A 0.5 μl sample was injected into the liner in splitless mode, and the liner was exchanged every ten samples. The injection temperature started at 50o C and ramped to 250o C by 12o C per sec. The GC column was a Rtx-5Sil MS at a constant flow of 1 ml min−1 helium gas. The column temperature started at 50o C for 1 min and then ramped to 330o C. Automatic mass spectral deconvolution was performed with peak detection of GC spectrum using The BinBase algorithm . After peak peaking, peak data were deposited BinBase DB with BinBase ID. BinBase settings: validity of chromatogram , unbiased retention index marker detection , retention index calculation by 5th order polynomial regression. Spectra are cut to 5% base peak abundance and matched to database entries from most to least abundant spectra using the following matching filters: equivalent to about ± 2 s retention time, unique ion must be included in apexing masses and present at > 3% of base peak abundance, mass spectrum similarity must fit criteria dependent on peak purity and signal/noise ratios . Failed spectra are automatically entered as new database entries if s/n > 25, purity < 1.0 and presence in the biological study design class was > 80%. Resulting .txt files were exported to a data server with absolute spectral intensities and further processed by a filtering algorithm implemented in the metabolomics BinBase DB .At the molecular level for resistance in initial infection stages,vertical farming equipments a lack of bacterial propagation, induction of vir, and incomplete integration of T-DNA result in crown gall resistance in grapevines . V. riparia that showed RR in our study had a broad range of crown gall resistance by particularly inhibiting the integration of A. vitis T-DNA into plant chromosome .
This type of resistance prevents the pathogen from infection, resulting in the reduced GI, leading to no or little gall development to have also lowered GDI, which may be applied as a major resistance mechanism for the four Vitis species of RR. For SR, GI was similar to SS, but GDI, to RR, suggesting the pathogen infection in SR occurs as in SS, but gall development is retarded as in RR to have moderate GSI between SS and RR. For SS, both A. vitis T-DNA integration into host genome and expression of encoded plant oncogenes and increase in phytohormone levels occur readily, resulting in the formation of tumors and their proliferation . Out of a total 134 metabolites produced in the grapevine stem internodes infected with A. vitis K306, 11 metabolites were significantly related with response types of 10 Vitis species examined in our study. At pre-inoculation stage, only one metabolite was significantly increased in RR relative to SR, while all 11 metabolites were significantly increased in either SS or RR at post inoculation stages, suggesting metabolic changes may occur preferentially around the infection sites after the pathogen infection, especially at initial stages of the pathogen infection with response type-related 8 metabolites, of which 6 related to SS but only 2 most significantly related to RR and SR . At post 2 , all three metabolites were more related to SS than RR or SR; however, at post 3 all three metabolites were more related to RR or SR than SR and/or SS. These all suggest that the metabolic changes occur actively in relation to susceptible responses at the initial stages after the pathogen infection, but in relation to resistant responses at later infection stages with full gall development.
An amine derivative, octopine, produced by the octopine-type A. vitis used in our study and two plant hormones, auxin and cytokinin, produced in the T-DNA transformed plant cells, drive the uncontrolled gall development, determining gall morphology depending on the ratios of the plant hormones . However, neither octopine and its amino acid component arginine nor the plant hormones were produced in a significant level around the infection sites in our study, suggesting the gall-inducing metabolites may hardly be transported out of the tumorigenic tissues. However, the component class most abundantly produced in relation to response types was amino acid in our study, including 4 amino acids produced at post 1 and one at post 3, which was significantly related to SS and RR, respectively. The functions of these amino acids in crown gall development have not been clearly understood, but amino acids are generally involved in the primary metabolism of all living organisms, and the developing tumor becomes a metabolic sink accumulating metabolites to be supplied in a priority for its growth by the induction of vascular tissue differentiation around gall surface . It was reported previously that three SS-related amino acids except tyrosine increase several folds in tumor tissue at later infection stage , suggesting also these SS-related amino acids should be related to the gall development, although the time for the production of the materials may differ depending on Agrobacterium species. Other component classes of the metabolites such as carbohydrates and carboxylic acids which were increased in SS at 2 and 7 days after inoculation, respectively, may also serve for the gall growth and differentiation by inducing vir genes; gallocatechin, a flavonoid, may be involved in the gall formation as a potential auxin transport regulator as in root nodulation and root-knot gall formation . Quinic acid, catechins, and stilbene are related to Pierce’s disease, showing these metabolites occur at great levels in xylem tissues of the grapevine infected with Xylella fastidiosa.
In plants, major resistance-related responses to Agrobacterium spp. occur at two periods of time, initially at the time of recognizing the tumorigenic pathogens and later during the time of the pathogenesis in which auxin and cytokinin cause an increase of ethylene that together with salicylic acid inhibits agrobacterial virulence . In our study, resveratrol at post 1 and valine and xylonic acid at post 3 were definitely differentiated as RR from the other response types . Resveratrol is a stilbenoid, a type of natural phenol, and a phytoalexin produced naturally by several plants in response to injury or pathogen infection . In grapevine, resveratrol is primarily found in the grape skin and produced as phytoalexin in grapes infected with the grey mold pathogen, Botrytis cinerea, of which the accumulation amounts vary with grapevine genotypes, their geographic origin, and exposure to fungal infection . Considering the biological characteristics of this stilbene compound , this metabolite may be produced in Vitis species with RR at a significant amount and act as a phytoalexin to inhibit the growth and virulence of the tumorigenic pathogen probably more at initial infection stages. For the other metabolites increased in Vitis spp. with RR at the later infection stages , valine , and xylonic acid ,vertical farms their roles in plant responses are not clearly understood. Alterations in the plant metabolism in response to different pathogen infections may function as either supporting the ongoing defense mechanism to lead an efficient resistance response or being exploited by the pathogen to facilitate infection as can be seen in plant glutamate metabolism . Thus, it is not an unusual thing that the metabolites in the same compound classes were differentiated in relation to the opposing response types; xylonic acid significantly related to RR at post 3, while quinic acid to SS at post 2; valine related to RR at post 3, while four amino acids to SS at post 1 in our study. Considering the same class metabolites concomitantly occurred in relation to different response types at different infection stages, the same class metabolites may play different roles depending on their requirements for in situ metabolism, leading to either compatible or incompatible responses to the pathogen infection In our study, sucrose contents were highest in grapevine stem internodes with SR at post 1 and post 3, which was significantly differentiated from SS at post 1 and RR at post 3, respectively . Considering the increased expression of sucrose degradation enzyme genes in gall tissues of Arabidopsis and its induction of Agrobacterium virulence genes , the increased sucrose contents may support its intake into the metabolic sink, crown gall tissue, contributing to the gall development in different degrees depending on infection stages . Host sucrose, synthesized from photosynthetic products in the cytoplasm of aerial plant parts, is transported to tumor cells apoplastically and the sucrose contents in phloem sap differ depending on the crown gall-developmental stages with higher around actively growing young galls than old matured ones .
In our study, the sucrose contents fluctuated with time after infection and were higher at post 1 than post 3, regardless of response types, suggesting the uptake of sucrose should be required for the gall development. Another carbohydrate cellobiose was significantly higher in RR relative to SR at preinfectional stages, but higher in SS than RR and SR at post 1, suggesting its roles in healthy plant tissues may alter oppositely in diseased plant tissues with uncontrolled gall development. In our study, metabolite profile analysis revealed the following aspects: Remarkable differential increases of metabolites occurred in internodes of Vitis species after A. vitis infection, most prevalently at two days after inoculation, and more related to susceptible type of response for 10 metabolites that are useful for the metabolic processes in gall growth and differentiation as nutritional compounds or plant hormone regulator. Among three metabolites definitely differentiated into the resistant response , resveratrol appeared to be importantly related to resistant responses as it is a well-known phytoalexin compound in several plant pathosystems. All of these aspects will provide important information that can be applied for the selection of grapevine cultivars resistant to the crown gall disease caused by A. vitis, and their use as root stocks for the control of the crown gall disease in the scions of the grapevines susceptible to A. vitis. Abscission is the process of organ separation, which plays a critical role in the plant life cycle. Organ shedding occurs at abscission zones , comprising small, densely cytoplasmic cells at the boundary between an organ and the main plant body. Abscission has evolved as a successful strategy to adapt to the environment in response to developmental and environmental cues. Abscission allows plants to detach nonfunctional or diseased organs and is also important for seed dispersal. The timing of abscission, especially of flower and fruit abscission, is of interest to agriculture. Breeding of appropriate abscission behavior has successfully solved crop production and yield problems such as grain shattering, cotton boll shedding, premature legume dehiscence, and mechanical harvest in tomato. It has been well-demonstrated that the timing of abscission is regulated by cross-talk between the phytohormones auxin and ethylene.Arabidopsis flower abscission is inhibited, for example in the ethylene-insensitive mutants ethylene resistant 1-1 and ethylene insensitive 2. In tomato too, organ abscission is inhibited in ethylene receptor and ethylene sensitivity mutants including EIN , Never ripe , Sletr1-1, and Sletr1-2. Auxin plays a critical role in controlling abscission. The consensus of many studies is that the continuous polar flow of auxin passing through the abscission zone inhibits abscission and that reduction of this flow initiates abscission by making the AZ sensitive to ethylene. The polar flow is thought to be a reflection of agradient in auxin concentration across the abscission zone. In a series of classic experiments, it was shown that application of indole-3-acetic acid , to the distal side of Phaseolus vulgaris leaf explants inhibited abscission, while an application to the proximal side accelerated the process. The nature of the auxin gradient continues to be the subject of discussion—researchers have proposed that the gradient might be in auxin concentration, auxin biosynthesis, auxin transport, and/or auxin response. In Arabidopsis, manipulation of auxin biosynthesis specifically within floral organ AZ demonstrates that reduction of auxin level makes the flower organ shed prematurely. However, the disruption of auxin signaling/ response in AZ delayed the shedding of floral organs, suggesting that a functional IAA signaling/response pathway in AZ cells is required for abscission initiation. Given the importance of auxin balance between the distal and proximal sides of the AZ for organ shedding, it is essential to understand how this auxin gradient is maintained for regulating the initiation of the abscission process in response to developmental and environmental cues.