Genome-wide identification of the AP2/ERF gene family from Limonium bicolor and functional characterization of LbAP2/ERF32 under salt stress.

AP2/ERF transcription factors (TFs) play important roles in plant growth and development, plant morphogenesis and response to environmental stresses. However, their biological roles in recretohalophytes are still not fully revealed. Limonium bicolor L. is a typical recretohalophyte, which secretes excessive salt ions through the salt glands on the epidermis. Here, 64 LbAP2/ERF genes were identified in L. bicolor genome, which were unevenly distributed on the eight chromosomes. Cis-elements related to growth and development, stress response and phytohormone response are distributed in multiple LbAP2/ERF promoters. Expression analysis indicated that LbAP2/ERF genes responsed to NaCl, PEG and ABA. And the salt gland density, salt secretion of leaves and overall salt tolerance of LbAP2/ERF32 silenced lines were significantly reduced. In agreement, the genes related to salt gland development and ion transport were significantly changed in LbAP2/ERF32-silenced lines. Our findings provided fundamental information on the structure and evolutionary relationship of LbAP2/ERF gene family in salt gland development and salt secretion of L. bicolor and gave theoretical guideline for further functional study of LbAP2/ERF genes in response to abiotic stress.

The WRKY gene family in the halophyte Limonium bicolor: identification, expression analysis, and regulation of salt stress tolerance.

KEY MESSAGE: 63 L. bicolor WRKY genes were identified and their informatics was analyzed. The results suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Salt stress, as a universal abiotic stress, severely inhibits the growth and development of plants. WRKY transcription factors play a vital role in plant growth and development, as well as in response to various stresses. Nevertheless, little is known of systematic genome-wide analysis of the WRKY genes in Limonium bicolor, a model recretohalophyte. In this study, 63 L. bicolor WRKY genes were identified (LbWRKY1-63), which were unevenly distributed across seven chromosomes and one scaffold. Based on the structural and phylogenetic characteristics, 63 LbWRKYs are divided into three main groups. Cis-elements in the LbWRKY promoters were related to growth and development, phytohormone responses, and stress responses. Colinearity analysis showed strong colinearity between LbWRKYs and GmWRKYs from soybean (Glycine max). Therefore, LbWRKY genes maybe have similar functions to GmWRKY genes. Expression analysis showed that 28 LbWRKY genes are highly expressed in roots, 9 in stems, 26 in leaves, and 12 in flowers and most LbWRKY genes responded to NaCl, ABA, and PEG6000. Silencing LbWRKY10 reduced salt gland density and salt secretion ability of leaves, and the salt tolerance of the species. Consistent with this, genes associated with salt gland development were markedly down-regulated in the LbWRKY10-silenced lines. Our findings suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Our research provides new insights into the functions of the WRKY family in halophytes.

Global analysis of key post-transcriptional regulation in early leaf development of Limonium bicolor identifies a long non-coding RNA that promotes salt gland development and salt resistance.

Limonium bicolor, known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils.

Global dynamics and cytokinin participation of salt gland development trajectory in recretohalophyte Limonium bicolor.

Salt gland is an epidermal Na+ secretory structure that enhances salt resistance in the recretohalophyte sea lavender (Limonium bicolor). To elucidate the salt gland development trajectory and related molecular mechanisms, we performed single-cell RNA sequencing of L. bicolor protoplasts from young leaves at salt gland initiation and differentiation stages. Dimensionality reduction analyses defined 19 transcriptionally distinct cell clusters, which were assigned into 4 broad populations-promeristem, epidermis, mesophyll, and vascular tissue-verified by in situ hybridization. Cytokinin was further proposed to participate in salt gland development by the expression patterns of related genes and cytological evidence. By comparison analyses of Single-cell RNA sequencing with exogenous application of 6-benzylaminopurine, we delineated 5 salt gland development-associated subclusters and defined salt gland-specific differentiation trajectories from Subclusters 8, 4, and 6 to Subcluster 3 and 1. Additionally, we validated the participation of TRIPTYCHON and the interacting protein Lb7G34824 in salt gland development, which regulated the expression of cytokinin metabolism and signaling-related genes such as GLABROUS INFLORESCENCE STEMS 2 to maintain cytokinin homeostasis during salt gland development. Our results generated a gene expression map of young leaves at single-cell resolution for the comprehensive investigation of salt gland determinants and cytokinin participation that helps elucidate cell fate determination during epidermis formation and evolution in recretohalophytes.

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.