Elevated transcription of transposable elements is accompanied by het-siRNA-driven de novo DNA methylation in grapevine embryogenic callus.

BACKGROUND: Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific ('terroir') differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue. RESULTS: We found that compared with leaf tissue, grapevine embryogenic callus cultures accumulate relatively high genome-wide CHH methylation, particularly across heterochromatic regions. This de novo methylation is associated with an abundance of transcripts from highly replicated TE families, as well as corresponding 24 nt heterochromatic siRNAs. Methylation in the TE-specific CHG context was relatively low over TEs located within genes, and the expression of TE loci within genes was highly correlated with the expression of those genes. CONCLUSIONS: This multi-'omics analysis of grapevine embryogenic callus in comparison with leaf tissues reveals a high level of genome-wide transcription of TEs accompanied by RNA-dependent DNA methylation of these sequences in trans. This provides insight into the genomic conditions underlying somaclonal variation and epiallele formation in plants regenerated from embryogenic cultures, which is an important consideration when using these tissues for plant propagation and genetic improvement.

From UVR8 to flavonol synthase: UV-B-induced gene expression in Sauvignon blanc grape berry.

The aim of this research was to determine the effect of development and UV-B on flavonols and the regulation of gene activity in Vitis vinifera L. var. Sauvignon blanc grapes. Particular emphasis was placed on gene activity associated with the low and high fluence UV-B responses. Flavonols, particularly quercetin and kaempferol glycosides, increased substantially upon fruit exposure due to UV-B, with spatial analysis locating the changes to the berry skin. Of five VvFLS genes in grapes, two (VvFLS4 and 5) were found to be transcriptionally active, with VvFLS4 also being responsive to UV-B but VvFLS5 was not. Of the transcription factors known to regulate FLS (VvMYB12, VvMYCA1 and VvWDRs), only VvMYB12 was found to be responsive to UV-B. A number of candidate genes associated with the low and high UV-B fluence responses were also studied (VvUVR8, VvHY5, VvCOP1 and VvCHS; PR genes and VvMAPK3; respectively). The genes associated with the low fluence response exhibited transcriptional regulation in line with reports from other species, while the PR genes and VvMAPK3 only appeared to be responsive in a high UV-B fluence environment. Together, these data supports the view flavonol biosynthesis in grape is stimulated predominantly through the low fluence UV-B response pathway.

The effects of cane girdling before budbreak on shoot growth, leaf area and carbohydrate content of Vitis vinifera L. Sauvignon Blanc grapevines.

The influence of restricting available carbohydrates (CHOs) on shoot growth was studied by cane girdling field grown Vitis vinifera L. Sauvignon Blanc grapevines before budbreak. Canes were girdled 5, 10 or 20cm from the terminal bud of the cane, and the shoot growth of the terminal bud was monitored over the course of a single growing season. A linear relationship was found between the initial rate of shoot growth and the amount of cane isolated by the girdle. A decrease in available CHOs during initial shoot growth appeared to inhibit the shoot's ability to produce new vegetative nodes past the point of discontinuity, resulting in a decrease in total leaf area due to incomplete leaf expansion. The transition from the vine's dependence on reserve CHOs to a net positive state appeared to occur when shoot growth reached a steady state. In the case of severe CHO restriction, no lateral growth occurred, suggesting the CHO status in the vine may play a role in lateral bud growth. The cross-sectional area of canes or shoots were shown to have a linear relationship to their CHO content, which allows for an estimation of the amount of CHOs required to obtain growth similar to the control treatment. Additionally, main shoot leaf area can be used to predict total CHO content in the shoot at harvest.

Ammonia-oxidizing bacteria and archaea grow under contrasting soil nitrogen conditions.

Nitrification is a key process of the nitrogen (N) cycle in soil with major environmental implications. The recent discovery of ammonia-oxidizing archaea (AOA) questions the traditional assumption of the dominant role of ammonia-oxidizing bacteria (AOB) in nitrification. We investigated AOB and AOA growth and nitrification rate in two different layers of three grassland soils treated with animal urine substrate and a nitrification inhibitor [dicyandiamide (DCD)]. We show that AOB were more abundant in the topsoils than in the subsoils, whereas AOA were more abundant in one of the subsoils. AOB grew substantially when supplied with a high dose of urine substrate, whereas AOA only grew in the Controls without the urine-N substrate. AOB growth and the amoA gene transcription activity were significantly inhibited by DCD. Nitrification rates were much higher in the topsoils than in the subsoils and were significantly related to AOB abundance, but not to AOA abundance. These results suggest that AOB and AOA prefer different soil N conditions to grow: AOB under high ammonia (NH(3)) substrate and AOA under low NH(3) substrate conditions.

Altering expression of the flavonoid 3'-hydroxylase gene modified flavonol ratios and pollen germination in transgenic Mitchell petunia plants.

Antisense technology was successfully used to reduce flavonoid 3'-hydroxylase (F3'H) gene expression and enzyme activity and to promote the accumulation of monohydroxylated flavonols in petunia flower tissue. The hydroxylation pattern of specific flavonoid groups is a target for modification because of the possible associated changes in a range of factors including colour, stress tolerance and reproductive viability. Petunia (cv. Mitchell) plants were transformed to express in the antisense orientation the sequences encoding the F3'H (asF3'H). Transformants showed a range of responses, in terms of the level of endogenous F3'H gene expression and the relative proportion of the monohydroxylated flavonol (kaempferol) glycosides that accumulated. Kaempferol glycosides increased from 7% of the total flavonols in flower limb tissue of the wild type plants, to 45% in the flower limb tissue of line 114, the transgenic line that also showed the greatest decrease in F3'H expression in flower tissue. In leaf tissue, the trend was for a decrease in total flavonol concentration, with the relative proportion of kaempferol glycosides varying from ~40 to 80% of the total flavonols. The changes in leaf tissue were not consistent with the changes observed in flower tissue of the same lines. Endogenous F3'H activity in flower limb tissue was not completely shut down, although an 80% decrease in enzyme activity was recorded for line 114. The residual F3'H activity was still sufficient that quercetin glycosides remained as the major flavonol form. Alteration of F3'H activity appears to have affected overall flavonoid biosynthesis. A decrease in total flavonol concentration was observed in leaf tissue and two other flavonoid biosynthetic genes were down-regulated. No morphological changes were observed in the transgenic plants; however, up to a 60% decrease in pollen germination was observed in line 13. Thus, the relatively small change in flavonoid biosynthesis induced by the asF3'H transgene, correlated with several other effects beyond just the specific biosynthetic step regulated by this enzyme.

Flavonoid gene expression and UV photoprotection in transgenic and mutant Petunia leaves.

The effects of UVB radiation on plant growth rate, gene expression and flavonoid content in wild-type, and in transgenic and mutant F3'H deficient Petunia lines have been studied for the first time. In wild-type Petunia, UVB induced an increase in total levels of flavonols and this was due to an up-regulation of several genes in the phenylpropanoid pathway. Furthermore, UVB induced a higher rate of production of dihydroxylated flavonols than mono-hydroxylated equivalents. Thus, the ratio of quercetin (ortho-dihydroxylated) to kaempferol (monohydroxylated) increased. In the F3H deficient mutant line, increasing UVB resulted in up-regulation of all of the basic flavonoid biosynthetic genes. Total flavonoids increased to levels significantly higher than in control plants, and the predominant flavonoid was kaempferol. The leaves of these plants grew at a significantly slower rate than comparably treated wild-type plants under ambient or enhanced UVB radiation. This suggests that the predominance of quercetin in the wild-type confers a protective advantage that is not matched in the mutant, even with higher overall flavonoid levels. In contrast, the antisense F3H construct produced an unexpected down-regulation of C4H, CHS and CHI transcription. This resulted in lower total flavonoid production in these plants. The growth rate of these plants was not impaired in UVB to a statistically significant extent, and the Q:K ratio did not change with increasing UVB radiation. This investigation has established a likely correlation between the effect of UVB on plant growth rate, the level of activity of the F3'H gene, and the proposed photoprotection afforded by an increased Q:K ratio.