Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.

Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-c...

Full description

Bibliographic Details
Main Authors: Daniela Richaud, Claudia Stange, Agata Gadaleta, Pasqualina Colasuonno, Roberto Parada, Andrés R Schwember
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2018-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0208948
id doaj-5ce5f0f958174444bf3213b88011a258
record_format Article
spelling doaj-5ce5f0f958174444bf3213b88011a2582021-03-03T21:03:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011312e020894810.1371/journal.pone.0208948Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.Daniela RichaudClaudia StangeAgata GadaletaPasqualina ColasuonnoRoberto ParadaAndrés R SchwemberIncreasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions.https://doi.org/10.1371/journal.pone.0208948
collection DOAJ
language English
format Article
sources DOAJ
author Daniela Richaud
Claudia Stange
Agata Gadaleta
Pasqualina Colasuonno
Roberto Parada
Andrés R Schwember
spellingShingle Daniela Richaud
Claudia Stange
Agata Gadaleta
Pasqualina Colasuonno
Roberto Parada
Andrés R Schwember
Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
PLoS ONE
author_facet Daniela Richaud
Claudia Stange
Agata Gadaleta
Pasqualina Colasuonno
Roberto Parada
Andrés R Schwember
author_sort Daniela Richaud
title Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
title_short Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
title_full Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
title_fullStr Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
title_full_unstemmed Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.
title_sort identification of lycopene epsilon cyclase (lcye) gene mutants to potentially increase β-carotene content in durum wheat (triticum turgidum l.ssp. durum) through tilling.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2018-01-01
description Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions.
url https://doi.org/10.1371/journal.pone.0208948
work_keys_str_mv AT danielarichaud identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
AT claudiastange identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
AT agatagadaleta identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
AT pasqualinacolasuonno identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
AT robertoparada identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
AT andresrschwember identificationoflycopeneepsiloncyclaselcyegenemutantstopotentiallyincreasebcarotenecontentindurumwheattriticumturgidumlsspdurumthroughtilling
_version_ 1714819027975536640