Identification and expression analysis of auxin-responsive GH3 family genes in Chinese hickory (Carya cathayensis) during grafting.

The GH3 genes play vital roles in auxin homeostasis by conjugating excess auxin to amino acids. However, how GH3 genes function during grafting in Chinese hickory (Carya cathayensis) is largely unknown. Here, based on the transcriptome database, a comprehensive identification and expression profiling analysis of 12 GH3 genes in Chinese hickory were performed. Phylogenetic analysis indicated that CcGH3-x exists in a specific subfamily. To understand the roles of CcGH3 genes, tissue-specific expression and the response to different phytohormones were determined. Expression profiles of GH3 genes of Chinese hickory during grafting were analysed. The data suggested that 10 CcGH3 genes were down-regulated at an early stage of grafting, indicating that auxin homeostasis regulated by the CcGH3 family might be inhibited at initial stages. At the completion of grafting, expression levels of members of the CcGH3 family were restored to normal levels. Endogenous auxin levels were also measured, and the data showed that free auxin decreased to the lowest level at an early stage of grafting, and then increased during grafting. Auxin amino acid conjugation increased at an early stage of grafting in rootstock, and then decreased with progression of the graft union. Our results demonstrate that the reduced expression of CcGH3 family genes during grafting might contribute to the release of free auxin, making an important contribution to the recovery of auxin levels after grafting.

Quantitative succinyl-proteome profiling of Chinese hickory (Carya cathayensis) during the grafting process.

BACKGROUND: Chinese hickory (Carya cathayensis) is a popular nut plant having high economic value. Grafting is applied to accelerate the transition from vegetative phase to reproductive phase. Lysine succinylation occurs frequently in the proteins associated with metabolic pathways, which may participate in the regulation of the grafting process. However, the exact regulatory mechanism underlying grafting process in Chinese hickory has not been studied at post-translational modification level. RESULTS: A comprehensive proteome-wide lysine succinylation profiling of Chinese hickory was explored by a newly developed method combining affinity enrichment and high-resolution LC-MS/MS. In total, 259 succinylation sites in 202 proteins were identified, representing the first comprehensive lysine succinylome in Chinese hickory. The succinylation was biased to occur in the cytosolic proteins of Chinese hickory. Moreover, four conserved succinylation motifs were identified in the succinylated peptides. Comparison of two grafting stages of Chinese hickory revealed that the differential expressed succinylated proteins were mainly involved in sugar metabolism, carbon fixation, amino acid metabolism and plant-pathogen interaction. Besides, seven heat shock proteins (HSPs) with 11 succinylation sites were also identified, all of which were observed to be up-regulated during the grafting process. CONCLUSIONS: Succinylation of the proteins involved in amino acid biosynthesis might be required for a successful grafting. Succinylated HSPs might play a role in stress tolerance of the grafted Chinese hickory plants. Our results can be a good resource for functional validation of the succinylated proteins and a starting point for the investigation of molecular mechanisms during lysine succinylation occurring at grafting site.

Analysis of transcriptome in hickory (Carya cathayensis), and uncover the dynamics in the hormonal signaling pathway during graft process.

BACKGROUND: Hickory (Carya cathayensis), a woody plant with high nutritional and economic value, is widely planted in China. Due to its long juvenile phase, grafting is a useful technique for large-scale cultivation of hickory. To reveal the molecular mechanism during the graft process, we sequenced the transcriptomes of graft union in hickory. RESULTS: In our study, six RNA-seq libraries yielded a total of 83,676,860 clean short reads comprising 4.19 Gb of sequence data. A large number of differentially expressed genes (DEGs) at three time points during the graft process were identified. In detail, 777 DEGs in the 7 d vs 0 d (day after grafting) comparison were classified into 11 enriched Gene Ontology (GO) categories, and 262 DEGs in the 14 d vs 0 d comparison were classified into 15 enriched GO categories. Furthermore, an overview of the PPI network was constructed by these DEGs. In addition, 20 genes related to the auxin-and cytokinin-signaling pathways were identified, and some were validated by qRT-PCR analysis. CONCLUSIONS: Our comprehensive analysis provides basic information on the candidate genes and hormone signaling pathways involved in the graft process in hickory and other woody plants.

Exogenous spermidine improved drought tolerance in Ilex verticillata seedlings.

Winterberry (Ilex verticillata (L.) A. Gray) is a recently introduced ornamental tree species in China that has not been closely investigated for its drought resistance. In this study, we used two-year-old cuttings from I. verticillata (L.) A. Gray and two representative varieties derived from it, I. verticillata 'Oosterwijk' and I. verticillata 'Jim Dandy', as materials to investigate how this plant responds to drought stress and whether exogenous spermidine (SPD) can alleviate the negative effects caused by drought stress. The results showed that as the degree of drought stress increased, the leaves of winterberry seedlings became chlorotic, and their edges became dry. Similarly, the relative water content, specific leaf weight, chlorophyll content, leaf nitrogen content, net photosynthetic rate, stomatal conductance and transpiration rate were significantly reduced, whereas the content of malondialdehyde continuously increased with the degree of drought stress. The activities of superoxide dismutase, peroxidase, and catalase increased under moderate drought stress and then decreased under severe drought stress. The levels of soluble sugar and abscisic acid continued to increase, while those of auxin and gibberellic acid decreased. When compared with individual drought stress, an increase in the amount of external SPD clearly alleviated the effect of drought stress on winterberry seedlings. The combined phenotypes and physiological indices of the winterberry leaves under drought stress conditions revealed that the drought resistance of the native species was significantly higher than its two varieties. This finding serves as an important theoretical foundation for the popularization and application of I. verticillata (L.) A. Gray and the two varieties.

Transcriptome Analysis Reveals the Response Mechanism of Digitaria sanguinalis, Arabidopsis thaliana and Poa annua under 4,8-Dihydroxy-1-tetralone Treatment.

4,8-dihydroxy-l-tetralone (4,8-DHT) is an allelochemical isolated from the outer bark of Carya cathayensis that acts as a plant growth inhibitor. In order to explore the mechanism of 4,8-DHT inhibiting weed activity, we treated three species of Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua with different concentrations of 4,8-DHT and performed phenotype observation and transcriptome sequencing. The results showed that with an increase in 4,8-DHT concentration, the degree of plant damage gradually deepened. Under the same concentration of 4,8-DHT, the damage degree of leaves and roots of Digitaria sanguinalis was the greatest, followed by Arabidopsis thaliana, while Poa annua had the least damage, and the leaves turned slightly yellow. Transcriptome data showed that 24536, 9913, and 1662 differentially expressed genes (DEGs) were identified in Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua, respectively. These DEGs were significantly enriched in photosynthesis, carbon fixation, glutathione metabolism, phenylpropanoid biosynthesis, and oxidative phosphorylation pathways. In addition, DEGs were also enriched in plant hormone signal transduction and the MAPK signal pathway in Arabidopsis thaliana. Further analysis showed that after 4,8-DHT treatment, the transcript levels of photosynthesis PSI- and PSII-related genes, LHCA/B-related genes, Rubisco, and PEPC were significantly decreased in Digitaria sanguinalis and Arabidopsis thaliana. At the same time, the transcription levels of genes related to glutathione metabolism and the phenylpropanoid biosynthesis pathway in Digitaria sanguinalis were also significantly decreased. However, the expression of these genes was upregulated in Arabidopsis thaliana and Poa annua. These indicated that 4,8-DHT affected the growth of the three plants through different physiological pathways, and then played a role in inhibiting plant growth. Simultaneously, the extent to which plants were affected depended on the tested plants and the content of 4,8-DHT. The identification of weed genes that respond to 4,8-DHT has helped us to further understand the inhibition of plant growth by allelochemicals and has provided a scientific basis for the development of allelochemicals as herbicides.

Agrobacterium rhizogenes: paving the road to research and breeding for woody plants.

Woody plants play a vital role in global ecosystems and serve as valuable resources for various industries and human needs. While many woody plant genomes have been fully sequenced, gene function research and biotechnological breeding advances have lagged behind. As a result, only a limited number of genes have been elucidated, making it difficult to use newer tools such as CRISPR-Cas9 for biotechnological breeding purposes. The use of Agrobacterium rhizogenes as a transformative tool in plant biotechnology has received considerable attention in recent years, particularly in the research field on woody plants. Over the past three decades, numerous woody plants have been effectively transformed using A. rhizogenes-mediated techniques. Some of these transformed plants have successfully regenerated. Recent research on A. rhizogenes-mediated transformation of woody plants has demonstrated its potential for various applications, including gene function analysis, gene expression profiling, gene interaction studies, and gene regulation analysis. The introduction of the Ri plasmid has resulted in the emergence of several Ri phenotypes, such as compact plant types, which can be exploited for Ri breeding purposes. This review paper presents recent advances in A. rhizogenes-mediated basic research and Ri breeding in woody plants. This study highlights various aspects of A. rhizogenes-mediated transformation, its multiple applications in gene function analysis, and the potential of Ri lines as valuable breeding materials.

Application of titanium regulates the functional components of photosynthetic apparatus in grafted seedlings of Carya cathayensis Sarg. under shade.

Light acts as a key environmental factor for normal growth and development of plants. Carya cathayensis Sarg. (hickory) faces low light conditions, especially those caused by cloudy or rainy days during the rapid growth period, which has caused adverse effects on its growth. In the current investigation, to alleviate the adverse effects of insufficient light on the cultivation of hickory, anti-hydrolyze stabilized ionic titanium (ASIT) was sprayed on the leaves of the three kinds of grafted seedlings and the non-grafted seedlings of hickory grown under different shade conditions. Results showed that the leaf mass per area and chlorophyll content of grafted hickory seedlings were increased after ASIT application. Rubisco content and photosynthetic rate (Pn) of seedlings grown under shading conditions were positively affected by ASIT treatment, especially on the 45th day of treatment, while the interaction effects of the two parameters between ASIT application and different shade treatments were significant. Titanium accumulation was the highest in roots, followed by leaves, and then in stems, while ASIT had the most significant effects on roots and leaves under 50 ± 5% shade. Severe shading inhibited growth and lead to serious destruction of chloroplast ultrastructure. In addition, the role of ASIT was rootstock-dependent, since ASIT had the weakest mitigation effect on the C/H grafted seedlings. To sum up, the application of ASIT to the grafted seedlings of hickory could improve its ability to resist shade stress.

Expression analysis of PIN family genes in Chinese hickory reveals their potential roles during grafting and salt stress.

Grafting is an effective way to improve Chinese hickory while salt stress has caused great damage to the Chinese hickory industry. Grafting and salt stress have been regarded as the main abiotic stress types for Chinese hickory. However, how Chinese hickory responds to grafting and salt stress is less studied. Auxin has been proved to play an essential role in the stress response through its re-distribution regulation mediated by polar auxin transporters, including PIN-formed (PIN) proteins. In this study, the PIN gene family in Chinese hickory (CcPINs) was identified and structurally characterized for the first time. The expression profiles of the genes in response to grafting and salt stress were determined. A total of 11 CcPINs with the open reading frames (ORFs) of 1,026-1,983 bp were identified. Transient transformation in tobacco leaves demonstrated that CcPIN1a, CcPIN3, and CcPIN4 were localized in the plasma membrane. There were varying phylogenetic relationships between CcPINs and homologous genes in different species, but the closest relationships were with those in Carya illinoinensis and Juglans regia. Conserved N- and C-terminal transmembrane regions as well as sites controlling the functions of CcPINs were detected in CcPINs. Five types of cis-acting elements, including hormone- and stress-responsive elements, were detected on the promoters of CcPINs. CcPINs exhibited different expression profiles in different tissues, indicating their varied roles during growth and development. The 11 CcPINs responded differently to grafting and salt stress treatment. CcPIN1a might be involved in the regulation of the grafting process, while CcPIN1a and CcPIN8a were related to the regulation of salt stress in Chinese hickory. Our results will lay the foundation for understanding the potential regulatory functions of CcPIN genes during grafting and under salt stress treatment in Chinese hickory.

Genome-wide identification and expression analysis of AUX/LAX family genes in Chinese hickory (Carya cathayensis Sarg.) Under various abiotic stresses and grafting.

Auxin is essential for regulating plant growth and development as well as the response of plants to abiotic stresses. AUX/LAX proteins are auxin influx transporters belonging to the amino acid permease family of proton-driven transporters, and are involved in the transport of indole-3-acetic acid (IAA). However, how AUX/LAX genes respond to abiotic stresses in Chinese hickory is less studied. For the first time identification, structural characteristics as well as gene expression analysis of the AUX/LAX gene family in Chinese hickory were conducted by using techniques of gene cloning and real-time fluorescent quantitative PCR. Eight CcAUX/LAXs were identified in Chinese hickory, all of which had the conserved structural characteristics of AUX/LAXs. CcAUX/LAXs were most closely related to their homologous proteins in Populus trichocarpa , which was in consistence with their common taxonomic character of woody trees. CcAUX/LAXs exhibited different expression profiles in different tissues, indicating their varying roles during growth and development. A number of light-, hormone-, and abiotic stress responsive cis-acting regulatory elements were detected on the promoters of CcAUX/LAX genes. CcAUX/LAX genes responded differently to drought and salt stress treatments to varying degrees. Furthermore, CcAUX/LAX genes exhibited complex expression changes during Chinese hickory grafting. These findings not only provide a valuable resource for further functional validation of CcAUX/LAXs, but also contribute to a better understanding of their potential regulatory functions during grafting and abiotic stress treatments in Chinese hickory.

Chromium Bioaccumulation and Its Impacts on Plants: An Overview.

Chromium (Cr) is an element naturally occurring in rocky soils and volcanic dust. It has been classified as a carcinogen agent according to the International Agency for Research on Cancer. Therefore, this metal needs an accurate understanding and thorough investigation in soil-plant systems. Due to its high solubility, Cr (VI) is regarded as a hazardous ion, which contaminates groundwater and can be transferred through the food chain. Cr also negatively impacts the growth of plants by impairing their essential metabolic processes. The toxic effects of Cr are correlated with the generation of reactive oxygen species (ROS), which cause oxidative stress in plants. The current review summarizes the understanding of Cr toxicity in plants via discussing the possible mechanisms involved in its uptake, translocation and sub-cellular distribution, along with its interference with the other plant metabolic processes such as chlorophyll biosynthesis, photosynthesis and plant defensive system.

Melatonin regulates the functional components of photosynthesis, antioxidant system, gene expression, and metabolic pathways to induce drought resistance in grafted Carya cathayensis plants.

The Chinese hickory (Carya cathayensis) is an economically important tree species popular for its nuts. However, the tree requires a long time to reach the nut-producing phase. To overcome this problem, grafting is widely used to reduce the time from the vegetative to the reproductive phase. This tree species also faces many environmental challenges due to climate change; drought is an important factor affecting growth and development. Here, we designed an experiment to assess the protective efficiency of melatonin in grafted Chinese hickory plants under drought stress. The results revealed that exogenously applied melatonin successfully recovered the growth of grafted Chinese hickory plants and improved photosynthetic efficiency. Exogenously applied melatonin also boosted the antioxidative defense system of the plants under drought stress, resulting in enhanced reactive oxygen species (ROS) scavenging. The accumulation of compatible solutes such as total soluble sugars and proline was also triggered by melatonin. Moreover, the analyses using metabolomics revealed that drought-stressed plants treated with melatonin regulated key metabolic pathways such as phenylpropanoid, chlorophyll and carotenoid biosynthesis, carbon fixation, and sugar metabolism. To further validate the physiological, biochemical, and metabolomic factors, we studied the molecular mechanisms by analyzing the expression of key genes involved in chlorophyll metabolism (chlorophyllase, CHLASE), antioxidative defense (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; peroxidase, POD), and phenylalanine ammonia-lyase (PAL). Exogenously applied melatonin significantly regulated the transcript levels of key genes involved in the biological processes mentioned above. Melatonin also showed crosstalk with other hormones (zeatin, gibberellin A14, 24-epibrassinolide, jasmonic acid, and abscisic acid) to regulate the physiological processes. The results of this study show that melatonin regulates biological processes at the metabolic and molecular levels to resist drought stress.

Gene expression of halophyte Kosteletzkya virginica seedlings under salt stress at early stage.

Gene differential expression of Kosteletzkya virginica seedlings under salt stress at two time points (2, 24 h) in roots and leaves was analyzed using the cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Polymorphic transcript-derived fragments (TDFs) among control plants and salt-treated plants were grouped into four main differential expression patterns: repression (A), de novo induction (B), up-regulation (C) and down-regulation (D). Among them, 34 differentially expressed gene fragments were homologous to known genes from other species and 4 were sequences with unknown functions. These differentially expressed genes can be classified into four groups according to their putative functions: (1) genes for re-establishing ion homeostasis and protecting the plant from stress damage; (2) genes involved in metabolism or energy and resuming plant growth and development under salt stress; (3) genes involved in regulation of gene expression; (4) genes for signal transduction. Changes of eight differentially expressed genes were confirmed by quantitative real time RT-PCR.

[Analysis of gene expression involved in the response to salt stress in the dicot halophyte Kosteletzkya virginica L. seedlings].

Kosteletzkya virginica L. Presl. is an obligate wetland species indigenous to southeastern US. Its niche in salt marsh foretells its high salinity tolerance. cDNA-AFLP technique was used to identify the gene transcriptional profiles of leaves and roots from K. virginica seedlings under salt stress in order to clarify the molecular architecture of stress tolerance in the dicot halophyte. Expression analysis over time intervals and under various salt stresses in leaves or roots showed that the quantitatively expressed pattern (in which genes were quantitatively up- or down-regulated under salt stress or fluctuate with different NaCl concentrations) was more prevalent than the qualitatively expressed pattern (in which genes were induced or silenced under salt stress) in K. virginica seedlings under salt stress. The qualitative pattern was appreciably more predominant than the quantitative one only in roots when exposed to salt stress for 2 h. Although each expression pattern was observed in leaves as well as in roots, the percentage of genes (i.e., up-/down-regulated or induced/silenced under salt stress) was dynamically changeable under salt stress at different time intervals. All these results indicated that there was no established formula of gene expression patterns in deciphering the sophisticated mechanism of plant salinity tolerance, considering that plants undergo a series of dynamically physiological and metabolic pathways in sensing and response to salt stress for different tissues and during different stages of stress. A number of Trivially distributed file system (TDFs) up-regulated or induced under salt stress from leaves and roots were sequenced, and the sequences were blasted against the NCBI non-redundant protein database using translated nucleotide query (Blastx). The TDFs from K. virginica seedlings involved in sensing and response to salt stress can be classified at least into three groups according to their putative functions: (1) genes for re-establishing ionic homeostasis or preventing from damage (specially genes for transporter); (2) genes for resuming plant growth and development under salt stress, such as key enzymes involved in energy synthesis or hormone regulatory pathway; (3) genes for signal transduction and so on. The relationship of expression patterns of these TDFs with the molecular mechanism of salt tolerance in K. virginica was discussed.

Identification and expression profiling of the Aux/IAA gene family in Chinese hickory (Carya cathayensis Sarg.) during the grafting process.

Auxin is an essential regulator in various aspects of organism growth and development. Members of the Aux/IAA family of genes encode short-lived nuclear proteins and mediate the responses of auxin-regulated gene expression. Here, the first identification and characterization of 22 cDNAs encoding the open reading frame of the Aux/IAA family in Chinese hickory (named as CcIAA) has been performed. The proteins encoded by these genes contain four whole or partially conserved domains of the Aux/IAA family. Phylogenetic analysis indicated that CcIAAs were unevenly distributed among eight different subgroups. The spatio-specific expression profiles showed that most of the CcIAAs preferentially expressed in specific tissues. Three CcIAA genes, including CcIAA11, CcIAA27a2 and CcIAAx, were predominantly expressed in stem. The predominant expression of CcIAA genes in stems might play important roles in vascular reconnection during the graft process. Furthermore, expression profiles of Aux/IAA genes during the grafting process of Chinese hickory have been analysed. Our data suggested that 19 CcIAAs were down-regulated and 3 CcIAAs (including CcIAA28, CcIAA8a and CcIAA27b) were induced, indicating their specializations during the grafting process. The involvement of CcIAA genes at the early stage after grafting gives us an opportunity to understand the role of auxin signalling in the grafting process.

Comparative Proteomic Analysis of the Graft Unions in Hickory (Carya cathayensis) Provides Insights into Response Mechanisms to Grafting Process.

Hickory (Carya cathayensis), a tree with high nutritional and economic value, is widely cultivated in China. Grafting greatly reduces the juvenile phase length and makes the large scale cultivation of hickory possible. To reveal the response mechanisms of this species to grafting, we employed a proteomics-based approach to identify differentially expressed proteins in the graft unions during the grafting process. Our study identified 3723 proteins, of which 2518 were quantified. A total of 710 differentially expressed proteins (DEPs) were quantified and these were involved in various molecular functional and biological processes. Among these DEPs, 341 were up-regulated and 369 were down-regulated at 7 days after grafting compared with the control. Four auxin-related proteins were down-regulated, which was in agreement with the transcription levels of their encoding genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the 'Flavonoid biosynthesis' pathway and 'starch and sucrose metabolism' were both significantly up-regulated. Interestingly, five flavonoid biosynthesis-related proteins, a flavanone 3-hyfroxylase, a cinnamate 4-hydroxylase, a dihydroflavonol-4-reductase, a chalcone synthase, and a chalcone isomerase, were significantly up-regulated. Further experiments verified a significant increase in the total flavonoid contents in scions, which suggests that graft union formation may activate flavonoid biosynthesis to increase the content of a series of downstream secondary metabolites. This comprehensive analysis provides fundamental information on the candidate proteins and secondary metabolism pathways involved in the grafting process for hickory.