Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

A seed-specific transcription factor, HSFA9, anticipates UV-B light responses by mimicking the activation of the UV-B receptor in tobacco.

Sunflower heat shock factor A9 (HSFA9, hereafter A9) is a transcription factor involved in seed desiccation tolerance and longevity. A9 also links the regulation of seed maturation with that of seedling photomorphogenesis through visible light receptors. Analyses in transgenic Nicotiana tabacum (tobacco) indicated that A9 also affects responses mediated by NtUVR8, the receptor of ultraviolet light B (UV-B). We compared the effects of A9 and UV-B illumination on the nuclear localization of GFP-NtUVR8 in Nicotiana benthamiana leaves. We also used co-immunoprecipitation and limited proteolysis for analyzing the interaction between A9 and NtUVR8. We found that A9, by binding to NtUVR8, induced structural changes that resulted in enhancing the nuclear localization of NtUVR8 by hindering its nuclear export. The localization of UVR8 is crucial for receptor activation and function in Arabidopsis, where UV-B-activated nuclear UVR8 binds the E3 ubiquitin ligase COP1, leading to enhanced UV-B responses and photoprotection. A9 similarly activated NtUVR8 by enhancing COP1 binding without UV-B light. Seedlings and dark-germinated seeds that overexpress A9 showed primed UV-B light stress protection. Our results unveil a UV-B-independent activation mechanism and a role for UVR8 in plant seeds that might contribute to early stress protection, facilitating seedling establishment.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

Heat Stress Factors Expressed during Seed Maturation Differentially Regulate Seed Longevity and Seedling Greening.

Heat Stress Factor A9 (A9), a seed-specific transcription factor contributing to seed longevity, also enhances phytochrome-dependent seedling greening. The RNA-seq analyses of imbibed-seed transcripts here reported indicated potential additional effects of A9 on cryptochrome-mediated blue-light responses. These analyses also suggested that in contrast to the A9 effects on longevity, which require coactivation by additional factors as A4a, A9 alone might suffice for the enhancement of photomorphogenesis at the seedling stage. We found that upon its seed-specific overexpression, A9 indeed enhanced the expected blue-light responses. Comparative loss-of-function analyses of longevity and greening, performed by similar expression of dominant-negative and inactive forms of A9, not only confirmed the additional greening effects of A9, but also were consistent with A9 not requiring A4a (or additional factors) for the greening effects. Our results strongly indicate that A9 would differentially regulate seed longevity and photomorphogenesis at the seedling stage, A9 alone sufficing for both the phytochrome- and cryptochrome-dependent greening enhancement effects.

SUMO-Dependent Synergism Involving Heat Shock Transcription Factors with Functions Linked to Seed Longevity and Desiccation Tolerance.

A transcriptional synergism between HaHSFA9 (A9) and HaHSFA4a (A4a) contributes to determining longevity and desiccation tolerance of sunflower (Helianthus annuus, L.) seeds. Potential lysine SUMOylation sites were identified in A9 and A4a and mutated to arginine. We show that A9 is SUMOylated in planta at K38. Although we did not directly detect SUMOylated A4a in planta, we provide indirect evidence from transient expression experiments indicating that A4a is SUMOylated at K172. Different combinations of wild type and SUMOylation site mutants of A9 and A4a were analyzed by transient expression in sunflower embryos and leaves. Although most of the precedents in literature link SUMOylation with repression, the A9 and A4a synergism was fully abolished when the mutant forms for both factors were combined. However, the combination of mutant forms of A9 and A4a did not affect the nuclear retention of A4a by A9; therefore, the analyzed mutations would affect the synergism after the mutual interaction and nuclear co-localization of A9 and A4a. Our results suggest a role for HSF SUMOylation during late, zygotic, embryogenesis. The SUMOylation of A9 (or A4a) would allow a crucial, synergic, transcriptional effect that occurs in maturing sunflower seeds.

Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed longevity.

The plant hormone auxin regulates growth and development by modulating the stability of auxin/indole acetic acid (Aux/IAA) proteins, which in turn repress auxin response factors (ARFs) transcriptional regulators. In transient assays performed in immature sunflower embryos, we observed that the Aux/IAA protein HaIAA27 represses transcriptional activation by HaHSFA9, a heat shock transcription factor (HSF). We also found that HaIAA27 is stabilized in immature sunflower embryos, where we could show bimolecular fluorescence complementation interaction between native forms of HaIAA27 and HaHSFA9. An auxin-resistant form of HaIAA27 was overexpressed in transgenic tobacco seeds, leading to effects consistent with down-regulation of the ortholog HSFA9 gene, effects not seen with the native HaIAA27 form. Repression of HSFs by HaIAA27 is thus likely alleviated by auxin in maturing seeds. We show that HSFs such as HaHSFA9 are targets of Aux/IAA protein repression. Because HaHSFA9 controls a genetic program involved in seed longevity and embryonic desiccation tolerance, our findings would suggest a mechanism by which these processes can be auxin regulated. Aux/IAA-mediated repression involves transcription factors distinct from ARFs. This finding widens interpretation of auxin responses.

Loss of function of the HSFA9 seed longevity program.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

The HaDREB2 transcription factor enhances basal thermotolerance and longevity of seeds through functional interaction with HaHSFA9.

BACKGROUND: Transcription factor HaDREB2 was identified in sunflower (Helianthus annuus L.) as a drought-responsive element-binding factor 2 (DREB2) with unique properties. HaDREB2 and the sunflower Heat Shock Factor A9 (HaHSFA9) co-activated the Hahsp17.6G1 promoter in sunflower embryos. Both factors could be involved in transcriptional co-activation of additional small heat stress protein (sHSP) promoters, and thus contribute to the HaHSFA9-mediated enhancement of longevity and basal thermotolerance of seeds. RESULTS: We found that overexpression of HaDREB2 in seeds did not enhance longevity. This was deduced from assays of basal thermotolerance and controlled seed-deterioration, which were performed with transgenic tobacco. Furthermore, the constitutive overexpression of HaDREB2 did not increase thermotolerance in seedlings or result in the accumulation of HSPs at normal growth temperatures. In contrast, when HaDREB2 and HaHSFA9 were conjointly overexpressed in seeds, we observed positive effects on seed longevity, beyond those observed with overexpression of HaHSFA9 alone. Such additional effects are accompanied by a subtle enhancement of the accumulation of subsets of sHSPs belonging to the CI and CII cytosolic classes. CONCLUSION: Our results reveal the functional interdependency of HaDREB2 and HaHSFA9 in seeds. HaDREB2 differs from other previously characterized DREB2 factors in plants in terms of its unique functional interaction with the seed-specific HaHSFA9 factor. No functional interaction between HaDREB2 and HaHSFA9 was observed when both factors were conjointly overexpressed in vegetative tissues. We therefore suggest that additional, seed-specific factors, or protein modifications, could be required for the functional interaction between HaDREB2 and HaHSFA9.

The dark side of green fluorescent protein.

Here, severe interference of chlorophyll with green fluorescent protein (GFP) fluorescence is described for medicago (Medicago truncatula), rice (Oryza sativa) and arabidopsis (Arabidopsis thaliana). This interference disrupts the proportional relationship between GFP content and fluorescence that is intrinsic to its use as a quantitative reporter. The involvement of chlorophyll in the loss of GFP fluorescence with leaf age was shown in vivo, by the removal of chlorophyll through etiolation or by ethanol extraction, and in vitro, by titration of a GFP solution with chlorophyll solutions of various concentrations. A substantial decrease in fluorescence in early development of medicago and rice leaves correlated with chlorophyll accumulation. In all three species tested, removal of chlorophyll yielded up to a 10-fold increase in fluorescence. Loss of GFP fluorescence in vitro was 4-fold greater for chlorophyll b than for chlorophyll a. Differences exist between plant species for the discrepancy between apparent GFP fluorescence and its actual level in green tissues. Substantial errors in estimating promoter activity from GFP fluorescence can occur if pigment interference is not considered.

Interaction of PvALF and VP1 B3 domains with the beta -phaseolin promoter.

The phas promoter is potently transcribed during embryogenesis but in vegetative tissues it is completely silenced by a rotationally positioned nucleosome. Ectopic expression in leaves of PvALF, a seed-specific transcription factor belonging to the plant-exclusive B3 domain-containing VP1/ABI3 family, leads to chromatin remodeling of the phas promoter, permitting transcriptional activation by the growth regulator abscisic acid (ABA). Specific interaction with RY elements present in 40-42 bp oligonucleotide probes has been shown in vitro for Arabidopsis ABI3 and the isolated B3 domain of maize VP1. Here, both in vivo and in vitro approaches were used to show physical interaction of the B3 domain of VP1 or PvALF to RY elements in the native phas promoter. In electrophoretic mobility shift assays, small changes in B3 domain concentration differentiated between RY element-specific and sequence non-specific DNA binding. Increased affinity of the PvALF B3 domain to RY elements was observed in the presence of histones and other basic proteins, possibly reflecting the ability of this B3 factor to interact with the phas promoter in its nucleosomal configuration.

A seed-specific heat-shock transcription factor involved in developmental regulation during embryogenesis in sunflower.

We report the cloning and functional characterization of the first heat-shock transcription factor that is specifically expressed during embryogenesis in the absence of environmental stress. In sunflower embryos this factor, HaHSFA9, trans-activated promoters with poor consensus heat-shock cis-elements, including that of the seed-specific Hahsp17.6G1 gene. Mutations that improved the heat-shock cis-element consensus at the Hahsp17.7G4 promoter impaired transient activation by HaHSFA9 in sunflower embryos. The same mutations did not affect heat-shock-induced gene expression of this promoter in transgenic tobacco plants but reduced the developmental activation by endogenous heat-shock transcription factors (HSFs) in seeds. Sunflower, and perhaps other plants such as tobacco, differs from the vertebrate animal systems in having at least one specialized HSF with expression and (or) activation patterns strictly restricted to embryos. Our results strongly indicate that HaHSFA9 is a transcription factor critically involved in the developmental activation of Hahsp17.6G1 and in that of similar target genes as Hahsp17.7G4.

Selective activation of the developmentally regulated Ha hsp17.6 G1 promoter by heat stress transcription factors.

Using two well-characterized heat stress transcription factors (Hsfs) from tomato (Lycopersicon peruvianum; LpHsfA1 and LpHsfA2), we analyzed the transcriptional activation of the Ha hsp17.6 G1 promoter in sunflower (Helianthus annuus) embryos. In this system, we observed transient promoter activation only with LpHsfA2. In contrast, both factors were able to activate mutant versions of the promoter with improved consensus Hsf-binding sites. Exclusive activation by LpHsfA2 was also observed in yeast (Saccharomyces cerevisiae) without other Hsfs and with a minimal Cyc1 promoter fused to the Ha hsp17.6 G1 heat stress cis-element. Furthermore, the same promoter mutations reproduced the loss of activation selectivity, as observed in sunflower embryos. The results of in vitro binding experiments rule out differential DNA binding of the two factors as the explanation for the observed differential activation capacity. We conclude that the specific sequence of this heat stress cis-element is crucial for Hsf promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding. In sunflower embryos, we also observed synergistic transcriptional activation by co-expression of LpHsfA1 and LpHsfA2. Mutational analyses of the Ha hsp17.6 G1 promoter, combined with in vitro binding assays, suggest that mixed oligomers of the two factors may be involved in promoter activation. We discuss the relevance of our observations for mechanisms of developmental regulation of plant heat stress protein genes.