Hydropriming changes temperature thresholds for seed germination of tree species from the Caatinga, a Brazilian tropical dry forest

Abstract: This study evaluates the effects of hydration and dehydration cycles (HD cycles) on seed germination of four Caatinga tree species (Anadenanthera colubrina var. cebil, Enterolobium contortisiliquum, Pityrocarpa moniliformis and Pterogyne nitens) subjected to different temperatures and determine the thermal upper and lower limits of germination. For this, seeds were subjected to 0, 1, 2 and 3 HD cycles and set to germinate at temperatures of 5, 10, 15, 25, 35, 40 and 45 °C. Germinability and t50 were calculated and the differences of these parameters were compared by two-way ANOVA. In addition, germination rate (GR = 1/t50), as well as ceiling (Tc), optimum (To) and base (Tb) temperatures were calculated to estimate the limits below or above which the seeds fail to germinate. We observed that the more HD cycles, the better the seed responds, especially at extreme temperatures. Seeds of all studied species showed To between 25 and 35 °C, Tb < 10 °C, and Tc > 40 °C. The slope of regression curves for germination rate showed that submitting seeds to HD cycles expands its thermal tolerance range, revealing that this seed pre-treatment can be efficient for species as it increases their tolerance to thermal stress.


INTRODUCTION
Knowledge on the environmental conditions that interfere with seed germination is essential for understanding the establishment of plant diversity. Among the environmental conditions that interfere with the germination process, temperature is one of the factors that most affect germinability, germination speed, and specific biochemical and enzymatic reactions that drive the entire germination process (Bewley et al., 2013).
Germination of non-dormant seeds occurs within a temperature range called cardinal temperatures, that is, ceiling (T c ), base (T b ) and optimum (T o ) temperatures at which a seed can germinate. The optimum temperature is considered the ideal temperature for the species to germinate since it results in the largest number of seeds germinated in a shorter period (Bewley et al., 2013). The optimum temperature range can be related to the species' site of occurrence, dispersion period, and plant germination strategies (Meiado et al., 2016). In the Caatinga, a Tropical Dry Forest occurring mainly in the Northeast region of Brazil, many species have optimum germination ranging from 30 to 35 °C, evidencing that these plants have higher tolerance to high temperatures compared to species from more humid ecosystems (Meiado et al., 2012;Nascimento et al., 2018;Dantas et al., 2020).
Changes in environment temperature can affect the permeability of membranes and sub or supra-optimum temperatures tend to reduce the speed of germination process and expose seeds to adverse factors for longer, reducing reproductive success (Bewley et al., 2013). Future climate scenarios predict, based on greenhouse gases emissions, higher stocasticity and lower volumes of rainfalls and increase in environmental temperatures (IPCC, 2014). These scenarios may compromise seed germination, early seedling development, and the establishment of plants in the nature. Even those species well adapted to hot and dry climates, such as the Caatinga, can be affected by future climate pessimistic scenarios, demanding in such arid environments that species become increasingly tolerant (Chen and Arora, 2013;Alvarado-Lopez et al., 2014).
In view of this scenario, new methods to increase the success of seed germination and seedling establishment have been created and improved, among them hydropriming, priming treatments, or hydration and dehydration cycles (HD cycles). These techniques consist of hydrating seeds just enough to enableinitial biochemical processes essential for germination, but not enough to promote the rootprotrusion (Alvarado-Lopez et al., 2014). Upon passing through such discontinuous hydration cycles, seeds have higher germinability in shorter period, along with the activation of some specific genes related to tolerance to environmental stresses, such as thermal stress, when seeds are allowed to germinate (Dubrovsky, 1996;Alvarado-Lopez et al., 2014).
Seeking to predict the germinative behavior of some species, researchers have resorted to experiments by using controlled conditions and models based on population parameters, such as degree-days (thermal time), which allows estimating the time required for seed germination in a given thermal regime based on germinative parameters (Cardoso, 2011;Nakao and Cardoso, 2015). Such models allow quantifying the germinative response amid temperature changes in the environment. Anadenanthera colubrina (Vell.) Brenan var. cebil (Griseb.) Altschul, Enterolobium contortisiliquum (Vell.) Morong, Pityrocarpa moniliformis (Benth.) Luckow and RW Jobson, and Pterogyne nitens Tul. are Caatinga tree species, considered early pioneer or secondary, fast growing and thus used in reforestation programs in degraded areas of the Caatinga (Queiroz, 2009). Thus, this study evaluates the effects of hydropriming, due to discontinuous hydration, on germinative behavior of these four species subjected to different temperatures. We also estimate thermal thresholds for germination by testing the hypothesis according to which a discontinuous hydration can increase the thermal range for germination by reducing T b and increasing T c .

MATERIAL AND METHODS
Four tree species of the Fabaceae family with wide distribution in the Caatinga were selected: Anadenanthera colubrina (Vell.) Brenan var. cebil (Griseb.) Altschul, Enterolobium contortisiliquum(Vell.) Morong, Pityrocarpa Journal of Seed Science, v.43, e202143004, 2021 moniliformis (Benth.) Luckow and RW Jobson, and Pterogyne nitensTul. Seeds were collected in Caatinga areas in the Northeast region of Brazil in 2014 and packed in cloth bags in a cold chamber (5 ± 2 °C), for no longer than six months, until experiments were performed. According to Köppen-Geiger climate classification system,the collection site is characterized by hot semi-arid climate (BSh), with maximum temperatures reaching more than 36 °C in dry seasons (Climate Data, 2020).

HD cycles and temperature treatments
Initially, seeds of E.contortisiliquum, P. moniliformis and P. nitens overcame their physical dormancy by immersing them in concentrated sulfuric acid (H 2 SO 4 ) for sixty, forty and five minutes, respectively. To promote HD cycles in seeds, hydration times were set based on previous studies on hydration curves, which corresponded to two hours for A. colubrina, seven hours for E. contortisiliquum, five hours for P. moniliformis, and eight hours for P. nitens. Thus, seeds were placed in trays containing two layers of germitest substrate paper and 200 mL of distilled water and incubated 25 °C during the mentioned hydration times.
The dehydration period of seeds of each species was set according to the time required by the seed to return to its initial biomass in an oven with forced air circulation at 40 °C. For P. moniliformis seeds, four hours of drying were necessary. The seeds of P. nitens dehydrated after six hours. Seeds of A. colubrina and E. contortisiliquum were dehydrated after eight hours of drying. Therefore, one HD cycle was the hydration followed by dehydration during specific times for each species. Seeds were subjected to 0, 1, 2 or 3 HD cycles, with similar dehydration periods for all cycles (Lima et al., 2018).
After undergoing the HD cycles, seeds were subjected to thermal stress treatments during germination. For each treatment, 100 seeds of each species were used, distributed in four replications of 25 seeds sown in 15-cm diameter Petri dishes containing filter paper moistened with 15 mL of distilled water. These were placed to germinate in B.O.D. (biological oxygen demand) germination chambers, in a 12-hour photoperiod (light intensity: 200 μmol.m -2 .s -1 ) and in seven constant temperature treatments (5, 10, 15, 25, 35, 40 and 45 °C). Germination was evaluated daily for 30 days, and the criterion for considering germinated seeds was primary root protrusion.

Germination parameters, modeling and statistical analysis
At the end of observations, the following were calculated: germinability (%) (arc-sin √% transformed, Marques et al., 2015) and time to obtain the germination of half the sample of germinated seeds (t 50 -days) according to the equation: where N is the final number of germinated seeds and nj is the cumulative number of germinated seeds by adjacent counts in times tj and ti, respectively, when ni < N/2 < nj) (Farooq et al., 2005).
Germination percentages for each temperature were plotted in function of time, creating accumulated germination curves adjusted for Boltzmann sigmoidal curves. The t 50 was estimated from each curve and the germination rate (GR) was calculated as the reciprocal of t 50 . The GR values were calculated separately for optimum sub and supra-temperatures (Covell et al., 1986). A linear regression analysis was performed to calculate the interception on the x-axis of both sub and supra-optimum temperatures, providing estimates of base and ceiling temperatures (T b and T c ) for germination, respectively, below and above which seeds fail to germinate (Covell et al., 1986). The interception of linear regression lines was used to estimate optimum temperature (T o ; Hardegree, 2006;Oliveira et al., 2019;Dantas et al., 2020).
The thermal time of the species that germinated at sub-optimum temperatures (θ sub ) and supra-optimum temperatures (θ supra ) were the reciprocal of the slope angle of each linear regression and were calculated according to the equations: where T is the germination temperature, T b is the base temperature, T c is the ceiling temperature, and t is the time since the imbibition started (Covell et al., 1986).
The results of germinability and t 50 were submitted to two-way ANOVA (HD cycles and temperature), and the means were compared by Tukey test. The normality of data residual and the homogeneity of variances were assessed using the Shapiro-Wilk and Levene tests, respectively, and all statistical analyses were performed using the software STATISTICA 13.0 at 0.05 significance (StatSoft, 2020).

RESULTS AND DISCUSSION
The period of germination is considered the most critical stage for survival and establishment of new plants, with this in mind researchers seek to understand the germinative behavior of species using models to quantify the seed response to some biological phenomena to better control this process (Cardoso, 2011). Figure 1 and Table 1 show, respectively, a temperature response germination model and its parameters for seeds (hydroprimed or not) of the four studied species. According to the model, seeds of all species managed to germinate in a wide temperature range (10-40 °C) and failed to do so at the temperatures 5 °C and 45 °C (    Regarding hydropriming, the more cycles resulted in better seed response, especially at extreme temperatures, both sub and supra-optimum, not having much effect when the seeds germinated at temperatures close to T o . Furthermore, the species showed different germination response pattern when subjected to extreme temperatures and to the HD cycles (Tables 1, 2; Figures 1, 2).
The temperature thresholds for seed germination were calculated from the GR plotted against temperature. The seeds of all species showed wide a thermal threshold, with an optimum temperature (T o ) between 25 °C and 35 °C, a base temperature (T b ) below 10 °C, and a ceiling temperature (T c ) above 40 °C (Figure 2). The slope of regression curves for germination rates showed seeds that undergone HD cycles, were even more tolerant to thermal stress and had increased thermal range. Parameters of curves are in Table 1: when y = 0, x = Tb and Tc (for sub and supra-optimal temperature curves, respectively). Angular coefficients of curves are θsub and θsupra (for sub and supra-optimal temperature curves, respectively). Seeds of A. colubrina showed germinability higher than 90% at all temperatures evaluated (F = 2.39, df = 4, p = 0.0610, Figure 1A) and discontinuous hydration did not confer greater higher germinabiliy in any temperature for seeds of this species (F = 0.80, df = 3, p = 0.4970). However, supra-optimal temperature threshold for germination was increased with 1 to 3 cycles of HD, and T c increased from 49.5 to 55 °C (Figure 2A).

Journal of Seed
Seeds of E. contortisiliquum subjected to HD cycles showed higher tolerance to sub-optimum temperatures (10 and 15 °C), with an increase of about 20% in seed germination (F = 8.18, df = 4, p < 0.0001, Figure 1B). Although HD cycles increased GR and altered the GR curves slope, decreasing thermal time (θ sub , θ supra ) these pre-treatments did not alter temperature limits for germination ( Figure 2B, Table 1).
Discontinuous hydration provided greater tolerance to seeds of P. moniliformis and P. nitens at all extreme temperatures evaluated, mainly after undergoing three HD cycles (F = 315.751, df = 4, p < 0.0001, Figure 1C; and F = 254.303, df = 4, p < 0.0001, Figure 1D, respectively). P. moniliformis seeds showed about 20 and 40% increase in germination at 10 and 40 °C, respectively ( Figure 1C). For P. nitens seeds, such increase corresponded to approximately 20% at the same temperatures ( Figure 1D). These two species also responded to HD cycles by increasing GR, altering the GR curves slope and, decreasing thermal time (θsub, θsupra), rather than altering temperature thresholds for germination ( Figures 2C, D; Table 1).
One of the main effects of HD cycles was to reduce the germination thermal requirements (thermal time) at sub and supra-optimum temperatures, which was observed in seeds of E. contortisiliquum and P. moniliformis (Table 1). The untreated seeds of E. contortisiliquum, germinated after 105 °C.day -1 and, after HD cycles, this was reduced to 37.2 Journal of Seed Science, v.43, e202143004, 2021 .day -1 . At supra-optimum temperatures, this change would be from 46.7 °C.day -1 to 18.5 °C.day -1 . The untreated seeds of P. moniliformis showed θsub and θsupra, respectively, 32.3 and 40.9 °C.day -1 and after 2 HD cycles required only 18.2 and 23.2 °C.day -1 . Seeds of A. colubrina however, did not show alterations in thermal requirements after undergoing HD cycles and P. nitens showed increase in thermal requirements after undergoing 1 and 2 HD cycles ( Table 1).
Seeds of all studied species were able to germinate over a wide temperature range, with optimum temperatures for seed germination at 25 (A. colubrina, P. moniliformis, P. nitens) and 35 °C (E. contortisiliquum), since, at these temperatures' germination was higher and/or faster. Previous studies have pointed out that the optimum temperature Journal of Seed Science, v.43, e202143004, 2021 for germination of tropical species occurs within a range of 20 to 35 °C and that the appropriate temperature for germination varies according to the region of occurrence of the species and the favorable time for the germination of its seeds, growth and plant development (Azerêdo et al., 2011;Oliveira et al., 2014;Ramos et al., 2018).
The results presented here suggest that the species can germinate and withstand high environmental temperatures, expanding their establishment and distribution to different habitats in the Caatinga. Regarding hydropriming treatment, the results showed that the responses of the seeds to the temperature treatments were affected by discontinuous hydration. HD cycles improved germination, mainly at extreme temperatures, both sub and supra-optimum (10 and 40 °C), not having much effects when the seeds were put to germinate at temperatures close to optimum (25 and 35 °C). These results corroborate those reported by Mendonça et al. (2005) with the species Triplaris americana L. (Polygonaceae), whose germination percentage at sub-optimum temperature (20 °C) was favored by osmotic conditioning. In addition to these authors, Jeller and Perez (2003) also found in seeds of Senna spectabilis (DC.) HS Irwin and Barneby var. excelsa (Schrad.) HS Irwin and Barneby (Fabaceae) that osmotic conditioning, in general, provided increases in seed performance under conditions of thermal stress both at a sub-optimum temperature (12 °C) and supra-optimum temperature (39 °C), revealing that this is also an efficient treatment (Jeller and Perez, 2003). Similar results showed that Urochloa sp. (Poaceae) primed seeds had their optimum temperature changed to higher temperatures and that effect of priming was more intense at non-optimum temperatures, widening its germination temperature range (Nakao and Cardoso, 2015). In addition, the authors observed that there is generally a higher increase in T c than decrease in T b which may explain the increase in optimal and ceiling temperatures of the species in this study occurring in the Caatinga ecosystem, naturally characterized by high temperatures.
The number of cycles also affected the response of seeds, and the passage through three cycles of HD provided higher germinability and shorter germination time compared to untreated seeds. This can be explained by the longer time of exposure to water, suggesting that the greater the number of cycles, the greater the progress of the initial physiological processes related to seed germination (Lima and Meiado, 2017). Hydropriming can be limited by the amount and time of exposure of seeds to water or conditioning agents. In natural conditions, seeds can undergo several short cycles of hydration and dehydration, which would lead to a greater tolerance to high temperatures at which seeds are exposed to germinate in this semiarid ecosystem (Lima and Meiado, 2017;2018a;2018b;Lima et al., 2018). Cycles of hydration and dehydration of seeds provide biochemical changes, such as mobilization of protein storage, mainly globulins and prolamins (Aragão et al., 2002), synthesis of stress proteins and late embryogenesis proteins (LEA) (Bray, 2004), and accumulation of organic solutes (Matias et al., 2015) allowing seeds to cope better with drought periods, such as the predicted by future climate scenarios (IPCC, 2014).
Seeds GR has been studied on different species subjected to several factors, such as storage time, water deficit and different temperatures, in order to obtain the germination thresholds Gomes et al., 2019;Daibes and Cardoso, 2020). In this study, the temperature thresholds, as well as the sub and supra-optimum thermal times of the species, showed that they can germinate in a wider temperature range after ondergoing HD cycles, obtaining with T b below 10 °C and T c above 40 °C. Agreeing to our results, studies on seeds native to the Caatinga reported that different species and all seed lots (from different harvests and provenances) germinated in a wide thermal range, with T b below 10 °C and/or T c above 40 °C Oliveira et al., 2019;Dantas et al., 2020).
According to Bewley et al. (2013), higher temperatures increase the water absorption and the enzymatic activities of the seeds, making them germinate more quickly, however extremely high environmental temperatures (above 40 °C) affect germination by denaturation enzymes and/or inducing water loss.
Other studies have already demonstrated the benefits of hydropriming in relation to the time required for seeds to germinate (Contreras-Quiroz et al., 2016;Lima and Meiado, 2017;2018a;2018b;Lima et al., 2018). In these works, carried out on species that also occur in the Caatinga seeds that ondergone HD cycles significantly decreased the germination time This acceleration of germination speed can be beneficial for seeds to germinate and for seedlings to establish themselves in more favorable times and not be exposed to other environmental adversities, such as water Journal of Seed Science, v.43, e202143004, 2021