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.

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.

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.

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.

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.

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.

Functions of Phytochrome in Rice Growth and Development

Phytochrome family mainly senses red and far-red light to regulate a range of developmental processes throughout the life cycle of plants. Rice phytochrome gene family is composed of three members known as PHYA, PHYB and PHYC. It has been elucidated that individual phytochromes display both unique and overlapping roles in rice photomorphogenesis by characterization of all rice phytochrome mutants including single mutants, all combinations of double mutants as well as triple mutants. Based on the published data and authors’ ongoing studies, current knowledge of rice phytochrome functions in regulating seedling de-etiolation, root gravitropic response and elongation, plant architecture, flowering time and fertility is summarized. Additionally, the important issues in the field of rice phytochromes are proposed.

Transcriptome Analysis of Ten-DPA Fiber in an Upland Cotton (<i>Gossypium hirsutum</i>) Line with Improved Fiber Traits from Phytochrome A1 RNAi Plants

Silencing phytochrome A1 gene (PHYA1) by RNA interference in Upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with improved fiber quality (longer, stronger and finer fiber). To reveal molecular mechanisms that govern fiber development with positive fiber traits, a study of global gene expression profiling of 10-DPA fibers in a PHYA1 RNAi line and its parent Coker 312 was conducted by high-throughput RNA sequencing. A comparative analysis of transcriptomes between the two lines had identified 142 genes that were differentially expressed in the 10-DPA fiber of the RNAi line. Gene Ontology analysis showed that these differentially expressed genes were mainly involved in metabolic pathways, heterocyclic/organic cyclic compound binding and multiple enzyme activities, and cell structures which were reported to play important roles in fiber development. Twenty-eight KEGG pathways were mapped for the 142 genes, and the pathways related to glycolysis/gluconeogenesis and pyruvate metabolism were the most abundant and followed by cytochrome P450-involved pathways, suggesting that fiber improvement could be through the regulation of proteins involved in cytochrome P450 pathways. Genes encoding WRKY transcription factors, sucrose synthase, xyloglucan endotransglucosylase hydrolase, udp-glucuronate: xylan alpha-glucuronosyltransferase, and genes involved in lipid metabolism and ABA/brassinosteroid signal transduction pathways were found differentially expressed in the RNAi line. These genes have direct impacts on cotton fiber quality. The results of this study elucidate molecular signatures and possible mechanisms of fiber improvement in the background of PHYA1 RNAi in cotton and should help for future fine-tuning and programming of cotton fiber development.

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.

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.

Carbon Nanoparticle Exerts Positive Growth Effects with Increase in Productivity by Down-Regulating Phytochrome B and Enhancing Internal Temperature in Rice

The effects of carbon nanoparticle(CNP)on rice variety Swarna(MTU7029)were investigated.CNP induced effects similar to shade avoidance response(SAR)of Arabidopsis,with increase in shoot length,root length,root number,cotyledon area,chlorophyll content and total sugar content in rice seedlings.In mature plants,CNP treatment resulted increase in plant height,number of productive tillers per plant,normalized difference vegetation index,quantum yield and root growth.A total of 320 mg of CNP per plant administered in four doses resulted in improved grain traits such as filled grain rate,100-grain weight,grain length/width ratio,hulling rate,milling rate and head rice recovery.Seeds from the CNP-treated plants showed increase in amylose,starch and soluble sugar contents compared to controls.Strikingly,CNP treatment showed an average of 17.5%increase in yield per plant.Upon investigation to the molecular mechanism behind CNP induction of SAR,a significant downregulation of phytochrome B transcript was found.Decrease in perception of red wavelengths led to responses similar to SAR.Increase in plant’s internal temperature by 0.5ºC±0.1ºC was recorded after CNP treatment.We suggest that the internalized CNP aggregates may serve to absorb extra photons thereby increasing the internal temperature of plants.Phytochrome B accounts the hike in internal temperature and initiates a feed-back reduction of its own transcription.We suggest that moderate SAR is beneficial for rice plants to improve agronomic traits and yield.It presents a potential non-transgenic method for improving rice yield by CNP treatment.

Enhancing Flower Stalk Emergence in Phalaenopsis by Red Light Supplementation

A key step in regulating the flowering of Phalaenopsis is to control the emergence of the flower stalk or spiking. Light quality is an important factor to regulate plant reproduction. We used either red or blue light emitting diodes (LEDs) to substitute for 10% photosynthetic photon flux emitted by white fluorescent tubes as the control (WC) at approximately 70 μmole·m-2·s-1 to examine the effects of enhanced red (WR) or blue (WB) light on spiking and the dawn-to-dusk fluctuations in the photosynthetic pigments and carbohydrates of Phalaenopsis aphrodite, which were grown in 7.5 cm diameter pots for six weeks. WR treatment significantly elevated the ratio of red to far-red light and the level of phytochrome photostationary state in concurrence with an increase in the spiking ratio and length, but had little effect on the concentrations of chlorophyll a and chlorophyll b in the leaf, levels of soluble sugars such as glucose, fructose, sucrose, and insoluble starch in the leaf and shortened stem, the locus of spiking, when compared to the other two treatments. Evidently, the spiking of Phalaenopsis is improved by dependent on the relative amount of active phytochrome expressed in the photostationary state.

Low temperature-mediated repression and far-red light-mediated induction determine morning FLOWERING LOCUS T expression levels

In order to flower in the appropriate season,plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis(Arabidopsis thaliana)FLOWERING LOCUS T(FT).In Arabidopsis,FT messenger RNA levels peak in the morning and evening under natural long-day conditions(LDs).However,the regulatory mechanisms governing morning FT induction remain poorly understood.The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light(R:FR)ratios and constant temperatures.Here,we demonstrate that ZEITLUPE(ZTL)interacts with the FT repressors TARGET OF EATs(TOEs),thereby repressing morning FT expression in natural environments.Under LDs with simulated sunlight(R:FR=1.0)and daily temperature cycles,which are natural LD-mimicking environmental conditions,FT transcript levels in the ztl mutant were high specifically in the morning,a pattern that was mirrored in the toe1 toe2 double mutant.Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning.Far-red light counteracted ZTL activity by decreasing its abundance(possibly via phytochrome A(phyA))while increasing GIGANTEA(GI)levels and negatively affecting the formation of the ZTL-GI complex in the morning.Therefore,the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction.Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.

Combinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis

A subfamily of four Phytochrome （phy）-Interacting bHLH transcription Factors （PIFs） collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoacti- vated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a tran- sition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process, To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChlP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied prefer- entially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mecha- nisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.