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Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors.

Reeves WM, Lynch TJ, Mobin R, Finkelstein RR - Plant Mol. Biol. (2011)

Bottom Line: The direct effects of ABI4 and ABI5 in ABA response remain largely undefined.Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences.Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

View Article: PubMed Central - PubMed

Affiliation: Molecular, Cellular, and Developmental Biology Department, University of California at Santa Barbara, Santa Barbara, CA 93106, USA.

ABSTRACT
The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

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Confirmation of ABA and ABI dependent gene targets. Wild-type (Col or Ws), transgenic (35S-GFP-ABI4 or 35S-ABI5) and mutant (abi4 and abi5-1) seedlings were grown for 11 days on GM, then treated for 3 h with either ABA, cycloheximide (CHX) or ABA + cycloheximide (A/X). Induction of ABI targets was assayed by Northern blot. a ABI4-regulated genes, b ABI5-regulated genes, and c genes regulated by both ABI4 and ABI5. For the ABI4-GFP transcript, the probe was specific to ABI4. An arrowhead indicates full length transcript and an asterisk indicates a truncated/partially degraded transcript
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Fig1: Confirmation of ABA and ABI dependent gene targets. Wild-type (Col or Ws), transgenic (35S-GFP-ABI4 or 35S-ABI5) and mutant (abi4 and abi5-1) seedlings were grown for 11 days on GM, then treated for 3 h with either ABA, cycloheximide (CHX) or ABA + cycloheximide (A/X). Induction of ABI targets was assayed by Northern blot. a ABI4-regulated genes, b ABI5-regulated genes, and c genes regulated by both ABI4 and ABI5. For the ABI4-GFP transcript, the probe was specific to ABI4. An arrowhead indicates full length transcript and an asterisk indicates a truncated/partially degraded transcript

Mentions: Induction of ABI4 targets was found to be very sensitive to the relative strength of ABI4 overexpression. After the microarray and initial validation analyses, ABI4 overexpression in the line used for these studies was silenced in subsequent generations. Consequently, we created new overexpression lines, with a GFP-ABI4 fusion under the control of the 35S promoter, for use in continuing analysis of the identified targets. Although ABI4 function in this line was weaker than in the original 35S:ABI4 line (Supp. Fig. 1), possibly due to either reduced expression of the fusion gene or altered conformation of the fusion protein, some ABI4 transcriptional targets were still induced (Fig. 1). However, the induction of several targets was significantly weaker in the 35S:GFP:ABI4 line compared to the original 35S:ABI4 plants (Supp. Fig. 2A).Fig. 1


Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors.

Reeves WM, Lynch TJ, Mobin R, Finkelstein RR - Plant Mol. Biol. (2011)

Confirmation of ABA and ABI dependent gene targets. Wild-type (Col or Ws), transgenic (35S-GFP-ABI4 or 35S-ABI5) and mutant (abi4 and abi5-1) seedlings were grown for 11 days on GM, then treated for 3 h with either ABA, cycloheximide (CHX) or ABA + cycloheximide (A/X). Induction of ABI targets was assayed by Northern blot. a ABI4-regulated genes, b ABI5-regulated genes, and c genes regulated by both ABI4 and ABI5. For the ABI4-GFP transcript, the probe was specific to ABI4. An arrowhead indicates full length transcript and an asterisk indicates a truncated/partially degraded transcript
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044226&req=5

Fig1: Confirmation of ABA and ABI dependent gene targets. Wild-type (Col or Ws), transgenic (35S-GFP-ABI4 or 35S-ABI5) and mutant (abi4 and abi5-1) seedlings were grown for 11 days on GM, then treated for 3 h with either ABA, cycloheximide (CHX) or ABA + cycloheximide (A/X). Induction of ABI targets was assayed by Northern blot. a ABI4-regulated genes, b ABI5-regulated genes, and c genes regulated by both ABI4 and ABI5. For the ABI4-GFP transcript, the probe was specific to ABI4. An arrowhead indicates full length transcript and an asterisk indicates a truncated/partially degraded transcript
Mentions: Induction of ABI4 targets was found to be very sensitive to the relative strength of ABI4 overexpression. After the microarray and initial validation analyses, ABI4 overexpression in the line used for these studies was silenced in subsequent generations. Consequently, we created new overexpression lines, with a GFP-ABI4 fusion under the control of the 35S promoter, for use in continuing analysis of the identified targets. Although ABI4 function in this line was weaker than in the original 35S:ABI4 line (Supp. Fig. 1), possibly due to either reduced expression of the fusion gene or altered conformation of the fusion protein, some ABI4 transcriptional targets were still induced (Fig. 1). However, the induction of several targets was significantly weaker in the 35S:GFP:ABI4 line compared to the original 35S:ABI4 plants (Supp. Fig. 2A).Fig. 1

Bottom Line: The direct effects of ABI4 and ABI5 in ABA response remain largely undefined.Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences.Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

View Article: PubMed Central - PubMed

Affiliation: Molecular, Cellular, and Developmental Biology Department, University of California at Santa Barbara, Santa Barbara, CA 93106, USA.

ABSTRACT
The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

Show MeSH