Earbox, an open tool for high-throughput measurement of the spatial organization of maize ears and inference of novel traits.

BACKGROUND: Characterizing plant genetic resources and their response to the environment through accurate measurement of relevant traits is crucial to genetics and breeding. Spatial organization of the maize ear provides insights into the response of grain yield to environmental conditions. Current automated methods for phenotyping the maize ear do not capture these spatial features. RESULTS: We developed EARBOX, a low-cost, open-source system for automated phenotyping of maize ears. EARBOX integrates open-source technologies for both software and hardware that facilitate its deployment and improvement for specific research questions. The imaging platform consists of a customized box in which ears are repeatedly imaged as they rotate via motorized rollers. With deep learning based on convolutional neural networks, the image analysis algorithm uses a two-step procedure: ear-specific grain masks are first created and subsequently used to extract a range of trait data per ear, including ear shape and dimensions, the number of grains and their spatial organisation, and the distribution of grain dimensions along the ear. The reliability of each trait was validated against ground-truth data from manual measurements. Moreover, EARBOX derives novel traits, inaccessible through conventional methods, especially the distribution of grain dimensions along grain cohorts, relevant for ear morphogenesis, and the distribution of abortion frequency along the ear, relevant for plant response to stress, especially soil water deficit. CONCLUSIONS: The proposed system provides robust and accurate measurements of maize ear traits including spatial features. Future developments include grain type and colour categorisation. This method opens avenues for high-throughput genetic or functional studies in the context of plant adaptation to a changing environment.

The growths of leaves, shoots, roots and reproductive organs partly share their genetic control in maize plants.

We have tested to what extent the growth ability of several organs of maize share a common genetic control. Every night, leaf elongation rate reaches a maximum value (LERmax ) that has a high heritability, is repeatable between experiments and is correlated with final leaf length. Firstly, we summarized quantitative trait loci (QTLs) of LERmax and of leaf length in three mapping populations. Among the 14 consensus QTLs (cQTLs) of leaf length, seven co-located with cQTLs of LERmax with consistent allelic effects. Nine cQTLs of LERmax (4% of the genome) were highly reliable and confirmed by introgression lines. We then compared these QTLs with those affecting the growths of leaves, shoots, roots or young reproductive organs, detected with the same mapping populations in three field experiments or in literature datasets. Five of the nine most reliable cQTLs of LERmax co-located with QTLs involved in the growth of other organs (but not in flowering time) with consistent allelic effects. Reciprocally, two-thirds of the 20 QTLs of growth of different organs co-located with cQTLs of LERmax . Hence, LERmax , as determined in a phenotyping platform, is an indicator of the growth ability of other organs of the plant in controlled or in-field conditions.

Modelling temperature-compensated physiological rates, based on the co-ordination of responses to temperature of developmental processes.

Temperature fluctuates rapidly and affects all developmental and metabolic processes. This often obscures the effects of developmental trends or of other environmental conditions when temperature fluctuates naturally. A method is proposed for modelling temperature-compensated rates, based on the coordination of temperature responses of developmental processes. In a data set comprising 41 experiments in the greenhouse, growth chamber, or the field, the temperature responses in the range of 6-36 degrees C for different processes were compared in three species, maize, rice, and Arabidopsis thaliana. Germination, cell division, expansive growth rate, leaf initiation, and phenology showed coordinated temperature responses and followed common laws within each species. The activities of 10 enzymes involved in carbon metabolism exhibited monotonous exponential responses across the whole range 10-40 degrees C. Hence, the temperature dependence of developmental processes is not explained by a simple relationship to central metabolism. Temperature-compensated rates of development were calculated from the equations of response curve, by expressing rates per unit equivalent time at 20 degrees C. This resulted in stable rates when temperatures fluctuated over a large range (for which classical thermal time was inefficient), and in time courses of leaf development which were common to several experiments with different temperature scenarios.

Are source and sink strengths genetically linked in maize plants subjected to water deficit? A QTL study of the responses of leaf growth and of Anthesis-Silking Interval to water deficit.

Leaf growth and Anthesis-Silking Interval (ASI) are the main determinants of source and sink strengths of maize via their relations with light interception and yield, respectively. They depend on the abilities of leaves and silks to expand under fluctuating environmental conditions, so the possibility is raised that they may have a partly common genetic determinism. This possibility was tested in a mapping population which segregates for ASI. Maximum leaf elongation rate per unit thermal time (parameter a) and the slopes of its responses to evaporative demand and soil water status (parameters b and c) were measured in greenhouse and growth chamber experiments, in two series of 120 recombinant inbred lines (RILs) studied in 2004 and 2005 with 33 RILs in common both years. ASI was measured in three and five fields under well-watered conditions and water deficit, respectively. For each RIL, the maximum elongation rate per unit thermal time was reproducible over several experiments in well-watered plants. It was accounted for by five QTLs, among which three co-localized with QTLs of ASI of well-watered plants. The alleles conferring high leaf elongation rate conferred a low ASI (high silk elongation rate). The responses of leaf elongation rate to evaporative demand and to predawn leaf water potential were linear, allowing each RIL to be characterized by the slopes of these response curves. These slopes had three QTLs in common with ASI of plants under water deficit. The allele for leaf growth maintenance was, in all cases, that for shorter ASI (maintained silk elongation rate). By contrast, other regions influencing ASI had no influence on leaf growth. These results may have profound consequences for modelling the genotype x environment interaction and for designing drought-tolerant ideotypes.

A model co-ordinating the elongation of all leaves of a sorghum cultivar was applied to both Mediterranean and Sahelian conditions.

Sorghum leaf development was analysed at plant level by analysing the time-course of elongation and identifying the beginning and end of the elongation phases of each leaf blade. This was done with destructive and non-destructive measurements in 14 experiments carried out during several growing periods in Southern France and Sahelian Africa. Elongation of each blade was characterized by the succession of a nearly exponential phase and a linear phase. For a given blade and provided that time was expressed in thermal units, initiation, beginning and end of the linear phase, and time-courses of elongation rate were strikingly similar in all experiments, except in environments with a maximum air temperature close to 40 degrees C and a maximum vapour pressure deficit close to 6 kPa. The relative elongation rate during the exponential phase declined with leaf number from 0.08 to 0.02 degrees Cd(-1), while the duration of this phase increased from 140 to 320 degrees Cd. By contrast, the absolute elongation rate during the linear phase was nearly constant from leaf 8 onwards. This phase was shorter than the exponential phase regardless of leaf position, but accounted for the largest part of blade length. A strict pattern of leaf development was observed at the whole plant level, whereby dates of elongation events and leaf and ligule appearance, represented on a thermal time scale, were linearly related to phytomer number. This pattern exhibited a simultaneous elongation cessation of the last-formed leaves and a mismatch between real and apparent (from leaf to ligule appearance) elongation duration.

The elongation rate at the base of a maize leaf shows an invariant pattern during both the steady-state elongation and the establishment of the elongation zone.

Spatial and temporal analyses of elongation and cell length of monocotyledon leaves have most often been performed during the period when leaves are visible and elongate at a constant rate (steady-state). In the present study, the focus was on the earlier stages, during the establishment of the elongation zone. Regardless of leaf development stage, the segment located between 0 and 35 mm from the leaf insertion point had a relative elongation rate that increased with distance from insertion point ('accelerating zone') while the segment located further than 35 mm had a relative elongation rate that decreased ('decelerating zone'). This stable pattern held for both young, non-emerged leaves, where it was restricted to the portion corresponding to the length of the blade, and for leaves during steady-state elongation. In the same way, the profile of cell length was essentially the same during early development and during steady-state elongation. The results of a temporal analysis of whole-leaf elongation rate, carried out in the field and in the greenhouse at different light intensities were consistent with a time-invariant pattern of elongation. Whole-leaf relative elongation rate increased with time until the leaf reached 30-40 mm length (although at different leaf ages depending on conditions), and declined afterwards. These results suggest that the patterns governing the elongation rate of a sector of a maize leaf are independent of the leaf developmental stage but depend on sector position only.

N. plumbaginifolia zeaxanthin epoxidase transgenic lines have unaltered baseline ABA accumulations in roots and xylem sap, but contrasting sensitivities of ABA accumulation to water deficit.

A series of transgenic lines of Nicotiana plumbaginifolia with modified expression of zeaxanthin epoxidase gene (ZEP) provided contrasting ABA accumulation in roots and xylem sap. For mild water stress, concentration of ABA in the xylem sap ([ABA](xylem)) was clearly lower in plants underexpressing ZEP mRNA (complemented mutants and antisense transgenic lines) than in wild-type. In well-watered conditions, all lines presented similar [ABA](xylem) and similar ABA accumulation rates in detached roots. Plants could, therefore, be grown under normal light intensities and evaporative demand. Both ZEP mRNA abundance and ABA accumulation rate in roots increased with water deficit in all transgenic lines, except in complemented aba2-s1 mutants in which the ZEP gene was controlled by a constitutive promoter which does not respond to water deficit. These lines presented no change in root ABA content either with time or dehydration. The increase in ZEP mRNA abundance in roots with decreasing RWC was more pronounced in detached roots than in whole plants, suggesting a difference in mechanism. In all transgenic lines, a linear relationship was observed between predawn leaf water potential and [ABA](xylem), which could be reproduced in several experiments in the greenhouse and in the growth chamber. It is therefore possible to represent the effect of the transformation by a single parameter, thereby allowing the use of a quantitative approach to assist understanding of the behaviour of transgenic lines.

Quantitative analysis of cell division in leaves: methods, developmental patterns and effects of environmental conditions.

In planta quantitative studies of cell cycle are necessary for examining the role of cell division in the response of plants to environmental conditions and to analyse the behaviour of transformed plants in this context. We present and discuss non-intrusive kinematic methods which allow estimating the duration of cell cycle with a high spatial resolution in the leaf. Different methods are proposed and discussed for monocotyledons and dicotyledons, and compared with methods involving the use of chemicals. In monocotyledon leaves, cell division is restricted to a limited zone near the leaf insertion point, twice as long in the mesophyll as in the epidermis. In dicotyledons, cell division occurs in the whole leaf with a uniform and constant cell cycle duration for a determinate number of cell cycles, representing about half of leaf development. Over several experiments, this number is well conserved in a given leaf zone in the absence of stresses, but larger near the leaf base than near the leaf tip. After that, cell cycle duration increases because cells are progressively blocked in G1 while the durations of S-G2-M phases do not change with time. Leaf temperature affects neither the distribution of nuclei in each phase of the cycle nor the number of cell cycles in a leaf. Water or light deficits both cause a partial blockage of nuclei in G1 during the stress only, thereby increasing cell cycle duration and decreasing final cell number. These results suggest that a strong developmental programme drives cell division in leaves, so a simple framework allows analysis of temporal patterns, of spatial gradients and of the effect of environmental conditions.

Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.

We have investigated the spatial distributions of cell division rate, p34(cdc2) kinase activity, and amount of p34(cdc2a) in maize (Zea mays) leaves grown at contrasting temperatures and soil water conditions. An original method for calculating cell division rate in all leaf tissues is proposed. In all studied conditions, cell division rate was stable and maximum in the first 2 cm beyond the leaf insertion point, declined afterward, and reached zero at 7 cm from the insertion point. The spatial distribution of p34(cdc2) kinase activity, expressed on a per cell basis, followed the same pattern. In contrast, the amount of p34(cdc2a) was maximum in the first centimeter of the leaf, declined afterward, but remained at 20% of maximum in more distal zones with a near-zero cell division rate. A mild water deficit caused a reduction in cell division rate and p34(cdc2) kinase activity by approximately 45% in all leaf zones, but did not affect the amount of p34(cdc2a). Growth temperature affected to the same extent cell division rate and p34(cdc2) kinase activity, but only if p34(cdc2) kinase activity was assayed at growth temperature, and not if a standard temperature was used in all assays. A common linear relationship between cell division rate and p34(cdc2) kinase activity applied to all causes of changes in cell division rate, i.e. cell aging, water deficit, or changes in temperature. It is shown that temperature has two distinct and additive effects on p34(cdc2) kinase activity; first, an effect on the rate of the reaction, and second, an effect on the amount of p34(cdc2a).

Co-Ordination of Cell Division and Tissue Expansion in Sunflower, Tobacco, and Pea Leaves: Dependence or Independence of Both Processes?

Temporal analyses of cell division and tissue expansion in pea, tobacco, and sunflower leaves reveal that both processes follow similar patterns during leaf development. Relative cell division and relative tissue expansion rates are maximal and constant during early leaf development, but they decline later. In contrast, relative cell expansion rate follows a bell-shaped curve during leaf growth. Cell division and tissue expansion have common responses to temperature, intercepted radiation, and water deficit. As a consequence, final leaf area and cell number remain highly correlated throughout a large range of environmental conditions for these different plant species, indicating that cell division and tissue expansion are co-ordinated during leaf development. This co-ordination between processes has long been explained by dependence between both processes. Most studies on dicotyledonous leaf development indicate that leaf expansion rate depends on the number of cells in the leaf. We tested this hypothesis with a large range of environmental conditions and different plant species. Accordingly, we found a strong correlation between both absolute leaf expansion rate and leaf cell number. However, we showed that this relationship is not necessarily causal because it can be simulated by the hypothesis of independence between cell division and tissue expansion according to Green's theory of growth (1976).

Spatial distributions of expansion rate, cell division rate and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature.

The spatial distributions of leaf expansion rate, cell division rate and cell size was examined under contrasting soil water conditions, evaporative demands and temperatures in a series of experiments carried out in either constant or naturally fluctuating conditions. They were examined in the epidermis and all leaf tissues. (1) Meristem temperature affected relative elongation rate by a constant ratio at all positions in the leaf. If expressed per unit thermal time, the distribution of relative expansion rate was independent of temperature and was similar in all experiments with low evaporative demand and no water deficit. This provides a reference distribution, characteristic of the studied genotype, to which any distribution in stressed plants can be compared. (2) Evaporative demand and soil water deficit affected independently the distribution of relative elongation rate and had near-additive effects. For a given stress, a nearly constant difference was observed, at all positions of the leaf, between the relative elongation rates of stressed plants and those of control plants. This caused a reduction in the length of the zone with tissue elongation. (3) Methods for calculating cell division rate in the epidermis and in all leaf tissues are proposed and discussed. In control plants, the zone with cell division was 30 mm and 60 mm long in the epidermis and in whole tissues, respectively. Both this length and relative division rate were reduced by soil water deficit. The size of epidermal and of mesophyll cells was nearly unaffected in the leaf zone with both cell division and tissue expansion, suggesting that water deficit affects tissue expansion rate and cell division rate to the same extent. Conversely, cell size of epidermis and mesophyll were reduced by water deficit in mature parts of the leaf.

Water deficit and spatial pattern of leaf development. Variability In responses can Be simulated using a simple model of leaf development

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695-703).

Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. A common pattern of development for all zones of a leaf and different leaves of a plant

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration. RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.

Control of Leaf Expansion Rate of Droughted Maize Plants under Fluctuating Evaporative Demand (A Superposition of Hydraulic and Chemical Messages?).

We have analyzed the possibility that chemical signaling does not entirely account for the effect of water deficit on the maize (Zea mays L.) leaf elongation rate (LER) under high evaporative demand. We followed time courses of LER (0.2-h interval) and spatial distribution of elongation rate in leaves of either water-deficient or abscisic acid (ABA)-fed plants subjected to varying transpiration rates in the field, in the greenhouse, and in the growth chamber. At low transpiration rates the effect of the soil water status on LER was related to the concentration of ABA in the xylem sap and could be mimicked by feeding artificial ABA. Transpiring plants experienced a further reduction in LER, directly linked to the transpiration rate or leaf water status. Leaf zones located at more than 20 mm from the ligule stopped expanding during the day and renewed expansion during the night. Neither ABA concentration in the xylem sap, which did not appreciably vary during the day, nor ABA flux into shoots could account for the effect of evaporative demand. In particular, maximum LER was observed simultaneously with a minimum ABA flux in the droughted plants, but with a maximum ABA flux in ABA-fed plants. All data were interpreted as the superposition of two additive effects: the first involved ABA signaling and was observed during the night and in ABA-fed plants, and the second involved the transpiration rate and was observed even in well-watered plants. We suggest that a hydraulic signal is the most likely candidate for this second effect.

Temperature Affects Expansion Rate of Maize Leaves without Change in Spatial Distribution of Cell Length (Analysis of the Coordination between Cell Division and Cell Expansion).

We have analyzed the way in which temperature affects leaf elongation rate of maize (Zea mays L.) leaves, while spatial distributions (observed at a given time) of cell length and of proportion of cells in DNA replication are unaffected. We have evaluated, in six growth chamber experiments with constant temperatures (from 13 to 34[deg]C) and two field experiments with fluctuating temperatures, (a) the spatial distributions of cell length and of leaf elongation rate, and (b) the distribution of cell division, either by using the continuity equation or by flow cytometry. Leaf elongation rate was closely related to meristem temperature, with a common relationship in the field and in the growth chamber. Cell division and cell elongation occurred in the first 20 and 60 mm after the ligule, respectively, at all temperatures. Similar quantitative responses to temperature were observed for local cell division and local tissue expansion rates (common x intercept and normalized slope), and both responses were spatially uniform over the whole expanding zone (common time courses in thermal time). As a consequence, faster cell elongation matched faster cell division rate and faster elongation was compensated for by faster cell displacement, resulting in temperature-invariant profiles of cell length and of proportion of dividing cells. Cell-to-cell communication, therefore, was not necessary to account for coordination.

Stomatal response to abscisic Acid is a function of current plant water status.

We investigated, under laboratory and field conditions, the possibility that increasing abscisic acid (ABA) concentrations and decreasing water potentials can interact in their effects on stomata. One experiment was carried out with epidermal pieces of Commelina communis incubated in media with a variety of ABA and polyethylene glycol concentrations. In the media without ABA, incubation in solutions with water potentials between -0.3 and -1.5 megapascals had no significant effect on stomatal aperture. Conversely, the sensitivity of stomatal aperture to ABA was trebled in solutions at -1.5 megapascals compared with sensitivity at -0.3 megapascals. The effect of the change in sensitivity was more important than the absolute effect of ABA at the highest water potential. In a field experiment, sensitivity of maize stomatal conductance to the concentration of ABA in the xylem sap varied strongly with the time of the day. We consider that the most likely explanation for this is the influence of a change in leaf or epidermal water potential that accompanies an increase in irradiance and saturation deficit as the day progresses. These observations suggest that epidermal water relations may act as a modulator of the responses of stomata to ABA. We argue that such changes must be taken into account in studies or modeling of plant responses to drought stress.

[Isoenzymes in two micromycetes species: taxonomic aspects]. / Isoenzymes chez deux espèces de micromycètes: aspect taxonomique.

Strains of different origins belonging to the two species of fungi imperfecti Cylindrocarpon ianothele and Calcarisporium arbuscula exhibit difference in their morphological or physiological appearances. We have attempted to determine if these variabilities could also be observed at the level of other phenotypic markers, the enzymes glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, octanol dehydrogenase and malate dehydrogenase. Isozymes were separated by polyacrylamide gel electrophoresis and demonstrated isozyme variability as a function of growth and physiological states of a given strain. At the same state of development, the different strains exhibited highly heterogeneous isozyme groups which appear difficult to use for taxonomic purposes.