PhenoWell®-A novel screening system for soil-grown plants.

As agricultural production is reaching its limits regarding outputs and land use, the need to further improve crop yield is greater than ever. The limited translatability from in vitro lab results into more natural growth conditions in soil remains problematic. Although considerable progress has been made in developing soil-growth assays to tackle this bottleneck, the majority of these assays use pots or whole trays, making them not only space- and resource-intensive, but also hampering the individual treatment of plants. Therefore, we developed a flexible and compact screening system named PhenoWell® in which individual seedlings are grown in wells filled with soil allowing single-plant treatments. The system makes use of an automated image-analysis pipeline that extracts multiple growth parameters from individual seedlings over time, including projected rosette area, relative growth rate, compactness, and stockiness. Macronutrient, hormone, salt, osmotic, and drought stress treatments were tested in the PhenoWell® system. The system is also optimized for maize with results that are consistent with Arabidopsis while different in amplitude. We conclude that the PhenoWell® system enables a high-throughput, precise, and uniform application of a small amount of solution to individually soil-grown plants, which increases the replicability and reduces variability and compound usage.

Drought affects the rate and duration of organ growth but not inter-organ growth coordination.

Drought at flowering and grain filling greatly reduces maize (Zea mays) yield. Climate change is causing earlier and longer-lasting periods of drought, which affect the growth of multiple maize organs throughout development. To study how long periods of water deficit impact the dynamic nature of growth, and to determine how these relate to reproductive drought, we employed a high-throughput phenotyping platform featuring precise irrigation, imaging systems, and image-based biomass estimations. Prolonged drought resulted in a reduction of growth rate of individual organs-though an extension of growth duration partially compensated for this-culminating in lower biomass and delayed flowering. However, long periods of drought did not affect the highly organized succession of maximal growth rates of the distinct organs, i.e. leaves, stems, and ears. Two drought treatments negatively affected distinct seed yield components: Prolonged drought mainly reduced the number of spikelets, and drought during the reproductive period increased the anthesis-silking interval. The identification of these divergent biomass and yield components, which were affected by the shift in duration and intensity of drought, will facilitate trait-specific breeding toward future climate-resilient crops.

Nocturnal gibberellin biosynthesis is carbon dependent and adjusts leaf expansion rates to variable conditions.

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA4 levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA4 by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

cis-Cinnamic acid is a natural plant growth-promoting compound.

Agrochemicals provide vast potential to improve plant productivity, because they are easy to implement at low cost while not being restricted by species barriers as compared with breeding strategies. Despite the general interest, only a few compounds with growth-promoting activity have been described so far. Here, we add cis-cinnamic acid (c-CA) to the small portfolio of existing plant growth stimulators. When applied at low micromolar concentrations to Arabidopsis roots, c-CA stimulates both cell division and cell expansion in leaves. Our data support a model explaining the increase in shoot biomass as the consequence of a larger root system, which allows the plant to explore larger areas for resources. The requirement of the cis-configuration for the growth-promoting activity of CA was validated by implementing stable structural analogs of both cis- and trans-CA in this study. In a complementary approach, we used specific light conditions to prevent cis/trans-isomerization of CA during the experiment. In both cases, the cis-form stimulated plant growth, whereas the trans-form was inactive. Based on these data, we conclude that c-CA is an appealing lead compound representing a novel class of growth-promoting agrochemicals. Unraveling the underlying molecular mechanism could lead to the development of innovative strategies for boosting plant biomass.

Drought resistance is mediated by divergent strategies in closely related Brassicaceae.

Droughts cause severe crop losses worldwide and climate change is projected to increase their prevalence in the future. Similar to the situation for many crops, the reference plant Arabidopsis thaliana (Ath) is considered drought-sensitive, whereas, as we demonstrate, its close relatives Arabidopsis lyrata (Aly) and Eutrema salsugineum (Esa) are drought-resistant. To understand the molecular basis for this plasticity we conducted a deep phenotypic, biochemical and transcriptomic comparison using developmentally matched plants. We demonstrate that Aly responds most sensitively to decreasing water availability with early growth reduction, metabolic adaptations and signaling network rewiring. By contrast, Esa is in a constantly prepared mode as evidenced by high basal proline levels, ABA signaling transcripts and late growth responses. The stress-sensitive Ath responds later than Aly and earlier than Esa, although its responses tend to be more extreme. All species detect water scarcity with similar sensitivity; response differences are encoded in downstream signaling and response networks. Moreover, several signaling genes expressed at higher basal levels in both Aly and Esa have been shown to increase water-use efficiency and drought resistance when overexpressed in Ath. Our data demonstrate contrasting strategies of closely related Brassicaceae to achieve drought resistance.

Functional analysis of Arabidopsis and maize transgenic lines overexpressing the ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

The role of HEXOKINASE1 in Arabidopsis leaf growth.

KEY MESSAGE: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine ß-synthase domains, as an interacting protein of HXK1.

Author Correction: The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis.

This corrects the article DOI: 10.1038/ncomms15235.

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis.

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes.

Strobilurins as growth-promoting compounds: how Stroby regulates Arabidopsis leaf growth.

Strobilurins are an important class of agrochemical fungicides used throughout the world on a wide variety of crops as protection against fungal pathogens. In addition to this protective role, they are reported to also positively influence plant physiology. In this study, we analysed the effect of Stroby® WG, a commercially available fungicide consisting of 50% (w/w) kresoxim-methyl (KM) as active strobilurin compound, on Arabidopsis leaf growth. Treatment of seedlings with Stroby resulted in larger leaves due to an increase in cell number. Transcriptome analysis of Stroby-treated rosettes demonstrated an increased expression of genes involved in redox homeostasis, iron metabolism and sugar transport. Stroby treatment strongly induced the expression of the subgroup Ib basic helix-loop-helix (bHLH) transcription factors, which have a role in iron homeostasis under iron-limiting conditions. Single loss-of-function mutants of three bHLHs and their triple bhlh039, bhlh100 and bhlh101 mutant did not respond to Stroby treatment. Although iron and sucrose content was not affected, nitric oxide (NO) levels and nitrate reductase (NR) activity were significantly increased in Stroby-treated rosettes as compared with control plants. In conclusion, we suggest that the Stroby-mediated effects on growth depend on the increased expression of the subgroup Ib bHLHs and higher NO levels.

Unlocking the potential of plant phenotyping data through integration and data-driven approaches.

Plant phenotyping has emerged as a comprehensive field of research as the result of significant advancements in the application of imaging sensors for high-throughput data collection. The flip side is the risk of drowning in the massive amounts of data generated by automated phenotyping systems. Currently, the major challenge lies in data management, on the level of data annotation and proper metadata collection, and in progressing towards synergism across data collection and analyses. Progress in data analyses includes efforts towards the integration of phenotypic and -omics data resources for bridging the phenotype-genotype gap and obtaining in-depth insights into fundamental plant processes.

Natural Variation of Molecular and Morphological Gibberellin Responses.

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling.

Leaf Growth Response to Mild Drought: Natural Variation in Arabidopsis Sheds Light on Trait Architecture.

Plant growth and crop yield are negatively affected by a reduction in water availability. However, a clear understanding of how growth is regulated under nonlethal drought conditions is lacking. Recent advances in genomics, phenomics, and transcriptomics allow in-depth analysis of natural variation. In this study, we conducted a detailed screening of leaf growth responses to mild drought in a worldwide collection of Arabidopsis thaliana accessions. The genetic architecture of the growth responses upon mild drought was investigated by subjecting the different leaf growth phenotypes to genome-wide association mapping and by characterizing the transcriptome of young developing leaves. Although no major effect locus was found to be associated with growth in mild drought, the transcriptome analysis delivered further insight into the natural variation of transcriptional responses to mild drought in a specific tissue. Coexpression analysis indicated the presence of gene clusters that co-vary over different genetic backgrounds, among others a cluster of genes with important regulatory functions in the growth response to osmotic stress. It was found that the occurrence of a mild drought stress response in leaves can be inferred with high accuracy across accessions based on the expression profile of 283 genes. A genome-wide association study on the expression data revealed that trans regulation seems to be more important than cis regulation in the transcriptional response to environmental perturbations.

Chloroplasts Are Central Players in Sugar-Induced Leaf Growth.

Leaves are the plant's powerhouses, providing energy for all organs through sugar production during photosynthesis. However, sugars serve not only as a metabolic energy source for sink tissues but also as signaling molecules, affecting gene expression through conserved signaling pathways to regulate plant growth and development. Here, we describe an in vitro experimental assay, allowing one to alter the sucrose (Suc) availability during early Arabidopsis (Arabidopsis thaliana) leaf development, with the aim to identify the affected cellular and molecular processes. The transfer of seedlings to Suc-containing medium showed a profound effect on leaf growth by stimulating cell proliferation and postponing the transition to cell expansion. Furthermore, rapidly after transfer to Suc, mesophyll cells contained fewer and smaller plastids, which are irregular in shape and contain fewer starch granules compared with control mesophyll cells. Short-term transcriptional responses after transfer to Suc revealed the repression of well-known sugar-responsive genes and multiple genes encoded by the plastid, on the one hand, and up-regulation of a GLUCOSE-6-PHOSPHATE TRANSPORTER (GPT2), on the other hand. Mutant gpt2 seedlings showed no stimulation of cell proliferation and no repression of chloroplast-encoded transcripts when transferred to Suc, suggesting that GPT2 plays a critical role in the Suc-mediated effects on early leaf growth. Our findings, therefore, suggest that induction of GPT2 expression by Suc increases the import of glucose-6-phosphate into the plastids that would repress chloroplast-encoded transcripts, restricting chloroplast differentiation. Retrograde signaling from the plastids would then delay the transition to cell expansion and stimulate cell proliferation.

Measurement of plant growth in view of an integrative analysis of regulatory networks.

As the regulatory networks of growth at the cellular level are elucidated at a fast pace, their complexity is not reduced; on the contrary, the tissue, organ and even whole-plant level affect cell proliferation and expansion by means of development-induced and environment-induced signaling events in growth regulatory processes. Measurement of growth across different levels aids in gaining a mechanistic understanding of growth, and in defining the spatial and temporal resolution of sampling strategies for molecular analyses in the model Arabidopsis thaliana and increasingly also in crop species. The latter claim their place at the forefront of plant research, since global issues and future needs drive the translation from laboratory model-acquired knowledge of growth processes to improvements in crop productivity in field conditions.

Cost saving potential in cardiovascular hospital costs due to reduction in air pollution.

OBJECTIVE: We describe a methodological framework to estimate potential cost savings in Belgium for a decrease in cardiovascular emergency admissions (ischemic heart disease (IHD), heart rhythm disturbances (HRD), and heart failure) due to a reduction in air pollution. METHODS: Hospital discharge data on emergency admissions from an academic hospital were used to identify cases, derive risk functions, and estimate hospital costs. Risk functions were derived with case-crossover analyses with weekly average PM10, PM2.5, and NO2 exposures. The risk functions were subsequently used in a micro-costing analysis approach. Annual hospital cost savings for Belgium were estimated for two scenarios on the decrease of air pollution: 1) 10% reduction in each of the pollutants and 2) reduction towards annual WHO guidelines. RESULTS: Emergency admissions for IHD and HRD were significantly associated with PM10, PM2.5, and NO2 exposures the week before admission. The estimated risk reduction for IHD admissions was 2.44% [95% confidence interval (CI): 0.33%-4.50%], 2.34% [95% CI: 0.62%-4.03%], and 3.93% [95% CI: 1.14%-6.65%] for a 10% reduction in PM10, PM2.5, and NO2 respectively. For Belgium, the associated annual cost savings were estimated at € 5.2 million, € 5.0 million, and € 8.4 million respectively. For HRD, admission risk could be reduced by 2.16% [95% CI: 0.14%-4.15%], 2.08% [95% CI: 0.42%-3.70%], and 3.46% [95% CI: 0.84%-6.01%] for a 10% reduction in PM10, PM2.5, and NO2 respectively. This corresponds with a potential annual hospital cost saving in Belgium of € 3.7 million, € 3.6 million, and € 5.9 million respectively. If WHO annual guidelines for PM10 and PM2.5 are met, more than triple these amounts would be saved. DISCUSSION: This study demonstrates that a model chain of case-crossover and micro-costing analyses can be applied in order to obtain estimates on the impact of air pollution on hospital costs.

Leaf responses to mild drought stress in natural variants of Arabidopsis.

Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress.

An international review of the frequency of single-bicycle crashes (SBCs) and their relation to bicycle modal share.

OBJECTIVES: To study cyclists' share of transport modes (modal share) and single-bicycle crashes (SBCs) in different countries in order to investigate if the proportion of cyclist injuries resulting from SBCs is affected by variation in modal share. METHODS: A literature search identified figures (largely from western countries) on SBC casualties who are fatally injured, hospitalised or treated at an emergency department. Correlation and regression analyses were used to investigate how bicycle modal share is related to SBCs. RESULTS: On average, 17% of fatal injuries to cyclists are caused by SBCs. Different countries show a range of values between 5% and 30%. Between 60% and 95% of cyclists admitted to hospitals or treated at emergency departments are victims of SBCs. The proportion of all injured cyclists who are injured in SBCs is unrelated to the share of cycling in the modal split. The share of SBC casualties among the total number of road crash casualties increases proportionally less than the increase in bicycle modal share. CONCLUSIONS: While most fatal injuries among cyclists are due to motor vehicle-bicycle crashes, most hospital admissions and emergency department attendances result from SBCs. As found in previous studies of cyclists injured in collisions, this study found that the increase in the number of SBC casualties is proportionally less than the increase in bicycle modal share.