Novel molecular insights into the anti‐oxidative stress response and structure–function of a salt‐inducible cyanobacterial Mn‐catalase

KatB, a salt‐inducible Mn‐catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological–biochemical function of KatB at the molecular level. Anabaena overexpressing the wild‐type KatB protein (KatBWT) detoxified H₂O₂ efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F‐2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X‐ray crystallography‐based analysis showed that F‐2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F‐2, through its interaction with F‐66 and W‐43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn‐catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.     

Development and use of anchored‐SSRs to study DNA polymorphism in bread wheat (Triticum aestivum L.)

In bread wheat, 21 anchored simple sequence repeat (SSR) primer pairs detecting SSR length polymorphism and 42 anchored SSR primers detecting microsatellite‐anchored fragment length polymorphisms (MFLPs) are reported. Eight bread wheat genotypes were used for detecting polymorphism. The number of alleles in SSR analysis ranged from two to six, with a mean of 2.9 alleles per SSR. The number of polymorphic bands in MFLP ranged from two to 40, with a mean of 12.74 polymorphic bands/primer combination, the SSRs with CT/GA motifs giving the highest level of polymorphism (a mean of 18.37 bands). The average value of polymorphic information content (PIC) was 0.473 for SSRs and 0.061 for MFLP.     

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells

While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G‐actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt‐ and Actin‐Resistant (HSAR‐hDNase I) have been developed previously. However, constitutive expression of HSAR‐hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR‐hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR‐hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR‐hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR‐hDNase I was both hyperactive and resistant to inhibition by salt and G‐actin, resulting in an enzyme having ca. 10‐fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523–533, 2017     

Enrichment of Triticum aestivum gene annotations using ortholog cliques and gene ontologies in other plants

Background

While the gargantuan multi-nation effort of sequencing T. aestivum gets close to completion, the annotation process for the vast number of wheat genes and proteins is in its infancy. Previous experimental studies carried out on model plant organisms such as A. thaliana and O. sativa provide a plethora of gene annotations that can be used as potential starting points for wheat gene annotations, proven that solid cross-species gene-to-gene and protein-to-protein correspondences are provided.

Results

DNA and protein sequences and corresponding annotations for T. aestivum and 9 other plant species were collected from Ensembl Plants release 22 and curated. Cliques of predicted 1-to-1 orthologs were identified and an annotation enrichment model was defined based on existing gene-GO term associations and phylogenetic relationships among wheat and 9 other plant species. A total of 13 cliques of size 10 were identified, which represent putative functionally equivalent genes and proteins in the 10 plant species. Eighty-five new and more specific GO terms were associated with wheat genes in the 13 cliques of size 10, which represent a 65% increase compared with the previously 130 known GO terms. Similar expression patterns for 4 genes from Arabidopsis, barley, maize and rice in cliques of size 10 provide experimental evidence to support our model. Overall, based on clique size equal or larger than 3, our model enriched the existing gene-GO term associations for 7,838 (8%) wheat genes, of which 2,139 had no previous annotation.

Conclusions

Our novel comparative genomics approach enriches existing T. aestivum gene annotations based on cliques of predicted 1-to-1 orthologs, phylogenetic relationships and existing gene ontologies from 9 other plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1496-2) contains supplementary material, which is available to authorized users.     

Pollen callus culture in Triticum aestivum

Summary Pollen shed between 4–8 d from anthers of Triticum aestivum cultured in liquid medium gave rise to calluses. Tillers were harvested at the mid-to late-unicellular pollen stages and chilled for 8 d at 4–5 °C before the anthers were dissected out. Pollen cultures gave about 6 times as many calluses on a per anther basis as anthers cultured on solid medium. With the most productive of 5 cultivars tested, pollen culture results in roughly one callus for each anther used, though the calluses formed by pollen culture were less productive for the regeneration of shoots than calluses derived from anthers cultured on solid medium. The ratio of green to albino shoots is roughly 1 1 for anther cultures but considerably less for pollen cultures.     

Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco

Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.     

Leaf wax of Triticum aestivum

Leaf waxes from spring wheat varieties Selkirk and Manitou contain hydrocarbons (6%, 10%), long chain esters (14%, 13%), free acids (5%, 8%), free alcohols (19%, 21%), β-diketone (16%, 20%), hydroxy β-diketones (8%, 10%), unidentified gum (29%, 16.5%) and minor amounts of diol diesters, glycerides and aldehydes. The major hydrocarbon is nonacosane and major esters are octacosyl esters of C₁₄–C₃₂ acids but C₂₀ and C₂₂ alcohol esters of trans 2-docosenoic and tetracosenoic acids are also present (Selkirk 20%, Manitou 10% of total esters). Previously unknown trans 2-docosen-1-ol is present as an ester (Selkirk 5%, Manitou 2.5% of total esters). Free acids are C₁₄–C₃₂ acids and trans 2-docosenoic and tetracosenoic acids (Selkirk 30%, Manitou 9% of free acids). Octacosanol is the principal free alcohol. Hentriacontane-14,16-dione is the β-diketone and the hydroxy β-diketones are a 1:1 mixture of 8- and 9- hydroxyhentriacontane-14,16-diones.     

The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.).

We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16,700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M x s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.     

小麦TaHDA19基因的克隆及胁迫表达分析

利用基因克隆技术从小麦（Triticum aestivum L.）品种‘科农199’中扩增得到一个RPD3/HDA1型组蛋白去乙酰化酶基因TaHDA19,采用生物信息学方法对该基因序列的结构特征进行分析,并对植株不同组织及不同胁迫条件下该基因的表达量进行检测。结果显示：TaHDA19的开放阅读框长1560 bp,共编码519个氨基酸;该基因的氨基酸序列存在组蛋白去乙酰化酶（HDAC）家族典型的结构域Hist-deacety1。该基因上游启动子区含有多种响应元件,如：光响应元件I-box和G-box,脱落酸响应元件ABRE和低温响应元件LTR等。‘科农199’TaHDA19基因的表达结果表明：该基因在根、茎、叶片和幼穗中均有表达,其中在叶片中表达量最高;采用脱落酸、氯化钠和PEG分别处理植株0、1、3、6、12、24 h后,基因的表达水平出现差异;在对植株的12个生育时期进行35℃和42℃热胁迫处理1 h后,该基因表达量均出现上调。研究结果表明TaHDA19可能在小麦响应非生物胁迫过程中发挥重要作用。     

Pollen-mediated gene flow in wheat (Triticum aestivum L.) in a semiarid field environment in Spain

Transgenic wheat (Triticum aestivum L.) varieties are being developed and field-tested in various countries. Concerns regarding gene flow from genetically modified (GM) crops to non-GM crops have stimulated research to estimate outcrossing in wheat prior to the release and commercialization of any transgenic cultivars. The aim is to ensure that coexistence of all types of wheat with GM wheat is feasible in accordance with current regulations. The present study describes the result of a field experiment under the semi-arid climate conditions of Madrid, Spain, at two locations (??La Canaleja?? and ??El Encin?? experimental stations) in Madrid over a 3-year period, from 2005 to 2007. The experimental design consisted of a 50?×?50?m wheat pollen source sown with wheat cultivars resistant to the herbicide chlortoluron (??Deganit?? and ??Castan?? respectively) and three susceptible receptor cultivars (??Abental??, ??Altria?? and ??Recital??) sown in replicated 1?×?1?m plots at different distances (0, 1, 3, 5, 10, 20, 40, 80 and 100?m) and four directions. Outcrossing rates were measured as a percentage of herbicide-resistant hybrids using an herbicide-screening assay. Outcrossing was greatest near the pollen source, averaging 0.029?% at 0?m distance at ??La Canaleja?? and 0.337?% at ??El Encin??, both below the 0.9?% European Union regulated threshold, although a maximum outcrossing rate of 3.5?% was detected in one recipient plot. These percentages declined rapidly as the distance increased, but hybrids were detected at different rates at distances of up to 100?m, the maximum distance of the experiment. Environmental conditions, as drought in 2004?C2005 and 2005?C2006, may have influenced the extent of outcrossing. These assays carried out in wheat under semi-arid conditions in Europe provide a more complete assessment of pollen-mediated gene flow in this crop.     

云南小麦(Triticum aestivum ssp yunnanense King)的抗旱性鉴定研究初报

云南小麦(Triticum aestivum ssp yunnanense King)产于云南省澜沧江、怒江下游,为云南省所特有,具有护颖坚硬、极难脱粒、穗轴受压力易折断等原始特性,又有耐瘠薄、抗旱、抗穗发芽、抗鸟害等多种优点.本文研究了云南小麦的苗期和成株期抗旱性.结果表明云南小麦的苗期抗旱性在不同品种间存在较大差异,但均不及云南普通小麦地方品种油榨地光头麦;而澜沧铁壳麦和永德铁壳麦的苗期抗旱性强于育成品种云麦42.云南小麦具有较强的成株期抗干旱能力,不同品种间差异不明显,其中云县铁壳麦超过油榨地光头麦,其它品种与育成品种云麦42接近.     

Molecular Characterization and Expression Analysis of Triticum aestivum Squamosa‐Promoter Binding Protein‐Box Genes Involved in Ear Development

Wheat (Triticum aestivum L.) is one of the most important crops in the world. Squamosa‐promoter binding protein (SBP)‐box genes play a critical role in regulating flower and fruit development. In this study, 10 novel SBP‐box genes (TaSPL genes) were isolated from wheat ((Triticum aestivum L.) cultivar Yanzhan 4110). Phylogenetic analysis classified the TaSPL genes into five groups (G1–G5). The motif combinations and expression patterns of the TaSPL genes varied among the five groups with each having own distinctive characteristics: TaSPL20/21 in G1 and TaSPL17 in G2 mainly expressed in the shoot apical meristem and the young ear, and their expression levels responded to development of the ear; TaSPL6/15 belonging to G3 were upregulated and TaSPL1/23 in G4 were downregulated during grain development; the gene in G5 (TaSPL3) expressed constitutively. Thus, the consistency of the phylogenetic analysis, motif compositions, and expression patterns of the TaSPL genes revealed specific gene structures and functions. On the other hand, the diverse gene structures and different expression patterns suggested that wheat SBP‐box genes have a wide range of functions. The results also suggest a potential role for wheat SBP‐box genes in ear development. This study provides a significant beginning of functional analysis of SBP‐box genes in wheat.     

Allelopathy in wheat (Triticum aestivum)

Wheat (Triticum aestivum) allelopathy has potential for the management of weeds, pests and diseases. Both wheat residue allelopathy and wheat seedling allelopathy can be exploited for managing weeds, including resistant biotypes. Wheat varieties differ in allelopathic potential against weeds, indicating that selection of allelopathic varieties might be a useful strategy in integrated weed management. Several categories of allelochemicals for wheat allelopathy have been identified, namely, phenolic acids, hydroxamic acids and short‐chain fatty acids. Wheat allelopathic activity is genetically controlled and a multigenic model has been proposed. Research is underway to identify genetic markers associated with wheat allelopathy. Once allelopathic genes have been located, a breeding programme could be initiated to transfer the genes into modern varieties for weed suppression. The negative impacts of wheat autotoxicity on agricultural production systems have also been identified when wheat straws are retained on the soil surface for conservation farming purposes. A management package to avoid such deleterious effects is discussed. Wheat allelopathy requires further study in order to maximise its allelopathic potential for the control of weeds, pests and diseases, and to minimise its detrimental effects on the growth of wheat and other crops.