DOI: 10.5061/dryad.v6wwpzh20
title: Similar transcriptomic responses to early and late drought stresses produce divergent phenotypes in sunflower (Helianthus annuus L.)

description: Cultivated sunflower (Helianthus annuus L.) exhibits numerous phenotypic
 and transcriptomic responses to drought. However, the ways in which these
 responses vary with differences in drought timing and severity are
 insufficiently understood. We used phenotypic and transcriptomic data to
 evaluate the response of sunflower to drought scenarios of different
 timing and severity in a common garden experiment. Using a semi-automated
 outdoor high-throughput phenotyping platform, we grew six oilseed
 sunflower lines under control and drought conditions. Our results reveal
 that similar transcriptomic responses can have disparate phenotypic
 effects when triggered at different developmental time points. Leaf
 transcriptomic responses, however, share similarities despite timing and
 severity differences (e.g., 523 differentially expressed genes (DEGs) were
 shared across all treatments), though increased severity elicits greater
 differences in expression, particularly during vegetative growth. Across
 treatments, DEGs were highly enriched for genes related to photosynthesis
 and plastid maintenance. A co-expression analysis identified a single
 module (M8) enriched in all drought stress treatments. Genes related to
 drought, temperature, proline biosynthesis, and other stress responses
 were overrepresented in this module. In contrast to transcriptomic
 responses, phenotypic responses were largely divergent between early and
 late drought. Early-stressed sunflowers responded to drought with reduced
 overall growth, but became highly water-acquisitive during recovery
 irrigation, resulting in overcompensation (higher aboveground biomass and
 leaf area) and a greater overall shift in phenotypic correlations, whereas
 late-stressed sunflowers were smaller and more water use-efficient. Taken
 together, these results suggest that drought stress at an earlier growth
 stage elicits a change in development that enables greater uptake and
 transpiration of water during recovery, resulting in higher growth rates
 despite similar initial transcriptomic responses.
description: Six sunflower maintainer (B) lines bred for oil-rich seeds from
 the SAM population (HA124, HA370, HA412HO, HA850, HAR4, and SF193, also
 known as XRQ) were grown at the Heliaphen outdoor high-throughput
 phenotyping platform at INRAE Toulouse (France) in 2018. Seeds were
 planted on 17 April 2018 in 10 L pots of Terreau Proveen PAM 2 substrate.
 Plants were fertilized with 300 mL of Peter’s Professional 17-07-27 and
 200 mL of Hortrilon per pot on 5 May, 25 May, and 13 June, and were
 provided Ortiva Top fungicide on 28 May and 15 June. Pots were maintained
 at specified soil moisture levels by measuring the pot weights daily and
 refilling them with water to return the pot weights to target levels. Soil
 around the plants was covered with a silicone cover to limit soil
 evaporation and was protected from rain using a cone-shaped polystyrene
 skirt. Drought treatment regimes were imposed by permitting pot weights to
 drop to specified levels for set periods of time.
 Plants were grown under one of five irrigation treatment groups.
 Control plants were provided daily water to maintain a pot weight equal to
 the recorded weight of the pot left to drain for two hours after full
 water saturation, defining a fraction of transpirable water (FTSW) of 1.
 The early moderate (EM) and early severe (ES) drought stress groups were
 subjected to drought stress during the vegetative growth phase from 7 May
 until target stress levels (EM = 0.4 FTSW; ES = 0.2 FTSW) were reached.
 Early drought stress target soil moisture levels were reached between 1
 June and 11 June. Following these early drought stress treatments, these
 plants were returned to and maintained at control levels of soil moisture
 (FTSW = 1) until the end of the experiment. The late moderate (LM) and
 late severe (LS) drought stress groups were subjected to drought stress
 during the reproductive growth phase starting on 12 June (plants initiated
 flowering between 18 June and 2 July). Late drought stress was maintained
 at target drought levels (LM = 0.4 FTSW; LS = 0.2 FTSW) until 3 August,
 after which all plants were denied irrigation and left to dry until
 harvest on 31 August. Thus, treatment groups for this study were control
 (C), early moderate (EM), early severe (ES), late moderate (LM), and late
 severe (LS). Six replicates of each combination of treatment group (n = 5)
 and genotype (n = 6) were grown, resulting in 180 plants, of which 174
 survived. The volume of water provided daily to each
 plant was measured automatically, permitting us to track the time series
 of water provision to each plant in each treatment group. One potential
 source of error introduced by this irrigation regime is that not all water
 provided to the plant was transpired. Some small but unmeasured portion of
 the water inevitably evaporated from the soil surface despite the use of
 silicone pot covers. More notably, error was introduced by water being
 taken up and stored in the plant. Water stored in the plant increases the
 pot weight, reducing the amount of water the automated system will provide
 in subsequent rewatering events, resulting in a soil moisture lower than
 targeted levels. The scale of this effect is, however, likely to be
 minimal due to the large difference in weight between the pot/soil and
 plant biomass. Several phenotypic traits (plant height,
 leaf number and area, collar diameter) were measured multiple times over
 the course of the experiment, producing the time-series data. Because the
 phenotyping platform (RapidoScan RS-C-025-1600-MOD) records a maximal
 plant height of 1.5 m, plant height data were truncated to those collected
 before 28 June, the date when some plants began to reach this height.
 Total leaf area was calculated for each plant by finding individual leaf
 areas for every other leaf and doubling the sum of those values.
 Other traits were collected at the end of the experiment after
 harvest and after 48 h dry-down at 80°C for yield components (seed number
 and weight, plant weight). Aboveground plant weight was divided into total
 seed weight, capitulum weight, and vegetative (leaf and stem) plant
 weight. Total water added was defined as the total water provided to a
 single plant over the course of the experiment. We defined water use
 efficiency (WUE) as the total dry plant weight (vegetative plant weight
 plus capitulum weight plus total seed weight) per total water added per
 plant. At the end of each stress treatment (12 June and
 3 August, respectively), dental putty imprints of both sides of leaves
 one-third of the way down the plant were taken for stomatal data
 collection. Clear nail polish was applied to the dental putty imprints and
 removed with clear tape. Images were taken from three or four 100×
 magnified areas per leaf print using a light microscope. Stomata were
 counted manually for each image and averaged per leaf side. Ten individual
 stomata per leaf side were imaged at 400× and measured for pore length and
 guard cell width (as a proxy for pore depth). Using these stomatal
 measurements (density, pore size, pore depth), we calculated the maximum
 anatomical stomatal conductance (gsmax hereafter). It should be noted
 that, given the duration and implementation of our drought treatment, the
 first sampled leaves began development before the early drought stress and
 finished during the early drought stress period, though the second sampled
 leaves fully developed during the drought stress period.