Familial variation in Pinus leiophylla Schiede ex Schltdl. & Cham. seedlings in response to drought: water and osmotic potential

 

Variación familial en plántulas de Pinus leiophylla Schiede ex Schltdl. & Cham. en respuesta a la sequía: potencial hídrico y osmótico

 

Natalia Castelán-Muñoz¹; Marcos Jiménez-Casas^*1; Humberto A. López-Delgado³; Hutziméngari Campos-García²; J. Jesús Vargas-Hernández¹

 

¹ Postgrado en Ciencias Forestales, Colegio de Postgraduados, Campus Montecillo. Carretera México-Texcoco km 36.5. C. P. 56230. Montecillo, Edo. de México.

² Postgrado en Recursos Genéticos y Productividad-Fisiología Vegetal, Colegio de Postgraduados, Campus Montecillo. Carretera México-Texcoco km 36.5. C. P. 56230. Montecillo, Edo. de México. Correo-e: marcosjc@colpos.mx Tel.: 595 95 20246 ext. 1454 (*Autor para correspondencia).

³ Laboratorio de Fisiología-Biotecnología, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Conjunto SEDAGRO. C. P. 52140. Metepec, Estado de México.

 

Received: October 3, 2014.
Accepted: August 11, 2015.

 

ABSTRACT

The seedling variation in four families of Pinus leiophylla with different origins was evaluated regarding the reaction to drought, considering water potential variables (ψ_a), osmotic potential variables (ψ₀), components and biomass allocation. The families of P. leiophylla are located in a seed orchard of the Colegio de Postgraduados in the State of Mexico. The study was done with the purpose of identifying the genotypes resistant to water stress. After 26 days without water, 50 % of the seedlings presented permanent decay in the apex of the stem, with ψ_a = -3.35 MPa and ψ₀ = -3.23 MPa, which represented a decrease of 596 and 112 %, respectively, due to drought. The accumulation of biomass was also significantly affected (P = 0.05) in the families assessed, with the exception of the family from San Rafael. On average, the biomass of the root of the seedlings in drought was 38 % smaller than that of the seedlings under normal circumstances. The P. leiophylla families from San Juan Tetla and Santa María Atepetzingo (both from the state of Puebla) presented a weaker response to the stress imposed, whereas the family from Tlalmanalco (State of Mexico) was the most affected.

Keywords: Water stress, water potential, osmotic potential, biomass allocation.

 

RESUMEN

La variación de plántulas de cuatro familias de Pinus leiophylla de diferentes procedencias se evaluó en respuesta a la sequía, considerando las variables potencial hídrico (ψ_a), potencial osmótico (ψ₀), componentes y asignación de biomasa. Las familias de P. leiophylla se localizan en un huerto semillero del Colegio de Postgraduados en el Estado de México. El estudio se hizo con el fin de identificar los genotipos resistentes al estrés hídrico. Después de 26 días sin riego, 50 % de las plántulas presentaron decaimiento permanente del ápice del tallo, con ψ_a = -3.35 MPa y ψ₀ = -3.23 MPa, lo que representó disminución de 596 y 112 %, respectivamente, por efecto de la sequía. La acumulación de biomasa también fue afectada significativamente (P = 0.05) en las familias evaluadas, a excepción de la procedente de San Rafael. En promedio, la biomasa de la raíz de las plántulas en sequía fue 38 % menor que las plántulas sin sequía. Las familias de P. leiophylla provenientes de San Juan Tetla y de Santa María Atepetzingo (ambas del estado de Puebla) presentaron mejor respuesta al estrés impuesto, mientras que la familia de Tlalmanalco (Estado de México) fue la más afectada.

Palabras clave: Estrés hídrico, potencial hídrico, potencial osmótico, asignación de biomasa.

 

INTRODUCTION

Generally, zones to be reforested are open or clear spaces with microclimates and low levels of humidity; therefore, the seedlings in the establishment phase frequently go through water stress. In this phase, the length of the roots is less than 40 cm, and they are therefore in the ground area where the loss of water is rapid due to the infiltration of precipitation, evaporation and competition of other plant species (Su, Li, Liu, & Xu, 2014). Plants under water stress present a series of physiological changes that can indicate damage or resistance or repair mechanisms. These changes can be observed in the water potential, photosynthesis, enzymatic activity, protoplasm, osmotic adjustment and levels of endogenous hormones (Zhu, Li, & Jia, 2011). Variation studies on the response of plants to water stress can provide important information for the identification of genotypes that are resistant to drought; in addition, it could ensure success for reforestation and restoration programs because any adjustment that decreases water content could determine plant survival (Young, Boshier, & Boyle, 2000).

Pinus leiophylla Schiede ex Schlechtendal & Chamisso is an important species that produces resin, included in reforestation and preservation programs of soil due to its ability to settle in poor and stony soils and to its relative resistance to abiotic stress factors (Dvorak, Hodge, & Kietzka, 2007; Jiménez & Zwiazek, 2014; Morales, Ramírez, Delgado, & López, 2010). Although P. leiophylla presents a wide natural distribution, its population in the central region of Mexico has been reduced due to the change of soil use (Morales et al., 2010; Richardson et al., 2007).

In 1991, the postgraduate college in Forest Sciences of the Colegio de Postgraduados (CP) established in Montecillo, State of Mexico a P. leiophylla seed orchard with 180 specimens of 12 different origins from the central region of the Trans-Mexican Volcanic Belt. The specimens were selected from a progeny study for their ex situ conservation and production of germplasm resistant to adverse factors through geographical hybridization. This study was done with material obtained from the same orchard. The objective was to determine the response of P. leiophylla to extreme drought and the subsequent recovery in water potential, osmotic potential, dry biomass and biomass allocation. The seedlings correspond to four families selected for their outstanding growth, resistance to Toumeyella pinícola (an incident plague in the orchard) and high quality in seed production.

 

MATERIALS AND METHODS

Plant material

The seeds were collected in 2009 from four families from the P. leiophylla seed orchard, whose origins were: San Rafael (SR) and Tlalmanalco (TL), State of Mexico; San Juan Tetla (JT) and Santa María (SM) Atepetzingo, Puebla. Table 1 shows some characteristics of each origin. The seeds germinated (October, 2011) and were transplanted to plastic containers (310 cm³) with a mixture of peat, pearlite and vermiculite (ratio 2:1:1). The seeds grew and were kept in greenhouse conditions, watering them with tap water two times per week and fertilizing (20-20-20) every 15 days. A total of 90 seedlings of one year of age were used in total for each of the four families, all in individual rhizotrons formed with two PVC tubes (20 x 10 cm) joined with coupling bands and a net backing. A substrate from the mixture of peat, pearlite and vermiculite was used (2:1:1), adding an extended-release fertilizer.

Experimental design

The experiment was developed from December 4, 2012 to January 17, 2013 in a greenhouse at the Montecillo Campus of the CP. The experiment was designed in complete random blocks with an arrangement of divided parcels, with three repetitions. The large parcel corresponded to the humidity level in the substrate and the small parcel corresponded to the family; eight treatments of the combination of the two humidity levels were assessed from the four families. For the control treatment (T), 45 seedlings from each family were kept under an optimal watering regime in order to conserve the substrate with a volumetric humidity (θ_a) between 65 and 85 %; whereas for the drought treatment (D), the other 45 seedlings had their last watering on day zero of the experiment at 95 % of θ_a. In the drought treatment, watering was suspended until 50 % of the plants presented permanent wilt signs (26 days after). When these signs were evident, the recovery watering close to 85 % of θ_a began and lasted for 17 days. The θ_a was monitored every third day with a time domain reflectometry sensor (WET Sensor, AT Delta-T Devices Ltd, United Kingdom) in the first 8 cm of the surface of the rhizotron, in one sample of the three plants per family per humidity level. The same plants were always used.

Evaluation of the variables and statistical analysis

The water potential of the stem (ψ_a) was obtained before sunrise through the pressure balance method (Scholander, Hammel, Bradstreet, & Hemmingsen, 1965) with a Scholander pressure chamber (model 3005, Soilmoisture Equipment Corp., USA). The osmotic potential (ψ₀) of the needles was calculated through the van't Hoff equation (Taiz & Zeiger, 2006):

where:

c = Solute concentration (mol·kg^-1)

R = General constant of the gases (0.00831 kg·MPa·mol^-1·K^-1)

T = Absolute temperature (K).

The solute concentration was measured at 20 °C in a vapor pressure osmometer (Vapro, Model-5520, Wescor Inc., USA) from 20 μL of needle sap. The measurements were done in four dates: day 0 (start of the experiment), day 17, day 26 (end of the drought period), and day 44 (last day of the recovery watering period). A random sample of four plants per treatment was used.

The dry biomass of each specimen comprised the root and the aerial part; these were dried on a stove at 70 °C until reaching a constant weight in an analytical scale with a 0.001 g precision (Scout Ohaus, USA). The biomass allocation was estimated with the biomass relation of the root/biomass of the aerial part (r/pa). The sampling (six plants per treatment) was carried out on the same dates as the ψ_a, with the exception of day 17 of the drought period.

All variables were analyzed with an ANOVA (P ≤ 0.05). The initial measurements were analyzed with a completely random design and the remaining sampling dates with a completely random block design with a divided parcel arrangement. Subsequently, the measurements were compared with a minimum significant difference (MSD, P = 0.05). The data was analyzed with the statistical analysis program Statistix 8 (2005).

 

RESULTS AND DISCUSSION

Water and osmotic state of P. leiophylla

Figure 1 shows the behavior of the water (ψ_a) and osmotic (ψ₀) potential in P. leiophylla during the 44 days of evaluation. At the start of the experiment, the seedlings presented a ψ_a of -0.38 at -0.43 MPa and a ψ₀ of -1.68 at -1.91 MPa. Table 2 shows that there was no statistical difference among the four families. The progressive water stress caused the gradual increase of the tension with which the water was retained in the xylem. This tension resulted in the foliar demand to replace the lost water to the atmosphere through transpiration during the day and the inability of the roots to absorb enough water from the ground (Tai & Zeiger, 2006).

On day 17, the average ψ_a of the control seedlings (between -0.44 and -0.53 MPa) was statistically equal among the families, whereas in drought conditions, the seedlings of the SM family presented a ψ_a statistically higher (less negative) than the rest of the families (Figure 1). The ψ_a of the seedlings in drought decreased 307, 373, 405 and 450 % in the SM, JT, SR and TL families, respectively, in relation to the control seedlings, indicating considerably severe water stress (Prieto et al., 2004). On the other hand, the ψ₀ was also statistically equal among families, whereas in the seedlings in drought it decreased 41, 42, 59 and 62 % in SM, SR, JT and TL, respectively, with relation to the control seedlings. On day 17, the ψ₀ had not yet decreased in such an abrupt manner as the ψ_a and both were close, which indirectly indicated that the loss of water content caused a decrease mainly in the turgor potential (Steudle, 1993). Turgor loss is extremely sensitive to water deficit; it is the first biophysical effect (Taiz & Zeiger, 2006), and in the case of guard cells, it is one of the factors that regulates the closure of stomata (Su et al., 2014).

After 26 days of treatment, the ψ_a of the seedlings in drought (between -2.6 and -5 MPa) reflected extreme water stress, considered in woody plants from -2.5 MPa (Landis, 1989). The difference of the W_a between seedlings in both levels of humidity increased significantly; the ψ_a decreased 522 % in SM, 553 % in JT, 589 % in SR and 720 % in TL compared to the control seedlings (in normal conditions). The foregoing could mean a relative resistance to drought of P. leiophylla in comparison to other conifers. The descent limit of ψ_a that the woody species resist without presenting irreversible physical damage varies with the species. In Pinus engelmannii Carr. seedlings that are five months old, decay of the apical sprout has been observed between -2.4 and -1.7 MPa (Prieto et al., 2004); while in olive varieties (Olea europaea L.) it has been registered up to 6.5 MPa after 21 days without water (Sofo, 2011). Regarding the ψ₀ in drought conditions, JT and SR presented a ψ₀ (-3.4 MPa) significantly smaller (P = 0.05) than the rest of the families (Figure 1). The ψ₀ of the control seedlings was statistically the same (P > 0.05) among the families (between -1.47 and -1.56 MPa). The decrease caused by the water deficit was 97 % for the SM family, 114 % for TL, 119 % for SR and 122 % for JT, which indicates a higher concentration of the solutes in the cells of the plants in drought. This could be due to the decrease in the cellular volume by the loss of water of the turgor pressure or the total increase of osmotically active substances (inorganic ions, organic acids, carbohydrates and amino acids) that could be associated with protective functions against the dehydration of the cell walls (Sander & Arndt, 2012). Major and Johnson (2001) found differences in the ψ₀ between families of Picea mariana Mill. trees; the most tolerant to water deficit with a higher osmotic adjustment presented lower P₀, even when there were no inter-family differences in the ψ_a. Shvaleva et al. (2005) evaluated clones of Eucalyptus globulus Labill. of 11 months of age and reported a decrease between 43 and 75 % of the ψ₀ (between -1.14 and -1.19 MPa) due to the effect of the water deficit.

The standard error of the seedlings under the drought treatment was higher in accordance to the availability of water and it decreased in the substrate. This indicates, up until a certain limit, that the stronger the intensity of the drought, the higher phenotypical variation in response to the stress between specimens of the same family (ψ_a and ψ₀) and between families of P. leiophylla (in the case of ψ₀). This behavior was also observed in E. globulus by Shvaleva et al. (2005).

On day 27, recovery watering began, and 17 days after, the last sampling procedure was done. Even though the differences of ψa were significant (P < 0.05) among the levels of humidity in all families, said potential decreased significantly in the tension units, indicating a recovery tendency. The ψa of the seedlings of the SM, TL, SR and JT families with water stress was 82, 90, 97 and 132 % smaller, respectively, than that of the control seedlings. The ψ₀ of the seedlings of the SM and SR families remained smaller (40 %) than that of their corresponding control, whereas the seedlings of the JT and TL families recovered completely.

Components and biomass allocation in P. leiophylla

Tables 3 and 4 indicate that at the start of the experiment, the seedlings of the four families of P. leiophylla had statistically the same biomass, both in the root and in the aerial part and in the r/pa relation (0.27). The values indicate that there was a smaller allocation of dry biomass for the root in comparison to the aerial part, which could be due to their growth in containers with limited volume.

The progressive water deficit of 26 days affected the accumulation of biomass in three families of P. leiophylla in drought conditions in comparison to the control (Table 5); it restricted the growth of the root (23 %) of the JT family seedlings, it facilitated the increase (36 %) of the root in the SM family and restricted the aerial part (27 %) of the TL seedlings. The SR family did not present any impact on their biomass. In the drought treatment, the JT family had a smaller biomass on the root and the SM family had a larger biomass, whereas in the control treatment, the seedlings of the SM family had a smaller root biomass. At the same time, the aerial part of the seedlings had statistically similar biomass on a familial level. Furthermore, the proportion in the biomass allocation between the root and the aerial part was not significantly affected (P < 0.05) in any of the families. In comparative studies of several species of pines, no significant changes were observed in the r/pa relation after a similar drought period (Prieto et al., 2004). However, a significant increase in the r/pa relation in favor of the radical system in P. leiophylla (Martínez, Vargas, López, & Muñoz, 2002), Eucalyptus microtheca F. Muell. (Susiluoto & Berninger, 2007), P. pinceana Gordo. (Martiñón, Vargas, López, Gómez, & Vaquera, 2010) and Quercus brantii Lindl. (Zolfaghari, Fayyaz, Nazari, & Valladares, 2013) seedlings has been reported. The increase can be caused by a longer period of water stress, which has been considered to be a resistance mechanism to drought, when maintaining a more beneficial balance between the absorption capacity and the use of water (Duan, Yin, & Li, 2005). In this study, the tendency to allocate more biomass to the root was observed in the SM family.

The results of this study were probably influenced by the period in which it was developed, since the cellular elongation of the apical meristems decreases in winter, in order to enter latency. In addition to the small evaluation period, Xu, Zhou, and Shimizu (2010) indicate that the regulation capacity through the asymmetric growth could be abruptly lost when the plants are subjected to extreme drought. This implies that there could be a soil humidity threshold in response to the biomass allocation during water stress. At the end of the recovery period, the seedlings showed a limitation in the accumulation of root and aerial biomass as a result of the previous drought period (Table 6), possibly determined by a decrease in the CO₂ assimilation rates, due to a reduced stomatal conductance (Ramachandra, Viswanatha, & Vivekanandan, 2004). Due to the previous drought period, the TL, SM, SR and JT family seedlings had a root that was 30, 36, 41 and 48 % smaller, respectively, and an aerial part that was 24, 12, 19, and 16 % smaller, respectively, in comparison to the seedlings from the control treatment. A decrease in the biomass allocation for almost all of the families, with the exception of TL, was also recorded. The difference in the r/pa relation of the JT, SM and SR in drought was 32, 27 and 28 % respectively, regarding the treatment under normal circumstances.

 

CONCLUSIONS

After 26 days without water, 50 % of the Pinus leiophylla seedlings of the four families withstood an extreme drought of up to -3.35 MPa of ψ_a without presenting any permanent wilt, showing a decrease of 112 % of the ψ₀ and 12 % in the accumulation of biomass in the aerial part, and a variable effect for each family on the root and the r/pa. The response to drought depended on the family. The JT family manifested a higher osmotic sensitivity; the SM took longer to be affected than the others, keeping the best water status at the end of the drought since it was the only one that increased the biomass of the root. On the other hand, the SR family showed the best recovery, even when the accumulation of biomass of the root and of the aerial part was limited. The seedlings of the TL family were the most affected even though they showed an efficient recovery capacity. Consequently, the JT and SM families could have the highest survival capacity in the field, under limited conditions of humidity.

 

ACKNOWLEDGEMENTS

To the Consejo Nacional de Ciencia y Tecnología (CONACYT) and the Colegio de Postgraduados for the funding given to carry out this study. To Dr. Carlos Trejo López, Dr. Armando Gómez Guerrero, Dr. Víctor M. Ordaz Chaparro and Luis Méndez Hidalgo for their counseling and the material and technical support they provided.

 

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