Xuanming Liu

Blue Light-Dependent Interaction of CRY2 with SPA1 Regulates COP1 activity and Floral Initiation in Arabidopsis

Cryptochromes are blue light receptors that mediate light regulation of gene expression in all major evolution lineages, but the molecular mechanism underlying cryptochrome signal transduction remains not fully understood. It has been reported that cryptochromes suppress activity of the multifunctional E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) to regulate gene expression in response to blue light. But how plant cryptochromes mediate light suppression of COP1 activity remains unclear. We report here that Arabidopsis CRY2 (cryptochrome 2) undergoes blue light-dependent interaction with the COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A 1 (SPA1). We demonstrate that SPA1 acts genetically downstream from CRY2 to mediate blue light suppression of the COP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO). We further show that blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 interaction. These results reveal for the first time a wavelength-specific mechanism by which a cryptochrome photoreceptor mediates light regulation of protein degradation to modulate developmental timing in Arabidopsis.

Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light

Plant photoreceptors mediate light suppression of the E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) to affect gene expression and photomorphogenesis. However, how photoreceptors mediate light regulation of COP1 activity remains unknown. We report here that Arabidopsis blue-light receptor cryptochrome 1 (CRY1) undergoes blue-light-dependent interaction with the COP1-interacting protein SPA1 (SUPPRESSOR OF PHYTOCHROME A). We further show that the CRY1-SPA1 interaction suppresses the SPA1-COP1 interaction and COP1-dependent degradation of the transcription factor HY5. These results are consistent with a hypothesis that photoexcited CRY1 interacts with SPA1 to modulate COP1 activity and plant development.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.

Arabidopsis Cryptochrome 2 Completes Its Posttranslational Life Cycle in the Nucleus

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light–dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light–dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light–dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light–dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light–dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.

Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2

Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a “closed” conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an “open” conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

Piercing and vacuum infiltration of the mature embryo: a simplified method for Agrobacterium-mediated transformation of indica rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T0) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T0 plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T1 transformants.

Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad-dependent photoreduction

Cryptochromes are blue-light receptors mediating various light responses in plants and animals. The photochemical mechanism of cryptochromes is not well understood. It has been proposed that photoactivation of cryptochromes involves the blue-light–dependent photoreduction of flavin adenine dinucleotide via the electron transport chain composed of three evolutionarily conserved tryptophan residues known as the “trp triad.” We investigated this hypothesis by analyzing the photochemical and physiological activities of Arabidopsis cryptochrome 2 (CRY2) mutations altered in each of the three trp-triad residues. We found that all trp-triad mutations of CRY2 tested lost photoreduction activity in vitro but retained the physiological and biochemical activities in vivo. Some of the trp-triad mutations of CRY2 remained responsive to blue light; others, such as CRY2W374A, became constitutively active. In contrast to wild-type CRY2, which undergoes blue-light–dependent interaction with the CRY2-signaling proteins SUPPRESSOR OF PHYA 1 (SPA1) and cryptochrome-interaction basic helix–loop–helix 1 (CIB1), the constitutively active CRY2W374A interacts with SPA1 and CIB1 constitutively. These results support the hypothesis that cryptochromes mediate blue-light responses via a photochemistry distinct from trp-triad–dependent photoreduction and that the trp-triad residues are evolutionarily conserved in the photolyase/cryptochrome superfamily for reasons of structural integrity rather than for photochemistry per se.

A Novel Chloroplast-Localized Pentatricopeptide Repeat Protein Involved in Splicing Affects Chloroplast Development and Abiotic Stress Response in Rice

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and modulate organellar gene expression by participating in various aspects of organellar RNA metabolism. In rice, the family contains 477 members, and the majority of their functions remain unclear. In this study, we isolated and characterized a rice mutant, white stripe leaf (wsl), which displays chlorotic striations early in development. Map-based cloning revealed that WSL encodes a newly identified rice PPR protein which targets the chloroplasts. In wsl mutants, PEP-dependent plastid gene expression was significantly down-regulated, and plastid rRNAs and translation products accumulate to very low levels. Consistently with the observations, wsl shows a strong defect in the splicing of chloroplast transcript rpl2, resulting in aberrant transcript accumulation and its product reduction in the mutant. The wsl shows enhanced sensitivity to ABA, salinity, and sugar, and it accumulates more H2O2 than wild-type. These results suggest the reduced translation efficiency may affect the response of the mutant to abiotic stress.

A Lectin Receptor Kinase Positively Regulates ABA Response During Seed Germination and Is Involved in Salt and Osmotic Stress Response

Lectin receptor-like kinases (LecRK) are widespread in higher plants; however, little is known about their physiological roles. In this study, At1g70130 (designated LecRK-b2), an Arabidopsis LecRK gene, has been investigated. LecRK-b2 was predominantly expressed during seed germination, and its expression was ceased following germination. The expression of LecRK-b2 was induced by abscisic acid (ABA), salt, and osmotic stress. LecRK-b2 loss-of-function mutation slightly reduced the ABA sensitivity during seed germination, and this reduced sensitivity was demonstrated not due to lower ABA accumulation level in the seeds. Dual-luciferase transient expression assay confirmed that the transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) could activate the luciferase under driving of LecRK-b2 promoter. LecRK-b2 transcription level was found to be down-regulated in abi3 during seed germination. Furthermore, LecRK-b2 loss-of-function mutation reduced the salt and osmotic sensitivity during early development stage of Arabidopsis. Taken together, these results suggest that LecRK-b2 functions as a positive regulator of the ABA response during the seed germination and is involved in salt and osmotic stress response in the early development stage.

FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

Significance Receptor-like kinase FERONIA (FER) not only serves as a receptor for growth-regulating rapid alkalinization factor (RALF) peptide but also acts as an important node in a variety of other signaling pathways, including plant responses to hormones, pathogens, and abiotic stresses. However, the mechanism underlying FER actions in these signaling cross-talks remain largely unknown. Our previous work identified a molecular relay that allows FER to inhibit abscisic acid (ABA) response through activation of a small G protein to enhance the activity of the clade A protein phosphatase type 2C (PP2C) ABA Insensitive 2 (ABI2), a repressor of ABA response. In this study, we found that ABI2 can directly interact and dephosphorylate FER, providing a feedback mechanism for RALF activation of FER. Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.