Flowering is the critical growth stage for adverse effects of salinity on the grain yield of sunflower

doi:10.21203/rs.3.rs-2312808/v1

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Flowering is the critical growth stage for adverse effects of salinity on the grain yield of sunflower

https://doi.org/10.21203/rs.3.rs-2312808/v1

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published 10 Aug, 2023

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Purpose Crop sensitivity to root zone salinity can vary over time during a cropping season, which can lead to severe damage when high sensitivity coincides with high soil salinity. The variation in salinity sensitivity of sunflower (Helianthus annuus L.) during its growth cycle is not known.

Methods Two pot experiments were conducted in sand culture with a complete nutrient solution to test the response of sunflower to salinity at different growth stages. Solutions were repeatedly flushed through pots in excess to maintain the specified salt concentrations in the soil solution. In Experiment 1, salt-sensitive growth stages were determined by applying solutions of increasing electrical conductivity (EC < 0.7, 2, 4 or 8 dS m^-1) throughout vegetative, flowering or grain filling stages based on well-defined phenology. In Experiment 2, the most sensitive stage to root-zone salinity was determined by exposing the plants to 10-day periods of salinity (EC < 0.7, 8, 16 and 24 dS m^-1) overlapping by 4 days starting from the 13-leaf to grain filling.

Results In both experiments, decreases in seed yield were associated with exposure of plants to elevated EC during the period when the disk flower was opening to ~95% anthesis, while grain filling was the least sensitive. In experiment 2, measurements of ion concentrations and rates of photosynthesis in leaves suggested that Na⁺ toxicity decreased yield due to decreases in the availability of photosynthates to flowers.

Conclusion To maximise sunflower yield in saline soils, crop management should minimise salinity stress from flower opening to anthesis.

leaf Na+/K+

Photosynthesis

Pollen development

Salt sensitivity

Yield components

In saline agricultural soils, the intensity of salinity in the root zone can vary over time, and has its most adverse effect on crop yield if high salinity coincides with sensitive growth stages. Previous studies on determinate (wheat; Triticum aestivum L. and sorghum; Sorghum bicolor (L.) Moench.) and indeterminate (cowpea; Vigna unguiculata (L.) Walp.) crops reported that the vegetative stage was more sensitive to root zone salinity than the flowering stage (Maas et al. 1986; Maas and Poss 1989a, b). However, in these studies, the vegetative stage also included the period of panicle or spikelet differentiation, hence the exact developmental stage that is most sensitive to saline water irrigation was not clearly identified. In these studies, crop growth was divided into intervals for saline irrigation based on three periods of equal numbers of days, and irrigation was gradually increased at the start and then gradually decreased before the end of each interval. By contrast to cowpea, wheat and sorghum, in rice the critical stage for saline water irrigation was determined to be the 3-leaf (V₃) to panicle initiation (R₀) stage (Zeng et al. 2001). Other studies on determinate and indeterminate crops have suggested that exposure to salinity, water stress and high evaporation during the young microspore stage can impair pollen development and consequently, decrease both seed set and yield (Astiz and Hernández 2013; Nguyen and Sutton 2009; Parish et al. 2012; Saini et al. 1984).

We believe that a key to understanding this apparent confusion in the period of greatest sensitivity of crops to salinity may be because of a relatively loose definition of the ‘vegetative’ and ‘flowering’ stages of crop development in previous experiments (discussed in more detail in the Discussion). Here we would assert that one of the keys to accurately determining the specific phase of greatest sensitivity to salinity is to have clear means of defining the stages of flower development and maturity. For example, Saini et al. (1984) classified seven stages of pollen development in wheat each covering a period of around 9 or 10 days, whereas in sunflower the period from 10 days before R1 (the appearance of the star-like shape in the inflorescence) to R5.8 (80% anthesis) covered around 37 days. Zeng et al. (2001) determined the timing of critical growth stages in rice by dividing the growth stages into five equal periods of 20 days based on phenology from unimbibed seed until the late milk stages.

In the coastal zone of the Ganges Delta, where salinity increases during the dry season, salinity tolerance at the post-flowering stages would be more useful than tolerance at the early stages of growth as the salinity of the soil solution generally increases with the progress of the season. Moreover, the Ganges Delta is a place where fresh water for irrigation is in short supply in the dry season (Mila et al. 2021). If crops are able to increase salt tolerance during the dry season, it may be possible to irrigate crops with slightly to moderately saline water. Detailed studies on salinity dynamics and water availability in river, canal, pond and groundwater during the dry winter season in the Ganges Delta (Mila et al. 2021) suggest that soil and water salinity become limiting to most crops from early February. Sunflower, a promising crop for this region (Paul et al. 2021), is generally planted after rice at the end of the monsoon season and may be irrigated with either brackish pond, canal or groundwater. However, it is not known whether the sensitivity to root zone salinity varies over the growth cycle in sunflower.

In the present study, two pot experiments were conducted to compare salt-sensitivity between the vegetative, flowering or grain filling stages, and to determine the specific timing of the most critical stage with six 10-day periods of exposure to salinity from the 13-leaf stage (V13) to the grain filling (R7.6) stage.

Study location, soil and weather conditions

Experiment 1 was conducted in a polythene shade house at Khulna University in Bangladesh in 2019 (see "Supplementary Information" Fig. S1a). PVC pots (diameter 0.25 m, height 0.75 m) contained washed sand with a field capacity (weight basis) of 16.2%, a bulk density of 1420 kg m^-3 and a water holding capacity (at saturation) of 230 mL kg^-1 of soil. The air temperature during the day ranged from 21.7 to 37.3^°C (mean = 30.9^°C) and the temperature at night ranged from 10.0 to 28.2^°C (mean = 19.8^°C) (see "Supplementary Information" Fig. S2a). The relative humidity varied between 22% and 100% with means during the day and night of 58% and 89% respectively (see "Supplementary Information" Fig. S2b). Total evaporation varied from 0.4 to 5.1 mm d^-1 (mean = 2.9 mm d^-1) (see "Supplementary Information" Fig. S2c).

Experiment 2 was conducted with pots in an open location1 (see "Supplementary Information" Fig. S1b) at Murdoch University, Perth in 2020–21. Plastic pots (diameter 0.30 m, height 0.27 m) contained 16 kg of air-dried sand with a field capacity (weight basis) of 19.6% and a bulk density of 1600 kg m^-3. The air temperature in the day ranged from 19.0 to 42.4^°C (mean = 29.6^°C); the temperature at night ranged from 6.5 to 24.7°C (mean = 16.2^°C) (see "Supplementary Information" Fig. S3a). The relative humidity varied between 9% and 97% with a mean of 32% and 76% during the day and night respectively (see "Supplementary Information" Fig. S3b). Total evaporation varied from 0.8 to 16.0 mm d^-1 (mean = 9.7 mm d^-1) (see "Supplementary Information" Fig. S3c).

Experimental design and crop management

Sunflower (Helianthus annuus L., cv. Hysun 33) was grown in Experiment 1 in a completely randomized block design with three saline irrigation levels (EC values of 2, 4 and 8 dS m^− 1) applied at three growth stages; control plants were grown with an EC < 0.7 dS m^− 1 throughout (Table 1). There were seven replications. The crop was sown on 17 January 2019. Growth stages were identified according to the method of Schneiter and Miller (1981). The vegetative stage started with the appearance of the first true leaf when the cotyledons were less than 4 cm in length (VE). The flowering stage started at least 7 days before the formation of the terminal bud, when the inflorescence resembled a many-pointed star (R1) (see “Supplementary Information” Fig. S4b). The grain filling stage started with the completion of 80% of anthesis (R5.8) (see “Supplementary Information” Fig. S4f). At the vegetative stage, irrigation with saline water began on the 12th day after sowing and continued until day 47 (Table 1). At the flowering stage, irrigation with saline water was imposed from day 48 to day 84 (Table 1). At the grain filling stage, saline irrigation was imposed from day 85 until harvest on day 106 (Table 1).

Table 1

Salinity (EC, dS m^-1) of the irrigation water used in Experiment 1 at the vegetative, flowering and grain filling stages^a
Treatment number	Vegetative (12–47 days)	Flowering (48–84 days)	Grain filling (85–106 days)
T₁	< 0.7	< 0.7	< 0.7
T₂	2	< 0.7	< 0.7
T₃	4	< 0.7	< 0.7
T₄	8	< 0.7	< 0.7
T₅	< 0.7	2	< 0.7
T₆	< 0.7	4	< 0.7
T₇	< 0.7	8	< 0.7
T₈	< 0.7	< 0.7	2
T₉	< 0.7	< 0.7	4
T₁₀	< 0.7	< 0.7	8

^aThermal time. Thermal time was calculated as the average of the daily minimum and maximum temperature minus a base temperature for sunflower (4.8°C, c.f. Granier and Tardieu 1998). For the vegetative, flowering and grain filling stages, the number of growing degree days were 257–1022, 1047–2035, and 2064–2707^°C, respectively.

Sunflower cv. AussieClear 20 was grown in Experiment 2 in a completely randomized block design with four water salinity levels (EC values < 0.7, 8, 16 and 24 dS m^− 1) applied to plants at six growth stages (Table 2), with three replicates. Seeds were sown on 24 October 2020. To avoid osmotic shock at 16 and 24 dS m^− 1 of saline irrigation, concentrations were increased by 8 dS m^− 1 per day until reaching the final concentration. The six 10-day periods of saline irrigation (< 0.7, 8, 16 and 24 dS m^− 1) were conducted from the end of vegetative to the grain filling stage. Visual images of the stage of growth at the start of each time of salinity application can be seen in the Supplementary Information Fig. S5. Timing 1 spanned the end of vegetative growth (V13; 13 true leaves) to the beginning of reproductive growth (R2; small disk with bracts). The six 10-day periods of saline irrigation, overlapping by 4 days between two successive periods, covering the whole of flowering up to R7.6 (large yellow colour at the back of the disk). The plants were harvested at physiological maturity between days 89 and 107 when the back of the sunflower head turned brown and the floral bracts were yellow and brown.

Table 2

Timing of the saline irrigation treatments in Experiment 2 based on phenology according to Schneiter and Miller (1981)^a
Timing of saline irrigation	Definition of stages during initiation and termination of salinity (Schneiter and Miller 1981)	Salinity period
Timing of saline irrigation		Day	Thermal time (°C days)
Timing 1	13-leaf (V13) to small disk (Before R2)	42–51	849–1073
Timing 2	Before R2 to > 2 cm nodal distance (R3)	48–57	883–1218
Timing 3	0.5–2.0 cm nodal distance (R2) to 50% disk opening (R4.5)	54–63	893–1385
Timing 4	Before opening of disk flower (R3.5) to 95% anthesis (R5.95)	60–69	939–1547
Timing 5	70% anthesis (R5.7) to small yellow colour at the back of the disk (R7)	66–75	996–1710
Timing 6	Wilting of ray flowers (R6) to large yellow colour at the back of the disk (R7.6)	72–81	1034–1859

^aThermal time was calculated in the same way as in Table 1.

In each experiment, three seeds were sown per pot at sowing; these were thinned to one plant per pot after establishment. The plants were irrigated with a complete nutrient solution containing 3.0 mM KNO₃, 2.5 mM Ca(NO₃)₂, 1.5 mM MgSO₄, 0.2 mM KH₂PO₄, 50 µM Fe–EDTA, 23 µM H₃BO₃, 5.0 µM MnSO₄, 0.40 µM ZnSO₄, 0.2 µM CuSO₄ and 0.1 µM Na₂MoO₄ (c.f. Maas and Poss 1989b), together with the salt treatments using NaCl and CaCl₂ in a 2:1 molar ratio. Experiment 1 was irrigated three times daily while Experiment 2 was irrigated in two equal splits during the morning and afternoon. All pots were freely drained after each irrigation event, and the salinity (EC) of irrigation water and drainage water were the same. The salt treatment was terminated by irrigating the pots with a non-saline nutrient solution until the salinity of the leachate was similar to that of the non-saline nutrient solution. At harvest, seed yield and yield components (diameter of the flower head, number of mature and immature seeds and 100-seed weight) were measured.

Relationship between relative yield and irrigation water salinity (EC) or solute potential (Ψ_s)

A nonlinear least-squares inversion curve (Van Genuchten 1983) was used to determine the salt sensitivity at different growth stages induced by EC or Ψ_s. This curve was:

Y_r = 1/[1+(C/C₅₀) ^ρ]

where, C = EC (or –Ψ_s), C₅₀ = the EC (or –Ψ_s) corresponding to a 50% yield reduction, ρ = an empirical constant [Exp (sC₅₀)] that specifies the steepness of the curve. The curves were fitted to the data in Genstat 18th edition (VSN International).

Leaf photosynthesis and transpiration

In Experiment 2, five of the youngest fully expanded leaves were randomly selected at the end of each timing treatment for measuring stomatal conductance, photosynthesis and transpiration using a portable photosynthesis system (LI–COR) between 11 am and 2 pm on sunny days. The live and dead leaf numbers were recorded after the completion of each timing of salinity and after 9 days of recovery. Plant height and disk diameter were recorded before and at the end of the salinity treatment, and 9 days after salinity removal. Changes in plant height and disk diameter were calculated after 10 days of salinity treatment and after 9 days of salinity recovery.

At harvest, photographic images of seed-set were collected after removing the disk florets.

At the end of each timing treatment, two fully expanded leaves (with no necrotic tissues) from each replication were collected, washed with DI water and blotted with tissue paper. The leaf samples were oven-dried at 60°C for 48 h or until reaching constant weight. The dried leaves were ground mechanically, and after nitric acid digestion the Na⁺ and K⁺ concentrations in these leaves were determined using inductively coupled plasma-optical emission spectrometry (model: 700 Series ICP–OES).

Statistical analysis

Two-way analysis of variance (ANOVA) of seed yield and yield components was done to evaluate the effects of salinity and growth stage and their interactions using Jamovi (0.9.2.6) and R software (Team 2013). A correlation matrix and regression analyses of seed yield, yield components, plant growth and crop stress parameters were conducted using Jamovi software and in Microsoft Excel.

[1] The open location was selected to enable the cross-pollination of flowers by bees.

Seed yield and yield components (Experiment 1)

Exposure of plants to an EC of 2–8 dS m^− 1 at flowering was more damaging to seed yield than exposure at the vegetative or grain filling stages (Fig. 1a). Compared to the control (EC < 0.7 dS m^− 1), the treatments of 2–8 dS m^− 1 at flowering, vegetative and grain filling stages decreased seed yields by 48–55%, 12–42% and 24–38%, respectively (Fig. 1a).

Based on Van Genuchten curves fitted to the data, there was a 30% decrease in seed yield (C₇₀) at the vegetative, flowering and grain filling stages at EC values of 4.3, 2.6 and 5.2 dS m^− 1 respectively, equivalent to solute potentials (Ψ_s) of – 0.21, – 0.13 and – 0.25 MPa respectively (Fig. 2a). The finding proved that the flowering stage was the most sensitive stage and grain filling was the least sensitive stage.

A similar sensitivity to salinity at the flowering stage was observed for yield components of mature seed number, 100-seed weight, disk diameter and total seed number (Fig. 1b–e). Compared to the controls, each of these yield components showed similar patterns of variation when exposed to EC values of 2–8 dS m^− 1 at the vegetative, flowering and grain filling stages (Fig. 1b–e). The yield component that responded differently was the percentage of immature seed: irrespective of timing, an EC of 8 dS m^− 1 decreased the percent of immature seed to 18–26% of control values (Fig. 1f). In addition, all salinity treatments (EC 2–8 dS m^− 1) at grain filling decreased the percent of immature seed from 100% (control) to 22–47% (Fig. 1f).

The yield components of mature seed number, 100-seed weight, disk diameter, total seed number and percent immature seed were significantly affected (P < 0.001) by EC and crop growth stage (Fig. 1b–f). In addition, there were significant interactions between EC and crop growth stage for mature seed number, total seed number and per cent immature seed (Fig. 1b, e and f). Table S3a in the Supplementary Information shows the correlation matrix between seed yield and yield attributes. All parameters were highly correlated (P < 0.001 and Pearson's r = 0.72–0.90) with seed yield except per cent immature seed. There were highly significant (P < 0.001) positive relationships between seed yield and both mature seed number and 100-seed weight (r² values between 0.49 and 0.81) (Fig. 3a and 3b). Multiple linear regression showed that the maximum variation in seed yield was accounted for (r² = 0.99) by the combination of mature seed number and 100-seed weight (Eq. 1):

Seed yield = – 12.84 + 0.041 x mature seed number + 3.17 x 100-seed weight (Eq. 1)

Yield response to timing of saline irrigation (Experiment 2)

The data for yield and yield components in experiment 2 are presented here in three forms. Figure 4 shows the average effects of the imposition of salinity at different growth stages on the yield and yield components of sunflower. Figure 5 presents a statistical summary of these results, showing the main effects of the timing of salinity and the level of salinity on yield and yield components. Finally, Fig. 2b shows the non-linear least-square inversion curves for the effects of the different timings of salinity on grain yield.

Data presented in Fig. 4 show that seed yield decreased at 8–16 dS m^− 1 with the progress of time of application (Fig. 4a). A similar effect was found for mature seed number, 100-seed weight and disk diameter (Fig. 4b–d). However, at 24 dS m^− 1 timing 3 had the most adverse effect on seed yield, mature seed number, disk diameter, total seed number and % immature seed (Fig. 4a–b, and Fig. 4d–f).

Highly saline irrigation (EC 24 dS m^-1) at timing 3 was most damaging and had lowest yield (1.2 g plant^-1), followed by timing 4 (7.9 g plant^-1) and timing 5 (11.9 g plant^-1) (Fig. 4a). A yield decrease of 87–99% occurred between timing 3 and timing 5 at EC of 24 dS m^-1 compared to the control (EC < 0.7 dS m^-1). At EC 24 dS m^-1, timing 3 produced ~ 58–65% more immature seed than timing 4 and timing 5 (Fig. 5f and the seed set image in Supplementary Information, Fig. S8d–S8f). Timing 3 also showed 98, 53 and 45% lower numbers of mature seed, total seed number and disk diameter, respectively, at 24 dS m^-1 compared with the control (Fig. 5b, 5e and 5d). However, at EC 8 and 16 dS m^-1, timing 4 and timing 5 had the lowest seed yields of 57.3 and 20.1 g plant^-1, respectively (Fig. 5a).

Figure 5 presents a statistical summary of the main effects of the timing of salinity application and the level of salinity for experiment 2. In overview, the data presented show that exposure to salinity at timings 4 and 5 gave the lowest yield (46.4 g plant^− 1) followed by timings 3 and 6 (54 g plant^− 1); the highest yield occurred with timing 1 (73 g plant^− 1) (Fig. 5a). Similar timing effects were found for mature seed number, 100-seed weight, disk diameter and total seed number (Fig. 5b–e).

The main points from Fig. 5 are supported by the analysis of non-linear least-square inversion curves (Fig. 2b). These showed that the seed yield responses to salinity for timings 4 and 5 were relatively similar, while the responses for timings 3 and 6 were also similar up to EC 14 dS m^− 1 (Fig. 2b). The EC value associated with a 30% decrease in yield (C₇₀) for timings 4 and 5 were 6.5 dS m^− 1 (or a Ψ_s of – 0.31 MPa). Similarly, C₇₀ values for timing 1, timing 2, timing 3 and timing 6 were 15.0, 9.8, 7.9 and 7.8 dS m^− 1 (or Ψ_s values of – 0.73, – 0.47, – 0.38 and – 0.38 MPa), respectively. Therefore, timings 4 and 5 were most sensitive, and timing 1 was the least sensitive stage based on yield reduction and lower C₇₀ values. The growth stages between the end of timing 3 and the start of timing 5 were covered by the growth stages of timing 4, R3.5 to R5.95 (Table 2).

It can be concluded that the period from before the opening of disk flowers (R3.5) to 95% anthesis (R5.95) was the critical part of flowering when salinity affected seed yield.

Seed yield was strongly correlated (P < 0.001) with all measured yield components. However, mature seed number gave the highest (Pearson's r = 0.93) correlation with yield (See "Supplementary Information" Table S3b). Significant (P < 0.001) positive relationships were also found for mature seed number and 100-seed weight with yield, with r² values ranging from 0.80 to 0.86 (Fig. 3c and 3d). Multiple linear regression of mature seed number and 100-seed weight accounted for 97% of the variation in seed yield (Eq. 2):

Seed yield = – 37.5 + 0.044 x mature seed number + 9.65 x 100-seed weight (Eq. 2)

Leaf photosynthesis, leaf Na⁺, K⁺ and Na⁺/K⁺ (Experiment 2)

Photosynthesis decreased with the increase of salinity from EC 8 to 24 dS m^− 1 at each timing relative to control (Fig. 6a). In addition, at EC values of 16 and 24 dS m^− 1 photosynthesis decreased progressively with time. With EC values of 16 and 24 dS m^− 1, there were major increases in Na⁺ and Na⁺/K⁺ from timing 3 onwards, but the effects at EC 8 dS m^− 1 were negligible (Fig. 6b and 6d).

The effect on photosynthesis of leaf Na⁺/K⁺ or Na⁺ at timings 4 and 5, relative to the maximum value in control plants is shown in Fig. 7. With timing 4 there were highly significant (P < 0.001 and r² = 0.93) negative relationships between photosynthesis and Na⁺/K⁺ or Na⁺ in leaves (Fig. 7a, b). With timing 5, there were similar negative relationships, but of lower significance (P < 0.01 and r² = 0.77) (Fig. 7c and d).

Seed yield in relation to photosynthesis, leaf Na⁺ and Na⁺/K⁺ (Experiment 2)

Figure 8 shows the effects of photosynthesis on relative seed yield and Na⁺/K⁺ on relative seed yield at timing 4 and 5. Increasing rates of photosynthesis had positive effects on relative seed yield (r² values of 0.91 for timing 4 and 0.93 for timing 5). Increasing Na⁺/K⁺ had negative effects on relative seed yield (r² values of 0.78 for timing 4 and 0.82 for timing 5). Based on the lines of best fit, relative seed yields decreased by 50% as the rate of photosynthesis decreased from ~ 20 to 10 mmol CO₂ m^− 2 s^− 1, and as the Na⁺/K⁺ increased from ~ 0 to 0.2–0.3. Table S4 in the Supplementary Information, shows the correlation matrix between seed yield and stress parameters.

Using a system that repeatedly flushed sand with excess irrigation to maintain stable soil solution EC values, we found that in sunflower, flowering was the most sensitive stage to salinity stress with EC values ranging from 2 to 8 dS m^− 1 in the soil solution. The decline in sunflower yield was most closely correlated with a decrease in mature seed number, although changes in 100-seed weight were also significant. Increasing salinity from 4 to 8 dS m^− 1 during the flowering stage increased the area without seeds at the centre of the disk (see "Supplementary Information" Fig. S9). A second experiment showed that the most sensitive phase of flowering to root-zone salinity was between R3.5 (before the opening of disk flower) and R5.95 (95% anthesis). These findings have particular implication for sunflower growth and irrigation management in environments with variable soil salinity during the growing season.

In Experiment 1, the fitting of non–linear least–square inversion curves showed that exposure to an EC of 2.6 dS m^− 1 (solute potential Ψ_s, – 0.13 MPa) at the flowering stage caused a 30% decrease in yield relative to the control (C₇₀). By contrast, previous studies (Maas et al. 1986; Maas and Poss 1989a, b) reported the vegetative stage to be the most sensitive to salinity of the irrigation water in sorghum, cowpea and wheat. These differences in the growth stage associated with greatest salt sensitivity between the present study and previous studies might relate to the definition of the start and end of the flowering stage. In the present study, sunflower growth stages were identified following Schneiter and Miller (1981) in which the start of flower initiation occurs 7 days before any visible appearance of the flower. Critically, it is during this period that early pollen development occurs. In the study on wheat, the flowering stage was defined as being from the appearance of the flag leaf up to anthesis, and in sorghum, the flowering stage was defined as being from the initiation of rapid stem elongation up to anthesis (Maas et al. 1986; Maas and Poss 1989b). In fact, in both these studies, the vegetative stage would therefore have included the period of spikelet/panicle differentiation. In the cowpea experiment, the ‘flowering stage’ was identified as starting with the appearance of the first floral bud, continuing up to pod development; in this study also the early stages of pollen development would have fallen into the ‘vegetative’ rather than the ‘flowering’ period. These studies in which critical events associated with flowering were included in the vegetative phase can be compared with another study in rice. Zeng et al. (2001) avoided the above confusion by classifying the vegetative stage as starting from 3-leaf to panicle initiation (R₀) while the flowering stage covered from R₀ to early booting (R₂). In this study, the critical stage for sensitivity to salinity was determined to be the 3-leaf (V₃) to panicle initiation (R₀) stage (Zeng et al. 2001). In another study on rice, 15-days of exposure to a high level of salinization (EC 18 dS m^− 1) at the panicle initiation and flowering stages reduced seed yield by 64 and 32% respectively compared to the non-saline control (Castillo et al. 2003).

Do other studies point to early flowering as being critical to the expression of yield? In cereals (wheat, rice, barley and sorghum), drought stress during spikelet differentiation affected spikelet number and initiation of flower formation on the spike, and further stress (during the young microspore stage) caused pollen sterility (Dolferus et al. 2011; Ji et al. 2010). The young microspore stage occurs well before anthesis. In the present experiment, we did not conduct histological examinations to define when the pollen meiosis stage occurred. However, we assume (based on Scheitner and Miller 1981) that this occurs before the opening of the disk flower; microsporogenesis is very quick and generally takes ~ 1–2 days. There are indications that abiotic stress (e.g. drought and temperature extremes) can affect the natural development and function of the tapetum, the inner layer of cells in the anther closest to the microspore mother cells (Parish et al. 2012). A similar sensitivity of sunflower tapetal development and pollen growth, might be responsible for the decrease in seed number per disk and yield in our study.

In Experiment 2, 10 days of saline irrigation from R3.5 to R5.95 during the flowering stage had more damaging effects on seed set and seed yield than saline irrigation from the end of the vegetative (13-leaf) to early flowering (before R2) stages or during late flowering to 100% of anthesis (R6) when a large portion of the back of the disk turned yellow (R7.6). This suggests that development within the disk from the R3.5 stage to 95% anthesis is highly sensitive to salinity, but the period after anthesis is more tolerant to salinity. Mark and Palmer (1981) outline five developmental stages of sunflower flowers (FS6–FS10) that correspond with the stages of R3.5–R4 given by Schneiter and Miller (1981). From FS6 to FS10, the development of the floret with bracts, the 5-lobed corolla and hairy floret bracts at the centre of the receptacle radius occurs and can be visible after cutting the disk. However, at R5.1 to R5.95, the development of anthers and fertilization occurs. Direct confirmation of pollen fertility/infertility as a consequence of different root-zone salinity timings would be valuable in future studies. In addition, future research involving the transfer of pollen from salt-affected to salt-unaffected disc florets (and vice versa) would help to more closely define the extent to which pollen development as opposed to corolla fertility might be directly impaired by salinity.

Our work has demonstrated that the adverse effects of salinity on grain yield are associated with decreased photosynthesis just before anthesis (Fig. 8a). In previous work, water stress-induced osmotic shock affected male gametophyte development in wheat by reducing sugar delivery to reproductive tissue due to a decrease in photosynthesis (Saini 1997). In rice, 3-days of water deficit at Ψ_s ≤ – 0.50 MPa during early stages of anther development (pollen mother cell to vacuolated microspore stage) reduced seed yield due to reduced pollen fertility (Nguyen and Sutton 2009). A strong correlation between grain set and the viability of young microspores indicated that a 50% reduction in viable pollen reduced grain set by ~ 59% (Nguyen and Sutton 2009). In wheat, the effect of heat stress (30°C for 3 days), and water stress (leaf water potential – 2.54 MPa) at the meiosis stage caused pollen abortion or total sterility due to failure of tapetal development and non-completion of the first mitosis of microsporogenesis (Saini et al. 1984). In our study, there were higher Na⁺ concentrations in the leaf tissue at high salinities (24 dS m^− 1 and from timing 3 onwards at 16 dS m^− 1) and photosynthesis decreased with the increase in salinity (Fig. 7a and 7b). Our results are consistent with the view that yield decreased at high salinities due to the changes in the availability of photosynthates to flowers correlated with Na⁺ toxicity. In other words, the sterility effect in salinity-affected sunflower that depressed seed number was caused by a lack of carbohydrate supply during pollen development. Support for this hypothesis could be gained by the supply of sugar to florets during exposure to root zone salinity whilst also measuring ion, sugar and carbohydrate concentrations in the affected florets.

The present experiments were done in open conditions with contrasting day and night environments and further variation during treatment periods (see "Supplementary Information" Table S1a and S1b) but the other studies discussed above were all done in controlled environments. Hence, there may have other effects like high temperature in conjunction with salinity that contributed to yield reduction. It might therefore be valuable as a confirmation of the present conclusions to grow sunflower and contrast it with a cereal in the same experimental setup, treating them at well-defined stages of reproductive development, with the collection of pollen viability data, to verify the critical timing of pollen development for root-zone salinity stress.

Continuous root-zone salinity during the flowering stage caused the greatest decrease in seed yield relative to exposure to salinity at the vegetative or grain filling stages. An EC of 2–8 dS m^− 1 at the vegetative, flowering and grain filling stages gave an average of 32, 52 and 30% reductions in seed yield, respectively, compared to the control. Relative seed yield decreased by 30% (C₇₀) with EC of 4.3, 2.6 and 5.2 dS m^− 1 during root-zone salinization at the vegetative, flowering and grain filling stages. Within the reproductive growth stage, saline irrigation (8 and 16 dS m^− 1) from the R3.5 to R5.95 growth stages was most harmful based on yield decrease and lower EC values associated with 30% decrease in relative yield. These results suggest that saline irrigation from R3.5 to R5.95 can decrease sunflower yield more severely than the same quality of irrigation water at preceding and following growth stages. In situations where there is insufficient fresh irrigation water to complete the lifecycle of sunflower it is suggested that non-saline water could be used at the flowering stage from R3.5 to R5.95 while slightly to moderately saline water could be used at other crop growth stages with less serious adverse impacts on seed yield.

Acknowledgements The authors are grateful to the Australian Centre for International Agricultural Research (Project LWR/2014/073) for financial support and for a John Allwright Fellowship to the principal author. We are thankful to Khulna University (Bangladesh) and Murdoch University (Western Australia) for their support in conducting these experiments. We are grateful for helpful comments on the manuscript from Dr Qifu Ma.

Funding The Australian Centre for International Agricultural Research provided project support for this work (Project LWR/2014/073) and a scholarship to the senior author.

Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Flowering is the critical growth stage for adverse effects of salinity on the grain yield of sunflower

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