Thermal-Biological Aspects of Seed Germination of Colubrina glandulosa Perkins Under Different Temperatures

The aim of this study was to determine the cardinal temperatures for germinating colubrina seeds, verify isothermal velocity variation based on the transition state model and calculate the ∆H variation as a function of temperature. Seeds were incubated at constant temperatures of 5, 10, 15, 20, 25, 30, 35 and 40 °C and alternating temperature from 20-30 °C in an 8-hour photoperiod. The variables analyzed were: G, PC, IVG, TMG, VMG, Fi, U, Z, CR, CPA, MSR and MSP. Arrhenius equation was linearized by logarithmic transformation, producing the graph of -RlnV × 1/T from the experimental values of velocity. A net enthalpy change (∆H) in relation to temperature was represented by the expression: ∆H = [RT(θ − T)·(Tm + TM)]/[(T − Tm)·(TM − T)]. The logarithm regression of the reaction rate on the reciprocal of the temperature fit best to the quadratic model. The distribution of ∆H with asymptotes close to Tm and TM indicated that the processes that occurred in the supra-optimal temperature range were of a different nature from those that occurred in the infra-optimal temperature range. The data showed |∆H| < 12 Kcal/mol in the optimal range and |∆H| > 30 Kcal/mol for temperatures of 10, 15 and 35 °C. The minimum and maximum temperature limits were 10 and 35 °C, respectively. Germination speed was related to temperature in a curvilinear manner. The germination process was endergonic and only occurred when energy was ≥ -38.35 Kcal/mol and ≤ 32.42 Kcal/mol.


Introduction
Colubrina glandulosa Perkins, popularly known as colubrina, is a rare heliophyte and selective hygrophyte tree species (family Rhamnaceae), which is native and distributed in South America, ranging from the coastal Brazil to Bolivia, Paraguay, and Peru.Its wood is suitable for civil and naval construction and external and hydraulic projects.The wood produces high quality charcoal and firewood.It also presents ornamental value, and is indicated for urban afforestation.Its flowers are honey-bearing and suppliers of nectar and pollen.This species has been recommended to help recover degraded ecosystems due to its rapid growth (Lorenzi, 2016).It has phytotherapeutic value, as the leaves and bark can be used as a fever reducer or for vitamin C deficiency.Carvalho (2005) classified this species in the initial secondary ecological group.
In view of the ancient logging and relictual situation of the colubrina populations, the seeds should receive special attention for conservation, and should be present in heterogeneous forests that are permanently preserved.A lack of specific information is available on the ecophysiology of seed germination of this species in the Rules for Seed Analysis (Brasil, 2009) and Instructions for Analysis of Seeds of Forest Species (Brasil, 2013).This species does not have established criteria for standardizing seedling production methods.
Temperature is one of the main environmental factors that govern seed germination, as it strongly influences both the rate of water imbibition by the seed and the biochemical reactions that determine the entire process (Oliveira, França, Torres, Nogueira, & Freitas, 2016).Consequently, temperature variations affect the speed, percentage, and uniformity of germination (Carvalho & Nakagawa, 2012).Each species has a temperature range where germination will occur and is considered optimal, where the efficiency of the process is total, and extreme limits of maximum and minimum tolerated by the seeds, above or below which, respectively, germinability cannot be measured (Bastos, Ferraz, Lima Junior, & Pritchard, 2017).Therefore, species with different geographic for germin Based on t the presen temperatur thermal in germinatio

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The isothe temperatur simulated of ±0.5 °C running w with scisso paper towe and contai The seeds germinated that the su ion period and ocess and faci hamber, at con od of 8 hours l was within the r lowed by wash rification treat ate on two shee eight (Brasil, 2 (Brasil, 2009) .The nsure , and 15 °C, the test was extended for another 7 days by transferring the seeds to the ideal temperature.The variables analyzed were: (a) Germinability: gi = (Σki = 1ni/N) × 100 (Carvalho, Santana, & Ranal, 2005); where, ni the number of seeds germinated in time i and N the total number of seeds placed to germinate.
(b) First germination count: It was performed together with the germination test by computing the percentage of normal seedlings obtained on the third day after the test started.
(c) Speed index of germination: IVG = G 1 /N 1 + G 2 /N 2 + … + G n /N n (Maguire, 1962); where, G 1 , G 2 , and G n are the number of seeds germinated in the first, second, and last count; and N 1 , N 2 , and N n are the number of sowed days at the first, second, and last count.
(d) Mean germination time: t = Σki = 1(niti)/Σki = 1ni; where, ti is the time from the start of the experiment to the i nth observation (days or hours); ni is the number of seeds germinated at time i (corresponding number or i nth observation); and k is the last day of germination.
(f) Relative germination frequency: Fi = ni/Σki = 1ni; where, ni is the number of seeds germinated per day and Σni is the total number of germinated seeds.
(g) Uncertainty index: U = -Σki = 1Filog 2 Fi ≍ Fi = ni/Σki = 1ni; where, Fi is the relative frequency of germination; ni is the number of seeds germinated at time i (corresponding number or i nth observation); and k is the last day of germination.
(h) Synchronicity index: (Primack, 1980); where, C n1,2 is the combination of seeds germinated in the ith time, and ni is the number of seeds germinated at time i.
(i) Length of aerial part and primary root: At the end of the germination test, the lengths of the primary root (from the base of the neck to the end of the primary root) and of the aerial part (from the collar to the apex of the seedling) of the normal seedlings in the experimental unit were measured using a graduated ruler.
(j) Aerial part dry mass and primary root: After the measurements, the roots and aerial part of the normal seedlings of the experimental unit were conditioned in Kraft paper bags and placed in a forced air circulating oven, regulated at 80 °C, until the samples reached constant weight (24 h).Then, dry mass was determined on a precision analytical balance (0.0001 g).
Based on the activated complex model, the graph with the coordinates y = -RlnV (V = experimental values of velocity) = A(1/T) × 10 5 , with R = 1,987 Kcal mol -1 and T in Kelvin, was constructed to explain variations in germination velocity over the entire thermal range.
From the Arrhenius equation ∂(-RlnV)/∂(1/T) = ΔH ≠ + RT, the net energy change (enthalpy) of germination activation was calculated for both the infra (V1) and the supra-optimal (V2), using the minimum (T m ) and maximum (T M ) germination temperatures as parameters (Labouriau & Osborn, 1984).Thus, in the range V1, , the net change in enthalpy (∆H ≠ ) as a function of temperature was represented by the expression: where, θ (harmonic mean of minimum and maximum temperatures) = [(2T m •T M )/(T m + T M )], and T the experimental temperature, following the physiological interpretation of the opposite signs of ΔH ≠ in the infra and supra-optimal bands of germination.
The experimental design was completely randomized, with four replicates of 25 seeds per treatment.Data were submitted to analysis of variance and the means were compared by Tukey's test at a 5% probability.A polynomial regression analysis was performed to test the linear and quadratic models for quantitative effects, and the most significant R 2 was selected.The statistical program used was Sisvar version 5.6 (Ferreira, 2011).
The stochastic model is: Y ij = μ i + ε ij (i = 1, … and j = 1, … r); where, i is the index referring to the treatment and j is the experimental unit.

Results and Discussion
The colubrina seeds germinated in the range 10 °C ≤ T ≤ 35 °C, with the minimum cardinal point at 5 °C < T < 10 °C, and the maximum at 35 °C < T < 40 °C, i.e., no germination occurred at 5 °C or 40 °C, resulting in physiological adaptation of the seeds to the environmental conditions for the species.The optimum temperature was 25 °C < T < 30 °C, which allowed high germinability at a lower germination time (TMG) (Table 1).Nevertheless, the seeds present some plasticity regarding this adaptive character, as this species occurs over several Brazilian states, in regions of transition between the Cerrado or Atlantic Forest biomes for the Caatinga.
Increasing the experimental temperature increased G, first count (PC) and germination speed index (IVG), within a certain limit, but temperatures > 30 °C caused a marked reduction in total germination until the maximum temperature was reached (35 °C) (Table 1).No seeds germinated at 35 °C, due to thermoinhibition, which can also cause thermal dormancy or loss of viability.On the other hand, a small number of seeds germinated (12%) at 10 °C during the 19 day incubation (Table 1), contributing to the proliferation of microorganisms harmful to the establishment of the seedlings.Low temperatures may have resulted in the gradual immobilization of seed reserves, gradually decreasing the percent germination (R. B. Note.Means followed by the same lowercase letter in the column do not differ from each other to a 5% probability by the Tukey test.
The Arrhenius curve is shown in Figure 2. The typical unimodal relationship between the logarithm of the reaction velocity and the reciprocal of the temperature better fit a quadratic regression model, where the decreasing part of the curve represented the supra-optimal thermal range, and the increasing part of the curve corresponded to the infra-optimal range.Based on the activated complex model and isothermal dependence of germination, the theoretical optimum temperature for velocity was 31.4 °C.
It should be emphasized that the energy barrier of activation encompasses both thermal and organizational transitions.The model assumes that an enzyme can exist in two states, such as active and inactive.According to Machado, Bortolin, Paranhos, and Silva (2016), products are formed when the enzyme is in its active state, which, in turn, is in equilibrium with the denatured or inactive form.As temperature increases, equilibrium shifts to the inactive enzymatic state.

Figur
The  4), which may extend the problem to the rest of the plant cycle, with effects on speed of development and production by area (Missio et al., 2017).According to Marcos-Filho (2015), injuries due to cooling are probably related to damage to the membrane system, because embryonic axes subjected to these conditions lose organic substances.Temperature has its main effect on the physical state of the cell membrane, particularly on lipid fluidity (Lopes & Franke, 2011).Some of the reactions that would generally culminate in the protrusion of the primary root proceeded normally at the maximum temperature, but subsequent normal seedling development did not occur (Table 4), possibly as a consequence of a lower rate of protein synthesis or other processes particularly sensitive to the temperature increase.This observation may be related to the loss of conformational structure of the enzymes at a given temperature, which also leads to loss of function or inactivation (Ataíde, Borges, & Leite Filho, 2016).
Temperature variations within the optimum range were the most adequate for seed germination and other aspects of initial development of the plant (Tables 1, 2, and 4), as there is a relationship between these temperatures and the biome where the seeds were produced.Note.Means followed by the same lowercase letter in the column do not differ from each other to a 5% probability by the Tukey test.

Conclusions
Colubrina seeds presented a wide range of tolerance to temperatures, with minimum and maximum limits of 10 and 35 °C, respectively.
The optimal temperature for colubrina seed germination was 30 °C.
Germination speed was in a curvilinear relationship with temperature.
The germination process was predominantly endergonic and occurred only when an energy ≥ -38.35 Kcal mol -1 and ≤ 32.42 Kcal mol -1 was reached.

Table 4 .
Root length (CR), length of the aireal part (CPA), root dry mass (MSR) and dry mass of the aireal part (MSP) of C. glandulosa seedlings, submitted to different temperatures