The ABA-INSENSITIVE-4 (ABI4) transcription factor links redox, hormone and sugar signaling pathways

Abstract

The cellular reduction-oxidation (redox) hub processes information from metabolism and the environment and so regulates plant growth and defense through integration with the hormone signaling network. One key pathway of redox control involves interactions with ABSCISIC ACID (ABA). Accumulating evidence suggests that the ABA-INSENSITIVE-4 (ABI4) transcription factor plays a key role in transmitting information concerning the abundance of ascorbate and hence the ability of cells to buffer oxidative challenges. ABI4 is required for the ascorbate-dependent control of growth, a process that involves enhancement of salicylic acid (SA) signaling and inhibition of jasmonic acid (JA) signaling pathways. Low redox buffering capacity reinforces SA- and JA-interactions through the mediation of ABA and ABI4 to fine-tune plant growth and defense in relation to metabolic cues and environmental challenges. Moreover, ABI4-mediated pathways of sugar sensitivity are also responsive to the abundance of ascorbate, providing evidence of overlap between redox and sugar signaling pathways.

The cellular redox hub, which is comprised of oxidants such as reactive oxygen species (ROS) and antioxidants such as ascorbate (vitamin C), has a decisive input into transcriptional controls within the cell. Ascorbate is the major low molecular weight antioxidant of plant cells¹ and participates in the regulation of plant growth and defense through interactions with the major hormone signaling pathways (Fig. 1). It has long been recognized that ascorbate is a multifunctional metabolite in plants with diverse roles in metabolism and development.^2,3 In the last decade we provided evidence that ascorbate could be a signaling molecule in plants.^4,5 However, there have been few insights into mechanisms by which plants sense the abundance of ascorbate and use this information to regulate growth and defense.¹ Previously, we have shown that the ascorbate-deficient (vtc1 and vtc2) mutants exhibit enhanced ABA- and SA-pathways.^4,6

Figure 1. The role of the redox hub in the integration of environmental and metabolic cues. The redox hub comprising overlapping and interconnected redox signals integrates environmental and metabolic signals and is influenced by the generation of reactive oxygen species (ROS) at a range of spatio-temporal scales. Key modulators of the redox hub are the major soluble antioxidants ascorbate and glutathione whose content and redox status are dependent on both environmental and metabolic conditions. Accumulating evidence links the activity of the redox hub with hormonal signaling resulting in plant resistance to biotic and abiotic stresses.

ABA is a well characterized systemic signal that moves from roots to shoots to reduce stomatal conduction and leaf growth at times of dehydration. ABA exerts responses in stomatal guard cells through the activation of NADPH oxidases and on gene expression through ABF/AREB basic leucine zipper transcription factors.⁷ However, ABA also participates in plant responses to biotic stresses.⁸ In this context, biotrophic pathogens are generally considered to be sensitive to SA-mediated defense responses while necrotrophic pathogens are controlled by JA and ET.^9,10 The vtc1 and vtc2 mutants exhibit increased resistance to biotrophic pathogens⁶ but they are considered to have enhanced sensitivity to necrotrophic pathogens. Wounding and JA can influence ascorbate accumulation^11. Moreover, recent evidence suggests that JA-mediated defense responses are modified by low ascorbate.¹² Such observations suggest that low ascorbate abundance or the decrease in redox state buffering capacity trigger changes in either hormone concentration or sensitivity (or both) that steer the adaptive plant responses toward SA defenses and away from JA pathways.

It has long been known that plant defense responses are greatly influenced by hormone crosstalk, in which different hormone signaling pathways can act antagonistically or synergistically. For example, SA antagonizes JA-dependent defenses, thereby prioritizing SA-dependent resistance over JA-dependent defense.^13,14 Studies on vtc1 and vtc2 mutants have shown that redox and ABA-signaling pathways can participate in the SA-JA interaction providing additional regulatory potential to flexibly tailor the plant’s adaptive response to a variety of environmental and metabolic cues.

Transcriptone Re-Programming

A comparison of the leaf transcriptomes of the abi4, vtc2 and abi4vtc2 mutants revealed that fewer transcripts were differentially expressed in abi4 relative to the wild type than in the other genotypes (Fig. 2). Twice as many transcripts were differentially expressed in vtc2 relative to Col0 and the abi4vtc2 double mutants showed substantially greater transcriptional re-programming than either of the single mutants with 768 transcripts constitutively induced and 515 repressed relative to the wild type (Fig. 2).

Figure 2. Common and genotype-specific transcripts that were differentially expressed in the abi4, vtc2 and abi4vtc2 mutants relative to Col0. Whole Arabidopsis rosettes were harvested following six weeks growth in controlled environment chambers, RNA extracted and microarray analysis performed as described.¹² Venn diagrams indicate genes that were commonly or uniquely differentially expressed in the three genotypes in comparison with Col0.

Comparisons of the patterns of gene expression in the abi4, vtc1 and vtc2 mutants relative to the wild type had revealed a high degree of similarity with over two-thirds of the transcripts commonly expressed.¹² The transcripts that were increased in a similar manner in the abi4, vtc1 and vtc2 mutants included mRNAs encoding a large number of defense-related proteins.¹²

A marked increase in the abundance of transcripts encoding components involved in both SA-dependent and SA-independent defense responses was observed in abi4 and vtc2 genotypes and in the abi4vtc2 double mutants.¹² In particular, the SA-responsive PR-1, PR-2, PR-4 and PR-5 transcripts were among the most highly expressed genes in the abi4 and vtc2 genotypes and in the abi4vtc2 double mutants (Fig. 3). However, some key components of the SA-defense pathways such as PAD4, EDS1 and EDS5 were induced in the vtc2 and abi4vtc2 mutants but not in the abi genotype. These data suggest that the induction of these SA-dependent defense pathways is not greatly influenced by the ABI4 transcription factor. Conversely, while marker genes for the JA/ET defense signaling pathways, such as PDF1.2a and PDF1.2b, were induced in the abi4, vtc2 and abi4vtc2 genotype, the increase in the abundance of these transcripts was markedly lower in vtc2 than in abi4 or abi4vtc2 (Fig. 3). This finding suggests that JA-dependent defense pathways are modulated by ABI4. ET-responsive transcripts, such as the member of the ET response factor subfamily B-3 of the ERF/AP2 transcription factor family (ATERF-2), which is a positive regulator of JA-responsive genes, such as PDF1.2,¹⁵ were induced in a similar manner in the abi4, vtc2 and abi4vtc2 genotype relative to the wild type.

Figure 3. Expression of transcripts encoding defense related proteins responsive to SA (PR1, PR2, PR4, PR5) or JA/ET (PDF1.2a, PDF1.2b) in abi4, vtc2 and abi4vtc2 mutant Arabidopsis relative to Col0. Whole Arabidopsis rosettes were harvested following six weeks growth in controlled environment chambers, RNA extracted and microarray analysis performed as described.¹²

Influences on Flowering

Like the effect ascorbate-dependent effects on vegetative growth, the ascorbate-dependent control of flowering was also dependent on ABI4 (Fig. 4). The vtc2 mutants displayed a late-flowering phenotype relative to the wild type (Fig. 4), as reported previously.⁶ The abi4 mutants flowered before the wild type and the vtc2 mutants (Fig. 4). Moreover, the abi4vtc2 double mutants flowered early and profusely (Fig. 4) despite having the same ascorbate levels as the vtc2 mutants.¹² The transcript profile of the abi4vtc2 double mutants relative to the vtc2 mutants might hold some clues regarding how ascorbate and ABI4 might contribute to the control of flowering. A number of transcripts that were increased in a similar manner in the abi4, vtc2 and abi4vtc2 mutants included mRNAs encoding proteins that are involved in the control of flowering time and circadian rhythms (Table 1). These include proteins that are involved in the control of flowering time, circadian rhythms and light signaling (Table 1). For example, mRNAs encoding CONSTANS-like 2, the NUCLEAR FACTOR Y, SUBUNIT B2 (NF-YB2) and CIRCADIAN CLOCK ASSOCIATED 1, which is a major regulator of the circadian rhythm and is involved in phytochrome control, were decreased in all three mutants relative to the wild type. Other transcripts that are under circadian control such as WNK6 and glycine-rich RNA-binding protein 8 were differentially regulated in the mutants relative to the wild type. However, a suite of transcripts were differentially expressed only in the abi4vtc2 double mutants relative to the wild type (Table 1).

Figure 4. A comparison of the phenotypes of the abi4, vtc2 and abi4vtc2 mutants relative to the wild type (Col0). All plants were grown in a controlled environment chamber for 7.5 weeks with a 10 h photoperiod as previously described.¹²

Table 1. Transcripts that are differentially-expressed in the abi4, vtc2 and abi4vtc2 mutants relative to Col0 that encode proteins involved in the control of flowering time, circadian rhythms and light signaling.

AGIa	Expression ratio relative to Col0b			TAIR annotationc
	abi4	vtc2	abi4vtc2
At3g02380	-3.9	-5.8	-6.1	COL2; constans-like 2
At5g47640	-2.1	-2.1	-2.3	NF-YB2; NUCLEAR FACTOR Y, SUBUNIT B2
At2g46830	-4.5	-4.8	-6.4	CCA1;CIRCADIAN CLOCK ASSOCIATED1
At3g18750	-2.3	-2.7	-2.8	WNK6 (WITH NO K ( = LYSINE) 6)
At4g39260	+2.1	+2.3	+2.9	GR-RBP8; RNA binding / nucleic acid binding nucleotide binding
At3g17609	-3.1	-3.5	-3.3	HYH; HY5-HOMOLOG
At5g24850	-2.6	-2.1	-3.3	CRY3; cryptochrome 3
At1g69720	+2.7	+2.7	+2.8	ho3; HEME OXYGENASE 3
At5g24120	-3.0	-2.9	-4.4	SIG5; SIGMA FACTOR E
At1g78600	+4.0	+4.3	+4.2	LZF1; LIGHT-REGULATED ZINC FINGER PROTEIN 1
At1g10370	-2.5	-5.1	-4.9	ERD9; EARLY-RESPONSIVE TO DEHYDRATION 9
At4g27440	+4.6	+3.7	+4.6	PORB; PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B
At4g13250	+2.2	+2.2	+2.1	NYC1; NON-YELLOW COLORING 1
At4g14690	-4.2	-3.9	-5.0	ELIP2; EARLY LIGHT-INDUCIBLE PROTEIN 2
At5g52570	-4.9	-6.7	-6.0	BETA-OHASE 2; BETA-CAROTENE HYDROXYLASE 2
At3g15270	-2.2	nsd	nsd	SPL5; SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 5
At1g25560	nsd	+2.2	nsd	TEM1; TEMPRANILLO 1
At5g15850	nsd	-2.1	nsd	COL1; Constans-like 1
At1g76570	nsd	-2.0	nsd	chlorophyll A-B binding family protein
At3g04910	nsd	nsd	+2.0	WNK1; WITH NO LYSINE (K) 1
At5g62430	nsd	nsd	-2.3	CDF1; CYCLING DOF FACTOR 1
At2g30520	nsd	nsd	-2.2	RPT2; ROOT PHOTOTROPISM 2
At4g37590	nsd	nsd	-2.5	NPY5; NAKED PINS IN YUC MUTANTS 5
At4g17230	nsd	nsd	+2.0	SCL13; Scarecrow-like 13
At4g25420	nsd	nsd	-2.2	GA20ox1; gibberellin 20-oxidase
At3g05120	nsd	nsd	+2.4	GID1A; GA INSENSITIVE DWARF1A
At2g40100	nsd	nsd	-2.4	LHCB4.3; light harvesting complex PSII
At1g16720	nsd	nsd	-2.5	HCF173; high chlorophyll fluorescence phenotype 173

^a Arabidopsis genome initiative number; ^b+, Transcript abundance enhanced compared with Col0; -, transcript abundance repressed compared with Col0. All ratios are expressed on a linear scale; ^cDatabase annotation of the protein product; ^dGene expression not significantly different from Col0.

A number of transcripts that were increased in a similar manner in the abi4, vtc2 and abi4vtc2 mutants included mRNAs encoding proteins that are involved in light signaling pathways that impact on control of flowering time and circadian rhythms. For example, the expression of HY5-HOMOLOG HYH, which is involved in phytochrome B signaling, was decreased in the abi4, vtc2 and abi4vtc2 genotypes relative to the wild type (Table 1), as was cryptochrome 3, which encodes a photoreceptor that recognizes and repairs UV lesions of DNA. Transcripts that are involved in light signaling pathways such as SIGMA FACTOR E (SIGE) and LIGHT-REGULATED ZINC FINGER PROTEIN 1 were altered in expression in the abi4, vtc2 and abi4vtc2 genotypes relative to Col0 (Table 1). While, these findings may reflect the suggested role of ABI4 in chloroplast- to nucleus- retrograde signaling pathways,¹⁶ little direct experimental support for this conclusion was obtained.¹²

Metabolite Re-Programming

The vtc2 and abi4vtc2 leaves had low levels of ascorbate relative to the wild type plants and the abi4 genotype.¹² The vtc2 and abi4vtc2 leaves also had significantly lower amounts of threonic acid, which is a product of ascorbate degradation¹⁷ than the wild type or the abi4 leaves. The vtc2 and abi4vtc2 leaves also had significantly lower amounts of inositol than the wild type plants (Fig. 5). This finding may give insights into the defense pathways that are triggered by low ascorbate but that do not require a functional ABI4 protein in the induction mechanism. Similarly, certain cinnamic acid and lignoceric acid derivatives were significantly lower in the vtc2 and abi4vtc2 leaves than the wild-type (Fig. 5).

Figure 5. Heatmap displaying relative metabolite content in Arabidposis Col0, abi4, vtc2 and abi4vtc2. Plants were grown for six weeks under controlled environments and individual leaves harvested from four replicate plants and immediately frozen on liquid nitrogen. Following lyophilisation, leaves were extracted and metabolites were derivatised for GC/MS as previously described.³⁰ The figure shows metabolite content in each of the four replicates relative to the centered mean across all samples as previously described.³¹

While the vtc2 leaves had significantly less sucrose, succinate and fumarate than those of the wild-type plants (Fig. 5) the metabolite profile suggests that primary metabolism was not greatly changed as a result of low ascorbate. However, glutamate levels were significantly lower in the leaves of the vtc2, abi4 and abi4vtc2 genotypes relative to the wild type, as were various fatty-acids and long-chain alcohols (Fig. 5). These observations may indicate that low ascorbate may exert some effects on metabolism.

The Impact of Redox Buffering Capacity on Hormone and Sugar Signaling Pathways

JA, ET, SA and ABA are major players in coordinating signaling networks involved in the adaptive response of plants to the biotic and abiotic environment.^8,18,19 The signaling cascades that are regulated by these hormones partly overlap and are interconnected in a complex network of cross-communicating hormone signaling pathways that are directly linked to the redox signaling hub of the plant cell. The enormous regulatory potential of such an interactive network allows plants to rapidly adapt to the environment and to utilize their resources in an energy-efficient manner.

One of the most striking features of the transcriptome and metabolome reprogramming observed in the vtc2, abi4 and abi4vtc2 mutants relative to the wild type is the marked effects of low ascorbate and hence redox buffering capacity on hormone and defense-related transcripts. In particular, low ascorbate enhances SA- and ABA-signaling pathways.^4,12 The significant overlap in transcriptome signatures of the vtc1, vtc2 and abi4 mutants revealed considerable overlap in the transcriptome reprogramming of defense pathways in these mutants including transcription factors such as ERF104 and ZAT 10.¹²

ABA has long been associated with abiotic stress responses²⁰ but it plays a much wider role in the responses of plants to biotic as well as biotic stresses.²¹ ABA and redox signaling are strongly linked through the activation of NADPH oxidases in both local and systemic responses to stresses that result in dehydration such as high light.²² The NADPH oxidase forms AtrbohD and AtrbohF are required for the ABA-dependent closure of leaf stomata.^23,24 The latter process is dependent on the upstream function of the protein phosphatase 2C encoded by ABA insensitive 1 (ABI1) and a downstream function of the protein phosphatase 2C encoded by ABA insensitive 2 (ABI2),²⁵ the MAP kinase AtMPK3²⁶ and other P38-like MAP kinases.²⁷ Ethylene-induced closure of stomata also requires AtrbohD and AtrbohF.²⁸

We have previously described a further link between ABA and redox signaling in plants that involves ascorbate.⁴ We have also shown that ABA signaling through the ABI4 transcription factor is required for ascorbate-dependent growth regulation.¹² These data suggest that enhanced SA signaling in the vtc2 mutants represses JA-signaling pathways through ABI4. Evidence in support of this conclusion comes from the changes in gene expression patterns in vtc2 and abi4vtc2 double mutants relative to Col0. In particular, transcripts encoding the ORA47 transcription factor were enhanced in the abi4 and vtc2 single mutants relative to Col0 but repressed in the abi4vtc2 double mutants.¹² Marker genes for downstream JA signaling pathways, such as PDF1.2a and PDF1.2b, were much higher in the abi4 and abi4vtc2 mutants than in vtc2 relative to Col0 (Fig. 3).

The analysis of abi4 and vtc2 single mutants relative to the abi4vtc2 double mutant also revealed a further link between the ABA, redox and sugar signaling pathways through ABI4. ABI4 plays an important role in sugar signaling, as well as ABA responses²⁹ as illustrated by the ability of abi4 seeds to germinate on media containing high levels of glucose whereas the wild type seeds showed a poor germination under these conditions.¹² While the germination of the vtc2 mutant seeds showed a similar sensitivity to high glucose as observed in wild type, the abi4vtc2 double mutants exhibited a glucose-sensitive phenotype.¹² Thus, the glucose-insensitivity arising from impaired ABI4 signaling was repressed when redox buffering capacity was low. The pronounced effect of ascorbate on sugar signaling suggests that the ability to sense the abundance of key metabolites is sensitive to redox regulation. Taken together these data show that the cellular redox rub, which is responsive to oxidative signaling triggered by a wide diversity of stresses as well as oxidants produced by metabolism, fine-tunes the plant responses to hormonal and metabolite/energy regulators, providing enhanced sensitivity, flexibility and balance.

Kerchev PI, Pellny TK, Vivancos PD, Kiddle G, Hedden P, Driscoll S, et al. The transcription factor ABI4 Is required for the ascorbic acid-dependent regulation of growth and regulation of jasmonate-dependent defense signaling pathways in Arabidopsis. Plant Cell. 2011;23:3319–34. doi: 10.1105/tpc.111.090100.