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.

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.

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.

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.

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.

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.

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.

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.

Transcriptional Programs Related to Phytochrome A Function in Arabidopsis Seed Germination

In Arabidopsis seeds, germination is promoted only by phytochromes, principally phytochrome B （phyB） and phytochrome A （phyA）. Despite the abundant information concerning the molecular basis of phyB signaling downstream of PIF1/PIL5, the signaling network inducing germination by phyA is poorly known. Here, we describe the influence of phyA on the transcriptome of Arabidopsis seeds when germination is induced by a far-red （FR） pulse. The expression of 11% of the genome was significantly regulated by phyA. Most of the genes were up-regulated and the changes noted late （i.e. 5 h after a FR pulse）, whereas changes in down-regulated genes were more abundant earlier （i.e. 0.5 h after a FR pulse）. Auxin- and GA-associated elements were overrepresented in the genes that were modified by phyA. A significant number of genes whose expression was affected by phyA had not been previously reported to be dependent on PIL5. Among them, homozygotic mutant seeds of MYB66, a SAUR-like protein, PIN7, and GASA4 showed an impaired promo- tion of germination by phyA. Natural variation at the transcriptional level was found in early signaling and GA metabolic genes, but not in ABA metabolic and expansin genes between Columbia and Landsberg erecta accessions. Although phyA and phyB/PIL5 signaling pathways share some molecular components, our data suggest that phyA signaling is partially independent of PIL5 when germination is promoted by very low fluences of light.

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.

Strigolactone-Regulated Hypocotyl Elongation Is Dependent on Cryptochrome and Phytochrome Signaling Pathways in Arabidopsis

Seedling development including hypocotyl elongation is a critical phase in the plant life cycle. Light regula- tion of hypocotyl elongation is primarily mediated through the blue light photoreceptor cryptochrome and red/far-red light photoreceptor phytochrome signaling pathways, comprising regulators including COP1, HY5, and phytochrome- interacting factors （PIFs）. The novel phytohormones, strigolactones, also participate in regulating hypocotyl growth. However, how strigolactone coordinates with light and photoreceptors in the regulation of hypocotyl elongation is largely unclear. Here, we demonstrate that strigolactone inhibition of hypocotyl elongation is dependent on cryp- tochrome and phytochrome signaling pathways. The photoreceptor mutants cry1 cry2, phyA, and phyB are hyposensi- tive to strigolactone analog GR24 under the respective monochromatic light conditions, while cop1 and pifl pif3 pif4 pif5 （pifq） quadruple mutants are hypersensitive to GR24 in darkness. Genetic studies indicate that the enhanced respon- siveness of cop1 to GR24 is dependent on HY5 and MAX2, while that of pifq is independent of HY5. Further studies demonstrate that GR24 constitutively up-regulates HY5 expression in the dark and light, whereas GR24-promoted HY5 protein accumulation is light- and cryptochrome and phytochrome photoreceptor-dependent. These results suggest that the light dependency of strigolactone regulation of hypocotyl elongation is likely mediated through MAX2-dependent promotion of HY5 expression, light-dependent accumulation of HY5, and PIF-regulated components.

Phytochrome-regulated Gene Expression

Identification of all genes involved in the phytochrome （phy）-medieted responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth end development. This article highlights end integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-reguleted genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an Initial subset of these genes. The data： （a） reveal considerable complexity in the channeling of the light signals through the different phy-femily members （phyA to phyE） to responsive genes; （b） identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, end, therefore, as potentially important regulators in the transcriptional-network hierarchy; and （c） identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetioletion.

The Role of Phytochrome in Stress Tolerance

It is well-documented that phytochromes can control plant growth and development from germination to flowering. Additionally, these photoreceptors have been shown to modulate both biotic and abiotic stress. This has led to a series of studies exploring the molecular and biochemical basis by which phytochromes modulate stresses, such as salinity, drought, high light or herbivory. Evidence for a role of phytrochromes in plant stress tolerance is explored and reviewed.

Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis

Light serves as a crucial environmental cue which modulates plant growth and development, and which is controlled by multiple photoreceptors including the primary red light photoreceptor,phytochrome B(phyB). The signaling mechanism of phyB involves direct interactions with a group of basic helix-loop-helix(bHLH) transcription factors, PHYTOCHROME-INTERACTING FACTORS(PIFs), and the negative regulators of photomorphogenesis, COP1 and SPAs. H2 A.Z is an evolutionarily conserved H2 A variant which plays essential roles in transcriptional regulation. The replacement of H2 A with H2 A.Z is catalyzed by the SWR1 complex. Here, we show that the Pfr form of phyB physically interacts with the SWR1 complex subunits SWC6 and ARP6. phyB and ARP6 coregulate numerous genes in the same direction,some of which are associated with auxin biosynthesis and response including YUC9, which encodes a rate-limiting enzyme in the tryptophandependent auxin biosynthesis pathway. Moreover,phyB and HY5/HYH act to inhibit hypocotyl elongation partially through repression of auxin biosynthesis. Based on our findings and previous studies, we propose that phyB promotes H2 A.Z deposition at YUC9 to inhibit its expression through direct phyB-SWC6/ARP6 interactions,leading to repression of auxin biosynthesis, and thus inhibition of hypocotyl elongation in red light.

Phytochrome B and AGB1 Coordinately Regulate Photomorphogenesis by Antagonistically Modulating PIF3 Stability in Arabidopsis

Phytochrome B (phyB), the primary red light photoreceptor, promotes photomorphogenesis in Arabidopsis by interacting with the basic helix-loop-helix transcriptional factor PIF3 and inducing its phosphorylation and degradation. Heterotrimeric G proteins are known to regulate various developmental processes in plants and animals. In Arabidopsis, the G-protein β subunit AGB1 is known to repress photomorphogenesis. However, whether and how phyB and AGB1 coordinately regulate photomorphogenesis are largely unknown. Here we show that phyB physically interacts with AGB1 in a red light-dependent manner and that AGB1 interacts directly with PIF3. Moreover, we demonstrate that the AGB1-PIF3 interaction inhibits the association of PIF3 with phyB, leading to reduced phosphorylation and degradation of PIF3, whereas the phyB-AGB1 interaction represses the association of PIF3 with AGB1, resulting in enhaneed phosphorylation and degradation of PIF3. Our results suggest that phyB and AGB1 antagonistically regulate PIF3 stability by dynamically interacting with each other and PIF3. This dynamic mechanism may allow plants to balanee phyB and G-protein signaling to optimize photomorphogenesis.

Stress responsive gene CIPK14 is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis

In this study, we show that CIPK14,a stress responsive CBL-interacting protein kinase gene,is involved in phytochrome A-mediated far-red light inhibition of greening in Arabidopsis seedlings. The CIPK14-impairment mutant cipk14 grown in continuous far-red (FR) light did not show greening when exposed to white light illumination for 15 h. By contrast, the FR-grown phytochrome A null mutant phyA greened within 0.5 h of exposure to white light. Although greening of Col-4 (wild-type) was not completely abolished by FR, it exhibited a significantly decreased greening capacity compared with that of phyA. Further analyses demonstrated that the expression of protochlorophyllide reductase (POR) genes was correlated with the greening ability of the genotypes. In addition, CIPK14 appeared to be regulated by both the circadian clock and PhyA. Taken together, these results suggest that CIPK14 plays a role in PhyA-mediated FR inhibition of seedling greening, and that a Ca-related kinase may be involved in a previously undefined branch point in the phytochrome A signaling pathway.

Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum)

The phytochrome B (PHYB) gene of Arabidopsis thaliana was introduced into cotton through Agrobacterium tumefaciens.Integration and expression of PHYB gene in cotton plants were confirmed by molecular evidence.Messenger RNA (mRNA) expression in one of the transgenic lines,QCC11,was much higher than those of control and other transgenic lines.Transgenic cotton plants showed more than a two-fold increase in photosynthetic rate and more than a four-fold increase in transpiration rate and stomatal conductance.The increase in photosynthetic rate led to a 46% increase in relative growth rate and an 18% increase in net assimilation rate.Data recorded up to two generations,both in the greenhouse and in the field,revealed that overexpression of Arabidopsis thaliana PHYB gene in transgenic cotton plants resulted in an increase in the production of cotton by improving the cotton plant growth,with 35% more yield.Moreover,the presence of the Arabidopsis thaliana PHYB gene caused pleiotropic effects like semi-dwarfism,decrease in apical dominance,and increase in boll size.

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.

Red Light-Induced Phytochrome Relocation into the Nucleus in Adiantum capillus-veneris

Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. In- tracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphyl, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.