Recent allopolyploidy alters Spartina microRNA expression in response to xenobiotic-induced stress.

Environmental contamination by xenobiotics represents a major threat for natural ecosystems and public health. In response, xenobiotic detoxification is a fundamental trait of organisms for developmental plasticity and stress tolerance, but the underlying molecular mechanisms remain poorly understood in plants. To decipher this process, we explored the consequences of allopolyploidy on xenobiotic tolerance in the genus Spartina Schreb. Specifically, we focused on microRNAs (miRNAs) owing to their central function in the regulation of gene expression patterns, including responses to stress. Small RNA-Seq was conducted on the parents S. alterniflora and S. maritima, their F1 hybrid S. x townsendii and the allopolyploid S. anglica under phenanthrene-induced stress (phe), a model Polycyclic Aromatic Hydrocarbon (PAH) compound. Differentially expressed miRNAs in response to phe were specifically identified within species. In complement, the respective impacts of hybridization and genome doubling were detected, through changes in miRNA expression patterns between S. x townsendii, S. anglica and the parents. The results support the impact of allopolyploidy in miRNA-guided regulation of plant response to phe. In total, we identified 17 phe-responsive miRNAs in Spartina among up-regulated MIR156 and down-regulated MIR159. We also describe novel phe-responsive miRNAs as putative Spartina-specific gene expression regulators in response to stress. Functional validation using Arabidopsis (L.) Heynh. T-DNA lines inserted in homologous MIR genes was performed, and the divergence of phe-responsive miRNA regulatory networks between Arabidopsis and Spartina was discussed.

Complete Nucleotide Sequence of Australian Tomato spotted wilt virus Isolate TSWV-QLD2.

The complete nucleotide (nt) sequence of an Australian isolate of Tomato spotted wilt virus was determined by deep RNA sequencing and deep small RNA sequencing. The tripartite genome consists of an 8,914-nt L segment, a 4,851-nt M segment, and a 2,987-nt S segment.

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences.

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.

Complete Nucleotide Sequence of an Australian Isolate of Turnip mosaic virus before and after Seven Years of Serial Passaging.

The complete genome sequence of an Australian isolate of Turnip mosaic virus was determined by Sanger sequencing. After seven years of serial passaging by mechanical inoculation, the isolate was resequenced by RNA sequencing (RNA-Seq). Eighteen single nucleotide polymorphisms were identified between the isolates. Both isolates had 96% identity to isolate AUST10.

The pineapple AcMADS1 promoter confers high level expression in tomato and Arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant.

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

BACKGROUND: Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. RESULTS: The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. CONCLUSIONS: We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Diversity of sequences and expression patterns among alleles of a sugarcane loading stem gene.

Modern sugarcane cultivars are highly polyploid and aneuploid hybrids, which are propagated as clones. Their complex genome structure comprises 100-130 chromosomes and 10-13 hom(e)ologous copies of most loci. There is preliminary evidence of very high heterozygosity, with implications for genetic improvement approaches ranging from marker-assisted selection to transgenics. Here, we report that sugarcane cultivar Q200 has at least nine alleles at the Loading Stem Gene (ScLSG) locus. Exon-intron structure is identical and the predicted protein products show at least 92 % identity, across sugarcane alleles and the Sorghum homologue Sb07g027880. There is substantial variation in the 5' UTR and promoter regions including numerous allele-specific nucleotide polymorphisms, insertions and deletions. We developed an allele-specific qRT-PCR method to undertake the first compelling test of allele-specific expression in polyploid sugarcane. Seven alleles distinguished by this method all showed peak expression in the sucrose-loading zone of the stem, but there was apparent variability in expression patterns across other tissues. The ScLSG2 and ScLSG5 alleles appear promising for specificity of expression in stems, relative to leaf, meristem, emerging shoot and root tissues. Within the stem, there was activity in parenchyma, vascular and rind tissues. This expression pattern is of interest in basic research and biotechnology aimed at enhanced sucrose content, engineering value-added products, and manipulation of stem biomass composition.

Sugarcane Loading Stem Gene promoters drive transgene expression preferentially in the stem.

Promoter regions of six sugarcane Loading Stem Gene (ScLSG) alleles were analyzed using bioinformatic and transgenic approaches. Stable transgene expression analyses, on multiple independent lines per construct, revealed differences between ScLSG promoters in absolute levels and in tissue-selectivity of luciferase reporter activity. Four promoters drove peak expression in the sucrose-loading zone and maintained substantial expression throughout mature stems. One drove a pattern of gradual increase along the stem maturation profile. In general, stem: root expression ratio increased with plant age. The ScLSG5 promoter had the fewest light-enhanced and root-expression motifs in bioinformatic analysis, and drove the highest level and specificity of transgene expression in stems. This indicates the potential to further improve the stem specificity of ScLSG promoter sequences by eliminating enhancers of expression in other tissues. An intron in the 5'UTR was important for expression strength. The ScLSG promoters will be useful for research and biotechnology in sugarcane, where the tailored expression of transgenes in stems is important for enhanced accumulation of sugar or value-added products, and for development as a bioenergy feedstock.

Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.

Microarray analysis of gene expression profiles in ripening pineapple fruits.

BACKGROUND: Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. RESULTS: Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. CONCLUSIONS: This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Reproduction in male swamp wallabies (Wallabia bicolor): puberty and the effects of season.

This study describes pubertal changes in testes and epididymides and seasonal changes in the adult male reproductive organs and plasma androgen concentrations of the swamp wallaby (Wallabia bicolor). Pre-pubescent males had testes with solid seminiferous cords and spermatogenesis only to the stage of gonocytes. Their epididymides had empty lumina along their entire length. The testes of three males undergoing puberty had some lumen formation and mitotic activity. Their epididymides were similar in appearance to those of adult males but were entirely devoid of any cells within the lumen of the duct. Three other pubescent males showed full lumen formation in the testes and spermatogenesis up to the elongating spermatid stage. Their epididymides were similar in appearance to those of adult males but with no spermatozoa in the duct. However, cells of testicular origin were found in the lumen of the duct in all regions suggesting that testicular fluids and immature germ cells shed into the rete testes flow through the seminiferous tubules into the epididymis before the release of mature testicular spermatozoa. The weights of testes and epididymides of adult males showed no change throughout the year but prostate weight and plasma androgen concentrations varied significantly with season, with maximums in spring and summer and minimums in winter. The volume fraction of Leydig cells and seminiferous tubules was significantly lower in winter than in summer; but, despite this, maturing spermatozoa were found in the testes throughout the year. Females in the area conceived year-round, suggesting that seasonal changes in the male reproductive tract did not prevent at least some males from breeding throughout the year.

Reproduction in female swamp wallabies, Wallabia bicolor.

The swamp wallaby (Wallabia bicolor) is a common, medium-sized, browsing macropodid marsupial that is unique in many ways. Relatively little is known about the reproductive biology of this species. Previous studies have proposed that the swamp wallaby has a pre-partum oestrus because the gestation period (x = 35.5 days, n = 4) is on average longer than the oestrus period (x = 31.0 days, n = 5) and the period from the removal of pouch young (RPY) to mating (x = 26.0 days, n = 3). In the current study, the period from RPY to birth was confirmed at x = 31.25 days (n = 4) in captive animals, consistent with a pre-partum oestrus. A growth curve for swamp wallaby pouch young was constructed from the progeny of captive animals to estimate the age and date of birth of young in a wild, culled population in South Gippsland, Victoria, and the reproduction of females in the wild throughout the year was examined. Young were born in every month of the year, with no statistically significant variation in the number of young born in each month. Females did not have a period of seasonal anoestrus and conceived throughout the year. Female swamp wallabies in South Gippsland bred continuously throughout the period of this study.

PineappleDB: an online pineapple bioinformatics resource.

BACKGROUND: A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences. DESCRIPTION: PineappleDB is an online, curated database providing integrated access to annotated expressed sequence tag (EST) data for cDNA clones isolated from pineapple fruit, root, and nematode infected root gall vascular cylinder tissues. The database currently houses over 5600 EST sequences, 3383 contig consensus sequences, and associated bioinformatic data including splice variants, Arabidopsis homologues, both MIPS based and Gene Ontology functional classifications, and clone distributions. The online resource can be searched by text or by BLAST sequence homology. The data outputs provide comprehensive sequence, bioinformatic and functional classification information. CONCLUSION: The online pineapple bioinformatic resource provides the research community with access to pineapple fruit and root/gall sequence and bioinformatic data in a user-friendly format. The search tools enable efficient data mining and present a wide spectrum of bioinformatic and functional classification information. PineappleDB will be of broad appeal to researchers investigating pineapple genetics, non-climacteric fruit ripening, root-knot nematode infection, crassulacean acid metabolism and alternative RNA splicing in plants.

Analysis of the asparagus (Asparagus officinalis) asparagine synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate Suc-repression.

In asparagus (Asparagus officinalis L.), increased levels of asparagine (Asn) and Asn synthetase (AS) transcript are detected during foliar senescence and in harvested spears, possibly triggered by signals from a reduced supply of carbohydrate. To identify cis-elements mediating this regulation, the asparagus AS gene promoter was isolated and analysed by DNA sequencing, followed by expression of AS::GUS (ß-glucuronidase) reporter-gene constructs in transgenic tissue, and electrophoretic mobility shift assays (EMSA). The 1958-base pair (bp) region of the AS promoter upstream of the translation initiation ATG (-1958 bp region) was sufficient to confer sucrose (Suc)-regulated expression on the GUS reporter gene in asparagus callus and protoplasts, which were transformed by particle bombardment and electroporation, respectively. Removal of Suc from callus or protoplast media resulted in the induction of GUS activity. Deletion analysis of this 1958-bp fragment identified elements in the -640 to -266bp region as important for both high GUS levels and mediating the Suc response. This was supported by EMSA results, which showed the formation of three nuclear protein-DNA complexes with the -558 to -284 bp fragment of the promoter. A 20-bp oligonucleotide, designed to match the sequence from -423 to -404 bp, was able to out-compete formation of one of these protein-DNA complexes, suggesting a specific interaction with this sequence. This region of the promoter, overlapping with the 20-bp oligonucleotide sequence, contains a 10-bp stretch identical to a sequence previously shown to mediate low Suc induction of an Oryza sativa (rice) α-amylase gene, and may thus represent a conserved Suc-responsive element.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals.

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the -1958 bp AS promoter linked to the ß-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in -1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than -405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least -640 bp of the AS promoter but not those with -523 bp or smaller promoter fragments. Fusion of the -640 to -523 bp region to a -381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

Expression analysis of four Pinus radiata male cone promoters in the heterologous host Arabidopsis.

Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the beta-glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils.

Maternal regulation of milk composition, milk production, and pouch young development during lactation in the tammar wallaby (Macropus eugenii ).

Specific changes in milk composition during lactation in the tammar wallaby (Macropus eugenii) were correlated with the ages of the developing pouch young (PY). The present experiment was designed to test the hypothesis that the sucking pattern of the PY determines the course of mammary development in the tammar wallaby. To test this hypothesis, groups of 60-day-old PY were fostered repeatedly onto one group of host mothers so that a constant sucking stimulus on the mammary gland was maintained for 56 days to allow the lactational stage to progress 42 days ahead of the age of the young. Analysis of the milk in fostered and control groups showed the timing of changes in the concentration of protein and carbohydrate were essentially unaffected by altering the sucking regime. The only change in milk protein secretion was a small delay in the timing of down-regulation of the secretion of whey acidic protein and early lactation protein in the host tammars. In addition, the rates of growth and development of the foster PY were significantly increased relative to those of the control PY because of ingesting more milk with a higher energy content and different composition than normal for their age. The present study demonstrates that the lactating tammar wallaby regulates both milk composition and the rate of milk production and that these determine the rates of PY growth and development, irrespective of the age of the PY.