Functions of Basic Helix-Loop-Helix (bHLH) Proteins in the Regulation of Plant Responses to Cold, Drought, Salt, and Iron Deficiency: A Comprehensive Review.

Abiotic stresses including cold, drought, salt, and iron deficiency severely impair plant development, crop productivity, and geographic distribution. Several bodies of research have shed light on the pleiotropic functions of BASIC HELIX-LOOP-HELIX (bHLH) proteins in plant responses to these abiotic stresses. In this review, we mention the regulatory roles of bHLH TFs in response to stresses such as cold, drought, salt resistance, and iron deficiency, as well as in enhancing grain yield in plants, especially crops. The bHLH proteins bind to E/G-box motifs in the target promoter and interact with various other factors to form a complex regulatory network. Through this network, they cooperatively activate or repress the transcription of downstream genes, thereby regulating various stress responses. Finally, we present some perspectives for future research focusing on the molecular mechanisms that integrate and coordinate these abiotic stresses. Understanding these molecular mechanisms is crucial for the development of stress-tolerant crops.

Physiological and Proteomic Responses of the Tetraploid Robinia pseudoacacia L. to High CO2 Levels.

The increase in atmospheric CO2 concentration is a significant factor in triggering global warming. CO2 is essential for plant photosynthesis, but excessive CO2 can negatively impact photosynthesis and its associated physiological and biochemical processes. The tetraploid Robinia pseudoacacia L., a superior and improved variety, exhibits high tolerance to abiotic stress. In this study, we investigated the physiological and proteomic response mechanisms of the tetraploid R. pseudoacacia under high CO2 treatment. The results of our physiological and biochemical analyses revealed that a 5% high concentration of CO2 hindered the growth and development of the tetraploid R. pseudoacacia and caused severe damage to the leaves. Additionally, it significantly reduced photosynthetic parameters such as Pn, Gs, Tr, and Ci, as well as respiration. The levels of chlorophyll (Chl a and b) and the fluorescent parameters of chlorophyll (Fm, Fv/Fm, qP, and ETR) also significantly decreased. Conversely, the levels of ROS (H2O2 and O2·-) were significantly increased, while the activities of antioxidant enzymes (SOD, CAT, GR, and APX) were significantly decreased. Furthermore, high CO2 induced stomatal closure by promoting the accumulation of ROS and NO in guard cells. Through a proteomic analysis, we identified a total of 1652 DAPs after high CO2 treatment. GO functional annotation revealed that these DAPs were mainly associated with redox activity, catalytic activity, and ion binding. KEGG analysis showed an enrichment of DAPs in metabolic pathways, secondary metabolite biosynthesis, amino acid biosynthesis, and photosynthetic pathways. Overall, our study provides valuable insights into the adaptation mechanisms of the tetraploid R. pseudoacacia to high CO2.

Effects of NaHCO3 Stress on Black Locust (Robinia pseudoacacia L.) Physiology, Biochemistry, and Rhizosphere Bacterial Communities.

Soil salinization has become an ecological and environmental problem that cannot be ignored. Tetraploid black locust (Robinia pseudoacacia L.) is a leguminous tree with characteristics of drought and saline-alkali tolerance. Rhizosphere bacteria are the primary functional microorganisms within the plant root system, and they play a crucial role in regulating plant growth and enhancing stress tolerance. However, there is still a lack of research on the effect of saline-alkali stress on the bacterial community structure in the rhizosphere of black locusts. In this study, we applied 0, 50, 100, and 150 mM NaHCO3 stress to diploid (2×) and tetraploid (4×) black locusts for 16 days. We used 16S rDNA sequencing to investigate the changes in the rhizosphere bacterial communities. Furthermore, we evaluated soil enzyme activity and plant physiological characteristics to explore the response of rhizosphere bacteria to NaHCO3 stress. The results demonstrated that the 4× plant exhibited superior alkali resistance compared to its 2× plant counterpart under NaHCO3 stress. Simultaneously, it was observed that low concentrations of NaHCO3 stress notably increased the abundance of rhizosphere bacteria in both plant types, while reducing their diversity. The impact of stress on the rhizosphere bacterial community weakened as the stress concentration increased. The application of NaHCO3 stress caused a significant change in the composition of the bacterial community in the rhizosphere. Additionally, alkaline salt stress influences the diversity of rhizosphere bacterial communities, which are linked to soil enzyme activities. These data will help us better understand the relationship between the dominant rhizosphere bacterial community and black locust. They will also provide a reference for further improving the alkali resistance of black locust by enhancing the soil bacterial community.

Effects of bleeding of Actinidia arguta (Sieb. & Zucc) Planch. ex miq. on its plant growth, physiological characteristics and fruit quality.

Bleeding is as particularly a serious phenomenon in Actinidia arguta and has important effects on this plant's growth and development. Here we used A. arguta to study the effects of bleeding on the growth and development of leaves and fruits after a bleeding episode. We detect and analyze physiological indices of leaves and fruit after bleeding. The result revealed that the relative electrical conductivity and malondialdehyde (MDA) of leaves increased in treatment. Nitro blue tetrazolium chloride (NBT) and 3,3-diaminobenzidine (DAB) staining revealed the accumulation of reactive oxygen species (ROS) in leaves after bleeding. The chlorophyll content and photosynthetic parameter of plants were also decreased. In fruits, pulp and seed water content decreased after the damage, as did fruit vitamin C (Vc), soluble sugar content, and soluble solids content (SSC); the titratable acid content did not change significantly. We therefore conclude that bleeding affects the physiological indices of A. arguta. Our study provides a theoretical basis for understanding the physiological changes of A. arguta after bleeding episodes and laying a timely foundation for advancing research on A. arguta bleeding and long-term field studies should be executed in order to gain insights into underlying mechanisms.

A Deacetylase CqSIRT1 Promotes WSSV Infection by Binding to Viral Envelope Proteins in Cherax quadricarinatus.

Sirtuin 1 (SIRT1), a member of the class III lysine deacetylases, exhibits powerful functional diversity in physiological processes and disease occurrences. However, the potential molecular mechanism underlying the role of SIRT1 during viral infection in crustaceans is poorly understood. Herein, SIRT1 was functionally characterized from the red claw crayfish Cherax quadricarinatus, which possesses typically conserved deacetylase domains and strong evolutionary relationships across various species. Moreover, gene knockdown of CqSIRT1 in crayfish haematopoietic tissue (Hpt) cell culture inhibited white spot syndrome virus (WSSV) late envelope gene vp28 transcription. In contrast, enhancement of deacetylase activity using a pharmacological activator promoted the replication of WSSV. Mechanically, CqSIRT1 was co-localized with viral envelope protein VP28 in the nuclei of Hpt cells and directly bound to VP28 with protein pulldown and co-immunoprecipitation assays. Furthermore, CqSIRT1 also interacted with another two viral envelope proteins, VP24 and VP26. To the best of our knowledge, this is the first report that WSSV structural proteins are linked to lysine deacetylases, providing a better understanding of the role of CqSIRT1 during WSSV infection and novel insights into the basic mechanism underlying the function of lysine deacetylases in crustaceans.

Potato Virus Y Strain N-Wi Offers Cross-Protection in Potato Against Strain NTN-NW by Superior Competition.

Potato virus Y (PVY) is one of the most economically important pathogens of potato. PVY exhibits different phenotypes in dissimilar potato cultivars. Previously, we observed that two recombinant isolates, PVYN-Wi-HLJ-BDH-2 (BDH) and PVYNTN-NW(SYR-II)-INM-W-369-12 (369), exhibited different virulence levels in potato cultivar Kexin 13 despite high genome sequence identity. Indeed, 369 induced severe necrosis and plant death in severe cases in Kexin 13 and severe mosaic in cultivar Yanshu 8, whereas BDH caused mainly mosaic symptoms on the plants of both cultivars. We hypothesized that preinfection of plants with BDH could cross-protect them from 369 infection, and not vice versa. Challenge inoculation, either by mechanical wounding or through grafting, with 369 on plants that were preinfected with BDH did not augment the symptom expression in both cultivars. Reverse transcription quantitative PCR analysis showed that, after challenge inoculation with 369, the titer of the isolate on BDH-preinfected plants remained at a low level (about 3 × 104 copy/µl) during the tested time course (0 h to 30 days). In contrast, in plants that were preinoculated with buffer (mock) and challenge inoculated with 369, the titer of 369 increased continuously until reaching its highest level of about 2 × 107 (Yanshu 8) and about 4 × 108 (Kexin 13) during the time course. Surprisingly, in plants that were preinfected with 369 and challenge inoculated with BDH, the accumulation of BDH reached nearly the same level as that in plants that were preinoculated with buffer and challenge inoculated with BDH. Taken together, these results suggest that PVYN-Wi mediated cross-protection against PVYNTN-NW(SYR-II) by superior competition and better fitness.

Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening.

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Leaf Color Change in Chokecherry (Padus virginiana).

Chokecherry (Padus virginiana L.) is an important landscaping tree with high ornamental value because of its colorful purplish-red leaves (PRL). The quantifications of anthocyanins and the mechanisms of leaf color change in this species remain unknown. The potential biosynthetic and regulatory mechanisms and the accumulation patterns of anthocyanins in P. virginiana that determine three leaf colors were investigated by combined analysis of the transcriptome and the metabolome. The difference of chlorophyll, carotenoid and anthocyanin content correlated with the formation of P. virginiana leaf color. Using enrichment and correlation network analysis, we found that anthocyanin accumulation differed in different colored leaves and that the accumulation of malvidin 3-O-glucoside (violet) and pelargonidin 3-O-glucoside (orange-red) significantly correlated with the leaf color change from green to purple-red. The flavonoid biosynthesis genes (PAL, CHS and CHI) and their transcriptional regulators (MYB, HD-Zip and bHLH) exhibited specific increased expression during the purple-red periods. Two genes encoding enzymes in the anthocyanin biosynthetic pathway, UDP glucose-flavonoid 3-O-glucosyl-transferase (UFGT) and anthocyanidin 3-O-glucosyltransferase (BZ1), seem to be critical for suppressing the formation of the aforesaid anthocyanins. In PRL, the expression of the genes encoding for UGFT and BZ1 enzymes was substantially higher than in leaves of other colors and may be related with the purple-red color change. These results may facilitate genetic modification or selection for further improvement in ornamental qualities of P. virginiana.

Physiological and Proteomic Responses to Drought in Leaves of Amygdalus mira (Koehne) Yü et Lu.

Various environmental stresses strongly influence plant development. Among these stresses is drought, which is a serious threat that can reduce agricultural productivity and obstruct plant growth. Although the mechanism of plants in response to drought has been studied extensively, the adaptive strategies of Amygdalus mira (Koehne) Yü et Lu grown in drought and rewatered habitats remain undefined. Amygdalus mira from the Tibetan Plateau has outstanding nutritional and medicinal values and can thrive in extreme drought. In this study, the physiological and proteomic responses in leaves of A. mira were investigated during drought and recovery period. The changes in plant growth, photosynthesis, enzymes, and non-enzymatic antioxidant under drought and rewatering were also analyzed in leaves. Compared with controls, A. mira showed stronger adaptive and resistant characteristics to drought. In addition, the proteomic technique was also used to study drought tolerance mechanisms in A. mira leaves. Differentially expressed proteins were identified using mass spectrometry. Accordingly, 103 proteins involved in 10 functional categories: cytoskeleton dynamics, energy metabolism, carbohydrate metabolism, photosynthesis, transcription and translation, transport, stress and defense, molecular chaperones, other materials metabolism, and unknown function were identified. These results showed that an increase of stress-defense-related proteins in leaves after drought treatment contributed to coping with drought. Importantly, A. mira developed an adaptive mechanism to scavenge reactive oxygen species (ROS), including enhancing antioxidant enzyme activities and non-enzymatic antioxidant contents, reducing energy, and adjusting the efficiency of gas exchanges. These results may help to understand the acclimation of A. mira to drought.

Transcriptome analysis of salt stress responsiveness in the seedlings of wild and cultivated Ricinus communis L.

Soil salinity is one of the major environmental factors, influencing agricultural productivity of crops. As a non-edible and ideal oilseed crop, castor (Ricinus communis L.) has great industrial value in biofuel, but molecular mechanisms of salt stress regulation are still unknown. In this study, the differentially expressed genes (DEGs) for differential salt tolerance in two castor cultivar (wild castor : Y, cultivated castor 'Tongbi 5': Z) were identified. 12 libraries were sampled for Illumina high-throughput sequencing to consider 132,426 nonredundant unigenes and 31,221 gene loci. Multiple phytohormones and transcription factors (TFs) were correlated with salt-tolerance and differently enriched in these two genotypes. The type 2C protein phosphatases (PP2C) homologs were all upregulated under salt stress. Importantly, IAA (1), DELLA (1) and Jasmonate zim domain (JAZ) (1) were also identified and found to be differentially expressed. Based on the co-expressed module by regulatory networks and heatmap analysis, ERF/AP2, WRKY and bHLH families were prominently participate in high salt stress response of wild and cultivated castor. Finally, these results highlight that the hub DEGs and families were more accumulated in cultivated castor than those in wild castor, providing novel insights into the salinity adaptive mechanisms and genetic improvement in castor.

Consistent habitat preference underpins the geographically divergent autumn migration of individual Mongolian common shelducks.

While many avian populations follow narrow, well-defined "migratory corridors," individuals from other populations undertake highly divergent individual migration routes, using widely dispersed stopover sites en route between breeding and wintering areas, although the reasons for these differences are rarely investigated. We combined individual GPS-tracked migration data from Mongolian-breeding common shelduck Tadorna tadorna and remote sensing datasets, to investigate habitat selection at inland stopover sites used by these birds during dispersed autumn migration, to explain their divergent migration patterns. We used generalized linear mixed models to investigate population-level resource selection, and generalized linear models to investigate stopover-site-level resource selection. The population-level model showed that water recurrence had the strongest positive effect on determining birds' occupancy at staging sites, while cultivated land and grassland land cover type had strongest negative effects; effects of other land cover types were negative but weaker, particularly effects of water seasonality and presence of a human footprint, which were positive but weak or non-significant, respectively. Although stopover-site-level models showed variable resource selection patterns, the variance partitioning and cross-prediction AUC scores corroborated high inter-individual consistency in habitat selection at inland stopover sites during the dispersed autumn migration. These results suggest that the geographically widespread distribution (and generally rarity) of suitable habitats explained the spatially divergent autumn migrations of Mongolian breeding common shelduck, rather than the species showing flexible autumn staging habitat occupancy.

The adaptation strategies of Herpetospermum pedunculosum (Ser.) Baill at altitude gradient of the Tibetan plateau by physiological and metabolomic methods.

BACKGROUND: Herpetospermum pedunculosum (Ser.) Baill is annual scandent herbs. They are used in the treatment of piles, inflammation of the stomach and the intestines. It can survive the extreme environment of the Tibetan Plateau (TP). However, the underlying mechanisms of this adaptation to H. pedunculosum from TP remain unclear. Here, we combined physiological and metabolomics methods to analyze H. pedunculosum response to altitude gradient differences. RESULTS: At high altitude, increases in the activities of Ascorbate peroxidase (APX), Glutathione reductase (GR), Dehydroascorbate reductase (DHAR), Monodehydroascorbate reductase (MDHAR), Superoxide dismutase (SOD) have been observed in leaves. Total Glutathion content, total Ascorbate content and the ASA (ascorbic acid)/docosahexaenoic acid (DHA) ration were highly elevated from low altitude to high altitude. In addition, high altitude induces decrease of the Anthocyanidin content (ANTH) and increase of abscisic acid content (ABA). The GC-MS analyses identified of 50 metabolites from leaves of H. pedunculosum. In addition, a metabolic network was constructed based on metabolomic datasets using a weighted correlation network analysis (WGCNA) approach. The network analysis uncovered 4 distinguished metabolic modules highly associated with I, II, III and IV respectively. Furthermore, the analysis successfully classified 50 samples into seven groups: carbohydrates, amino acids, organic acids, lipid components, polyamine, secondary metabolism and others. CONCLUSIONS: In the present study, the content of parts of amino acid components increased in samples collected at higher altitudes, and most of metabolites, including carbohydrates and organic acids were assigned to the carbon metabolic pathway comprising reductive pentose phosphate pathway, glycolysis and TCA cycle, indicating the direct relationship between adaptability and the carbon metabolic pathway and amino acids in H. pedunculosum response to high altitude. The results of this study laid the foundation of the molecular mechanism on H. pedunculosum from high altitude.

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

BACKGROUND: Polyploidy 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. RESULTS: Mitochondrial 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. CONCLUSIONS: Tetraploid 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.

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

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.

Cell-based immunotherapy with cytokine-induced killer (CIK) cells: From preparation and testing to clinical application.

Cell-based immunotherapy holds promise in the quest for the treatment of cancer, having potential synergy with surgery, chemotherapy and radiotherapy. As a novel approach for adoptive cell-based immunotherapy, cytokine-induced killer (CIK) cells have moved from the 'bench to bedside'. CIK cells are a heterogeneous subset of ex-vitro expanded, polyclonal T-effector cells with both natural killer (NK) and T-cell properties, which present potent non-major histocompatibility complex-restricted cytotoxicity against a variety of tumor target cells. Initial clinical studies on CIK cell therapy have provided encouraging results and revealed synergistic antitumor effects when combined with standard therapeutic procedures. At the same time, issues such as inadequate quality control and quantity of CIK cells as well as exaggerated propaganda were continuously emerging. Thus, the Ministry of Health in China stopped CIK cell therapy in May 2016, which was a major setback for the innovation of CIK cell-based immunotherapy. Thus, it is very important to modify technical criteria to develop a standardized operation procedure (SOP) and standardized system for evaluating antitumor efficacy in a safe way.

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

BACKGROUND: Plants 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). RESULTS: Several 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. CONCLUSIONS: The 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.

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

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 (Vk), 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.

Selection of a marker gene to construct a reference library for wetland plants, and the application of metabarcoding to analyze the diet of wintering herbivorous waterbirds.

Food availability and diet selection are important factors influencing the abundance and distribution of wild waterbirds. In order to better understand changes in waterbird population, it is essential to figure out what they feed on. However, analyzing their diet could be difficult and inefficient using traditional methods such as microhistologic observation. Here, we addressed this gap of knowledge by investigating the diet of greater white-fronted goose Anser albifrons and bean goose Anser fabalis, which are obligate herbivores wintering in China, mostly in the Middle and Lower Yangtze River floodplain. First, we selected a suitable and high-resolution marker gene for wetland plants that these geese would consume during the wintering period. Eight candidate genes were included: rbcL, rpoC1, rpoB, matK, trnH-psbA, trnL (UAA), atpF-atpH, and psbK-psbI. The selection was performed via analysis of representative sequences from NCBI and comparison of amplification efficiency and resolution power of plant samples collected from the wintering area. The trnL gene was chosen at last with c/h primers, and a local plant reference library was constructed with this gene. Then, utilizing DNA metabarcoding, we discovered 15 food items in total from the feces of these birds. Of the 15 unique dietary sequences, 10 could be identified at specie level. As for greater white-fronted goose, 73% of sequences belonged to Poaceae spp., and 26% belonged to Carex spp. In contrast, almost all sequences of bean goose belonged to Carex spp. (99%). Using the same samples, microhistology provided consistent food composition with metabarcoding results for greater white-fronted goose, while 13% of Poaceae was recovered for bean goose. In addition, two other taxa were discovered only through microhistologic analysis. Although most of the identified taxa matched relatively well between the two methods, DNA metabarcoding gave taxonomically more detailed information. Discrepancies were likely due to biased PCR amplification in metabarcoding, low discriminating power of current marker genes for monocots, and biases in microhistologic analysis. The diet differences between two geese species might indicate deeper ecological significance beyond the scope of this study. We concluded that DNA metabarcoding provides new perspectives for studies of herbivorous waterbird diets and inter-specific interactions, as well as new possibilities to investigate interactions between herbivores and plants. In addition, microhistologic analysis should be used together with metabarcoding methods to integrate this information.

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 responses of diploid and tetraploid black locust (Robinia pseudoacacia L.) subjected to salt stress.

Tetraploid black locust (Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×. However, 4× developed diverse responses under salt stress. A comparative proteomic analysis revealed that 41 and 37 proteins were differentially expressed in 2× and 4×, respectively. These proteins were mainly involved in photosynthesis, stress and defense, energy, metabolism, transcription/translation, and transportation. Distinct patterns of protein changes between 2× and 4× were analyzed. Collectively, our results suggest that the plants showed significantly different responses to salt stress based on ploidy level of the plant. The 4× possessed a better salt protection mechanism than that of 2×, suggesting salt tolerance in the polyploid plant.