A role for GIGANTEA

Abstract

The initiation of flowering in Arabidopsis is retarded or abolished by environmental stresses. Focusing on salt stress, we provide a molecular explanation for this well-known fact. A protein complex consisting of GI, a clock component important for flowering and SOS2, a kinase activating the [Na⁺] antiporter SOS1, exists under no stress conditions. GI prevents SOS2 from activating SOS1. In the presence of NaCl, the SOS2/GI complex disintegrates and GI is degraded. SO2, together with the Ca²⁺-activated sensor of sodium ions, SOS3, activates SOS1. In gi mutants, SOS1 is constitutively activated and gi plants are more highly salt tolerant than wild type Arabidopsis. The model shows GI as a transitory regulator of SOS pathway activity whose presence or amount connects flowering to environmental conditions.

To survive, plant species had to adjust to changes in the environment over geological times, or to adapt to niche environments characterized by extreme, unfavorable conditions. Prime examples are crops, most of which originated in subtropical areas of the earth. They have been altered to thrive in a range of geographical latitudes, under different light intensities and light periods, and different temperature, humidity and types of soil. Crucial points determining wild plant reproductive success and farmer’s yield is the seasonal progression guiding growth, flowering and seed or fruit set and ripening. The plant model Arabidopsis, a long-day plant like many cereals (barley, wheat, oat, etc), spinach, potatoes, radish, onion, sugar beet and horticultural crops (carnation, rapeseed for canola oil) blooms as days get longer and flowers when exposed to light in excess of 12 h.¹ Plants measure day length to decide on flowering and the transition from the vegetative to the reproductive stage.² A plant internal biological clock, the circadian clock, is at the basis of this photoperiodism. This clock is also a known requisite for plants to cope with changing environments and to sustain a number of biological functions.³

Arabidopsis GIGANTEA (GI), encoded by a single gene,⁴ is confined to plant species, and has so far been found in all plants.^5-8 The GI protein functions in circadian clock maintenance and the elicitation of photoperiod-dependent flowering.^9-12 This is accomplished by day time accumulation of the GI protein followed by proteasome-dependent degradation during the night.¹³ GI transcript expression is itself under control by the circadian clock.^11,12 GI mainly controls flowering by regulating the time of day during which two other crucial components of the photoperiod-dependent flowering pathway are expressed. One is CO (Constans), a nuclear zinc finger protein,^14-16 the second being FT (Flowering Locus T), a floral integrator encoding a RAF-kinase-inhibitor-like protein.¹⁷gi mutants exhibit lower transcript expressions and changed rhythms of two circadian clock oscillators, CCA1 (circadian clock associated 1) and LHY (late elongated hypocotyl). In addition, gi mutants flower late compared with wild type in long days.⁹

GI seems to be involved in other important biological functions such as sucrose metabolism¹⁸ and cell wall deposition.¹⁹ Moreover, gi recessive mutants show defects in abiotic stress responses accompanied by pleiotropic phenotypes and tolerance to paraquat-induced oxidative stress, which is light dependent.²⁰ The longer hypocotyl of gi mutants under constant red light suggests GI involvement in PhyB-mediated red light signaling,⁴ and the gi mutant is sensitive to low temperature.^21,22 Also, gi plants contain high starch levels suggesting starch accumulation and the initiation of flowering to be regulated by GI.²³

Although precise biochemical functions for GI have not been defined it appears that GI is part of a network receiving inputs on environmental cues and transmitting them to modulate circadian timing for growth and development. We have recently substantiated this notion based on the recognition that the presence of GI and its absence in gi mutants are crucial for the initiation of flowering in relationship to the plants tolerance to high salinity (Fig. 1).

Figure 1.gi mutants exhibit increased salt tolerance. Three-wk-old plants were irrigated every other day with NaCl solution for two wk.

Salt stress delays flowering in Arabidopsis wild type as a result of reduced transcript levels for CO and FT.²⁴ Flowering of gi in the absence of salt stress is similar to the retardation of flowering in wild type after salt treatment. However, GI protein amount is drastically decreased upon salt treatment. In fact, salt promotes 26S proteasome dependent degradation of the GI protein. The resistance against salt of gi mutants is not due to increased high levels of salt-induced osmoprotectants, such as proline based on expression of, P5CS1 (delta 1-pyrroline-5-carboxylate synthetase) and the ABA independent transcription factor, DREB2A (dehydration-responsive element binding protein 2A). Rather, increased tolerance is based on enhanced activity of SOS1 (salt overly sensitive 1), a Na⁺/H⁺ antiporter.

The process is regulated by protein:protein interactions between three proteins: GI, SOS2, a kinase and the SOS1 antiporter protein localized to the plasma membrane. In the absence of NaCl, GI protein directly binds to the SOS2 kinase which is then prevented from phosphorylating and thus activating the antiporter activity of SOS1. The salt stress dependent degradation of GI frees SOS2 to interact with SOS3, a Ca²⁺ activated/binding protein. SOS1 phosphorylation by the SOS2/SOS3 complex then establishes export of sodium ions from cells that leads to enhanced salt tolerance

We propose a new model for the functioning of GI as a link between the clock and vegetative vs. reproductive growth depending on a changing environment (Fig. 2). The model establishes a precise function for GI that is supported by dynamic changes in protein-protein interactions. This model extends an existing salt stress response network including transcription mediated gene expression and Na⁺ export from cells by the three components of the well-studied SOS pathway

Figure 2. The interaction between GI and components of the SOS pathway. GI acts as a negative regulator of SOS1 activity in the absence of elevated [NaCl]. NaCl-induced degradation of GI leads to SOS2-initiated activation of SOS1 by phosphorylation, while SOS1 is constitutively active in the absence of GI (gi). Salt-dependent degradation of GI or its absence retard or abolish the progression toward flowering.

GI is a very large protein of about 130 kDa, has no known or characterized domains. It is not found in other kingdoms. Functions of GI can so far only be described biochemically by finding interactors. Molecular interactors for GI include F-box proteins, FKF1 (flavin-binding, kelch repeat, F-box 1) in photoperiod-dependent flowering²⁵ and ZTL (zeitlupe) in the circadian clock.²⁶ Another GI interactor is a GA (gibberellic acid) signaling negative regulator, SPY (spindly), O-linked N-acetylglucosamine transferase²⁷ whose mutants spy-1 and spy-3 are resistant to drought and high salinity.^27,28 The pleiotropic phenotypes of gi mutants suggest various associating partnerships in diverse biological regulations.

This leads to questions about the significance of GI’s involvement indifferent signaling networks. Sequestration of GI by SOS2 in the form of a negative regulatory circuit interrupts a futile biochemical reaction in the absence of salt stress, while at the same time preserving GI to participate in other functions. The changing environment—salt stress—leads to the degradation of GI, which eliminates its functioning in its other reactions. This may in fact enable other reactions to proceed for which GI is a negative regulator while interrupting circuits for which GI is required as an interacting partner.

Salt tolerance is dramatically increased in the presence of NaCl as GI is degraded. As a consequence of the absence of GI (gi) or following its salt-dependent degradation flowering is delayed or abolished. In contrast, salt tolerant gi plants show accelerated growth in high NaCl. This indicates the presence of functional salt exclusion or export mechanisms in a species that is considered highly salt-sensitive, possibly demonstrating higher order decision making processes superseding the biochemical machinery in plants. The mechanism exemplifies an ability of plants to employ synthesized proteins whose accumulation is regulated according to the time of day to predict and deal with changing environmental conditions

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.