Global transcriptome and gene co-expression network analyses reveal regulatory and non-additive effects of drought and heat stress in grapevine.

Despite frequent co-occurrence of drought and heat stress, the molecular mechanisms governing plant responses to these stresses in combination have not often been studied. This is particularly evident in non-model, perennial plants. We conducted large scale physiological and transcriptome analyses to identify genes and pathways associated with grapevine response to drought and/or heat stress during stress progression and recovery. We identified gene clusters with expression correlated to leaf temperature and water stress and five hub genes for the combined stress co-expression network. Several differentially expressed genes were common to the individual and combined stresses, but the majority were unique to the individual or combined stress treatments. These included heat-shock proteins, mitogen-activated kinases, sugar metabolizing enzymes, and transcription factors, while phenylpropanoid biosynthesis and histone modifying genes were unique to the combined stress treatment. Following physiological recovery, differentially expressed genes were found only in plants under heat stress, both alone and combined with drought. Taken collectively, our results suggest that the effect of the combined stress on physiology and gene expression is more severe than that of individual stresses, but not simply additive, and that epigenetic chromatin modifications may play an important role in grapevine responses to combined drought and heat stress.

GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience.

The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.

Gene body demethylation increases expression and is associated with self-pruning during grape genome duplication.

A colchicine-induced autotetraploid grapevine exhibiting potentially valuable agronomic traits for grape production and breeding, including self-pruning, was identified. This study investigated DNA methylation variation and its role in gene expression during self-pruning in the autotetraploid grapevine. We used RNA-Seq to estimate differentially expressed genes between diploid and autotetraploid grapevine shoot tips. The genes showing increases in the autotetraploid were mainly related to stress response pathways, whereas those showing decreases in the autotetraploid were related to biological metabolism and biosynthesis. Whole-genome bisulfite sequencing was performed to produce single-base methylomes for the diploid and autotetraploid grapevines. Comparison between the methylomes revealed that they were conserved in CG and CHG contexts. In the autotetraploid grapevine, hypodifferentially methylated regions (DMRs) and hyper-DMRs in the gene body increased or decreased gene expression, respectively. Our results indicated that a hypo-DMR in the ACO1 gene body increased its expression and might promote self-pruning. This study reports that hypo-DMRs in the gene body increase gene expression in plants and reveals the mechanism underlying the changes in the modifications affecting gene expression during genome duplication. Overall, our results provide valuable information for understanding the relationships between DNA methylation, gene expression, and autotetraploid breeding in grape.

A whole canopy gas exchange system for the targeted manipulation of grapevine source-sink relations using sub-ambient CO2.

BACKGROUND: Elucidating the effect of source-sink relations on berry composition is of interest for wine grape production as it represents a mechanistic link between yield, photosynthetic capacity and wine quality. However, the specific effects of carbohydrate supply on berry composition are difficult to study in isolation as leaf area or crop adjustments can also change fruit exposure, or lead to compensatory growth or photosynthetic responses. A new experimental system was therefore devised to slow berry sugar accumulation without changing canopy structure or yield. This consisted of six transparent 1.2 m3 chambers to enclose large pot-grown grapevines, and large soda-lime filled scrubbers that reduced carbon dioxide (CO2) concentration of day-time supply air by approximately 200 ppm below ambient. RESULTS: In the first full scale test of the system, the chambers were installed on mature Shiraz grapevines for 14 days from the onset of berry sugar accumulation. Three chambers were run at sub-ambient CO2 for 10 days before returning to ambient. Canopy gas exchange, and juice hexose concentrations were determined. Net CO2 exchange was reduced from 65.2 to 30 g vine- 1 day- 1, or 54%, by the sub-ambient treatment. At the end of the 10 day period, total sugar concentration was reduced from 95 to 77 g L- 1 from an average starting value of 23 g L- 1, representing a 25% reduction. Scaling to a per vine basis, it was estimated that 223 g of berry sugars accumulated under ambient supply compared to 166 g under sub-ambient, an amount equivalent to 50 and 72% of total C assimilated. CONCLUSIONS: Through supply of sub-ambient CO2 using whole canopy gas exchange chambers system, an effective method was developed for reducing photosynthesis and slowing the rate of berry sugar accumulation without modifying yield or leaf area. While in this case developed for further investigations of grape and wine composition, the system has broader applications for the manipulation and of study of grapevine source-sink relations.

Fast Phenomics in Vineyards: Development of GRover, the Grapevine Rover, and LiDAR for Assessing Grapevine Traits in the Field.

This paper introduces GRover (the grapevine rover), an adaptable mobile platform for the deployment and testing of proximal imaging sensors in vineyards for the non-destructive assessment of trunk and cordon volume and pruning weight. A SICK LMS-400 light detection and ranging (LiDAR) radar mounted on GRover was capable of producing precise (±3 mm) 3D point clouds of vine rows. Vineyard scans of the grapevine variety Shiraz grown under different management systems at two separate locations have demonstrated that GRover is able to successfully reproduce a variety of vine structures. Correlations of pruning weight and vine wood (trunk and cordon) volume with LiDAR scans have resulted in high coefficients of determination (R² = 0.91 for pruning weight; 0.76 for wood volume). This is the first time that a LiDAR of this type has been extensively tested in vineyards. Its high scanning rate, eye safe laser and ability to distinguish tissue types make it an appealing option for further development to offer breeders, and potentially growers, quantified measurements of traits that otherwise would be difficult to determine.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Abscisic Acid Down-Regulates Hydraulic Conductance of Grapevine Leaves in Isohydric Genotypes Only.

Plants evolved different strategies to cope with water stress. While isohydric species maintain their midday leaf water potential (ΨM) under soil water deficit by closing their stomata, anisohydric species maintain higher stomatal aperture and exhibit substantial reductions in ΨM It was hypothesized that isohydry is related to a locally higher sensitivity of stomata to the drought-hormone abscisic acid (ABA). Interestingly, recent lines of evidence in Arabidopsis (Arabidopsis thaliana) suggested that stomatal responsiveness is also controlled by an ABA action on leaf water supply upstream from stomata. Here, we tested the possibility in grapevine (Vitis vinifera) that different genotypes ranging from near isohydric to more anisohydric may have different sensitivities in these ABA responses. Measurements on whole plants in drought conditions were combined with assays on detached leaves fed with ABA. Two different methods consistently showed that leaf hydraulic conductance (Kleaf) was down-regulated by exogenous ABA, with strong variations depending on the genotype. Importantly, variation between isohydry and anisohydry correlated with Kleaf sensitivity to ABA, with Kleaf in the most anisohydric genotypes being unresponsive to the hormone. We propose that the observed response of Kleaf to ABA may be part of the overall ABA regulation of leaf water status.

The Synthesis and Accumulation of Resveratrol Are Associated with Veraison and Abscisic Acid Concentration in Beihong (Vitis vinifera × Vitis amurensis) Berry Skin.

Resveratrols are polyphenolic secondary metabolites that can benefit human health, and only occur in a few plant families including Vitaceae. It has been reported that abscisic acid (ABA) can induce veraison (the onset of grape berry ripening) and may induce the accumulation of resveratrol in berry skin. However, the relationships between ABA, veraison, the accumulation of anthocyanins and the accumulation of resveratrol in the berry are poorly understood. This study attempted to answer this question through an investigation of the effect of applied ABA and fluridone (a synthetic inhibitor of ABA) on the biosynthesis and accumulation of ABA, anthocyanin, and resveratrol in Beihong (Vitis vinifera × Vitis amurensis) berry skin. Under natural conditions, resveratrol concentration was very low before 91 DAA (days after anthesis), i.e., 2 weeks after veraison, however, it increased sharply from this point to 126 DAA (maturity). Exogenous ABA applications all resulted in an increase in berry skin ABA and anthocyanin concentration, irrespective of the developmental stage at which the treatment occurred (20 and 10 days pre-veraison, veraison or 7 days post-veraison), thereby advancing veraison. In contrast, resveratrol concentration increased only when ABA was applied at 10 days pre-veraison or at veraison. As a result, the accumulation of resveratrol was associated with veraison in grape berry skin and this accumulation, together with that of anthocyanins, was associated with ABA concentration. The response of resveratrol biosynthesis in the berry skin to manipulation of ABA varied during berry development and was less sensitive to ABA than the response of anthocyanin biosynthesis.

The outcome of patients with low risk gestational trophoblastic neoplasia treated with single agent intramuscular methotrexate and oral folinic acid.

BACKGROUND: Gestational trophoblastic neoplasia (GTN) persisting despite local treatment requires chemotherapy. In 2000, the revised International Federation of Gynaecology and Obstetrics (FIGO)/World Health Organisation (WHO) staging system was introduced, classifying patients as at 'low' or 'high' risk for resistance to single agent treatment. PATIENTS AND METHODS: We have evaluated the complete response rates of patients with low risk GTN treated with 2 weekly intramuscular (IM) methotrexate 50mg four doses days 1, 3, 5, 7 and oral folinic acid 15 mg days 2, 4, 6, 8 (MTX/FA). Patient data between January 2000 and December 2011 were collated and the relationships between FIGO/WHO risk score and outcomes evaluated. RESULTS: Two hundred and eighty nine patients were treated with single agent IM MTX/FA and assessed for treatment response. 29/36 (81%) patients with a FIGO/WHO total score of 6 developed resistance to MTX/FA compared with 87/253 (34%) patients with a score of 0-5 (p ≤ 0.0001). Significantly higher rates of resistance were found for patients with an hCG level of >100,000 iu/l compared to an hCG level of <100,000 iu/l (84% versus 34% p ≤ 0.0001). All patients were eventually cured with chemotherapy or surgical salvage. CONCLUSIONS: Patients with low risk GTN that have a FIGO/WHO score of 6 or hCG level of >100,000 iu/l have high rates of resistance to MTX/FA and require further treatment. Revision of the FIGO/WHO scoring system may be appropriate to enable selection of more effective first line chemotherapy.

The relationship of maternal age to molar pregnancy incidence, risks for chemotherapy and subsequent pregnancy outcome.

The national registration and treatment service for molar pregnancies in the UK allows for the collection of accurate data on this relatively rare diagnosis. In England and Wales, between 2000 and 2009, 5,793 patients with complete moles and 7,790 with partial moles were registered, compared with a total of 8,242,511 conceptions. The overall molar pregnancy incidence was 1 for every 607 conceptions (complete mole 1:1,423; partial mole 1:1,058), but with major variations with age. For complete moles, the risk varied from < 1:1,000 for ages 18-40, to 1:156 for women aged 45 and 1:8 for those aged 50 and above. The overall risk of requiring chemotherapy after a complete mole was 13.6% and 1.1% for partial mole, while the risk of a further molar pregnancy in the next conception was 1:68 but each of these figures have considerable variations with age. These modern statistics on molar pregnancy risks and outcomes should be of value to clinicians and their patients, while discussing this rare diagnosis.

Hyperthyroidism and human chorionic gonadotrophin production in gestational trophoblastic disease.

BACKGROUND: Gestational trophoblastic disease (GTD) is a rare complication of pregnancy, ranging from molar pregnancy to choriocarcinoma. Patients with persistent disease require treatment with chemotherapy. For the vast majority, prognosis is excellent. Occasionally, GTD is complicated by hyperthyroidism, which may require treatment. This is thought to occur due to molecular mimicry between human chorionic gonadotrophin (HCG) and thyroid-stimulating hormone (TSH), and hence cross-reactivity with the TSH receptor. Hyperthyroidism usually resolves as the GTD is successfully treated and correspondingly HCG levels normalise. METHODS: This paper reviews cases of GTD treated over a 5-year period at one of the three UK centres and identifies the prevalence of hyperthyroidism in this population. Four cases with clinical hyperthyroidism are discussed. RESULTS: On review of the 196 patients with gestational trophoblastic neoplasia treated with chemotherapy in Sheffield since 2005, 14 (7%) had biochemical hyperthyroidism. Of these, four had evidence of clinical hyperthyroidism. CONCLUSION: Concomitant biochemical thyroid disease in patients with GTD is relatively common, and measurement of thyroid function in patients with persistent GTD is, therefore, important. The development of hyperthyroidism is largely influenced by the level of HCG and disease burden, and usually settles with treatment of the persistent GTD. However, rarely the thyroid stimulation can have potentially life-threatening consequences.

Changes in nutritional value of cyanogenic trifolium repens grown at elevated atmospheric CO2.

Global food security in a changing climate depends on both the nutritive value of staple crops as well as their yields. Here, we examined the direct effect of atmospheric carbon dioxide on the toxicity of the important pasture crop, Trifolium repens L. (clover). Shoots of T. repens contain cyanogenic glycosides that break down to release toxic hydrogen cyanide when damaged. The ability of animals to tolerate cyanogenic compounds is dependent, in part, on their overall protein intake. We grew T. repens communities at ambient and approximately twice-ambient CO(2) in a controlled environment greenhouse experiment. We found that the ratio of total cyanogenic glycosides to total protein ratio was nearly two times higher in leaves of T. repens grown at elevated CO(2). This study highlights the importance of assessing the nutritive value of this and other plants in response to rising CO(2) so that steps can be taken to address any adverse consequences for herbivores.

Impact of the revised FIGO/WHO system on the management of patients with gestational trophoblastic neoplasia.

OBJECTIVE: To study the effect of a change in disease scoring systems on the management of patients with gestational trophoblastic neoplasia (GTN) in our supra-regional treatment centre. METHODS: We reviewed disease characteristics and treatment outcomes in 632 GTN patients managed at our centre from 1973 to 2006. Two disease scoring systems were used sequentially, the Sheffield modification of the Charing Cross Scoring System (SCCSS) before 2000, and the revised FIGO/modified WHO system (FIGO 2000) thereafter. RESULTS: Using the SCCSS 573 (90.7%) patients were classified as low risk (LR) and 59 (9.3%) as high risk (HR). With FIGO 2000, 587 (92.9%) were LR and 45 (7.1%) HR. For LR patients, the complete response (CR) to first line single agent chemotherapy was 77% before 2000 and 61.6% from 2000 to 2006. For HR patients, the CR rates with first line chemotherapy were 79.5% and 75% respectively. The higher threshold for assigning a patient as HR using FIGO 2000 had an impact on the success of treatment; only 7/19 patients (37%) who were scored 6 by FIGO 2000, and thus treated as LR with methotrexate/folinic acid, achieved a CR. CONCLUSION: In our experience, the revised FIGO/modified WHO scoring system has down scored some patients who would have been considered as high risk with the previous scoring system. A trend to lower CR with first line chemotherapy and an increase in the need for second line chemotherapy was seen.

Nitrogen in cell walls of sclerophyllous leaves accounts for little of the variation in photosynthetic nitrogen-use efficiency.

Photosynthetic rate per unit nitrogen generally declines as leaf mass per unit area (LMA) increases. To determine how much of this decline was associated with allocating a greater proportion of leaf nitrogen into cell wall material, we compared two groups of plants. The first group consisted of two species from each of eight genera, all of which were perennial evergreens growing in the Australian National Botanic Gardens (ANBG). The second group consisted of seven Eucalyptus species growing in a greenhouse. The percentage of leaf biomass in cell walls was independent of variation in LMA within any genus, but varied from 25 to 65% between genera. The nitrogen concentration of cell wall material was 0.4 times leaf nitrogen concentration for all species apart from Eucalyptus, which was 0.6 times leaf nitrogen concentration. Between 10 and 30% of leaf nitrogen was recovered in the cell wall fraction, but this was independent of LMA. No trade-off was observed between nitrogen associated with cell walls and the nitrogen allocated to ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco). Variation in photosynthetic rate per unit nitrogen could not be explained by variation in cell wall nitrogen.

Phosphorus availability and elevated CO2 affect biological nitrogen fixation and nutrient fluxes in a clover-dominated sward.

The response of biological nitrogen fixation (BNF) to elevated CO(2) was examined in white clover (Trifolium repens)-dominated swards under both high and low phosphorus availability. Mixed swards of clover and buffalo grass (Stenotaphrum secundatum) were grown for 15 months in 0.2 m2 sand-filled mesocosms under two CO2 treatments (ambient and twice ambient) and three nutrient treatments [no N, and either low or high P (5 or 134 kg P ha(-1)); the third nutrient treatment was supplied with high P and N (240 kg N ha(-1))]. Under ambient CO2, high P increased BNF from 410 to 900 kg ha(-1). Elevated CO2 further increased BNF to 1180 kg ha(-1) with high P, but there was no effect of CO2 on BNF with low P. Allocation of N belowground increased by approx. 50% under elevated CO2 irrespective of supplied P. The results suggest that where soil P availability is low, elevated CO2 will not increase BNF, and pasture quality could decrease because of a reduction in aboveground N.

The role of repeat uterine evacuation in the management of persistent gestational trophoblastic disease.

OBJECTIVE: To evaluate the role of second (and third) uterine evacuation in the management of persistent gestational trophoblastic disease (GTD). METHODS: This was an observational study of all cases registered over a 10-year period at the Trophoblastic Disease Centre at Weston Park Hospital, Sheffield. Five hundred and forty-four of 4050 women registered during 1991-2000 underwent a second uterine evacuation following a presumptive diagnosis of persistent GTD. The reason for evacuation, hCG level prior to the procedure, histological appearances of evacuated products and the clinical outcome (in terms of the need for chemotherapy) were determined. RESULTS: After a second uterine evacuation 368 patients (68%) completed the follow-up programme without further evidence of persistent disease or need for chemotherapy. If the diagnosis of persistent GTD was confirmed solely on the basis of elevated hCG levels then 171 of 282 (60%) patients did not require chemotherapy. Chemotherapy was more likely where there was histological evidence of persistent trophoblastic disease and where the urinary hCG was >1500 IU/L at the time of the repeat evacuation. Twenty-eight of 60 patients (46%) undergoing a third evacuation required chemotherapy. CONCLUSION: Second uterine evacuation can be a useful therapeutic option for patients with presumed persistent trophoblastic disease not mandating immediate chemotherapy, particularly where the hCG level is <1500 IU/L. Patients with documented persistent trophoblastic disease on histological examination of the second evacuation sample are more likely to require chemotherapy. Third evacuation is not now recommended.

Is there a relationship between treatment for infertility and gestational trophoblastic disease?

BACKGROUND: The aim of the study was to record the incidence of treatment for infertility prior to development of gestational trophoblastic disease (GTD). METHODS AND RESULTS: A retrospective analysis was undertaken of 231 consecutive women receiving chemotherapy for persistent GTD at Weston Park Hospital, Sheffield, from 1991 to 2001. Three patients in this group had received treatment for infertility prior to their molar pregnancy. In a control group of 226 patients not requiring treatment for persistent GTD, four had had treatment for infertility just before their molar pregnancy, and in a further control group of 208 'normal' pregnancies, eight patients had had treatment for infertility prior to conception. CONCLUSION: We conclude that we can demonstrate no relationship between infertility treatment and subsequent development of GTD.

Low-risk persistent gestational trophoblastic disease treated with low-dose methotrexate: efficacy, acute and long-term effects.

The aim of this study was to evaluate the efficacy and toxicity of low-dose methotrexate with folinic acid rescue in a large series of consecutively treated patients with low-risk persistent gestational trophoblastic disease. Between January 1987 and December 2000, 250 patients were treated with intramuscular methotrexate (50 mg on alternate days 1, 3, 5, 7) with folinic acid (7.5 mg orally on alternate days 2, 4, 6, 8) rescue. The overall complete response rate without recurrence was 72% for first-line treatment and 95% for those who required second-line chemotherapy. Eight women (3.2%) had recurrence following remission and two (0.8%) had new moles. Two women (0.8%) died of their disease giving an overall cure of 99%. Only 10 women (4%) experienced grade III/IV toxicity during the first course of treatment and 13 women (5.2%) subsequently. Toxicity included mucositis and stomatitis, pleuritic chest pain, thrombocytopenia, uterine bleeding, abdominal pain, liver function changes, rash and pericardial effusion. A total of 59 women (23.6%) required second-line chemotherapy; 48 women had methotrexate resistance, eight had methotrexate toxicity and an empirical decision to change therapy was made in three. In all, 11 women (4.4%) had a hysterectomy before, during or after treatment; 141 women (56.4%) became pregnant following treatment: in 128 (90.7%), the outcome was successful. Methotrexate with folinic acid rescue is an effective treatment for low-risk persistent trophoblastic disease. It has minimal severe toxicity, excellent cure rates and does not appear to affect fertility.

Gestational trophoblastic disease in the Asian population of Northern England and North Wales.

OBJECTIVE: To determine the incidence and trends of gestational trophoblastic disease in the Asian population of Northern England and North Wales.A prospective observational study. SETTING: Trophoblastic Screening and Treatment Centre, Weston Park Hospital. POPULATION: A total of 3660 women registered with gestational trophoblastic disease between 1991 and 1999. MAIN OUTCOME MEASURES: 1. The incidence of gestational trophoblastic disease in Asian and non-Asian population. 2. Trend in incidence over study period. RESULTS: Three hundred and twenty-two Asian patients were registered. The incidence of gestational trophoblastic disease in the northern part of England and Wales averaged 1 per 714 live births. The incidence of gestational trophoblastic disease in the Asian population was 1.95 times higher than in the non-Asian population (1 per 387 live births vs 1 per 752 live births). There was an excess of molar pregnancies in the extreme maternal age groups; the incidence in these women was twice as high as in the whole reproductive cohort. The ratio of partial to complete hydatidiform mole increased from 0.9 in the lower age to 2.6 in the older age group. There appeared to be a slowly rising trend in the incidence of gestational trophoblastic disease; the increase was higher in the Asian than in the non-Asian population. The ratio of partial to complete moles increased with age in both populations. CONCLUSION: Asian women are at increased risk of having molar pregnancies. Molar pregnancies are more common at the extremes of reproductive age. The setting up of regional or national registration centres has helped to provide more accurate estimates of the true incidence of the disease.