Fanjuan Meng

Physiological and proteomic responses to salt stress in chloroplasts of diploid and tetraploid black locust ( Robinia pseudoacacia L.)

Salinity is an important abiotic stressor that negatively affects plant growth. In this study, we investigated the physiological and molecular mechanisms underlying moderate and high salt tolerance in diploid (2×) and tetraploid (4×) Robinia pseudoacacia L. Our results showed greater H2O2 accumulation and higher levels of important antioxidative enzymes and non-enzymatic antioxidants in 4× plants compared with 2× plants under salt stress. In addition, 4× leaves maintained a relatively intact structure compared to 2× leaves under a corresponding condition. NaCl treatment didn’t significantly affect the photosynthetic rate, stomatal conductance or leaf intercellular CO2 concentrations in 4× leaves. Moreover, proteins from control and salt treated 2× and 4× leaf chloroplast samples were extracted and separated by two-dimensional gel electrophoresis. A total of 61 spots in 2× (24) and 4× (27) leaves exhibited reproducible and significant changes under salt stress. In addition, 10 proteins overlapped between 2× and 4× plants under salt stress. These identified proteins were grouped into the following 7 functional categories: photosynthetic Calvin-Benson Cycle (26), photosynthetic electron transfer (7), regulation/defense (5), chaperone (3), energy and metabolism (12), redox homeostasis (1) and unknown function (8). This study provides important information of use in the improvement of salt tolerance in plants.

Physiological and proteomic analyses of the drought stress response in Amygdalus Mira (Koehne) Yü et Lu roots

BackgroundPlants are oftentimes exposed to many types of abiotic stresses. Drought is one of the main environmental stresses which limits plant growth, distribution and crop yield worldwide. Amygdalus mira (Koehne) Yü et Lu is an important wild peach, and it is considered an ideal wild peach germplasm for improving cultivated peach plants. Because of the loss of genetic variation, cultivated peach plants are sensitive to biotic and abiotic stresses. Wild peach germplasm can offer many useful genes for peach improvement. Responses to drought by withholding water have been studied in Amygdalus mira (Koehne) Yü et Lu roots. In this study, plants were divided into well-watered (control) and water-stressed (treatment) groups, and the treatment group did not receive water until the recovery period (day 16).ResultsSeveral physiological parameters, including root water content and root length, were reduced by drought stress and recovered after rewatering. In addition, the relative conductivity, the levels of proline, MDA and H2O2, and the activities of ROS scavenging enzymes (POD, APX and CAT) were increased, and none of these factors, except the level of proline, recovered after rewatering. In total, 95 differentially expressed proteins were revealed after drought. The identified proteins refer to a extensive range of biological processes, molecular functions and cellular components, including cytoskeleton dynamics (3.16% of the total 95 proteins), carbohydrate and nitrogen metabolism (6.33% of the total 95 proteins), energy metabolism (7.37% of the total 95 proteins), transcription and translation (18.95% of the total 95 proteins), transport (4.21% of the total 95 proteins), inducers (3.16% of the total 95 proteins), stress and defense (26.31% of the total 95 proteins), molecular chaperones (9.47% of the total 95 proteins), protein degradation (3.16% of the total 95 proteins), signal transduction (7.37% of the total 95 proteins), other materials metabolism (5.26% of the total 95 proteins) and unknown functions (5.26% of the total 95 proteins). Proteins related to defense, stress, transcription and translation play an important role in drought response. In addition, we also examined the correlation between protein and transcript levels.ConclusionsThe interaction between enzymatic and non-enzymatic antioxidants, the levels of proline, MDA, H2O2 and the relative conductivity, and the expression level of proteins in drought-treated plants all contribute to drought resistance in Amygdalus mira (Koehne) Yü et Lu.

Evaluation of genetic diversity on Prunus mira Koehne by using ISSR and RAPD markers

Peach (Prunus mira L.; 2n = 16), which originated in China, is a fruit tree widely cultivated all over the world with high economic value. Prunus mira Koehne is a wild species distributed in Tibet with special medicinal and ecological values. To conserve its breeding and germ plasm resources, two DNA-based molecular markers, inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD), were used for genetic diversity evaluations of 48 samples from three regions, including Tibet (XZ), Henan Province (HN) and Heilongjiang Province (HLJ), in this study. High levels of polymorphism were observed: 77.80% (ISSR) and 72.73% (RAPD), which indicated that ISSR and RAPD were efficient methods to detect the genetic diversity of P. mira Koehne. The samples from Tibet preserved higher genetic diversity and most genetic variations occurred among populations. There was no significant relationship observed between the genetic distance of populations and their geographic distribution. For conservation of P. mira Koehne, ex situ conservation is a suitable strategy that needs to be established in the future.

Genetic diversity of strawberry (Fragaria ananassa Duch.) from the Motuo County of the Tibet Plateau determined by AFLP markers

Strawberry (Fragaria ananassa Duch.) is one of the most important berries in the world. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic diversity of strawberry in Motuo County of the Tibet Plateau. Seventy strawberry samples grown in Motuo County were collected for AFLP analysis using 256 primer combinations. A set of 10 primer combinations produced 374 clearly identifiable AFLP loci. The polymorphism ranged from 50% to 64% with an average of 58.8%. Neis genetic differentiation index for AFLP was 0.75, and the estimate of gene flow was less than 1 (Nm, 0.41). The unweighted pair group method with arithmetic mean dendrogram and Bayesian analysis clustered 70 samples into six groups. These findings clearly indicated high genetic diversity in wild strawberry from the Motuo County. These results could lay the foundation for further breeding and conservation of strawberry.

cDNA- AFLP analysis of the response of tetraploid black locust ( Robinia pseudoacacia L.) to salt stress

Soil salinity is a major abiotic stress that limits plant growth. In this study, to understand the tolerance mechanism associated with salinity stress in tetraploid black locust ( Robinia pseudoacacia L.), 2-yearold plants were treated by salinity. cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to study the gene expression of tetraploid black locust in response to 5, 10 and 15 days after salinity stress (300 mM NaCl) treatment. One hundred and ten (110) transcript derived fragments (TDFs) with up-regulation expression in leaves of tetraploid black locust in response to salinity stress were characterized and classified into seven groups. The putative functions of these TDFs were related to energy metabolism, material metabolism, signal transduction, transcription factor, stress and defense-related proteins and transport facilitation. The expression patterns of six genes having direct or indirect relation with salt stress response were analyzed through qRT-PCR. The possible roles of these genes are discussed. These data may enhance the understanding of the salinity tolerance mechanisms in polyploid plants. Keywords : cDNA-AFLP, salt stress, gene expression, qRT-PCR; tetraploid black locust.

Comparative mitochondrial proteomic, physiological, biochemical and ultrastructural profiling reveal factors underpinning salt tolerance in tetraploid black locust (Robinia pseudoacacia L.)

BackgroundPolyploidy is an important phenomenon in plants because of its roles in agricultural and forestry production as well as in plant tolerance to environmental stresses. Tetraploid black locust (Robinia pseudoacacia L.) is a polyploid plant and a pioneer tree species due to its wide ranging adaptability to adverse environments. To evaluate the ploidy-dependent differences in leaf mitochondria between diploid and tetraploid black locust under salinity stress, we conducted comparative proteomic, physiological, biochemical and ultrastructural profiling of mitochondria from leaves.ResultsMitochondrial proteomic analysis was performed with 2-DE and MALDI-TOF-MS, and the ultrastructure of leaf mitochondria was observed by transmission electron microscopy. According to 2-DE analysis, 66 proteins that responded to salinity stress significantly were identified from diploid and/or tetraploid plants and classified into 9 functional categories. Assays of physiological characters indicated that tetraploids were more tolerant to salinity stress than diploids. The mitochondrial ultrastructure of diploids was damaged more severely under salinity stress than that of tetraploids.ConclusionsTetraploid black locust possessed more tolerance of, and ability to acclimate to, salinity stress than diploids, which may be attributable to the ability to maintain mitochondrial structure and to trigger different expression patterns of mitochondrial proteins during salinity stress.

The effects of elevated CO2 (0.5%) on chloroplasts in the tetraploid black locust (Robinia pseudoacacia L.)

Abstract Some ploidy plants demonstrate environmental stress tolerance. Tetraploid (4×) black locust (Robinia pseudoacacia L.) exhibits less chlorosis in response to high CO 2 than do the corresponding diploid (2×) plants of this species. We investigated the plant growth, anatomy, photosynthetic ability, chlorophyll (chl) fluorescence, and antioxidase activities in 2× and 4× black locusts cultivated under high CO 2 (0.5%). Elevated CO 2 (0.5%) induced a global decrease in the contents of total chl, chl a, and chl b in 2× leaves, while few changes were found in the chl content of 4× leaves. Analyses of the chl fluorescence intensity, maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), K‐step (V k), and J‐step (VJ) revealed that 0.5% CO 2 had a negative effect on the photosynthetic capacity and growth of the 2× plants, especially the performance of PSII. In contrast, there was no significant effect of high CO 2 on the growth of the 4× plants. These analyses indicate that the decreased inhibition of the growth of 4× plants by high CO 2 (0.5%) may be attributed to an improved photosynthetic capacity, pigment content, and ultrastructure of the chloroplast compared to 2× plants.

Short‐term effect of elevated CO2 concentration (0.5%) on mitochondria in diploid and tetraploid black locust (Robinia pseudoacacia L.)

Abstract Recent increases in atmospheric CO 2 concentration have affected the growth and physiology of plants. In this study, plants were grown with 0.5% CO 2 for 0, 3, and 6 days. The anatomy, fluorescence intensity of H2O2, respiration rate, and antioxidant activities of the mitochondria were analyzed in diploid (2×) and tetraploid (4×) black locust (Robinia pseudoacacia L.). Exposure to 0.5% CO 2 resulted in clear structural alterations and stomatal closure in the mitochondria. Reduced membrane integrity and increased structural damage were observed in 2× plants at 6 days. However, after 0.5% CO 2 treatment, little structural damage was observed in 4× plants. Under severe stress, H2O2 and malondialdehyde were dramatically induced in both 2× and 4× plants. Proline remains unchanged at an elevated CO 2 concentration in 4× plants. Moreover, the total respiration and alternative respiration rates decreased in both 2× and 4× plants. In contrast, the cytochrome pathway showed no decrease in 2× plants and even increased slightly in 4× plants. The antioxidant enzymes and nonenzymatic antioxidants, which are related to the ascorbate–glutathione pathway, were inhibited following CO 2 exposure. These analyses indicated that 4× and 2× plants were damaged by 0.5% CO 2 but the former were more resistant than the latter, and this may be due to increases in antioxidant enzymes and nonenzymatic antioxidants and stabilized membrane structure.

Application of RAGA-PPC model on the optimal harvest time of Puccinellia tenuiflora

In order to ascertain the optimal harvest time of Puccinellia tenuiflora in practical production scientifically, PPC (Projection Pursuit Classification) model based on RAGA (Real coding based Accelerating Genetic Algorithm) was applied. Projection direction was optimized and high-dimension data were converted into low-dimension space with PPC, value of projection indexes could be obtained. Based on these projection function values, the sequence of samples from the big to the small was gained, at the same time, subjective impact on weight matrix farthest could be avoided with PPC model. The results showed that the initial biloom stage have the biggest value of projection indexes, which was 2.2572, and its comprehensive evaluation was the best based on the data from 2007 to 2008. Puccinellia tenuiflora harvested in the initial bloom stage had higher nutritive value, higher output and lower coarse fibre content. This model offered a new method to solve the evaluating problem of the optimal harvest time of Puccinellia tenuiflora.

The effects of exotic and native poplars on rhizosphere soil microbe and enzyme activity

The relationship between tree species and soil microbial communities has attracted much attention in ecology. However, how different poplars species affect soil microbial community and soil enzymes activities are not well studied. Random amplified polymorphic DNA (RAPD) method was used to assess the effects of six plant species on soil microbial community. The results indicate that respirations were significantly higher in planted soil than that in control soil. The order of values of soil respiration rates was: eluosiyang > jiayang > xiaoqingyang > xinganyang > fenglanyang > xiaoyeyang > control. The different poplars species could change soil enzyme activities. The lowest phenol coxidase value was found for xiaoqingyang. The value of acid phosphomonoesterase was particularly high in the rhizosphere of eluosiyang, where it was 1.2 folds higher than that of control soil. In contrast to the other two enzymes, β-glucosidase activity did not differ significantly among parts of poplar species (p>0.05). From the dendrogram, cluster analysis unweighted pair-group method with arithmetic averages (UPGMA) resulted in a dendrogram with four main groups. Group I included jiayang, fenlangyang and xiaoyeyang. Eluosiyang and xinganyang were clustered into Group II. Group III included xiaoqingyang. Group IV contained control. Key words : Exotic poplar, native poplar, rhizosphere soil, enzyme activities, random amplified polymorphic DNA (RAPD).