Roles of JnRAP2.6-like from the transition zone of black walnut in hormone signaling.
ABSTRACT: An EST sequence, designated JnRAP2-like, was isolated from tissue at the heartwood/sapwood transition zone (TZ) in black walnut (Juglans nigra L). The deduced amino acid sequence of JnRAP2-like protein consists of a single AP2-containing domain with significant similarity to conserved AP2/ERF DNA-binding domains in other species. Based on multiple sequence alignment, JnRAP2-like appears to be an ortholog of RAP2.6L (At5g13330), which encodes an ethylene response element binding protein in Arabidopsis thaliana. Real-time PCR revealed that the JnRAP2-like was expressed most abundantly in TZ of trees harvested in fall when compared with other xylem tissues harvested in the fall or summer. Independent transgenic lines over-expressing JnRAP2-like in Arabidopsis developed dramatic ethylene-related phenotypes when treated with 50 µM methyl jasmonate (MeJA). Taken together, these results indicated that JnRAP2-like may participate in the integration of ethylene and jasmonate signals in the xylem and other tissues. Given the role of ethylene in heartwood formation, it is possible JnRAP2-like expression in the transition zone is part of the signal transduction pathway leading to heartwood formation in black walnut.
Project description:Taiwania (Taiwania cryptomerioides) is an important tree species in Taiwan because of the excellent properties of its wood and fascinating color qualities of its heartwood (HW), as well as the bioactive compounds therein. However, limited information is available as to the HW formation of this species. The objective of this research is to analyze the differentially expressed genes (DEGs) during the HW formation process from specific Taiwania xylem tissues, and to obtain genes that might be closely associated with this process. The results indicated that our analyses have captured DEGs representative to the HW formation process of Taiwania. DEGs related to the terpenoid biosynthesis pathway were all up-regulated in the transition zone (TZ) to support the biosynthesis and accumulation of terpenoids. Many DEGs related to lignin biosynthesis, and two DEGs related to pinoresinol reductase (PrR)/pinoresinol lariciresinol reductase (PLR), were up-regulated in TZ. These DEGs together are likely involved in providing the precursors for the subsequent lignan biosynthesis. Several transcription factor-, nuclease-, and protease-encoding DEGs were also highly expressed in TZ, and these DEGs might be involved in the regulation of secondary metabolite biosynthesis and the autolysis of the cellular components of ray parenchyma cells in TZ. These results provide further insights into the process of HW formation in Taiwania.
Project description:Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny.
Project description:The procambium and cambium are meristematic tissues from which vascular tissue is derived. Vascular initials differentiate into phloem towards the outside of the stem and xylem towards the inside. A small peptide derived from CLV-3/ESR1-LIKE 41 (CLE41) is thought to promote cell divisions in vascular meristems by signalling through the PHLOEM INTERCALLATED WITH XYLEM (PXY) receptor kinase. pxy mutants, however, display only small reductions in vascular cell number, suggesting a mechanism exists that allows plants to compensate for the absence of PXY. Consistent with this idea, we identify a large number of genes specifically upregulated in pxy mutants, including several AP2/ERF transcription factors. These transcription factors are required for normal cell division in the cambium and procambium. These same transcription factors are also upregulated by ethylene and in ethylene-overproducing eto1 mutants. eto1 mutants also exhibit an increase in vascular cell division that is dependent upon the function of at least 2 of these ERF genes. Furthermore, blocking ethylene signalling using a variety of ethylene insensitive mutants such as ein2 enhances the cell division defect of pxy. Our results suggest that these factors define a novel pathway that acts in parallel to PXY/CLE41 to regulate cell division in developing vascular tissue. We propose a model whereby vascular cell division is regulated both by PXY signalling and ethylene/ERF signalling. Under normal circumstances, however, PXY signalling acts to repress the ethylene/ERF pathway.
Project description:Ethylene-responsive factors (ERFs) are commonly considered to play an important role in pathogen defense responses. However, only few of ERF members have been characterized in Sea island cotton (Gossypium barbadense). Here, we reported a novel AP2/ERF transcription factors gene, named GbERFb which was cloned and identified from Sea island cotton by RACE. The expression of GbERFb was significantly induced by treatments with ethylene, Methyl jasmonate, salicylic acid, wounding, H2O2 and Verticillium dahliae (V. dahliae) infection. Bioinformatics analysis showed that GbERFb protein containing a conserved ERF DNA binding domain and a nuclear localization signal sequence, belonged to IXb subgroup of the ERF family. Further experiments demonstrated that GbERFb could bind the GCC box cis-acting element and interact with GbMAPKb (MAP kinase) directly in yeast. Over-expression of GbERFb in tobacco could increase the disease resistance to V. dahliae. The results suggest that the GbERFb, a new AP2/ERF transcription factor, could enhance the resistance to V. dahliae and be useful in improvement of crop resistance to pathogenes.
Project description:Auxin is necessary for the inhibition of root growth induced by aluminium (Al) stress, however the molecular mechanism controlling this is largely unknown. Here, we report that YUCCA (YUC), which encodes flavin monooxygenase-like proteins, regulates local auxin biosynthesis in the root apex transition zone (TZ) in response to Al stress. Al stress up-regulates YUC3/5/7/8/9 in the root-apex TZ, which we show results in the accumulation of auxin in the root-apex TZ and root-growth inhibition during the Al stress response. These Al-dependent changes in the regulation of YUCs in the root-apex TZ and YUC-regulated root growth inhibition are dependent on ethylene signalling. Increasing or disruption of ethylene signalling caused either enhanced or reduced up-regulation, respectively, of YUCs in root-apex TZ in response to Al stress. In addition, ethylene enhanced root growth inhibition under Al stress was strongly alleviated in yuc mutants or by co-treatment with yucasin, an inhibitor of YUC activity, suggesting a downstream role of YUCs in this process. Moreover, ethylene-insensitive 3 (EIN3) is involved into the direct regulation of YUC9 transcription in this process. Furthermore, we demonstrated that PHYTOCHROME INTERACTING FACTOR4 (PIF4) functions as a transcriptional activator for YUC5/8/9. PIF4 promotes Al-inhibited primary root growth by regulating the local expression of YUCs and auxin signal in the root-apex TZ. The Al-induced expression of PIF4 in root TZ acts downstream of ethylene signalling. Taken together, our results highlight a regulatory cascade for YUCs-regulated local auxin biosynthesis in the root-apex TZ mediating root growth inhibition in response to Al stress.
Project description:Our recent studies have demonstrated multiple health-promoting benefits from black walnut kernels. These biological functions of black walnuts are likely associated with their bioactive constituents. Characterization of phenolic compounds found in black walnut could point out underexplored bioactive activities of black walnut extracts and promote the development of novel applications of black walnut and its by-products. In the present study, we assessed bioactivity profiles of phenolic compounds identified in the kernels of black walnuts using a high-throughput screening (HTS) approach. Black walnut phenolic compounds were evaluated in terms of their total antioxidant capacity, antioxidant response element (ARE) induction, and anticancer activities. The anticancer activities were identified by evaluating the effects of the phenolic compounds on the growth of the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). Out of 16 phenolic compounds tested, several compounds (penta-O-galloyl-?-d-glucose, epicatechin gallate, quercetin, (-)-epicatechin, rutin, quercetin 3-?-d-glucoside, gallic acid, (+)-catechin, ferulic acid, syringic acid) exerted antioxidant activities that were significantly higher compared to Trolox, which was used as a control. Two phenolic compounds, penta-O-galloyl-?-d-glucose and quercetin 3-?-d-glucoside, exhibited antiproliferative activities against both the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). The antioxidant activity of black walnut is likely driven not only by penta-O-galloyl-?-d-glucose but also by a combination of multiple phenolic compounds. Our findings suggested that black walnut extracts possibly possess anticancer activities and supported that penta-O-galloyl-?-d-glucose could be a potential bioactive agent for the cosmetic and pharmaceutical industries.
Project description:With respect to the biosynthesis of plant alkaloids, that of benzylisoquinoline alkaloids (BIAs) has been the most investigated at the molecular level. Previous investigations have shown that the biosynthesis of BIAs is comprehensively regulated by WRKY and bHLH transcription factors, while promoter analyses of biosynthesis enzyme-encoding genes have also implicated the involvement of members of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily. To investigate the physiological roles of AP2/ERF transcription factors in BIA biosynthesis, 134 AP2/ERF genes were annotated using the draft genome sequence data of Eschscholzia californica (California poppy) together with transcriptomic data. Phylogenetic analysis revealed that these genes could be classified into 20 AP2, 5 RAV, 47 DREB, 60 ERF and 2 Soloist family members. Gene structure, conserved motif and orthologous analyses were also carried out. Gene expression profiling via RNA sequencing in response to methyl jasmonate (MeJA) indicated that approximately 20 EcAP2/ERF genes, including 10 group IX genes, were upregulated by MeJA, with an increase in the expression of the transcription factor-encoding gene EcbHLH1 and the biosynthesis enzyme-encoding genes Ec6OMT and EcCYP719A5. Further quantitative RT-PCR confirmed the MeJA responsiveness of the EcAP2/ERF genes, i.e., the increased expression of 9 group IX, 2 group X and 2 group III ERF subfamily genes. Transactivation activity of group IX EcAP2/ERFs was also confirmed by a luciferase reporter assay in conjunction with the promoters of the Ec6OMT and EcCYP719A5 genes. The physiological roles of AP2/ERF genes in BIA biosynthesis and their evolution in the regulation of alkaloid biosynthesis are discussed.
Project description:Black walnut (Juglans nigra L.) is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. Although consumption of black walnut has been linked to multiple health-promoting effects (e.g., antioxidant, antimicrobial, anti-inflammatory), the bioactive compounds have not been systematically characterized. In addition, the associations between different black walnut cultivars and their health-promoting compounds have not been well established. In this study, the kernels of twenty-two black walnut cultivars selected for nut production by the University of Missouri Center for Agroforestry (Columbia, MO, USA) were evaluated for their antibacterial activities using agar-well diffusion assay. Among the selected cultivars, four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against a Gram-positive bacterium (Staphylococcus aureus), whereas other cultivars showed no effect on the inhibition of this bacterium. The antibacterial compounds showing the strongest activity were isolated with bioassay-guided purification and identified using a metabolomics approach. Six antibacterial bioactive compounds responsible for antimicrobial activity were successfully identified. Glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are novel antibacterial compounds identified in the kernels of black walnuts. The metabolomics approach provides a simple and cost-effective tool for bioactive compound identification.
Project description:<h4>Background and aims</h4>It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression.<h4>Methods</h4>Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed.<h4>Key results</h4>Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens.<h4>Conclusions</h4>AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.
Project description:The walnut shell is a hard and protective layer that provides an essential barrier between the seed and its environment. The shell is based on only one unit cell type: the polylobate sclerenchyma cell. For a better understanding of the interlocked walnut shell tissue, we investigate the structural and compositional changes during the development of the shell from the soft to the hard state. Structural changes at the macro level are explored by X-ray tomography and on the cell and cell wall level various microscopic techniques are applied. Walnut shell development takes place beneath the outer green husk, which protects and delivers components during the development of the walnut. The cells toward this outer green husk have the thickest and most lignified cell walls. With maturation secondary cell wall thickening takes place and the amount of all cell wall components (cellulose, hemicelluloses and especially lignin) is increased as revealed by FTIR microscopy. Focusing on the cell wall level, Raman imaging showed that lignin is deposited first into the pectin network between the cells and cell corners, at the very beginning of secondary cell wall formation. Furthermore, Raman imaging of fluorescence visualized numerous pits as a network of channels, connecting all the interlocked polylobate walnut shells. In the final mature stage, fluorescence increased throughout the cell wall and a fluorescent layer was detected toward the lumen in the inner part. This accumulation of aromatic components is reminiscent of heartwood formation of trees and is suggested to improve protection properties of the mature walnut shell. Understanding the walnut shell and its development will inspire biomimetic material design and packaging concepts, but is also important for waste valorization, considering that walnuts are the most widespread tree nuts in the world.