Modes of Action of Phytochromes in Establishing Different Phototropic Responsiveness of Maize Coleoptiles

Previous studies have examined the effects of red light (R) on phototropism of maize ( Zea mays L. cv. Royaldent Hit 85) coleoptiles. The R effect on time-dependent phototropism (TDP) was further studied by characterizing its fluence-response relationship. The results showed the R effect was a low-fluence-response, unlike those on pulse-induced phototropisms that show a very-low-fluence-response mode. A subsequent pulse of far-red light (FR) could reverse the R effect. TDP responsiveness, however, recovered as the following FR was extended, The FR-dependent increase in TDP responsiveness was obtained even coleoptiles were pretreated only with FR. It suggested that TDP responsiveness could also be established in response to a FR signal. The fluence-response relationship for the effect of FR was then investigated. The effect of FR depended on the time of irradiation and required high photon fluences. Because reciprocity was invalid at the higher fluence range, the effect of FR would be a high-irradiance-response mode. Relation between phytochrome action modes and possible multiple pathways for phototropic signal transduction was analyzed based on the experiment results.

Seed Germination in Tomato: A Focus on Interaction between Phytochromes and Gibberellins or Abscisic Acid

Separately, it is well-documented that phytochromes (phys), gibberellin (GA) and abscisic acid (ABA) strongly control the seed germination in tomato. However, we hipothesized that phys interact with GA or ABA during this response. Thus, we make an analysis of seed germination of ABA deficient (sit), GA constitutive response (pro), phytochrome deficient (au) mutants as well as, specially, au sit and au pro double mutants of tomato?incubated in the dark or light conditions during 120 h [12 h intervals (i)]. Compared to au, which severely?reduced percentage germination (Gi%) and pro, which did not alter Gi%, au pro showed in the light enhanced Gi% and germination speed index (GSI) besides the reduced average germination time (AGT). Moreover, in the dark, germination of au pro was similar to pro. These results indicate that the mechanisms by which GA modulate germination in tomato are light dependent through the phy signaling, whereas intermediary values of Gi%, GSI and AGT in dark and light of au sit compared to au and sit single mutants indicate an additive effect of the au and sit mutations, suggesting that ABA and phy may act through the parallel signaling pathway.

CRISPR/Cas9-engineered mutation to identify the roles of phytochromes in regulating photomorphogenesis and flowering time in soybean

Soybean(Glycine max)responds to ambient light variation by undergoing multiform morphological alterations,influencing its yield potential and stability in the field.Phytochromes(PHYs)are plant-specific red(R)and far-red(FR)light photoreceptors mediating photomorphogenesis and photoperiodic flowering.As an ancient tetraploid,soybean harbors four PHYA,two PHYB,and two PHYE paralogs.Except for GmPHYA2/E4 and GmPHYA3/E3,which have been identified as photoperiod-dependent flowering repressors,the functions of GmPHYs are still largely unclear.We generated a series of individual or combined mutations targeting the GmPHYA or GmPHYB genes using CRISPR/Cas9 technology.Phenotypic analysis revealed that GmPHYB1 mediates predominantly R-light induced photomorphogenesis,whereas GmPHYA2/E4 and GmPHYA3/E3,followed by GmPHYA1 and GmPHYB2,function redundantly and additively in mediating FR light responses in seedling stage.GmPHYA2/E4 and GmPHYA3/E3,with weak influence from GmPHYA1 and GmPHYA4,delay flowering time under natural long-day conditions.This study has demonstrated the diversified functions of GmPHYAs and GmPHYBs in regulating light response,and provides a core set of phytochrome mutant alleles for characterization of their functional mechanisms in regulating agronomic traits of soybean.

Phytochromes are Involved in Elongation of Seminal Roots and Accumulation of Dry Substances in Rice Seedlings

Phytochromes have been reported to play important roles in seedling de-etiolation and flowering in rice.To identify the roles of phytochromes in regulating root growth and accumulation of dry substances,the lengths of seminal roots and the dry weights of seedlings were measured in the wild type as well as the phytochrome A（phyA） and phytochrome B（phyB） mutants grown under different conditions.When the whole seedlings were exposed to white light,the elongation of the seminal roots was significantly photoinhibited in the wild type,whereas this inhibitory effect was clearly reduced in the phyA and phyB mutants.When the roots of the seedlings were blocked from white light,the phyA and phyB mutants exhibited significantly longer seminal roots than the wild type.These results suggest that both the root-localized and shoot-localized PHYA and PHYB are involved in the photoinhibition of seminal root elongation in rice seedlings.By measuring the dry weights of roots and shoots,it is revealed that PHYB positively regulates the accumulation of dry substances in shoots,however,PHYA exerts the contrary effects on the accumulation of dry substances in roots and shoots of rice seedlings.

Green light mediates atypical photomorphogenesis by dual modulation of Arabidopsis phytochromes B and A

Although green light(GL)is located in the middle of the visible light spectrum and regulates a series of plant developmental processes,the mechanism by which it regulates seedling development is largely unknown.In this study,we demonstrated that GL promotes atypical photomorphogenesis in Arabidopsis thaliana via the dual regulations of phytochrome B(phyB)and phyA.Although the Pr-to-Pfr conversion rates of phyB and phyA under GL were lower than those under red light(RL)in a fluence rate-dependent and time-dependent manner,long-term treatment with GL induced high Pfr/Pr ratios of phyB and phyA.Moreover,GL induced the formation of numerous small phyB photobodies in the nucleus,resulting in atypical photomorphogenesis,with smaller cotyledon opening angles and longer hypocotyls in seedlings compared to RL.The abundance of phyA significantly decreased after short-and long-term GL treatments.We determined that four major PHYTOCHROME-INTERACTING FACTORs(PIFs:PIF1,PIF3,PIF4,and PIF5)act downstream of phyB in GL-mediated cotyledon opening.In addition,GL plays opposite roles in regulating different PIFs.For example,under continuous GL,the protein levels of all PIFs decreased,whereas the transcript levels of PIF4 and PIF5 strongly increased compared with dark treatment.Taken together,our work provides a detailed molecular framework for understanding the role of the antagonistic regulations of phyB and phyA in GL-mediated atypical photomorphogenesis.

Phytochromes Regulate SA and JA Signaling Pathways in Rice and Are Required for Developmentally Controlled Resistance to Magnaporthe grisea

Old leaves of wild-type rice plants （Oryza sativa L. cv. Nipponbare） are more resistant to blast fungus （Magnaporthe grisea） than new leaves. In contrast, both old and new leaves of the rice phytochrome triple mutant （phyAphyBphyC） are susceptible to blast fungus. We demonstrate that pathogenesis-related class 1 （PR1） proteins are rapidly and strongly induced during M. grisea infection and following exogenous jasmonate （JA） or salicylic acid （SA） exposure in the old leaves, but not in the new leaves of the wild-type. In contrast, the accumulation of PR1 proteins was significantly attenuated in old and new leaves of the phyAphyBphyC mutant. These results suggest that phytochromes are required for the induction of PR1 proteins in rice. Basal transcription levels of PRla and PRlb were substantially higher in the wildtype as compared to the phyAphyBphyC mutant, suggesting that phytochromes also are required for basal expression of PR1 genes. Moreover, the transcript levels of genes known to function in SA- or JA-dependent defense pathways were regulated by leaf age and functional phytochromes. Taken together, our findings demonstrate that phytochromes are required in rice for age-related resistance to M. grisea and may indirectly increase PR1 gene expression by regulating SA- and JA-dependent defense pathways.

Thermal Reversion of Plant Phytochromes

Phytochromes are red/far-red reversible photoreceptors essential for plant growth and development.Phytochrome signaling is mediated by the physiologically active far-red-absorbing Pfr form that can be inactivated to the red-absorbing Pr ground state by light-dependent photoconversion or by light-independent thermal reversion,also termed dark reversion.Although the term“dark reversion”is justified by historical reasons and frequently used in the literature,“thermal reversion”more appropriately describes the process of light-independent but temperature-regulated Pfr relaxation that not only occurs in darkness but also in light and is used throughout the review.Thermal reversion is a critical parameter for the light sensitivity of phytochrome-mediated responses and has been studied for decades,often resulting in contradictory findings.Thermal reversion is an intrinsic property of the phytochrome molecules but can be modulated by intra-and intermolecular interactions,as well as biochemical modifications,such as phosphorylation.In this review,we outline the research history of phytochrome thermal reversion,highlighting important predictions that have been made before knowing the molecular basis.We further summarize and discuss recent findings about the molecular mechanisms regulating phytochrome thermal reversion and its functional roles in light and temperature sensing in plants.

A LexA-based yeast two-hybrid system for studying light- switchable interactions of phytochromes with their interacting partners

Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light environment.Phytochromes exist in vivo in two forms,the inactive Pr form and the active Pfr form,that are interconvertible by treatments with R or FR light.It is believed that phytochromes transduce light signals by interacting with their signaling partners.A GAL4-based lightswitchable yeast two-hybrid(Y2H)system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components.However,several pairs of interactions between phytochromes and their interactors,such as the phyACOP1 and phyA-TZP interactions,were demonstrated by other assay systems but were not detected by this GAL4 Y2H system.Here,we report a modified LexA Y2H system,in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein.The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin(PCB)extracted from Spirulina.The well-defined interaction pairs,including phyA-FHY1 and phyB-PIFs,are well reproducible in this system.Moreover,we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions.Together,our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.

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.

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

In Arabidopsis,although studies have demonstrated that phytochrome A(phyA)and phyB are involved in blue light signaling,how blue light-activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1(COP1)-SUPPRESSOR OF PHYA-105(SPA1)E3 complex remains largely unknown.Here,we show that phyA responds to early and weak blue light,whereas phyB responds to sustainable and strong blue light.Activation of both phyA and phyB by blue light inhibits SPA1 activity.Specifically,blue light irradiation promoted the nuclear import of both phytochromes to stimulate their binding to SPA1,abolishing SPA1’s interaction with LONG HYPOCOTYL 5(HY5)to release HY5,which promoted seedling photomorphogenesis.

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.

Carotenoids and Chlorophyll are not the Chromophores of Blue Light-Induced Prehaustoria in Dodder （Cuscuta campestris） Seedlings

Previous studies demonstrated that cryptochromes are involved in blue light-induced coiling and prehaustoria development in young de-etiolated dodder seedlings. In this study, we suggest that carotenoids and chlorophyll are not the blue light absorbing chromophores involved in the mediation of prehaustoria development to blue light. Norflurazon-bleached dodder segments coiled and formed prehaustoria under blue light. However, norflurazon significantly reduced prehaustoria number （62%） under a mixture of red and far-red light, suggesting that phytochromes could be altered by norflurazon.

Immunoassay of Phytochrome A Content in Photoperiod-sensitive Genic Male-sterile Rice

A rice mutant (Oryza sativa L. Nongken 58S) 'photoperiodsensitive male-sterile rice' (PGMR) is sterile under long day-length (LD), but fertile in short days-length (SD). Phytochrome (Phy) has proved to be the photoreceptor involved in photoperiod-induced male sterile process in PGMR.To speculate how phytochrome functions, PhyA levels in leaf extracts of PGMRand wild-type rice (Oryza sativa L. Nongken 58) under different photoperiods were measured by means of a sandwich enzyme-linked immunosorbent assay (ELISA). The top two leaves of treated rice plants were sampled at the end of the last dark phase for 10 cycles at photoperiod-sensitive stage for fertility alteration in the mutant. Compared with LD treatment, the SD treatment resulted in 39% increase of relative PhyA content (per unit fresh weight) in PGMR, but only in 18% increase in its wild-type. In addition, after 10 cycles of end of far-red irradiations of day,the heading and flowering dates of PGMR were delayed for 2 days, while its male fertility almost remained unchanged. It is well known that the end of day far-red responses are mediated by PhyB.These results suggested that in PGMR PhyA took part in regulation of male sterility, hut PhyB was involved in mediating flowering time.

The molecular mechanism of shade avoidance in crops--How data from Arabidopsis can help to identify targets for increasing yield and biomass production

In order to prevent or counteract shading,plants enact a complex set of growth and developmental adaptations when they sense a change in light quality caused by other plants in their vicinity.This shade avoidance response（SAR）typically includes increased stem elongation at the expense of plant fitness and yield,making it an undesirable trait in an agricultural context.Manipulating the molecular factors involved in SAR can potentially improve productivity by increasing tolerance to higher planting density.However,most of the investigations of the molecular mechanism of SAR have been carried out in Arabidopsis thaliana,and it is presently unclear in how far results of these investigations apply to crop plants.In this review,current data on SAR in crop plants,especially from members of the Solanaceae and Poaceae families,are integrated with data from Arabidopsis,in order to identify the most promising targets for biotechnological approaches.Phytochromes,which detect the change in light caused by neighboring plants,and early signaling components can be targeted to increase plant productivity.However,they control various photomorphogenic processes not necessarily related to shade avoidance.Transcription factors involved in SAR signaling could be better targets to specifically enhance or suppress SAR.Knowledge integration from Arabidopsis and crop plants also indicates factors that could facilitate the control of specific aspects of SAR.Candidates are provided for the regulation of plant architecture,flowering induction and carbohydrate allocation.Yet to-be-elucidated factors that control SAR-dependent changes in biotic resistance and cell wall composition are pointed out.This review also includes an analysis of publicly available gene expression data for maize to augment the sparse molecular data available for this important species.

Heterotrimeric G-protein is involved in phytochrome A-mediated cell death of Arabidopsis hypocotyls

The heterotrimeric guanine nucleotide-binding protein （G-protein） has been demonstrated to mediate various signaling pathways in plants. However, its role in phytochrome A （phyA） signaling remains elusive. In this study, we discover a new phyA-mediated phenotype designated far-red irradiation （FR） preconditioned cell death, which occurs only in the hypocotyls of FR-grown seedlings following exposure to white light （WL）. The cell death is mitigated in the Gα mutant gpal but aggravated in the Gβ mutant agbl in comparison with the wild type （WT）, indicative of antagonistic roles of GPA1 and AGBI in the phyA-mediated cell-death pathway. Further investigation indicates that FR-induced accumulation of nonphotoconvertible protochlorophyllide （Pchlide^633）, which generates reactive oxygen species （ROS） on exposure to WL, is required for FR-preconditioned cell death. Moreover, ROS is mainly detected in chloroplasts using the fluorescent probe. Interestingly, the application of H2O2 to dark-grown seedlings results in a phenotype similar to FR-preconditioned cell death. This reveals that ROS is a critical mediator for the ceil death. In addition, we observe that agb1 is more sensitive to H2O2 than WT seedlings, indicating that the G-protein may also modify the sensitivity of the seedlings to ROS stress. Taking these results together, we infer that the G-protein may be involved in the phyA signaling pathway to regulate FR-preconditioned cell death ofArabidopsis hypocotyls. A possible mechanism underlying the involvement of the G-protein in phyA signaling is discussed in this study.

Light regulation of horticultural crop nutrient uptake and utilization

Plants demonstrate dynamic changes in molecular structures under fluctuating light conditions.Accumulating evidence suggests that light plays a vital role in plant growth and morphogenesis.In particular,light has a role in the absorption and utilization of nutrients in plants.Despite significant progress in understanding the mechanism of nutrient acquisition and assimilation,how light affects and regulates ion uptake remains a question.Studies in model plants,Arabidopsis thaliana,suggest that light affects the nutrient utilization in roots through a complex regulatory network;nonetheless,the molecular mechanisms underlying the various effects of light on these processes in crop plants remain fragmentary.In this review,we discuss the light effects(light quality,light intensity,and photoperiod)on nutrient uptake and utilization in horticultural crops for optimizing crop productivity and increasing fertilizer use efficiency.

Creation of two hyperactive variants of phytochrome B1 for attenuating shade avoidance syndrome in maize

Increasing the planting density of maize is an effective measure to improve its yield.However,plants under high planting density tend to trigger shade avoidance syndrome(SAS),reducing lodging resistance and ultimately yield drop.Phytochrome B(phyB)plays a dominant role in mediating shade avoidance response.This study constructed two hyperactive mutated alleles of maize PHYB1:ZmPHYB1^(Y98F)(mimicking Y104F of AtPHYB)and ZmPHYB1^(Y359F)(mimicking Y361F of AtPHYB).Ectopic expression of ZmPHYB1^(Y98F) and ZmPHYB1^(Y359F) under the control of the ZmPHYB1 promoter in the Arabidopsis phyB-9 background rendered enhanced activity on complementing the phyB-9 related phenotypes compared with ZmPHYB1^(WT).Moreover,similar to the behavior of ZmPHYB1^(WT),ZmPHYB1Y98F and ZmPHYB1^(Y359F) proteins are localized to the nucleus after red light exposure,and could interact with PIF proteins of maize.In addition,expression of ZmPHYB1^(Y98F) and ZmPHYB1^(Y359F) variants under the control of the native ZmPHYB1 promoter attenuated SAS of maize seedlings subjected to simulated shade treatment.It effectively reduced mature maize’s plant height and ear height in field conditions.The results combined demonstrate the utility of ZmPHYB1^(Y98F) and ZmPHYB1^(Y359F) for attenuating SAS and breeding high density-tolerant varieties of maize.

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.

Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles

The phytochrome gene family, which is in Arabidopsis thaliana, consists of phytochromes A-E（phyA to phyE）, regulates plant responses to ambient light environments. PhyA and phyB have been characterized in detail, but studies on phyC to phyE have reported discrepant functions. In this study, we show that phyD regulates the Arabidopsis gravitropic response by inhibiting negative gravitropism of hypocotyls under red light condition. PhyD had only a limited effect on the gravitropic response of roots in red light condition. PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was dependent upon the red light fluence rate.