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ZmbZIP60 mRNA is spliced in maize in response to ER stress.

Li Y, Humbert S, Howell SH - BMC Res Notes (2012)

Bottom Line: Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus.Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb.Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Sciences Institute, Iowa State University, Ames, IA 50014, USA.

ABSTRACT

Background: Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR) in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA.

Results: A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT) or tunicamycin (TM) or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP) genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb.

Conclusions: The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

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Induction of ZmbZIP60 splicing by ER stress agents. ZmbZIP60 splicing in maize seedlings was detected using a flanking primers RT-PCR assay (FP) or a specific primer assay for spliced mRNA (SPS) or unspliced mRNA (SPU). Both unspliced (slower migrating) and spliced (faster migrating) forms appear in the FP assay and the migration position of both forms is indicated by arrows. Seven-day-old seedlings were treated by (A) immersing their roots in 2.5 mM DTT solution or (B) topically spraying the seedlings with the same solution. RNA was extracted from roots and shoots. (C) Seedlings were treated with 100 μM cyclopiazonic acid (CPA) or with 5 μg/ml tunicamycin (TM). RNA was obtained from roots. Con = control (No treatment).
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Figure 3: Induction of ZmbZIP60 splicing by ER stress agents. ZmbZIP60 splicing in maize seedlings was detected using a flanking primers RT-PCR assay (FP) or a specific primer assay for spliced mRNA (SPS) or unspliced mRNA (SPU). Both unspliced (slower migrating) and spliced (faster migrating) forms appear in the FP assay and the migration position of both forms is indicated by arrows. Seven-day-old seedlings were treated by (A) immersing their roots in 2.5 mM DTT solution or (B) topically spraying the seedlings with the same solution. RNA was extracted from roots and shoots. (C) Seedlings were treated with 100 μM cyclopiazonic acid (CPA) or with 5 μg/ml tunicamycin (TM). RNA was obtained from roots. Con = control (No treatment).

Mentions: Having a mRNA splicing assay in hand, we are able to test various stress agents for their ability to elicit the splicing response in corn seedlings. Seeds were germinated on moist filter paper. Typically, stress agents such as DTT or tunicamycin are used in the laboratory to elicit ER stress responses. They serve as proxies for environmental stresses by interfering with protein folding in the ER. In our experiments, the agents were either added to a hydroponic solution or topically applied by spraying directly onto the seedlings. When DTT was added to the hydroponic medium, spliced forms of ZmbZIP60 were detected by the FP assay in root tissue 30 min after treatment and increased thereafter (Figure 3A). The SPS assay is more sensitive, and it can be clearly seen that spliced forms are induced by 30 min after DTT treatment. There does appear to be some increase in the unspliced form in roots following DTT treatment, but that effect was not reproducible. No splicing was observed in the shoots.


ZmbZIP60 mRNA is spliced in maize in response to ER stress.

Li Y, Humbert S, Howell SH - BMC Res Notes (2012)

Induction of ZmbZIP60 splicing by ER stress agents. ZmbZIP60 splicing in maize seedlings was detected using a flanking primers RT-PCR assay (FP) or a specific primer assay for spliced mRNA (SPS) or unspliced mRNA (SPU). Both unspliced (slower migrating) and spliced (faster migrating) forms appear in the FP assay and the migration position of both forms is indicated by arrows. Seven-day-old seedlings were treated by (A) immersing their roots in 2.5 mM DTT solution or (B) topically spraying the seedlings with the same solution. RNA was extracted from roots and shoots. (C) Seedlings were treated with 100 μM cyclopiazonic acid (CPA) or with 5 μg/ml tunicamycin (TM). RNA was obtained from roots. Con = control (No treatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369818&req=5

Figure 3: Induction of ZmbZIP60 splicing by ER stress agents. ZmbZIP60 splicing in maize seedlings was detected using a flanking primers RT-PCR assay (FP) or a specific primer assay for spliced mRNA (SPS) or unspliced mRNA (SPU). Both unspliced (slower migrating) and spliced (faster migrating) forms appear in the FP assay and the migration position of both forms is indicated by arrows. Seven-day-old seedlings were treated by (A) immersing their roots in 2.5 mM DTT solution or (B) topically spraying the seedlings with the same solution. RNA was extracted from roots and shoots. (C) Seedlings were treated with 100 μM cyclopiazonic acid (CPA) or with 5 μg/ml tunicamycin (TM). RNA was obtained from roots. Con = control (No treatment).
Mentions: Having a mRNA splicing assay in hand, we are able to test various stress agents for their ability to elicit the splicing response in corn seedlings. Seeds were germinated on moist filter paper. Typically, stress agents such as DTT or tunicamycin are used in the laboratory to elicit ER stress responses. They serve as proxies for environmental stresses by interfering with protein folding in the ER. In our experiments, the agents were either added to a hydroponic solution or topically applied by spraying directly onto the seedlings. When DTT was added to the hydroponic medium, spliced forms of ZmbZIP60 were detected by the FP assay in root tissue 30 min after treatment and increased thereafter (Figure 3A). The SPS assay is more sensitive, and it can be clearly seen that spliced forms are induced by 30 min after DTT treatment. There does appear to be some increase in the unspliced form in roots following DTT treatment, but that effect was not reproducible. No splicing was observed in the shoots.

Bottom Line: Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus.Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb.Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Sciences Institute, Iowa State University, Ames, IA 50014, USA.

ABSTRACT

Background: Adverse environmental conditions produce ER stress and elicit the unfolded protein response (UPR) in plants. Plants are reported to have two "arms" of the ER stress signaling pathway-one arm involving membrane-bound transcription factors and the other involving a membrane-associated RNA splicing factor, IRE1. IRE1 in yeast to mammals recognizes a conserved twin loop structure in the target RNA.

Results: A segment of the mRNA encoding ZmbZIP60 in maize can be folded into a twin loop structure, and in response to ER stress this mRNA is spliced, excising a 20b intron. Splicing converts the predicted protein from a membrane-associated transcription factor to one that is targeted to the nucleus. Splicing of ZmbZIP60 can be elicited in maize seedlings by ER stress agents such as dithiothreitol (DTT) or tunicamycin (TM) or by heat treatment. Younger, rather than older seedlings display a more robust splicing response as do younger parts of leaf, along a developmental gradient in a leaf. The molecular signature of an ER stress response in plants includes the upregulation of Binding Protein (BIP) genes. Maize has numerous BIP-like genes, and ER stress was found to upregulate one of these, ZmBIPb.

Conclusions: The splicing of ZmbZIP60 mRNA is an indicator of ER stress in maize seedlings resulting from adverse environmental conditions such as heat stress. ZmbZIP60 mRNA splicing in maize leads predictively to the formation of active bZIP transcription factor targeted to the nucleus to upregulate stress response genes. Among the genes upregulated by ER stress in maize is one of 22 BIP-like genes, ZmBIPb.

Show MeSH
Related in: MedlinePlus