Introduction

Water availability is one of the essential factors determining crop yield (Stewart and Lal 2018). In the context of progressing climate change, periods with precipitation deficits are expected to occur more often (Pokhrel et al. 2021). There is no doubt that this will affect the metabolic functions of plants (Fàbregas and Fernie 2019), photosynthesis (Zargar et al. 2017) and, eventually, their growth (Attia et al. 2015) and productivity (Trethowan and Reynolds 2007). Therefore, to maintain crops in dry regions, it is necessary to not only obtain cultivars with increased tolerance to drought or an ability to use water more efficiently (Gadzinowska et al. 2019), but also cultivars with a high potential of regeneration during rehydration after drought stress (Hura et al. 2015, 2018; Ostrowska et al. 2023a, b). Efficient regeneration, e.g., of cereals, after drought means fast recovery of plants manifested by the formation of additional lateral stems with ears, which will reduce grain yield loss (Hura et al. 2019).

Oxidative stress is an important component of senescence. The degradation of chlorophyll causes an increase in the production of free radicals such as hydrogen peroxide (H2O2) (Jajic et al. 2015). A key element in the effective regeneration of plants after drought is slowing down of their senescence accelerated during the drought (Patharkar and Walker 2019). Senescence is observable in the progressing yellowing and drying of plant leaves, starting from the lowest leaves upward, toward the flag leaf (Hura et al. 2012; Ostrowska et al. 2023b). The level of chlorophyll is one of the biochemical indicators of the progressing plant senescence during drought (Munné-Bosch and Alegre 2004; Vijayalakshmi et al. 2024). A number of studies confirmed lower chlorophyll levels in dehydrated leaves of cereals (Liu et al. 2018; Gadzinowska et al. 2019, 2021; Baccari et al. 2020; Rustioni and Bianchi 2021), which was often accompanied by a lower activity of the photosynthetic apparatus (Li et al. 2019) or limited photosynthesis (Wang et al. 2019).

Research demonstrated that the accumulation of H2O2 during drought accelerates the process of senescence (Bieker et al. 2012), which reduces plant yield (Verma et al. 2004). Nonetheless, it should be emphasized that H2O2, apart from exerting toxic effects on plant cell structures, is also a regulator of many biological processes (Swanson and Gilroy 2010). It has been confirmed to regulate growth, control cell cycle and programed cell death, stimulate the activity of MAP kinases, limit pathogen invasion, modulate abiotic stress tolerance, or play an important role during wounding stress and hormone signaling (Gechev et al. 2006; Garg and Manchanda 2009; Peer et al. 2013; Huang et al. 2019; Mittler et al. 2022; Tyagi et al. 2022). The main source of reactive oxygen species (ROS) in plants are chloroplasts in photosynthetic cells, mitochondria in which the respiratory processes occur (Zhao et al. 2020), and peroxysomes indispensable for crucial metabolic reactions, such as fatty acid β-oxidation, photorespiration, the glyoxylate cycle, and generation–degradation of H2O2 (Corpas et al. 2001). Despite the importance of H2O2 in plant biology, our knowledge, particularly that of the importance of this molecule in plant regeneration after a drought stress, is still full of gaps and many functional relationships remain to be explained.

Therefore, the aim of this study was to analyze the relationships between the level of hydrogen peroxide and efficiency of the recovery during rehydration after water stress. We assumed that the drought-induced increased accumulation of H2O2 could be maintained during rehydration and, in this way, continue to stimulate, rather than slow down, plant senescence. The study involved two DH lines of winter triticale with different regeneration potential, as manifested by the formation of different numbers of lateral stems during rehydration after water stress.

Materials and Methods

Plant Materials

Two double haploid (DH) lines of winter triticale, derived from F1 hybrid “Grenado” × “Zorro” (Dyda et al. 2022) were selected for the study. “Grenado” and “Zorro” were registered by Strzelce Plant Breeders Ltd (Plant Breeding and Acclimatization Institute Group, Poland) and Danko Plant Breeders Ltd, respectively. The DH lines were obtained at the Department of Cell Biology of Institute of Plant Physiology Polish Academy of Science (IPP PAS) in Kraków by the anther culture method according to Wędzony (2003). The selected lines, GZDH88 and GZDH27, are characterized by similar growth rate and they do not produce lateral stems with ears in optimum soil water content conditions and also under drought stress. During rehydration after water stress, they produce different numbers of lateral stems with ears. The GZDH27 line usually produces three lateral stems with ears during rehydration after water stress, whereas the line GZDH88 in the same conditions usually produces one lateral stem with ear.

Plant Growth Conditions

The seeds of both lines were sown into plastic pots (3.7 l, 6 plants per pot) filled with a mixture of soil and sand (1:3; v/v). The seedlings at the 1-leaf stage were vernalized in cool chambers for seven weeks at + 3 °C (± 1 °C) and subjected to illumination of photosynthetic photon flux density (PPFD) about 150 μmol m−2 s−1, photoperiod 10 h light/14 h dark. The plants at the 3-leaf stage were transferred into greenhouse chambers (air temperature 26–28/18 °C day/night, relative air humidity about 40%, PPFD about 250 μmol m−2 s−1 at the level of the top leaf). The plants were irrigated with full-strength Hoagland’s nutrient solution (Hoagland 1948) once a week.

Drought and Rehydration Treatments

Soil drought was induced when the flag leaf was well visible and developed (BBCH scale 39). Over seven days, water content in the pots was reduced to 30–35% by ceasing to water the plant and was maintained at this level for the next two weeks. In the control pots, water content was maintained at 70–75%. The soil water content in the pots was measured gravimetrically (daily between 8.00 a.m. and 10.00 a.m.), taking into account the plant weight. After 21 days of reduced watering, the soil water content was restored to 70–75% (DH lines at the heading stage) (Fig. 1S).

Measurements

Analyses were performed on the 14th day of drought, counted from the day when water content in the pots reached the value of 30–35%, on the 5th day of rehydration in the flag leaves of the main stems (lateral stem started to come out in both lines), and on the 25th day of rehydration in the flag leaves of the lateral stems. The leaves were collected for experiments under light conditions.

Midday relative water content (RWC) was calculated from the following equation: RWC (%) = (fw − dw)/(tw − dw) × 100, where fw is the fresh weight, tw is the turgid weight (leaves were soaked in freshly deionized water for 24 h in darkness at 5 °C), and dw is the dry weight (Turner 1981).

Stomatal conductance was measured in the central part of the flag leaves, using a leaf porometer Decagon Devices SC-1 (Pullman, WA, USA).

Chlorophyll content of the flag leaves was measured in the central part of the flag leaves, using Chlorophyll Content Meter CL-01 (Hansatech Instruments Ltd., England).

Analysis of H2O2 content was performed according to Ishikawa et al. (1993). Flag leaves were homogenized in an extraction buffer (1.4 ml) containing potassium phosphate buffer (50 mM, pH 7.5), trichloroacetic acid (5%), EDTA (1 mM), and polyvinylpyrrolidone (1% w/v). The reaction mixture consisted of 2.5 ml homovanillic acid (1.25 mM), 2.5 µl of horseradish peroxidase (1380 U mg−1) and 20 µl of leaf extract. Hydrogen peroxide content was determined with a Perkin-Elmer LS 50B spectrofluorometer (Norwalk, CT, USA). The samples were excited at 315 nm and fluorescence was detected between 400 and 450 nm. The slit width of excitation and emission monochromators was set at 10 nm.

Chlorophyll fluorescence measurements were performed with a fluorometer FMS 2 (Hansatech Instruments, Kings Lynn, UK). The leaves were adapted to darkness for 25 min. The maximum potential PSII efficiency (Fv/Fm) and non-photochemical quenching (qN) were calculated according to van Kooten and Snel (1990). Photochemical quenching coefficient (qP), efficiency of excitation transfer to open PSII centers (\(F_{{\text{v}}}^{\prime } /F_{{\text{m}}}^{\prime }\)), PSII quantum efficiency (ФPSII) and the electron transport rate (ETR) were calculated as in Genty et al. (1989).

For both triticale DH lines, we analyzed the grain yield per plant and the number of lateral stems with ears developed during rehydration.

Statistical Analysis

Statistical analysis was carried out using Statistica v. 13.0 package (Statsoft Inc., Tulsa, OK, USA). The Duncan multiple range test at the probability level of 0.05 was performed to estimate the significance of differences between treatment means. Differences between two means were compared by the Student’s t test. The Pearson correlation coefficient between measured parameters was tested at a probability of p = 0.05.

Results

The values of RWC dropped notably on the last day of soil drought, to 75.3% in the GZDH27 line and 73.5% in the GZDH88 line. On the fifth day of rehydration, RWC in the flag leaves (92.0% for GZDH27, 91.2% for GZDH88) was similar to that of the control (92.6% for GZDH27, 90.6% for GZDH88) (Table 1).

Table 1 Changes in relative water content—RWC [%], stomatal conductance—gS [mmol H2O m−2 s−1], chlorophyll level—Chl [SPAD units], and hydrogen peroxide content—H2O2 [µmol g−1 (DW)] during soil drought and rehydration in two DH lines of winter triticale, GZDH27 and GZDH88
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Stomatal conductance in the GZDH27 line exposed to soil drought was limited considerably from 333.1 to 31.4 mmol H2O m−2 s−1, and in the GZDH88 line from 335.8 to 30.9 mmol H2O m−2 s−1. On the fifth day of rehydration, only for the GZDH88 line stomatal conductance was considerably lower (209.9 mmol H2O m−2 s−1) than that of the control (340.5 mmol H2O m−2 s−1) (Table 1).

The chlorophyll content in the dehydrated flag leaves of both DH lines was much lower too, and for GZDH27 decreased by 27.1% and for GZDH88 by 31.6%. Its low levels were observed also on the fifth day of rehydration in the GZDH88 line (55.5% of the control) (Table 1). Figure 1 shows the progressing senescence of the leaves collected from various parts of the main stem of both winter triticale DH lines on the fifth day of rehydration. The rate of senescence, as shown by yellowing, browning, and drying of the leaves from the bottom toward the flag leaf, was clearly faster in the GZDH88 line.

Fig. 1
figure 1

Senescence progress in the GZDH27 and GZDH88 lines on the fifth day of rehydration after drought, when both lines started to grow lateral stems. A flag leaves, B leaves sampled from the middle part of the main stem, C leaves sampled from the lower part of the main stem

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The content of H2O2 in the flag leaves increased considerably on the last day of soil drought from 0.96 to 2.10 µmol g−1 in the GZDH27 line, and from 1.02 to 2.45 µmol g−1 in the GZDH88 line. On the fifth day of rehydration, much higher H2O2 levels (2.34 µmol g−1) were also observed in the GZDH88 line in comparison with the control (0.97 µmol g−1) (Table 1).

The dehydrated flag leaves in both DH lines, in comparison with the control, showed a significant decrease in the maximum potential PSII efficiency—Fv/Fm (for GZDH27 by 1.9%, for GZDH88 by 2.6%), efficiency of excitation transfer to open PSII centers—\(F_{{\text{v}}}^{\prime } /F_{{\text{m}}}^{\prime }\) (for GZDH27 by 15.4%, for GZDH88 by 17.7%), PSII quantum efficiency—ФPSII (for GZDH27 by 45.7%, for GZDH88 by 48.1%), photochemical quenching—qP (for GZDH27 by 38.5%, for GZDH88 by 36.8%), and the electron transport rate—ETR (for GZDH27 by 55.4%, for GZDH88 by 70.0%). In the same conditions, we observed an increase in non-photochemical quenching—qN (for GZDH27 by 5.9%, for GZDH88 by 11.0%) in both DH lines. Only in the GZDH88 line on the fifth day of rehydration the activity of the photosynthetic apparatus was still much lower than that of the control (Table 2).

Table 2 Changes in the maximum potential PSII efficiency (Fv/Fm), efficiency of excitation transfer to open PSII centers (\(F_{{\text{v}}}^{\prime } /F_{{\text{m}}}^{\prime }\)), PSII quantum efficiency (ФPSII), photochemical quenching coefficient (qP), non-photochemical quenching (qN), and the electron transport rate (ETR) during soil drought and rehydration in two DH lines of winter triticale, GZDH27 and GZDH88
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During rehydration, the GZDH27 line produced approximately three lateral stems with ears (LSN), and the GZDH88 line usually had one lateral stem with ear (Table 3). The flag leaves of the lateral stems in GZDH88 plants showed higher activity of the photosynthetic apparatus during rehydration than those of the GZDH27 line. In the GZDH88 line, we observed a significant increase in the values of five parameters of fluorescence (Fv/Fm by 2%, \(F_{{\text{v}}}^{\prime } /F_{{\text{m}}}^{\prime }\) by 16%, ФPSII by 47%, qP by 29%, and ETR by 134%) and a decrease in qN (by 6%) in comparison with the GZDH27 line. We did not observe any significant differences between the examined DH lines, with regard to RWC, gS, Chl, and H2O2 (Table 3).

Table 3 Lateral stem number—LSN [LSN/plant] generated during rehydration after drought
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We found a significant correlation between the content of hydrogen peroxide in the flag leaves of the main stem on the one hand, and chlorophyll level (r = − 0.76, p = 0.0016, r2 = 0.58) (Fig. 2A) and the number of produced lateral stems with ears (r = − 0.78, p = 0.00004, r2 = 0.61) on the other (Fig. 2B). Lower levels of H2O2 in the GZDH27 line were accompanied by high chlorophyll content and higher number of lateral stems. In the GZDH88 line, high levels of hydrogen peroxide were accompanied by low chlorophyll levels and lower number of lateral stems.

Fig. 2
figure 2

Correlations between the content of H2O2 in the flag leaves of the main stem, and chlorophyll level—Chl (A) and the number of lateral stems with ears—LSN (B). White circles—GZDH27 line, black circles—GZDH88 line. The lines represent linear adjustment at a probability level p < 0.05

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Grain yield analysis showed that the soil drought significantly reduced yield in both DH lines of winter triticale (Fig. 3). On the other hand, the GZDH88 line, producing a single lateral stem with ear during rehydration, was more effective in regeneration after the soil drought and, in this case, yield losses were lower than in the GZDH27 line, which in the same conditions produced as many as three lateral stems with ears. In the case of the GZDH88 line, productivity of lateral stems considerably limited the yield loss, whereas the productivity of lateral stems in the GZDH27 line was significantly lower. In the GZDH27 line, the main stem showed much higher productivity than the three lateral stems (Fig. 3).

Fig. 3
figure 3

Analysis of grain yield in two DH lines of winter triticale, GZDH27 and GZDH88, under optimum hydration (C) and after drought (D). MS main stem, LS lateral stem. Means indicated with the same letters were not significantly different in the Duncan test at 0.05 probability level. Mean values ± SE (n = 18)

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Figure 4 shows grain from the ears of the main stems and lateral stems for the GZDH22 and GZDH88 lines. Drought and rehydration treatments revealed clear differences both in the number and size of the grains from the ears on both types of stems in comparison with the control. The grains from the ears of lateral stems in the GZDH88 line were smaller and their number was lower than in the GZDH27 line. The grains from the ears of the main stems also varied, both in size and number per ear.

Fig. 4
figure 4

Examples of grain yield from the ears of the main and lateral stems in the lines GZDH27 (A) and GZDH88 (B). C control, D drought, R rehydration, MS main stem, LS1/LS2/LS3 lateral stems, 1/2/3 grain yield for three plants of the GZDH27 and GZDH88 lines

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Discussion

There is not enough information in the literature on the physiological, biochemical, and molecular foundations of the formation of lateral stems in cereals, as well as on their productivity during regeneration after soil drought. The potential of lateral stem production during rehydration can possibly limit yield loss (Hura et al. 2015, 2019). Specifically, this applies to situations when the photosynthetic activity of leaves, including the key flag leaf, was permanently limited during drought, for instance, as a result of an advanced senescence of the main stem (Wang et al. 2017). The emergence of lateral stems during rehydration after drought requires large energy expenditure (Peng et al. 2022; Torrecillas et al. 1995). Therefore, plant recovery depends not only on the extent of damage, caused by the soil drought but also on the optimum regeneration of plants after the soil drought ceases (Couchoud et al. 2020). The term "optimum regeneration after drought" is to be understood as a sustainable distribution of primary and secondary metabolites between the repair and regeneration processes during rehydration (Hagedorn et al. 2016; Patono et al. 2022).

In this study, despite the same level of dehydration of the flag leaves in both DH lines, as assessed by RWC measurements, a high level of hydrogen peroxide was maintained in the GZDH88 line on the fifth day of rehydration (Table 1), which was accompanied by a decrease in chlorophyll level (Table 1). This indicates that drought-initiated accelerated senescence of the main stem continued also during rehydration in the GZDH88 line. Chlorophyll degradation lowers photosynthetic activity, thus limiting carbon fixation and sugar transport to the new, growing plant organs (Sultana et al. 2021). Another consequence of the accelerated senescence is lowered crop yield (Joshi et al. 2019). Also, senescence can continue during rehydration following soil drought cessation (Hura et al. 2015, 2018).

It was demonstrated in earlier papers that hydrogen peroxide affects plant senescence during drought (Prochazkova et al. 2001; Yang et al. 2023). Arabidopsis mutants characterized by overproduction of hydrogen peroxide, for example, cpr5 (CONSTITUTIVE EXPRESSION OF PR GENES5) and jub1 (JUNGBRUNNEN1), demonstrated premature senescence of the leaves (Jing et al. 2008; Wu et al. 2012). On the other hand, senescence due to water stress or plant age was delayed in the mutants with lower accumulation of H2O2, such as ntl4 (NAC WITH TRANSMEMBRANE MOTIF 1-LIKE4) and aaf-KO (ARABIDOPSIS A-FIFTEEN knock-out) (Chen et al. 2012; Lee et al. 2012). Vanacker et al. (2006) reported that higher levels of H2O2 tend to increase protein and lipid oxidation rate, leading to numerous functional disturbances typical of senescence. Moreover, it should be emphasized that hydrogen peroxide was perceived mainly as a toxic molecule, formed as a byproduct of aerobic cellular metabolism (Liao et al. 2012; Smirnoff and Arnaud 2019). Nonetheless, it is also indicated that H2O2 is an important signal molecule that regulates various developmental processes, such as programmed cell death (PCD), stomatal movements, senescence and cell differentiation, as well as plant morphogenesis (Neill et al. 2002).

In the present study, we showed that high H2O2 content in the flag leaves of the GZDH88 line was accompanied by low chlorophyll levels (Fig. 2A), and a limited number of lateral stems produced during rehydration in comparison with the GZDH27 line (Fig. 2B). Therefore, it was to be expected that the GZDH27 line, which produced up to three lateral stems during rehydration (Table 3), would have higher regenerative potential. Rather unexpectedly, the higher regenerative potential was demonstrated for the GZDH88 line producing a single lateral stem, which resulted in lower yield loss (Fig. 3). Most probably, the formation of as many as three lateral stems during rehydration required too much energy from GZDH27 plants, as the result of which both efficient production of carbohydrates and their effective allocation into the ears of lateral stems could not be guaranteed (Zavala and Ravetta 2001; Obeso 2002; Guo et al. 2021). That is why the grains of three lateral stems of GZDH27 plants were smaller in size and number (Fig. 4B) in comparison with those from the single lateral stem of the GZDH88 line (Fig. 4B). The limited productivity of the GZDH27 line was indicated also by the results of chlorophyll fluorescence measurements in the flag leaves of lateral stems, which demonstrated a significantly lower activity of the photosynthetic apparatus in this line in comparison with the GZDH88 one (Table 3). Other studies confirmed that the activity of the photosynthetic apparatus, photosynthesis efficiency, and plant productivity may be limited during rehydration after water stress due to excessive production of reactive oxygen species (Hura et al. 2015; Ostrowska et al. 2023a, b). The number of lateral stems can be associated with the regulating effect of hydrogen peroxide on morphogenesis (Neill et al. 2002; Hong et al. 2018; Liu et al. 2022). A persistent high content of H2O2 in the flag leaves of the GZDH88 line on the fifth day of rehydration, when lateral stems started to appear in both lines, could be the decisive factor why only one lateral stem was formed. An increase in the content of hydrogen peroxide during rehydration could be the result of intensified metabolic processes after cell rehydration (Weissman et al. 2005; Oliver et al. 2020). Moreover, the persistent low activity of the photosynthetic apparatus of flag leaves in the GZDH88 line on the fifth day of rehydration (Table 2) could, in our opinion, have resulted in a privileged distribution of metabolites into the lateral stems and in their limited exploitation in the main stem because of its rapid senescence.

Conclusions

To maintain triticale in dry regions, it is necessary to obtain cultivars with a high potential of regeneration during rehydration after drought stress. The two studied DH lines of winter triticale did not show any differences in their response to drought but their responses to rehydration after the soil drought were drastically different. In the GZDH88 line, rehydration did not limit the drought-induced senescence, which was manifested by a higher content of hydrogen peroxide and lower chlorophyll levels in the flag leaves, and resulted in the formation of only one lateral stem. Nonetheless, the production of three lateral stems during rehydration in the GZDH27 line reduced the yield loss to a lower degree than in the GZDH88 line. This is to be associated with high energy expenditure required by GZDH27 plants for the effective production of carbohydrates and their effective allocation into the ears of lateral stems. Hence, the formation of three lateral stems with ears during rehydration after drought does not guarantee a limitation of grain yield loss. Our study demonstrated that the formation of as many as three lateral stems with ears during rehydration is not indicative of high plant regenerative potential. It should be also underlined, that the increased accumulation of hydrogen peroxide, which had been induced by soil drought, can be maintained during rehydration, thus stimulating plant senescence. Future research is needed to evaluate a greater number of triticale genotypes under drought stress and rehydration during various stages of a plants development.