Physiological adaptive traits are a potential allele reservoir for maize genetic progress under challenging conditions.

Combined phenomic and genomic approaches are required to evaluate the margin of progress of breeding strategies. Here, we analyze 65 years of genetic progress in maize yield, which was similar (101 kg ha-1 year-1) across most frequent environmental scenarios in the European growing area. Yield gains were linked to physiologically simple traits (plant phenology and architecture) which indirectly affected reproductive development and light interception in all studied environments, marked by significant genomic signatures of selection. Conversely, studied physiological processes involved in stress adaptation remained phenotypically unchanged (e.g. stomatal conductance and growth sensitivity to drought) and showed no signatures of selection. By selecting for yield, breeders indirectly selected traits with stable effects on yield, but not physiological traits whose effects on yield can be positive or negative depending on environmental conditions. Because yield stability under climate change is desirable, novel breeding strategies may be needed for exploiting alleles governing physiological adaptive traits.

Identification of Key Tissue-Specific, Biological Processes by Integrating Enhancer Information in Maize Gene Regulatory Networks.

Enhancers are key players in the spatio-temporal coordination of gene expression during numerous crucial processes, including tissue differentiation across development. Characterizing the transcription factors (TFs) and genes they connect, and the molecular functions underpinned is important to better characterize developmental processes. In plants, the recent molecular characterization of enhancers revealed their capacity to activate the expression of several target genes. Nevertheless, identifying these target genes at a genome-wide level is challenging, particularly for large-genome species, where enhancers and target genes can be hundreds of kilobases away. Therefore, the contribution of enhancers to plant regulatory networks remains poorly understood. Here, we investigate the enhancer-driven regulatory network of two maize tissues at different stages: leaves at seedling stage (V2-IST) and husks (bracts) at flowering. Using systems biology, we integrate genomic, epigenomic, and transcriptomic data to model the regulatory relationships between TFs and their potential target genes, and identify regulatory modules specific to husk and V2-IST. We show that leaves at the V2-IST stage are characterized by the response to hormones and macromolecules biogenesis and assembly, which are regulated by the BBR/BPC and AP2/ERF TF families, respectively. In contrast, husks are characterized by cell wall modification and response to abiotic stresses, which are, respectively, orchestrated by the C2C2/DOF and AP2/EREB families. Analysis of the corresponding enhancer sequences reveals that two different transposable element families (TIR transposon Mutator and MITE Pif/Harbinger) have shaped part of the regulatory network in each tissue, and that MITEs have provided potential new TF binding sites involved in husk tissue-specificity.

Drought affects abortion of reproductive organs by exacerbating developmentally driven processes via expansive growth and hydraulics.

Abortion of reproductive organs is a major limiting factor of yield under water deficit, but is also a trait selected for by evolutionary processes. The youngest reproductive organs must be prone to abortion so older organs can finish their development in case of limited resources. Water deficit increases natural abortion via two developmentally driven processes, namely a signal from the first fertilized ovaries and a simultaneous arrest of the expansive growth of all ovaries at a precise stage. In maize (Zea mays) subjected to water deficits typically encountered in dryland agriculture, these developmental mechanisms account for 90% of drought-associated abortion and are irreversible 3 d after silk emergence. Consistently, transcripts and enzyme activities suggest that the molecular events associated with abortion affect expansive growth in silks whereas ovaries maintain a favourable carbon status. Abortion due to carbon starvation is only observed for severe drought scenarios occurring after silking. Both kinetic and genetic evidence indicates that vegetative and reproductive structures share a partly common hydraulic control of expansive growth. Hence, the control of expansive growth of reproductive structures probably has a prominent effect on abortion for mild water deficits occurring at flowering time, while carbon starvation dominates in severe post-flowering drought scenarios.

A robot-assisted imaging pipeline for tracking the growths of maize ear and silks in a high-throughput phenotyping platform.

BACKGROUND: In maize, silks are hundreds of filaments that simultaneously emerge from the ear for collecting pollen over a period of 1-7 days, which largely determines grain number especially under water deficit. Silk growth is a major trait for drought tolerance in maize, but its phenotyping is difficult at throughputs needed for genetic analyses. RESULTS: We have developed a reproducible pipeline that follows ear and silk growths every day for hundreds of plants, based on an ear detection algorithm that drives a robotized camera for obtaining detailed images of ears and silks. We first select, among 12 whole-plant side views, those best suited for detecting ear position. Images are segmented, the stem pixels are labelled and the ear position is identified based on changes in width along the stem. A mobile camera is then automatically positioned in real time at 30 cm from the ear, for a detailed picture in which silks are identified based on texture and colour. This allows analysis of the time course of ear and silk growths of thousands of plants. The pipeline was tested on a panel of 60 maize hybrids in the PHENOARCH phenotyping platform. Over 360 plants, ear position was correctly estimated in 86% of cases, before it could be visually assessed. Silk growth rate, estimated on all plants, decreased with time consistent with literature. The pipeline allowed clear identification of the effects of genotypes and water deficit on the rate and duration of silk growth. CONCLUSIONS: The pipeline presented here, which combines computer vision, machine learning and robotics, provides a powerful tool for large-scale genetic analyses of the control of reproductive growth to changes in environmental conditions in a non-invasive and automatized way. It is available as Open Source software in the OpenAlea platform.

The growth of vegetative and reproductive structures (leaves and silks) respond similarly to hydraulic cues in maize.

The elongation of styles and stigma (silks) of maize (Zea mays) flowers is rapid (1-3 mm h(-1) ), occurs over a short period and plays a pivotal role in reproductive success in adverse environments. Silk elongation rate was measured using displacement transducers in 350 plants of eight genotypes during eight experiments with varying evaporative demand and soil water status. Measured time courses revealed that silk elongation rate closely followed changes in soil water status and evaporative demand, with day-night alternations similar to those in leaves. Day-night alternations were steeper with high than with low plant transpiration rate, manipulated via evaporative demand or by covering part of the leaf area. Half times of changes in silk elongation rate upon changes in evaporative demand or soil water status were 10-30 min, similar to those in leaves. The sensitivity of silk elongation rate to xylem water potential was genetically linked to that of leaf elongation rate. Lines greatly differed for these sensitivities. These results are consistent with a common hydraulic control of expansive growth in vegetative and reproductive structures upon changes in environmental conditions via a close connection with the xylem water potential. They have important implications for breeding, modelling and phenotyping.

Is Change in Ovary Carbon Status a Cause or a Consequence of Maize Ovary Abortion in Water Deficit during Flowering?

Flower or grain abortion causes large yield losses under water deficit. In maize (Zea mays), it is often attributed to a carbon limitation via the disruption of sucrose cleavage by cell wall invertases in developing ovaries. We have tested this hypothesis versus another linked to the expansive growth of ovaries and silks. We have measured, in silks and ovaries of well-watered or moderately droughted plants, the transcript abundances of genes involved in either tissue expansion or sugar metabolism, together with the concentrations and amounts of sugars, and with the activities of major enzymes of carbon metabolism. Photosynthesis and indicators of sugar export, measured during water deprivation, suggested sugar export maintained by the leaf. The first molecular changes occurred in silks rather than in ovaries and involved genes affecting expansive growth rather than sugar metabolism. Changes in the concentrations and amounts of sugars and in the activities of enzymes of sugar metabolism occurred in apical ovaries that eventually aborted, but probably after the switch to abortion of these ovaries. Hence, we propose that, under moderate water deficits corresponding to most European drought scenarios, changes in carbon metabolism during flowering time are a consequence rather than a cause of the beginning of ovary abortion. A carbon-driven ovary abortion may occur later in the cycle in the case of carbon shortage or under very severe water deficits. These findings support the view that, until the end of silking, expansive growth of reproductive organs is the primary event leading to abortion, rather than a disruption of carbon metabolism.

Ovary Apical Abortion under Water Deficit Is Caused by Changes in Sequential Development of Ovaries and in Silk Growth Rate in Maize.

Grain abortion allows the production of at least a few viable seeds under water deficit but causes major yield loss. It is maximum for water deficits occurring during flowering in maize (Zea mays). We have tested the hypothesis that abortion is linked to the differential development of ovary cohorts along the ear and to the timing of silk emergence. Ovary volume and silk growth were followed over 25 to 30 d under four levels of water deficit and in four hybrids in two experiments. A position-time model allowed characterizing the development of ovary cohorts and their silk emergence. Silk growth rate decreased in water deficit and stopped 2 to 3 d after first silk emergence, simultaneously for all ovary cohorts, versus 7 to 8 d in well-watered plants. Abortion rate in different treatments and positions on the ear was not associated with ovary growth rate. It was accounted for by the superposition of (1) the sequential emergence of silks originating from ovaries of different cohorts along the ear with (2) one event occurring on a single day, the simultaneous silk growth arrest. Abortion occurred in the youngest ovaries whose silks did not emerge 2 d before silk arrest. This mechanism accounted for more than 90% of drought-related abortion in our experiments. It resembles the control of abortion in a large range of species and inflorescence architectures. This finding has large consequences for breeding drought-tolerant maize and for modeling grain yields in water deficit.

Water deficits uncouple growth from photosynthesis, increase C content, and modify the relationships between C and growth in sink organs.

In plants, carbon (C) molecules provide building blocks for biomass production, fuel for energy, and exert signalling roles to shape development and metabolism. Accordingly, plant growth is well correlated with light interception and energy conversion through photosynthesis. Because water deficits close stomata and thus reduce C entry, it has been hypothesised that droughted plants are under C starvation and their growth under C limitation. In this review, these points are questioned by combining literature review with experimental and modelling illustrations in various plant organs and species. First, converging evidence is gathered from the literature that water deficit generally increases C concentration in plant organs. The hypothesis is raised that this could be due to organ expansion (as a major C sink) being affected earlier and more intensively than photosynthesis (C source) and metabolism. How such an increase is likely to interact with C signalling is not known. Hence, the literature is reviewed for possible links between C and stress signalling that could take part in this interaction. Finally, the possible impact of water deficit-induced C accumulation on growth is questioned for various sink organs of several species by combining published as well as new experimental data or data generated using a modelling approach. To this aim, robust correlations between C availability and sink organ growth are reported in the absence of water deficit. Under water deficit, relationships weaken or are modified suggesting release of the influence of C availability on sink organ growth. These results are interpreted as the signature of a transition from source to sink growth limitation under water deficit.

Floret initiation, tissue expansion and carbon availability at the meristem of the sunflower capitulum as affected by water or light deficits.

• The co-ordination between floret initiation and meristem expansion, and their relationships with carbon availability, were studied and quantified in sunflower (Helianthus annuus) plants subjected to light or water shortages. • Meristem size, number of floret primordia, primordium size, rate of plant biomass accumulation, leaf area, photosynthetic rate, and soluble sugar content in the capitulum were measured until completion of floret initiation. • Although treatments differentially affected tissue expansion and biomass acquisition, a common relationship between the final number of florets and the rate and duration of meristem expansion was conserved. In the absence of water deficit, changes in relative expansion rate in the meristem paralleled changes in soluble sugar content. Water deficit reduced tissue expansion both in leaves and in the capitulum, and induced the accumulation of soluble sugars in the meristem. Use of these sugars at re-watering was associated with increased meristem growth and higher floret numbers compared with control plants. • Floret initiation and meristem tissue expansion remained strongly co-ordinated under all studied circumstances, and both depended on local carbon availability when water supply was unlimited. Transient water deficits favoured reproductive meristem growth and floret production. Equations accounting for these results constitute a framework for phenotyping the response to drought.

Phenotyping the development of leaf area in Arabidopsis thaliana.

The study of leaf expansion began decades ago and has covered the comparison of a wide range of species, genotypes of a same species and environmental conditions or treatments. This has given rise to a large number of potential protocols for today's leaf development biologists. The final size of the leaf surface of a plant results from the integration of many different processes (which may be quantified by various developmental variables) at different organizational levels, such as, the duration and the rate of leaf production by the plant, the duration and the rate of individual leaf expansion, and also cell production and expansion in the leaf. There is much evidence to suggest that the magnitude of a variable at one organizational scale cannot be inferred to another scale because of different feedbacks from one scale to another. This chapter offers a series of protocols, which are the most commonly used in plant developmental biology, to assess quantitatively leaf expansion both at the scale of the shoot and the individual leaf. The protocols described here are for the comparison of Arabidopsis thaliana genotypes, but can be easily adapted to compare leaf expansion under different environmental conditions and in other dicotyledonous plants.

Drought-induced changes in anthesis-silking interval are related to silk expansion: a spatio-temporal growth analysis in maize plants subjected to soil water deficit.

The growth and emergence of maize silks has a considerable importance in yield determination under drought conditions. Spatial and temporal patterns of the rates of tissue expansion and of cell division were characterized in silks of plants subjected to different soil water potentials. In all cases, silk development consisted of four phases: (1) cell division and tissue expansion occurred together uniformly all along the silk; (2) cell division progressively ceased from tip to base, while expansion remained spatially uniform including during the phase (3) after the cessation of cell division; and (4) as the silk emerged from the husks, expansion ceased in the emerged portion, probably because of direct evaporative demand, while the relative growth rate progressively decreased in the enclosed part. The rates of tissue expansion and cell division were reduced with water deficit, resulting in delayed silk emergence. The duration of cell division was not affected, and in all cases, the end of cell division in the silk coincided with anther dehiscence. The duration of phase 3, between the end of cell division and the arrest of cell growth in silk apex, considerably increased with water deficit. It corresponded to the anthesis-silking interval used by breeders to characterize the response of cultivars to stress.

How does the meristem of sunflower capitulum cope with tissue expansion and floret initiation? A quantitative analysis.

The coordination between floret initiation and tissue expansion has been studied and quantified in the apical meristem of sunflower (Helianthus annuus) plants grown under different light availability. A method was developed to quantify tissue expansion in the meristem during floret initiation from measurements of meristem area, number of florets and primordium size. Initially, floret initiation and tissue expansion occurred simultaneously at the meristem surface. The duration of this phase remained unchanged across environments, whereas the rate of tissue expansion varied greatly. Floret initiation rate depended on meristem initial size and tissue-expansion rate. Thereafter, floret initiation continued without tissue expansion in the meristem, resulting in a rapid decrease of meristem area. A set of equations was proposed to predict floret initiation rate and floret number as a function of the rates of tissue expansion in the meristem before and during floret initiation. This formalism demonstrated the role of tissue expansion in determining the final number of florets, and provided a framework to analyse the response of floret initiation to genotype and environment.

A whole-plant analysis of the dynamics of expansion of individual leaves of two sunflower hybrids.

Common features in the time-course of expansion of leaves which considerably differed in final area, due to phytomer position, growing conditions and genotype, were identified. Leaf development consisted of two phases of exponential growth, followed by a third phase of continuous decrease of the relative expansion rate. The rate and the duration of the first exponential phase were common to all phytomers, growing conditions and genotypes. Leaves differed in the rate and the duration of the second exponential phase. The decrease of the relative expansion rate during the third phase depended on neither genotype nor growing conditions. It was phytomer-dependent and was deduced from the rate of the second phase via a parameter common to all cases studied. Differences in final leaf area among growing conditions were linked to different expansion rates during the second exponential phase. The duration of the phases at any given phytomer position was the same for the two hybrids in different growing conditions. The dates of developmental events (initiation, end of the two exponential phases, full expansion), and the rate of the second exponential phase, were related to phytomer position, defining a strict pattern of leaf development at the whole plant level. Using this framework simplified the analysis of the response of leaf expansion to genotype and environment.

Individual leaf development in Arabidopsis thaliana: a stable thermal-time-based programme.

In crop species, the impact of temperature on plant development is classically modelled using thermal time. We examined whether this method could be used in a non-crop species, Arabidopsis thaliana, to analyse the response to temperature of leaf initiation rate and of the development of two leaves of the rosette. The results confirmed the large plant-to-plant variability in the studied isogenic line of the Columbia ecotype: 100-fold differences in leaf area among plants sown on the same date were commonly observed at a given date. These differences disappeared in mature leaves, suggesting that they were due to a variability in plant developmental stage. The whole population could therefore be represented by any group of synchronous plants labelled at the two-leaf stage and followed during their development. Leaf initiation rate, duration of leaf expansion and maximal relative leaf expansion rate varied considerably among experiments performed at different temperatures (from 6 to 26 degrees C) but they were linearly related to temperature in the range 6-26 degrees C, with a common x-intercept of 3 degrees C. Expressing time in thermal time with a threshold temperature of 3 degrees C unified the time courses of leaf initiation and of individual leaf development for plants grown at different temperatures and experimental conditions. The two leaves studied (leaf 2 and leaf 6) had a two-phase development, with an exponential phase followed by a phase with decreasing relative elongation rate. Both phases had constant durations for a given leaf position if expressed in thermal time. Changes in temperature caused changes in both the rate of development and in the expansion rate which mutually compensated such that they had no consequence on leaf area at a given thermal time. The resulting model of leaf development was applied to ten experiments carried out in a glasshouse or in a growth chamber, with plants grown in soil or hydroponically. Because it predicts accurately the stage of development and the relative expansion rate of any leaf of the rosette, this model facilitates precise planning of sampling procedures and the comparison of treatments in growth analyses.

Leaf senescence induced by mild water deficit follows the same sequence of macroscopic, biochemical, and molecular events as monocarpic senescence in pea.

We have compared the time course of leaf senescence in pea (Pisum sativum L. cv Messire) plants subjected to a mild water deficit to that of monocarpic senescence in leaves of three different ages in well-watered plants and to that of plants in which leaf senescence was delayed by flower excision. The mild water deficit (with photosynthesis rate maintained at appreciable levels) sped up senescence by 15 d (200 degrees Cd), whereas flower excision delayed it by 17 d (270 degrees Cd) compared with leaves of the same age in well-watered plants. The range of life spans in leaves of different ages in control plants was 25 d (340 degrees Cd). In all cases, the first detected event was an increase in the mRNA encoding a cysteine-proteinase homologous to Arabidopsis SAG2. This happened while the photosynthesis rate and the chlorophyll and protein contents were still high. The 2-fold variability in life span of the studied leaves was closely linked to the duration from leaf unfolding to the beginning of accumulation of this mRNA. In contrast, the duration of the subsequent phases was essentially conserved in all studied cases, except in plants with excised flowers, where the degradation processes were slower. These results suggest that senescence in water-deficient plants was triggered by an early signal occurring while leaf photosynthesis was still active, followed by a program similar to that of monocarpic senescence. They also suggest that reproductive development plays a crucial role in the triggering of senescence.