A novel xylanase,XynA4-2, from thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus</Emphasis> sp. A4 with potential applications in the brewing industry

A xylanase gene, xynA4-2, was obtained from the genome sequence of thermoacidophilic Alicyclobacillus sp. A4 and expressed in Escherichia coli BL21 (DE3). xynA4-2 encodes a mature protein of 411 residues with a calculated molecular weight of 46.8 kDa. Based on the amino acid sequence similarities (highest identity of 61%), the enzyme was confined into glycoside hydrolase family 10. The purified recombinant XynA4-2 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was stable over a broad pH range, retaining more than 90% of the original activity at pH 5.8–12.0, 37°C for 1 h. The substrate specificity of XynA4-2 was relatively narrow, exhibiting 100, 93, and 35% of the relative activity towards birchwood xylan, oat spelt xylan, and wheat arabinoxylan, respectively. Supplementation of XynA4-2 to mash caused the reduction of mash filtration rate (5.6%) and viscosity (4.0%). When combined with the commercial glucanase from Sunson, higher reduction was detected in the filtration rate (12.0%) and viscosity (17.2%). These favorable properties make XynA4-2 a good candidate in the brewing industry.     

Can the Study of Natural Vegetation Succession Assist in the Control of Soil Erosion on Abandoned Croplands on the Loess Plateau,China?

In the Loess Plateau, China, arable cultivation of slope lands is common and associated with serious soil erosion. Planting trees or grass may control erosion, but planted species may consume more soil water and can threaten long‐term ecosystem sustainability. Natural vegetation succession is an alternative ecological solution to restore degraded land, but there is a time cost, given that the establishment of natural vegetation, adequate to prevent soil erosion, is a longer process than planting. The aims of this study were to identify the environmental factors controlling the type of vegetation established on abandoned cropland and to identify candidate species that might be sown soon after abandonment to accelerate vegetation succession and establishment of natural vegetation to prevent soil erosion. A field survey of thirty‐three 2 × 2–m plots was carried out in July 2003, recording age since abandonment, vegetation cover, and frequency of species together with major environmental and soil variables. Data were analyzed using correspondence analysis, classification tree analysis, and species response curves. Four vegetation types were identified and the data analysis confirmed the importance of time since abandonment, total P, and soil water in controlling the type of vegetation established. Among the dominant species in the three late‐successional vegetation types, the most appropriate candidates for accelerating and directing vegetation succession were King Ranch bluestem (Bothriochloa ischaemum) and Lespedeza davurica (Leguminosae). These species possess combinations of the following characteristics: tolerance of low water and nutrient availability, fibrous root system and strong lateral vegetative spread, and a persistent seed bank.     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

Identification of new sources of aluminum resistance in wheat

Aluminum (Al) toxicity is a major constraint for wheat production in acidic soils. An Al resistance gene on chromosome 4DL that traces to Brazilian wheat has been extensively studied, and can provide partial protection from Al damage. To identify potentially new sources of Al resistance, 590 wheat accessions, including elite wheat breeding lines from the United States and other American and European countries, landraces and commercial cultivars from East Asia, and synthetic wheat lines from CIMMYT, Mexico, were screened for Al resistance by measuring relative root elongation in culture with a nutrient solution containing Al, and by staining Al-stressed root tips with hematoxylin. Eighty-eight wheat accessions demonstrated at least moderate resistance to Al toxicity. Those selected lines were subjected to analysis of microsatellite markers linked to an Al resistance gene on 4DL and a gene marker for the Al-activated malate transporter (ALMT1) locus. Many of the selected Al-resistant accessions from East Asia did not have the Al-resistant marker alleles of ALMT1, although they showed Al resistance similar to the US Al-resistant cultivar, Atlas 66. Most of the cultivars derived from Jagger and Atlas 66 have the Al-resistant marker alleles of ALMT1. Cluster analysis separated the selected Al-resistant germplasm into two major clusters, labeled as Asian and American–European clusters. Potentially new germplasm of Al resistance different from those derived from Brazil were identified. Further investigation of Al resistance in those new germplasms may reveal alternative Al-resistance mechanisms in wheat. Electronic supplementary material The online version of this article (doi:contains supplementary material, which is available to authorized users. Responsible Editor: Thomas B. Kinraide.     

RNAi knockdown of Oryza sativa root meander curling gene led to altered root development and coiling which were mediated by jasmonic acid signalling in rice

Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice.     

Novel quantitative trait loci (QTL) for Fusarium head blight resistance in wheat cultivar Chokwang

Fusarium head blight (FHB) is one of the most destructive diseases in wheat. This study was to identify new quantitative trait loci (QTL) for FHB resistance and the molecular markers closely linked to the QTL in wheat cultivar Chokwang. The primers of 612 simple sequence repeats (SSRs) and 12 target-region-amplified polymorphism (TRAP) marker were analyzed between resistant (Chokwang) and susceptible (Clark) parents. One hundred and seventy-two polymorphic markers were used to screen a population of 79 recombinant inbred lines (RILs) derived from the cross of Chokwang and Clark. One major QTL, Qfhb.ksu-5DL1, was identified on chromosome 5DL. The SSR marker Xbarc 239 was mapped in the QTL region, and also physically located to the bin of 5DL1-0.60-0.74 by using Chinese Spring deletion lines. Another QTL Qfhb.ksu-4BL1was linked to SSR Xbarc 1096 and tentatively mapped on 4BL. A QTL on 3BS, Qfhb.ksu-3BS1, was also detected with marginal significance in this population. Different marker alleles for these QTL were detected between Chokwang and Sumai 3 and its derivatives. These results suggested that Chokwang contains new QTL for FHB resistance that are different from those in Sumai 3. Pyramiding resistance QTL from various sources may enhance FHB resistance in wheat cultivars.     

Genetic evidence for functional dependency of p18Ink4c on Cdk4

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.     

Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.     

Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans -Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.