The gene NCED1 encodes 9-cis-epoxycarotenoid dioxygenase, which catalyzes oxidative cleavage of 9-cis-epoxycarotenoids neoxanthin and violaxanthin to xanthoxin, a key step in the biosynthesis of abscisic acid in higher plants. In the present study, the complete NCED1 of 1 917 bp was cloned and characterized from rice (Oryza sativa L. cv. N22) as no earlier reports were available for its characterization from indica cultivar. The NCED1 had no intron and encoded a protein of 639 amino acids with a predicted molecular mass of 68.62 kD and pI of 6.07. The aliphatic index and grand average of hydropathicity were found to be 77.04 and -0.148, respectively. Multiple alignment analysis revealed that the sequence shared a high identity with the Oryza sativa japonica group (100 %) followed by Triticum aestivum (90 %), Hordeum vulgare (90 %), and Zea mays (89 %). The enzyme had a RPE65 domain of 476 amino acid residues. The RPE65 domain requires Fe(II) as a cofactor coordinated with 4 histidine residues and 3 glutamic acid residues. The phylogenic tree shows that NCED1 of japonica rice and NCED1 of indica rice were in the same group. They might have been evolved from a common ancestor. Analysis with a PSORT III tool shows that NCED is a chloroplastic protein. The real-time quantitative PCR and RNA-sequencing studies show that the expression of NCED1 was progressively reduced with increasing water stress, and a negative correlation between expression of OsNCED1 and severity of stress was established. Further, NCED1 expression negatively correlated with abscisic acid (ABA) accumulation under water stress whereas in some other species its expression increased along with ABA accumulation. This might be due to feedback inhibition of the ABA biosynthesis in rice.

Trehalose is a nonreducing disaccharide that is involved in the regulation of plant responses to a variety of environmental stresses. Trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP) are two key enzymes in trehalose synthesis and they are widely distributed in higher plants. At present, TPS family genes have been systematically identified and analyzed in many plant species, but the TPP family genes have been rarely studied. In this study, ten TPS and six TPP genes in cannabis (Cannabis sativa L.) were identified at the genomic level. The phylogenetic tree of TPS and TPP family members in cannabis, Arabidopsis, and rice was constructed, and all the genes were divided into three subgroups: Class I, Class II, and Class III. The number of exons and motif types among Class I members was exactly the same, as were Class II members, but the gene structure and motif types of Class III members were slightly different. There were four pairs of CsTPSs and CsTPPs that had gene duplication, indicating that gene duplication events played an important role in the amplification of TPS and TPP families in cannabis. The results of expression analysis under abiotic stresses showed that 68.75 % of CsTPS and CsTPP genes were significantly induced by at least one abiotic stress. Among these genes, the expression of CsTPS1, CsTPS9, and CsTPPA was highest under at least one abiotic stress. These three genes may play a key role in abiotic stress responses. Most of the CsTPS and CsTPP genes that are closely located in the evolutionary tree have the same or similar functions. To our knowledge, this is the first paper that systematically reports the TPS and TPP gene families in cannabis.

Martaban camphor [Cinnamomum parthenoxylon (Jack) Meisn] is a woody tree in India, China, Indonesia, Thailand, and Vietnam and has been widely utilized for commercial purposes. It is threatened due to fragmented habitats, over-deforestation, and oil extraction. To conserve this species, the investigation of genetic diversity and population structure of this species is essential. Herein, we analyzed 192 adult trees from eight populations covering its natural distribution range in Vietnam using ten polymorphic EST-SSR markers. Medium levels of genetic diversity (R = 2.7, H_o = 0.399, H_e = 0.426) and genetic differences between populations (F_st = 0.223) were determined. Two populations, Cuc Phuong and Xuan Nha have undergone recent bottlenecks. These results indicated that anthropogenic activities may be the major factor for the low heterozygosity and influenced the number of alleles in all C. parthenoxylon populations. Clustering analyses revealed three genetic clusters that related to gene flow between different areas. We proposed in situ conservation for some populations with high levels of allelic richness, genetic diversity, or private alleles. The collecting of the seeds of the remaining populations for ex-situ conservation could be performed.

Leek is an economically important vegetable. In model plants, the CONSTANS (CO) and CONSTANS-like (COL) genes play central roles in plant flowering modulation. However, none of leek CO homolog has been identified, because of limited gene resources obtained in this crop. Here, we reported the transcriptome analysis of leek, along with the identification of putative leek CONSTANS-like (COL) (ApCOL) genes. A total of 189 713 non-redundant transcripts were de novo assembled by using about 128.9 million clean sequence reads, of which, 48 621 were achieved for functional annotation. Thereafter, the search for putative ApCOL genes against the assembled transcripts was performed, and 17 genes were identified. The 17 putative ApCOL proteins, together with 16 function-known COL proteins published for other species, were subjected to phylogenetic analysis and divided into four groups. Some putative ApCOL members showed high sequence similarity with published COL proteins involved in flowering regulation. Expression analysis revealed that, among the 17 putative ApCOL genes, eight, two, and three genes showed higher expression in leaves, cauloids, and roots, respectively. The discovery of putative ApCOL genes and the characterization of their expression patterns will provide a basis for future clarification of their functions in leek growth and development.

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method used for unraveling gene functions. As an attractive alternative to mutant collections or stable transgenic plants, it has been widely used in reverse-genetics studies owing to its ease use and quick turnaround time. Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana. However, the conventional vector construction is difficult and the efficiencies of the infection methods are low. Here, we improved the vector construction and viral infection methods, inserted an inverted-repeat fragment of the phytoene desaturase gene into a TYMV-derived vector by homologous recombination and transformed Brassica rapa with plasmid DNA harboring a cDNA copy of the TYMV genome through particle bombardment. An apparent photobleaching phenotype was detected and efficient VIGS was induced. An 80-bp fragment was sufficient to produce VIGS in leaves, stems, roots, flowers, siliques, and stalks of B. rapa. Because TYMV has a wide host range in Brassica, the VIGS system described here will contribute to the improvement of high-throughput technology and efficient functional research in B. rapa and other Brassicaceae crops.

Strawberry tree (Arbutus unedo) is a small perennial tree that grows spontaneously in the Mediterranean basin, Ireland, and Portugal. In this work, strawberry tree clones were established in vitro from epicormic shoots obtained from a young tree, an adult tree, and from a seedling. They were propagated by axillary shoot buds proliferation on solid and in liquid media, and also in a modified De Fossard medium with 9 µM benzylaminopurine. The organogenesis from calli obtained from apical leaves of the in vitro grown shoots from the three genotypes was carried out in the same basal liquid medium supplemented with 9 µM thidiazuron. Micropropagation through organogenesis in liquid medium proved to be more efficient than the other tested methods (considering the number of shoots produced), but the shoots were showing hyperhydricity. Shoots were sucessufully rooted on medium with indole-3-butyric acid and acclimatized ex vitro with rates higher than 90 %. Six month-old plants from the most proliferative genotype (AU1) and propagated in vitro by different methods were submitted to drought stress (no watering for 10 d) and several morphological and physiological parameters were evaluated and compared to a control group (watered to 70 % field capacity). No significant differences were found in plant biomass, root length, and plant height, however, slight differences were observed in water potential, net photosynthetic rate, intercellular CO₂ concentration, and stomatal conductance between the plantlets propagated on solid or liquid medium. In general, the responses to drought stress imposed were was similar in plants micropropagated by different propagation methods.

Tea plant (Camellia sinensis) is a typical plant that accumulates abundant aluminum (Al) and fluoride (F). Al and F play vital roles in the growth and development in tea plant. MYB (myeloblastosis) is one of the largest transcription factor families in plant, which plays an important role in the stress responses. As the largest and multifunctional subfamily of MYB, R2R3-MYB subfamily participates in multiple abiotic stresses in plant. However, there are few studies about R2R3-MYB in tea plant, especially in the accumulation of Al and F during its growth. Here, we identified 43 CsMYB genes from the transcriptome database of tea plant and analyzed the relative expression in Al and F treatments. According to the Arabidopsis thaliana classification, 43 CsMYB genes are divided into 18 subgroups via phylogenetic tree analysis. All 43 CsMYB proteins have the typical R2R3-MYB domain by MEME prediction. Moreover, 9 CsMYB genes (CsMYB11, CsMYB17, CsMYB29, CsMYB38 to CsMYB43) that related to abiotic stress were selected from 43 CsMYB genes for a further study, and the relative expressions showed that they are tissue-specifically expressed. In addition, the results of different concentrations of Al treatments showed that CsMYB11, -17, -29, -38 and -41 are stimulated by Al treatment. The results of different concentrations of F treatments showed that the CsMYB11, -17, -38, -39, -40, -41, -42 and -43 participate in F accumulation of C. sinensis roots. Our research establishes a solid foundation for further investigation into the molecular functions of R2R3-MYB transcription factors in C. sinensis.

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.

Dendrobium williamsonii and Dendrobium cariniferum (Orchidaceae) are endangered perennial herbs, and they are very similar in morphology. Chloroplast genome sequencing technology provides a powerful tool for molecular analysis to get more infomation for phylogenetic analysis and identification of Dendrobium species. In this study, the complete chloroplast genomes of Dendrobium williamsonii and Dendrobium cariniferum were assembled and characterized using Illumina NovaSeq 6000. The genome sizes are 159 695 and 159 479 bp, including pairs of inverted repeats (27 055 and 27 024 bp) each separated by small single-copy regions (18 451 and 18 488 bp) and large single-copy regions (87 134 and 86 943 bp). The chloroplast genome overall GC content was 37.11% and 37.13%, respectively. Each chloroplast genome encoded the same number (147) of genes, including 88 protein-coding genes, 51 tRNA genes, and 8 rRNA genes. Comparative analysis of chloroplast genomes revealed the high degree of divergence included accD-psaL and ycf4 -cemA. The phylogenetic tree showed the two Dendrobium species formed only one small clade. A pair of primers that could effectively identify the two Dendrobium species were also screened. This study will provide theoretical basis for species identification, genetic breeding, and evolution of Dendrobium.

Tetracentron sinense Oliv, the only tall deciduous tree in the family Tetracentraceae, is listed as a national second-grade key protected plant in China. To reveal the effect of associated species, irradiance, and altitudes on photosynthetic capacity of T. sinense, photosynthetic physiological characteristics of T. sinense and its associated species Acer pictum and Pterocarya stenoptera were measured by a Li-6400 portable photosynthetic meter. The light saturation point (LSP), the maximum net photosynthetic rate of the P_N-PAR (P_Nmax), carboxylation efficiency (CE), the maximum net photosynthetic rate of the P_N-CO₂ (P^*_Nmax), carbon dioxide compensation point (CCP) and light respiration rate (R_p) of T. sinense in forest gap (FG) were higher than those in forest edge (FE) and understory (US). In FE, the net photosynthetic rate (P_N), light compensation point (LCP), LSP, P^*_Nmax of T. sinense were lower than those of Pterocarya stenoptera, while the LSP, P_Nmax, and saturation point of carbon dioxide (C_iast) of T. sinense in US were lower than those of Acer pictum and Pterocarya stenoptera. The specific leaf area (SLA) of T. sinense decreased with reduction in the irradiance. With increasing altitude, the P_Nmax, LSP, and SLA of young individuals of T. sinense (YT) increased; the LCP of YT or the LSP of mature individuals of T. sinense (MT) increased first and then decreased. The results showed that 1) the photosynthetic capacity and adaptability of T. sinense were better in FG than that in FE and US; 2) the photosynthetic capacity of T. sinense in FE and US was weaker than that of its associated species, and its ecological range of light adaptation was also narrower than that of its associated species, placing T. sinense at a competitive disadvantage, which may be one of the important reasons for its poor regeneration; and 3) the environmental conditions at higher altitude can contribute to the growth and survival of T. sinense. Therefore, active artificial intervention should be undertaken to expand area of forest gap for T. sinense and transplant its seedlings to higher altitude to promote growth and population regeneration of T. sinense.

Garlic mustard (Alliaria petiolata) is an herbaceous biennial plant native to Europe. In Ukraine, in addition to becoming a serious invader, garlic mustard can serve as a host to several viruses, which may affect agricultural crops. In view of this, the purpose of the study was to identify the virome of garlic mustard growing in Ukraine. Plant samples collected in Kyiv regions were tested for the presence of cucumber mosaic virus (CMV), turnip mosaic virus (TuMV), turnip yellow mosaic virus (TYMV), watermelon mosaic virus II (WMV), and turnip crinkle virus (TCV) by serological and/or molecular methods. According to the results found in the present study, symptomatic A. petiolata obtained in 2021 were infected with CMV (60%), TuMV (20%), or co-infected with CMV + TuMV (20%). TYMV, WMV II, and TCV were not detected in any of the collected samples. The cDNA fragments encoded the coat protein (CP) gene of CMV and TuMV were sequenced and named as CMV-Ap and TuMV-Ap, respectively. In phylogenetic analysis, the CMV-Ap closely resembled the German isolate MW582807 (Sarracenia sp.), with 99.8% nucleotide identity and belongs to subgroup II of CMV. In the phylogenetic tree, TuMV-Ap clustered with isolates AP017803, AP017764, AP017791, and JQ073722, and represented the highest identity (98.6%) to Iranian isolate IRNTRa9 (AP017803) from Rapistrum rugosum and Turkish isolate TUR49 (AP017872) from Raphanus raphanistrum. The sequences of CMV-Ap and TuMV-Ap were deposited in the GenBank under Accession Numbers MZ540213 and OM799323, respectively. The results obtained in the study indicate the important role of infected garlic mustard as alternative host and natural reservoir of CMV and TuMV from which these economically important viruses can spread to other wild and cultivated plants. This is the first molecular evidence of TuMV infection in A. petiolata from Ukraine.

Two ginseng species Panax vietnamensis and Panax stipuleanatus are precious medicinal plants restricted in several Vietnam provinces. They are very limited and endangered due to degraded habitats and over-harvesting. To preserve these two species, we used eight nuclear microsatellite markers to investigate genetic variability from the nine populations with 246 individuals for these two ginseng species. Our findings showed a moderate genetic heterozygosity in two species, P. vietnamensis (H_E = 0.386) and P. stipuleanatus (H_E = 0.342). Deficiency of heterozygosity was observed in all the studied populations of P. vietnamensis and three populations of P. stipuleanatus. Some populations had high allelic richness for both species. Private alleles were determined in all the studied populations of P. vietnamensis and two P. stipuleanatus populations. Genetic differentiation was low in two ginseng species. However, habitat loss, over-utilization and over-harvesting can be the main causes of reduced genetic heterozygosity. Neighbor-joining tree and discriminant analysis of principal components detected three major genetic groups. Finally, based on our findings, we propose in situ conservation of populations with high expected heterozygosity, allelic richness, and private alleles. Seed collection should be performed for ex-situ conservation as genetic pools in the future.

Microsatellite markers were developed for Dicksonia sellowiana (Dicksoniaceae), an overexploited and endangered tree-fern species native to Brazil. From an enriched genomic library, 11 primer pairs were selected and used to characterize 36 D. sellowiana individuals from six Brazilian populations. Eight primer pairs amplified dinucleotide and hexanucleotide repeats with two to ten alleles per locus; three primer pairs were monomorphic. For the set of polymorphic markers, the mean observed and expected heterozygosity ranged from 0.29 to 0.44 and from 0.27 to 0.56, respectively. Eight of the primer pairs were also successfully amplified for Cyathea vestita (Cyatheaceae). These molecular markers can be useful tools for genetic studies aiming to analyze the impact of deforestation and overexploitation on the population structure and genetic diversity of fern species from various botanical families.

Dehydration responsive element binding factor (DREB) is believed to be a stress-tolerance enhancer in plants. In the present study, a cold-binding factor (CBF)/DREB homologous gene NnDREB1 (XP_010242642.1) was isolated from lotus roots using rapid amplification of cDNA ends (RACE) and reverse transcription (RT)-PCR methods. Analysis of the deduced amino acid sequence and phylogeny classified NnDREB1 into the A-1 group of the DREB1 subfamily. Expression profiling using a quantitative PCR method revealed that NnRDEB1 was significantly induced by NaCl, mannitol, and polyethylene glycol, but not by low temperature and abscisic acid. To evaluate function of NnRDEB1, Arabidopsis thaliana was transformed with the NnDREB1 gene in a binary vector construct. The transgenic plants exhibited higher resistance to drought compared with the wild-type plants in terms of survival rates, dry and fresh masses, and chlorophyll content. In addition, overexpression of NnDREB1 resulted in higher germination rates compared with the wild type plants on MS medium containing mannitol. The expressions of downstream target stressrelated genes, including cold-regulated15B (COR15B), rare cold inducible 2B (RCI2B) and repeat domain 26 (RD26), were activated in the transgenic plants. Taken together, the results suggest that NnDREB1 might be an important protein in lotus root drought tolerance.

Pathogenesis-related (PR) proteins play key roles in plant disease resistance. Here, we isolated and characterized pathogenesis-related PR2 gene encoding β-1,3-glucanase from Brassica juncea and named it BjPR2 (GenBank accession number DQ359125). The amino acid sequence of BjPR2 showed ~99 % similarity with β-1,3-glucanase of Brassica rapa, B. napus, and B. oleracea. BjPR2 transcription was rapidly increased after Alternaria brassicae infection, salicylic acid application, and wounding, but the induction was delayed in response to jasmonic acid. To investigate the transcriptional regulation of BjPR2 gene, its promoter was isolated. In silico analysis of BjPR2 promoter showed cis-regulatory elements upstream of TATA and CAAT boxes responsive to defense, hormones, wounding, and plant developmental stage. Homozygous Arabidopsis thaliana lines were developed with plasmid construct having β-glucuronidase (GUS) reporter gene driven by BjPR2 promoter. The analysis of GUS protein in Arabidopsis lines showed that BjPR2 promoter drived distinct pattern of pathogen inducible expression after fungal infection (Alternaria brassicae, Erysiphe orontii), phytohormones, and wounding. It also showed age dependent and organ specific expressions. BjPR2 promoter drove strong GUS activity in Arabidopsis seedlings and showed organ specific expression at the later growth stages (lateral organ junctions, leaf serrate, base of siliques, and receptacle). Due to stress-inducible and tissue specific nature, the BjPR2 promoter can serve as a potential candidate in genetic engineering.

Woody plants faced multiple abiotic stresses in forest plantation that can influence their growth and development. Dalbergia odorifera T. Chen is a vulnerably endangered tree species, and references about its responses to abiotic stresses are very rare in literature. Furthermore, the mechanisms underlying the abiotic stress tolerance in plants induced by exogenous glycine betaine (GB) remains unclear. Indeed, the alternative oxidase (AOX) is one of the major components of antioxidant enzymatic machinery, and there are no studies that focused on the effects of GB on the amount of AOX protein in plants under drought or cold stresses. Thus, the aim of this study was to investigate the effect of exogenous GB on the phenotype, osmoprotectants, photosynthetic pigments, and antioxidant systems in D. odorifera under cold and drought stresses. The layout of the trial was a factorial experiment in a completely randomized design using two factors including abiotic stress (drought and cold) and GB. Moreover, the principal component analysis based on the measured parameters manifested how the selected ROS scavengers were dispersed throughout the treatments. The results showed huge beneficial impacts of GB on the phenotypic traits of D. odorifera; GB also influences positively the antioxidant machinery, photosynthetic pigments, redox-homeostasis, and water status in D. odorifera seedlings under both stresses. Moreover, exogenous GB affects more the AOX pathway in D. odorifera under cold stress than under drought stress.

Thioredoxins (Trx) are small multifunctional redox proteins that contain thioredoxin conserved domain and active site WCXXC. The Trx family has an important role in multiple processes, including electron transport, seed germination, redox regulation, biotic and abiotic stresses resistance, etc. Although Trx genes have been extensively characterized in some plants, they have not been reported in peanut until now. The identification of AhTrx genes provides potential candidate genes for studying their effects and regulatory mechanisms in peanut (Arachis hypogaea L.) growth and development, especially under aluminium (Al) stress. It is also helpful to further analyze the Al resistance pathway in plants. Seventy AhTrx genes were identified using a genome-wide search method and conservative domain analysis. Then the basic physicochemical properties, phylogenetic relationship, gene structure, chromosomal localization, and promoter prediction were studied by the bioinformatic methods. Furthermore, the expressions of AhTrx genes under different Al treatment times in two peanut cultivars were tested using a real-time quantitative polymerase chain reaction. Seventy AhTrx genes were identified and characterized. Phylogenetic tree analysis showed that all AhTrx members could be classified into 9 groups with different conserved domains. Motif 1 was found to exist in every sequence, with an active site. Furthermore, the gene structures showed that the AhTrx family was complicated and changeable during evolution. The chromosomal localization indicated that the distribution and density of the Trx family on 20 peanut chromosomes were uneven. Predictive promoter analysis indicated that AhTrx proteins might play a role in phytohormones synthesis and stress response. Finally, the expression patterns of the AhTrx genes showed that every gene was differently expressed under Al treatment in different peanut cultivars, some were obvious, others had no significant difference, some were at a high level, while others were at a low level. This study systematically identifies the Trx gene family in peanut, providing some candidates for further study on its effects and regulatory mechanism under Al stress in peanut.

Seasonal growth is characteristic for many tree species including almond. Varying conditions during the season are responsible for growth cessation, bud set, dormancy entry, cold hardening, and bud burst. Here, we report the characterization of an almond homologue of the Arabidopsis GIGANTEA (AtGI) gene (designated as PdGI, GenBank accession No. KJ502316). We propose a role for this gene in the transition to dormancy and cold acclimation. The complementary DNA (cDNA) sequence of PdGI was 4 322 bp long and contained an open reading frame of 3 512 bp. The deduced amino acid sequence of PdGI shared 76 % identity with AtGI. The expression of PdGI at ambient day/night temperatures of 22/20 ºC was differentially regulated under a 16-h or 12-h photoperiod, increasing during the day and decreasing after dusk. However, this diurnal regulation was disrupted when plants were transferred to cold (12 ºC) conditions. In addition, we have assessed the expression of PdGI and putative almond homologues of the downstream target genes CONSTANS (PdCO-like) and FLOWERING LOCUS T (PdFT-like) in flower buds and shoots from adult trees during the bud break period in autumn and early winter. Our results show a clear increase in transcript abundance towards anthesis, suggesting a role of these genes in flower development.

We isolated PaAP3, a homolog of the class B MADS-box transcription factor gene APETALA3 (AP3), from the monoecious plant London plane tree (Platanus acerifolia Willd.). PaAP3 encodes a protein that shares good levels of identity with class B genes from Arabidopsis thaliana (35 and 51 % identity with PISTILLATA (PI) and AP3, respectively), and also with class B genes of other woody species (59 % identity with PTD from Populus trichocarpa and 66 % with TraAP3 from Trochodendron aralioides). Reverse transcription polymerase chain reaction showed that PaAP3 was expressed in both the female and male flowers of P. acerifolia, but almost no signal was detected in the vegetative tissues or mature embryos. The PaAP3 expression in male flowers showed a relationship with developmental stage. There was a small transient increase during differentiation of the flower primordia in June, but maximal levels occurred during December when flower development appeared arrested. Increased PaAP3 expression was also detected in March of the following year, corresponding to meiotic divisions of the microspore mother cells, but this was lost by April when the pollen was mature.

Agarwood, the resin part of Aquilaria spp., is valued in medicine, perfumes, and incense. The most important components of agarwood are sesquiterpenes, which are produced only when a healthy tree is wounded. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes in Aquilaria sinensis (Lour.) Gilg, and it is a typical wound-inducible synthase. To elucidate its regulatory mechanism at the transcriptional level, a 978-bp sequence upstream of the translation initiation codon ATG of the promoter for ASS1 was cloned. Computational analysis revealed that this promoter contained many known cis-elements including several defense related transcriptional factor-binding boxes. To functionally validate the promoter, a 5' truncated fragment fused with the β-glucuronidase (GUS) reporter gene was used for generating stable transgenic Arabidopsis plants. The spatial and temporal expression patterns of GUS in transgenic Arabidopsis showed that the promoter of ASS1 was induced by mechanical wound and mainly expressed in vascular bundles. Subcellular localization showed that ASS1 localized in the nucleus and plasma membrane. Here, identification of the ASS1 promoter not only lays a foundation for studying its transcriptional regulation, but also provides clues for studying the synthesis mechanism of agarwood sesquiterpenes.

Masson pine (Pinus massoniana Lamb.) is an important tree species of high economic value in southern China, but osmotic stress threatens its growth and development. In this study, physiological measurements and RNA-Seq analysis were used to clarify the physiological and molecular responses of P. massoniana under osmotic stress. Osmotic treatment caused cell membrane damage and reactive oxygen species (ROS) accumulation in the tree seedlings, but it also increased their antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities and osmotic substances (soluble sugars, proline, and trehalose) content so as to adjust to osmotic stress conditions. A total of 1 789 differentially expressed genes (DEGs) were identified by transcriptome sequencing, of which 962 were up-regulated and 827 genes down-regulated. A series of stress-induced genes associated with signal transduction, ROS-scavenging, osmotic regulation, late embryogenesis abundant (LEA) protein, pentatricopeptide repeat-containing protein, and transcription factors' regulation were distinguishable. This detailed investigation of the stress-responsive genes and pathways provides new insight into molecular mechanism of abiotic stress response in P. massoniana. Further, this study's data can contribute to genetic engineering or molecular breeding efforts to enhance osmotic resistance in P. massoniana stands.

The Eucalyptus globulus is one of the important forest tree species which reveals enhanced tolerance to multiple abiotic stresses. The enhanced tolerance of E. globulus against low temperature is linked to the presence of enhanced accumulation of lysine-rich dehydrin proteins. The accumulation of dehydrin protein of Mr 10 kDa in response to multiple abiotic stresses prevents cells from dehydration. It is encoded by a novel dehydrin-10 (DHN-10) gene. In the present study, we have used the DHN-10 gene of E. globulus of Pakistani origin, which was cloned in a bacterial expression vector pET30(a) and sequenced. We have found the 315 bp long coding sequence of this gene which has been enrolled as Eucalyptus GZJ-2018 DHN-10 gene under GenBank accession number MG948256.1. The in silico studies have identified several differences of this gene from the earlier enrolled DHN-10 genes: the studied gene possessed higher amphipathic character because of the presence of five additional electrically charged amino acids (two positively charged histidine, one negatively charged glutamate, and two negatively charged aspartate residues) and one extra lysine residue. The studied DHN-10 gene has been successfully expressed in the BL21-DE3 expression strain of Escherichia coli and 10 kDa protein has been detected on the nitrocellulose membrane. Our study is the first report of the sub-cloning of the DHN-10 gene and its expression outside the Eucalyptus cell.

Aristotelia chilensis (Mol.) Stuntz is an evergreen species endemic to Chile. It grows in open areas or under tree canopy, and its leaves emerge in early spring and summer. The objective of this study was to determine changes in photosynthetic parameters, total phenol content (TPC), antioxidant activity, and anatomy of apical and basal leaves of A. chilensis during the year. Photosynthesis performance was determined by measuring electron transport rate (ETR), the quantum efficiency of photosystem II (F_v/F_m), photochemical quenching (qP), and non-photochemical quenching (NPQ) with a fluorimeter. Leaf extracts were analysed to determine TPC and antioxidant activity. The maximum ETR and qP were recorded in spring and summer when the photosynthetically active radiation (PAR) at midday was higher (1901 and 1968 µmol m^-2 s^-1, respectively) than in other parts of a year. The F_v/F_m had typical physiological values in both types of leaves (about 0.8 in all the seasons). Also the NPQ was not influenced by the kind of leaves and season of the year. In concordance, the basal spring leaves had the highest TPC values. In contrast, the highest values of antioxidant activity were recorded in basal winter leaves followed by basal spring leaves. The results suggested that an increase in PAR (spring) positively affected the antioxidant activity and TPC, which correlated with higher ETR and qP values. The apical leaves showed morphological adaptations during the year and areas of intercellular spaces and palisade parenchyma were larger than in the basal leaves.

Pogostemon cablin (Blanco) Benth is a medicinal plant rich in terpenoids and flavonoids. Currently, the molecular mechanism of flavonoid biosynthesis in P. cablin remains unclear. In this study, three copies of the phenylalanine ammonia lyase (PAL) gene designated as PcPAL1 - PcPAL3, one copy of the chalcone synthase (CHS) gene referred to as PcCHS, and four copies of the CHS-like gene referred to as PcCHSL1 - PcCHSL4 were isolated from P. cablin. PcPAL1, PcPAL2, and PcPAL3 comprised 2 136, 2 136, and 2 148 bp full-length open reading frames (ORFs) encoding 711, 711, and 715 amino acids, respectively. PcCHS, PcCHSL1, PcCHSL2, PcCHSL3, and PcCHSL4 contained 1 173, 1 176, 1 179, 1 173, and 1 170 bp ORFs encoding 390, 391, 392, 390, and 389 amino acids, respectively. A phylogenetic analysis indicates that PcPAL2 was closer to PcPAL3 than PcPAL1, and that PcCHS was clustered with other plant CHS genes but independently to the PcCHSLs subgroup. A sequence analysis shows that PcCHSLs diverged in functional residues when compared to plant CHSs including PcCHS, suggesting that PcCHSLs may be divergent in tertiary structures. A quantitative reverse-transcription polymerase chain reaction analysis indicates that the expression profiles of PcPALs, PcCHS, and PcCHSLs in leaves and stems differed among four cultivars. Our results suggest that divergence in sequence and expression profiles of the tested genes may contribute to the diversity in flavonoids among the four cultivars.

Phylogenetic analysis has become a common step in characterization of gene and protein sequences. However, despite the availability of numerous affordable and more-or-less intuitive software tools, construction of biologically relevant, informative phylogenetic trees remains a process involving several critical steps that are inherently non-algorithmic, i.e., dependent on decisions made by the user. These steps involve, but are not limited to, setting the aims of the phylogenetic study, choosing sequences to be analyzed, and selecting methods employed in sequence alignment construction, as well as algorithms and parameters used to construct the actual phylogenetic tree. This review aims towards providing guidance for these decisions, as well as illustrating common pitfalls and problems occurring during phylogenetic analysis of plant gene sequences.

The amount and composition of phytohormones, sugars, and some other leaf characteristics depending on a crop load were evaluated in apple (Malus domestica Borkh. cv. Ligol grafted on P 60 rootstock) trees in order to prevent biennial bearing. The crop load was adjusted to 12 (control, unthinned), 8, 4, and 0 (non-fruiting) inflorescences (or fruits) per cm² of trunk cross-sectional area (TCSA). Inflorescences were removed in May before flowering. Phytohormones were analyzed in axillary buds and leaves in September. Results show that, in contrast to the unthinned trees, thinning to 4 fruits cm^-2(TCSA) resulted in a significant decrease of yield per tree, but a significant increase of fruit mass, return bloom, and leaf area. The heavy crop load resulted in suppressed bloom in the following year. Composition and content of phytohormones was changed considerably. Moreover, thinning resulted in an increased hexose accumulation. Such data suggest that flowering inhibition depended on the phytohormones that were exported to buds and on sugar-hormone signalling cross-talk.

An essential step in the carotenoid biosynthesis pathway is the formation of phytoene by phytoene synthase (PSY). In this study, three new genes coding PSYs (CpPSYA, CpPSYB, and CpPSYC) were cloned from Cucurbita pepo and their expression patterns analysed in three cultivars of summer squash which had a different carotenoid content. The gene sequences had a high similarity with those from other plant species, and their predicted proteins were significantly different from each other. A phylogenetic analysis indicates that CpPSYA and CpPSYB shared a high homology and were also homologous with PSYs from others cucurbits, whereas CpPSYC was more closely related to orthologues from strawberry and carrot. An expression analysis revealed that CpPSYA had a higher expression in flowers compared to leaves and showed a differential expression during fruit development. The amount of CpPSYA transcript was higher in fruits with a higher carotenoid content than in those with a lower carotenoid content. However, CpPSYB and CpPSYC showed a relatively high expression in leaves, and their expression in fruits varied among the different cultivars and fruit tissues. These results suggest that the CpPSY genes were under different regulatory mechanisms and they may have different roles in C. pepo.

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.

Multidrug and toxic compound extrusion (MATE) proteins is a newly characterized transporter family in plants. However, knowledge of this family in systematic classification, molecular evolution, and expression patterns in plants is limited. In this study, MATE gene sequence, structure, and names as well as MATE protein size and subcellular localization in rice were analyzed using bioinformatics tools, chromosome localizations, and gene clusters. The function of MATE proteins was further elucidated on a basis of phylogenetic relationships. Using available transcriptomic data, the expression pattern and function of MATE were different in the selected organs and developments stages of rice. In addition, the relative abundance of OsMATE1 transcripts increased 3 h after copper treatment and so it was identified as a candidate gene for Cu tolerance in rice. This research provided basic data for further studies on MATE genes in rice and theoretical information about the biological function of MATE proteins.

The WRKY belongs to an important plant specific transcription factor families which are involved in response to various environmental stresses, as well as in growth and developmental processes. In the present report, a genome-wide identification and characterization of WRKY gene family in barley led to revision of HvWRKYs to 93 members. The phylogenetic tree was also reconstructed based on the full-length WRKY protein sequences in barley and Arabidopsis. HvWRKYs were classified into three major groups (I, II, and III) and group II was further divided to 5 subgroups (a to e). HvWRKYs were named after this classification. Interestingly, some specific motifs were discovered in subgroups IIa, IIb, and III. Analyzing the available microarray data revealed eight candidate WRKY genes which were up-regulated under drought and salinity stresses compared to the optimum conditions at seedling stage in barley. Expression profiles of these WRKY genes were validated by quantitative real-time PCR. All the investigated candidate genes (HvWRKY_IIc2, HvWRKY_III11, HvWRKY_IIb2, HvWRKY_IId4, HvWRKY_III23, HvWRKY_IIa5 and HvWRKY_IIc19) except for HvWRKY_I8 were significantly up-regulated by drought stress at the seedling stage in drought-tolerant genotype, indicating their role in drought tolerance. We hope the presented information would be helpful toward achieving drought tolerant cultivars through genetic engineering or molecular breeding.