Highly efficient Agrobacterium-based transformation system for callus cells of the C3 halophyte Suaeda salsa

Genetic manipulation technologies have been limited in the halophyte Suaeda salsa L. due to the lack of an efficient transformation system. Here, we examined factors affecting transformation and developed an efficient transformation system at the cell level using S. salsa hypocotyl as starting material. S. salsa hypocotyl explants from 10-day-old seedlings were precultured for 2 days on a hygromycin (hyg)-free callus induction medium (CIM) and then inoculated with Agrobacterium tumefaciens suspension at a concentration of 0.5 at OD₆₀₀ for 5–10 min. After cocultivation with A. tumefaciens for 4 days in the dark, followed by selection on carbenicillin (carb) for 3 days, explants were placed on CIM containing 10 mg l⁻¹ hyg and 500 mg l⁻¹ carb with three to four consecutive subcultures for up to 45 days. β-Glucuronidase assays showed an average transformation frequency of 62.89%. Gene integration was confirmed by polymerase chain reaction analysis and Northern blot analysis. To our knowledge, this is the first study to show Agrobacterium-mediated transformation in the C₃ halophyte S. salsa.     

The leaf tonoplast V-H(+)-Atpase activity of a C3 halophyte Suaeda salsa is enhanced by salt stress in a Ca-dependent mode

Suaeda salsa seedlings grown in Hoagland nutrient solution were treated with different concentrations of NaCl combined with two levels of Ca2+ (0 and 20 mmol/L) to study the effect of Ca2+ nutrition on the growth and activity of leaf tonoplast V-H(+)-ATPase. Increase of Ca2+ concentration in the solution markedly increased the relative growth quantity of S. salsa seedlings and Ca2+ and K+ concentration in the leaf cell sap under NaCl stress. The leaf V-H(+)-ATPase activity was significantly increased with increasing NaCl concentration under high Ca2+ application (20 mmol/L), but little changed under Ca2+ starvation (0 mmol/L). Western blot analysis showed that the leaf V-H(+)-ATPase of S. salsa was at least composed of A, B, D and c subunits, and their protein amounts were not affected by NaCl treatments under Ca2+ starvation (0 mmol/ L) with an exception of 100 mmol/L NaCl, but increased under high Ca2+ application (20 mmol/L). There was a positive correlation between activity of V-H(+)-ATPase and the protein amounts of the subunits. The results suggest that Ca2+ nutrition played an important role in the salt tolerance of S. salsa, and that enhancement of V-H(+)-ATPase activity under salt stress was Ca2(+)-dependent.     

辽河口盐地碱蓬湿地景观破碎化及驱动机制

湿地景观破碎化的加剧严重影响了湿地生物多样性保护和湿地生态系统服务功能的维持,本研究以辽河口盐地碱蓬(Suaeda salsa)湿地为研究对象,基于GIS平台构建1985—2019年间湿地景观数据库,定量分析湿地景观破碎化特征及其驱动因素。结果表明:1985—2019年间,辽河口盐地碱蓬湿地呈退化趋势,具体表现为湿地面积萎缩,湿地景观破碎化加剧。盐地碱蓬湿地面积在1988年达到最大值4158.81 hm~2,景观聚集度较高,此后,湿地景观破碎度出现先增大后减小再增大的波动变化,2003年和2019年盐地碱蓬湿地景观破碎化最为严重,破碎度分别达到5.90和7.89;影响辽河口盐地碱蓬湿地景观破碎化主要驱动因素为人为开发活动和水文过程;景观破碎化的整体上升趋势与道路修建、农田开发、水产养殖、径流量和输沙量有较好的空间对应关系,道路修建、农田开发和水产养殖面积的增加以及年径流量的减少是导致盐地碱蓬湿地景观破碎化的主导因素。     

The combined effect of salt stress and heat shock on proteome profiling in Suaeda salsa

Under natural conditions or in the field, plants are often subjected to a combination of different stresses such as salt stress and heat shock. Although salt stress and heat shock have been extensively studied, little is known about how their combination affects plants. We used proteomics, coupled with physiological measurements, to investigate the effect of salt stress, heat shock, and their combination on Suaeda salsa plants. A combination of salt stress and heat shock resulted in suppression of CO₂ assimilation and the photosystem II efficiency. Approximately 440 protein spots changed their expression levels upon salt stress, heat shock and their combination, and 57 proteins were identified by MS. These proteins were classified into several categories including disease/defense, photosynthesis, energy production, material transport, and signal transduction. Some proteins induced during salt stress, e.g. choline monooxygenase, chloroplastic ATP synthase subunit beta, and V-type proton ATPase catalytic subunit A, and some proteins induced during heat shock, e.g. heat shock 70 kDa protein, probable ion channel DMI1, and two component sensor histidine kinase, were either unchanged or suppressed during a combination of salt stress and heat shock. In contrast, the expression of some proteins, including nucleoside diphosphate kinase 1, chlorophyll a/b binding protein, and ABC transporter I family member 1, was specifically induced during a combination of salt stress and heat shock. The potential roles of the stress-responsive proteins are discussed.     

Molecular Cloning and Characterization of a Vacuolar H+-pyrophos-phatase Gene, SsVP, from the Halophyte Suaeda salsa and its Overexpression Increases Salt and Drought Tolerance of Arabidopsis

The chenopodiaceae Suaeda salsa L. is a leaf succulent euhalophyte. Shoots of the S. salsa are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. S. salsa does not have salt glands or salt bladders on its leaves. Thus, this plant must compartmentalize the toxic Na⁺ in the vacuoles. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole. In this work, we isolated the cDNA of the vacuolar membrane proton-translocating inorganic pyrophosphatase (H⁺-PPase) from S. salsa. The SsVP cDNA contains an uninterrupted open reading frame of 2292 bp, coding for a polypeptide of 764 amino acids. Northern blotting analysis showed that SsVP was induced in salinity treated leaves. The activities of both the V-ATPase and the V-PPase in Arabidopsis overexpressing SsVP-2 is higher markedly than in wild-type plant under 200 mM NaCl and drought stresses. The Overexpression of SsVP can increase salt and drought tolerance of transgenic Arabidopsis. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Shanli Guo, Haibo Yib: These authors contributed equally to this work     

Salt tolerance in the halophyte Suaeda maritima L. Dum.

Osmotic potentials and individual epidermal cell turgor pressures were measured in the leaves of seedlings of Suaeda maritima growing over a range of salinities. Leaf osmotic potentials were lower (more negative) the higher the salt concentration of the solution and were lowest in the youngest leaves and stem apices, producing a gradient of osmotic potential towards the apex of the plant. Epidermal cell turgor pressures were of the order of 0.25 to 0.3 MPa in the youngest leaves measured, decreasing to under 0.05 MPa for the oldest leaves. This pattern of turgor pressure was largely unaffected by external salinity. Calculation of leaf water potential indicated that the gradient between young leaves and the external medium was not altered by salinity, but with older leaves, however, this gradient diminished from being the same as that for young leaves in the absence of NaCl, to under 30% of this value at 400 mM NaCl. These results are discussed in relation to the growth response of S. maritima.     

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na⁺/H⁺ antiporter) and Arabidopsis AVP1 (vacuolar H⁺-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K⁺/Na⁺ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H₂O₂ generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.Feng-Yun Zhao and Xue-Jie Zhang contributed equally to this work     

真盐生植物盐地碱蓬根系边缘细胞在耐盐中的作用初探

以黄河三角洲潮间带盐地碱蓬种子生成的幼苗为材料,研究了NaCl胁迫对盐地碱蓬生长与根系边缘细胞的影响。盐地碱蓬的第一个边缘细胞几乎与根尖同步产生,当根长达到13mm时,边缘细胞数目达到最大值。NaCl胁迫抑制边缘细胞的活性,但低浓度的NaCl处理增加边缘细胞的数目。低浓度NaCl处理时果胶甲基酯酶（PME）的活性比对照有明显增加,超氧化物歧化酶（SOD）活性随着NaCl浓度的增加呈现先上升后下降的趋势,低浓度NaCl可以增加盐地碱蓬根内过氧化氢酶（CAT）的活性,NaCl处理时间和处理浓度都对过氧化物酶（POD）活性的影响不明显。这些结果表明,盐地碱蓬至少部分通过增加调控活性氧（ROS）水平增加PME活性及根系边缘细胞数目来抵抗NaCl胁迫。     

NaCl和Na2CO3对盐地碱蓬胁迫效应的比较

在相同的Na 浓度(如100 mmol/L)下,NaCl处理促进碱蓬植株干重增加,提高根系活力,而Na2CO3处理导致植株干重减少,根系活力降低;与NaCl胁迫相比,Na2CO3胁迫下叶片内Na 含量上升和K 含量下降幅度更大,叶肉细胞质Na 含量和叶内脯氨酸含量增加幅度更大,而V-H -ATPase(液泡膜H -ATPase)和V-H -PPase (液泡膜H -PPase)增加幅度较少;与NaCl胁迫不同,Na2CO3胁迫下SOD(超氧化物歧化酶)活性不是增加,而是降低,与此相一致,MDA(丙二醛)含量大幅度增加.上述结果表明,碱蓬对Na2CO3胁迫的抗性低于对NaCl的抗性,这可能与Na2CO3胁迫引起的Na 、K 离子严重失衡、活性氧清除能力降低有关.     

KCl和NaCl处理对盐生植物碱蓬幼苗生长和水分代谢的影响

研究了用不同浓度KCl和等浓度NaCl处理的盐生植物碱蓬幼苗水分代谢的变化.结果表明,NaCl处理显著促进碱蓬生长,根吸水增加,含水量高于对照.而相同浓度KCl处理却严重抑制碱蓬生长,含水量显著下降,400 mmol/L的 KCl处理后6 d,地上部分发生萎蔫,尔后死亡.KCl处理伤害碱蓬的原因之一是抑制根系吸水,而对蒸腾速率无显著影响,即水分吸收速率与蒸腾速率比值下降,植株缺水引起伤害.     

The effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of Suaeda salsa under saline conditions

Suaeda salsa, a leaf succulent shrub in the family Chenopodiaceae, is one of the most important halophytes in China. Suaeda salsa produces dimorphic seeds (soft brown seeds and hard black seeds). Seeds of S. salsa were collected from the coastal salt flats near Huanghua City, China. Experiments were conducted to determine the salinity-alleviating effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of S. salsa. Brown seeds had a higher germination rate than black seeds in all experiments. Black seeds were more sensitive to salt in the absence of light in comparison to brown seeds. Brown seeds absorbed water more quickly in comparison to black seeds and were found to be more tolerant of salt stress. Our results showed that 1-aminocyclopropane-1-carboxylate (ACC, the immediate precursor of ethylene), nitrite, GA₄ and BA improved seed germination in the presence of salt. However, nitrate, GA₁, GA₃ failed to alleviate salt stress. ABA inhibited seed germination and seedling growth. Possible mechanisms involved in the alleviation of salt stress in S. salsa seeds and the ecological adaptation of the seeds to the environment are discussed.     

NaCl胁迫对2种表型盐地碱蓬种子萌发的渗透效应和离子效应研究

采用高低2个浓度的NaCl、LiCl及等渗甘露醇溶液处理紫红色表型(紫色型)和绿色表型(绿色型)盐地碱蓬种子,通过测定它们的种子萌发率、吸胀速率和胚内离子含量,研究NaCl胁迫对2种表型种子萌发的离子效应和渗透效应.结果表明:(1)2种表型盐地碱蓬种子萌发率在高浓度(300 mmol/L)和低浓度(100 mmol/L)NaCl处理下均显著降低,紫色型种子萌发率在低浓度下显著低于绿色型,而在高浓度下却显著高于绿色型;绿色型种子萌发率在高浓度(30 mmol/L)和低浓度(10 mmol/L)LiCl处理下均未受到显著影响,但紫色型种子萌发率却均极显著降低;2种表型盐地碱蓬种子萌发率在低浓度等渗甘露醇处理下均极显著低于低浓度NaCl处理,而高浓度等渗甘露醇处理却均与高浓度NaCl处理无显著差异.(2)2表型种盐地碱蓬种子的吸胀速率在低浓度NaCl处理下没有受到显著影响,但高浓度NaCl处理及与之等渗的高浓度甘露醇处理下都显著降低,而且紫色型种子的吸胀速率在等渗甘露醇处理时显著高于绿色型.(3)2种表型盐地碱蓬种子胚中的Na 含量和Na /K 在对照和低浓度NaCl处理下无显著差异,但紫色型种子胚中的Na 、K 含量在高浓度NaCl处理时都显著高于对照,且K 含量增加的幅度远大于Na 含量,导致紫色型种子胚中的Na /K 显著低于绿色型.研究发现,盐地碱蓬种子萌发在低浓度NaCl胁迫下主要受离子效应抑制,而高浓度NaCl胁迫下则主要受渗透效应抑制,紫色型种子萌发率在高浓度NaCl胁迫下高于绿色型的原因之一是前者能维持更低的Na /K 比.     

盐和重金属复合胁迫对翅碱蓬萌发与生长的影响及调控措施

为探究Zn、Cu与盐复合胁迫对翅碱蓬(Suaeda salsa)萌发生长的影响机理及其调控措施,以翅碱蓬为研究对象,采用水培试验方法,测定Zn、Cu和盐复合胁迫条件下翅碱蓬种子发芽率、萌发速率和幼苗渗透调节物质含量等指标,分析1.5 mg/L吲哚乙酸(IAA)、100 mg/L赤霉素(GA)和0.3%硝酸钾(KNO₃)处理对Zn、Cu与盐复合胁迫条件下翅碱蓬萌发与生长的影响。结果表明：(1) Zn、Cu与高盐复合胁迫极显著降低翅碱蓬种子的发芽率,中高浓度的Zn、Cu与盐复合胁迫极显著降低翅碱蓬种子的萌发速率,Zn、Cu和盐复合胁迫对翅碱蓬种子的萌发生长影响表现为低促高抑效应,影响因子间存在明显的协同效应;(2)Zn、Cu和盐复合胁迫条件下,随着盐浓度的升高,翅碱蓬幼苗体内过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)3种抗氧化酶活性呈先升高后降低的变化趋势,Zn、Cu和高盐复合胁迫使翅碱蓬幼苗体内丙二醛(MDA)含量增加近2.5倍;(3)1.5 mg/L IAA溶液浸种12 h可显著提高Zn、Cu与高盐复合胁迫条件下翅碱蓬种子的发芽率和萌发速...     

Enhanced Tonoplast H+-ATPase Activity and Superoxide Dismutase Activity in the Halophyte Suaeda salsa Containing High Level of Betacyanin

In this study, high-betacyanin Suaeda salsa seedlings were developed and used to explore whether the betacyanin accumulation is related to salinity tolerance in S. salsa. After 8 days of culture, betacyanin content decreased markedly in both high-betacyanin S. salsa seedlings and the control under nonsalt stress, but the decreases were suppressed by NaCl treatments. Betacyanin content in high-betacyanin seedlings was much higher than that in the control throughout the salt treatments. Growth of S. salsa plants was significantly promoted by NaCl treatments, and the fresh weight of high-betacyanin seedlings was much higher than that of the control when grown in 400 mmol L⁻¹ NaCl. Similar cell sap osmolarity and K⁺/Na⁺ ratios were observed in high-betacyanin seedlings and the control. No obvious differences in V-ATPase (tonoplast H⁺-ATPase) activity, leaf SOD (superoxide dismutase) activity, and total chloroplast SOD (including thylakoid-bound SOD and stroma SOD) activity were detected between high-betacyanin seedlings and the control under nonsalt stress conditions. However, V-ATPase hydrolytic activity increased dramatically in S. salsa seedlings when subjected to different levels of NaCl, and the increases in V-ATPase activity in high-betacyanin seedlings were much higher than that in the control. No clear pattern was observed for NaCl-dependent activity changes of P-ATPase (plasma membrane H⁺-ATPase) and V-PPase (tonoplast H⁺-pyrophosphatase). Similar changes were demonstrated in leaf SOD activity and chloroplast SOD activity under salt stress. Both leaf SOD activity and chloroplast SOD activity were markedly enhanced with the increase of NaCl or with time, especially thylakoid-bound SOD activity. Furthermore, the increases in chloroplast SOD activity and thylakoid-bound SOD activity were much higher in high-betacyanin seedlings than that in the control at different levels of NaCl treatment. The higher V-ATPase activity, chloroplastic SOD activity, and thylakoid-bound SOD activity demonstrated in high-betacyanin seedlings, but lower in the control, suggest that high-betacyanin S. salsa seedlings may have higher potential to be energized by the electrochemical gradient for ion uptake into the vacuole and to scavenge O₂^−• in situ produced in the chloroplasts, which may lead to higher salt tolerance than the control under salt stress. Thus, betacyanin may be involved in salt tolerance of S. salsa.     

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

盐胁迫下两草种SOD和POD及脯氨酸动态研究

在NaCl、KCl、MgSO₄及其复合盐胁迫下, 研究草坪草-金牌美达丽(Lolium perenne)和猎狗(Fesluca elata) 中脯氨酸含量、超氧化物歧化酶和过氧化物酶活性的变化。结果表明:不同盐分、不同浓度胁迫下脯氨酸含量呈波动式变化, 其峰值出现在较高浓度下, 说明在一定范围内盐胁迫强度越大脯氨酸积累越明显;SOD 的最高值出现在低浓度下, 而最低值无此特点, 不同盐胁迫下的最高值、最低值相差不多, 说明L.perenne 和F.elata 相差不明显;POD 活性随盐浓度增加呈波动性变化, L.perenne (除KCl)和F.elata (除MgSO₄)最高值均出现在低浓度下, L.perenne 的最低值集中在高浓度, 而F.elata 的最低值则集中在低浓度, F.elata在高浓度胁迫下, 处于一个比较稳定的水平, 高于或接近CK, 且各值比较接近, 而在L.perenne 中却没有这一结果。     

Salt tolerance in the halophyte Suaeda maritima (L.) Dum. The influence of the salinity of the culture solution on leaf starch and phosphate content

Abstract. The starch concentration in mature leaves of the halophyte Suaeda maritima increased from 4.7 to 7.3 mg mg⁻¹ chlorophyll when sodium chloride (680 mol M⁻³) was added to the solution in which the plants were grown. This effect of salinity on the starch: chlorophyll ratio was greater in young than in old leaves. Electron micrographs showed the starch to be in the chloroplasts and this was confirmed by measurements on isolated chloroplasts. Total phosphorus concentration (mg mg⁻¹ chlorophyll) in leaves of all ages from plants of S. maritima decreased on salinization of the growth medium suggesting an inverse relationship between phosphorus and starch concentrations. However, although leaf starch concentration varied with leaf age, phosphorus concentration did not. The cause of starch accumulation in chloroplasts at salinities which are optimal for growth (340 mol m⁻³) remains unclear.     

Molecular Cloning and Different Expression of a Vacuolar Na+/H+ antiporter gene in Suaeda salsa Under Salt Stress

A Na⁺/H⁺ antiporter catalyzes the transport of Na⁺ and H⁺ across the tonoplast membrane. We isolated a vacuolar Na⁺/H⁺ antiporter cDNA (SsNHX1) clone from a euhalophyte, Suaeda salsa. The nuclear sequence contains 2262 bp with an open reading frame of 1665 bp. The deduced amino acid sequence is similar to that of AtNHX1 and OsNHX1 in rice, with the highest similarities within the predicted transmembrane segments and an amiloride-binding domain. Northern blot analysis shows that the expression of the S. salsa gene was increased by salt stress. The results suggest that the SsNHX1 product is likely a Na⁺/H⁺ antiporter and may play important roles in the salt tolerance of S. salsa. This revised version was published online in July 2006 with corrections to the Cover Date.