Study On Response Mechanism Of Prunus Triloba Lindl. To Alkaline Stress

The saline-alkalinization of soil is widely distributed both in the whole world and in China,and the central hill area in the Sichuan Basin is the concentrated area of alkaline soil and is the main planting region for Chinese plum?Prunus salicina Lindl.?in Sichuan Province of China.Due to the high pH value of the soil,the iron chlorosis,inhibiting the growth of plants,causing leaves fall early,and having a serious impact on fruit yield and quality,is common among plum trees in this area,which has become an important limiting factor in the development of plum industry in this area.Plum tree mainly depends on grafting to reproduce,so the alkaline tolerance of plum trees is actually dependent on the rootstock alkaline tolerance.At present,plum tree mainly uses the wild peach?Prunus persica Lindl.?as its rootstock,but the wild peach possesses a major drawback of poor saline-alkaline resistance.Prunus triloba Lindl.,another rootstock of plum tree,is a specialty and naturally salt-alkaline-tolerant plant,and it grows well in saline-alkaline soils with a pH of 8.8?0.3%salt content?,and it also has good grafting affinity with Chinese plum.Therefore,we selected P.triloba as the test material for alkaline stress treatment in our study,and observed and analyzed the microstructure,the physiological and biochemical indices,the photosynthetic characteristics,and the differentially expressed genes of its leaves under the alkaline stress,respectively.To our knowledge,this is the first physiological and transcriptomic analysis of P.triloba in response to alkaline stress.We obtained a large number of transcript sequences and data,which will enrich genomic resources for P.triloba and deepen our understanding of molecular and physiological alkaline tolerance mechanisms in P.triloba.Moreover,our results also provide new insights into understanding alkaline acclimation mechanisms in Chinese plum?P.salicina?trees.All those above will have a positive and far-reaching significance for the cultivation of new varieties of fruit trees with strong alkaline resistance and the development and utilization of alkaline soil in the future.The main results of this study are as follows:1.Effects of alkaline stress on the leaf microstructure of P.triloba.The upper leaf blades of the test materials on 0 d and 12 d after the alkaline stress were sampled to observe the blade structure through the microscope.The results showed that the leaves treated with alkaline stress had the following changes compared to the control leaves:the thickness of the blade became thicker,some large cells of the upper and lower epidermis became slightly flat;the mesophyll structure became more compact;the palisade tissue was significantly thicker,tissue cells became slender,the sponge tissue cells became slightly smaller,the organizational structure became tighter,the leaf room space was significantly reduced,the ratio of the thickness of the palisade tissue to the thickness of the sponge tissue increased;leaf stomatal density increased to a greater extent,the stomatal volume significantly became smaller,the degree of stomatal opening is reduced or in a semi-closed state;the leaf cuticle thickness increased.It can be seen that during the process of the tissue morphogenesis of the P.triloba leaves,leaf blade would change its orientation due to the change of soil condition,and form the microstructure corresponding to the alkaline stress environment,thus enhancing the ability of alkaline resistance of P.triloba.2.Effects of alkaline stress on P.triloba seedling physiological indices.The 10physiological indexes were determined using the leaf blades of P.triloba on day 0 d,3 d,6d,9 d and 12 d after the alkaline stress.The results showed that as the duration of alkaline stress increased,the relative water content?RWC?of leaves declined gradually from82.11%to 51.24,the leaf relative electrical conductivity?REC?increased gradually,the total chlorophyll content on 12 d decreased by 58.05%compared to 0 d,and the content of malondialdehyde?MDA?content increased by 135.39%compared to 0 d,which suggested that with increasing duration of alkaline stress,the cells of P.triloba leaves dehydrated,the chlorophyll degradated accelerately,and the plasma membrane permeability gradually increased.Osmotic protective substances had the following changes:the soluble sugar accumulation of leaves was significantly increased and increased with the prolongation of alkaline stress,the content of soluble protein increased first and then decreased as the duration of alkaline stress increased,the content of proline was significantly higher than that of the control,reached 4.47 times of the control at 12 d after the alkaline stress.These suggested that P.triloba accumulated these osmotic substances in its body to reduce cell osmotic potential,allowing cells to maintain normal water absorption and stabilize the structure and function of the large biomolecules.The activities of superoxide dismutase?SOD?,peroxidase?POD?,and catalase?CAT?in the leaves of P.triloba seedlings all increased with an increasing duration of alkaline stress.The biggest increase in activity was exhibited by POD,followed by SOD and then CAT,suggesting that the increase in the activity of these protective enzymes could reduce the accumulation of active oxygens in the plant and the oxidative damage induced by alkaline stress.In summary,P.triloba can be adjusted by different physiological and biochemical regulation to self-protection and adaptation under alkaline stress.3.Effects of alkaline stress on P.triloba seedling photosynthetic parameters.The photosynthetic parameters of the test materials under alkaline stress and control materials on the 12th day were measured every 2 hours from 7:00 am to 19:00 pm.The diurnal changes of net photosynthetic rate?Pn?of the control and test materials both showed significant bimodal curves,and the daily Pn of the treatment was 33.10%lower than that of the control;the change of stomatal conductance?Gs?was consistent with the diurnal variation of Pn;the change trend of the internal CO₂ concentration?Ci?was opposite to that of Pn,showing that the diurnal variation of Ci in the morning and evening was higher,and at noon was lower;the diurnal variation of transpiration rate?Tr?of the control and test materials both showed“single peak”curves,while the daily Tr of the test was lower than that of the control,the peak of the test was significantly reduced,and the peak appeared slightly ahead of time.The water use efficiency?WUE?of the test materials was higher than that of the control within one day,and the daily average of WUE of the test materials was 1.14 times that of the control.In conclusion,P.triloba adjust the photosynthesis through the degree of stomatal opening,controlling CO₂ access,under alkaline stress.In order to ensure that the Pn was not significantly affected by alkaline stress,P.triloba would try to reduce its Tr that leads to the increase of WUE.This is an important mechanism of P.triloba for adapting to adversity through changes in photosynthetic physiology.4.Effects of alkaline stress on the differentially expressed genes of P.triloba.To further understand the molecular response mechanism of P.triloba under short-term alkaline stress,we used Illumina HiSeq 2500 platform to perform a large-scale transcriptome analysis of P.triloba leaves under the alkaline stress at 0 h,1 h,3 h,6 h and 12 h,in order to identify alkaline stress-related differentially expressed genes?DEGs?and main putative simple sequence repeat?SSR?markers.Approximately 53.0 million high-quality clean reads were generated from 59.6 million raw reads,and a total of124,786 unigenes were obtained after de novo assembly of P.triloba transcriptome data.After alkaline stress treatment,a total of 8,948 unigenes were identified as DEGs.Based on these DEGs,a Gene Ontology?GO?enrichment analysis was conducted,suggesting that28 genes may play an important role in the early alkaline stress response.In addition,analysis of DEGs with the Kyoto Encyclopedia of Genes and Genomes?KEGG?revealed that pathways were significant at different treatment time points.Moreover,a total of14,073 alkaline stress-related SSR markers with 1–6 repeat motifs were identified.Similar expression patterns was found between the quantitative real-time PCR?qRT-PCR?results and the RNA-Seq data for seven alkaline-related genes,confirming the reliability of the RNA-Seq results.It was found that under the condition of alkaline stress,the expression of some genes of P.salicina could be started or strengthened to improve its resistance and adaptability to adverse environment,which was its response to the adversity at the molecular level.