Maize cryptochromes 1a1 and 1a2 promote seedling photomorphogenesis and shade resistance in Zea mays and Arabidopsis

Maize growth and development are regulated by light quality,intensity and photoperiod.Cryptochromes are blue/ultraviolet-A light receptors involved in stem elongation,shade avoidance,and photoperiodic flowering.To investigate the function of cryptochrome 1(CRY1) in maize,where it is encoded by Zm CRY1,we obtained two Zm CRY1a genes(Zm CRY1a1 and Zm CRY1a2),both of which share the highest similarity with other gramineous plants,in particular rice CRY1a by phylogenetic analysis.In Arabidopsis,overexpression of Zm CRY1a genes promoted seedling de-etiolation under blue and white light,resulting in dwarfing of mature plants.In seedlings of the maize inbred line Zong 31(Zm CRY1aOE),overexpression of Zm CRY1a genes caused a reduction in the mesocotyl and first leaf sheath lengths due to down-regulation of genes influencing cell elongation.In mature transgenic maize plants,plant height,ear height,and internode length decreased in response to overexpression of Zm CRY1a genes.Expression of Zm CRY1a were insensitive to low blue light(LBL)-induced shade avoidance syndrome(SAS) in Arabidopsis and maize.This prompted us to investigate the regulatory role of the gibberellin and auxin metabolic pathways in the response of Zm CRY1a genes to LBL treatment.We confirmed a link between Zm CRY1a expression and hormonal influence on the growth and development of maize under LBL-induced SAS.These results reveal that Zm CRY1a has a relatively conservative function in regulating maize photomorphogenesis and may guide new strategies for breeding high density-tolerant maize cultivars.

The dual-action mechanism of Arabidopsis cryptochromes

Photoreceptor cryptochromes (CRYs) mediate blue-light regulation of plant growth and development. It has been reported that Arabidopsis CRY1and CRY2 function by physically interacting with at least 84 proteins, including transcription factors or co-factors, chromatin regulators, splicing factors, messenger RNA methyltransferases, DNA repair proteins, E3 ubiquitin ligases, protein kinases and so on. Of these 84 proteins, 47 have been reported to exhibit altered binding affinity to CRYs in response to blue light, and 41 have been shown to exhibit condensation to CRY photobodies. The blue light-regulated composition or condensation of CRY complexes results in changes of gene expression and developmental programs. In this mini-review, we analyzed recent studies of the photoregulatory mechanisms of Arabidopsis CRY complexes and proposed the dual mechanisms of action, including the “Lock-and-Key” and the “Liquid-Liquid Phase Separation (LLPS)” mechanisms. The dual CRY action mechanisms explain, at least partially, the structural diversity of CRY-interacting proteins and the functional diversity of the CRY photoreceptors.

The involvement of the N-terminal PHR domain of Arabidopsis cryptochromes in mediating light signaling

Light is a key environmental cue that fundamentally regulates all aspects of plant growth and development,which is mediated by the multiple photoreceptors including the blue light photoreceptors cryptochromes(CRYs).In Arabidopsis,there are two well-characterized homologous CRYs,CRY1 and CRY2.Whereas CRYs are flavoproteins,they lack photolyase activity and are characterized by an Nterminal photolyase-homologous region(PHR)domain and a C-terminal extension domain.It has been established that the C-terminal extension domain of CRYs is involved in mediating light signaling through direct interactions with the master negative regulator of photomorphogenesis,COP1.Recent studies have revealed that the N-terminal PHR domain of CRYs is also involved in mediating light signaling.In this review,we mainly summarize and discuss the recent advances in CRYs signaling mediated by the N-terminal PHR domain,which involves the N-terminal PHR domain-mediated dimerization/oligomerization of CRYs and physical interactions with the pivotal transcription regulators in light and phytohormone signaling.

Photooligomerization Determines Photosensitivity and Photoreactivity of Plant Cryptochromes

Plant and non-plant species possess cryptochrome(CRY)photoreceptors to mediate blue light regulation of development or the circadian clock.The blue light-dependent homooligomerization of Arabidopsis CRY2 is a known early photoreaction necessary for its functions,but the photobiochemistry and function of light-dependent homooligomerization and heterooligomerization of cryptochromes,collectively referred to as CRY photooligomerization,have not been well established.Here,we show that photooligomerization is an evolutionarily conserved photoreaction characteristic of CRY photoreceptors in plants and some non-plant species.Our analyses of the kinetics of the forward and reverse reactions of photooligomerization of Arabidopsis CRY1 and CRY2 provide a previously unrecognized mechanism underlying the different photosensitivity and photoreactivity of these two closely related photoreceptors.We found that photooligomerization is necessary but not sufficient for the functions of CRY2,implying that CRY photooligomerization is presumably accompanied by additional function-empowering conformational changes.We further demonstrated that the CRY2-CRY1 heterooligomerization plays roles in regulating functions of Arabidopsis CRYs in vivo.Taken together,these results suggest that photooligomerization is an evolutionarily conserved mechanism determining the photosensitivity and photoreactivity of plant CRYs.

An early-morning gene network controlled by phytochromes and cryptochromes regulates photomorphogenesis pathways in Arabidopsis

Light perception at dawn plays a key role in coordinating multiple molecular processes and in entraining the plant circadian clock.The Arabidopsis mutant lacking the main photoreceptors,however,still shows clock entrainment,indicating that the integration of light into the morning transcriptome is not well understood.In this study,we performed a high-resolution RNA-sequencing time-series experiment,sampling every 2 min beginning at dawn.In parallel experiments,we perturbed temperature,the circadian clock,photoreceptor signaling,and chloroplast-derived light signaling.We used these data to infer a gene network that describes the gene expression dynamics after light stimulus in the morning,and then validated key edges.By sampling time points at high density,we are able to identify three light-and temperature-sensitive bursts of transcription factor activity,one of which lasts for only about 8 min.Phytochrome and cryptochrome mutants cause a delay in the transcriptional bursts at dawn,and completely remove a burst of expression in key photomorphogenesis genes(HY5 and BBX family).Our complete network is available online(http://www-users.york.ac.uk/∼de656/dawnBurst/dawnBurst.html).Taken together,our results show that phytochrome and cryptochrome signaling is required for fine-tuning the dawn transcriptional response to light,but separate pathways can robustly activate much of the program in their absence.

Identification of medaka magnetoreceptor and cryptochromes

Magnetoreception is a hallmark ability of animals for orientation and migration via sensing and utilizing geomagnetic fields.Magnetoreceptor(MagR) and cryptochromes(Cry) have recently been identified as the basis for magnetoreception in Drosophila.However,it has remained unknown whether MagR and Cry have conserved roles in diverse animals.Here we report the identification and expression of magr and cry genes in the fish medaka(Oryzias latipes).Cloning and sequencing identified a single magr gene,four cry genes and one cry-like gene in medaka.By sequence alignment,chromosomal synteny and gene structure analysis,medaka cry2 and magr were found to be the orthologs of human Cry2 and Magr,with cry1 aa and crylab being coorthologs of human Cry1.Therefore,magr and cry2 have remained as single copy genes,whereas cry1 has undergone two rounds of gene duplication in medaka.Interestingly,magr and cry genes were detected in various stages throughout embryogenesis and displayed ubiquitous expression in adult organs rather than specific or preferential expression in neural organs such as brain and eye.Importantly,magr knockdown by morpholino did not produce visible abnormality in developing embryos,pointing to the possibility of producing viable magr knockouts in medaka as a vertebrate model for magnet biology.

Supplemental blue and red light promote lycopene synthesis in tomato fruits

Lycopene, one of the strongest natural antioxidants known and the main carotene in ripe tomato, is very important for human health. Light is well known to be one of the most important environmental stimuli influencing lycopene biosynthesis; specifically, red light induces higher lycopene content in tomato. However, whether blue light promotes lycopene synthesis remains elusive and exactly how light stimulation promotes lycopene synthesis remains unclear. We applied supplemental blue and red lighting on tomato plants at anthesis to monitor the effect of supplemental blue and red lighting on lycopene synthesis. Our results showed that supplemental blue/red lighting induced higher lycopene content in tomato fruits; furthermore, we found that the expression of key genes in the lycopene synthesis pathway was induced by supplemented blue/red light. The expression of light signaling components, such as red-light receptor phytochromes(PHYs), blue-light receptor cryptochromes(CRYs) and light interaction factors, phytochrome-interacting factors(PIFs) and ELONGATED HYPOCOTYL 5(HY5) were up-or down-regulated by blue/red lighting. Thus, blue and red light increased lycopene content in tomatoes by inducing light receptors that modulate HY5 and PIFs activation to mediate phytoene synthase 1(PSY1) gene expression. These results provide a sound theoretical basis for further elucidation of the light regulating mechanism of lycopene synthesis in tomatoes, and for instituting a new generation of technological innovations for the enhancement of lycopene accumulation in crop production.

Integration of light signaling with photoperiodic flowering and circadian rhythm

Plants become photosynthetic through de-etiolation, a developmental process regulated by red/far-red light-absorbing phytochromes and blue/ultraviolet A light-absorbing cryptochromes. Genetic screens have identified in the last decade many far-red light signaling mutants and several red and blue light signaling mutants, suggesting the existence of distinct red, far-red, or blue light signaling pathways downstream of phytochromes and cryptochromes. However, genetic screens have also identified mutants with defective de-etiolation responses under multiple wavelengths. Thus, the opti- mal de-etiolation responses of a plant depend on coordination among the different light signaling pathways. This review intends to discuss several recently identified signaling components that have a potential role to integrate red, far-red, and blue light signalings. This review also highlights the recent discoveries on proteolytic degradation in the desensitization of light signal transmission, and the tight connection of light signaling with photoperiodic flowering and circadian rhythm. Studies on the controlling mechanisms of de-etiolation, photoperiodic flowering, and circadian rhythm have been the fascinating topics in Arabidopsis research. The knowledge obtained from Arabidopsis can be readily applied to food crops and ornamental species, and can be contributed to our general understanding of signal perception and transduction in all organisms.

Jiao-tai-wan Up-regulates Hypothalamic and Peripheral Circadian Clock Gene Cryptochrome and Activates PI3K/AKT Signaling in Partially Sleep-deprived Rats

This study aims to explore the effect and mechanism of Jiao-tai-wan （JTW） on systemic and tissue-specific inflammation and insulin resistance in obesity-resistant （OR） rats with chronic partial sleep deprivation （PSD）. OR rats with PSD were orally given JTW and Estazolam for 4 weeks. The amount of food intake and metabolic parameters such as body weight increase rate, fasting plasma glucose （FPG）, fasting insulin （FINS）, homeostasis model assessment-insulin resistance （HOMA-IR） and plasma inflammatory markers were measured. The expression levels of circadian proteins cryptochrome 1 （Cryl） and cryptochrome 2 （Cry2） in hypothalamus, adipose and liver tissues were also determined. Meanwhile, the mRNA expression of inflammatory markers, activity of nuclear factor kappa B （NF-κB） p65 protein, as well as the expression levels of insulin signaling pathway proteins in hypothalamus, adipose and liver tissues were measured. Additionally, cyclic adenosine 3＇, 5＇-monophosphate （cAMP） and activity of vasodilator-stimulated phosphoprotein （VASP） in hypothalamus tissue were measured. JTW significantly decreased the body weight increase rate and food intake, ameliorated systemic inflammation and insulin resistance. JTW effectively ameliorated inflammation and increased PI3K/AKT signaling activation in hypothalamus, adipose and liver. Interestingly, all these changes were associated with the up-regulation of circadian gene Cryl and Cry2 protein expression. We also found that in hypothalamus tissue of PSD rats, down-regulation of Cryl and Cry2 activated cAMP/PKA signaling and then led to inflammation, while JTW inhibited this signaling. These results suggested that JTW has the beneficial effect on ameliorating inflammation and insulin resistance in partially sleep-deprived rats by up-regulating Cry expression.

Optogenetic activation of intracellular signaling based on light-inducible protein-protein homo-interactions

Dynamic protein-protein interactions are essential for proper cell functioning.Homointeraction events—physical interactions between the same type of proteins—represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways.Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms.The emerging optogenetic technology,based on genetically encoded light-sensitive proteins,provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision.Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.

Effects of Suppressing OsCRY1a Gene Expression on Rice Agronomic Traits

Using primers designed according to the published sequence of rice OsCRY1a gene, we obtained part of the gene fragment by PCR and constructed an RNA interference expression vector with it. To down-regulate the expression level of the gene or lead to the loss-of-function of the gene, the vector was then introduced into rice via Agrobacterium-mediated transformation. Based on the performance of the transgenic plants, the functions of the gene were analyzed and deduced. The results indicated that suppressing the expression of the gene retarded flowering for 16 d in rice with the plant height and grain length significantly increasing whereas other important agronomic traits observed remained unchanged apparently.

<i>Pinus taeda</i>cDNA Microarray as a Tool for Candidate Gene Identification for Local Red/Far-Red Light Adaptive Response in <i>Pinus sylvestris</i>

Light quality response is a vital environmental cue regulating plant development. Conifers, like angiosperms, respond to the changes in light quality including the level of red (R) and far-red (FR) light, which follows a latitudinal cline. R and FR wavelengths form a significant component of the entire plant life cycle, including the initial developmental stages such as seed germination, cotyledon expansion and hypocotyl elongation. With an aim to identify differentially expressed candidate genes, which would provide a clue regarding genes involved in the local adaptive response in Scots pine (Pinus sylvestris) with reference to red/far-red light;we performed a global expression analysis of Scots pine hypocotyls grown under two light treatments, continuous R (cR) and continuous FR (cFR) light;using Pinus taeda cDNA microarrays on bulked hypocotyl tissues from different individuals, which represented different genotypes. This experiment was performed with the seeds collected from northern part of Sweden (Ylinen, 68?N). Interestingly, gene expression pattern with reference to cryptochrome1, a blue light photoreceptor, was relatively high under cFR as compared to cR light treatment. Additionally, the microarray data analysis also revealed expression of 405 genes which was enhanced under cR light treatment;while the expression of 239 genes was enhanced under the cFR light treatment. Differentially expressed genes were re-annotated using Blast2GO tool. These results indicated that cR light acts as promoting factor whereas cFR antagonises the effect in most of the processes like C/N metabolism, photosynthesis and cell wall metabolism which is in accordance with former findings in Arabidopsis. We propose cryptochrome1 as a strong candidate gene to study the adaptive cline response under R and FR light in Scots pine as it shows a differential expression under the two light conditions.

蓝光受体CRY2化身“暗黑舞者”

隐花色素(CRY)是调节植物光反应的蓝光受体。CRY在黑暗中以无活性的单体形式存在,吸收光子后构象变化并发生寡聚化,同时改变了其与互作蛋白间的亲和力,进而调控光反应蛋白的转录或稳定性以调节植物的生长发育。最近的一项研究发现了CRY2的一个精巧作用机制,CRY不仅可被蓝光“激活”,还可被黑暗信号“激活”,从而构建起光信号和暗信号依赖的光受体信号转导更节能的模式。他们发现CRY2即便在黑暗中也能抑制根尖分生组织中的细胞分裂,调控根的伸长,并控制大量基因的表达。FL1和FL3与细胞分裂基因的染色质结合以促进其转录。需要说明的是,只有黑暗中的CRY2单体可与FL1/FL3相互作用,从而抑制后者促进根伸长的功能,蓝光则解除该抑制作用。这一发现重塑了人们对光受体的认识,为理解植物感知和响应不同信号以调节生长和适应性提供了全新的视角,对深入理解基因的功能极具启发意义。

Light-Regulated Stomatal Aperture in Arabidopsis

The stomatal pores of plant leaves, situated in the epidermis and surrounded by a pair of guard cells, allow CO2 uptake for photosynthesis and water loss through transpiration. Blue light is one of the dominant environmental signals that control stomatal movements in leaves of plants in a natural environment. This blue light response is mediated by blue/UV A light-absorbing phototropins （phots） and cryptochromes （crys）. Red/far-red light-absorbing phytochromes （phys） also play a role in the control of stomatal aperture. The signaling components that link the perception of light signals to the stomatal opening response are largely unknown. This review discusses a few newly discovered nuclear genes, their function with respect to the phot-, cry-, and phy-mediated signal transduction cascades, and possible involve- ment of circadian clock.

Long-distance blue light signalling regulates phosphate deficiency-induced primary root growth inhibition

Although roots are mainly embedded in the soil, recent studies revealed that light regulates mineral nutrient uptake by roots. However, it remains unclear whether the change in root system architecture in response to different rhizosphere nutrient statuses involves light signaling. Here, we report that blue light regulates primary root growth inhibition under phosphate-deficient conditions through the cryptochromes and their downstream signaling factors. We showed that the inhibition of root elongation by low phosphate requires blue light signal perception at the shoot and transduction to the root. In this process, SPA1 and COP1 play a negative role while HY5 plays a positive role. Further experiments revealed that HY5 is able to migrate from the shoot to root and that the shoot-derived HY5 autoactivates root HY5 and regulates primary root growth by directly activating the expression of LPR1, a suppressor of root growth under phosphate starvation. Taken together, our study reveals a regulatory mechanism by which blue light signaling regulates phosphate deficiency-induced primary root growth inhibition, providing new insights into the crosstalk between light and nutrient signaling.

Molecular Cloning and Expression Analysis of Cryptochrome Gene Ps CRY2in Tree Peony

Cryptochromes are blue/ultraviolet-A(UV-A) light receptors involved in regulating various aspects of plant growth and development.Investigations of the structure and functions of cryptochromes in plants have largely focused on herbaceous plants. However, few data on the function of CRY2 are available in woody plants. In this study, a cryptochrome 2(CRY2) gene was isolated from Paeonia suffruticosa by Reverse Transcription Polymerase Chain Reaction(RT-PCR). Sequence alignment and motif analysis showed that the deduced amino acids contained a PHR domain near the amino terminus and a CCT domain near the carboxy terminus. Ps CRY2 showed high identity with At CRY2 of Arabidopsis.Phylogenetic analysis indicated that it was closely related to Citrus sinensis. Gene expression analysis revealed that the highest expression levels of Ps CRY2 occurred in the bud and seed embryo of P. suffruticosa, followed by the roots, stems, and leaves. Ps CRY2 was upregulated during the entire process of bud differentiation, whereas this was downregulated during the early stage of bud development and upregulated in the middle and late stages. The highest level of Ps CRY2 expression was observed in the big bell-like flower buds. These results suggested that Ps CRY2 plays an important role in both bud differentiation and bud development. The expression patterns of Ps CRY2 in the buds of ‘Luoyanghong' and ‘Qiufa1' were similar, whereas that in the buds of ‘Qiufa 1' was significantly higher than in the buds of ‘Luoyanghong'. The buds of plants subjected to different photoperiod treatments exhibited variations in Ps CRY2 expression patterns. The expression of Ps CRY2 decreased during bud sprouting and in the small bell-like flower buds that were subjected to short-day photoperiod compared to that observed under long-day photoperiod.

Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis

Light is a key environmental cue that inhibits hypocotyl cell elongation through the blue and red/far-red light photoreceptors cryptochrome- and phytochrome-mediated pathways in Arabidopsis. In contrast, as a pivotal endogenous phytohormone auxin promotes hypocotyl elongation through the auxin receptors TIR1/AFBs-mediated degradation of AUX/IAA proteins （AUX/IAAs）. However, the molecular mechanisms underlying the antagonistic interaction of light and auxin signaling remain unclear. Here, we report that light inhibits auxin signaling through stabilization of AUX/IAAs byblue and red light-dependent interactions of cryptochrome 1 （CRY1） and phytochrome B with AUX/IAAs, respectively. Blue light-triggered interactions of CRY1 with AUX/IAAs inhibit the associations of TIR1 with AUX/IAAs, leading to the repression of auxin- induced degradation of these proteins. Our results indicate that photoreceptors share AUX/IAAs with auxin receptors as the same direct downstream signaling components. We propose that antagonistic regulation of AUX/IAA protein stability by photoreceptors and auxin receptors allows plants to balance light and auxin signals to optimize their growth.

Therapeutic regulation of autophagy in hepatic metabolism

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ thatgoverns body energy metabolism. Autophagy’s role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.

CRYPTOCHROME 1 Is Implicated in Promoting R Protein-Mediated Plant Resistance to Pseudomonas syringae in Arabiclopsis

Plants have evolved complex mechanisms to defend themselves against pathogens. It has been shown that several defense responses are influenced by light, and the red/far-red light photoreceptor phytochromes （PHY） modulate plant defense responses in Arabidopsis. Blue light receptor cryptochromes （CRY） work together with PHY to regulate many light-controlled responses, including photomorphogenesis, floral induction, and entrainment of the circadian clock. We report here that the Arabidopsis blue light photoreceptor CRY1 positively regulates inducible resistance to Pseudomonas syringae under continuous light conditions. By challenging plants with R syringae pv. tomato （Pst.） DC3000 carrying avrRpt2, we demonstrate that effector-triggered local resistance is down-regulated in the cry1 mutant, leading to more pathogen multiplication. In plants overexpressing CRY1 （CRYl-ovx）, however, local resistance is significantly up-regulated. We also show that systemic acquired resistance （SAR） is positively regulated by CRY1, and that salicylic acid （SA）-induced pathogenesis-related gene PR-1 expression is reduced in the cry1 mutant, but enhanced in CRYl-ovx plants. However, our results in- dicate that CRY1 only modestly influences SA accumulation and has no effect on hypersensitive cell death. These results suggest that CRY1 may positively regulate R protein-mediated resistance to P. syringae with increased PR gene expression.

GmCRY1s modulate gibberellin metabolism to regulate soybean shade avoidance in response to reduced blue light

Soybean is an important legume crop that displays the classic shade avoidance syndrome(SAS),including exaggerated stem elongation,which leads to lodging and yield reduction under density farming conditions.Here,we compared the effects of two shade signals,low red light to far-red light ratio(R:FR)and low blue light(LBL),on soybean status and revealed that LBL predominantly induces excessive stem elongation.We used CRISPR-Cas9-engineered Gmcry mutants to investigate the functions of seven cryptochromes(GmCRYs)in soybean and found that the four GmCRY1s overlap in mediating LBL-induced SAS.Lightactivated GmCRY1s increase the abundance of the bZlP transcription factors STF1 and STF2,which directly upregulate the expression of genes encoding GA2 oxidases to deactivate GA1 and repress stem elongation.Notably,GmCRY1b overexpression lines displayed multiple agronomic advantages over the wild-type control under both dense planting and intercropping conditions.Our study demonstrates the integration of GmCRY1-mediated signals with the GA metabolic pathway in the regulation of LBL-induced SAS in soybean.It also provides a promising option for breeding lodging-resistant,high-yield soybean cultivars in the future.