Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds.
Nonogaki Mariko,Sall Khadidiatou,Nambara Eiji,Nonogaki Hiroyuki
The Plant journal : for cell and molecular biology
Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology.
Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.
Bao Gegen,Zhuo Chunliu,Qian Chunmei,Xiao Ting,Guo Zhenfei,Lu Shaoyun
Plant biotechnology journal
Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality.
Cloning and functional analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits.
Zhang Mei,Leng Ping,Zhang Guanglian,Li Xiangxin
Journal of plant physiology
Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.
Functional characterization of the BnNCED3 gene in Brassica napus.
Xu Peipei,Cai Weiming
Plant science : an international journal of experimental plant biology
Abscisic acid (ABA) has been implicated in plant adaptation to various environmental stresses and the regulation of seed dormancy, leaf senescence and organ abscission progression. The cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) family proteins is a critical step in the regulation of abscisic acid (ABA) synthesis in plants. In the present study, the NCED family gene BnNCED3 was isolated from Brassica napus. BnNCED3 encodes a 592-amino acid protein with high amino acid sequence similarity to the Arabidopsis AtNCED3 protein. Expression pattern assays revealed that BnNCED3 is ubiquitously expressed at different levels in all the examined organs. Furthermore, the overexpression of BnNCED3 contributed to ABA accumulation and NO and ROS generation in transgenic Arabidopsis plants, thereby enhancing abiotic stress tolerance. These experiments also indicated the involvement of BnNCED3 in the control of plant development in transgenic Arabidopsis, such as the inhibition of seed germination, lateral root initiation, early phase changes and the enhancement of ABA-associated leaf senescence. Together, these results indicated that BnNCED3 is at least partly involved in both stress adaptation and plant development through the regulation of ABA biosynthesis.
Myb10-D confers PHS-3D resistance to pre-harvest sprouting by regulating NCED in ABA biosynthesis pathway of wheat.
Lang Jing,Fu Yuxin,Zhou Yong,Cheng Mengping,Deng Min,Li Maolian,Zhu Tingting,Yang Jian,Guo Xiaojiang,Gui Lixuan,Li Linchuan,Chen Zhongxu,Yi Yingjin,Zhang Lianquan,Hao Ming,Huang Lin,Tan Chao,Chen Guoyue,Jiang Qiantao,Qi Pengfei,Pu Zhien,Ma Jian,Liu Zehou,Liu Yujiao,Luo Ming-Cheng,Wei Yuming,Zheng Youliang,Wu Yongrui,Liu Dengcai,Wang JiRui
The New phytologist
Pre-harvest sprouting (PHS), the germination of grain before harvest, is a serious problem resulting in wheat yield and quality losses. Here, we mapped the PHS resistance gene PHS-3D from synthetic hexaploid wheat to a 2.4 Mb presence-absence variation (PAV) region and found that its resistance effect was attributed to the pleiotropic Myb10-D by integrated omics and functional analyses. Three haplotypes were detected in this PAV region among 262 worldwide wheat lines and 16 Aegilops tauschii, and the germination percentages of wheat lines containing Myb10-D was approximately 40% lower than that of the other lines. Transcriptome and metabolome profiling indicated that Myb10-D affected the transcription of genes in both the flavonoid and abscisic acid (ABA) biosynthesis pathways, which resulted in increases in flavonoids and ABA in transgenic wheat lines. Myb10-D activates 9-cis-epoxycarotenoid dioxygenase (NCED) by biding the secondary wall MYB-responsive element (SMRE) to promote ABA biosynthesis in early wheat seed development stages. We revealed that the newly discovered function of Myb10-D confers PHS resistance by enhancing ABA biosynthesis to delay germination in wheat. The PAV harboring Myb10-D associated with grain color and PHS will be useful for understanding and selecting white grained PHS resistant wheat cultivars.
Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene and the role of ABA on fruit ripening.
Zhang Mei,Yuan Bing,Leng Ping
Plant signaling & behavior
In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence, six 740 bp cDNAs (LeNCED1, LeNCED2, PpNCED1, VVNCED1, DKNCED1 and CMNCED1) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, were cloned from fruits of tomato, peach, grape, persimmon and melon using an RT-PCR approach. A Blast homology search revealed a similarity of amino acid 85.76% between the NCEDs. A relationship between ABA and ethylene during ripening was also investigated. At the mature green stage, exogenous ABA treatment increased ABA content in flesh, and promoting ethylene synthesis and fruit ripening, while treatment with nordihydroguaiaretic acid (NDGA), inhibited them, delayed fruit ripening and softening. However, ABA inhibited the ethylene synthesis obviously while NDGA promoted them when treated the immature fruit with these chemicals. At the breaker, NDGA treatment cannot block ABA accumulation and ethylene synthesis. Based on the results obtained in this study, it was concluded that ABA plays different role in ethylene synthesis system in different stages of tomato fruit ripening.
Identification and functional analysis of 9-cis-epoxy carotenoid dioxygenase (NCED) homologs in G. hirsutum.
Pei Xinxin,Wang Xiaoyang,Fu Guoyong,Chen Baojun,Nazir Mian Faisal,Pan Zhaoe,He Shoupu,Du Xiongming
International journal of biological macromolecules
9-cis-epoxy carotenoid dioxygenase (NCED) is a fundamental enzyme, which plays an essential role in the process of organ development and stress resistance by regulating abscisic acid (ABA) synthesis in plant. In this study, a total of 7, 7, 14 and 14 NCED genes were identified from the genomes of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic tree showed that all forty-two NCED genes could be classified into three groups in cotton genus. Collinear analysis revealed that the NCED genes in G. hirsutum were not amplified by tandem repeats after polyploidy events. The function of NCED genes was evaluated between two accessions with contrasting plant height. The results showed that expression of the NCED genes in dwarf accession was higher than that in taller ones. GhNCED1-silenced cotton plants confirmed that suppression of NCED genes could increase the plant height, but reduce the resistance abilities to drought and salt stress. Our study systematically identified the homologs of NCED genes and their functions in cotton, which could provide new genetic resources for improving plant height and stress in future cotton breeding.
Double NCED isozymes control ABA biosynthesis for ripening and senescent regulation in peach fruits.
Wang Pengfei,Lu Siyuan,Zhang Xueying,Hyden Brennan,Qin Lijie,Liu Lipeng,Bai Yangyang,Han Yan,Wen Zhiliang,Xu Jizhong,Cao Hongbo,Chen Haijiang
Plant science : an international journal of experimental plant biology
During ripening, peach fruits (Prunus persica L. Batsch) rapidly progress to the senescent stage, resulting in a brief shelf life. Abscisic acid (ABA) plays an important role in regulating the ripening process, both in climacteric and non-climacteric fruits. A key enzyme for ABA biosynthesis in higher plants is 9-cis-epoxycarotenoid dioxygenase (NCED). In this study, two NCED isozymes, PpNCED1 and PpNCED5, were identified in peach fruits. While both NCED genes had similar transcriptional patterns (up-regulation) at the beginning of peach ripening, PpNCED5 showed a consistently lower expression level than PpNCED1. During the post-harvest stage, gene expression of PpNCED1 declined, while PpNCED5 expression increased relative to PpNCED1 expression. Considering the dynamic process of ABA accumulation during fruit ripening and senescence in peach, this study indicates that both NCED genes cooperatively control ABA biosynthesis in peach fruits. Moreover, spatio-temporal expression and transcriptional response to hormone and abiotic stress suggested that there is functional divergence between PpNCED1 and PpNCED5 genes in peach. A carotenoid-rich callus system was used to verify the function of PpNCED1 and PpNCED5. In the transgenic callus system, both PpNCED1 and PpNCED5 isozymes promoted ABA biosynthesis, which likely accelerated cell senescence through activating ROS signals. The results from this study provide evidence supporting an ABA biosynthetic regulation process via the two NCED genes in peach fruit, and suggest a mechanism of ABA-induced fruit ripening and senescence.
Genome-wide identification and expression analysis of the NCED family in cotton (Gossypium hirsutum L.).
Li QingHua,Yu XianTao,Chen Long,Zhao Gang,Li ShiZhou,Zhou Hao,Dai Yu,Sun Na,Xie YongFei,Gao JunShan,Li DaHui,Sun Xu,Guo Ning
Abscisic acid (ABA) is an important plant hormone that plays multiple roles in regulating growth and development as well as in stress responses in plants. The NCED gene family includes key genes involved in the process of ABA synthesis. This gene family has been found in many species; however, the function of the NCED gene family in cotton is unclear. Here, a total of 23 NCED genes (designated as GhNCED1 to GhNCED23) were identified in cotton. Phylogenetic analysis indicated that the identified NCED proteins from cotton and Arabidopsis could be classified into 4 subgroups. Conserved motif analysis revealed that the gene structure and motif distribution of proteins within each subgroup were highly conserved. qRT-PCR and ABA content analyses indicated that NCED genes exhibited stage-specific expression patterns at tissue development stages. GhNCED5, GhNCED6 and GhNCED13 expression was similar to the change in ABA content, suggesting that this gene family plays a role in ABA synthesis. These results provide a better understanding of the potential functions of GhNCED genes.