Transgenerational effects of temporal drought stress on spring barley morphology and functioning

Accepted Manuscript Title: Transgenerational effects of temporal drought stress on spring barley morphology and functioning Author: Artur Nosalewicz Joanna Sieci´nska Magdalena ´ Smiech Magdalena Nosalewicz Dariusz Wi˛acek Alicja Pecio Damian Wach PII: DOI: Reference:

S0098-8472(16)30152-6 http://dx.doi.org/doi:10.1016/j.envexpbot.2016.07.006 EEB 3095

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Environmental and Experimental Botany

Received date: Revised date: Accepted date:

25-3-2016 15-7-2016 15-7-2016

´ Please cite this article as: Nosalewicz, Artur, Sieci´nska, Joanna, Smiech, Magdalena, Nosalewicz, Magdalena, Wi˛acek, Dariusz, Pecio, Alicja, Wach, Damian, Transgenerational effects of temporal drought stress on spring barley morphology and functioning.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Transgenerational effects of temporal drought stress on spring barley morphology and functioning Artur Nosalewicz1*, Joanna Siecińska1, Magdalena Śmiech2, Magdalena Nosalewicz1, Dariusz Wiącek1, Alicja Pecio3, Damian Wach3 1

Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290

Lublin, Poland 2

Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Postępu

36A, Jastrzębiec, 05-552 Magdalenka, Poland 3

Institute of Soil Science and Plant Cultivation - State Research Institute,

Czartoryskich 8, 24-100 Puławy, Poland *corresponding author e-mail: [email protected] Short title: Transgenerational effects of drought on spring barley

Highlights Transgenerational effects of drought on barley shoots and roots were observed Drought affecting two barley generations increased the contribution of thin roots Seed-derived nutrients explained the observed transgenerational effects of drought

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Abstract Drought stress affects plants leaving a long-lasting imprint that may be passed onto progeny, affecting its growth and functioning. The aim of the research was to analyse the transgenerational effect of intense drought on alterations in functioning and changes in the growth of shoot and roots of spring barley. Barley seeds from a maternal generation that were affected by intense drought stress at the flag leaf stage or grown in the optimum conditions throughout the vegetation period were used as a sowing material to analyse the growth of a progeny generation at drought or in soil at optimum soil moisture. The difference in water availability during the growth of the progeny generation significantly affected all measured indicators of plant growth and functioning. However, significant differences were also found between offspring growing in the same conditions but characterised by different water availability during the growth of the maternal generation. The differences between plants with different stress histories in the maternal generation were more pronounced in barley offspring grown at drought than at optimum soil water 2

availability. The drought induced during the growth of the barley maternal generation decreased the shoot-to-root mass ratio, enhanced growth of thin roots, and reduced the number of thick roots in the progeny grown at drought, compared to plants that were grown in the same conditions but without drought in the maternal generation. Maintenance of relatively long roots at low allocation of biomass to roots at drought in the progeny of the stressed maternal generation may be an adaptive response that can minimise carbon cost of water and nutrient acquisition. The results obtained may have implications in the prediction of responses of crops and ecosystems to drought and in proper design of scientific experiments on plant responses to water availability.

Keywords: water deficit; stress imprint; drought memory; seed provisioning; seed minerals; root topology; biomass distribution

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Abbreviations O-O – optimum soil water potential maintained during the growth of maternal and progeny generations; O-DS – optimum soil water potential maintained during the growth of maternal generations; drought stress induced during the growth of the progeny generation, DS-O - drought stress induced during the growth of the maternal generation; optimum soil water potential maintained during the growth of the progeny generation; DS-DS - drought stress induced during the growth of the maternal and progeny generations;

1. INTRODUCTION Drought is the major abiotic stress that negatively influences agricultural yields worldwide, especially when stress occurs during reproductive growth stages (Wang et al., 2014). According to

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predicted climate changes (IPCC 2007), the frequency and intensity of drought events will increase in the near future. To cope with drought stress, plants respond with complex physiological and biochemical changes that influence their growth and morphology. Relatively rapid physiological changes may be followed by alterations in shoot and root growth, morphology, and anatomy that affect plant functioning in a longer time scale. As shown in several studies, e.g. Herman and Sultan (2011), Kou et al. (2011), Metz et al. (2015), some of these responses to the stressor may be delayed or even passed on to subsequent generations. Experimental observations indicate mechanisms of the so-called “plant memory” (Verhoeven and Grup, 2012). Among the possible few (Herman et al., 2012), the major mechanisms that may alter the growth of a progeny in response to stresses experienced by the maternal generation are seed-derived nutrients and epigenetic changes at the DNA level associated with cytosine methylation. These mechanisms were reported to affect the morphology and growth of the offspring generation (Johannes et al., 5

2009, Verhoeven and Gurp, 2012, Nadeem et al., 2013). The effect of drought on the seed mineral composition has been shown in several studies (Bourgeois et al., 2009, Esmaeilian et al., 2012, Lamont and Groom, 2013). Plant metabolism affected by the stressful maternal environment was found to influence accumulation of nutrients and increase the variability of post-germination growth of plants with the same genetic background (Vessal et al., 2012, Crisp et al., 2016). However, the knowledge of the role of these mechanisms in seedling drought avoidance is still incomplete. The research of Metz et al. (2015) showed that performance parameters associated with drought tolerance such as the seed number, total aboveground mass, time to flowering, and production of fruit in Biscutella didyma offspring originating from parents grown under drought were not improved. On the other hand, Garg et al. (2015) have shown recently that rice cultivars with contrasting drought responses showed significantly different DNA methylation patterns, affecting expression of genes responsible for resistance to abiotic stresses.

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Mechanisms that allow passing on the information about exposure to stress to subsequent generations may be advantageous for organisms if the perceived stress repeats during the growth of the next generation. These changes may be advantageous and help in offspring adaptation to adverse growth conditions as well as increase their fitness to parental growth conditions. The root system size and rooting depth are known to represent the most important determinants of water and nutrient acquisition; roots are also important in overall plastic responses to adverse growth conditions (Malamy, 2005). The plant response to drought often involves changes in the root size and architecture. Adaptation of plants to drought stress includes deeper root and higher root density in deep layers (Benjamin and Nelsen, 2005; Zeid and Shedeed, 2006). Significant correlations between drought stress and DNA methylation in root tips have been shown by Labra et al. (2002). Sultan (2003) indicates that the root growth rate in offspring altered by parental growth conditions may be helpful in offspring establishment.

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Knowledge of the range of interactions between plants and the environment on a transgenerational scale is important both for understanding the functioning of natural ecosystems and for cultivation of crop varieties with increased resistance to abiotic stresses (Nicotra et al., 2010, Walter et al., 2106). The existence of mechanisms of the stress imprint and the level of the resulting alterations in plant growth may lead to increased stability of agricultural ecosystems by increasing their resistance to weather perturbations (Walter et al., 2013). Currently, there are no reports concerning alterations in the root system morphology and topology induced by stress experienced by the plant maternal generation, despite the key role of the plant root system in response to drought. These changes may be highly important not only in increasing the accuracy of prediction of crop adaptation to climate change, but also in proper design of scientific experiments on plant response to stresses. Therefore, the aim of the study was to analyse changes in barley growth, morphology of the root

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system, and overall plant functioning that may be induced by drought stress perceived by maternal plant generations.

2. MATERIAL AND METHODS 2.1. Growth conditions, plant, and soil material The maternal generation of spring barley (Hordeum vulgare, cv. Sebastian) was grown in greenhouse conditions (E 21o 39’, N 51o 21’) in 22-cm diameter and 20 cm-high pots filled with light loamy soil. The average conditions during the growth period were as follows: light intensity 415 mol photons m-2s-1, 10.5 h light period during plant growth and 11.6 h during the stress, air temperature 19,4oC, and relative air humidity 68,5%. The soil was fertilized in accordance with the soil concentrations of N, P, K, S, Mg, B, Cu, Mn, and Fe and plant requirements. The soil size fractions determined with a laser diffractometer were as follows: 7.13% of clay, 70.93% of silt, and 21.94% of sand (Ryżak and Bieganowski, 2011) Mastersizer 2000 (Malvern, UK).

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The maternal generations were grown at (i) optimum soil water potential throughout the vegetation period (O) or at (ii) optimum soil water potential with drought stress (DS) induced by withholding of watering at the full flag leaf stage (BCH 45-47), 50 days after sowing. Soil water potentials in the treatment with optimum water potential and at water deficit in drought treatment were equal to -16 and -320 kPa, respectively. The soil water potential was maintained by drip irrigation steered by a computer system (Adviser Company, Poland) and corrected using an electronic balance. The water stresses in the maternal generation lasted for 14 days (when the soil water potential was within a range of – 320 to -260 kPa); afterwards the water potential in the treatments was increased to -16 kPa by watering to the specified weight. Seeds obtained from a self-pollinated maternal generation grown at two water regimes were used to produce a progeny generation in growth chambers (KK1200, POL-EKO, Poland). The conditions during the progeny growth were as follows: day and night temperatures 24oC (14 hours) and 18oC, respectively, photosynthetic 10

active radiation 240 mol photons m-2s-1, and relative air humidity 60 %. Pots with a diameter of 75 mm and a height of 250 mm were filled with the same soil as that used during the growth of the maternal plants. Calculated soil doses were packed into 2.5 cm deep soil layers using a cylindrical piston with a diameter equal to the inner diameter of the PCV cylinders to obtain the bulk density 1.40 Mg m-3. The progeny was sown 6 months after harvesting of the seeds. The initial number of 5 seedlings per one pot was reduced to one soon after germination. The progeny with and without drought stress applied in the maternal generation were grown in two different conditions: optimum soil water potential (DS-O and O-O) or water potential decreasing from initially optimal soil water availability to -320 kPa at 18-21st DAS, corresponding to intense drought stress (DS-DS and ODS). The water stresses in the progeny generation lasted for 8-9 days (when the soil water potential was within a range of – 320 to -260 kPa). The soil water potential was maintained at the specified level by daily watering to the specified weight. 2.2. Seed mineral analysis 11

In order to determine the content of elements, the seeds obtained from maternal generation were subjected to digestion in a microwave mineralizer (Berghoff Speedwave Four) in Teflon vessels DAP 100. A mixture of 8 ml 65% HNO3 and 2 ml 30% H2O2 was used for mineralization. The resulting solutions were analysed by ICP-OES (Thermo Scientific iCAP Series 6500) equipped with a charge injection device (CID) detector and TEVA software. A multi-element standard solution for ICP-OES containing Fe, Mg, and P in 5% HNO3 (1000 ppm, Analityk-46, Inorganic Ventures, US) and a multi-element standard solution containing Mn and Zn in 10% HNO3 (100 ppm, Analityk-47, Inorganic Ventures, US) were used for standardization (Maillard et al., 2015). 2.3. Analysis of roots and shoots The impact of the water stress on the maternal generation was evaluated by measurements of photosynthesis and leaf conductance. The measurements were performed on the first fully developed leaf in a leaf cuvette of the gas exchange system GFS-3000 with the standard

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measuring head 3010-S (Walz, Germany) at 24oC, 60% RH, light intensity 234 molm-2s-1, and 400ppm CO2. Analyses of progeny roots and shoots were performed at day 21 after sowing. Intact root systems were gently washed on a fine sieve to remove soil. Then, the roots were spread out in the cuvette with a thin layer of water and scanned at 1200dpi using a flatbed scanner (Epson Expression 10000 XL, Seiko Epson Corp., Japan). Images were analysed using WinRHIZO (Win RHIZO Pro v. 2007d, Regent Instrument Inc., Canada) for the average root length and diameter, root length, volume, and number of tips (T) in the diameter classes. Dry mass of progeny shoots and roots was determined after drying at 105oC until constant mass was reached. The transpiration in the maternal and progeny generations was calculated by subtraction of daily loss of mass in pots with and without plants at the same soil moisture. The proline concentration in the first fully developed leaves was determined on day 21 after sowing with methods described by Bates et al. (1973). 2. 4. Statistics 13

Generally, each measurement was performed in twelve replications unless indicated otherwise. The mean and the standard error of the mean were used to compare the data. Prior to ANOVA, the data distribution was analysed for normality with the Shapiro-Wilk test. Statistical analysis of the results was done using confidence tests with an ANOVA analysis of variance (STATISTICA 12). The means were compared with the HSD Tukey test.

3. RESULTS 3.1. The effect of drought on the barley maternal generation and properties of the seeds The drought induced during the growth of the maternal generation severely restricted plant functioning, as indicated by measurements of photosynthesis, transpiration, and leaf conductance during the maximum intensity of the stress at the flag leaf stage (Table 1).

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The insignificant reduction of grain yield was accompanied by a slight increase in the average mass of seeds in response to the drought (Table 2). Chemical analysis, however, showed significant (p<0.01) differences between seeds from plants grown in the different conditions in the concentration of all analysed mineral nutrients resulting from differences in water availability during the growth of the maternal generation. Phosphorus was the only nutrient with a lower concentration in the seeds of the stressed maternal generation. All the other nutrients exhibited a significant increase in their concentrations, i.e. total nitrogen by 14.6%, magnesium by 9.4%, iron by 24.7%, and zinc by 13.0%, in the seeds of the stressed plants.

3.2. The transgenerational effects on plant morphology and root topology The differences in water availability during the growth of the progeny, irrespective of the water conditions in the maternal generation, resulted in statistically significant differences (p<0.0010.01) in all analysed indicators of growth and functioning, including 15

shoot and root biomass, root morphology and topology, transpiration, and proline concentration (Table 3). However, to pursue the aim of the research, we focused only on the differences in the progeny generation between plants that were growing in the same conditions but were characterised by a different stress history during the maternal generation. The summary of the interactions between the maternal and offspring environments in barley, analysed by ANOVA, is shown in table 3.

Water availability in the maternal generation affected the growth of shoots and biomass distribution between shoots and roots of the progeny, as shown in figure 1. Generally, both these indicators of the plant growth rate in the optimum soil water conditions and in the stress were lower in treatments affected by the drought stress in the maternal generation. However, these alterations in plant growth were significant only between the progeny generation growing at drought stress (O-DS and DS-DS). 16

The dissimilarities in the plant growth conditions applied during the growth of both subsequent generations influenced various root characteristics. Plants without water stress in the maternal generation, grown in the optimum soil conditions (O-O), had the longest roots (total root length 466.6 cm); the shortest roots, i.e. 235.5 cm, were noted for the O-DS treatment (Table. 4). However, no significant differences in the total root length were observed in the progeny grown in the same conditions. Statistically significant differences in the average root diameter (p<0.001) and the root surface area (p<0.05) were noticed in the progeny grown at drought. Both these parameters were lower in the progeny exposed to drought stress in the maternal generation (DS-DS) than in the O-DS treatment.

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Analyses of the root length distribution in the various diameter classes (Fig. 2) revealed some additional differentiation in the morphology of the studied plants. Irrespective of the progeny growth conditions, plants exposed to drought stress in the maternal generation had a higher proportion of fine roots than plants growing in the optimum conditions in the maternal generation. This effect was statistically significant for the root diameter below 0.25 mm only for the progeny grown at water deficit. Additional significant differences in root distribution were noted for thicker roots. Roots with diameters in the range of 0.15-0.30 and 0.65-0.75 in plants growing in the optimum conditions and 0.55 – 0.80 mm in the drought treatment were longer in the offspring of plants that had not been exposed to drought stress.

Analyses of an intact root system showed that the number of root tips (Fig. 3), a trait related to root branching, was significantly affected by water availability during the growth of the maternal 18

generation. Offspring growing at drought stress with drought stress in the maternal generation (DS-DS) had the lowest number of root tips with diameters above 0.5 mm in all treatments, including plants growing in the same conditions but differing in stress experienced by the maternal generation (O-DS). This shows that, at drought stress, barley plants affected by drought in their previous generation produced a lower number of thick lateral roots, consuming high amounts of assimilates, than plants growing in the same conditions, but without the exposure to drought in the previous generation. This observation is in agreement with the measurements of the root volume (Fig. 4). The lowest root volume was noticed for fractions with a diameter above 0.5 mm in the DS-DS treatment, but the differences between the two drought treatments were not significant. Significant differences were noted between plants grown in the optimum conditions; they were characterised by the highest volume of roots with a diameter ≥0.5 mm. The drought experienced by the maternal generation of the DS-O treatment significantly (p<0.05) reduced the volume of the thickest root fraction (Fig.4. C), compared to O-O. The 19

changes observed within the roots with a diameter above 0.5 mm as well as in the volume and number of tips are important, as this fraction accounted for more than 80% of a volume of the whole root system, irrespective of the treatment. 3.4. Transpiration and proline concentration

Data on transpiration (Fig. 5 A), which is closely related to daily plant water uptake, calculated for the 21st day of the experiment show that plants growing in the optimum soil water conditions transpired approximately seven times more water than plants growing in dry soil, irrespective of the treatment. In both treatments with drought stress in the previous generation, plants transpired by 21.2% (DS-O) and 5.4% (DS-DS) lower amounts of water from soil than plants in the corresponding The proline concentration in the leaves (Fig. 5 B) increased almost up to two times in plants growing at the water stress (O-DS and DS-DS), compared to the optimum conditions. The concentration of 20

this amino acid was slightly lower in the treatments with the drought stress imprint, but the differences were not statistically significant.

4. DISCUSSION Intense abiotic stress may induce many changes in plant functions, including epigenetic modifications, morphological changes, and accumulation of protective proteins and metabolites, which increase the probability of survival in adverse conditions. These effects of exposure to stress have transgenerational consequences as well (Mirouze and Paszkowski, 2011, Mune-Bosh and Alegre 2013). The underlying mechanisms involve epigenetic modifications resulting from differential DNA methylation (Johannes et al., 2009, Herman and Sultan, 2011; Verhoeven and van Gurp, 2012). However, the transgenerational impact of drought on early plant growth can also be at least partly explained by seed provisioning and changes in the concentration and amount of mineral nutrients stored in seeds (Zas et al. 2013, Larios et al., 2015, Crisp et al., 2016).

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The concentration of mineral nutrients in seeds is affected by water availability, especially during reproductive stages (Samarah et al., 2004). The significant differences in the measured concentration of seed minerals (Table 2) may be partly responsible for the observed transgenerational effects of drought stress. Generally, the increasing concentration of mineral nutrients, which is an effect of water deficit during seed development, is consistent with observations reported by Farahani et al. (2011). Their experiment on barley demonstrated that water deficit stimulated accumulation of mineral nutrients in seeds. In general, the importance of macro and micronutrients for root growth follows the order P > Mg > N > Zn > Fe (Fageria and Moreira, 2011). At low availability of each of the nutrients, reduced root growth is observed, with the exception of P. Phosphorous affects strongly the growth of the plant root system (Wu et al., 2014) but its deficiency has been shown to stimulate the growth of thin roots (Niu et al., 2013). It is well known that seed-derived nitrogen and phosphorus account for a significant fraction of plant biomass at early growth stages, helping during seedling establishment and early growth, 22

especially when nutrient availability is limited by environmental factors (Muraoka et al., 1997, Ding et al. 2010). The significantly lower amount of P in the seeds originating from the stressed plants could be one of the factors increasing the root-to-shoot mass ratio and causing an increase in the length of thin roots. These plant characteristics have been described by Li et al. (2007) as adaptive traits facilitating growth in an environment that is low in P. On the other hand, higher amounts of P stored in seeds originating from unstressed plants may decrease plant P deficit during early plant growth, as shown by Nadeem et al. (2013). In our experiment, phosphorous was at an optimum concentration in soil with an optimum moisture level but its availability decreased with the increasing intensity of drought (He and Dijkstra, 2014). Nitrogen, i.e. a major component of seed proteins, present at substantially higher concentrations in seeds from drought-stressed plants was reported to be affected by environmental conditions, increasing plant plastic response to adverse conditions through changes in plant morphology and physiology (Bourgeois et al., 2009). The important role of seed-derived nitrogen and phosphorus in 23

seedling establishment was also stressed by Lamont and Groom (2013); both nutrients ensure sufficient carbon supply for deeper rooting, which helps young plants to avoid drought. In our experiment, the progeny characterised by the lowest shoot biomass (DS-DS) were able to maintain the rooting depth at a level noted for plants with significantly higher shoot biomass. Studies conducted by Germain et al. (2013) on a grass species Avena barbata yielded similar findings about the impact of water stress on the parental generation, i.e. increased seed mass and seed nitrogen concentration improving seedling performance in comparison to that in plants from wet-grown parents. The decrease in the progeny shoot dry mass (Fig. 1 A) in response to drought in the maternal generation noted for the DS-O and DS-DS treatments might be helpful in plant adaptation to adverse environmental conditions. Li et al. 2009 observed an opposite response of P deficiency in rice shoots and roots. Shoot growth was inhibited and at the same time root growth was stimulated at P deficiency. This observation may partly explain the lower shoot 24

biomass accompanying the slightly longer roots in DS-DS (lower seed P concentration) than O-DS (higher seed P concentration). The alterations in offspring size and development are one of the observed transgenerational effects in plant response to drought (Herman and Sultan, 2011). Studies on grasslands conducted by Wang et al. (2007) have shown that biomass-dependent resistance to drought is an important but underemphasized phenomenon. In field conditions, water scarcity during drought may result in increased competition for water, especially in high biomass communities. It was also shown that the important variation in grassland biomass was strongly associated with precipitation rates in the past years (Wiegand et al., 2004). According to Herman et al. 2012, plastic responses to limited resources experienced by the maternal generation can “preadapt” the offspring for functioning under the same stresses. Similarly, a decrease in the SDM/RDM ratio (Fig 1 B) is regarded as a positive indicator of adaptation to drought (Nicotra et al., 2010). In our experiment, such adaptation was significantly enhanced (lower value of SDM/RDM) in plants whose maternal generation had been 25

exposed to drought. Water shortages induce alterations in partitioning of assimilates between shoots and roots in favour of roots, which allows increasing the rooting depth and acquiring water from deeper soil (Nosalewicz and Lipiec, 2014). Our findings are important, as they describe transgenerational effects of drought that are ascribed in the available literature mostly to only one plant generation. Walter et al. (2013) underline that changes in the SDM/RDM ratio is one of the morphological responses with implications for future stress. Increased contribution of thin roots in plants affected by drought in two subsequent generations (DS-DS) allows efficient uptake of mineral nutrients. The efficiency of thin roots arises from the reduced use of nutrients and carbohydrates, compared to that of thick roots (Eissenstat and Volder, 2005, Zhang et al., 2012). The root system architecture is also important in controlling soil water extraction during drought; it is controlled by genes but is also strongly modified by environmental factors. Epigenetic

mechanisms

may

also

be

involved

in

transgenerational effects of abiotic stresses on root growth and 26

morphology, as shown in many recent studies (Boyko et al. 2010, Agboola et al., 2012, Bian et al. 2013, Cortijo et al., 2014). However, the precise role of these mechanisms is still debatable. There are no studies concerning transgenerational effects of drought directly on plant transpiration. Tricker et al. (2013), however, have demonstrated that the response of Arabidopsis thaliana stomatal genes to low relative humidity related to plant water use were heritable but the relation of the observed plant response was not easy to explain as improvement of plant resilience to abiotic stress. The limited volume of soil in the experiment could possibly be a reason why the observed transgenerational effects of drought, e.g. the differences in biomass distribution between shoots and roots and root properties (surface area), did not affect transpiration. However, the higher total root length accompanied by the lower shoot dry mass, as noted in DS-DS in comparison to O-DS, may be a factor helping plant in efficient water use. Proline accumulation during drought is a typical plant response to water stress (Seki et al., 2007, Mohammadkhani and Heidari, 2008; 27

Man et al. 2011). It allows adjusting many plant functions, i.e. cell turgor and stomatal opening, thereby increasing plant tolerance to drought. Zhang et al. (2013) demonstrated that a progeny of a stressed rice generation (osmotic stress) accumulated a higher amount of proline than a progeny of an unstressed generation. However, the transgenerational effect of proline accumulation in response to water deficit was not observed in our experiment. Tucic et al. (2011) have summarised various studies and reported that plant morphological adaptive diversity is not always accompanied by biochemical adaptation. Kinoshita and Seki (2014) suggest that future research should provide insight in the role of stress memory in crop adaptation; however, until now, investigations have been conducted mostly on model plants. The plasticity of barley described in this study, which may potentially contribute to an increase in the survivability of seedlings in an adverse environment, is an important consequence of the transgenerational plant responses to water deficits. Based on the presented results, it seems that the observed variability of plant 28

response to single stress regarded often as an “environmental noise” in many experiments (Mune Bosh and Alegre, 2013) might be decreased by taking into account stresses experienced by previous plant generations. Our results show that the intense water stress occurring at the flag leaf stage significantly affects the growth of a subsequent barley generation. The alterations in plant morphology and topology in response to the stress experienced by the maternal generation were evident to a greater extent in the progeny exposed to drought than daughter plants grown in optimum conditions. These results may have implications in prediction of responses of crops and ecosystems to climate change and in proper design of scientific experiments on plant responses to drought.

Acknowledgments The work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought.”

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Fig. 1. Shoot dry mass (SDM) and shoot-to-root dry mass ratio (SDM/RDM) of barley progeny grown at different water regimes during two subsequent plant generations. O – optimum soil water potential; DS - drought stress. The growth conditions of progeny generation are indicated on the horizontal axis. Different colours indicate water availability during the growth of the maternal generation. The values are the means of 12 replications ±S.E. * indicates a significant effect of the maternal environment (p<0.01). Fig. 2. Root lengths within the root diameter classes of the progeny generation grown at optimum soil water (A) and at drought stress (B) affected by water availability during maternal growth indicated by different colours. The values are the means of 12 replications ±S.E, * indicates significant differences among treatments at p<0.05, ** at p<0.01.

Fig. 3. Number of root tips in the root diameter classes of the progeny: D≤ 0.05 mm (A); 0.05
in Fig. 1.

43

Fig. 4. Root volume in the root diameter classes: D≤ 0.05 mm (A); 0.05
Fig. 5. Transpiration (A) and proline concentration (B) in leaves of progeny affected by different water availability levels in two subsequent plant generations. The values are the means of 12 (transpiration) or 4 (proline) replications ±S.E. No significant effects of the maternal environment on transpiration and proline concentration were observed; the effect of progeny environment is presented in Table 3. Explanations of abbreviations as in fig. 1.

44

B

Fig 2.

45

0

Root diameter (mm)

* *

>0.95

0.90-0.95

0.85-0.90

>0.95

0.90-0.95

0.85-0.90

0.80-0.85

* *

0.80-0.85

80

0.75-0.80

0.70-0.75

0.65-0.70

** ** *

0.75-0.80

*

0.70-0.75

** 0.55-0.60

O

0.65-0.70

6

0.55-0.60

0.50-0.55

0.45-0.50

Progeny

0.50-0.55

** ** ** ** * 0.40-0.45

8

0.45-0.50

A 0.35-0.40

DS

0.40-0.45

0

0.35-0.40

SDM/RDM

0.04

0.30-0.35

0.25-0.30

SDM (g)

*

0.30-0.35

0.25-0.30

0.20-0.25

80

0.15-0.20

O

0.20-0.25

0.10-0.15

0.05-0.10

<0.05

Root length (cm) 0.08

0.15-0.20

0.10-0.15

40

0.05-0.10

<0.05

Root length (cm) 0.12

O (maternal) DS (maternal) O (maternal) DS (maternal)

*

4

2

0 DS

Progeny

Fig. 1.

O (maternal) DS (maternal)

40

0

Root diameter (mm) O (maternal)

DS (maternal)

DS (maternal)

O

2500 2000 1500 1000 500 0

Number of tips

Number of tips

Number of tips

O (maternal)

5000 4000 3000 2000 1000 0

DS

O

Progeny

50 40 30 20 10 0 O

DS

DS Progeny

Progeny

A

*

B

C

Fig 3.

DS (maternal)

0.0006 0.0004 0.0002 0 O

DS

0.015

*

0.01 0.005 0 O

Progeny

A

0.02

Root volume (cm3)

Root volume (cm3)

Root volume (cm3)

O (maternal) 0.0008

C

46

DS Progeny

Progeny

Fig 4.

*

O

DS

B

0.1 0.08 0.06 0.04 0.02 0

Proline concentration (g gDW-1)

Transpiration (g H2O d-1)

O (maternal) DS (maternal)

15 10 5

O (maternal) DS (maternal)

15 10 5 0

0 O

O

DS

DS Progeny

Progeny

A

20

B

Fig 5.

47

Table 1. Photosynthesis rate (Pn) transpiration (E), stomatal conductance (GH2O) at the time corresponding to the maximum intensity of the stress in the maternal generation Water availability

Pn

E

(mol m-2s- (mmol m-2s-

(g H20 d-1)

1)

1)

(maternal) Optimum

GH2O

7.07 a (0.35) 138.50

a

8.00 a (0.18)

b

1.48 b (0.05)

(48.61) Drought stress

-0.47 (0.27)

b

16.30 (1.27)

Different letters indicate significant differences (p<0.001) between treatments; numbers in brackets indicate standard error, n=6.

48

Table 2. Selected properties of seeds obtained from the maternal generation grown in the optimum conditions and at drought stress. Water

Grains Mass of

Concentration (n=6)

availabili

per

1000

Ntot

ty

one

seeds

(% DM)

(materna plant, l)

P

Mg

Fe

Zn

(ppm dry matter)

(g)

n=60

Optimu

19.36 57.67a

1.92 a

4712 a

1354 a

31.77 a

38.10 a

m

a

(1.08)

(0.05)

(16)

(8)

(0.26)

(0.49)

Drought

17.06 58.15a

2.20 b

4613 b

1482 b

39.63 b

43.04 b

stress

a

(1.04)

(0.07)

(17)

(22)

(0.50)

(0.28)

(0.31)

(1.19) Different letters indicate significant differences (p<0.01) between treatments; numbers in brackets indicate standard error.

49

Table. 3. p values form the ANOVA analysis for the effect of drought in the maternal (M) and progeny (P) generations on various barley characteristics. Code SDM

SDM

RL

RD

RA

RV

Trans

/

Prol.

p.

RDM

D<0.05

0.05
D>0.5

0.5 M

**

**

NS

***

*

NS

**

**

NS

NS

P

***

***

***

***

***

***

**

***

***

***

MxP

NS

NS

*

*

NS

NS

*

NS

NS

NS

SDM – shoot dry mass, SDM/RDM shoot-to-root dry mass ratio, RLroot length, RD – root diameter, RA – root surface area, RV – volume of roots in the diameter classes (mm), Transp. – transpiration, Prol. – proline concentration; * p< 0.05; ** - P<0.01, ***p <0.001

50

Table 4. Selected root characteristics of barley affected by different water regimes applied in two subsequent plant generations Water availability

Length

Diameter

Surface

Rooting

(cm)

(mm)

area

depth*

(cm2)

(cm)

Maternal Proge ny O

O

DS

DS

O

DS

O

DS

466.6a

0.393a

57.92a

22.34a(1.3

(28.2)

(0.025)

(1.69)

1)

235.5b

0.523b

38.47bc

20.40a(1.3

(11.8)

(0.009)

(5.52)

5)

378.9a

0.354a

41.79b

19.34a(1.6

(44.3)

(0.027)

(5.16)

7)

245.5b

0.408a

31.46c

19.80a(1.2

(16.1)

(0.005)

(2.00)

3)

Different letters indicate significant differences (p<0.05) between treatments; numbers in brackets indicate standard error. *assumed to be equal to the length of the longest root.

51