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 abundant 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 four 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 scRNA-seq with exogenous application of 6-benzylaminopurine, we delineated five salt gland development-associated sub-clusters and defined salt gland specific differentiation trajectories from sub-clusters 8, 4, or 6 to sub-cluster 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.

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.

An uncharacterized gene Lb1G04794 from Limonium bicolor promotes salt tolerance and trichome development in Arabidopsis.

Halophytes can grow and reproduce in high-salinity environments, making them an important reservoir of genes conferring salt tolerance. With the expansion of saline soils worldwide, exploring the mechanisms of salt tolerance in halophytes and improving the salt tolerance of crops have become increasingly urgent. Limonium bicolor is a halophyte with salt glands that secrete excess Na+ through leaves. Here, we identified an uncharacterized gene Lb1G04794, which showed increased expression after NaCl treatment and was high during salt gland development in L. bicolor. Overexpression of Lb1G04794 in L. bicolor showed promoted salt gland development, indicating that this gene may promote salt gland differentiation. Transgenic Arabidopsis strains overexpressing Lb1G04794 showed increased trichomes and decreased root hairs under normal conditions. Compared with wild type (WT), root growth in the transgenic lines was less inhibited by NaCl treatment. Transgenic seedlings accumulated less fresh/dry weight reductions under long-term salt treatment, accompanied by lower Na+ and malondialdehyde accumulation than WT, indicating that these transgenic lines behave better growth and undergo less cellular damage under NaCl stress. These results were consistent with the low expression levels of salt-tolerance marker genes in the transgenic lines upon salt stress. We conclude that the unknown gene Lb1G04794 positively regulated salt gland development, and promoted salt tolerance of Arabidopsis, offering a new direction for improving salt tolerance of non-halophytes and crops.

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.

Coupled Development of Salt Glands, Stomata, and Pavement Cells in Limonium bicolor.

Salt-resistant plants have different mechanisms to limit the deleterious effects of high salt in soil; for example, recretohalophytes secrete salt from unique structures called salt glands. Salt glands are the first differentiated epidermal structure of the recretohalophyte sea lavender (Limonium bicolor), followed by stomata and pavement cells. While salt glands and stomata develop prior to leaf expansion, it is not clear whether these steps are connected. Here, we explored the effects of the five phytohormones salicylic acid, brassinolide, methyl jasmonate, gibberellic acid, and abscisic acid on the development of the first expanded leaf of L. bicolor and its potential connection to salt gland, stomata, and pavement cell differentiation. We calculated the total number of salt glands, stomata, and pavement cells, as well as leaf area and pavement cell area, and assessed the correlations between these parameters. We detected strong and positive correlations between salt gland number and pavement cell area, between stomatal number and pavement cell area, and between salt gland number and stomatal number. We observed evidence of coupling between the development of salt glands, stomata, and pavement cells in L. bicolor, which lays the foundation for further investigation of the mechanism behind salt gland development.

Roles of Phytohormones and Their Signaling Pathways in Leaf Development and Stress Responses.

Phytohormones participate in various processes over the course of a plant's lifecycle. In addition to the five classical phytohormones (auxins, cytokinins, gibberellins, abscisic acid, and ethylene), phytohormones such as brassinosteroids, jasmonic acid, salicylic acid, strigolactones, and peptides also play important roles in plant growth and stress responses. Given the highly interconnected nature of phytohormones during plant development and stress responses, it is challenging to study the biological function of a single phytohormone in isolation. In the current Review, we describe the combined functions and signaling cascades (especially the shared points and pathways) of various phytohormones in leaf development, in particular, during leaf primordium initiation and the establishment of leaf polarity and leaf morphology as well as leaf development under various stress conditions. We propose a model incorporating the roles of multiple phytohormones in leaf development and stress responses to illustrate the underlying combinatorial signaling pathways. This model provides a reference for breeding stress-resistant crops.