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DNA methylation is changed and associates with gene expression alterations in plant response to phosphate starvation (Pi-), a common stress that impacts plant growth and productivity. However, in the horticultural model species Solanum lycopersicum (tomato), the dynamics of DNA methylation and its relationship with changes in gene transcription and alternative splicing (AS) under Pi- are unknown. Here, we performed integrative methylome and transcriptome analyses of tomato seedlings under Pi-deficient and -sufficient conditions. We found Pi- caused a slight increase in the overall methylation level, with millions of differentially methylated cytosines (DmCs) and a few hundred differentially methylated regions (DMRs). We also identified thousands of differentially expressed (DE) and differential AS (DAS) genes induced by Pi-, and found that DmCs were more abundant in non-expressed genes than in DE or DAS genes. Moreover, DNA methylation alterations weakly correlated with transcription changes but not with DAS events, and hyper-CHH-DMRs overlapping with transposable elements (TEs) were enriched in a subset of Pi starvation response (PSR) genes. We propose that changes in DNA methylation may be associated with the differential expression of some PSR genes, but that most of these changes probably control the expression of nearby TEs, rather than directly affecting the transcription or AS of PSR genes. Besides, the pattern of methylation changes upon Pi- may largely be shaped by TE distributions. Together, our study provides comprehensive insights into the association of DNA methylation with gene transcription and AS under Pi- in tomato and may contribute to unveiling novel roles of epigenetic mechanisms in plant stress response.

C2H2 zinc finger proteins (ZFPs) play important roles in plant development and response to abiotic stresses, and have been studied extensively. However, there are few studies on ZFPs in mangroves and mangrove associates, which represent a unique plant community with robust stress tolerance. MpZFP1, which is highly induced by salt stress in the mangrove associate Millettia pinnata, was cloned and functionally characterized in this study. MpZFP1 protein contains two zinc finger domains with conserved QALGGH motifs and targets to the nucleus. The heterologous expression of MpZFP1 in Arabidopsis increased the seeds’ germination rate, seedling survival rate, and biomass accumulation under salt stress. The transgenic plants also increased the expression of stress-responsive genes, including RD22 and RD29A, and reduced the accumulation of reactive oxygen species (ROS). These results indicate that MpZFP1 is a positive regulator of plant responses to salt stress due to its activation of gene expression and efficient scavenging of ROS.

Salt stress is a major increasing threat to global agriculture. Pongamia (Millettia pinnata), a semi-mangrove, is a good model to study the molecular mechanism of plant adaptation to the saline environment. Calcium signaling pathways play critical roles in the model plants such as Arabidopsis in responding to salt stress, but little is known about their function in Pongamia. Here, we have isolated and characterized a salt-responsive MpCML40, a calmodulin-like (CML) gene from Pongamia. MpCML40 protein has 140 amino acids and is homologous with Arabidopsis AtCML40. MpCML40 contains four EF-hand motifs and a bipartite NLS (Nuclear Localization Signal) and localizes both at the plasma membrane and in the nucleus. MpCML40 was highly induced after salt treatment, especially in Pongamia roots. Heterologous expression of MpCML40 in yeast cells improved their salt tolerance. The 35S::MpCML40 transgenic Arabidopsis highly enhanced seed germination rate and root length under salt and osmotic stresses. The transgenic plants had a higher level of proline and a lower level of MDA (malondialdehyde) under normal and stress conditions, which suggested that heterologous expression of MpCML40 contributed to proline accumulation to improve salt tolerance and protect plants from the ROS (reactive oxygen species) destructive effects. Furthermore, we did not observe any measurable discrepancies in the development and growth between the transgenic plants and wild-type plants under normal growth conditions. Our results suggest that MpCML40 is an important positive regulator in response to salt stress and of potential application in producing salt-tolerant crops.

Alteration of fruit quality caused by environmental stress is a common but largely unresolved issue for plant cultivation and breeding practices. Phosphorus (P) deficiency may interfere with a variety of metabolic processes whose intermediate products are correlated with important fruit quality traits. However, how low P stress affects fruit quality has not been investigated in detail. In this study, we assessed the contents of major metabolites associated with tomato fruit quality under two low P treatments that started at the seedling or flowering stage. The major pigments and the key organic acids related to fruit sourness were differentially over-accumulated as fruit ripened under two low P treatments compared to those under the control treatment, while the total content of soluble sugars contributing to fruit sweetness was substantially reduced under both treatments. These changes were largely attributed to the alteration of enzyme activities in the relevant metabolic pathways. In particular, we found that low P stress from different developmental stages had differential effects on the activation of γ-aminobutyric acid shunt that were likely responsible for the preferential accumulation of different organic acids in tomato fruits. Our study suggested that low P stress strongly affected tomato fruit quality and the effects appeared to be variable under different regimes of low P conditions.

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half, PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Macronutrient elements including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S) are required in relatively large and steady amounts for plant growth and development. Deficient or excessive supply of macronutrients from external environments may trigger a series of plant responses at phenotypic and molecular levels during the entire life cycle. Among the intertwined molecular networks underlying plant responses to macronutrient stress, noncoding RNAs (ncRNAs), mainly microRNAs (miRNAs) and long ncRNAs (lncRNAs), may serve as pivotal regulators for the coordination between nutrient supply and plant demand, while the responsive ncRNA-target module and the interactive mechanism vary among elements and species. Towards a comprehensive identification and functional characterization of nutrient-responsive ncRNAs and their downstream molecules, high-throughput sequencing has produced massive omics data for comparative expression profiling as a first step. In this review, we highlight the recent findings of ncRNA-mediated regulation in response to macronutrient stress, with special emphasis on the large-scale sequencing efforts for screening out candidate nutrient-responsive ncRNAs in plants, and discuss potential improvements in theoretical study to provide better guidance for crop breeding practices.

Pongamia (Millettia pinnata syn. Pongamia pinnata) is a multipurpose biofuel tree which can withstand a variety of abiotic stresses. Commercial applications of Pongamia trees may substantially benefit from improvements in their oil-seed productivity, which is governed by complex regulatory mechanisms underlying seed development. MicroRNAs (miRNAs) are important molecular regulators of plant development, while relatively little is known about their roles in seed development, especially for woody plants. In this study, we identified 236 conserved miRNAs within 49 families and 143 novel miRNAs via deep sequencing of Pongamia seeds sampled at three developmental phases. For these miRNAs, 1327 target genes were computationally predicted. Furthermore, 115 differentially expressed miRNAs (DEmiRs) between successive developmental phases were sorted out. The DEmiR-targeted genes were preferentially enriched in the functional categories associated with DNA damage repair and photosynthesis. The combined analyses of expression profiles for DEmiRs and functional annotations for their target genes revealed the involvements of both conserved and novel miRNA-target modules in Pongamia seed development. Quantitative Real-Time PCR validated the expression changes of 15 DEmiRs as well as the opposite expression changes of six targets. These results provide valuable miRNA candidates for further functional characterization and breeding practice in Pongamia and other oilseed plants.

Abstarct The electrosensing of chlorzoxazone (CHZ) was studied for the first time at nanostructured Au/graphene modified glassy carbon electrode (AuNPs/Gr/GCE) employing different voltammetric techniques viz. cyclic, linear sweep and differential pulse voltammetries. The results indicated that the voltammetric response of CHZ was improved distinctly at the AuNPs/Gr/GCE electrode than that at bare GCE. In order to carry out the CHZ determination, the dependency of the response current on supporting electrolyte pH, sweep rate and CHZ concentration were inspected to optimize the investigational conditions. The surface characterizations of the Au/graphene modified and unmodified glassy carbon electrodes were carried out. The anodic peak showed a process of diffusion-control. The number of electrons transferred was calculated to be two. The determination of [CHZ] using the modified electrode was studied over the range of 1.0 × 10−7 M to 10.0 × 10−7 M which showed the LOD value to be 1.164 × 10−8 M. The study of excipients showed that the method owns selectivity. A differential pulse voltammetry which is fast and simple, showed a good accuracy and precision, for the purpose of determining the [CHZ] in biomedical applications like pharmaceutical formulation and also in pathological sample testing. In addition, this type of work can find its usage towards scientific purposes that involves electrical measurements, particularly in industrial quality management, environmental pollution management, as it involves simple sample preparation, appreciable repeatability, reproducibility and also high sensitivity.

Figure S1. Sequence saturation analysis for the nine sequencing libraries. Figure S2. Pearsonâ s correlation analysis of the RPKM values of all nine samples. Figure S3. Principal component analysis of the RPKM values of all nine samples. Figure S4. Linear regression analysis between gene expression ratios obtained from RNA-Seq and qRT-PCR data. Table S7. Primers for 10 lipid-metabolism-related unigenes in qRT-PCR analyses. (DOCX 600 kb)

The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

The installation position of the external mirror, a very significant safety parts of vehicles, directly affects the driver’s field of view. To determine the position of the external mirror is a complicated task, factors need to be considered such as styling, ergonomic, safety, engineering design, etc. In the process of vehicle development, the external mirror is often arranged after the modeling schemes is determined. However, it is not easy to get a good visual field and human comfort subject to the constraints of the styling features. Therefore, it is necessary to give out the position of the external mirror and the field of vision in the early stage of modeling. In order to obtain a good view, the ideal position of the outside mirror is packaged considering of ergonomic, and the minimum visible dimension of the mirror is designed on the basis of vision conformance, which avoid the safety hazard and massive amounts of modification caused by improper design of the external mirror.

Pongamia (Millettia pinnata) is a promising biofuel crop with multiple merits. Breeding of ideal Pongamia germplasm for industrial application demands substantial progress in molecular biology of this legume species, which has been largely hampered by the paucity of its genomic data. In this study, we constructed and characterized a comprehensive seed transcriptome by the high-throughput Illumina sequencing technology. We obtained over 83 million high-quality reads, which were processed and assembled into 53,586 unigenes with a mean length of 787 bp. Among these unigenes, 39,602 (73.90 %) and 24,078 (44.93 %) showed significant similarity to proteins in the NCBI non-redundant and the Swiss-Prot protein databases, respectively. Of the annotated unigenes, 30,619 (57.14 %) were classified into 56 Gene Ontology categories. Furthermore, 21,905 (40.88 %) unigenes were assigned to 128 pathways in the Kyoto Encyclopedia of Genes and Genomes pathway database. A set of 364 unigenes involved in five pathways closely related to oil biosynthesis and accumulation were screened out as candidates for future functional analyses. On the other hand, 5710 expressed sequence tag-simple sequence repeats (EST-SSRs) were identified in 4951 unigenes with a density of one SSR every 7.39-kb sequence. One hundred EST-SSRs were randomly selected to validate amplification and to assess polymorphism among 12 Pongamia individuals. Eighty-two primer pairs successfully amplified DNA fragments and 17 of them detected polymorphism, in which the polymorphism information content values ranged from 0.14 to 0.57. This transcriptome dataset will serve as a valuable basis for studies on functional genomics, molecular genetics and molecular breeding of Pongamia.

Semi-mangroves form a group of transitional species between glycophytes and halophytes, and hold unique potential for learning molecular mechanisms underlying plant salt tolerance. Millettia pinnata is a semi-mangrove plant that can survive a wide range of saline conditions in the absence of specialized morphological and physiological traits. By employing the Illumina sequencing platform, we generated ~192 million short reads from four cDNA libraries of M. pinnata and processed them into 108,598 unisequences with a high depth of coverage. The mean length and total length of these unisequences were 606 bp and 65.8 Mb, respectively. A total of 54,596 (50.3%) unisequences were assigned Nr annotations. Functional classification revealed the involvement of unisequences in various biological processes related to metabolism and environmental adaptation. We identified 23,815 candidate salt-responsive genes with significantly differential expression under seawater and freshwater treatments. Based on the reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses, we verified the changes in expression levels for a number of candidate genes. The functional enrichment analyses for the candidate genes showed tissue-specific patterns of transcriptome remodelling upon salt stress in the roots and the leaves. The transcriptome of M. pinnata will provide valuable gene resources for future application in crop improvement. In addition, this study sets a good example for large-scale identification of salt-responsive genes in non-model organisms using the sequencing-based approach.

Millettia pinnata L. is a leguminous tree with great potential in biodiesel applications and also a typical semi-mangrove. In this review, we presented several aspects about the recent research progress in molecular biology of M. pinnata. We descrived several types of molecular markers used to assess the genetic diversity and phylogeny of this species, genome and transcriptome analyses based on high-throughput sequencing platform accomplished for this species, and several gene and genomic sequences of this species isolated for further research. Finally, based on the current research progress, we proposed some orientations for future molecular biology research on M. pinnata.

Avicennia marina is a widely distributed mangrove species that thrives in high-salinity habitats. It plays a significant role in supporting coastal ecosystem and holds unique potential for studying molecular mechanisms underlying ecological adaptation. Despite and sometimes because of its numerous merits, this species is facing increasing pressure of exploitation and deforestation. Both study on adaptation mechanisms and conservation efforts necessitate more genomic resources for A. marina. In this study, we used Illumina sequencing of an A. marina foliar cDNA library to generate a transcriptome dataset for gene and marker discovery. We obtained 40 million high-quality reads and assembled them into 91,125 unigenes with a mean length of 463 bp. These unigenes covered most of the publicly available A. marina Sanger ESTs and greatly extended the repertoire of transcripts for this species. A total of 54,497 and 32,637 unigenes were annotated based on homology to sequences in the NCBI non-redundant and the Swiss-prot protein databases, respectively. Both Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed some transcriptomic signatures of stress adaptation for this halophytic species. We also detected an extraordinary amount of transcripts derived from fungal endophytes and demonstrated the utility of transcriptome sequencing in surveying endophyte diversity without isolating them out of plant tissues. Additionally, we identified 3,423 candidate simple sequence repeats (SSRs) from 3,141 unigenes with a density of one SSR locus every 8.25 kb sequence. Our transcriptomic data will provide valuable resources for ecological, genetic and evolutionary studies in A. marina.

Genomic regions that are unusually divergent between closely related species or racial groups can be particularly informative about the process of speciation or the operation of natural selection. The two sequenced genomes of cultivated Asian rice, Oryza sativa, reveal that at least 6% of the genomes are unusually divergent. Sequencing of ten unlinked loci from the highly divergent regions consistently identified two highly divergent haplotypes with each locus in nearly complete linkage disequilibrium among 25 O. sativa cultivars and 35 lines from six wild species. The existence of two highly divergent haplotypes in high divergence regions in species from all geographical areas (Africa, Asia, and Oceania) was in contrast to the low polymorphism and low linkage disequilibrium that were observed in other parts of the genome, represented by ten reference loci. While several natural processes are likely to contribute to this pattern of genomic variation, domestication may have greatly exaggerated the trend. In this hypothesis, divergent haplotypes that were adapted to different geographical and ecological environments migrated along with humans during the development of domesticated varieties. If true, these high divergence regions of the genome would be enriched for loci that contribute to the enormous range of phenotypic variation observed among domesticated breeds., Synopsis The coexistence of high and low divergence regions in the genomes of two incipient species can be informative about the process of speciation. For example, it may indicate a long period of continual gene flow during species formation. In the conventional view of speciation by geographical separation, there is little intermingling in the process, and the level of divergence should be relatively uniform across the genome. Domesticated plants and animals are excellent materials for studying speciation because the process of domestication may often exaggerate the forces that drive speciation. These forces include selection (artificial rather than natural) and admixture among diverging varieties mediated by humans. In this study, the authors analyzed the whole genome sequences between the two subspecies of domesticated rice. These subspecies have developed partial reproductive isolation. By studying the entire genomic patterns, as well as the detailed population genetics of 20 loci among 60 lines of cultivars and wild rice, the authors observed regions of unusually high divergence, which occupy more than 6% of the whole genome. Hence, the formation of domesticated rice resembles a process in which previously divergent populations/races were brought together, sorted, and re-assembled. How much this process may echo the formation of species in nature is discussed.

The extent of molecular differentiation between domesticated animals or plants and their wild relatives is postulated to be small. The availability of the complete genome sequences of two subspecies of the Asian rice, Oryza sativa (indica and japonica) and their wild relatives have provided an unprecedented opportunity to study divergence following domestication. We observed significantly more amino acid substitutions during rice domestication than can be expected from a comparison among wild species. This excess is disproportionately larger for the more radical kinds of amino acid changes (e.g. Cys--Tyr). We estimate that approximately a quarter of the amino acid differences between rice cultivars are deleterious, not accountable by the relaxation of selective constraints. This excess is negatively correlated with the rate of recombination, suggesting that 'hitchhiking' has occurred. We hypothesize that during domestication artificial selection increased the frequency of many deleterious mutations.

The most remarkable morphological specializations of mangroves are vivipary, salt secretion, and aerial roots. There has been a long debate on whether the complex traits vivipary and secreters have a single origin, the answer to which has profound implications for the mechanism of evolution in mangroves. We took a large and representative sample across mangroves and sequenced the 18S rRNA, rbcL, and matR genes. Together with the outgroups, our data yielded a high resolution phylogeny which allowed us to gain much needed insight into the distributions of the two characters and address their evolutionary origins. For each character, its ancestral state in the phylogeny was estimated by the maximum likelihood method. Overall evidence is in favor of a multiple origin for both vivipary and salt secretion in mangroves.

The inherent replicative mode of transposition endows retrotransposons with considerable advantages as genetic tools in plant genome analysis. Here we present a high-throughput sequence-specific amplification polymorphism (S-SAP) method based on copia-like retrotransposons to fulfill the increasing desire of screening large numbers of samples in plants. Classic approach for digestion, ligation and pre-amplification was combined with optimized fluorescent multiplex PCR for simultaneously selective amplifying S-SAP fragments, and multiple S-SAPs were subsequently detected by capillary electrophoresis using ABI PRISM 3700 capillary instruments. Comparisons of results from multiplex PCR with simplex PCR, and from capillary electrophoresis with slab-gel electrophoresis demonstrated that this method is an efficient, economical, and accurate means for high-throughput and large-scale genotyping retrotransposon variation in plants.