The RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor.

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named  RING  ZINC  FINGER PROTEIN  1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.

The NAC gene family in the halophyte Limonium bicolor: Identification, expression analysis, and regulation of abiotic stress tolerance.

NAC transcription factors regulate plant growth, development, and stress responses. However, the number, types, and biological functions of Limonium bicolor LbNAC genes have remained elusive. L. bicolor secretes excessive salt ions through salt glands on its stems and leaves to reduce salt-induced damage. Here, we identified 63 NAC members (LbNAC1-63) in L. bicolor, which were unevenly distributed across eight chromosomes. Cis-elements in the LbNAC promoters were related to growth and development, stress responses, and phytohormone responses. We observed strong colinearity between LbNACs and GmNACs from soybean (Glycine max). Thus, LbNAC genes may share similar functions with GmNAC genes. Expression analysis indicated that 16 LbNAC genes are highly expressed in roots, stems, leaves, and flowers, whereas 17 LbNAC genes were highly expressed throughout salt gland development, suggesting that they may regulate this developmental stage. Silencing LbNAC54 in L. bicolor decreased salt gland density, salt secretion from leaves, and overall salt tolerance. In agreement, genes related to salt gland development were significantly downregulated in LbNAC54-silenced lines. Our findings shed light on LbNAC genes and help elucidate salt gland development and salt secretion in L. bicolor. Our data also provide insight into NAC functions in halophytes.

The transmembrane protein LbRSG from the recretohalophyte Limonium bicolor enhances salt gland development and salt tolerance.

Salt glands are the unique epidermal structures present in recretohalophytes, plants that actively excrete excess Na+ by salt secretory structures to avoid salt damage. Here, we describe a transmembrane protein that localizes to the plasma membrane of the recretohalophyte Limonium bicolor. As virus-induced gene silencing of the corresponding gene LbRSG in L. bicolor decreased the number of salt glands, we named the gene Reduced Salt Gland. We detected LbRSG transcripts in salt glands by in situ hybridization and transient transformation. Overexpression and silencing of LbRSG in L. bicolor pointed to a positive role in salt gland development and salt secretion by interacting with Lb3G16832. Heterologous LbRSG expression in Arabidopsis enhanced salt tolerance during germination and the seedling stage by alleviating NaCl-induced ion stress and osmotic stress after replacing or deleting the (highly) negatively charged region of extramembranous loop. After screened by immunoprecipitation-mass spectrometry and verified using yeast two-hybrid, PGK1 and BGLU18 were proposed to interact with LbRSG to strengthen salt tolerance. Therefore, we identified (highly) negatively charged regions in the extramembrane loop that may play an essential role in salt tolerance, offering hints about LbRSG function and its potential to confer salt resistance.

Exogenous 6-BA enhances salt tolerance of Limonium bicolor by increasing the number of salt glands.

KEY MESSAGE: Exogenous 6-BA can increase endogenous hormone content, improve photosynthesis, decrease Na+ by increasing leaf salt gland density and salt secretion ability, and reduce ROS content so that it can promote L. bicolor growth. 6-benzyl adenine (6-BA) is an artificial cytokinin and has been widely applied to improving plant adaptation to stress. However, it is rarely reported that 6-BA alleviates salt damage of halophytes. In this paper, we treated Limonium bicolor seedlings, a recretohalophyte with high medicinal and ornamental values, with 300 mM NaCl and different concentrations of 6-BA (0.5, 1.0, and 1.5 mg/L) and measured plant growth, physiological index, the density of salt gland, and the salt secretion ability of leaves. The results showed that exogenous applications 1.0 mg/L 6-BA significantly improved plant growth and photosynthesis, increased cytokinin and auxins contents, K+ and organic soluble matter contents, the activities of SOD, CAT, APX, and POD, and decreased Na+, H2O2, and O2- contents compared to that treated with 300 mM NaCl. Further research showed that exogenous 6-BA significantly increased the density of salt gland and the salt secretion ability of leaves by upregulating the expression of the salt gland developmental genes, therefore, can secrete more excess Na+, and thus reduces the Na+ concentration in leaves, which can alleviate Na+ damage to the species. In all, exogenous 1.0 mg/L 6-BA can increase endogenous hormone, improve photosynthesis, decrease Na+ by increasing secretion ability, and reduce ROS content of L. bicolor so that it can improve the growth. These results above systematically prove the new role of 6-BA in salt tolerance of L. bicolor.

Molecular characterization of a novel cytorhabdovirus infecting Plumbago indica L.

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3' leader-N-(P')-P-P3-M-G-P6-L-5' trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus.

Characteristics of plastid genomes in the genus Ceratostigma inhabiting arid habitats in China and their phylogenomic implications.

BACKGROUND: Ceratostigma, a genus in the Plumbaginaceae, is an ecologically dominant group of shrubs, subshrub and herb mainly distributed in Qinghai-Tibet Plateau and North China. Ceratostigma has been the focal group in several studies, owing to their importance in economic and ecological value and unique breeding styles. Despite this, the genome information is limited and interspecific relationships within the genus Cerotastigma remains unexplored. Here we sequenced, assembled and characterized the 14 plastomes of five species, and conducted phylogenetic analyses of Cerotastigma using plastomes and nuclear ribosomal DNA (nrDNA) data. RESULTS: Fourteen Cerotastigma plastomes possess typical quadripartite structures with lengths from 164,076 to 168,355 bp that consist of a large single copy, a small single copy and a pair of inverted repeats, and contain 127-128 genes, including 82-83 protein coding genes, 37 transfer RNAs and eight ribosomal RNAs. All plastomes are highly conservative and similar in gene order, simple sequence repeats (SSRs), long repeat repeats and codon usage patterns, but some structural variations in the border of single copy and inverted repeats. Mutation hotspots in coding (Pi values > 0.01: matK, ycf3, rps11, rps3, rpl22 and ndhF) and non-coding regions (Pi values > 0.02: trnH-psbA, rps16-trnQ, ndhF-rpl32 and rpl32-trnL) were identified among plastid genomes that could be served as potential molecular markers for species delimitation and genetic variation studies in Cerotastigma. Gene selective pressure analysis showed that most protein-coding genes have been under purifying selection except two genes. Phylogenetic analyses based on whole plastomes and nrDNA strongly support that the five species formed a monophyletic clade. Moreover, interspecific delimitation was well resolved except C. minus, individuals of which clustered into two main clades corresponding to their geographic distributions. The topology inferred from the nrDNA dataset was not congruent with the tree derived from the analyses of the plastid dataset. CONCLUSION: These findings represent the first important step in elucidating plastome evolution in this widespread distribution genus Cerotastigma in the Qinghai-Tibet Plateau. The detailed information could provide a valuable resource for understanding the molecular dynamics and phylogenetic relationship in the family Plumbaginaceae. Lineage genetic divergence within C. minus was perhaps promoted by geographic barriers in the Himalaya and Hengduan Mountains region, but introgression or hybridization could not be completely excluded.

Ovule Transcriptome Analysis Discloses Deregulation of Genes and Pathways in Sexual and Apomictic Limonium Species (Plumbaginaceae).

The genus Limonium Mill. (sea lavenders) includes species with sexual and apomixis reproductive strategies, although the genes involved in these processes are unknown. To explore the mechanisms beyond these reproduction modes, transcriptome profiling of sexual, male sterile, and facultative apomictic species was carried out using ovules from different developmental stages. In total, 15,166 unigenes were found to be differentially expressed with apomictic vs. sexual reproduction, of which 4275 were uniquely annotated using an Arabidopsis thaliana database, with different regulations according to each stage and/or species compared. Gene ontology (GO) enrichment analysis indicated that genes related to tubulin, actin, the ubiquitin degradation process, reactive oxygen species scavenging, hormone signaling such as the ethylene signaling pathway and gibberellic acid-dependent signal, and transcription factors were found among differentially expressed genes (DEGs) between apomictic and sexual plants. We found that 24% of uniquely annotated DEGs were likely to be implicated in flower development, male sterility, pollen formation, pollen-stigma interactions, and pollen tube formation. The present study identifies candidate genes that are highly associated with distinct reproductive modes and sheds light on the molecular mechanisms of apomixis expression in Limonium sp.

Enhanced Na+ and Cl- sequestration and secretion selectivity contribute to high salt tolerance in the tetraploid recretohalophyte Plumbago auriculata Lam.

MAIN CONCLUSION: Enhanced secretion of Na+ and Cl- in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl-, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata. Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300 mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl- in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl- secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl-(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.

A first look at sea-lavenders genomics - can genome wide SNP information tip the scales of controversy in the Limonium vulgare species complex?

BACKGROUND: Sea-lavenders (Limonium Mill., Plumbaginaceae) are a cosmopolitan group of diploid and polyploid plants often adapted to extreme saline environments, with a mostly Tethyan distribution, occurring in the Mediterranean, Irano-Turanian, Euro-Siberian and in the New World. The halophylic Limonium vulgare polyploid complex in particular, presents a large distribution throughout extreme salt-marsh habitats and shows little morphological but high taximetric variation, frequently blurring species delimitation. In this work we pursue three main goals: assert whether SNP data from polyploid individuals has the resolution to distinguish the seven sampled species, to better understand how genetically structured Limonium vulgare is, and attempt to identify specific molecular mechanisms for the differentiation between L. maritimum and L. vulgare. For this purpose, 95 individuals were genotyped using Genotyping by Sequencing (GBS), which were assembled as two independent datasets using IPYRAD. All analyses performed downstream of assembly were fully automated. Phylogenetic inference, PCA, and admixture plots were used to infer answers to the study's main goals. RESULTS: Close to 10,000 SNPs were obtained for each dataset. Phylogenetic analyses reveal that polyploid data can be used to infer species relationships. Population structure analyses suggest a genetically structured L. vulgare. A set of 34 SNPs were found to be fully segregated between L. vulgare and L. maritimum, two of which are potentially linked to proteins that might be involved in the speciation process. CONCLUSION: Despite polyploid data analyses shortcomings, GBS generated SNPs have the resolution to discern all seven included species. Limonium vulgare revealed pronounced genetic structure along a geographical north-south cline. L. maritimum always appears as a distinct genetic entity. Segregated SNPs between L. vulgare and L. maritimum indicate salinity response and morphological trait control genes as potentially interesting to follow up for studying these species' divergence process.

Bracelet salt glands of the recretohalophyte Limonium bicolor: Distribution, morphology, and induction.

Halophytes complete their life cycles in saline environments. The recretohalophyte Limonium bicolor has evolved a specialized salt secretory structure, the salt gland, which excretes Na+ to avoid salt damage. Typical L. bicolor salt glands consist of 16 cells with four fluorescent foci and four secretory pores. Here, we describe a special type of salt gland at the base of the L. bicolor leaf petiole named bracelet salt glands due to their beaded-bracelet-like shape of blue auto-fluorescence. Bracelet salt glands contain more than 16 cells and more than four secretory pores. Leaf disc secretion measurements and non-invasive micro-test techniques indicated that bracelet salt glands secrete more salt than normal salt glands, which helps maintain low Na+ levels at the leaf blade to protect the leaf. Cytokinin treatment induced bracelet salt gland differentiation, and the developed ones showed no further differentiation when traced with a living fluorescence microscopy imager, even though new salt gland development and leaf expansion were observed. Transcriptome revealed a NAC transcription factor gene that participates in bracelet salt gland development, as confirmed by its genome editing and overexpression in L. bicolor. These findings shed light on bracelet salt gland development and may facilitate the engineering of salt-tolerant crops.

Functional genomics-enabled characterization of CYP81B140 and CYP81B141 from Plumbago zeylanica L. substantiates their involvement in plumbagin biosynthesis.

MAIN CONCLUSION: Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.

Lb1G04202, an Uncharacterized Protein from Recretohalophyte Limonium bicolor, Is Important in Salt Tolerance.

With global increases in saline soil, it has become increasingly important to decipher salt-tolerance mechanisms and identify strategies to improve salt tolerance in crops. Halophytes complete their life cycles in environments containing ≥200 mM NaCl; these remarkable plants provide a potential source of genes for improving crop salt tolerance. Recretohalophytes such as Limonium bicolor have salt glands that secrete Na+ on their leaf epidermis. Here, we identified Lb1G04202, an uncharacterized gene with no conserved domains, from L. bicolor, which was highly expressed after NaCl treatment. We confirmed its expression in the salt gland by in situ hybridization, and then heterologously expressed Lb1G04202 in Arabidopsis thaliana. The transgenic lines had a higher germination rate, greater cotyledon growth percentage, and longer roots than the wild type (WT) under NaCl treatments (50, 100 and 150 mM). At the seedling stage, the transgenic lines grew better than the WT and had lower Na+ and malonyldialdehyde accumulation, and higher K+ and proline contents. This corresponded with the high expression of the key proline biosynthesis genes AtP5CS1 and AtP5CS2 under NaCl treatment. Isotonic mannitol treatment showed that Lb1G04202 overexpression significantly relieved osmotic stress. Therefore, this novel gene provides a potential target for improving salt tolerance.

The genome of the recretohalophyte Limonium bicolor provides insights into salt gland development and salinity adaptation during terrestrial evolution.

Halophytes have evolved specialized strategies to cope with high salinity. The extreme halophyte sea lavender (Limonium bicolor) lacks trichomes but possesses salt glands on its epidermis that can excrete harmful ions, such as sodium, to avoid salt damage. Here, we report a high-quality, 2.92-Gb, chromosome-scale L. bicolor genome assembly based on a combination of Illumina short reads, single-molecule, real-time long reads, chromosome conformation capture (Hi-C) data, and Bionano genome maps, greatly enriching the genomic information on recretohalophytes with multicellular salt glands. Although the L. bicolor genome contains genes that show similarity to trichome fate genes from Arabidopsis thaliana, it lacks homologs of the decision fate genes GLABRA3, ENHANCER OF GLABRA3, GLABRA2, TRANSPARENT TESTA GLABRA2, and SIAMESE, providing a molecular explanation for the absence of trichomes in this species. We identified key genes (LbHLH and LbTTG1) controlling salt gland development among classical trichome homologous genes and confirmed their roles by showing that their mutations markedly disrupted salt gland initiation, salt secretion, and salt tolerance, thus offering genetic support for the long-standing hypothesis that salt glands and trichomes may share a common origin. In addition, a whole-genome duplication event occurred in the L. bicolor genome after its divergence from Tartary buckwheat and may have contributed to its adaptation to high salinity. The L. bicolor genome resource and genetic evidence reported in this study provide profound insights into plant salt tolerance mechanisms that may facilitate the engineering of salt-tolerant crops.

Melatonin increases growth and salt tolerance of Limonium bicolor by improving photosynthetic and antioxidant capacity.

BACKGROUND: Soil salinization is becoming an increasingly serious problem worldwide, resulting in cultivated land loss and desertification, as well as having a serious impact on agriculture and the economy. The indoleamine melatonin (N-acetyl-5-methoxytryptamine) has a wide array of biological roles in plants, including acting as an auxin analog and an antioxidant. Previous studies have shown that exogenous melatonin application alleviates the salt-induced growth inhibition in non-halophyte plants; however, to our knowledge, melatonin effects have not been examined on halophytes, and it is unclear whether melatonin provides similar protection to salt-exposed halophytic plants. RESULTS: We exposed the halophyte Limonium bicolor to salt stress (300 mM) and concomitantly treated the plants with 5 µM melatonin to examine the effect of melatonin on salt tolerance. Exogenous melatonin treatment promoted the growth of L. bicolor under salt stress, as reflected by increasing its fresh weight and leaf area. This increased growth was caused by an increase in net photosynthetic rate and water use efficiency. Treatment of salt-stressed L. bicolor seedlings with 5 µM melatonin also enhanced the activities of antioxidants (superoxide dismutase [SOD], peroxidase [POD], catalase [CAT], and ascorbate peroxidase [APX]), while significantly decreasing the contents of hydrogen peroxide (H2O2), superoxide anion (O2•-), and malondialdehyde (MDA). To screen for L. bicolor genes involved in the above physiological processes, high-throughput RNA sequencing was conducted. A gene ontology enrichment analysis indicated that genes related to photosynthesis, reactive oxygen species scavenging, the auxin-dependent signaling pathway and mitogen-activated protein kinase (MAPK) were highly expressed under melatonin treatment. These data indicated that melatonin improved photosynthesis, decreased reactive oxygen species (ROS) and activated MAPK-mediated antioxidant responses, triggering a downstream MAPK cascade that upregulated the expression of antioxidant-related genes. Thus, melatonin improves the salt tolerance of L. bicolor by increasing photosynthesis and improving cellular redox homeostasis under salt stress. CONCLUSIONS: Our results showed that melatonin can upregulate the expression of genes related to photosynthesis, reactive oxygen species scavenging and mitogen-activated protein kinase (MAPK) of L. bicolor under salt stress, which can improve photosynthesis and antioxidant enzyme activities. Thus melatonin can promote the growth of the species and maintain the homeostasis of reactive oxygen species to alleviate salt stress.

A novel gene LbHLH from the halophyte Limonium bicolor enhances salt tolerance via reducing root hair development and enhancing osmotic resistance.

BACKGROUND: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance. RESULTS: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance. CONCLUSION: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.

Molecular cloning and characterization of type III polyketide synthase from Plumbago zeylanica.

Two cDNAs encoding type Ш polyketide synthase (PKS1) and chalcone synthase (CHS, PKS2), were cloned from fresh leaves of Plumbago zeylanica L. (P. zeylanica). Their heterologous expression revealed that PKS1 catalyzed the formation of five α-pyrones from three to six acetate units by accepting acetyl-CoA and malonyl-CoA. In contrast, PKS2 catalyzed the formation of naringenin and bisnoryangonin by accepting p-coumaroyl-CoA and malonyl-CoA. Naringenin is thought to be involved in the biosynthesis of various bioactive flavonoids. PKS2 can be used to molecular breeding to enhance the production of these useful secondary metabolites via its overexpression.[Formula: see text].

Heterologous expression of the Limonium bicolor MYB transcription factor LbTRY in Arabidopsis thaliana increases salt sensitivity by modifying root hair development and osmotic homeostasis.

Arabidopsis thaliana TRY is a negative regulator of trichome differentiation that promotes root hair differentiation. Here, we established that LbTRY, from the recretohalophyte Limonium bicolor, is a typical MYB transcription factor that exhibits transcriptional activation activity and locates in nucleus. By in situ hybridization in L. bicolor, LbTRY may be specifically positioned in salt gland of the expanded leaves. LbTRY expression was the highest in mature leaves and lowest under NaCl treatment. For functional assessment, we heterologously expressed LbTRY in wild-type and try29760 mutant Arabidopsis plants. Epidermal differentiation was remarkably affected in the transgenic wild-type line, as was increased root hair development. Complementation of try29760 with LbTRY under both 35S and LbTRY specific promoter restored the wild-type phenotype. qRT-PCR analysis suggested that AtGL3 and AtZFP5 promote root hair cell fate in lines heterologously producing LbTRY. In addition, four genes (AtRHD6, AtRSL1, AtLRL2, and AtLRL3) involved in root hair initiation and elongation were upregulated in the transgenic lines. Furthermore, LbTRY specifically increased the salt sensitivity of the transgenic lines. The transgenic and complementation lines showed poor germination rates and reduced root lengths, whereas the mutant unexpectedly fared the best under a range of NaCl treatments. Under salt stress, the transgenic seedlings accumulated more MDA and Na+ and less proline and soluble sugar than try29760. Thus, when heterologously expressed in Arabidopsis, LbTRY participates in hair development, similar to other MYB proteins, and specifically reduces salt tolerance by increasing ion accumulation and reducing osmolytes. The expression of salt-tolerance marker genes (SOS1, SOS2, SOS3 and P5CS1) was significant reduced in the transgenic lines. More will be carried by downregulating expression of TRY homologs in crops to improve salt tolerance.

Exogenous melatonin enhances salt secretion from salt glands by upregulating the expression of ion transporter and vesicle transport genes in Limonium bicolor.

BACKGROUND: Salt, a common environmental stress factor, inhibits plant growth and reduces yields. Melatonin is a pleiotropic molecule that regulates plant growth and can alleviate environmental stress in plants. All previous research on this topic has focused on the use of melatonin to improve the relatively low salt tolerance of glycophytes by promoting growth and enhancing antioxidant ability. It is unclear whether exogenous melatonin can increase the salt tolerance of halophytes, particularly recretohalophytes, by enhancing salt secretion from the salt glands. RESULTS: To examine the mechanisms of melatonin-mediated salt tolerance, we explored the effects of exogenous applications of melatonin on the secretion of salt from the salt glands of Limonium bicolor (a kind of recretohalophyte) seedlings and on the expression of associated genes. A pretreatment with 5 µM melatonin significantly improved the growth of L. bicolor seedlings under 300 mM NaCl. Furthermore, exogenous melatonin significantly increased the dry weight and endogenous melatonin content of L. bicolor. In addition, this treatment reduced the content of Na+ and Cl- in leaves, but increased the K+ content. Both the salt secretion rate of the salt glands and the expression level of genes encoding ion transporters (LbHTK1, LbSOS1, LbPMA, and LbNHX1) and vesicular transport proteins (LbVAMP721, LbVAP27, and LbVAMP12) were significantly increased by exogenous melatonin treatment. These results indicate that melatonin improves the salt tolerance of the recretohalophyte L. bicolor via the upregulation of salt secretion by the salt glands. CONCLUSIONS: Our results showed that melatonin can upregulate the expression of genes encoding ion transporters and vesicle transport proteins to enhance salt secretion from the salt glands. Combining the results of the current study with previous research, we formulated a novel mechanism by which melatonin increases salt secretion in L. bicolor. Ions in mesophyll cells are transported to the salt glands through ion transporters located at the plasma membrane. After the ions enter the salt glands, they are transported to the collecting chamber adjacent to the secretory pore through vesicle transport and ions transporter and then are secreted from the secretory pore of salt glands, which maintain ionic homeostasis in the cells and alleviate NaCl-induced growth inhibition.

Differential transcriptome and metabolome analysis of Plumbago zeylanica L. reveal putative genes involved in plumbagin biosynthesis.

Plumbagin is a pharmacologically active naphthoquinone present in the Plumbago zeylanica L. having important medicinal properties. The root of P. zeylanica is rich and primary tissue of the plumbagin biosynthesis and accumulation. The complete biosynthetic pathway of plumbagin in plant is still obscure. The present study attempts to understand the plumbagin biosynthetic pathway with the help of differential transcriptome and metabolome analysis of P. zeylanica leaf and root. The transcriptome data showed co-expression of Aldo-keto reductase (PzAKR), Polyketide cyclase (Pzcyclase) and Cytochrome P450 (PzCYPs) transcripts along with the Polyketide synthase (PzPKS) transcripts. Their higher expression in root as compared to leaf supports their possible involvement in plumbagin biosynthesis. The metabolome data of leaf and root revealed naphthalene derivative isoshinanolone that could be potential precursor of plumbagin. Pathway elucidation and transcriptome data of P. zeylanica, will enable and accelerate research on naphthoquinone biosynthesis in plants.

Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production.

Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7-9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 µM acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and ½ MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. ½ MS-B5 liquid medium containing 30 g L-1 sucrose incubated in the dark resulted in the efficient biomass production of transformed hairy roots (12.5 g fresh weight, 1.8 g dry weight) with 3.2 mg g-1 DW plumbagin accumulation. This procedure provides a framework for large-scale cultivation of hairy roots for plumbagin production. This is the first report describing the establishment of P. europaea hairy root culture with special emphasis on plumbagin production.