The transcriptome of NaCl-treated Limonium bicolor leaves reveals the genes controlling salt secretion of salt gland.[Pubmed: 26936070]

Limonium bicolor, a typical recretohalophyte that lives in saline environments, excretes excessive salt to the environment through epidermal salt glands to avoid salt stress. The aim of this study was to screen for L. bicolor genes involved in salt secretion by high-throughput RNA sequencing. We established the experimental procedure of salt secretion using detached mature leaves, in which the optimal salt concentration was determined as 200 mM NaCl. The detached salt secretion system combined with Illumina deep sequencing were applied. In total, 27,311 genes were annotated using an L. bicolor database, and 2040 of these genes were differentially expressed, of which 744 were up-regulated and 1260 were down-regulated with the NaCl versus the control treatment. A gene ontology enrichment analysis indicated that genes related to ion transport, vesicles, reactive oxygen species scavenging, the abscisic acid-dependent signaling pathway and transcription factors were found to be highly expressed under NaCl treatment. We found that 102 of these genes were likely to be involved in salt secretion, which was confirmed using salt-secretion mutants. The present study identifies the candidate genes in the L. bicolor salt gland that are highly associated with salt secretion. In addition, a salt-transporting pathway is presented to explain how Na(+) is excreted by the salt gland in L. bicolor. These findings will shed light on the molecular mechanism of salt secretion from the salt glands of plants.

Identification and functional analysis of the autofluorescent substance in Limonium bicolor salt glands.[Pubmed: 26397201]

Limonium bicolor is a typical recretohalophyte with salt glands for the secretion of excess salts into the environment. We observed that L. bicolor salt glands showed obvious blue autofluorescence under UV excitation (330-380 nm). The aim of the present study was to identify and clarify a role for this autofluorescent substance in salt secretion. Sudan IV staining showed that the autofluorescent substance was localized in the cuticle of the salt glands. Moreover, the primary autofluorescent substance was identified as ferulic acid after treatment with 0.1 M ammonium hydroxide solution, alkaline and enzymatic hydrolysis. Additional experiments using two mutants exhibiting increased (fii) and decreased (fid) salt gland fluorescence indicated that the fluorescence intensity of salt glands under UV excitation was positively correlated with the content of ferulic acid in the cuticle, strongly suggesting that the primary autofluorescent substance in the salt glands was ferulic acid. Salt gland secretion was determined using leaf discs, and the results showed that the Na(+) secretion rate per single salt gland was also positively correlated with the content of ferulic acid in the cuticle, suggesting that ferulic acid in the cuticle was directly involved in salt secretion of salt gland.

K(+) accumulation in the cytoplasm and nucleus of the salt gland cells of Limonium bicolor accompanies increased rates of salt secretion under NaCl treatment using NanoSIMS.[Pubmed: 26259195]

Recretohalophytes with specialized salt-secreting structures (salt glands) can secrete excess salts from plant, while discriminating between Na(+) and K(+). K(+)/Na(+) ratio plays an important role in plant salt tolerance, but the distribution and role of K(+) in the salt gland cells is poorly understood. In this article, the in situ subcellular localization of K and Na in the salt gland of the recretohalophyte Limonium bicolor Kuntze is described. Samples were prepared by high-pressure freezing (HPF), freeze substitution (FS) and analyzed using NanoSIMS. The salt gland of L. bicolor consists of sixteen cells. Higher signal strength of Na(+) was located in the apoplast of salt gland cells. Compared with control, 200 mM NaCl treatment led to higher signal strength of K(+) and Na(+) in both cytoplasm and nucleus of salt gland cells although K(+)/Na(+) ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Moreover, the rate of Na(+) secretion per salt gland of L. bicolor treated with 200 mM NaCl was five times that of controls. These results suggest that K(+) accumulation both in the cytoplasm and nucleus of salt gland cells under salinity may play an important role in salt secretion, although the exact mechanism is unknown.

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation.[Pubmed: 25651944]

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt-tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty-six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt-secretion capacity in crops.

Chitinase genes LbCHI31 and LbCHI32 from Limonium bicolor were successfully expressed in Escherichia coli and exhibit recombinant chitinase activities.[Pubmed: 24385885]

The two chitinase genes, LbCHI31 and LbCHI32 from Limonium bicolor, were, respectively, expressed in Escherichia coli BL21 strain. The intracellular recombinant chitinases, inrCHI31 and inrCHI32, and the extracellular exrCHI31 and exrCHI32 could be produced into E. coli. The exrCHI31 and exrCHI32 can be secreted into extracellular medium. The optimal reaction condition for inrCHI31 was 5 mmol/L of Mn²⁺ at 40°C and pH 5.0 with an activity of 0.772 U using Alternaria alternata cell wall as substrate. The optimal condition of inrCHI32 was 5 mmol/L of Ba²⁺ at 45°C and pH 5.0 with an activity of 0.792 U using Valsa sordida cell wall as substrate. The optimal reaction condition of exrCHI31 was 5 mmol/L of Zn²⁺ at 40°C and pH 5.0, and the activity was 0.921 U using the A. alternata cell wall as substrate. Simultaneously, the optimal condition of exrCHI32 was 5 mmol/L of K⁺ at 45°C and pH 5.0, with V. sordida cell wall as the substrate, and the activity was 0.897 U. Furthermore, the activities of extracellular recombinant enzymes on fungal cell walls and compounds were generally higher than those of the intracellular recombinant enzymes. Recombinant exrCHI31 and exrCHI32 have better hydrolytic ability on cell walls of different fungi than synthetic chitins and obviously showed activity against A. alternata.

A glycine-rich RNA-binding protein can mediate physiological responses in transgenic plants under salt stress.[Pubmed: 21573794]

Glycine-rich RNA-binding proteins (GRPs) are involved in post-transcriptional regulation of genes, which have been found to play a role in stress response. However, whether GRPs can mediate some physiological responses related to salt stress tolerance is still not known. In the present study, we investigated the role of GRPs in salt stress-induced physiological responses by generating transgenic tobacco lines overexpressing a GRP (LbGRP1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild type (WT) tobacco, the transgenic plants showed significantly improved superoxide dismutase and catalase activities under salt stress conditions. Levels of proline in the transgenic plants were significantly higher than those in the WT plants grown under NaCl stress conditions. Furthermore, Na(+) content and Na(+)/K(+) ratio in the transgenic plants were lower than those in the WT plants under both normal growth and stress conditions. These results suggested that overexpression of the LbGRP1 gene can affect some physiological processes associated with salt tolerance of plants. Therefore, we hypothesize that LbGST1 can enhance stress resistance by mediating some physiological pathways.

A DREB gene from Limonium bicolor mediates molecular and physiological responses to copper stress in transgenic tobacco.[Pubmed: 20951468]

The DRE-binding (DREB) transcription factors play an important role in regulating stress-related genes. In the present study, a novel DREB gene (LbDREB) from Limonium bicolor was cloned. To characterize the function of DREB in heavy metal stress tolerance, LbDREB-transformed tobacco plants were generated and subjected to CuSO(4) stress. Analysis of the role of LbDREB in tolerance to copper stress in transgenic tobacco showed that overexpression of LbDREB increased the contents of soluble protein and proline, and elevated the ratio of K to Na under CuSO(4) stress. Moreover, overexpression of LbDREB can up-regulate some stress-related genes, including Cu/Zn superoxide dismutase (Cu/Zn SOD), peroxidases (PODs), late embryogenesis abundant (LEA), and lipid transfer proteins (LTP). These results suggest that LbDREB can enhance plant copper tolerance by up-regulating a series of stress-related genes, thereby mediating physiological processes associated with stress tolerance in plants.