The Role of γ-Aminobutyric Acid (GABA) in the Occurrence of Adventitious Roots and Somatic Embryos in Woody Plants.

The occurrence of adventitious roots and somatic embryos is a crucial step in micropropagation that frequently limits the application of this technique in woody plants. Recent studies demonstrated that they can be negatively or positively regulated with γ-aminobutyric acid (GABA), which is a four-carbon non-proteinous amino acid that not only acts as a main inhibitory neurotransmitter in mammals. It has been reported that GABA affects plant growth and their response to stress although its mode of action is still unclear. This review dealt with the effects of GABA on adventitious root formation and growth as well as on somatic embryogenesis. Furthermore, we focused on discussing the interaction of GABA with phytohormones, such as auxin, ethylene, abscisic acid, and gibberellin, as well as with the carbon and nitrogen metabolism during adventitious root development. We suggested that research on GABA will contribute to the application of micropropagation in the recalcitrant fruit and forest species.

Acclimation Strategy of Masson Pine (Pinus massoniana) by Limiting Flavonoid and Terpenoid Production under Low Light and Drought.

Low light and drought often limit the growth and performance of Masson pines (Pinus massoniana) in the subtropical forest ecosystem of China. We speculated that stress-induced defensive secondary metabolites, such as flavonoids and terpenoids, might influence the growth of Masson pines, considering the existence of tradeoffs between growth and defense. However, the mechanisms of Masson pines responsive to low light and drought at the levels of these two metabolites remain unclear. In the present work, the compositions of flavonoids and terpenoids, as well as their biosynthetic pathways, were revealed through metabolome and transcriptome analyses, respectively, coupled with a study on carbon allocation using a 13CO2-pulse-labeling experiment in two-year-old seedlings under low light (LL), drought (DR), and their combined stress (DL) compared to a control (CK). A total of 35 flavonoids and derivatives (LL vs. CK: 18; DR vs. CK: 20; and DL vs. CK: 18), as well as 29 terpenoids and derivatives (LL vs. CK: 23; DR vs. CK: 13; and DL vs. CK: 7), were differentially identified in the leaves. Surprisingly, most of them were decreased under all three stress regimes. At the transcriptomic level, most or all of the detected DEGs (differentially expressed genes) involved in the biosynthetic pathways of flavonoids and terpenoids were downregulated in phloem and xylem under stress treatments. This indicated that stress treatments limited the production of flavonoids and terpenoids. The reduction in the 13C allocation to stems might suggest that it is necessary for maintaining the growth of Masson pine seedlings at the whole-plant level by attenuating energetic resources to the biosynthetic pathways of flavonoids and terpenoids when facing the occurrence of adverse environments. Our results provide new insight into understanding the acclimation strategy of Masson pines or other conifers in adverse environments.

Effects of citric acid on antioxidant system and carbon-nitrogen metabolism of Elymus dahuricus under Cd stress.

Exogenous citric acid (CA), which acts as an important intermediate product of the tricarboxylic acid (TCA) cycle, can enhance the TCA cycle activity and activate the branched operation of the TCA cycle, thus providing energy required for resistance to adverse conditions. However, the effects of CA application on TCA cycle-related metabolism under cadmium (Cd) were less reported. To investigate the effects of CA on the Cd tolerance of Dahurian wildrye grass (Elymus dahuricus), the growth, Cd accumulation, antioxidant systems and metabolic pathways of leaves and roots were investigated by a potted soil experiment with Cd (50 mg/kg) and CA (4 mmol/L) treatments. The results showed that Cd stress seriously affected growth and induced the production of reactive oxygen in clover leaves and roots, leading to membrane peroxidation and activation of the antioxidant defense system. Exogenous CA could not only effectively relieve the inhibition of Cd stress on growth and reduce the amount of reactive oxygen by increasing the antioxidant capacities but could also promote an increase in root Cd content. Metabolomic results showed that the application of CA increased the contents of sugars, sugar alcohols, and resistant substances, and promoted the metabolism of amino acids including γ-aminobutyric acid (GABA). These alterations contributed the significant enhancement of the Cd resistance, which may be related to the changes in the TCA cycle activity and the metabolism of the shikimic acid pathway in leaves and roots as well as GABA shunt in roots.

Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology.

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

Chinese chestnut yellow crinkle disease influence microbiota composition of chestnut trees.

CONTEXT: Candidatus-phytoplasma castaneae has been found as the causal agent of the Chinese chestnut yellow crinkle disease. However, the ecological impact of the disease on microbiota of chestnut trees is unknown. AIMS: The study aim was to clarify difference attributes in microbial community structure of asymptomatic and symptomatic chestnut leaves and twigs for chestnut tree health in orchard. METHODS: Sample collections were conducted with both symptomatic and asymptomatic chestnut trees. Total DNA was extracted. Fungal ITS rDNA and bacterial 16S rDNA were amplified. The PCR products were sequenced with Illumina HiSeq. Platform. RESULTS: A total number of 852 fungal and 1156 bacterial OTUs (operational taxonomic units) were detected. The asymptomatic samples had a higher fungal and bacterial diversity than symptomatic ones. Non-metric multidimensional scaling (NMDS) analysis showed microbial communities among symptomatic and asymptomatic leaves and twigs samples formed individual cluster. Overall, Ascomycota and Proteobacteria were the most abundant fungal and bacterial phyla, respectively. Significantly different taxa playing key roles for each microbiota structure were identified. In symptomatic trees, microbial groups of plant pathogens were more abundant. CONCLUSION: Our results demonstrated that the phytoplasma pathogen may exert significant influence on the microbial community structure. The study will provide further fundamental clues for the little studied phytoplasma pathogens effects on host microbiota, phytoplasma pathogen control strategies.

[Response of photosynthesis and carbon/nitrogen metabolism to drought stress in Chinese chestnut 'Yanshanzaofeng' seedlings]. / æ¿æ —"燕山早丰"å¹¼è‹—å ‰åˆä¸Žç¢³æ°®ä»£è°¢å¯¹å¹²æ—±èƒè¿«çš„å“åº”.

Drought is a main factor affecting the growth and yield of Chinese chestnut trees in Yan-shan Mountains. To investigate the responses of chestnut seedlings to drought stress, the growth and physiological indices, including photosynthetic characteristics, biomass, proline, malondialdehyde, carbon and nitrogen contents were measured in roots, stems, and leaves after the Chinese chestnut 'Yanshanzaofeng' seedlings in the pots were treated by simulating drought for 22 days. The results showed that, compared with the normal irrigation, water contents in the roots, stems and leaves were decreased by 18.3%, 29.0% and 62.8%, respectively, accompanied by the considerable increases in the contents of proline (355.0%-1586.7%) and malondialdehyde except in the stems (41.1%-81.3%). The non-photochemical quenching coefficiency and net photosynthetic rate in the leaves were significantly decreased by 49.4% and 77.4%, respectively. The contents of non-structural carbohydrates were increased by 21.4% in stems and 69.5% in leaves, but that in roots did not change. The contents of nitrate were increased by 28.9% in stems and 26.8% in leaves, but that in roots did not change. Ammonium nitrogen was increased by 16.2%, 12.9% and 217.6% in roots, stems, and leaves, but being statistically significant in the leaves. These results indicated that drought stress led to serious damage to 'Yanshanzaofeng' chestnut seedlings, which inhibited photosynthetic performance, but they could improve their adaptation to drought stress by enhancing carbon and nitrogen metabolism. Our results provide a reference for the breeding and cultivation of drought resistance of the local Chinese chestnut resources.

A full-length transcriptome and gene expression analysis reveal genes and molecular elements expressed during seed development in Gnetum luofuense.

BACKGROUND: Gnetum is an economically important tropical and subtropical gymnosperm genus with various dietary, industrial and medicinal uses. Many carbohydrates, proteins and fibers accumulate during the ripening of Gnetum seeds. However, the molecular mechanisms related to this process remain unknown. RESULTS: We therefore assembled a full-length transcriptome from immature and mature G. luofuense seeds using PacBio sequencing reads. We identified a total of 5726 novel genes, 9061 alternative splicing events, 3551 lncRNAs, 2160 transcription factors, and we found that 8512 genes possessed at least one poly(A) site. In addition, gene expression comparisons of six transcriptomes generated by Illumina sequencing showed that 14,323 genes were differentially expressed from an immature stage to a mature stage with 7891 genes upregulated and 6432 genes downregulated. The expression of 14 differentially expressed transcription factors from the MADS-box, Aux/IAA and bHLH families was validated by qRT-PCR, suggesting that they may have important roles in seed ripening of G. luofuense. CONCLUSIONS: These findings provide a valuable molecular resource for understanding seed development of gymnosperms.

Effects of exogenous γ-Aminobutyric acid on the regulation of respiration and protein expression in germinating seeds of mungbean (Vigna radiata) under salt conditions

BACKGROUND: γ-Aminobutyric acid (GABA) bypasses the TCA cycle via GABA shunt, suggesting a relationship with respiration. However, little is known about its role in seed germination under salt conditions. RESULTS: In this study, exogenous GABA was shown to have almost no influence on mungbean seed germination, except 0.1 mM at 10 h, while it completely alleviated the inhibition of germination by salt treatment. Seed respiration was significantly inhibited by 0.1 and 0.5 mM GABA, but was evidently enhanced under salt treatment, whereas both were promoted by 1 mM GABA alone or with salt treatment. Mitochondrial respiration also showed a similar trend at 0.1 mM GABA. Moreover, proteomic analysis further showed that 43 annotated proteins were affected by exogenous GABA, even 0.1 mM under salt treatment, including complexes of the mitochondrial respiratory chain. CONCLUSIONS: Our study provides new evidence that GABA may act as a signal molecule in regulating respiration of mungbean seed germination in response to salt stress.

Exogenous GABA promotes adaptation and growth by altering the carbon and nitrogen metabolic flux in poplar seedlings under low nitrogen conditions.

Nitrogen (N) deficiency adversely affects tree growth. Additionally, γ-aminobutyric acid (GABA) is closely associated with growth and stress responses because of its effects on carbon (C) and N metabolism. However, little is known about its roles related to plant adaptations to N-deficient conditions. In this study, we analyzed the effects of GABA (0, 2 and 10 mM) applications on the growth traits and physiological responses of poplar (Populus alba × P. glandulosa '84K') seedlings under high N (HN) and low N (LN) conditions. We found that the added GABA interacted with N to affect more than half of the studied parameters, with greater effects in LN plants than in HN plants. Under LN conditions, the GABA application tended to increase poplar growth, accompanied by increased xylem fiber cell length and xylem width. In stems, exogenous GABA increased the abundance of non-structural carbohydrates (starch and sugars) and tricarboxylic acid cycle intermediates (succinate, malate and citrate), but had the opposite effect on the structural C contents (hemicellulose and lignin). Meanwhile, exogenous GABA increased the total soluble protein contents in leaves and stems, accompanied by significant increases in nitrate reductase, nitrite reductase and glutamine synthetase activities in leaves, but significant decreases in those (except for the increased glutamate synthetase activity) in stems. A multiple factorial analysis indicated that the nitrate assimilation pathway substantially influences poplar survival and growth in the presence of GABA under LN conditions. Interestingly, GABA applications also considerably attenuated the LN-induced increase in the activities of leaf antioxidant enzymes, including peroxidase and catalase, implying that GABA may regulate the relative allocation of C and N for growth activities by decreasing the energy cost associated with stress defense. Our results suggest that GABA enhances poplar growth and adaptation by regulating the C and N metabolic flux under N-deficient conditions.

Comparative study of alleviation effects of DMTU and PCIB on root growth inhibition in two tall fescue varieties under cadmium stress.

In plants, tolerance to cadmium (Cd) stress is closely related to indole-3-acetic acid (IAA) and hydrogen peroxide (H2O2). However, it is unclear whether Cd-resistant and -sensitive varieties respond differently to Cd stress. In this study, the effects of dimethylthiourea (DMTU, a H2O2 scavenger) and p-chlorophenoxy isobutyric acid (PCIB, an IAA signaling inhibitor) on root growth, endogenous hormones and antioxidant system were investigated to decipher how DMTU and PCIB treatments alleviate the inhibition of root elongation in Cd-resistant (Commander) and -sensitive (Crossfire III) tall fescue varieties under Cd stress. Both varieties subjected to 10 µM Cd treatments for 12 h presented a substantial decrease in root elongation coupled with a reduction in brassinosteroid (BR) and zeatin riboside (ZR) contents, but the changes in IAA and abscisic acid (ABA) contents under Cd stress were opposite in the two varieties. In addition, the H2O2 content and antioxidant enzyme activities significantly increased in both varieties. However, pretreatment with PCIB or DMTU mitigated the inhibition of root elongation caused by Cd, accompanied by the significant changes of aforementioned physiological parameters. PCIB significantly reduced the IAA content in 'Commander', while DMTU significantly increased the IAA content in 'Crossfire III' and effectively relieved the inhibition of root elongation. But both treatments decreased the Cd-induced H2O2 accumulation. These results indicated that DMTU or PCIB can alleviate the Cd-inhibited root elongation in two varieties whose resistance differed under Cd stress, but they presented differences in the response of hormones, especially IAA, which may be due to the different adaptation mechanisms of two varieties in response to Cd stress.

Effects of GABA and Vigabatrin on the Germination of Chinese Chestnut Recalcitrant Seeds and Its Implications for Seed Dormancy and Storage.

Recalcitrant chestnut seeds are rich in γ-aminobutyric acid (GABA), which negatively regulates adventitious root development by altering carbon/nitrogen metabolism. However, little is known regarding the role of this metabolite in chestnut seeds. In this study, we investigated the effects of GABA changes on the germination of chestnut seeds treated with exogenous GABA and vigabatrin (VGB, which inhibits GABA degradation). Both treatments significantly inhibited seed germination and primary root growth and resulted in the considerable accumulation of H2O2, but the endogenous GABA content decreased before germination at 48 h. Soluble sugar levels increased before germination, but subsequently decreased, whereas starch contents were relatively unchanged. Changes to organic acids were observed at 120 h after sowing, including a decrease and increase in citrate and malate levels, respectively. Similarly, soluble protein contents increased at 120 h, but the abundance of most free amino acids decreased at 48 h. Moreover, the total amino acid levels increased only in response to VGB at 0 h. Accordingly, GABA and VGB altered the balance of carbon and nitrogen metabolism, thereby inhibiting chestnut seed germination. These results suggested that changes to GABA levels in chestnut seeds might prevent seed germination. The study data may also help clarify the dormancy and storage of chestnut seeds, as well as other recalcitrant seeds.

Responses of GABA shunt coupled with carbon and nitrogen metabolism in poplar under NaCl and CdCl2 stresses.

The γ-aminobutyric acid (GABA) shunt is closely associated with plant tolerance; however, little is known about its mechanism. This study aimed to decipher the responses of the GABA shunt and related carbon-nitrogen metabolism in poplar seedlings (Populus alba × Populus glandulosa) treated with different NaCl and CdCl2 concentrations for 30 h. The results showed that the activities of glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) were activated, as well as α-ketoglutarate dehydrogenase (α-KGDH) and succinate dehydrogenase (SDH) activities were enhanced by NaCl and CdCl2 stresses, except for SDH under CdCl2 stress. Meanwhile, the expression levels of GADs, GABA-Ts SDHs, succinyl-CoA ligases (SCSs), and succinic acid aldehyde dehydrogenases (SSADHs) were also increased. Notably, significant increases in the key components of GABA shunt, Glu and GABA, were observed under both stresses. Soluble sugars and free amino acids were enhanced, whereas citrate, malate and succinate were almost inhibited by both NaCl and CdCl2 stresses except that citrate was not changed or just increased by 50-mM NaCl stress. Thus, these results suggested that the carbon-nitrogen balance could be altered by activating the GABA shunt when main TCA-cycle intermediates were inhibited under NaCl and CdCl2 stresses. This study can enhance the understanding about the functions of the GABA shunt in woody plants under abiotic stresses and may be applied to the genetic improvement of trees for phytoremediation.

GABA negatively regulates adventitious root development in poplar.

γ-Aminobutyric acid (GABA) influences plant growth, but little is known about how this metabolite regulates adventitious root (AR) development. Here, we investigate the effects of GABA on ARs using poplar lines overexpressing glutamate decarboxilase 2 (GAD2) and by treating poplar stem cuttings with exogenous GABA or vigabatrin (VGB; a specific GABA transaminase inhibitor). Endogenous GABA accumulation not only inhibited AR growth, but it also suppressed or delayed AR formation. Anatomical observations revealed that the GABA and VGB treatments resulted in a 1 d delay in the formation of AR primordia and the appearance of ARs. This delay coincided with changes in primary metabolism, including transient increases in hexose and amino acid levels. GABA-dependent changes in the expression of genes related to hormone synthesis and signalling, as well as analysis of hormone levels revealed that ethylene-dependent pathways were decreased at the earliest stage of AR formation. In contrast, auxin and abscisic acid were increased at 1-5 d as well as GA4 over a 5 d period of AR formation. These results demonstrate that GABA plays a crucial role in AR development. Evidence is presented demonstrating that GABA can interact with hormone-related pathways as well as carbon/nitrogen metabolism. These findings also elucidate the functions of GABA in plant development.

γ-Aminobutyric acid is closely associated with accumulation of flavonoids.

γ-Aminobutyric acid (GABA) is an important neurotransmitter in mammals whose receptor is reported to be regulated by flavonoids. In plants, it is considered to be at the intersection of carbon and nitrogen metabolism, but its relationship with flavonoid metabolism remains unclear. Our recent RNA-seq analysis showed that expression of flavonoid biosynthetic genes was influenced in poplar by the blockage of α-ketoglutarate dehydrogenase (α-KGDH) activity and the application of GABA under NaCl stress, accompanied by the changes in GABA shunt activity. Here, we further found that the flavonoid accumulation was significantly affected by blocking the activities of α-KGDH and GABA transaminase as well as applying exogenous GABA, coupled with the changes of endogenous GABA contents. Key genes involved in the flavonoid biosynthetic pathway were also significantly influenced, including two PALs, 4CL, and two CHSs. Our results suggest that the GABA shunt is closely associated with the metabolism of flavonoids, which would benefit future understanding of GABA's roles in carbon allocation by regulating the pathway of flavonoid biosynthesis under normal or stress conditions.

Significance of Photosynthetic Characters in the Evolution of Asian Gnetum (Gnetales).

Gnetum is a genus in the Gnetales that has a unique but ambiguous placement within seed plant phylogeny. Previous studies have shown that Gnetum has lower values of photosynthetic characters than those of other seed plants, but few Gnetum species have been studied, and those that have been studied are restricted to narrow taxonomic and geographic ranges. In addition, the mechanism underlying the lower values of photosynthetic characters in Gnetum remains poorly understood. Here, we investigated the photosynthetic characters of a Chinese lianoid species, i.e., Gnetum parvifolium, and co-occurring woody angiosperms growing in the wild, as well as seedlings of five Chinese Gnetum species cultivated in a greenhouse. The five Gnetum species had considerably lower values for photosynthesis parameters (net photosynthetic rate, transpiration rate, intercellular CO2 concentration, and stomatal conductance) than those of other seed plant representatives. Interrelated analyses revealed that the low photosynthetic capacity may be an intrinsic property of Gnetum, and may be associated with its evolutionary history. Comparison of the chloroplast genomes (cpDNAs) of Gnetum with those of other seed plant representatives revealed that 17 coding genes are absent from the cpDNAs of all species of Gnetum. This lack of multiple functional genes from the cpDNAs probably leads to the low photosynthetic rates of Gnetum. Our results provide a new perspective on the evolutionary history of the Gnetales, and on the ecophysiological and genomic attributes of tropical biomes in general. These results could also be useful for the breeding and cultivation of Gnetum.

Inhibition of α-ketoglutarate dehydrogenase activity affects adventitious root growth in poplar via changes in GABA shunt.

MAIN CONCLUSION: Blocking α-ketoglutarate dehydrogenase results in up-regulation of γ-aminobutyric acid (GABA) shunt activity, and inhibits the growth of poplar adventitious roots (ARs), indicating that AR growth is closely associated with GABA shunt. γ-Aminobutyric acid (GABA) shunt starts from α-ketoglutarate in the tricarboxylic acid cycle, which is thought to represent the cross road between carbon and nitrogen metabolism. Previous studies (Araújo et al. 2012b, Plant Cell 24: 2328-2351) have shown that blocking α-ketoglutarate dehydrogenase (α-KGDH) affects the GABA shunt activity, and inhibits growth. However, its effects on the growth of adventitious roots (ARs) are unclear. In this study, the growth of ARs in tissue-cultured 84K poplar (Populus alba × Populus glandulosa cv. '84K') was significantly inhibited when succinyl phosphate (SP), a specific inhibitor of α-KGDH, was supplied. The inhibition of ARs was associated with significant changes in the levels of soluble sugars, organic acids, and amino acids, and was coupled with the up-regulation of the GABA shunt activity at the transcriptional and translational levels. Exogenous GABA also inhibited AR growth following the increase of the endogenous GABA level. Transcriptomic analyses further showed that genes related to cell wall carbon metabolism and phytohormone (indoleacetic acid, ABA, and ethylene) signaling were affected by the changes of GABA shunt activity, resulting from the α-KGDH inhibition. Thus, our study indicates that the inhibition of poplar AR growth by blocking α-KGDH is closely associated with GABA shunt, which would benefit a better understanding of GABA's roles in plant development and stress response.

Roles of γ-aminobutyric acid on salinity-responsive genes at transcriptomic level in poplar: involving in abscisic acid and ethylene-signalling pathways.

MAIN CONCLUSION: γ-Aminobutyric acid (GABA) affected ABA and ethylene metabolic genes and signal components in salt-treated poplar, indicating its potential role in signal pathways of ABA and ethylene during salt stress. GABA is a small signalling molecule that accumulates rapidly in plants exposed to various stresses. However, the relationship between GABA and other signalling molecules, such as hormones, remains unclear. Here, in the poplar woody plant under 200-mM NaCl conditions, the application of low (0.25 mM) and high (10 mM) exogenous GABA, compared to 0 mM, affected the accumulation of hydrogen peroxide and hormones, including ABA and ethylene, in different manners. Transcriptomic analysis demonstrated that 1025 differentially expressed genes (DEGs; |log2Ratio| ≥ 1.5) were widely affected by exogenous GABA under salt stress. A clustering analysis revealed that GABA could rescue or promote the effects of salt stress on gene expression. Among them, 146 genes involved in six hormone-signalling pathways were enriched, including 22 ABA- and 50 ethylene-related genes. Quantitative expression of selected genes involved in hormone-related pathways showed that ABA metabolic genes (ABAG, ABAH2, and ABAH4), ethylene biosynthetic genes (ACO1, ACO2, ACO5, ACOH1, ACS1, and ACS7) and receptor genes (PYL1, PYL2, PYL4, and PYL6) were regulated by exogenous GABA, even at a 0.1 mM level. The production of ABA was negatively correlated with ABAH expression levels at different GABA concentrations. The increase of endogenous GABA, resulting from inhibitor (succinyl phosphonate) of α-ketoglutarate dehydrogenase, affected the PYLs levels. Thus, GABA may be involved in ABA- and ethylene-signalling pathways. Our data provide a better understanding of GABA's roles in the plant responses to environmental stresses.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Transcriptome differences between 20- and 3, 000-year-old Platycladus orientalis reveal that ROS are involved in senescence regulation

Background: Platycladus orientalis has an extremely long life span of several thousands of years, attracting great interests in the mechanisms involved in such successful senescence regulation and resistance at physiological and molecular levels. Results: The levels of reactive oxygen species (ROS) were higher in 3,000-year-old than in 20-year-old P. orientalis, and the activities of GR and GSH demonstrated the same trend. We produced and analyzed massive sequence information from pooled samples of P. orientalis through transcriptome sequencing, which generated 51,664 unigenes with an average length of 475 bp. We then used RNA-seq analysis to obtain a high-resolution age­course profile of gene expression in 20- and 3,000-year-old P. orientalis individuals. Totally, 106 differentially expressed genes were obtained, of which 47 genes were downregulated and 59 upregulated in the old tree. These genes were involved in transcription factors, hormone-related responses, ROS scavengers, senescence-related responses, stress response, and defense and possibly play crucial roles in tackling various stresses in the 3,000-year-old P. orientalis during its life time. The expression patterns of genes related to ROS homeostasis further indicated that the high ability of ROS scavenging could be helpful for the 3,000-year-old P. orientalis to resist senescence. Conclusions: This study provides a foundation for the elucidation of senescence resistance through molecular studies and the discovery of useful genes in P. orientalis.