were detected in both the culture medium and cells. To optimize the yield of compound 1a, we showed that: (1) the optimal concentration of added podophyllotoxin (1) was 33 mg L^-1; higher concentrations caused cell toxicity; (2) the stage of the cell cycle (lag/log/stationary) at which podophyllotoxin was added only marginally affected the yield of compound 1a; the optimal addition time was after lag phase, in which the yield of compound 1a reached ca. 76%, and (3) biotransformation of podophyllotoxin (1) was relatively slow; podophyllotoxin fed at 4 days after subculture resulted in yields of compound 1a of ca. 56, 64 and 76% after an additional 3, 6 and 10 days of incubation, respectively. Product 1a was purified and identified as isopicropodophyllone (1a) based on MS and NMR data.     

Opportunities of marker‐assisted selection for rice fragrance through marker–trait association analysis of microsatellites and gene‐based markers

Developing fragrant rice through marker‐assisted/aided selection (MAS) is an economical and profitable approach worldwide for the enrichment of an elite genetic background with a pleasant aroma. The PCR‐based DNA markers that distinguish the alleles of major fragrance genes in rice have been synthesised to develop rice scent biofortification through MAS. Thus, the present study examined the aroma biofortification potential of these co‐dominant markers in a germplasm panel of 189 F₂ progeny developed from crosses between a non‐aromatic variety (MR84) and a highly aromatic but low‐yielding variety (MRQ74) to determine the most influential diagnostic markers for fragrance biofortification. The SSRs and functional DNA markers RM5633 (on chromosome 4), RM515, RM223, L06, NKSbad2, FMbadh2‐E7, BADEX7‐5, Aro7 and SCU015RM (on chromosome 8) were highly associated with the 2AP (2‐acetyl‐1‐pyrroline) content across the population. The alleles traced via these markers were also in high linkage disequilibrium (R² > 0.70) and explained approximately 12.1, 27.05, 27.05, 27.05, 25.42, 25.42, 20.53, 20.43 and 20.18% of the total phenotypic variation observed for these biomarkers, respectively. F₂ plants harbouring the favourable alleles of these effective markers produced higher levels of fragrance. Hence, these rice plants can be used as donor parents to increase the development of fragrance‐biofortified tropical rice varieties adapted to growing conditions and consumer preferences, thus contributing to the global rice market.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

异源过表达OsATG8b基因提高转基因拟南芥的 氮/碳胁迫耐受性和产量

氮素是参与植物生长发育的一种重要元素, 对植物的产量和品质具有重要作用。自噬是真核生物中一种保守的细胞组分降解-循环再利用途径, 在植物生长发育和籽粒形成期间的氮素再动员过程中发挥作用。我们鉴定到水稻(Oryza sativa)自噬核心基因OsATG8b, 并获得2个独立的35S-OsATG8b转基因拟南芥(Arabidopsis thaliana)纯合株系。研究表明OsATG8b基因响应低氮胁迫处理, 过表达OsATG8b基因促进转基因拟南芥的生长发育, 使莲座叶增大, 单株产量显著提高(15.16%)。进一步研究表明, 过表达OsATG8b能够显著增强缺氮胁迫下转基因拟南芥叶片中的自噬活性, 从而有效缓解氮胁迫和碳胁迫对转基因拟南芥造成的生长抑制。因此, OsATG8b是提高氮素利用效率和产量的候选基因。