Study On The Role Of Vesicle Trafficking In Salt Secretion Of Salt Glands Of Limonium Bicolor

Soil salinization is a major problem worldwide, with more than950millionhectares of land being salt-affected, and salt-affected land is increasing every year dueto unsuitable irrigation. Soil salinity severely reduces agricultural yields andproductivity and threatens economic development, food and energy safety. Accordingto salinity tolerance, plants are divided into halophytes and nonhalophytes.Halophytes can survive to reproduce in an environment where the salt concentration isaround200mmol/L NaCl or more, and they can be further divided intorecretohalophytes, euhalophytes, and pseudo-halophytes. The unique features ofrecretohalophytes are specialized salt-secreting structures, including salt glands andsalt bladders, which can secrete excess salts from the cells of metabolically activetissues to the surface of the leaves of stems. This ability allows the plants to adapt tosalinity-affected habitats. Salt glands in plants play an important role in regulating ionbalance, maintaining the stability of osmotic pressure, and enhancing salinitytolerance. Salt glands are excretory organs scattered on leaf surfaces and stems thatare adapted for dealing with salt accumulation in plants. These excretory organs arehighly evolved structures, which by their ability to secrete salt, enable plants to growon saline-alkali soil without adversely suffering from salt damage. At present, theresearch on mechanism of plant salt gland secretion focused on anatomy andphysiology, little progress has been made in development and molecular mechanismof the plant salt gland. If the molecular mechanism is known, the key genes involvingplant salt secretion will be cloned and gene-modified crops having salt secretionability may be generated, which can grow in the saline area. Therefore, it is of greatsignificance to understand the mechanism of salt secretion of salt glands for thedevelopment and utilization of saline and alkaline land worldwide in the future.Limonium bicolor is a typical recretohalophyte with multicellular salt glandstructure, which is distributed in coastal and salinized area and considered as a propermaterial to study the salt gland of dicotyledons and as “pioneer plant” for improvingand utilizing saline soils. Therefore, the study on the secretion characteristics and molecular mechanism of L. bicolor salt gland is very important.In this thesis, ultrastructure, salt secretion property, vesicle-trafficking related genesand mutants of salt galnds were investigated. The main results were as follows:1. Ultrastructure features of L. bicolor salt glands.HPF and FS were successfully to fix the leaves and outstanding preservation of saltglands ultrastructure was obtained, as demonstrated by the clearly identifiable intactsalt glands. The salt gland of L. bicolor consists of sixteen cells, including four outercup cells, four inner cup cells, four accessory cells, and four secretory cells. Nuclei,numerous mitochondria, small vesicles, Golgi, rough endoplasmic reticulum, outersurface cuticle are clearly identifiable, the nuclei, stained with the DNA-specificfluorescent dye DAPI, in these cells are typically large with very prominent nucleoli.A plasmodesma in a wall between two secretory cells is also clearly visible. The saltgland is encapsulated by a thick cuticular layer except along region of the wallsbetween the collecting cells and the outer cup cells, which extended over the secretorycells creating a cavity between the cuticular layer and the cell wall of the secretorycell. Each of the secretory cells has a pore, the secretory pore, on its surface. Thesecretion of salts to the outer surface of the leaf may occur through the secretory poresof the salt gland within the cuticle. Salt gland cells contain numerous and well-definedmitochondria and are rich in ER, but lack a conspicuous large central vacuole. Instead,the cells contain a series of smaller “vacuole-like” membrane vesicles, many of whichare seen in close proximity to the cell membrane. More important, the tonoplast of the“vacuole-like” vesicles often appears to fuse with the plasmalemma in agreement witha vesicle-mediated secretion process. As the salt accumulates in the salt glands, itbecomes compartmentalized within the microvacuoles and is secreted when themicrovacuoles fuse with the plasmalemma.2. Determination of ions secretion rate in leaf discs.Fully expanded leaves were thoroughly rinsed with distilled water to remove all saltfrom the surface that had been secreted previously, and10-mm-diameter discs werepunched from the leaves. The discs were dried quickly on both sides using absorbentpaper then placed in70mm diameter Petri dishes containing30mL NaCl solution. The abaxial leaf disc surface was covered with mineral oil to allow volumetricanalyses of the secretion fluids. With time, secretory droplets appeared under the oilabove the salt glands. The experiment was conducted over a24-h period at roomtemperature (20°C) with a12h photoperiod. Secretory droplets were collected with amicropipette. The volume of secretory droplets (V) per leaf disc was determined andtheir ionic concentration (C) measured by the Dionex ICS-1100ion chromatographysystem. The ion secretion rate (pmol/gland/h) was calculated as [V×C/(number ofsalt gland per leaf disc×time)] where number of salt gland per leaf disc wascalculated as density of salt gland (number/cm2)×area of leaf disc (cm2) and timewas duration of the secretion (from covering with mineral oil to collecting thedroplets: hours). Leaf discs can be used to research the characteristics of ion secretionof single salt gland under NaCl treatment and to screen mutants involved in the saltgland salt secretion.3. Pathway and characteristics of ion secretion of salt glands of L. bicolor.Scanning electron microscope micrographs showed that each of the secretory cellshad a pore in the center of the cuticle, and the rice grain-like secretions were observedabove the pore. The chemical composition of secretions from secretory pores wasmainly NaCl using environmental scanning electron microscope technique.Non-invasive micro-test technology was used to directly measure ion secretion rate ofsalt gland, and secretion rates of Na+and Cl-were greatly enhanced by a200mmol/LNaCl treatment. However, epidermal cells and stoma showed little secretion of ions.In conclusion, our results provide evidence that the salt glands of L. bicolor have foursecretory pores and that NaCl is secreted through these pores of salt gland.4. K+accumulation in the cytoplasm and nucleus of the salt gland cells of L. bicoloraccompanies increased rates of salt secretion under NaCl treatment.Samples were prepared by HPF and FS and analysed using NanoSIMS. Highersignal strength of the Na+was located in the apoplast of salt gland cells. Comparedwith control plants grown in the absence of NaCl,200mmol/L NaCl treatment led tohigher signal strength of K+and Na+in both cytoplasm and nucleus of salt gland cellsalthough K+/Na+ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Contrastly, NaCl led to a marked reduction in leaf K+and K+/Na+ratio of L. bicolorseedlings. Moreover, the rate of Na+secretion per salt gland of L. bicolor treated with200mmol/L 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 mayplay an important role in salt secretion.5. Role of vesicle trafficking in salt secretion of salt glands and cloning of key genes.BFA has often been used as an inhibitor of secretion and vacuolar protein transportin plant cells. First of all, the determination of Na+, Cl-, and K+secretion rate of L.bicolor leaf discs under different BFA-treated concentrations (0,50,100,150,200μg/ml) and different treated time (20,40,60min). Then, non-invasive micro-testtechnology was used to directly measure Na+secretion rate of individual salt glands inleaf epidermis of L. bicolor. Last, effects of200μg/ml BFA on the morphology ofgolgi in the salt glands was examined. The results showed that BFA has a significantinhibition on the salt secretion of salt glands and disintegration of golgi in the cellswith BFA treatment. Part of5genes highly related to vesicle transport according tohigh-throughput sequencing has been cloned. Then, effects of different concentrationsof NaCl solution (0and200mmol/L) on the relative expression level of the5genesby real-time PCR were conducted, three of them were up-regulated and two of themwere down-regulated by NaCl treatment. All these results indicate that vesicletrafficking involved in the salt secretion of salt glands.6. Mutants involved in the salt gland salt secretion have obtained.N+ion beam implantation and60Co gamma radiation mutagenesis were applied inthe seeds of L. bicolor to get mutants involved in the salt gland salt secretion. Weobtained32mutants with abnormal salt gland density for M0generation and also withincreased/decreased salt secretion rate per salt galnd after measured by leaf discsecretion model. The mutants involved in salt secretion of salt gland laid thefoundation for further study of molecular mechanisms of salt secretion in the future.The innovations of this thesis were as follows:1. HPF and FS were successfully to fix the leaves and outstanding preservation of saltglands ultrastructure was obtained, in particular, many small membrane vesicles near or fused with plasma membrane were detected, which suggested that vesicletrafficking possibly participated salt secretion process and laid the structurefoundation for the research of the mechanism of plant salt gland secretion.2. Scanning electron microscope and environmental scanning electron microscopetechnique was firstly used to study the pathway and characteristics of ion secretion ofsalt glands of L. bicolor. The results provide evidence that the salt glands have foursecretory pores and that NaCl is secreted through these pores of salt gland.3. K+accumulation both in the cytoplasm and nucleus of salt gland cells undersalinity may play an important role in salt secretion using NanoSIMS.4. Vesicle trafficking involved in the salt secretion of salt glands because BFA has asignificant inhibition on the salt secretion of salt glands and disintegration of golgi inthe cells with BFA treatment, the relative expression level of5vesicle-related geneswas regulated by NaCl treatment (3of them up-regulated and2down-regulated),which further indicates that vesicle trafficking possibly participated salt secretionprocess.5.32lines of mutants have been obtained, among them15lines show higher saltsecretion rate and17lines show lower salt secretion rate compared with wild type.These mutants are very important for further study on molecular mechanisms of saltsecretion of salt glands.