An efficient protein extraction method applied to mangrove plant Kandelia obovata leaves for proteomic analysis

Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

Transcriptomic Analyses Reveal the Effect of Nitric Oxide on the Lateral Root Development and Growth of Mangrove Plant Kandelia Obovata

Background and aims Kandelia obovata, a dominant mangrove species in China, produces complex buttress roots and prop roots in intertidal wetlands where high quantities of nitric oxide (NO) are produced by reducing sediments. NO, a key signaling molecule, participates in an array of plant physiological and developmental processes. However, it is unclear whether NO functions in K. obovata root system establishment. Methods Here, we used a transcriptomic approach to investigate the potential role of NO in the regulation of K. obovata lateral root development and growth. Transcript profiles and bioinformatics analyses were used to characterize potential regulatory mechanisms. Results NO enhanced K. obovata lateral root development and growth in a dose-dependent manner. RNA-seq analysis identified 1,593 differentially expressed genes (DEGs), of which 646 and 947 were up- and down-regulated in roots treated with NO donor. Functional annotation analysis demonstrated that the starch and sucrose pathway was significantly induced in response to NO. A suite of DEGs involved in hormone signal transduction and cell wall metabolism was also differentially regulated by NO. Taken together, our results suggest that a complex interaction between energy metabolism, multiple hormone signaling pathways, and cell wall biosynthesis is required for the NO regulation on lateral root development and growth in mangrove plant K. obovata. Conclusion NO appears to contribute to the formation of the unique root system of mangrove plants.

Microcosm Study on Allelopathic Effects of Leaf Litter Leachates and Purified Condensed Tannins from Kandelia obovata on Germination and Growth of Aegiceras corniculatum

Kandelia obovata (Ko) and Aegiceras corniculatum (Ac) are common and dominant plant species in mangrove wetlands in South China, which are distributed in similar tidal zones along the coastline. The present study aimed to determine the allelopathic effects of leaf litter leachates (LLLs) from Ko and their purified condensed tannins (PCTs) on the germination and growth of Ac by mangrove microcosms. Replicate pots containing five different levels of LLLs and PCTs were separately prepared and propagules of Ac were placed in each treatment. Both LLLs and PCTs significantly inhibited the germination and growth of Ac, especially at high concentrations. The final germination rates of the roots and stems and the numbers of fine roots declined continuously, while other growth indicators, including the lengths of fine roots and nutritive roots and the biomasses of roots, stems, and leaves first increased and then decreased with increasing levels. These results indicate that LLLs from the leaf litter of Ko, in particular their PCTs, exerted an inhibition effect on propagule germination and seedling growth of Ac, and the inhibitory effects were concentration dependent. This study suggested that condensed tannins from leaf litter, acting as allelochemicals, could regulate the natural regeneration of a mangrove forest.

Effects of Ecological Restoration Using Non-Native Mangrove Kandelia obovata to Replace Invasive Spartina alterniflora on Intertidal Macrobenthos Community in Maoyan Island (Zhejiang, China)

Spartina alterniflora has extensively invaded the coastline of China, including in Maoyan Island of Zhejiang Province. Ecological restoration has been conducted using non-native mangrove Kandelia obovata to replace S. alterniflora in an attempt to restore the impacted intertidal zones. To illustrate the ecological effectiveness of the restoration projects, macrobenthos communities were studied among different habitats within the restored areas, including one non-restored S. alterniflora marsh (SA) and three differently-aged restored K. obovata stands planted in 2003, 2009, and 2011 respectively (KF14, KF8, and KF6). Besides, one unvegetated mudflat (MF) adjacent to the non-restored S. alterniflora marsh and one K. obovata forest transplanted in 2006 (RKF) at a previously barren mudflat without invasion history of S. alterniflora were set as reference sites. A total of 69 species of macrobenthos were collected from Maoyan Island, and the species richness was dominated by gastropoda (23 species), polychaeta (18 species), and malacostraca (16 species). There was no significant difference between the six sites in terms of the abundance of macrobenthos, with the average values of abundance peaking in KF6 (734.7 ind m−2) and being lowest in RKF (341.3 ind m−2). The six sites had significant differences in terms of the biomass of macrobenthos. The KF8 site contained the highest average biomass (168.3 g m−2), whereas the MF site had the lowest (54.3 g m−2). The Shannon-Wiener diversity index and Pielou’s evenness index of the macrobenthos did not exhibit significant differences among the six sites. However, the results of permutational multivariate analysis of variance (PERMANOVA) revealed significant spatial differences in the macrobenthos community structure between the sites. Since KF14 shared a similar macrobenthos community structure with RKF, while representing a strikingly different structure from SA, we infer that ecological restoration using K. obovata can restore the macrobenthos community to resemble to a normally planted K. obovata forest about 15 years after restoration.

Multi-omics analyses on Kandelia obovata reveal its response to transplanting and genetic differentiation among populations

Background Restoration through planting is the dominant strategy to conserve mangrove ecosystems. However, many of the plantations fail to survive. Site and seeding selection matters for planting. The process of afforestation, where individuals were planted in a novel environment, is essentially human-controlled transplanting events. Trying to deepen and expand the understanding of the effects of transplanting on plants, we have performed a seven-year-long reciprocal transplant experiment on Kandelia obovata along a latitudinal gradient. Results Combined phenotypic analyses and next-generation sequencing, we found phenotypic discrepancies among individuals from different populations in the common garden and genetic differentiation among populations. The central population with abundant genetic diversity and high phenotypic plasticity had a wide plantable range. But its biomass was reduced after being transferred to other latitudes. The suppressed expression of lignin biosynthesis genes revealed by RNA-seq was responsible for the biomass reduction. Moreover, using whole-genome bisulfite sequencing, we observed modification of DNA methylation in MADS-box genes that involved in the regulation of flowering time, which might contribute to the adaptation to new environments. Conclusions Taking advantage of classical ecological experiments as well as multi-omics analyses, our work observed morphology differences and genetic differentiation among different populations of K. obovata, offering scientific advice for the development of restoration strategy with long-term efficacy, also explored phenotypic, transcript, and epigenetic responses of plants to transplanting events between latitudes.

In vitro Bioassay of Allelopathic Activities of a Mangrove Tree, Kandelia obovata, and Fast-growing Trees, Betula platyphylla and Populus alba, Using Protoplast Co-culture Method

Allelopathic activities of a salt-tolerant and low-temperature tolerant mangrove tree, Kandelia obovata, which grows in brackish water regions of sub-tropical areas, and two fast-growing trees, Betula platyphylla and Populus alba, which grow in the temperate area, were examined by two in vitro bioassay methods, the sandwich method using dried leaves and the protoplast co-culture method using leaf protoplasts. Lettuce root growth examined by the sandwich method, was inhibited 50% by 50 mg dried mature leaves of K. obovata. In the protoplast co-culture method, inhibition rates of cell division of lettuce protoplasts were 31% and 69% by leaf protoplasts of K. obovata at densities of 1 × 104 mL-1 and 5 × 104 mL-1, respectively. These results were compared with the inverse relationship between allelopathic activities and salt tolerance of mangrove plants of different families. B. platyphylla showed 37% inhibition by the sandwich method using dried young leaves, but only 10% inhibition at 5 × 104 mL-1 by the protoplast co-culture method using leaf protoplasts of B. platyphylla. Dried young leaves of P. alba showed 66% inhibition, but the leaf protoplasts at the density of 5 × 104 mL-1 showed highly stimulatory activity. Abscisic acid, of which contents in leaf protoplasts of three tree species varies from high to low in relation to salt tolerance and recalcitrance of tissue culture, was discussed as a putative allelochemical.

Microcosm Study on Allelopathic Effects of Leaf Litter Leachates and Purified Condensed Tannins from Kandelia obovata on Germination and Growth of Aegiceras corniculatum

Kandelia obovata (Ko) and Aegiceras corniculatum (Ac) are common and dominant plant species in mangrove wetlands in South China, and distribute in the similar tidal zones along the coastline. The present study aimed to determine the allelopathic effects of leaf litter leachates (LLLs) from Ko and their purified condensed tannins (PCTs) on the germination and growth of Ac by mangrove microcosms. Replicate pots containing five different levels of LLLs and PCTs were separately prepared and propagules of Ac were placed in each treatment. Both LLLs and PCTs significantly inhibited the germination and growth of Ac, especially in high levels. The final germination rates of roots, stems, and the number of fine roots declined continuously while other growth indicators, including the lengths of fine roots, nutritive roots, the biomasses of roots, stems, leaves, increased firstly and then decreased with increasing levels. These results indicated that LLLs from the leaf litter of Ko, in particular, their PCTs exerted an inhibition effect on propagule germination and seedling growth of Ac, and the inhibitory effects were concentration dependent. This study suggested that condensed tannins from leaf litter, acting as allelochemicals, could regulate the natural regeneration of a mangrove forest.