Outline
Comptes Rendus

Molecular biology and genetics/Biologie et génétique moléculaires
Detection of phytochrome-like genes from Rhazya stricta (Apocynaceae) using de novo genome assembly
Comptes Rendus. Biologies, Volume 336 (2013) no. 11-12, pp. 521-529.

Abstract

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 Rstricta recovered 45,641 contigs greater than 1000 bp 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 Rstricta is due to RsphyA gene taking over the role of phyB.

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DOI: 10.1016/j.crvi.2013.10.009
Keywords: phyA, PhyB, phyC, phyE, BLASTp, Alkaloids

Jamal S.M. Sabir 1; Nabih A. Baeshen 1; Ahmed M. Shokry 1, 2; Nour O. Gadalla 1, 3; Sherif Edris 1, 4; Mohammed H. Mutwakil 1; Ahmed M. Ramadan 1, 2; Ahmed Atef 1; Magdy A. Al-Kordy 1, 3; Osama A. Abuzinadah 1; Fotouh M. El-Domyati 1, 4; Robert K. Jansen 1, 5; Ahmed Bahieldin 1, 4

1 Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, Jeddah 21589, Saudi Arabia
2 Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt
3 Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Egypt
4 Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt
5 Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA
@article{CRBIOL_2013__336_11-12_521_0,
     author = {Jamal S.M. Sabir and Nabih A. Baeshen and Ahmed M. Shokry and Nour O. Gadalla and Sherif Edris and Mohammed H. Mutwakil and Ahmed M. Ramadan and Ahmed Atef and Magdy A. Al-Kordy and Osama A. Abuzinadah and Fotouh M. El-Domyati and Robert K. Jansen and Ahmed Bahieldin},
     title = {Detection of phytochrome-like genes from {\protect\emph{Rhazya} stricta} {(Apocynaceae)} using \protect\emph{de novo} genome assembly},
     journal = {Comptes Rendus. Biologies},
     pages = {521--529},
     publisher = {Elsevier},
     volume = {336},
     number = {11-12},
     year = {2013},
     doi = {10.1016/j.crvi.2013.10.009},
     language = {en},
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%0 Journal Article
%A Jamal S.M. Sabir
%A Nabih A. Baeshen
%A Ahmed M. Shokry
%A Nour O. Gadalla
%A Sherif Edris
%A Mohammed H. Mutwakil
%A Ahmed M. Ramadan
%A Ahmed Atef
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%A Osama A. Abuzinadah
%A Fotouh M. El-Domyati
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Jamal S.M. Sabir; Nabih A. Baeshen; Ahmed M. Shokry; Nour O. Gadalla; Sherif Edris; Mohammed H. Mutwakil; Ahmed M. Ramadan; Ahmed Atef; Magdy A. Al-Kordy; Osama A. Abuzinadah; Fotouh M. El-Domyati; Robert K. Jansen; Ahmed Bahieldin. Detection of phytochrome-like genes from Rhazya stricta (Apocynaceae) using de novo genome assembly. Comptes Rendus. Biologies, Volume 336 (2013) no. 11-12, pp. 521-529. doi : 10.1016/j.crvi.2013.10.009. https://comptes-rendus.academie-sciences.fr/biologies/articles/10.1016/j.crvi.2013.10.009/

Original version of the full text

1 Introduction

Plant development and performance is strongly influenced by environment. Light is one of the most important factors affecting plants because it is involving in the regulation of seed germination, establishment of seedlings, determination of growth habit, and the transition to flowering [1]. Plants have a collection of photoreceptors perceiving information (light quality and quantity, and duration) about their light environment [2,3]. Red (R) and far-red (FR) light are important environmental signals in the regulation of plant development, with major roles in seedling de-etiolation, neighbor detection, and photoperiodism [4,5]. Plant R and FR photoreceptors, the phytochromes, are soluble chromoproteins consisting of a 120–130 kDa apoprotein and a linear tetrapyrrole chromophore. A small gene family phy, encoding the phytochrome apoproteins (chromoproteins), functions in the absorption of R and FR light and the transduction of intracellular signals during light-regulated plant development [6].

Plants contain multiple, distinct phytochrome species that are the products of a divergent gene family [7]. Physiological and biochemical experiments in earlier studies indicated that plants contain two different forms of phytochromes [8–10]. The first form is frequently referred to as type-I (or light labile) phytochrome, predominating in etiolated tissue, while the second form is referred to as type-II (or light-stable) phytochrome, prevalent in light-grown tissue [11–13].

The phy gene family was first identified in Arabidopsis and subsequently in many other plant species [1,7,14–16]. In Arabidopsis, there are five phytochrome genes, phyABCDE [17]. The product of the phyA gene, phyA, is light labile and predominates in etiolated seedlings where it accumulates to relatively high levels. phyB and phyC proteins are more light-stable, with phyB are common in light-grown tissues [18]. Arabidopsis may not be representative of flowering plants because additional phy loci related to phyA and phyB evolved independently several times in eudicots. Additionally, monocots may lack orthologs of Arabidopsis phyD and phyE [19]. Etiolated seedlings of phyB mutants are deficient in several responses to red light [20,21]. Light-grown seedlings of phyB mutants have an elongated, early flowering phenotype of the shade-avoidance syndrome of wild-type seedlings grown under low R/FR. The phyB mutant seedlings display attenuated responses to the low R/FR or to the end of day (EOD) far-red light, leading to the proposal that phyB may play a key role in the shade-avoidance response [22,23]. In addition, Hirschfeld et al. [13] indicated that the level of phyC apoprotein is strongly reduced in Arabidopsis phyB mutants. This observation suggests that phenotypes associated with phyB mutant genes result from the attenuation of phyC signaling. Some shade-avoidance responses of the phyB mutant to the low R/FR or to EOD far-red light are severely attenuated [22], although other responses, such as reduction in leaf area and acceleration of flowering, are clearly retained [24,25]. Devlin et al. [26] demonstrated that phyA–phyB double mutants respond to EOD far-red light by the acceleration of flowering and by the promotion of elongated internodes between rosette leaves. These responses are reversible by subsequent treatment with red light, indicating that one or more of phyC, phyD, or phyE control flowering time and internode elongation [26,27]. Unexpectedly, Hu et al. [28] revealed both light-dependent and -independent roles for phytochromes to regulate the Arabidopsis circadian clock, indicating dual roles for phytochromes to arrest and/or promote the progression of plant development in response to the prevailing light environment. Phytochrome was also shown to function in circadian clock adjustment to plant iron status through a new retrograde pathway that involves a plastid-encoded protein [29].

Rhazya stricta Decne is a robust plant with erect stems and upright thick and smooth leaves placed close together on the stem [30]. It is common in the Arabian Peninsula and the Indian subcontinent. Leaves are used to make a tonic with a peculiar bitter taste. Rhazya stricta is used traditionally in Asia for the treatment of different types of diseases, such as skin diseases, stomach diseases, and hypertension [31]. The leaves, flowers and fruits are also used in joint infections and for cancer [32]. Phytochemical analyses have identified more than 100 alkaloids [33] with several pharmacological properties. There is considerable evidence that light can affect the production of alkaloids. For example, Catharanthus roseus L. is affected by light in the production of the dimeric indole alkaloids, vinblastine and vincristine. The biosynthesis and accumulation of vindoline in the intact plant is controlled by tissue-specific, development regulated, and light-dependent factors [34]. Several lines of evidence showed that phytochromes are involved in the light-regulated biosynthesis of these dimeric indole alkaloids. Also, Höft et al. [35] demonstrated that the alkaloid content in the leaves of Tabernaemontana pachysiphon Stapf. (Apocynaceae) is influenced by light intensity.

In this study, phytochrome-like genes of phyA, phyC and phyE were characterized from de novo assembled genome contigs from next generation sequencing in the wild plant species Rhazya stricta. Bioinformatic analyses of these data confirmed the recovery of three full-length phytochrome-like genes.

2 Materials and methods

2.1 Isolation of nuclear DNA

Extraction of total DNA was performed using the modified procedure of Gawel and Jarret [36]. Three samples of discs collected from the upper leaves of R. stricta were frozen in liquid nitrogen (approximately 500 mg of tissue each). To remove RNA contaminants, RNase A (10 mg/mL, Sigma, USA) was added to the DNA samples and incubated at 37 °C for 30 min. Estimation of the DNA concentration in different samples was done by measuring optical density at 260 nm according to the equation:

Purified DNA samples were sent to Beijing Genomics Institute (BGI), Shenzhen, China for sequencing.

2.2 Sequence filtering and bioinformatic analysis

The raw sequence data were obtained using the Illumina python pipeline v. 1.3. For obtained libraries, only high quality reads (quality > 20) were retained. Then, de novo assembly of the short single-end read dataset was performed using assembler Velvet, which has been developed for the assembly of short reads using the de Bruijn graph algorithm [37].

2.3 Basic local alignment search tool (BLAST)

BLAST finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases, and calculates the statistical significance of matches based on pair-wise alignment method. It can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families (http://www.ncbi.nlm.nih.gov/BLAST).

2.4 Eukaryotic GeneMark.hmm program

GeneMark.hmm E-3.9 (http://exon.gatech.edu/eukhmm.cgi, A. Lukashin and M. Borodovsky, unpublished) was used for gene finding and annotation of R. stricta phytochrome-like genes. The statistical model of genomic sequence organization employed in the GeneMark.hmm algorithm is a HMM with duration [38] or a hidden semi-Markov model (HSMM).

2.5 Sequence alignment and estimation of phylogenetic relationships

MUSCLE [39] was used for multiple-sequence alignment. GenBank accession numbers for phytochrome protein sequences data aligned and analyzed in this study are displayed in Table 1. Maximum likelihood was used to build a tree where the evolutionary rates are free to differ in different lineages. ML trees were constructed using amino acid sequences and the Jones–Taylor–Thornton (JTT) substitution model. To evaluate the reliability of the inferred trees, bootstrap analysis using 1000 replicates was carried out. All the phylogenetic analyses were performed using MEGA 5.2 [40].

Table 1

Accession number, description of the genes and organism (Latin name), for phytochrome genes used in this study.

Accession No. Description Latin Name
XM_637404 Dictyostelium discoideum AX4 prolyl 4-hydroxylase (phyA) mRNA, complete cds Dictyostelium discoideum AX4
XM_004362186 Dictyostelium fasciculatum prolyl 4-hydroxylase (phyA) mRNA, complete cds Dictyostelium fasciculatum
AF547224 Synthetic construct phytase (phyA) mRNA, complete cds synthetic construct
EU786166 Aspergillus japonicus strain BCC18313 PhyA (phyA) mRNA, complete cds Aspergillus japonicas
EU786167 Aspergillus niger strain BCC18081 PhyA (phyA) mRNA, complete cds Aspergillus niger
XM_002561048 Penicillium chrysogenum Wisconsin 54-1255 phytase phyA from patent WO2003038111-A2-Penicillium chrysogenum (phyA) mRNA, complete cds Penicillium chrysogenum Wisconsin 54-1255
AJ310697 Agrocybe pediades mRNA for Phytase (phyA gene) Agrocybe pediades
AJ310700 Trametes pubescens mRNA for Phytase (phyA gene) Trametes pubescens
AB042805 Aspergillus oryzae phyA mRNA for phytase, complete cds Aspergillus oryzae
AJ310696 Peniophora lycii mRNA for Phytase (phyA gene) Peniophora lycii
GU120223 Aspergillus sp. A25 phytase (phyA) mRNA, complete cds Aspergillus sp. A25
AJ543399 Trichoderma harzianum phyA gene for phytase Trichoderma harzianum
JF412664 Amphicarpaea edgeworthii phytochrome B (PhyB) mRNA, complete cds Amphicarpaea edgeworthii
JQ771614 Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) phytochrome C (PhyC) mRNA, complete cds Arabidopsis thaliana
DQ208423 Solanum tuberosum phytochrome A (phyA) mRNA, complete cds Solanum tuberosum
AB743571 Fragaria x ananassa phyA mRNA for phytochrome A, complete cds Fragaria x ananassa
XM_002271635 PREDICTED: Vitis vinifera phytochrome E (PhyE), mRNA Vitis vinifera
GU994130 Amblyopyrum muticum cultivar Ae45 phytochrome A (PhyA) mRNA, complete cds Amblyopyrum muticum
GU994114 Triticum monococcum cultivar BO1 phytochrome A (PhyA) mRNA, complete cds Triticum monococcum
AB743572 Fragaria x ananassa phyB mRNA for phytochrome B, complete cds Fragaria x ananassa
XM_003555718 Glycine max phytochrome type A-like (LOC100790763), mRNA Glycine max
EU428746 Glycine max phytochrome A-1 (phyA) mRNA, complete cds Glycine max
EU428748 Glycine max phytochrome A-3 (phyA) mRNA, complete cd. Glycine max
EU428747 Glycine max phytochrome A-2 (phyA) mRNA, complete cds Glycine max
NM_117721 Arabidopsis thaliana phytochrome D (PhyD) mRNA, complete cds Arabidopsis thaliana
AY348568 Orobanche minor phytochrome A (phyA) mRNA, complete cds Orobanche minor
AY348569 Monotropastrum globosum phytochrome A (phyA) mRNA, complete cds Monotropastrum globosum
X75412 P. crispum phyA mRNA for phytochrome A Petroselinum crispum
AF544028 AY078504 Stellaria longipes ecotype prairie phytochrome B (PhyB) mRNA, complete cds Stellaria longipes
NM_001251357 Glycine max phytochrome A (PhyA), mRNA Glycine max
L34844 Soybean phytochrome A (phyA) mRNA, complete cds Glycine max
NM_117923 Arabidopsis thaliana phytochrome E (PHYE) mRNA, complete cds Arabidopsis thaliana
U39787 Ipomoea nil phytochrome E (PhyE) mRNA, complete cds Ipomoea nil
NM_122975 Arabidopsis thaliana phytochrome C (PhyC) mRNA, complete cds Arabidopsis thaliana
EU428751 Glycine max phytochrome B-3 (phyB) mRNA, complete cds Glycine max
XM_003533109 Glycine max phytochrome B-like (LOC100799831), mRNA Glycine max
NM_001123784 Arabidopsis thaliana phytochrome A (PhyA) mRNA, complete cds Arabidopsis thaliana
U56731 Sorghum bicolor phytochrome C (PhyC) mRNA, complete cds Sorghum bicolor
XM_002991073 Selaginella moellendorffii hypothetical protein (PhyB), mRNA Selaginella moellendorffii
AF544029 Stellaria longipes ecotype prairie phytochrome C (PhyC) mRNA, complete cds Stellaria longipes
U56729 Sorghum bicolor phytochrome A (PhyA) mRNA, complete cds Sorghum bicolor
XM_002278574 PREDICTED: Vitis vinifera phytochrome A (PhyA), mRNA Vitis vinifera
NM_100828 Arabidopsis thaliana phytochrome A (PhyA) mRNA, complete cds Arabidopsis thaliana
XM_002318877 Populus trichocarpa phytochrome (phya), mRNA Populus trichocarpa
AJ001914 Lycopersicon esculentum mRNA for phytochrome A, type 1 Solanum lycopersicum
AB109891 Oryza sativa Japonica Group PHYA mRNA for phytochrome A, complete cds Oryza sativa Japonica Group
AJ001318 Populus tremula x Populus tremuloides mRNA for phytochrome A Populus tremula x Populus tremuloides
AB036762 Armoracia rusticana phyA mRNA for phytochrome A, complete cds Armoracia rusticana
AJ001915 Lycopersicon esculentum mRNA for phytochrome A, type 2 Solanum lycopersicum
NM_001247561 Solanum lycopersicum alternative transcript type 3 (phyA), mRNA Solanum lycopersicum
AJ001916 Lycopersicon esculentum mRNA for phytochrome A type 3 Solanum lycopersicum
AY348567 Cuscuta pentagona phytochrome A (phyA) mRNA, complete cds Cuscuta pentagona
EU428752 Glycine max phytochrome B-4 (phyB) mRNA, complete cds Glycine max
AB018442 Oryza sativa Japonica Group mRNA for phytochrome C, complete cds Oryza sativa Japonica Group
NM_127435 Arabidopsis thaliana phytochrome B (PhyB) mRNA, complete cds Arabidopsis thaliana
AY345120 Cyrtosia septentrionalis phytochrome A (phyA) mRNA, complete cds Cyrtosia septentrionalis
AY888046 Triticum aestivum putative phytochrome B (PhyB) mRNA, complete cds Triticum aestivum
AB264087 Lotus japonicus phyb mRNA for phytochrome b, complete cds Lotus japonicas

3 Results

3.1 Next generation sequencing and BLAST searches for phytochrome-like gene family

A total of 73,841,902 single-end short DNA sequence reads was generated for R. stricta using the HiSeq 2000 Illumina platform (Illumina, San Diego, CA). The raw sequencing reads have been deposited at EMBL (accession number PRJEB4739). De novo assembly using Velvet generated 714,083 contigs and the 45,641 contigs greater than 1000 bp were used in constructing a local database. Five sequences belonging to Arabidopsis thaliana phytochrome gene family (i.e. AtphyABCDE, accession nos. NM_100828.3, NM_127345.3, NM_122975.2, NM_117721.1 and NM_117923.7, respectively) were obtained from GenBank and used for searching the local database to identify contigs with phytochrome-like sequences using BLAST. The BLAST searches resulted in the identification of three phytochrome-like contigs in R. stricta. The first contig (number 152,341) was 4955 bp long and had an average depth of coverage of 15.32 and corresponds to AtphyA-like sequence. The second contig (number 6,028) was 7216 bp long with an average coverage of 13.25 and represents an AtphyC-like. The third contig (number 66,070) was 7000 bp long with an average coverage of 12.61 and corresponds to an AtphyE-like sequence (Table 2).

Table 2

Description of the three detected Atphy-like sequences in Rhazya stricta genome contigs greater than 1000 bp.

Subject Query Score Identities E-value
PhyA (NM_100828.3) Contig no. 52,341 1266 1593/2037 (78%) 0.0
phyC (NM_122975.2) Contig no. 6,028 893 1243/1737 (72%) 0.0
phyE (NM_117923.7) Contig no. 6,070 850 1186/1654 (72%) 0.0

3.2 Annotation of the three phytochrome gene sequences

GeneMark.hmm was used to identify exon-, intron- and protein-like structures in three phytochrome-like sequences of the R. stricta contigs. Each contig consisted of one phytochrome-like gene comprised of three exons and two introns (Fig. 1, Table 3). Contigs 152,341, 6,028 and 66,070 were submitted to GenBank (accession numbers: phyA–HG380749; phyC–HG380750; phyE–HG380751). BLASTn and BLASTp of the GenBank nucleotide and protein databases were performed using the predicted mRNA and protein sequence of each contig to search for homologous sequences.

Fig. 1

The relative position for the three phytochrome-like genes (blue), i.e. RsphyA-like (a) RsphyC-like (b) and RsphyE-like (c), and exons (yellow) within the three contigs. Color online.

Table 3

Annotation results of the three contigs with Rsphy-like sequences showing number, type, range and length of exons (bp).

Gene Contig No. Exon No. Strand Exon Type Exon range (bp) Exon length (bp)
Start End
phyA 15,2341 3 Terminal 252 270 19
phyA 15,2341 2 Internal 379 1195 817
phyA 15,2341 1 Initial 1372 3418 2047
phyC 6,028 1 + Initial 923 2960 2038
phyC 6,028 2 + Internal 4035 4851 817
phyC 6,028 3 + Terminal 6854 7154 301
phyE 66,070 1 + Initial 1570 2667 1098
phyE 66,070 2 + Internal 2737 3619 883
phyE 66,070 3 + Terminal 5050 5852 803

Rhazya stricta phyA-like (or RsphyA-like) mRNA sequence (contig 152,341) had the highest homology to the phyA coding sequence of Wrightia coccinea with query coverage equal to 49% and an E-value of 0.0. Protein sequence searches indicated that RsphyA-like sequence is most similar to phyA of Solanum tuberosum with query coverage equal to 96% and an E-value of 0.0. Both results confirmed that contig 152,341 likely contains a full-length copy of the RsphyA-like gene (Table 3 and Fig. 1a). Searches of mRNA sequence predicted from the second contig (6,028) identified a Rhazya stricta phyC-like (or RsphyC-like) sequence that had the highest sequence identity to the phyC coding sequence of Vitis vinifera with query coverage equal to 98% and an E-value of 0.0. The protein sequence query identified the highest identity to phyC of Vitis riparia with query coverage equal to 99% and an E-value of 0.0. These results confirmed that contig 6,028 likely contains a full-length RsphyC-like gene (Table 3 and Fig. 1b). Finally, using the mRNA sequence predicted from the third contig (66,070), R. stricta phyE-like (or RsphyE-like) sequence was found to have high sequence identity to the phyE coding sequence of Ipomoea nil with query coverage equal to 96% and an E-value of 0.0. Protein sequence queries indicated that this contig had the highest identity with phyE of Ipomoea nil with query coverage equal to 97% and an E-value of 0.0. Both results confirmed that contig 66,070 likely contains a full-length copy of the RsphyE-like gene (Table 3 and Fig. 1c).

Protein sequences of the three identified R. stricta phy-like genes were analyzed against the pfam database (http://pfam.sanger.ac.uk/) to detect the protein family and conserved domains. The analysis revealed the presence of a phytochrome protein family (accession number PF00360). The domains summary of the predicted proteins is shown in Tables 4–6 and Fig. 2.

Table 4

Pfam search results show relative position, bit score and E-value for the identified protein family and domains within R. stricta phyA-like identified protein.

Family Description Entry type Clan Envelope Alignment HMM HMM length Bit score E-value
Start End Start End From To
N-terminal domain NTE N-terminal extension Region N/A 1 65 1 65 N/A N/A 65 60.8 4.00E−09
PAS_2 PAS fold Domain CL0183 66 182 66 182 2 110 110 135.8 7.30E−40
GAF GAF domain Domain CL0161 215 398 215 398 1 154 154 142.4 1.20E−41
PHY Phytochrome region Family CL0161 409 588 409 586 1 181 183 234.8 3.60E−70
C-terminal domain PAS PAS fold Domain CL0183 616 731 617 731 2 113 113 78.8 2.50E−22
PAS PAS fold Domain CL0183 746 871 747 869 2 111 113 82.0 2.40E−23
HisK Histidine kinase-related (phospho-acceptor) Domain CL0025 891 953 894 948 4 61 68 28.5 1.10E−06
Table 5

Pfam search results shows relative position, bit score and E-value for the identified protein family and domains within R. stricta phyC-like identified protein.

Family Description Entry type Clan Envelope Alignment HMM HMM length Bit score E-value
Start End Start End From To
N-terminal domain NTE N-terminal extension Region N/A 1 67 1 67 N/A N/A 67 137 1.00E−35
PAS_2 PAS fold Domain CL0183 68 184 68 184 1 110 110 145 1.00E−42
GAF GAF domain Domain CL0161 218 396 221 395 5 153 154 133.7 5.70E−39
PHY Phytochrome region Family CL0161 407 586 407 584 1 181 183 234.9 3.30E−70
C-terminal domain PAS PAS fold Domain CL0183 613 728 614 728 2 113 113 67.2 9.80E−19
PAS PAS fold Domain CL0183 743 868 745 866 3 111 113 58.5 4.70E−16
HisK Histidine kinase-related (phospho-acceptor) Domain CL0025 892 952 893 950 6 66 68 27.7 1.90E−06
Table 6

Pfam search results shows relative position, bit score and E-value for the identified protein family and domains within R. stricta phyE-like identified protein.

Family Description Entry type Clan Envelope Alignment HMM HMM length Bit score E-value
Start End Start End From To
N-terminal domain NTE N-terminal extension Region N/A 1 67 1 67 N/A N/A 67 138 3.00E−36
PAS_2 PAS fold Domain CL0183 83 196 83 196 1 110 110 136.7 3.70E−40
GAF GAF domain Domain CL0161 229 371 231 368 3 126 154 104.8 4.30E−30
PHY Phytochrome region Family CL0161 387 566 387 563 1 180 183 237.3 6.10E−71
C-terminal domain PAS PAS fold Domain CL0183 593 709 594 709 2 113 113 85.3 2.30E−24
PAS PAS fold Domain CL0183 724 846 725 844 2 111 113 57 1.40E−15
HisK Histidine kinase-related (phospho-acceptor) Domain CL0025 865 924 869 921 5 59 68 27.9 1.70E−06
Fig. 2

Domain structure of the three phytochrome-like proteins of R. stricta displayed as a Pfam diagram. The figure shows relative position for the identified protein family and N-terminal and C-terminal domains within the three identified proteins, i.e. RsphyA-like (a) RsphyC-like (b) and RsphyE-like (c). Domains are connected by a flexible hinge region (H). Color online.

3.3 Multi-sequence alignment (MSA) and phylogenetic analysis

The best BLASTp search hits were used to perform multi-sequence alignment. This resulted in 30 sequences from 23 different species. Alignment of the 30 sequences was obtained by gap open penalty of 10 and gap extension penalty of one. Alignment of R. stricta phytochromes-like proteins and the 30 amino acid sequences from the BLASTp search showed high sequence identity (Table 7). Phylogenetic relationships among these sequences showed that each phytochrome-like gene of the R. stricta grouped with the same type of subunit in the other plant species (Fig. 3).

Table 7

Information for 30 plant phytochrome proteins used for multi-sequence alignment.

No. Accession Description Latin name Length (aa)
1 AAF66603 phytochrome C Oryza sativa Indica Group 1137
2 AAP06790 phytochrome C1 apoprotein Zea mays 1135
3 AAR33026 phytochrome C Sorghum bicolor subsp. Verticilliflorum 1135
4 AAU06208 phytochrome C Triticum aestivum 1139
5 ABA46868 phytochrome A Solanum tuberosum 1123
6 ABB13327 phytochrome C Hordeum vulgare subsp. vulgare 1139
7 ACC60969 phytochrome A Vitis riparia 1124
8 ACC60971 phytochrome C Vitis riparia 1123
9 ACC60972 phytochrome E Vitis riparia 1124
10 AEA50880 phytochrome A Populus tremula 958
11 AEK26583 phytochrome A Populus tremula 1109
12 BAA99410 phytochrome A Armoracia rusticana 1122
13 BAH79238 phytochrome A Cardamine nipponica 1122
14 BAH79243 phytochrome A Cardamine resedifolia 1122
15 BAM67032 phytochrome A Chrysanthemum seticuspe f. boreale 1121
16 BAN14712 phytochrome E Lotus japonicas 943
17 P33530 Phytochrome A1 Nicotiana tabacum 1124
18 P55004 Phytochrome E Ipomoea nil 1115
19 XP_002271671 PREDICTED: phytochrome E Vitis vinifera 1124
20 XP_002512596 phytochrome A Ricinus communis 1124
21 XP_002519749 phytochrome B Ricinus communis 1131
22 XP_003535030 PREDICTED: phytochrome E-like Glycine max 1120
23 XP_003559446 PREDICTED: phytochrome C-like Brachypodium distachyon 1140
24 XP_003595571 Phytochrome E Medicago truncatula 1122
25 XP_004144620 PREDICTED: phytochrome C-like Cucumis sativus 1119
26 XP_004147430 PREDICTED: phytochrome E-like Cucumis sativus 1134
27 XP_004232975 PREDICTED: phytochrome E-like Solanum lycopersicum 1137
28 XP_004243570 PREDICTED: phytochrome C-like Solanum lycopersicum 1118
29 XP_004295419 PREDICTED: phytochrome E-like Fragaria vesca subsp. vesca 1162
30 XP_004303565 PREDICTED: phytochrome C-like Fragaria vesca subsp. vesca 1122
Fig. 3

Phylogenetic analysis of 33 phytochromes including those of R. stricta phytochrome proteins. Color online.

4 Discussion

Using local BLASTn searches, three phy-like genes (i.e., RsphyA-, RsphyC- and RsphyD-like) were identified in Rhazya stricta. By applying more sensitive protein search tools, such as GeneMark.hmm, it was possible to identify the start and stop codons as well as the exon and intron boundaries of phy-like homologues in R. stricta (Fig. 1). Domain searches confirmed that both the C-terminal and N-terminal domains are conserved for all three Rhazya phy-like genes (Fig. 2). phyA and phyB proteins have been shown to be the primary mediators of the phytochrome-mediated development (reviewed in Mathews [41]).

The fact that phyB gene was not detected in Rstricta could be the result of the protein sequence being too divergent from Arabidopsis to be detected by BLAST. This explanation seems unlikely because of the high amino acid sequence identity between the three detected phytochrome genes of Rhazya and other angiosperms (96–99%). Alternatively, phyB protein may be absent from Rhazya and one of the other subunits has taken over its function. Rhazya stricta grows in extremely high light intensities in the deserts of western Asia and these conditions may have affected the evolutionary history of phytochrome genes in this plant species. Earlier studies of single and double mutants for phyA and phyB genes have demonstrated redundancy of function between both genes [42], supporting the hypothesis that that phyA may have taken over the function of phyB in Rhazya. Other studies on transcriptional profiling of etiolated phyB mutants subjected to red (R) wavelengths showed similar response to that of the wild-type controls [43,44]. These results suggest that other phytochrome family member(s) may be predominantly responsible for perception and transduction of R light in the absence of phyB protein. Recently, phyA protein was shown to play a dominant role in regulating rapid gene expression responses to R light [45]. These and other more recent studies indicate a shuttle role for phyA protein in response to R light, which is normally masked in the presence of phyB [46]. Thus, we propose that absence of phyB gene in R. stricta has resulted in RsphyA taking over the role of phyB.

Phytochromes play both unique and overlapping roles across the life stages of plants to regulate a range of developmental processes especially at seed germination and seedling stages. The latter stage is known for accumulating the higher levels of alkaloids useful for different pharmaceutical purposes in another medicinally important plant Pinellia ternata (Araceae) [47]. The present study provides valuable information for future manipulations of phytochrome gene expression at the seedling stage to enhance resources for better understanding alkaloids and their potential medicinal/pharmaceutical use in Rhazya stricta.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

The authors gratefully acknowledge the financial support from King Abdulaziz University (KAU) Vice President for Educational Affairs Prof. Dr. Abdulrahman O. Alyoubi and the KAU Deanship of Scientific Research, Jeddah, Saudi Arabia, represented by the Unit of Strategic Technologies Research through the Project number 431/008-D for the Project entitled: “Environmental Meta-Genomics and Biotechnology of Rhazya stricta and its Associated Microbiota”.


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