The Arabidopsis transcription factor ABIG1 relays ABA signaled growth inhibition and drought induced senescence

One way for plants to withstand drought is to slow, or stop, new growth thereby conserving resources until better times return (Dolferus, 2014). Growth arrest coincides with predictable dry periods during the life cycle – such as in seed development or seasonal bud dormancy. Growth arrest or reduction can also occur in response to sporadic drought (e.g. Marc and Palmer, 1976; Verelst et al., 2013) More extreme than growth arrest is a response in which parts of the plant are sacrificed, as for instance in drought induced leaf senescence, allowing the plant to recover nutrients and to reduce the water cost of maintaining the leaf (Munné-Bosch et al., 2004).

Among plant hormones, abscisic acid (ABA) is the best known for its ability to allow plants to withstand drought. In response to desiccation, levels of the ABA biosynthetic enzyme AtNCED3 in the vascular parenchyma increase in Arabidopsis (Endo et al., 2008). This causes an increase in ABA which causes plants to close stomatal apertures, thus reducing water loss (Schroeder et al., 2001). Increases in ABA concentration during seed development impose maturation and dormancy on seeds (Finkelstein, 2013). ABA acts by binding to PYR-like co-receptors and bringing them together with PP2C family phosphatases (Cutler et al., 2010). This frees downstream SNRK2 kinases from repression by the PP2C phosphatase, allowing the kinase to modify protein targets at the plasma membrane - to alter turgor in guard cells - or to modify a set of transcription factors - to promote maturation and dormancy in the developing seed.

The role of ABA in controlling vegetative growth is less clear. Exogenously applied ABA slows growth of roots and shoots: it promotes dormancy in axillary buds (Shimizu-Sato and Mori, 2001), inhibits formation and growth of leaves (Marc and Palmer, 1976; Verelst et al., 2013) and inhibits root growth (DeSmet et al., 2003). ABA also can promote drought induced leaf senescence (Munné-Bosch and Alegre, 2004). Paradoxically, ABA appears to be required for growth as well. This conclusion is based on the observation that biosynthetic mutants with reduced levels of ABA are smaller than wild type plants (Barrero et al., 2005). Supplementation with ABA restores growth of plants grown under well watered conditions indicating the growth defect is not a secondary consequence of water loss. Taken at face value, these observations indicate that ABA promotes growth as well as inhibits it.

In this paper, we describe a transcription factor that is required for inhibition of growth in response to ABA. We propose that this factor does not act on the canonical ABA events of stomatal closure and seed dormancy but rather in a branch through which environmental stresses act through ABA to restrict growth.

A tenet of systems biology is that agents inhabiting the same node of a regulatory network share a biological function. REVOLUTA (REV) and KANADI1 (KAN1), opposing regulators of leaf polarity and meristem formation, also oppositely control components of the ABA signaling pathway (Reinhart et al., 2013). A connection between the ad/abaxial developmental and ABA signaling networks had not been predicted based on mutant phenotypes, and the biological relevance of the connection is not understood. Nevertheless, since ABA signaling genes are prominent among the small set of ORK genes (genes oppositely regulated by the REV and KAN transcription factors (Reinhart et al., 2013; Figure 1A), we reasoned that other genes oppositely regulated by them may also play a role in ABA signaling in the plant.

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The set of eight ORK genes (OPPOSITELY REGULATED BY REVOLUTA AND KANADI) includes two genes involved in ABA signaling: PYL6 and CIPK12. PYL6 encodes a member of the family of ABA receptors (Park et al., 2009). CIPK12 encodes a member of a family of SNRK3 kinases that play a role in the ABA regulatory pathway (Qin et al., 2008; Lumba et al., 2014). Except forZFP8, which plays a role in trichome development (Gan et al., 2007), the function of the other five genes at this regulatory node is unknown.

HOMEOBOX FROM ARABIDOPSIS THALIANA 22 (AT4G37790/ HAT22, Figure 1B) was chosen for study because it shows rapid and robust opposite regulation by REV and KAN1, because it is highly conserved among land plants and because its function in the plant is largely unknown. HAT22 is one of 10 Class II HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPII) genes (Ciarbelli et al., 2008). We propose to rename this gene ABA INSENSITIVE GROWTH 1 (ABIG1) to better reflect its function in the plant (see below).

abig1/hat22 mutants display normal stomatal closure and germination responses

abig1-1 mutant seeds and plants do not behave the same as canonical ABA resistant mutants. In contrast to canonical ABA insensitive (abi) mutants (Koorneef et al., 1984; Finkelstein, 1994), abig1-1 mutants were similar to wild type seeds in their inability to germinate in the presence of ABA, a potent germination inhibitor (Figure 2A). Another characteristic of ABA signaling defective mutants is a failure of stomatal closure in response to ABA application. ABA applied to the epidermis of wild-type and abig1-1 mutant leaves caused similar decreases in stomatal apertures (Figure 2B).

Figure 2

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abig1/hat22 mutants are resistant to ABA mediated growth inhibition of shoot tissues

To determine if ABIG1 affects responses to ABA after germination, we transferred seedlings at seven days from standard MS medium to MS medium supplemented with ABA. Under this treatment regimen ABA inhibited growth and caused leaf yellowing in wild type seedlings (Figure 3). ABA caused fewer leaves to develop at the shoot apex, and caused leaves to have shorter petioles and smaller leaf blades. abig1-1 mutants showed less response to ABA than wild type for leaf but not root growth: abig1 mutants differed in their response to ABA with regard to leaf number (p(genotype by [ABA]) <0.0001) and petiole length (p(genotype by [ABA]) <0.02) but not root length (p(genotype by [ABA]) = 0.285) as determined using a 2 way ANOVA analysis. We conclude that the wild type ABIG1 function is required for ABA-mediated inhibition of leaf production and petiole growth. These experiments were repeated with a second loss of function allele, abig1-4, and similar results were obtained (Figure 1—figure supplement 1; Figure 3—figure supplement 1).

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Overexpression of ABIG1/HAT22 mimics ABA application

Induced overexpression of ABIG1 (achieved by placing the ABIG1 cDNA under the control of an estradiol inducible XVE transcription factor [Zuo et al., 2000]) mimicked application of ABA to wild type plants. Leaves developed with small blades and short petioles, leaves were yellow, and fewer leaves were made (Figure 4). Thus, increased ABIG1 mRNA levels are sufficient to cause growth inhibition and leaf yellowing. We note that this is consistent with results by Köllmer et al. (2011) who found a modest increase in leaf senescence when ABIG1 (HAT22) was expressed under control of the 35S promoter. The more severe yellowing and seedling arrest phenotype seen in our experiments may be because the conditional nature of the estradiol induced promoter makes it possible for us to recover transgenic plants with high expression levels.

Figure 4

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In summary, examination of loss and gain of function mutants in ABIG1 support the hypothesis that this transcription factor contributes to ABA induced growth inhibition and leaf yellowing in the plant, and is sufficient to mimic the effect of exogenous ABA on growth inhibition and leaf yellowing. Such behavior is consistent with a model in which ABA inhibits growth of the shoot by causing increased levels of ABIG1 expression. Addition of increasing concentrations of ABA to estradiol treated XVE:ABIG1 plants did not make the phenotype more extreme than when estradiol alone was added (Figure 4C–E). This supports a model in which the major role for ABA in regulating plant growth is through its role in promoting the ABIG1 transcript accumulation.

ABIG1/HAT22 mutants show less leaf yellowing in response to drought

The finding that ABIG1 mRNA levels increase in response to both ABA and drought and that ABIG1 is necessary for ABA mediated growth inhibition and leaf yellowing suggested that ABIG1 might play a role in response to drought. To test this, plants were grown two per pot with one wild type and one mutant in each pot. At 34 days post-germination, just prior to bolting, the pots were split into two groups. The first group was well watered for the duration of the experiment while water was withheld from the second group. Under well-watered conditions, abig1-1 mutants had a lower percentage of yellow, senesced leaves than wild-type (Figure 5). For one experimental replicate, the amount of chlorophyll present in leaves was measured (Figure 5G). It was found to decrease significantly more in the wild type than in the mutant.

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After 17 days of withholding water, this difference became significantly more pronounced with the percent yellow or senesced leaves more than doubling in the wild type but only increasing modestly in the homozygous abig1-1 mutant (p(treatment × genotype) = 0.0008; Figure 5B,D). Under water withheld conditions, wild type plants made fewer side branches than under well watered conditions while abig1 mutants continued to produce the same number of side branches (p(treatment × genotype) = 0.0114; Figure 5E). The wild type plants were unable to remain upright while the mutant plants remained erect (Figure 5B ; 100% erect mutant plants (n = 9) vs 37.5% erect wild type plants (n = 9) in one experiment with similar results in 3 additional replicated experiments). Wild type plants also showed less extensive root systems than the abig1-1 mutants (Figure 5C). This experiment was repeated multiple (greater than three) times with similar results.

Similar results were obtained with the abig1-4 allele except that the abig1-4 mutants made the same number of side branches as wild type under dry conditions (Figure 5—figure supplement 1). Since both the Ds insertion in abig1-1 and the T-DNA insertion in abig1-4 disrupt the gene close to its 5 prime end, this phenotypic difference is unlikely due to a difference in allele strength but rather to differences in genetic background. In support of this, the phenotype of the abig1-1 mutation is similar to that of abig1-4 when it is outcrossed to wild type Columbia, the background which abig1-4 is in (Figure 5—figure supplement 3). We conclude that the ABIG1 transcription factor accelerates leaf senescence and acts to prevent the plant from remaining erect under dry conditions. This result was unexpected since ABA resistant mutants isolated in the past have been associated with drought sensitivity rather than drought resistance (see e.g. Wang et al., 2009).

Genes regulated by ABIG1 are enriched for stress related genes including ABA and ethylene response loci

To determine what genes the ABIG1 transcription factor controls, we treated XVE:ABIG1 plants and wild-type plants with 5 microMolar estradiol for 0, 60 and 120 min. Plants were flash frozen at each time point and RNA was isolated and subjected to RNA-seq. This treatment resulted in robust up-regulation of ABIG1 mRNA (Figure 4B; Supplementary file 1).

Using the CUFFDIFF application (Trapnell et al., 2013) we first identified genes differentially expressed after estradiol treatment (p adjusted <0.01; |FC|>1.5 (log base 2)). We then removed any genes that showed a (time of treatment by genotype) interaction p value greater than 0.05 (Two way-ANOVA). This limits the set to genes that respond differently in wild-type vs. XVE:ABIG1. We also removed genes for which one or more time points resulted in a 'no test' call by CUFFDIFF. The final list includes 18 up-regulated mRNAs (Figure 6A and Supplementary file 1) and 14 down-regulated mRNAs (Figure 6B and Supplementary file 1).

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Down-regulated, but not up-regulated, genes, showed statistically significant over-representation for Gene Ontology (GO) terms associated with response to stress, response to abiotic or biotic stimulus, and signal transduction (Figure 6C). The interaction of the EAR domain of ABIG1 with the TOPLESS co-repressor (Causier et al., 2012; T. Liu and M.K.Barton, unpublished), and the demonstrated ability of HD-ZIPII factors to repress their own transcription (Ohgishi et al., 2001; Ciarbelli et al., 2008) are evidence that ABIG1 acts a repressor. Direct targets of ABIG1 are therefore expected to fall among the genes down-regulated by ABIG1 induction.

The promoters (500 bp upstream of the ATG) of the down regulated genes are enriched for bHLH, MADS and a homeodomain transcription factor binding sites (p=1.6E-4, p=1.5E-2 and p=1.9E-2 respectively; P-Scan (Zambelli et al., 2009)). The strong representation of additional types of transcription factor binding sites, especially bHLH binding sites, within the down-regulated genes points to combinatorial regulation of these loci by ABIG1 and as yet unspecified bHLH proteins.

Promoters for up-regulated genes were enriched for bZIP transcription factor binding sites (p = 4.1E-3).

Notable among the regulated genes are: CYP707A3 which encodes an enzyme that catabolizes ABA (Umezawa et al., 2006); GRF5 which regulates leaf senescence and chloroplast number (Vercruyssen et al., 2015); BFN1, a gene encoding a nuclease involved in leaf and stem senescence (Farage-Barhom [remove (Farage-Barhorn] (Farage-Barhom et al., 2011); ATHB5, a gene implicated in drought resistance (Johannesson et al., 2003)and TOPLESS RELATED 3 (TPR3), a member of the TOPLESS co-repressor gene family and a likely partner of the ABIG1 transcription factor (Causier et al., 2012; Long et al., 2006).

Because several plant hormones influence ABA sensitivity and drought tolerance we queried the dataset with gene lists for genes involved in biosynthesis and/or signaling for the plant hormones abscisic acid, ethylene, jasmonate and cytokinin. These hormones were chosen because they have been implicated in promoting (ABA, jasmonate and ethylene) or preventing (cytokinin) senescence (Kim et al., 2015; Pré et al., 2008; Buchanan-Wollaston, 1996). We also included a set of genes associated with chlorophyll degradation. Six genes passed this filter (Supplementary file 1; Figure 6D). In addition to the ABA catabolic enzyme gene, CYP707A2, induction of ABIG1 also regulates At2g17820/HK1, a gene encoding a receptor that mediates ABA, drought and salt stress responses (Wollbach et al., 2008) and the gene encoding the ethylene receptor ERS2. Finally, two transcription factor genes in the jasmonate pathway, ORA59 and TIFY7 were down-regulated in this experiment. The identities of these regulated genes support a model in which ABIG1 promotes drought-induced senescence in part by changing the amount of ABA in the plant while also altering the sensitivity of the cell to ABA, ethylene and jasmonate. While all of these changes occur rapidly (within hours) after the induction of ABIG1 mRNA synthesis, the effects may nonetheless be indirect since these hormone pathways are known to cross-regulate one another. It is therefore unclear which of these genes are primary, direct targets of ABIG1 binding and which are secondary, indirect targets.

SAG113/HAI1/AT5g59220 encodes a PP2C phosphatase homologous to those in the core ABA signaling pathway. Its transcription is increased rapidly both in response to senescence and in response to ABA (Zhang and Gan, 2012). As such, it is a good candidate for regulation by ABIG1. However, the transcript corresponding to this locus did not show regulation by induced ABIG1 in the above experiment (Figure 6). We also did not observe a requirement for ABIG1 in ABA mediated up-regulation of SAG113/HAI1 (Figure 5— figure supplement 2). In addition, ABIG1 transcript induction did not alter levels of AtNAP (AT1G69490; Zhang and Gan, 2012) , a known positive regulator of SAG113/HAI1 through which ABA is thought to act. Thus, ABA up-regulation of SAG113/HAI1likely occurs through a parallel pathway. Such a parallel pathway may be primarily responsible for up-regulation of rapidly regulated targets while ABIG may be primarily involved in regulation of down-regulated genes

Alterations in cytokinin metabolism, particularly increases in cytokinin activity, are associated with both drought tolerance and with inhibition of senescence (Peleg et al., 2011; Kuppu et al., 2013). While we found no evidence for altered transcription of cytokinin pathway genes in response to transiently induced ABIG1 transcription (above), we did find that abig1-1 mutant seedlings have higher steady state levels of mRNA from the CYP735A2/At1G67110 cytokinin biosynthetic gene (Figure 6 —figure supplement 1). This change in steady state values is likely an indirect effect of reduced ABIG1 action since no change in CYP735A2 levels were observed within the first two hours of ABIG1 induction. Interestingly, while the levels of CYP735A2 remained steady in wild type in response to exogenous ABA, elevated CYP735A2 mRNA levels decreased in the abig1 mutant in response to exogenous ABA (Figure 6—figure supplement 1). Thus, not only do some ABA responses remain intact in abig1 mutants, the physiological and developmental context of the abig1 mutant also results in novel responses to ABA.

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Author details

1. Tie Liu

   Department of Plant Biology, Carnegie Institution for Science, Stanford, United States

   Contribution

   TL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Adam D Longhurst

   Department of Plant Biology, Carnegie Institution for Science, Stanford, United States

   Contribution

   ADL, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Franklin Talavera-Rauh

   Department of Plant Biology, Carnegie Institution for Science, Stanford, United States

   Contribution

   FT-R, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Samuel A Hokin

   Department of Plant Biology, Carnegie Institution for Science, Stanford, United States

   Contribution

   SAH, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. M Kathryn Barton

   Department of Plant Biology, Carnegie Institution for Science, Stanford, United States

   Contribution

   MKB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   kbarton@CarnegieScience.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5516-1835

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