Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron...Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.展开更多
The Ib subgroup of the bHLH gene family in Arabidopsis contains four members （AtbHLH38, AtbHLH39, AtbHLHIO0, and AtbHLH101）. AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deft-ciency...The Ib subgroup of the bHLH gene family in Arabidopsis contains four members （AtbHLH38, AtbHLH39, AtbHLHIO0, and AtbHLH101）. AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deft-ciency induced transcription factor （FIT）, directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLHI01 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.展开更多
Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red rati...Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red ratio is interpreted as shading by neighbor plants and induces cell elongation--a phenomenon called shade-avoidance syndrome （SAS）. PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation （CHIP） assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 （PARIOX） are insensitive to gibberellin （GA） or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid （BR） and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PARIOX. These results indicate that PAR1-PRE1 and PAR1-PIF4 het- erodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.展开更多
Dear Editor,In plants, the floral transition is flexibly controlled by various environmental conditions and endogenous developmental cues. In Arabidopsis, six major flowering pathways respond to changes in these facto...Dear Editor,In plants, the floral transition is flexibly controlled by various environmental conditions and endogenous developmental cues. In Arabidopsis, six major flowering pathways respond to changes in these factors （Fornara et al., 2010）. The photoperiod, vernalization, and ambient pathways monitor exogenous signals from the environment such as day length, minimum winter temperature, and ambient temperature （Fornara et al., 2010）. By contrast, the autonomous, gibberellin, and age pathways respond to endogenous cues linked to developmental status （Fornara et al., 2010）. Accumulating evidence indicates that the six flowering pathways converge in a network to regulate floral integrator genes FLOWERING LOCUS T （FT）, TWIN SISTER OF FT （TSF）, and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 （Fornara et al., 2010）.展开更多
Plants have evolved sophisticated genetic networks to regulate iron （Fe） homeostasis for their survival. Several classes of plant hormones including jasmonic acid （JA） have been shown to be involved in regulating ...Plants have evolved sophisticated genetic networks to regulate iron （Fe） homeostasis for their survival. Several classes of plant hormones including jasmonic acid （JA） have been shown to be involved in regulating the expression of iron uptake and/or deficiency-responsive genes in plants. However, the molecular mechanisms by which JA regulates iron uptake remain unclear. In this study, we found that JA negatively modulates iron uptake by downregulating the expression of FIT （bHLH29）, bHLH38, bHLH39, bHLHIO0, and bHLHI01 and promoting the degradation of FIT protein, a key regulator of iron uptake in Arabidopsis. We further demonstrated that the subgroup IVa bHLH proteins, bHLH18, bHLH19, bHLH20, and bHLH25, are novel interactors of FIT, which promote JA-induced FIT protein degradation. These four IVa bHLHs function redundantly to antagonize the activity of the Ib bHLHs （such as bHLH38） in regulating FIT protein stability under iron deficiency. The four IVa bHLH genes are primarily expressed in roots, and are inducible by JA treatment. Moreover, we found that MYC2 and JAR1, two critical components of the JA signaling pathway, play critical roles in mediating JA suppression of the expression of FIT and Ib bHLH genes, whereas they differentially modulate the expression of bHLH18, bHLH19, bHLH20, and bHLH25 to regulate FIT accumulation under iron deficiency. Taken together, these results indicate that by transcriptionally regulating the expression of different sets of bHLH genes JA signaling promotes FIT degradation, resulting in reduced expression of iron-uptake genes, IRT1 and FRO2, and increased sensitivity to iron deficiency. Our data suggest that there is a multilayered inhibition of iron-deficiency response in the presence JA in Arabidopsis.展开更多
In complex, constantly changing environments, plants have developed astonishing survival strategies. These elaborated strategies rely on rapid and precise gene regulation mediated by transcription factors （TFs）. TFs...In complex, constantly changing environments, plants have developed astonishing survival strategies. These elaborated strategies rely on rapid and precise gene regulation mediated by transcription factors （TFs）. TFs represent a large fraction of plant genomes and among them, MYBs and basic helix-loop-helix （bHLHs） have unique inherent properties specific to plants. Proteins of these two TF families can act as homo- or heterodimers, associate with proteins from other protein families, or form MYB/bHLH complexes to regulate distinct cellular processes. The ability of MYBs and bHLHs to interact with multiple protein part- ners has evolved to keep up with the increased metabolic complexity of multi-cellular organisms. Associ- ation and disassociation of dynamic TF complexes in response to developmental and environmental cues are controlled through a plethora of regulatory mechanisms specifically modulating TF activity. Regulation of TFs at the protein level is critical for efficient and precise control of their activity, and thus provides the mechanistic basis for a rapid on-and-off switch of TF activity. In this review, examples of post-translational modifications, protein-protein interactions, and subcellular mobilization of TFs are discussed with regard to the relevance of these regulatory mechanisms for the specific activation of MYBs and bHLHs in response to a given environmental stimulus.展开更多
Ginseng(Panax ginseng C.A. Meyer) is one of the best-selling herbal medicines, with ginsenosides as its main pharmacologically active constituents. Although extensive chemical and pharmaceutical studies of these compo...Ginseng(Panax ginseng C.A. Meyer) is one of the best-selling herbal medicines, with ginsenosides as its main pharmacologically active constituents. Although extensive chemical and pharmaceutical studies of these compounds have been performed, genome-wide studies of the basic helix-loop-helix(b HLH) transcription factors of ginseng are still limited. The b HLH transcription factor family is one of the largest transcription factor families found in eukaryotic organisms, and these proteins are involved in a myriad of regulatory processes. In our study, 169 bHLH transcription factor genes were identified in the genome of P. ginseng, and phylogenetic analysis indicated that these PGb HLHs could be classified into 24 subfamilies. A total of 21 RNA-seq data sets, including two sequencing libraries for jasmonate(JA)-responsive and 19 reported libraries for organ-specific expression analyses were constructed. Through a combination of gene-specific expression patterns and chemical contents,6 PGbHLH genes from 4 subfamilies were revealed to be potentially involved in the regulation of ginsenoside biosynthesis. These 6 PGbHLHs, which had distinct target genes, were further divided into two groups depending on the absence of MYC-N structure. Our results would provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factor action in P.ginseng.展开更多
基金The authors thank ProfMary Lou Guerinot （Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire） for providing IRT1 peptide antibody and for the critical reading of the manuscript. We are also grateful to Drs Zhentao Lin and Yongfu Fu （Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing） for providing the BiFC assay system and technical supporting. This work was supported by the National Natural Science Foundation of China （Grant nos, 30530460 and 30521001） and the Ministry of Science and Technology of China （Grant nos, 2005cb20904 and 2006AA 10A 105） and Chinese Academy of Sciences （Grant no. KSCX2-YW-N- 001） as well as by the Harvest Plus-China Program.
文摘Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.
文摘The Ib subgroup of the bHLH gene family in Arabidopsis contains four members （AtbHLH38, AtbHLH39, AtbHLHIO0, and AtbHLH101）. AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deft-ciency induced transcription factor （FIT）, directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLHI01 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.
文摘Phytochromes （Phy） and phytochrome-interacting factor （PIF） transcription factors constitute a major signal- ing module that controls plant development in response to red and far-red light. A low red：far-red ratio is interpreted as shading by neighbor plants and induces cell elongation--a phenomenon called shade-avoidance syndrome （SAS）. PAR1 and its closest homolog PAR2 are negative regulators of SAS; they belong to the HLH transcription factor family that lacks a typical basic domain required for DNA binding, and they are believed to regulate gene expressions through DNA binding transcription factors that are yet to be identified. Here, we show that light signal stabilizes PAR1 protein and PAR1 interacts with PIF4 and inhibits PIF4-mediated gene activation. DNA pull-down and chromatin immunoprecipitation （CHIP） assays showed that PAR1 inhibits PIF4 DNA binding in vitro and in vivo. Transgenic plants overexpressing PAR1 （PARIOX） are insensitive to gibberellin （GA） or high temperature in hypocotyl elongation, similarly to the pifq mutant. In addition to PIF4, PAR1 also interacts with PRE1, a HLH transcription factor activated by brassinosteroid （BR） and GA. Overexpression of PRE1 largely suppressed the dwarf phenotype of PARIOX. These results indicate that PAR1-PRE1 and PAR1-PIF4 het- erodimers form a complex HLH/bHLH network regulating cell elongation and plant development in response to light and hormones.
文摘Dear Editor,In plants, the floral transition is flexibly controlled by various environmental conditions and endogenous developmental cues. In Arabidopsis, six major flowering pathways respond to changes in these factors （Fornara et al., 2010）. The photoperiod, vernalization, and ambient pathways monitor exogenous signals from the environment such as day length, minimum winter temperature, and ambient temperature （Fornara et al., 2010）. By contrast, the autonomous, gibberellin, and age pathways respond to endogenous cues linked to developmental status （Fornara et al., 2010）. Accumulating evidence indicates that the six flowering pathways converge in a network to regulate floral integrator genes FLOWERING LOCUS T （FT）, TWIN SISTER OF FT （TSF）, and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 （Fornara et al., 2010）.
基金This work was supported by the Ministry of Agriculture of China （grant no. 2016ZX08009003-005） and the National Natural Science Foundation of China （grant no. 31471930）.
文摘Plants have evolved sophisticated genetic networks to regulate iron （Fe） homeostasis for their survival. Several classes of plant hormones including jasmonic acid （JA） have been shown to be involved in regulating the expression of iron uptake and/or deficiency-responsive genes in plants. However, the molecular mechanisms by which JA regulates iron uptake remain unclear. In this study, we found that JA negatively modulates iron uptake by downregulating the expression of FIT （bHLH29）, bHLH38, bHLH39, bHLHIO0, and bHLHI01 and promoting the degradation of FIT protein, a key regulator of iron uptake in Arabidopsis. We further demonstrated that the subgroup IVa bHLH proteins, bHLH18, bHLH19, bHLH20, and bHLH25, are novel interactors of FIT, which promote JA-induced FIT protein degradation. These four IVa bHLHs function redundantly to antagonize the activity of the Ib bHLHs （such as bHLH38） in regulating FIT protein stability under iron deficiency. The four IVa bHLH genes are primarily expressed in roots, and are inducible by JA treatment. Moreover, we found that MYC2 and JAR1, two critical components of the JA signaling pathway, play critical roles in mediating JA suppression of the expression of FIT and Ib bHLH genes, whereas they differentially modulate the expression of bHLH18, bHLH19, bHLH20, and bHLH25 to regulate FIT accumulation under iron deficiency. Taken together, these results indicate that by transcriptionally regulating the expression of different sets of bHLH genes JA signaling promotes FIT degradation, resulting in reduced expression of iron-uptake genes, IRT1 and FRO2, and increased sensitivity to iron deficiency. Our data suggest that there is a multilayered inhibition of iron-deficiency response in the presence JA in Arabidopsis.
文摘In complex, constantly changing environments, plants have developed astonishing survival strategies. These elaborated strategies rely on rapid and precise gene regulation mediated by transcription factors （TFs）. TFs represent a large fraction of plant genomes and among them, MYBs and basic helix-loop-helix （bHLHs） have unique inherent properties specific to plants. Proteins of these two TF families can act as homo- or heterodimers, associate with proteins from other protein families, or form MYB/bHLH complexes to regulate distinct cellular processes. The ability of MYBs and bHLHs to interact with multiple protein part- ners has evolved to keep up with the increased metabolic complexity of multi-cellular organisms. Associ- ation and disassociation of dynamic TF complexes in response to developmental and environmental cues are controlled through a plethora of regulatory mechanisms specifically modulating TF activity. Regulation of TFs at the protein level is critical for efficient and precise control of their activity, and thus provides the mechanistic basis for a rapid on-and-off switch of TF activity. In this review, examples of post-translational modifications, protein-protein interactions, and subcellular mobilization of TFs are discussed with regard to the relevance of these regulatory mechanisms for the specific activation of MYBs and bHLHs in response to a given environmental stimulus.
文摘Ginseng(Panax ginseng C.A. Meyer) is one of the best-selling herbal medicines, with ginsenosides as its main pharmacologically active constituents. Although extensive chemical and pharmaceutical studies of these compounds have been performed, genome-wide studies of the basic helix-loop-helix(b HLH) transcription factors of ginseng are still limited. The b HLH transcription factor family is one of the largest transcription factor families found in eukaryotic organisms, and these proteins are involved in a myriad of regulatory processes. In our study, 169 bHLH transcription factor genes were identified in the genome of P. ginseng, and phylogenetic analysis indicated that these PGb HLHs could be classified into 24 subfamilies. A total of 21 RNA-seq data sets, including two sequencing libraries for jasmonate(JA)-responsive and 19 reported libraries for organ-specific expression analyses were constructed. Through a combination of gene-specific expression patterns and chemical contents,6 PGbHLH genes from 4 subfamilies were revealed to be potentially involved in the regulation of ginsenoside biosynthesis. These 6 PGbHLHs, which had distinct target genes, were further divided into two groups depending on the absence of MYC-N structure. Our results would provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factor action in P.ginseng.
