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Identification and analysis of HD-ZIP transcription factors that regulate salt gland development and salt tolerance in Limonium bicolor.

Soil salinity severely constrains agricultural production. Elucidating the salt-tolerance mechanisms of halophytes can provide innovative approaches for improving the salt tolerance of crop plants. In this study, we performed genome-wide identification and analysis of 36 LbHDZ genes encoding homeodomain-leucine zipper (HD-ZIP) transcription factors in Limonium bicolor, a typical recretohalophyte that excretes excess salt ions through specialized salt glands. Expression profiling across different stages of salt gland development, as well as in various tissues under salt stress, indicated that multiple LbHDZ genes are involved in regulating salt gland development and salt tolerance. Among these genes, LbHDZ14 (a member of the HD-ZIP II subfamily) exhibited sustained high expression during the critical period of salt gland formation, while its transcript levels were significantly downregulated in leaves and roots under salt stress. Subsequent experiments demonstrated that LbHDZ14 is localized in the nucleus and negatively regulates salt gland density and salt tolerance by directly binding to the promoter of LbGDSL, a positive regulator of salt gland development. In conclusion, this study reveals the expression patterns of LbHDZ genes in L. bicolor, characterizes the functional mechanism of LbHDZ14, further elucidates the regulatory network underlying salt gland development, and provides candidate genes for enhancing crop salt tolerance.

Plumbaginaceae

Transcription factor LbUBC positively regulates salt gland development and salt tolerance by directly binding to the LbTTG1 promoter and repressing its transcription.

KEY MESSAGE:: LbUBC enhances salt tolerance by promoting salt gland development via repressing LbTTG1, revealing a synergisticregulatory mechanism in Limonium bicolor. In the context of increasingly severe soil salinization, salt-tolerant genetic resources from halophytes show great application potential. In particular, the recretohalophyte Limonium bicolor, which possesses specialized salt gland structures, has become a key model for deciphering the molecular mechanisms underlying salt tolerance and salt gland development. In this study, using LbTTG1-overexpressing and -silenced lines, we demonstrate that LbTTG1 negatively regulates salt-gland development and salt tolerance. Through yeast one-hybrid, EMSA, and dual-luciferase assays, Lb7G33228 (LbUBC) was screened and verified as an upstream transcriptional regulator of LbTTG1. LbUBC enhances salt tolerance in L. bicolor by positively regulating salt-gland development, verified using LbUBC silence and overexpression strains. Interestingly, LbUBC represses the expression of its downstream target LbTTG1, thereby releasing the inhibitory effect of LbTTG1 on salt-gland development. In this manner, LbUBC positively regulates salt-gland development, achieving a dynamic balance in the regulation of salt-gland development and salt tolerance in L. bicolor. This study reveals a synergistic regulatory mechanism involving multiple genes, offering new insights for comprehensively dissecting the molecular regulatory network of salt-gland development.

Salt Tolerance

Regulation of hepatic transport of bile salt. Effect of protein synthesis inhibition on excretion of bile salts and their binding to liver surface membrane fractions.

The overall transport of bile salts across the hepatocyte is characterized as a carrier-mediated process whose rate-limiting step is biliary secretion. Specific bile salt binding proteins have been identified in liver surface membrane fractions and were postulated to represent the initial interaction in bile salt translocation across both the sinusoidal and canalicular membranes. To test this hypothesis, cycloheximide was administered to rats to inhibit hepatic protein synthesis. 16 h after cycloheximide administration [14C]leucine incorporation into hepatic protein was inhibited by 93% at 1 h and 47% at 12 h. However, values of liver function tests were not increased, although serum albumin, serum alanine amino-transferase, and alkaline phosphatase were significantly decreased. Light and electron microscopy did not demonstrate necrosis or fat accumulation. The latter demonstrated minimal disorganization of rough endoplasmic reticulum and occasional lamellar whorls. 16 h after cycloheximide administration bile salt independent bile flow, basal bile salt excretion, and basal bile flow were unaltered, but the maximum bile salt transport capacity was reduced to 62% of control and 24 h later to 38%. Decreased bile salt transport was reversible, for it returned to control values after 48 h, when hepatic protein synthesis was also normal. Maximum bromosulfophthalein (BSP) transport, on the other hand, was reduced after 16 h to only 85% of control. Both bile salt and BPS maximum transport capacities decreased with time during inhibition of protein synthesis, apparently following first order kinetics. It was estimated that their half-lives are 20 h for bile salt transport and 55 h for BSP transport. These different turnover rates suggest that cycloheximide does not decrease active transport through generalized hepatic dysfunction or alteration of high energy sources possibly required for transport. The maximum number of [14C]cholic acid binding sites in liver surface membrane fractions was determined by an ultrafiltration assay. They were reduced to 68% of control after 16 h of cycloheximide and to 25% after 24 h. This reduction in the number of binding sites is apparently selective, for the activities of the liver surface membrane enzymes (Na+-K+)ATPase, Mg++-ATPase, and 5'-nucleotidase were not significantly changed. The associated alterations in bile salt transport and the maximum number of binding sites after cycloheximide administration suggests that these receptors may be the bile salt carriers.

Animals

Bile salt metabolism. II. Bile salts and disease.

Alterations of bile salt metabolism have been shown in numerous diseases. Liver damage results in elevated serum bile salt concentrations which may be useful as a sensitive index of hepatocellular disease. Changes in the relative proportions of the individual bile salts in serum occur with cholestasis. Urinary excretion of bile salts, largely in the form of sulphates, increases as a compensatory mechanism. Ileal disease or resection causes bile salt melabsorption. The increase in colonic bile salts produces a watery diarrhoea while the decrease in duodenal levels may cause steatorrhoea. Cholelithiasis may result from alteration in the relative proportions of cholesterol, lecithin and bile salts in bile. The mechanism apparently differs in various conditions predisposing to gallstone formation. A primary alteration of bile salt metabolism has been postulated in several other conditions. Considerable interest centres on the importance of metabolites of bile salts in the pathogenesis of colonic carcinoma. Chenodeoxycholic acid is a successful though costly treatment for selected patients with cholesterol gallstones. Bile salt binding agents, such as cholestyramine, are extremely useful especially in the control of pruritus in patients with cholestasis.

Animals

Effect of sugars on salt reception in true slime mold Physarum polycephalum. Physicochemical interpretation of interaction between salt and sugar receptions.

Interaction between salt and sugar receptions in plasmodium of Physarum polycephalum was studied by using double-chamber method. Effect of sugars on salt reception was evaluated by measuring membrane potential and the motive force of tactic movement of the slime mold, where salt concentration in one compartment was increased successively with a fixed sugar concentration. Results are summarized as follows: (1) The presence of D-glucose, D-mannose, D-maltose, or sucrose in medium led to increase of the threshold concentration Cth, for salts (chlorides and nitrates of Li, Na, K), whereas D-ribose decreased the threshold for salt reception. D-galactose showed no appreciable effect on Cth of every salt species examined. No change in Cth for salt reception was observed until concentration of sugars exceeded their respective thresholds. (2) Double logarithmic plots of Cth for salts against sugar concentration followed different straight lines for different cations, whose slopes being closely correlated with the effects of lyotropic number of anions in the absence of sugars. (3) Plots of log Cth against the reciprocal of the absolute temperature, 1/T, gave linear relations, and the slopes of the straight line became small with increase of sugar concentration above their respective thresholds. Experimental results obtained here suggest that the structure of water at the interface of cell membrane plays an indispensable role in the interaction between salt and sugar receptions.

Anions

Histochemical enzyme activity correlated to the structural segmentation of the proximal convoluted tubule in salt-depleted and salt-loaded rat kidneys.

In salt-depleted and salt-loaded rat kidneys a study was made of the structural segmentation of the proximal convoluted tubule (PCT) and the histochemical activity of non-specific acid and alkaline phosphatases and succinate dehydrogenase in the same segments. No quantitative structural or segmental alterations were observed, but significant changes in enzyme activity occured. These comprised: 1) A decrease in activity of acid phosphatase in segment 1 and the transitional zone in salt-depleted kidneys, and an increase in enzyme activity in segment 2 in salt-loaded kidneys. 2) a decrease in alkaline phosphatase activity in segment 2 in both salt-depleted and salt-loaded kidneys and 3) a decrease in succinate dehydrogenase activity in segment 2 in salt-depleted kidneys, and an increase in activity in the same segment in salt-loaded kidneys. Thus long-term variation in sodium intake are followed by segment-correlated variations in the activity of acid and alkaline phosphatase and succinate dehydrogenase in the PCT.

Acid Phosphatase

The effect of angiotensin-converting enzyme inhibition on regional blood flow in salt-depleted and salt-loaded normotensive conscious rats.

The effect of angiotensin-converting enzyme inhibition on regional blood flow was studied in a total of 21 normotensive Wistar rats fed on either low or high salt diet. A new potent angiotensin-converting enzyme inhibitor (CEI), SQ 14,225 was administered intravenously in a dose of 2 mg/Kg to the conscious animals, and changes in fractional distribution of cardiac output were determined with a microsphere method. Prior to administration of CEI, there was no significant difference in mean arterial pressure (MAP) or regional blood flow between salt-depleted and salt-loaded rats. With CEI, MAP did not change significantly in either group. Fractional distribution of cardiac output increased to the kidneys (p less than 0.002), and decreased to the stomach, spleen, and skeletal muscle (p less than 0.02, p less than 0.002, and p less than 0.01, respectively) in the salt-depleted group, while a pattern of blood flow distribution was not changed in the salt-loaded group. These results suggest that angiotensin II plays an important role in regulating regional blood flow in salt-depleted conscious animals, but not in salt-loaded ones.

Angiotensin-Converting Enzyme Inhibitors

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate.

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.

Bile Acids and Salts

Comparison of the bile salts of frogs with those of their tadpoles. Bile-salt changes during the metamorphosis of Rana Catesbeiana Shaw.

1. The bile salts of three frog species of the genus Ptychadena and of Rana catesbeiana have been compared with those of their tadpoles. For R. catesbeiana comparison was made of the bile salts in at least ten of the recognized stages of tadpole metamorphosis. 2. In all cases, adult bile salts were more complex than those of the tadpoles. 3. In R. catesbeiana after stage 18, 26-deoxy-5 alpha-ranol was hydroxylated to form 5 alpha-ranol (27-nor-5 alpha-cholestane-3 alpha, 7 alpha, 12 alpha, 24 xi, 26-pentol) and at least two other bile alcohols appeared in solvolysed bile salts. 4. Tadpole bile salts were not found to be biochemically more primitive than those of fully metamorphosed frogs; in some, but not all, cases tadpole bile alcohols could be regarded as biochemical precursors of those in the adult frogs. 5. Detailed evidence for the structure of the bile salts from mass-spectral fragmentation patterns has been deposited as Supplementary Publication SUP 50097 (2 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Animals

Diffusion coefficients of single bile salt and bile salt-mixed lipid micelles in aqueous solution measured by quasielastic laser light scattering.

Diffusion coefficients of single bile salt micelles and mixed binary micelles consisting of single bile salts and phosphatidylcholine in the molar ratio 3 : 1 have been measured using quasielastic laser light scattering spectroscopy. Among single systems, marked concentration dependence of the diffusion coefficient was observed only in those containing sodium taurodeoxycholate. Micellar size correlates with bile salt molecular structure in a manner similar to that observed by earlier workers with some minor differences to towards a more consistent ordering. Phosphatidylcholine produces small increases in micellar size in most systems depending on bile salt structure, and seems most plausibly interpreted as simple swelling of the single species micelles. In some systems, when phosphatidylcholine is added, size or shape changes are indicated. Diffusion coefficients were also studied in the ternary systems bile salt-phosphatidylcholine-cholesterol which were saturated and metastably super-saturated with respect to cholesterol. The Stokes radii in both systems were the same within experimental error, 35 A. Our results indicate that intermicellar interactions significantly contributed to the measured diffusion coefficients in the ternary systems as well as in the single sodium taurodeoxycholate and binary sodium taurodeoxycholate-phosphatidylcholine systems in the concentration range studied.

Bile Acids and Salts

Bile salt metabolism. I. The physiology of bile salts.

Bile salts are synthesized in the liver from cholesterol, conjugated with glycine or taurine and secreted in bile with cholesterol and lecithin. The molar concentrations of these three lipids determine solubility of cholesterol in bile. Within the gastrointestinal lumen bile salts play an essential role in lipid absorption and faty transport. An efficienct entero-hepatic circulation maintains hepatic bile salt secretion and provides a "feed-back" control of the bile salt and cholesterol metabolism. Potentially hepatotoxic lithocholic acid formed in the intestinal lumen by bacterial action on chenodeoxycholic acid is sulphated in the liver thus decreasing intestinal reabsorption. The total faecal excretion of bile salts balances hepatic synthesis and represents a major catabolic path in cholesterol metabolism.

Animals

Enhancement of solubility of drug salts by hydrophilic counterions: properties of organic salts of an antimalarial drug.

Judicious choice of the salt form of a drug can greatly affect the aqueous solubility and formulation of the compound. The objective of this work was to demonstrate the effect of various counteranions on the aqueous solubility of the antimalarial agent alpha-(2-piperidyl)-3, 6-bis(trifluoromethyl)-9-phenanthrenemethanol. Several organic salts of this drug were studied. The methods of synthesis, the apparent aqueous solubilities, and in vitro dissolution tests for these salts are reported. The lactate salt was 200 times as soluble as the hydrochloride salt. This enhanced solubility suggests that parenteral administration of this drug may now be feasible.

Antimalarials

Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa.

When unfertilized hamster eggs are placed in highly concentrated solutions of neutral salts (e.g., 2 M ammonium sulfate), the egg proper, or vitellus, shrinks, creating a large perivitelline space; the zona pellucida remains unchanged in appearance under the light microscope. When these eggs are inseminated, many spermatozoa attach to and penetrate the zona. The specificity as well as several physical and chemical characteristics of the zona seem to remain unchanged during storage of the eggs in salt solutions. The properties of the human zona pellucida which allow the attachment and penetration of human spermatozoa are also retained in concentrated salt solutions. Shipment of salt-stored human eggs at ambient temperature to any part of the world is feasible and inexpensive. The present study suggests that salt-stored eggs can be used as substitutes for fresh living eggs in the preliminary assessment of fertilizing capacity of spermatozoa when collection of a large number of fresh unfertilized eggs, particularly in humans, is not practical.

Animals

Determination of the faecal excretion of labelled bile salts after i.v. administration of 14C-cholic acid. An evaluation of the bile salt malabsorption before and after surgery in patients with Crohn's disease.

By measuring total faecal radioactivity, correlated to 24-hour enterohepatic circulation, following i.v. administration of 14C-cholic acid, bile salt malabsorption was evaluated before and/or after surgery in 80 patients with Crohn's disease localized to the ileum and/or the colon and the results related to the length of ileum diseased or resected. Before operation bile salt malabsorption was observed only in patients with inflammation of the terminal ileum, but no significant correlation was found between bile salt excretion and the extent of ileal disease. In patients subjected to ileal resection with sacrifice of the ileocaecal valve, bile salt malabsorption correlated strongly to the length of ileum resected. This correlation was about the same in ileostomy patients and in patients subjected to restorative operation. We concluded that determination of 14C in faeces is a more sensitive test than the Schilling test and the faecal fat excretion test in reflecting ileal dysfunction, at least in patients with ileal resections.

Adult

Association products and conformations of salt-dissociated and acid-extracted histones. A two-phase procedure for isolating salt-dissociated histones.

We present an extremely rapid and efficient method for the separation of salt-dissociated histones from DNA in which the macromolecular components of chicken erythrocyte chromatin are partitioned in a two-phase system of the water-soluble, nonionic polymers, poly(ethylene glycol) and dextran. We have compared the association products and conformations of salt-dissociated histones purified with the two-phase procedure and histones that had been extracted with 0.4 M H2SO4. In the gel chromatography system of D. R. vander Westhuyzen and C. von Holt (1971), FEBS Lett. 14, 333-337] the association products of salt-dissociated and acid-extracted histones are indistinguishable. Furthermore, the circular dichroism spectra of histones prepared with the two methods are identical within experimental error. These results indicate that histones extracted, with sulfuric acid can adopt conformations at least very similar to those of salt-dissociated preperties of total erythrocyte histones are the same in 2 M NaCl as those of these histones bound to DNA in chromatin in 1 mM Tris-Cl (pH 7.5). This result and the studies of Weintraub et al. [Weintraub, H., Palter, K., and Van Lente, F. (1975), Cell 6, 68-110] on the patterns of tryptic digest products of histones strongly suggest that in 2 M NaCl the histones exist in conformations very similar to their conformations when bound to DNA. The concept of native histone conformations is discussed in light of our results.

Animals

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles.

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.

Bile Acids and Salts

Interaction of uncharged bile salt derivatives with the ileal bile salt transport system.

Two series of uncharged conjugated bile salt derivatives, N-conjugates of ethanolamine and 3-amino-1,2-propanediol were studied for interaction with the ileal bile salt transport system. Evidence for interaction is threefold. 1) In everted gut sac experiments more material was removed from the mucosal compartment when ileal sacs were used. 2) These derivatives inhibited the in vitro transport of taurocholate. 3) In vivo intestinal perfusion demonstrated greater absorption from ileum than from jejunum. Number three demonstrates that such interactions are followed by transmucosal movement. Their uphill transport was less than taurocholate transport. The Na(+) requirement for cholyl-3-amino-1,2-propanediol interaction with the system was greater than for taurocholate. This observation is similar to that previously observed with taurodehydrocholate, which had a greater Na(+) requirement for transport than taurocholate. Therefore removal of the anionic charge, as well as distortion of steroid shape, increases the Na(+) requirement for substrate interaction with the transport system. These observations support our hypothesis that this interaction involves two recognition components; one includes the steroid moiety, the other a coulombic interaction between the anionic bile salt and a cationic membrane site. Additionally the membrane would have an anionic group to accomodate the Na(+). Both factors (steroidal and coulombic) operate for optimal substrate attachment. Simultaneously the system's affinity for Na(+) increases and active transport then proceeds.

Animals

Two natriuretic substances in extracts of urine from normal man when salt-depleted and salt-loaded.

When the extra-cellular fluid volume is expanded, the subsequent rise in urinary sodium excretion that occurs is due in part to a change in the concentration of some circulating natriuretic substance. Two natriuretic substances with different characteristics on Sephadex chromatography have been previously been identified separately by different workers. Extracts prepared from the urine of 31 normal subjects were tested for these two natriuretic materials in the normal conscious water-loaded rat. Two natriuretic fractions were found. The larger of the two was prepared on G50 Sephadex, and the smaller on G25 Sephadex. The natriuresis produced by the larger material was slow to develop and persisted for two hours. The natriuresis produced by the smaller material was maximal in the first 20 min, declined rapidly within the next 40 min, and tended to rise again during the subsequent 60 min. The amount of natriuretic activity that could be extracted from the freeze-dried urine was diminished by high concentrations of sodium chloride. The natriuretic activity of both materials was greater in the urine of the subjects when they were salt-loaded than when they were salt-depleted. The urine of salt-depleted subjects contained significant amounts of natriuretic material.

Animals