PHYB Gene Research Articles

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

Mutation of phytochrome B promotes resistance to sheath blight and saline-alkaline stress via increasing ammonium uptake in rice.

Phytochrome B (PhyB), a red-light receptor, plays important roles in diverse biological processes in plants; however, its function in NH4 + uptake and stress responses of plants is unclear. Here, we observed that mutation in indeterminate domain 10 (IDD10), which encodes a key transcription factor in NH4 + signaling, led to NH4 + -sensitive root growth in light but not in the dark. Genetic combinations of idd10 and phy mutants demonstrated that phyB, but not phyA or phyC, suppressed NH4 + -sensitive root growth of idd10. PhyB mutants and PhyB overexpressors (PhyB OXs) accumulated more and less NH4 + , respectively, compared with wild-type plants. Real time quantitative polymerase chain reaction (RT-qPCR) revealed that PhyB negatively regulated NH4 + -mediated induction of Ammonium transporter 1;2 (AMT1;2). AMT1 RNAi plants with suppressed AMT1;1, AMT1;2, and AMT1;3 expression exhibited shorter primary roots under NH4 + conditions. This suggested that NH4 + uptake might be positively associated with root growth. Further, PhyB interacted with and inhibited IDD10 and brassinazole-resistant 1 (BZR1). IDD10 interacted with BZR1 to activate AMT1;2. NH4 + uptake is known to promote resistance of rice (Oryza sativa) to sheath blight (ShB) and saline-alkaline stress. Inoculation of Rhizoctonia solani demonstrated that PhyB and IDD10 negatively regulated and AMT1 and BZR1 positively regulated resistance of rice to ShB. In addition, PhyB negatively regulated and IDD10 and AMT1 positively regulated resistance of rice to saline-alkaline stress. This suggested that PhyB-IDD10-AMT1;2 signaling regulates the saline-alkaline response, whereas the PhyB-BZR1-AMT1;2 pathway modulates ShB resistance. Collectively, these data prove that mutation in the PhyB gene enhances the resistance of rice to ShB and saline-alkaline stress by increasing NH4 + uptake.

Molecular regulation and genetic control of rice thermal response

Global warming threatens food security. Rice (Oryza sativa L.), a vital food crop, is vulnerable to heat stress, especially at the reproductive stage. Here we summarize putative mechanisms of high-temperature perception (via RNA secondary structure, the phyB gene, and phase separation) and response (membrane fluidity, heat shock factors, heat shock proteins, and ROS (reactive oxygen species) scavenging) in plants. We describe how rice responds to heat stress at different cell-component levels (membrane, endoplasmic reticulum, chloroplasts, and mitochondria) and functional levels (denatured protein elimination, ROS scavenging, stabilization of DNA and RNA, translation, and metabolic flux changes). We list temperature-sensitive genetic male sterility loci available for use in rice hybrid breeding and explain the regulatory mechanisms associated with some of them. Breeding thermotolerant rice species without yield penalties via natural alleles mining and transgenic editing should be the focus of future work.

Subfunctionalization of phytochrome B1/B2 leads to differential auxin and photosynthetic responses.

Gene duplication and polyploidization are genetic mechanisms that instantly add genetic material to an organism's genome. Subsequent modification of the duplicated material leads to the evolution of neofunctionalization (new genetic functions), subfunctionalization (differential retention of genetic functions), redundancy, or a decay of duplicated genes to pseudogenes. Phytochromes are light receptors that play a large role in plant development. They are encoded by a small gene family that in tomato is comprised of five members: PHYA, PHYB1, PHYB2, PHYE, and PHYF. The most recent gene duplication within this family was in the ancestral PHYB gene. Using transcriptome profiling, co‐expression network analysis, and physiological and molecular experimentation, we show that tomato SlPHYB1 and SlPHYB2 exhibit both common and non‐redundant functions. Specifically, PHYB1 appears to be the major integrator of light and auxin responses, such as gravitropism and phototropism, while PHYB1 and PHYB2 regulate aspects of photosynthesis antagonistically to each other, suggesting that the genes have subfunctionalized since their duplication.

Suppressor of bri1-120 mutant allele revealed interrelated and independent actions of brassinosteroid and light signaling

Brassinosteroids (BRs) are plant hormones that affect diverse aspects of plant development. Various BR-biosynthetic or BR-signaling mutants contribute to BR functions and signaling events in many plant species. The BR receptor brassinosteroid-Insensitive 1 (BRI1) plays critical roles in BR signaling. We previously identified a weak bri1 mutant allele, bri1-120, that has a mutation site in the extracellular domain of BRI1. Here, genetic suppressor screening revealed that a PHYB gene mutation led to suppression of ethyl methanesulfonate (EMS)-mutagenized bri1-120. The morphology of bri1-120phyB-1 indicated that compact and rounded phenotypes of bri1-120 were suppressed. However, BR sensitivity of the bri1-120phyB-1 was only recovered in hypocotyl elongation, and overexpression of PHYB in bri1-120 did not enhance bri1-120 phenotypes. To further investigate the relationship between BR and light signalings, we examined the seed germination pattern and hypocotyl growth of bri1-120phyB-1 as compared to that of each single mutant under various light conditions. Seed germination in bri1-120phyB-1 was higher than in both the single mutants. Hypocotyl length in bri1-120phyB-1 was intermediate between that of bri1-120 and phyB-1, whereas sensitivity to red light in bri1-120phyB-1 remained the same as in phyB-1. These results suggest that BR and light signalings affect diverse cellular responses both together and independently, depending on the specific cellular processes.

Development, genetic mapping and QTL association of cotton PHYA, PHYB, and HY5-specific CAPS and dCAPS markers

BackgroundAmong SNP markers that become increasingly valuable in molecular breeding of crop plants are the CAPS and dCAPS markers derived from the genes of interest. To date, the number of such gene-based markers is small in polyploid crop plants such as allotetraploid cotton that has A- and D-sub-genomes. The objective of this study was to develop and map new CAPS and dCAPS markers for cotton developmental-regulatory genes that are important in plant breeding programs.Results Gossypium hirsutum and G. barbadense, are the two cultivated allotetraploid cotton species. These have distinct fiber quality and other agronomic traits. Using comparative sequence analysis of characterized GSTs of the PHYA1, PHYB, and HY5 genes of G. hirsutum and G. barbadense one PHYA1-specific Mbo I/Dpn II CAPS, one PHYB-specific Alu I dCAPS, and one HY5-specific Hinf I dCAPS cotton markers were developed. These markers have successfully differentiated the two allotetraploid genomes (AD1 and AD2) when tested in parental genotypes of ‘Texas Marker-1’ (‘TM-1’), ‘Pima 3–79’ and their F1 hybrids. The genetic mapping and chromosome substitution line-based deletion analyses revealed that PHYA1 gene is located in A-sub-genome chromosome 11, PHYB gene is in A-sub-genome chromosome 10, and HY5 gene is in D-sub-genome chromosome 24, on the reference ‘TM-1’ x ‘Pima 3–79’ RIL genetic map. Further, it was found that genetic linkage map regions containing phytochrome and HY5-specific markers were associated with major fiber quality and flowering time traits in previously published QTL mapping studies.ConclusionThis study detailed the genome mapping of three cotton phytochrome genes with newly developed CAPS and dCAPS markers. The proximity of these loci to fiber quality and other cotton QTL was demonstrated in two A-subgenome and one D-subgenome chromosomes. These candidate gene markers will be valuable for marker-assisted selection (MAS) programs to rapidly introgress G. barbadense phytochromes and/or HY5 gene (s) into G. hirsutum cotton genotypes or vice versa.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0448-4) contains supplementary material, which is available to authorized users.

Introduction of the Arabidopsis PHYB gene increases resistance of photosynthetic apparatus in transgenic Solanum tuberosum plants to UV-B radiation

The influence of phytochrome B overproduction in 50- to 60-day-old transgenic potato plants (Solanum tuberosum L., lines Dara 5 and Dara 12 with moderate and intense expression of PHYB, respectively) on the resistance of photosynthetic apparatus to UV-B irradiation was investigated. In plants unexposed to UV-B, there was no significant difference in photosynthetic rates (P n) and fluorescence parameters (F v/F m, qN, qP) between the nontransformed (NT) line and Dara-5 and Dara-12 lines, whereas the content of photosynthetic pigments per 1 cm2 leaf area was higher in the transgenic plants. Irradiation with UV-B resulted in the decrease in photosynthetic rate in NT plants by 35–45%, whereas in Dara-12 line this rate was lowered by only 20–25%. Exposure to UV-B reduced the amplitudes of both fast and slow components of delayed fluorescence (DF), which indicated the diminished efficiency of photosystem II (PSII). The decrease in the maximum amplitude of the slow DF component was markedly lower in Dara-12 (19%) than in NT line (33%). The maximal photochemical quantum yield of PSII (F v/F m ratio) in plants exposed to UV-B was also suppressed stronger in NT line than in Dara-12. The line Dara-5 had intermediate position among other lines in terms of UV-B resistance of photosynthesis and PSII activity, but it was closer to NT than to Dara-12. Thus, the potato plants actively expressing the gene of Arabidopsis phytochrome B apoprotein (PHYB) demonstrated a higher resistance of photosynthetic apparatus to UV-B radiation compared to nontransformed plants. The elevated UV-B tolerance is most likely related to the increased leaf content of chlorophyll, carotenoids, and flavonoids.

The Phytochrome B/Phytochrome C Heterodimer Is Necessary for Phytochrome C-Mediated Responses in Rice Seedlings

BackgroundPhyC levels have been observed to be markedly lower in phyB mutants than in Arabidopsis or rice wild type etiolated seedlings, but the mechanism of this phenomenon has not been fully elucidated.ResultsIn the present study, we investigated the mechanism by which phyB affects the protein concentration and photo-sensing abilities of phyC and demonstrated that rice phyC exists predominantly as phyB/phyC heterodimers in etiolated seedlings. PHYC-GFP protein was detected when expressed in phyA phyC mutants, but not in phyA phyB mutants, suggesting that phyC requires phyB for its photo-sensing abilities. Interestingly, when a mutant PHYB gene that has no chromophore binding site, PHYB(C364A), was introduced into phyB mutants, the phyC level was restored. Moreover, when PHYB(C364A) was introduced into phyA phyB mutants, the seedlings exhibited de-etiolation under both far-red light (FR) and red light (R) conditions, while the phyA phyB mutants were blind to both FR and R. These results are the first direct evidence that phyC is responsible for regulating seedling de-etiolation under both FR and R. These findings also suggest that phyB is indispensable for the expression and function of phyC, which depends on the formation of phyB/phyC heterodimers.SignificanceThe present report clearly demonstrates the similarities and differences in the properties of phyC between Arabidopsis and rice and will advance our understanding of phytochrome functions in monocots and dicots.

Detection of phytochrome-like genes from Rhazya stricta (Apocynaceae) using de novo genome assembly

Phytochrome-like genes in the wild plant species Rhazya stricta Decne were characterized using a de novo genome assembly of next generation sequence data. Rhazya stricta contains more than 100 alkaloids with multiple pharmacological properties, and leaf extracts have been used to cure chronic rheumatism, to treat tumors, and in the treatment of several other diseases. Phytochromes are known to be involved in the light-regulated biosynthesis of some alkaloids. Phytochromes are soluble chromoproteins that function in the absorption of red and far-red light and the transduction of intracellular signals during light-regulated plant development. De novo assembly of the nuclear genome of R. stricta recovered 45,641 contigs greater than 1000bp long, which were used in constructing a local database. Five sequences belonging to Arabidopsis thaliana phytochrome gene family (i.e., AtphyABCDE) were used to identify R. stricta contigs with phytochrome-like sequences using BLAST. This led to the identification of three contigs with phytochrome-like sequences covering AtphyA-, AtphyC- and AtphyE-like full-length genes. Annotation of the three sequences showed that each contig consists of one phytochrome-like gene with three exons and two introns. BLASTn and BLASTp results indicated that RsphyA mRNA and protein sequences had homologues in Wrightia coccinea and and Solanum tuberosum, respectively. RsphyC-like mRNA and protein sequence were homologous to Vitis vinifera and Vitis riparia. RsphyE-like mRNA coding and protein sequences were homologous to Ipomoea nil. Multiple-sequence alignment of phytochrome proteins indicated a homology with 30 sequences from 23 different species of flowering plants. Phylogenetic analysis confirmed that each R. stricta phytochrome gene is related to the same phytochrome gene of other flowering plants. It is proposed that the absence of phyB gene in R. stricta is due to RsphyA gene taking over the role of phyB.

Comprehensive phylogenetic analyses of the Ruppia maritima complex focusing on taxa from the Mediterranean

Recent molecular phylogenetic studies reported high diversity of Ruppia species in the Mediterranean. Multiple taxa, including apparent endemics, are known from that region, however, they have thus far not been exposed to phylogenetic analyses aimed at studying their relationships to taxa from other parts of the world. Here we present a comprehensive phylogenetic analyses of the R. maritima complex using data sets composed of DNA sequences of the plastid genome, the multi-copy nuclear ITS region, and the low-copy nuclear phyB gene with a primary focus on the Mediterranean representatives of the complex. As a result, a new lineage, "Drepanensis", was identified as the seventh entity of the complex. This lineage is endemic to the Mediterranean. The accessions included in the former "Tetraploid" entity were reclassified into two entities: an Asia-Australia-Europe disjunct "Tetraploid_α" with a paternal "Diploid" origin, and a European "Tetraploid_γ" originating from a maternal "Drepanensis" lineage. Another entity, "Tetraploid_β", is likely to have been originated as a result of chloroplast capture through backcrossing hybridization between paternal "Tetraploid_α" and maternal "Tetraploid_γ". Additional discovery of multiple tetraploidizations as well as hybridization and chloroplast capture at the tetraploid level indicated that hybridization has been a significant factor in the diversification of Ruppia.

Effects of inositol, inositol-generating phytase B applied alone, and in combination with 6-phytase A to phosphorus-deficient diets on laying performance, eggshell quality, yolk cholesterol, and fatty acid deposition in laying hens

Phytase B, a product of Aspergillus niger phyB gene expressed in Trichoderma reesei, which increased myo-inositol concentrations in 20 mM sodium phytate solution 7.5-fold during 120-min incubation, a combination of phytase B with 6-phytase A, and pure myo-inositol were tested as feed supplements in Bovans Brown laying hens. In the 2-factorial experiment (2×5), birds from wk 50 to 62 were fed 2 basal diets, corn-soybean (CSM) or wheat-soybean (WSM), using 12 one-hen cages per treatment. For both basal diets, the dietary treatments included negative control (0.08% nonphytate P in CSM, 0.13% nonphytate P in WSM; NC); internal control groups, NC+0.04% nonphytate P from monocalcium phosphate, MCP (IC); NC+0.1% of myo-inositol (Inos), NC+phytase B at 1,300 units of phytase B-acid phosphatase activity (AcPU)/kg (PhyB), NC+phytase B at 1,300 AcPU/kg+6-phytase A at 300 FTU/kg (PhyA+B). Feed intake, laying performance, and eggshell quality were determined. The total lipid and cholesterol contents as well as fatty acid profile were assessed in egg yolks collected from hens fed CSM diets, as was fatty acid profile. The hens fed the WSM diet consumed significantly more feed, laid a higher mass of eggs daily with higher mean weights, and had a higher hen-day egg production than the birds receiving the CSM diets. Similarly, higher values for yolk weights, shell weights, shell thickness, shell density, and breaking strengths were determined in the eggs laid by the hens fed the WSM diets. In hens fed either the CSM diets with phytase B alone, or in combination with 6-phytase A, enhanced feed intakes, egg mass, and hen-day egg production were recorded. Phytases also enhanced the eggshell quality parameters in the hens fed both variants of the diets. Phytase B alone, or in combination with 6-phytase A, reduced the total lipid and cholesterol concentrations in egg yolks collected from the hens fed the CSM diets, whereas the combination of both phytases improved the n-6:n-3 polyunsaturated fatty acids ratio. In the CSM diets, the supplemental myo-inositol suppressed feed intakes, reduced egg production, had no effect on eggshell quality and reduced the deposition of eicosanoid fatty acids in yolks. When comparing the effects of feeding MCP, myo-inositol, and phytases on the nutritional quality of the eggs laid by the hens fed phosphorus-deficient CSM diets, it seems that the enhancements in nutritional quality cannot be attributed solely to higher phosphorus level or higher concentrations of myo-inositol.

Effects of phytase B on laying performance, eggshell quality and on phosphorus and calcium balance in laying hens fed phosphorus-deficient maize-soybean meal diets

A study was conducted to evaluate the effects of dietary supplementation of phytase B (product of the Aspergillus niger phyB gene expressed in Trichoderma reesei) on feed intake, laying performance, eggshell quality, and on phosphorus and calcium balance in laying hens. Seventy-two, 40 weeks old Hy Line Brown hens were fed for 14 weeks the following four phosphorus-deficient (0.12% nonphytate phosphorus, NPP), maize-soybean meal-based diets: (1) calcium-deficient (2.8% Ca) control diet; (2) diet 1 + phytase B at the activity of 2.5 acid phosphatase units (AcPU/kg); (3) control diet (3.8% Ca); (4) diet 3 + phytase B at the activity of 2.5 AcPU/ kg. Each dietary treatment was fed to 18 cages of hens, 1 hen/cage kept in individual cages. Hens fed the NPP- and Ca-deficient diets consumed more feed (P < 0.01) and excreted less calcium (P < 0.01) than those receiving P-deficient diets with the standard calcium level. There were no effects of calcium level on feed utilization, egg mass, egg weight, and eggshell breaking strength. Egg production, although numerically higher in hens fed low Ca diets with no enzyme added, failed to be significantly different due to the low number of hens investigated and therefore the measurement should be considered as preliminary and supplementary. Phytase B increased mean egg weight by about 7% in layers fed the NPP- and Ca-deficient diet (Ca × phytase B interaction, P < 0.05), increased shell breaking strength, particularly at the standard calcium level, significantly enhanced amounts of calcium retained by layers and amounts of  phosphorus retained by hens fed the Ca-deficient diets. Additionally, phytase B improved Ca retention at both dietary Ca levels and phosphorus retention in hens fed the Ca-deficient diets. Results of the study indicate that the efficacy of phytase B in NPP-deficient diets is strongly influenced by the dietary calcium level and the enzyme may modulate egg weight, eggshell quality, phosphorus and calcium retention in laying hens fed low-NPP, maize-soybean meal-based diets.

Cloning and Expression Analysis of <I>TaPhyB3</I> in <I>Triticum aesti-vum</I>

As an important regulator in growth,development,and metabolic activities of plant,light is perceived by a variety of photoreceptors that control developmental processes,such as germination,photomorphogenesis,flowering,and senescence.Phytochromes play a pivotal role in plant adaptability to ambient environment.Gene AtPhyB has been found to be involved in the response to red light.At present,PhyB genes have been cloned in various plants,and the expression patterns and functions of the gene family have been studied in Arabidopsis thaliana,rice (Oryza sativa L.),and maize (Zea mays L.),but not in wheat (Triticum aestivum L.).The objectives of this study were to clone the full length of PhyB and study its structure and expression under different lights.The full-length cDNA sequence of PhyB,encoding the apoprotein of phytochrome B,was cloned from the wheat cultivar Chinese Spring.This gene is located on chromosome 4D and designated TaPhyB3.This gene possesses four extrons and three introns and the open reading frame TaPhyB3 is 3 501 bases in length,which encodes a predicted protein of 1 166 amino acids.The conserved domains of PhyB gene family,i.e.,DAF-DOMAIN,PHYTOCHROME REGION,PAS-A DOMAIN,PAS-B DOMAIN,HISTIDINE RELATED DOMAIN 1,and HISTIDINE RELATED DOMAIN 2,were also observed in the predicted protein sequence.The alignment analysis of amino acid sequence showed that TaPhyB3 shared 93% or 90% identity with the PHYBs of rice or maize,respectively,but only 73% with that of Arabidopsis.After treated with continuous darkness,far-red,red,blue,and white lights for 7 d,young seedlings of Chinese Spring were sampled for TaPhyB3 expression analysis using real-time reverse transcription polymerase chain reaction (RT-PCR).TaPhyB3 expression levels in the wheat seedlings under far-red,red,blue,and white lights were 2.2,7.7,7.4,and 37.3 times as high as that in the seedlings under darkness.When exposed to white light for 90 d,the TaPhyB3 expression was detected in root,stem,leaf,and spike.However,the gene was mainly expressed in above-ground organs of wheat seedling,and the expression level in leaf was 11.4 times as high as that in root.The expression level of TaPhyB3 is speculated to positively correlate with the degree of the seedling photomorphogenesis.

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.

Photoperiod modifies the diurnal expression profile of VvPHYA and VvPHYB transcripts in field-grown grapevine leaves

Despite the crucial role that phytochromes (Phys) play in light perception and in the entrainment of the circadian clock to local time, the photoperiodic regulation of PHYA and PHYB gene expression has been poorly studied, especially in woody perennials. Here the dynamic of Vitis vinifera PHYA (VvPHYA) and PHYB (VvPHYB) transcript accumulation was studied in field-grown grapevine leaves throughout daily cycles under decreasing natural photoperiods. Given that in grapevine the entrance of buds into endodormancy (ED) is a photoperiod-driven process, increases in BR(50) values, a parameter that measures the depth of dormancy in single bud cuttings assays was used to determine the critical daylength at which grapevine discriminates between long day (LD) and short day (SD) photoperiod. Therefore, we monitored the daily expression profile of VvPHYA and VvPHYB transcripts before, during and after the defined critical daylength. Results showed that under LD photoperiod (21 December, daylength 14 h 40 min) the abundance of both transcripts oscillated with diurnal rhythms, attaining maximum and minimum levels before dawn and after dusk, respectively. However, under SD photoperiod (12 April, daylength 11 h 40 min) the rhythmic expression disappeared, and both transcripts expressed uniformly at high levels. Our results showing that photoperiod regulates VvPHYA and VvPHYB gene expression, contrast with those reported in Arabidopsis and in other herbaceous plants in which PHYA and PHYB gene expression is regulated by the circadian clock.

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the PHYB Gene from Arabidopsis

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.

포도 세포현탁배양계에서 Phytochrome 신호전달에 의해 조절되는 안토시아닌 생합성

포도 현탁배양계를 이용하여 안토시아닌 생합성 경로에 관여하는 phytochrome 신호 전달에 관하여 연구하였다. 그 결과 세포의 생중량과 세포수의 생장은 배양 시 광의 유무 및 단색광의 종류와 관계없이 배양 4일째부터 8일째까지 급격히 증가하였고 8일 이후부터 정상기에 들어서서 최대 생장을 보였다. 그러나 안토시아닌 색소 축적은 청색광에서 가장 높은 효과를 나타내었으며 적색광에서도 높았으나 원적색광하에서는 암소같이 색소생성이 억제되었다. 배양시기에 따른 색소생성에 미치는 광 조사의 효과는 대수증가기인 배양4일째에 조사한 단색광간에 차이가 인정되지 않았으나, 정상기인 배양 7일째 조사한 단색광의 효과는 그 종류에 따라 색소 축적 패턴에 큰 차이를 나타내는 것으로 보아 배양 7일째가 효과적이었다. Phytochrome의 광가역적 효과를 알아보기 위하여 암배양 7일째세포에 다양한 조건의 단색광 (B/R, B/FR, B/R/FR)을 가역적으로 처리했을 경우, 청색광 단독 처리한 구와 비슷한 양의 색소 축적양상을 보였으며, B/R/R 조건의 단색광이 가역적으로 처리된 세포주에서 가장 많은 색소 축적을 보였다. 그러나 배양 7일째 R/FR 광가역 처리시에는 적색광 단독 처리에 비해 색소 축적이 억제되었으나, R/FR/R 가역처리시에는 적색광의 색소 축적양과 동일한 수준으로 회복되었다. 안토시아닌 생합성의 주요효소인 PAL과 CHS의 발현분석에서 PAL의 발현은 7일째 청색광이 처리된 세포주에서 조금 증가하는 현상을 보였으나, 적색광과 근적외광 처리구에서는 거의 반응이 없었다. CHS의 발현은 단색광 처리에서 높은 변화를 보였으며, 광을 처리한 7일째 높은 발현을 보이다 12일 이후 다시 증가하는 패턴을 보였다. 또한 적색광과 원적색광이 가역적으로 처리된 세포주에서 CHS 발현양도 가역적으로 증가하였다. PHYA와 PHYB는 포도세포에서 단일유전자로 존재하였으며, 단색광 처리에 따라 색소축적이 이루어질 때 이들 이들 두 유전자의 발현도 증가하였다. This experiment was carried out to confirm that phytochrome regulates anthocyanin bio-synthesis during cell suspension culture system of grape or not. In suspension culture of grape, maximum accumulation of anthocyanin was observed at the stationary phase under continuous white light condition. From mono-chromatic light interruption for 24h at the 4th or 7th day on the suspension cultured cells, the anthocyanin accumulation was highly enhanced at the light interruption at 7th day than 4th day under all monochromatic light treatment. However, the cell growth patterns were not affected by any light treatment. In the darkness, the anthocyanin synthesis was very low but remarkably increased by blue light or red light irradiation. However, the increase of anthocyanin accumulation by blue or red light was suppressed by far-red light in the suspension cells of grape. This suppression by far-red light on the anthocyanin synthesis also observed on the cells treated red or far-red light alternatively. These results implied that phytochrome regulation system may be involved in the anthocyanin biosynthesis of the suspension grape cells. By RNA expression analysis, chalcone synthase (CHS) gene was expressed highly by blue and red light but low by far-red light. The synergistic increase of CHS gene expression was also observed at the treatment of blue light followed by red for 24h. This result may explain the increase of anthocyanin accumulation in B/R treatment. Although the expression of phytochrome gene (PHYA or PHYB) was not highly increased by all light treatment (blue, red, and far-red light) the expression of both PHYA gene and PHYB gene was increased a little in cells treated red or far-red light. In grape suspension cells, the red light enhanced the anthocyanin synthesis, whereas the far-red light was suppressed. Although it was not confirmed whether or not phytochrome gene is activated in anthocyanin accumulating grape cells, we believed that anthocyanin biosynthesis in grape cells may be regulated under phytochrome signal transduction system.

Distinct regulation of CAB and PHYB gene expression by similar circadian clocks

Phytochrome B (phyB) is a major phytochrome active in light-grown plants. The circadian clock controls the expression of the PHYB gene. We have used the luciferase reporter gene (LUC) to monitor the rhythmic expression of PHYB in photoreceptor and clock-associated mutant backgrounds. Surprisingly, we found that PHYB and CAB expression have different free-running periods, indicating that separate circadian clocks control these genes. The effects of mutations show that the clocks share common components. This suggests that they are copies of the same clock mechanism in different locations, most likely in different cell layers. Furthermore, we show that phyB is required for a negative feedback loop that strongly antagonises the expression of PHYB. Compared to a system with only one clock, this regulatory complexity might allow the phase of peak expression for one clock-controlled gene to alter, relative to other genes or to changing environmental conditions.

Molecular and phenotypic specificity of an antisense PHYB gene in Arabidopsis.

The family of phytochrome photoreceptors plays an essential role in regulating plant growth and development in response to the light environment. An antisense PHYB transgene has been introduced into wild-type Arabidopsis and shown to inhibit expression of the PHYB sense mRNA and the phyB phytochrome protein 4- to 5-fold. This inhibition is specific to phyB in that the levels of the four other phytochromes, notably the closely related phyD and phyE phytochromes, are unaffected in the antisense lines. Antisense-induced reduction in phyB causes alterations of red light effects on seedling hypocotyl elongation, rosette leaf morphology, and chlorophyll content, similar to the phenotypic changes caused by phyB null mutations. However, unlike the phyB mutants, the antisense lines do not flower early compared to the wild type. Furthermore, unlike the phyB mutants, the antisense lines do not show a reduction in phyC level compared to the wild type, making it possible to unequivocally associate several of the photomorphogenic effects seen in phyB mutants with phytochrome B alone. These results indicate that an antisense transgene approach can be used to specifically inhibit the expression and activity of a single member of the phytochrome family and to alter aspects of shade avoidance responses in a targeted manner.

The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26

The apple rootstock M26 (Malus domestica) was infected with a binary vector system of Agrobacterium tumefaciens carrying the neomycin phosphotransferase II and Arabidopsis phyB genes. Thirteen transformed clones were obtained from 329 infected leaves. Five of the clones had a single copy integration, six clones had two copies, one clone had five copies and one of the clones had eight copies of the phyB gene integrated. No differences in rooting were found between transformed and untransformed plants. The stem length was reduced in nine of the 13 transgenic clones, and shoot, root and plant dry weights were reduced in all transformed clones compared with untransformed control plants. Northern analysis showed that the Arabidopsis phyB gene was expressed in the transformed clones.