Role of HY5 in abscisic acid response in seeds and seedlings

Abstract

Seed dormancy offers plants an adaptive advantage that is crucial to their successful colonization of land. The decision to make the transition from a dormant seed to a photoautotrophic seedling is a result of a complex interaction of internal hormonal signals and external stimuli such as water, temperature and light. We recently showed that HY5, a well-characterized component in the light signaling pathway, also mediates abscisic acid (ABA) response during seed germination, early seedling growth and root development in Arabidopsis. We proposed that HY5 regulates these ABA responses partly by directly activating the transcription factor gene ABI5. By analyzing the premature germination of hy5 and abi5 single and double mutants, here we demonstrated that HY5 also positively controls seed maturation and dormancy, likely through direct activation of the ABI5 gene. The contrasting role of light regulation of seed development and germination may be important for the adaptation of plants to the environment.

The Light-Signaling Component HY5 Mediates ABA Response in Seeds and Seedlings

Light affects many aspects of plant development including seed germination, phototropism, de-etiolation and flowering.¹ Light regulates these processes at least partly through its regulation of the metabolism and signal transduction of phytohormones. Experimental evidence has indicated that classic plant growth hormones, including auxin, brassinosteriods, ethylene and gibberellic acid, are involved in light control of plant development.² Nonetheless, the role of ABA in light signaling is much less clear, although light affects the metabolism of ABA³ and the expression of certain ABA-responsive genes. In a recent study,⁴ we reported an interaction between the light signaling pathway and the ABA signaling pathway during seed germination and early seedling development. Our work demonstrated that (1) the well-studied light signaling regulator, HY5, also mediates ABA response in late embryogenesis-abundant (LEA) gene expression, seed germination, seedling establishment and root growth; (2) HY5 mediates these ABA responses partly by regulating the transcription of the seed/seedling-specific ABA signaling regulator ABI5, (3) ABA specifically enhances HY5 binding to the ABI5 chromatin and; (4) ABI5 sensitizes light repression of hypocotyl elongation. These results raise interesting questions on the interaction of light signaling and ABA signaling and its potential significance in plant response to the environment.

HY5 Controls the Expression of ABI5 and ABI5-Targeted Genes

The Arabidopsis HY5 is a positive regulator of seedling photomorphogenesis downstream of diverse photoreceptors.^5–7 HY5 encodes a transcription factor of the basic leucine zipper (bZIP) domain family and controls the expression of many light-responsive genes. The ABI5 locus also encodes a bZIP transcription factor whose accumulation inhibits seed germination and early seedling establishment.^8,9 ABI5 is expressed during seed maturation, seed germination and young seedling stages. Its expression pattern partly overlaps with that of HY5. Both HY5 protein and ABI5 protein are also subjected to proteolysis during the early seedling stage. These observations suggest that HY5 regulation of ABI5 may occur within a restricted developmental window and that there must be other factors controlling the activation of ABI5 by HY5. Indeed, in tobacco BY-2 protoplasts, we did not find clear activation of ABI5 promoter-driven reporter gene by HY5. This indicates that seed or early seedling-specific factors such as ABI3, LEC1 and FUS3^9–13 are also needed for ABI5 activation. Among others, ABI3 was previously suggested to act upstream of ABI5.^11,14 Nonetheless, in our yeast two-hybrid assays, HY5 did not physically interact with ABI3 (Chen H and Xiong L, unpublished), suggesting that these two components may not be in physical contact with one another if they are within a common ABI5 transcriptional activation complex. Further identification of the components in this complex will be important for revealing the signaling network controlling seed development and seed germination.

Similar to ABI5, the downstream target genes of ABI5 may also be tightly regulated. Recent microarray experiments with the abi5 as well as hy5 mutants have identified genes potentially regulated by these transcription factors (reviewed in refs. ¹⁵ and ¹⁶). However, the low level of transcript abundance for some of these genes in seedlings and the sensitivity of their regulation to developmental stages and tissue types make it difficult to interpret some of the data obtained in these microarray assays. While the transcript levels for ABI5 and its target genes in dry seeds are abundant, the level of ABI5 in young seedlings is rather low and often difficult to detect. Since promoter-reporter genes may overcome the potential posttranscriptional regulation of transcripts while allowing the detection of the promoter activation at the tissue level, we used the ABI5-GUS reporter to examine the regulation of the ABI5 by HY5. Our studies indicated that the ABI5 promoter was regulated by HY5 under different light regimes as well as under stress conditions.⁴

Significance of the HY5-ABI5 Regulon for Seedlings Under Abiotic Stress

Light has been well studied for its role in promoting seed germination.^17–19 The discovery that HY5 positively regulates the expression of ABI5, whose accumulation inhibits seed germination, is counterintuitive. This raises the question as to the biological significance for this regulation. ABI5 accumulation was suggested as a ‘checkpoint’ for germinated embryos.²⁰ Since ABI5 expression can be enhanced by stress and ABA through HY5, together with increased stability of the ABI5 protein, the resulting accumulation of ABI5 would reinstall dormancy in germinated embryos and enable them to survive the stress.

When the ABI5 gene was overexpressed in the wild type, the transgenic plants had shorter hypocotyls. This indicates that ABI5 enhances light response. ABA was previously shown to inhibit the elongation of hypocotyls.²¹ Stress and its induced ABA accumulation may thus have a role in preventing the over elongation of young seedlings to reduce seedling lodging and increase their stress tolerance. In addition, HY5 mediates ABA inhibition of lateral root growth⁴ and thus may potentially promote primary root growth. This changed root architecture will facilitate seedling establishment and water uptake in case of drought stress,²² although it is not all clear whether ABI5 is involved in this process since overexpression of ABI5 in the hy5 background did not rescue the ABA insensitivity of lateral root growth.⁴ Recently, blue light was shown to increase root growth and drought resistance of seedlings.²³ It is thus likely that light regulation of root development may be a common phenomenon important for seedling adaptation to environmental stress.

A Role of HY5 in Seed Dormancy Establishment

In contrast to its ability to break seed dormancy and promote germination, light is much less known for its possible role in dormancy induction and maintenance. Since HY5 is highly expressed in floral tissues,²⁴ and the ABI5 promoter-GUS reporter activity is much lower in both flowers and developing seeds of hy5 than in the wild type,⁴ we reasoned that HY5 protein may play a role in seed development or dormancy. To examine this possibility, a premature germination test was carried out with hy5⁴ (Col-0 background), abi5²⁵ (Ws background) and the hy5 abi5 double mutant using the method as described.²⁶ To our surprise, we found that freshly harvested hy5 seeds had significantly reduced dormancy (Fig. 1A and B). About 50 percent of hy5 seeds harvested at 14 days after pollination (DAP) were able to germinate. In contrast, none of the wild-type seeds harvested at the same time germinated. The germination rate of hy5 seeds from 14 DAP is even higher than that of wild-type seeds from 16 DAP. Due to the significant difference in dormancy between Ws and Col-0 (Fig. 1 and ref. 22), we cannot directly compare the dormancy of hy5 and abi5 mutants. In a previous study using a slightly different method to measure dormancy, it was concluded that the abi5 mutation does not significantly affect dormancy.²⁵ In our study, we found that abi5 has reduced dormancy (about a two-day difference) compared to its wild-type Ws (Fig. 1). The fact that no additive effect between hy5 and abi5 was observed suggests that HY5 and ABI5 act in the same pathway in controlling seed dormancy, which is consistent with the notion that HY5 regulates the expression of ABI5 during seed development.⁴

Figure 1.

Reduced dormancy of hy5 seeds. (A) Premature germination rate of Col-0 (filled square), hy5 (open square), Ws (filled triangle), abi5 (in Ws background, open triangle), and hy5 abi5 double mutant (open circle). Siliques at different developmental stage (days after pollination) were harvested, cut open and plated on 0.4% water-agar plates. Plates were incubated under continuous light at 22–24°C for three days before counting seed germination under a microscope. Numbers are average of three independent experiments with around 40 seeds each. (B) Pictures of seeds of 16 DAP Col-0, hy5, Ws, abi5 and hy5 abi5 double mutant, three days after harvesting and plating on water-agar plates. (C) Seed storage proteins in Col-0, hy5, Ws and abi5. Seed storage protein was extracted as described.²⁷ Fifty seeds were extracted with 150 µl extraction buffer and 10 µl of the extracts were loaded each lane. Proteins were resolved by 12% SDS-PAGE and visualized by Coomassie blue staining.

The Arabidopsis LEC1, LEC2, FUS3 and ABI3 genes control different aspects of seed development, and loss of function in these genes leads to the precocious growth of root and apical meristem, alterations in storage product accumulation, and reduction in the establishment of desiccation tolerance and dormancy.^12,17,26 Although hy5 mutant seeds have reduced dormancy, the seed storage proteins are not altered (Fig. 1C), suggesting that HY5 may work downstream of these seed maturation factors or act in a parallel pathway in controlling dormancy. Indeed, despite no evidence for direct regulation, ABI5 mRNA accumulation is downregulated in abi3, fus3 and lec1 mutants.^9,12 Furthermore, hy5 seeds from 12 DAP did not germinate after seven-day's incubation (data not shown), while germination is evident as early as 10 DAP for lec1, fus3 and abi3.²⁶ In line with the requirement of HY5 for ABI5 transcript accumulation in fresh or dry seeds,⁴ there is no alteration in storage proteins in abi5 seeds either (Fig. 1C).

Light as a signal is well known for its indispensable role in breaking seed dormancy during germination¹⁷ and for seedling photomorphogenesis. The current study suggests that light may play a much broader and contrasting role during seed development, seed germination and seedling establishment. Further unraveling the molecular mechanisms underlying these processes will be important to understand how seed development and germination coordinate with plant adaptation to the environment.

Addendum to: Chen H, Zhang J, Neff MM, Hong SW, Zhang H, Deng XW, Xiong L. Integration of light and abscisic acid signaling during seed germination and early seedling development. Proc Natl Acad Sci U S A. 2008;105:4495–4500. doi: 10.1073/pnas.0710778105.