Comparative Analysis of Transposable Elements in Strawberry Genomes of Different Ploidy Levels.

Transposable elements (TEs) make up a large portion of plant genomes and play a vital role in genome structure, function, and evolution. Cultivated strawberry (Fragaria x ananassa) is one of the most important fruit crops, and its octoploid genome was formed through several rounds of genome duplications from diploid ancestors. Here, we built a pan-genome TE library for the Fragaria genus using ten published strawberry genomes at different ploidy levels, including seven diploids, one tetraploid, and two octoploids, and performed comparative analysis of TE content in these genomes. The TEs comprise 51.83% (F. viridis) to 60.07% (F. nilgerrensis) of the genomes. Long terminal repeat retrotransposons (LTR-RTs) are the predominant TE type in the Fragaria genomes (20.16% to 34.94%), particularly in F. iinumae (34.94%). Estimating TE content and LTR-RT insertion times revealed that species-specific TEs have shaped each strawberry genome. Additionally, the copy number of different LTR-RT families inserted in the last one million years reflects the genetic distance between Fragaria species. Comparing cultivated strawberry subgenomes to extant diploid ancestors showed that F. vesca and F. iinumae are likely the diploid ancestors of the cultivated strawberry, but not F. viridis. These findings provide new insights into the TE variations in the strawberry genomes and their roles in strawberry genome evolution.

Analysis of Delta(9) fatty acid desaturase gene family and their role in oleic acid accumulation in Carya cathayensis kernel.

Carya cathayensis, commonly referred to as Chinese hickory, produces nuts that contain high-quality edible oils, particularly oleic acid (18:1). It is known that stearoyl-ACP desaturase (SAD) is the first key step converting stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the aminolevulinic acid (ALA) biosynthetic pathway and play an important role in OA accumulation. Thus far, there is little information about SAD gene family in C. cathayensis and the role of individual members in OA accumulation. This study searched the Chinese Hickory Genome Database and identified five members of SAD genes, designated as CcSADs, at the whole genome level through the comparison with the homologous genes from Arabidopsis. RNA-Seq analysis showed that CcSSI2-1, CcSSI2-2, and CcSAD6 were highly expressed in kernels. The expression pattern of CcSADs was significantly correlated with fatty acid accumulation during the kernel development. In addition, five full-length cDNAs encoding SADs were isolated from the developing kernel of C. cathayensis. CcSADs-green fluorescent protein (GFP) fusion construct was infiltrated into tobacco epidermal cells, and results indicated their chloroplast localization. The catalytic function of these CcSADs was further analyzed by heterologous expression in Saccharomyces cerevisiae, Nicotiana benthamiana, and walnut. Functional analysis demonstrated that all CcSADs had fatty acid desaturase activity to catalyze oleic acid biosynthesis. Some members of CcSADs also have strong substrate specificity for 16:0-ACP to synthesize palmitoleic acid (C16:1, PA). Our study documented SAD gene family in C. cathayensis and the role of CcSSI2-1, CcSSI2-2, and CcSAD6 in OA accumulation, which could be important for future improvement of OA content in this species via genetic manipulation.

High Light Intensity Triggered Abscisic Acid Biosynthesis Mediates Anthocyanin Accumulation in Young Leaves of Tea Plant (Camellia sinensis).

There is increasing interest in the production and consumption of tea (Camellia sinensis L.) processed from purple-leaved cultivar due to their high anthocyanin content and health benefits. However, how and why seasonal changes affect anthocyanin accumulation in young tea leaves still remains obscured. In this study, anthocyanin and abscisic acid (ABA) contents in young leaves of Zifuxing 1 (ZFX1), a cultivar with new shoots turning to purple in Wuyi Mountain, a key tea production region in China, were monitored over four seasons. Young leaves produced in September were highly purplish, which was accompanied with higher anthocyanin and ABA contents. Among the environmental factors, the light intensity in particular was closely correlated with anthocyanin and ABA contents. A shade experiment also indicated that anthocyanin content significantly decreased after 168 h growth under 75% shade, but ABA treatment under the shade conditions sustained anthocyanin content. To confirm the involvement of ABA in the modulation of anthocyanin accumulation, anthocyanin, carotenoids, chlorophyll, ABA, jasmonic acid (JA), and salicylic acid (SA) in the young leaves of four cultivars, including ZFX1, Zijuan (ZJ), wherein leaves are completely purple, Rougui (RG) and Fudingdabaicha (FDDB) wherein leaves are green, were analyzed, and antioxidant activities of the leaf extracts were tested. Results showed that ABA, not other tested hormones, was significantly correlated with anthocyanin accumulation in the purple-leaved cultivars. Cultivars with higher anthocyanin contents exhibited higher antioxidant activities. Subsequently, ZFX1 plants were grown under full sun and treated with ABA and fluridone (Flu), an ABA inhibitor. ABA treatment elevated anthocyanin level but decreased chlorophyll contents. The reverse was true to those treated with Flu. To pursue a better understanding of ABA involvement in anthocyanin accumulation, RNA-Seq was used to analyze transcript differences among ABA- or Flu-treated and untreated ZFX1 plants. Results indicated that the differentially expressed genes in ABA or Flu treatment were mainly ABA signal sensing and metabolism-related genes, anthocyanin accumulation-related genes, light-responsive genes, and key regulatory MYB transcription factors. Taking all the results into account, a model for anthocyanin accumulation in ZFX1 cultivar was proposed: high light intensity caused reactive oxygen stress, which triggered the biosynthesis of ABA; ABA interactions with transcription factors, such as MYB-enhanced anthocyanin biosynthesis limited chlorophyll and carotenoid accumulation; and transport of anthocyanin to vacuoles resulting in the young leaves of ZFX1 with purplish coloration. Further research is warranted to test this model.

A Comparative Genomics Approach for Analysis of Complete Mitogenomes of Five Actinidiaceae Plants.

Actinidiaceae, an economically important plant family, includes the Actinidia, Clematoclethra and Saurauia genus. Kiwifruit, with remarkably high vitamin C content, is an endemic species widely distributed in China with high economic value. Although many Actinidiaceae chloroplast genomes have been reported, few complete mitogenomes of Actinidiaceae have been studied. Here, complete circular mitogenomes of the four kiwifruit species and Saurauia tristyla were assembled. Codon usage, sequence repeats, RNA editing, gene transfers, selective pressure, and phylogenetic relationships in the four kiwifruit species and S. tristyla were comparatively analyzed. This research will contribute to the study of phylogenetic relationships within Actiniaceae and molecular barcoding in kiwifruit.

Relationship between the Grade and the Characteristic Flavor of PCT (Panyong Congou Black Tea).

Panyong Congou black tea (PCT) is one of the most representative and historically famous Congou black teas in China and has been gaining more and more attention for its beneficial health properties. Currently, four grades of PCT are available, based on the raw leaf materials and consumer palatability. The chemical profiles distinguishing different grades of PCT are yet to be defined, nor has the relationship with grade been evaluated. In the present study, chemometric analysis showed that epigallocatechin (EGC), catechin (C), polyphenols, gallic acid (GA), and free amino acids are grade related bio-markers of PCT. These compounds are associated with the sweet and mellow aftertaste of PCT. A total of 34 volatile components were identified, of which the three component types with the highest relative percentages were alcohols (51.34-52.51%), ketones (27.31-30.28%), and aldehydes (12.70-13.18%). Additionally, our results revealed that sweet floral and fruity aromas were positively correlated with six volatile organic compounds (VOCs), 1-pentanol, propyl hexanoate, linalool, cyclohexanone, hexanal, and 2,5-dimethylpyrazine. Clear discrimination was achieved using orthogonal projections to latent structures discriminant analysis (OPLS-DA). The findings provide vital information on the characteristic flavor of each grade of PCT.

Genome-Wide Expression Profiling Analysis of Kiwifruit GolS and RFS Genes and Identification of AcRFS4 Function in Raffinose Accumulation.

The raffinose synthetase (RFS) and galactinol synthase (GolS) are two critical enzymes for raffinose biosynthesis, which play an important role in modulating plant growth and in response to a variety of biotic or abiotic stresses. Here, we comprehensively analyzed the RFS and GolS gene families and their involvement in abiotic and biotic stresses responses at the genome-wide scale in kiwifruit. A total of 22 GolS and 24 RFS genes were identified in Actinidia chinensis and Actinidia eriantha genomes. Phylogenetic analysis showed that the GolS and RFS genes were clustered into four and six groups, respectively. Transcriptomic analysis revealed that abiotic stresses strongly induced some crucial genes members including AcGolS1/2/4/8 and AcRFS2/4/8/11 and their expression levels were further confirmed by qRT-PCR. The GUS staining of AcRFS4Pro::GUS transgenic plants revealed that the transcriptionlevel of AcRFS4 was significantly increased by salt stress. Overexpression of AcRFS4 in Arabidopsis demonstrated that this gene enhanced the raffinose accumulation and the tolerance to salt stress. The co-expression networks analysis of hub transcription factors targeting key AcRFS4 genes indicated that there was a strong correlation between AcNAC30 and AcRFS4 expression under salt stress. Furthermore, the yeast one-hybrid assays showed that AcNAC30 could bind the AcRFS4 promoter directly. These results may provide insights into the evolutionary and functional mechanisms of GolS and RFS genes in kiwifruit.

Genome-Wide Identification of TLP Gene Family and Their Roles in Carya cathayensis Sarg in Response to Botryosphaeria dothidea.

Hickory (Carya cathayensis) is a critical tree species of the genus Carya from the Juglandaceae family that contains nutrient-rich nuts. Due to large-scale soil degradation, the pests and diseases of hickory are becoming more and more serious. Thaumatin-like proteins (TLPs) are vital proteins involved in the complex defense process of plant pathogens. In this study, 40 CcTLP genes were identified genome-widely and phylogenetically grouped into three subfamilies. The sequence of CcTLPs had a conservative pattern, such as eight stable disulfide bonds, REDDD, and G-X-[GF]-X-C-X-T-[GA]-D-C-X(1,2)-G-X-(2,3)-C structure. In total, 57 cis-elements related to stress-responsive, light-responsive, phytohormone-responsive, and plant-responsive were discovered. Under salicylate (SA), methyl jasmonate (MeJA), and ethephon (ETH) treatments, the expressions of CcTLP28, CcTLP29, CcTLP30, CcTLP31, CcTLP32, CcTLP33, CcTLP37, CcTLP38, and CcTLP39 had different patterns. This is an indication that most of the TLP genes were upregulated by SA and downregulated by MeJA. Notably, seven TLP genes were significantly upregulated under the Botryosphaeria dothidea inoculation, especially CcTLP31, with an over 20-fold change. Nine genes were shown by subcellular localization analysis to be located at the plasma membrane and cytoplasm. The knowledge of the disease-resistant function of the CcTLP family in hickory is promoted by these results. A foundation reference for the molecular breeding of this plant in the future is provided by our findings.

Integrated transcriptome and proteome analysis of developing embryo reveals the mechanisms underlying the high levels of oil accumulation in Carya cathayensis Sarg.

Hickory (Carya cathayensis Sarg.) is an extraordinary nut-bearing deciduous arbor with high content of oil in its embryo. However, the molecular mechanism underlying high oil accumulation is mostly unknown. Here, we reported that the lipid droplets and oil accumulation gradually increased with the embryo development and the oil content was up to ~76% at maturity. Furthermore, transcriptome and proteome analysis of developing hickory embryo identified 32,907 genes and 9857 proteins. Time-series analysis of gene expressions showed that these genes were divided into 12 clusters and lipid metabolism-related genes were enriched in Cluster 3, with the highest expression levels at 95 days after pollination (S2). Differentially expressed genes and proteins indicated high correlation, and both were enriched in the lipid metabolism. Notably, the genes involved in biosynthesis, transport of fatty acid/lipid and lipid droplets formation had high expression levels at S2, while the expression levels of other genes required for suberin/wax/cutin biosynthesis and lipid degradation were very low at all the sampling time points, ultimately promoting the accumulation of oil. Quantitative reverse-transcription PCR analysis also verified the results of RNA-seq. The co-regulatory networks of lipid metabolism were further constructed and WRINKLED1 (WRI1) was a core transcriptional factor located in the nucleus. Of note, CcWRI1A/B could directly activate the expression of some genes (CcBCCP2A, CcBCCP2B, CcFATA and CcFAD3) required for fatty acid synthesis. These results provided in-depth evidence for revealing the molecular mechanism of high oil accumulation in hickory embryo.

Analysis of lipidomics profile of Carya cathayensis nuts and lipid dynamic changes during embryonic development.

Hickory (Carya cathayensis) nuts contain higher amount of lipids, and possess high nutritional value and substantial health benefits. However, their lipid composition and dynamic changes during embryogenesis have not been thoroughly investigated. Therefore, lipidomics profile and lipid dynamic changes during embryonic development were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Totally, 544 kinds of lipids were identified in mature hickory nuts with higher proportions of glycerolipids (59.94%) and glycerophospholipids (38.66%). Notably, diacylglycerols showed gradual uptrends, which corresponded with total glycerolipid and glycerophospholipid at middle and late stage of embryogenesis, suggesting the pivotal role of diacylglycerols in the accumulation of glycerolipids and glycerophospholipids. Moreover, triacylglycerols, diacylglycerols, phosphatidylethanolamines and phosphatidylcholines had high relative content with abundance of unsaturated fatty acids, specifically oleic acid, linoleic acid and linolenic acid, localized mainly at sn-2 lipid position. Together, our study provides innovative perspectives for studying the nutritional benefits of hickory nut lipids.

Comparison analysis of widely-targeted metabolomics revealed the variation of potential astringent ingredients and their dynamic accumulation in the seed coats of both Carya cathayensis and Carya illinoinensis.

Pecan and hickory nuts are two of consumers' favorite ones. Pecan seeds can be eaten fresh, while hickory ones must remove astringency before eating. Here, we reported that total phenols, flavonoids and condensed tannins of hickory seeds were reduced after de-astringent treatments. They gradually increased with development, showing higher levels in hickory seed coat at mid-late periods than that in pecan's. Widely-targeted metabonomics analysis of developing testa identified 424 kinds of components, including 101, 38, 58, 27 classes of flavonoids, tannins, phenolic acids, organic acids and others, showing 16 different changing trends. Notably, most kinds of flavonoids, hydrolysable tannins and phenolic acids at maturity were more than that of pecan's, while oligomeric condensed tannins were opposite. Gene expression analysis provided further explanations for their dynamic accumulation. These results unraveled potential astringent components in hickory testa and preliminary molecular mechanisms of their dynamic changes, offering theoretical basis for the targeted de-astringency.

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck).

'Liuyuezaoyou' is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck 'Guanximiyou'. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

Genome-Wide Identification of Tannase Genes and Their Function of Wound Response and Astringent Substances Accumulation in Juglandaceae.

Tannins are important polyphenol compounds with different component proportions in different plant species. The plants in the Juglandaceae are rich in tannins, including condensed tannins and hydrolyzable tannins. In this study, we identified seven tannase genes (TAs) responsible for the tannin metabolism from walnut, pecan, and Chinese hickory, and three nut tree species in the Juglandaceae, which were divided into two groups. The phylogenetic and sequence analysis showed that TA genes and neighboring clade genes (TA-like genes) had similar sequences compared with other carboxylesterase genes, which may be the origin of TA genes produced by tandem repeat. TA genes also indicated higher expressions in leaf than other tissues and were quickly up-regulated at 3 h after leaf injury. During the development of the seed coat, the expression of the synthesis-related gene GGTs and the hydrolase gene TAs was continuously decreased, resulting in the decrease of tannin content in the dry sample of the seed coat of Chinese hickory. However, due to the reduction in water content during the ripening process, the tannin content in fresh sample increased, so the astringent taste was obvious at the mature stage. In addition, the CcGGTs' expression was higher than CiGGTs in the initiation of development, but CcTAs continued to be down-regulated while CiTA2a and CiTA2b were up-regulated, which may bring about the significant differences in tannin content and astringent taste between Chinese hickory and pecan. These results suggested the crucial role of TAs in wound stress of leaves and astringent ingredient accumulation in seed coats of two nut tree species in the Juglandaceae.

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses.

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis "Xuegan" plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri.

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

Phylloremediation of Air Pollutants: Exploiting the Potential of Plant Leaves and Leaf-Associated Microbes.

Air pollution is air contaminated by anthropogenic or naturally occurring substances in high concentrations for a prolonged time, resulting in adverse effects on human comfort and health as well as on ecosystems. Major air pollutants include particulate matters (PMs), ground-level ozone (O3), sulfur dioxide (SO2), nitrogen dioxides (NO2), and volatile organic compounds (VOCs). During the last three decades, air has become increasingly polluted in countries like China and India due to rapid economic growth accompanied by increased energy consumption. Various policies, regulations, and technologies have been brought together for remediation of air pollution, but the air still remains polluted. In this review, we direct attention to bioremediation of air pollutants by exploiting the potentials of plant leaves and leaf-associated microbes. The aerial surfaces of plants, particularly leaves, are estimated to sum up to 4 × 108 km2 on the earth and are also home for up to 1026 bacterial cells. Plant leaves are able to adsorb or absorb air pollutants, and habituated microbes on leaf surface and in leaves (endophytes) are reported to be able to biodegrade or transform pollutants into less or nontoxic molecules, but their potentials for air remediation has been largely unexplored. With advances in omics technologies, molecular mechanisms underlying plant leaves and leaf associated microbes in reduction of air pollutants will be deeply examined, which will provide theoretical bases for developing leaf-based remediation technologies or phylloremediation for mitigating pollutants in the air.

Titanium as a Beneficial Element for Crop Production.

Titanium (Ti) is considered a beneficial element for plant growth. Ti applied via roots or leaves at low concentrations has been documented to improve crop performance through stimulating the activity of certain enzymes, enhancing chlorophyll content and photosynthesis, promoting nutrient uptake, strengthening stress tolerance, and improving crop yield and quality. Commercial fertilizers containing Ti, such as Tytanit and Mg-Titanit, have been used as biostimulants for improving crop production; however, mechanisms underlying the beneficial effects still remain unclear. In this article, we propose that the beneficial roles Ti plays in plants lie in its interaction with other nutrient elements primarily iron (Fe). Fe and Ti have synergistic and antagonistic relationships. When plants experience Fe deficiency, Ti helps induce the expression of genes related to Fe acquisition, thereby enhancing Fe uptake and utilization and subsequently improving plant growth. Plants may have proteins that either specifically or nonspecifically bind with Ti. When Ti concentration is high in plants, Ti competes with Fe for ligands or proteins. The competition could be severe, resulting in Ti phytotoxicity. As a result, the beneficial effects of Ti become more pronounced during the time when plants experience low or deficient Fe supply.

Volatiles Emitted at Different Flowering Stages of Jasminum sambac and Expression of Genes Related to α-Farnesene Biosynthesis.

Fresh jasmine flowers have been used to make jasmine teas in China, but there has been no complete information about volatile organic compound emissions in relation to flower developmental stages and no science-based knowledge about which floral stage should be used for the infusion. This study monitored volatile organic compounds emitted from living flowers of Jasminum sambac (L.) Ait. 'Bifoliatum' at five developmental stages and also from excised flowers. Among the compounds identified, α-farnesene, linalool, and benzyl acetate were most abundant. Since α-farnesene is synthesized through the Mevalonate pathway, four genes encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase, and terpene synthase were isolated. Their expression patterns in living flowers at the five stages and in excised flowers coincided with the emission patterns of α-farnesene. Application of lovastatin, a HMGR inhibitor, significantly reduced the expression of the genes and greatly decreased the emission of α-farnesene. The sweet scent was diminished from lovastatin-treated flowers as well. These results indicate that α-farnesene is an important compound emitted from jasmine flowers, and its emission patterns suggest that flowers at the opening stage or flower buds 8 h after excision should be used for the infusion of tea leaves.