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. 2024 Jan 16;13(2):261.
doi: 10.3390/plants13020261.

Physiological and Molecular Responses of Pyrus pyraster Seedlings to Salt Treatment Analyzed by miRNA and Cytochrome P450 Gene-Based Markers

Viera Paganová  1 , Marek Hus  1 , Helena Lichtnerová  1 , Jana Žiarovská  2 , Dagmar Moravčíková  2 , Matúš Kučka  2 , Katarína Ražná  2 , Aqsa Abbas  2
Affiliations

Physiological and Molecular Responses of Pyrus pyraster Seedlings to Salt Treatment Analyzed by miRNA and Cytochrome P450 Gene-Based Markers

Viera Paganová et al. Plants (Basel). .

Abstract

Physiological and molecular marker-based changes were studied in the tissues of two-year-old Pyrus pyraster (L.) Burgsd. seedlings under salt treatment. For 60 days, 5 mL of 100 mM NaCl solution was applied to each plant per day to a cumulative volume of 300 mL in the substrate. In response to osmotic stress, the seedlings increased their water use efficiency (WUE) on day 20 of regular NaCl application and maintained a stable net photosynthetic rate (An) per unit area. Under conditions of increasing salinity, the young plants maintained a balanced water regime of the leaf tissues (Ψwl). The seedlings invested mass to their root growth (R/S), retained a substantial portion (72%) of Na+ ions in the roots, and protected their leaves against intoxication and damage. A significant decrease in the leaf gas exchange parameters (gs, E, An) was manifested on day 60 of the experiment when the cumulative NaCl intake was 300 mL per plant. The variability in the reactions of the seedlings to salinity is related to the use of open-pollinated progeny (54 genotypes) in the experiment. Lus-miR168 showed tissue- and genotype-specific genome responses to the applied stress. Polymorphic miRNA-based loci were mostly detected in the root samples on the 20th and 35th days of the experiment. The cumulative effect of the salt treatment was reflected in the predominance of polymorphic loci in the leaves. We can confirm that miRNA-based markers represent a sensitive detection tool for plant stress response on an individual level. The screening and selection of the optimal type of miRNA for this type of research is crucial. The cytochrome P450-Based Analog (PBA) techniques were unable to detect polymorphism among the control and treated seedlings, except for the primer pair CYP2BF+R, where, in the roots of the stressed plant, insertions in the amplicons were obtained. The expression ratios of cytochrome P450 in the salt-stressed plants were higher in the roots in the case of 20/100 mL and in the leaves with higher doses. The observed physiological and molecular responses to salinity reflect the potential of P. pyraster seedlings in adaptation to osmotic and ionic stress.

Keywords: DNA polymorphism; PBA technique; adaptability; miRNA-based markers; salinity; woody plant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Samples of the control (A) and salt-treated (B) two-year-old seedlings of P. pyraster at the end of the experiment.
Figure 2
Figure 2
The content and distribution of Na+ ions in the plant tissues (leaves, stems, roots) of P. pyraster grown for 60 days under salt treatment with 100 mM NaCl solution. The values above the bars indicate distribution to the plant organs as a percentage of the total Na+ ion uptake.
Figure 3
Figure 3
The water potential of leaf tissue (Ψwl) (left) and relative water content (RWC) (right) of P. pyraster seedlings measured at the beginning of the experiment (0 mL) and during the salt treatment with 100 mM NaCl solution. A multiple comparison of the means (n = 9) was performed using the Tukey honest significant difference (HSD) test. The same letters indicate insignificant differences between measurements. The solid line indicates the median, the multiple symbol (×) shows the mean. The relationship between the plant parameters and the increasing volume of NaCl intake was analyzed by simple regression.
Figure 4
Figure 4
Box plot for the leaf gas exchange parameters (gs, E, An, WUE) for seedlings of P. pyraster at the beginning of experiment (0 mL) and during the salt treatment with 100 mM NaCl solution. A multiple comparison of the means (n = 7) was performed using the Tukey honest significant difference (HSD) test. The different letters indicate significant differences between measurements. The solid line indicates the median, the multiple symbol (×) shows the mean. The relationship between the plant parameters and the increasing volume of NaCl intake was analyzed by simple regression.
Figure 5
Figure 5
MiRNA loci detected by lus-miR168-based primers in leaf and root samples of the control (C1 and C2) and treatment (V1, V2 and V3) variants. C1—1st day of the experiment, without treatment; C2—60th day of the experiment, without treatment; V1—20th day of the experiment, 100 mL of 100 mM NaCl; V2—35th day of the experiment, 175 mL of 100 mM NaCl; V3—60th day of the experiment, 300 mL of 100mM NaCl.
Figure 6
Figure 6
MiRNA-based stress-induced cDNA polymorphism detected by lus-miR168 primers. Representative figure of three biological replicates treated with 100 mM NaCl on 35th day of experiment. L—leaf sample, R—root sample. The numbers 29, 31, and 47 represent the three genotypes (biological replicates) of salt-treated seedlings.
Figure 7
Figure 7
MiRNA loci detected by cca-miR160-based primers in both the analyzed tissue of control and treated variants. M—DNA size marker, L—leaf sample, R—root sample; 12 indicates the duplicated genotype of the control variant and 32 indicates the duplicated genotype of the treated seedlings.
Figure 8
Figure 8
Comparison of expression ratios of P. pyraster cytochrome P450 in salt-stressed plants compared to control plants.
Figure 9
Figure 9
Comparison of expression ratios of Pyrus pyraster cytochrome P450 in control plants before and after the time of salt stress treatment.
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