Dataset Information

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

ABSTRACT:

Background

Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state ¹³C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state.

Results

We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-¹³C₆ glucose feeding revealed nearly 100% ¹³C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano.

Conclusions

Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state ¹³C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant.

SUBMITTER: Koley S

PROVIDER: S-EPMC6526604 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

Koley Somnath S Raorane Manish L ML Junker Björn H BH

Plant methods 20190520

<h4>Background</h4>Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state <sup>13</sup>C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady st ...[more]

PMID: 31139238

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Project description:BackgroundCryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may not be safe or appropriate for all patients. Therefore, methods to mature ITT ex vivo are needed.ObjectivesAim to investigate the feasibility of inducing in vitro spermatogenesis from ITT cryopreserved for pediatric patients prior to initiation of gonadotoxic therapy.Materials and methodsCryopreserved-thawed ITT from prepubertal and peripubertal patients were cultured for 7, 16, and 32 days in medium with no hormones or supplemented with 5 IU/L FSH, 1 IU/L hCG, or 5IU/L FSH+1 IU/L hCG. Samples were evaluated histologically to assess tissue integrity, and immunofluorescence staining was performed to identify VASA (DDX4)+ germ cells, UCHL1+ spermatogonia, SYCP3+ spermatocytes, CREM+ spermatids, SOX9+ Sertoli cells. Proliferation (KI67) and apoptosis (CASPASE3) of germ cells and Sertoli cells were also analyzed. Sertoli and Leydig cell maturation was evaluated by AR and INSL3 expression as well as expression of the blood testis barrier protein, CLAUDIN11, and testosterone secretion in the culture medium.ResultsIntegrity of seminiferous tubules, VASA+ germ cells and SOX9+ Sertoli cells were maintained up to 32 days. The number of VASA+ germ cells was consistently higher in the peripubertal groups. UCHL1+ undifferentiated spermatogonia and SOX9+ Sertoli cell proliferation was confirmed in most samples. SYCP3+ primary spermatocytes began to appear by day 16 in both age groups. Sertoli cell maturation was demonstrated by AR expression but the expression of CLAUDIN11 was disorganized. Presence of mature and functional Leydig cells was verified by INSL3 expression and secretion of testosterone. Gonadotropin treatments did not consistently impact the number or proliferation of germ cells or somatic cells, but FSH was necessary to increase testosterone secretion over time in prepubertal samples.ConclusionITT were maintained in organotypic culture for up to 32 days and spermatogonia differentiated to produce primary spermatocytes in both pre- and peripubertal age groups. However, complete spermatogenesis was not observed in either group.

Dataset Information

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

Background

Results

Conclusions

Publications

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

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