Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.     

Differential usage of storage carbohydrates in the CAM bromeliad Aechmea 'Maya' during acclimation to drought and recovery from dehydration

CAM requires a substantial investment of resources into storage carbohydrates to account for nocturnal CO₂ uptake, thereby restricting carbohydrate partitioning to other metabolic activities, including dark respiration, growth and acclimation to abiotic stress. Flexible modulation of carbon flow to the different competing sinks under changing environmental conditions is considered a key determinant for the growth, productivity and ecological success of the CAM pathway. The aim of the present study was to examine how shifts in carbohydrate partitioning could assure maintenance of photosynthetic integrity and a positive carbon balance under conditions of increasing water deprivation in CAM species. Measurements of gas exchange, leaf water relations, malate, starch and soluble sugar (glucose, fructose and sucrose) contents were made in leaves of the CAM bromeliad Aechmea ‘Maya’ over a 6‐month period of drought and subsequently over a 2‐month period of recovery from drought. Results indicated that short‐term influences of water stress were minimized by elevating the level of respiratory recycling, and carbohydrate pools were maintained at the expense of export for growth while providing a comparable nocturnal carbon gain to that in well‐watered control plants. Longer term drought resulted in a disproportionate depletion of key carbohydrate reserves. Sucrose, which was of minor importance for providing substrate for the dark reactions under well‐watered conditions, became the major source of carbohydrate for nocturnal carboxylation as drought progressed. Flexibility in terms of the major carbohydrate source used to sustain dark CO₂ uptake is therefore considered a crucial factor in meeting the carbon and energy demands under limiting environmental conditions. Recovery from CAM‐idling was found to be dependent on the restoration of the starch pool, which was used predominantly for provision of substrate for nocturnal carboxylation, while net carbon export was limited. The conservation of starch for the nocturnal reactions might be adaptive with regard to responding efficiently to a return of water stress.     

Malate accumulation in different organs of Mesembryanthemum crystallinum L. following age-dependent or salinity-triggered CAM metabolism

Different organs of Mesembryanthemum crystallinum exhibit differing levels of CAM (Crassulacean acid metabolism), identifiable by quantification of nocturnal malate accumulation. Shoots and also basal parts of young leaves were observed to accumulate high concentrations of malate. It was typically found in mature leaves and especially prominent in plants subjected to salt stress. Small amount of nocturnal malate accumulation was found in roots of M. crystallinum plants following age-dependent or salinity-triggered CAM. This is an indication that malate can be also stored in non-photosynthetic tissue. Measurements of catalase activity did not produce evidence of the correlation between activity of this enzyme and the level of malate accumulation in different organs of M. crystallinum although catalase activity also appeared to be dependent on the photoperiod. In all material collected at dusk catalase activity was greater than it was observed in the organs harvested at dawn.     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Environment or development? Lifetime net CO2 exchange and control of the expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

The relative influence of plant age and environmental stress signals in triggering a shift from C(3) photosynthesis to Crassulacean acid metabolism (CAM) in the annual halophytic C(3)-CAM species Mesembryanthemum crystallinum was explored by continuously monitoring net CO(2) exchange of whole shoots from the seedling stage until seed set. Plants exposed to high salinity (400 mm NaCl) in hydroponic culture solution or grown in saline-droughted soil acquired between 11% and 24% of their carbon via net dark CO(2) uptake involving CAM. In contrast, plants grown under nonsaline, well-watered conditions were capable of completing their life cycle by operating in the C(3) mode without ever exhibiting net CO(2) uptake at night. These observations are not consistent with the widely expressed view that the induction of CAM by high salinity in M. crystallinum represents an acceleration of preprogrammed developmental processes. Rather, our study demonstrates that the induction of the CAM pathway for carbon acquisition in M. crystallinum is under environmental control.     

Diel shifts in carboxylation pathway and metabolite dynamics in the CAM bromeliad Aechmea 'Maya' in response to elevated CO2

Background and Aims

The deployment of temporally separated carboxylation pathways for net CO₂ uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO₂ world. The present study examined how long-term exposure to elevated CO₂ influences the relative contributions that C₃ and C₄ carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle.

Methods

Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea ‘Maya’ after exposure to 700 µmol mol⁻¹ CO₂ for 5 months.

Key Results

There was a 60 % increase in 24-h carbon gain under elevated CO₂ due to a stimulation of daytime C₃ and C₄ carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO₂ taken up under elevated CO₂ was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO₂ there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate.

Conclusions

Despite the increased size of the soluble sugar storage pool under elevated CO₂, there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. ‘Maya’. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO₂ in this CAM bromeliad.Key words: Aechmea ‘Maya’, carbon budgets, elevated CO₂, gas exchange, metabolite dynamics, PEPC, photosynthetic plasticity, Rubisco     

Measuring whole plant CO2 exchange with the environment reveals opposing effects of the gin2–1 mutation in shoots and roots of Arabidopsis thaliana

Using a cuvette for simultaneous measurement of net photosynthesis in above ground plant organs and root respiration we investigated the effect of reduced leaf glucokinase activity on plant carbon balance. The gin2–1 mutant of Arabidopsis thaliana is characterized by a 50% reduction of glucokinase activity in the shoot, while activity in roots is about fivefold higher and similar to wild type plants. High levels of sucrose accumulating in leaves during the light period correlated with elevated root respiration in gin2–1. Despite substantial respiratory losses in roots, growth retardation was moderate, probably because photosynthetic carbon fixation was simultaneously elevated in gin2–1. Our data indicate that futile cycling of sucrose in shoots exerts a reduction on net CO₂ gain, but this is over-compensated by the prevention of exaggerated root respiration resulting from high sucrose concentration in leaf tissue.     

Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum

The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.     

The respiratory energy requirements involved in nocturnal carbohydrate export from starch-storing mature source leaves and their contribution to leaf dark respiration

The present study explores the potential contribution of theenergy requirements associated with nocturnal carbohydrate exportto (1) the fraction of dark respiration correlating with leafnitrogen concentration and (2) the dark respiration of maturesource leaves. To this end, we determined the nocturnal carbohydrate-exportrates from leaves with an optimal nitrogen supply, and the correlationbetween the nitrogen concentration and the dark respirationof leaves. The specific energy costs of carbohydrate exportfrom starch-storing source leaves were determined both experimentallyand theoretically. The present estimate of the specific energycost involved in carbohydrate export as obtained by linear regression(0.70 mol CO₂ [mol sucrose]^–1), agrees well with bothliterature data obtained by different methods (0.47 to 1.26mol CO₂ [mol sucrose]^–1) and the theoretically calculatedrange for starch-storing species (0.40 to 1.20 mol CO₂ [molsucrose]^–1). The conversion of starch in the chloroplastto sucrose in the cytosol is a major energy-requiring process.Maximally 42 to ‘107’% of the slope of the relationshipbetween respiration rate and organic nitrogen concentrationof primary bean leaves, may be ascribed to the energy costsassociated with nocturnal export of carbohydrates. Total energycosts associated with export were derived from the product ofthe specific costs of carbohydrate export and the export rates,either measured on full-grown (primary) leaves of potato andbean or derived from the literature. These export costs account,on average, for 29% of the dark respiration rate in starch-storingspecies. We conclude that nocturnal carbohydrate export is amajor energy-requiring process in starch-storing species Key words: Carbohydrate export, leaf dark respiration, nitrogen concentration, respiratory costs, specific energy cost     

Metabolome and water status phenotyping of Arabidopsis under abiotic stress cues reveals new insight into ESK1 function

Metabolomic investigation of the freezing-tolerant Arabidopsis mutant esk1 revealed large alterations in polar metabolite content in roots and shoots. Stress metabolic markers were found to be among the most significant metabolic markers associated with the mutation, but also compounds related to growth regulation or nutrition. The metabolic phenotype of esk1 was also compared to that of wild type (WT) under various environmental constraints, namely cold, salinity and dehydration. The mutant was shown to express constitutively a subset of metabolic responses which fits with the core of stress metabolic responses in the WT. But remarkably, the most specific metabolic responses to cold acclimation were not phenocopied by esk1 mutation and remained fully inducible in the mutant at low temperature. Under salt stress, esk1 accumulated lower amounts of Na⁺ in leaves than the WT, and under dehydration stress its metabolic profile and osmotic potential were only slightly impacted. These phenotypes are consistent with the hypothesis of an altered water status in esk1 , which actually exhibited basic lower water content (WC) and transpiration rate (TR) than the WT. Taken together, the results suggest that ESK1 does not function as a specific cold acclimation gene, but could rather be involved in water homeostasis.     

Growth,physiological, biochemical and ionic responses of pistachio seedlings to mild and high salinity

Key message

Depending on salt concentrations, different mechanisms are involved in the tolerance of pistachio and an acclimation to salinity conditions occurs in the leaves that develop in the presence of salt.

Abstract

Pistachio (Pistacia vera L.) is a salt tolerant species that is considered an alternative crop for cultivation in salinzied orchard soils. In this work, 12-week-old pistachio seedlings cultivated in soil under greenhouse conditions were treated with five levels of salinity including control (0.63 dSm^?1), low (2 and 4 dSm^?1) and high (8 and 10 dSm^?1) salt concentrations for further 12 weeks. Plant growth parameters were not affected by mild salinity; a significant reduction was only observed from 8 dSm^?1. Considerable differences were observed between the young and mature leaves regarding osmotic and ionic stress effects of salt. Main compatible solutes were proline in mature leaves, proline and soluble sugars in young leaves, and soluble sugars and amino acids, other than proline, in roots. Concentration and content of Na in the leaves were not significantly increased at low levels of salinity and the K:Na and Ca:Na ratio of leaves were affected only by higher salt concentrations. Using the sequential extraction procedure for cell wall isolation, we observed that both absolute and relative amounts of Na in the cell wall fraction increased under low salinity, while decreased under higher levels of salt supply. Stable water relations, photochemistry and CO₂ assimilation rates particularly of young leaves, as well as ion homeostasis were mechanisms for maintenance of plants growth under mild salinity. Under severe saline conditions, the impaired ability of mature leaves for synthesis of assimilates, preferent allocation of carbohydrates to roots for maintenance of osmotic homeostasis and finally, reduction of protein synthesis caused growth inhibition in pistachio.     

Spatio-temporal leaf growth patterns of Arabidopsis thaliana and evidence for sugar control of the diel leaf growth cycle

Leaf growth dynamics are driven by diel rhythms. The analysis of spatio-temporal leaf growth patterns in Arabidopsis thaliana wild type and mutants of interest is a promising approach to elucidate molecular mechanisms controlling growth. The diel availability of carbohydrates is thought to affect diel growth. A digital image sequence processing (DISP)-based noninvasive technique for visualizing and quantifying highly resolved spatio-temporal leaf growth was adapted for the model plant A. thaliana. Diel growth patterns were analysed for the wild type and for a mutant with altered diel carbohydrate metabolism. A. thaliana leaves showed highest relative growth rates (RGRs) at dawn and lowest RGRs at the beginning of the night. Along the lamina, a clear basipetal gradient of growth rate distribution was found, similar to that in many other dicotyledonous species. The starch-free 1 (stf1) mutant revealed changed temporal growth patterns with reduced nocturnal, and increased afternoon, growth activity. The established DISP technique is presented as a valuable tool to detect altered temporal growth patterns in A. thaliana mutants. Endogenous changes in the diel carbohydrate availability of the starch-free mutant clearly affected its diel growth rhythms.     

A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum L

A strongly increased ATP/ADP ratio was found during the nocturnal phase I in crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants. Conversely, during the daytime phase III in CAM-performing plants the ATP/ADP ratio dropped to a similar level to that of C3 plants, cytochrome c oxidase activity was stimulated and mitochondrial Mn-superoxide dismutase activity was strongly increased. The findings suggest that a salinity-induced C3-CAM transition might be an efficient energy-conserving strategy for M. crystallinum plants, in which the strong nocturnal ATP production seems to be, at least partially, independent from the coupled mitochondrial electron transport.     

NaCl胁迫下抗盐突变体和野生型小麦Na~ 、K~ 累积的差异分析

对抗盐突变体和野生型小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）在盐胁迫下Ｎａ＋、Ｋ＋的累积状况进行了比较研究。结果表明,ＮａＣｌ胁迫下突变体根和叶中Ｎａ＋的相对累积较野生型显著地少,同时Ｎａ＋的净累积速率显著地低。这种Ｎａ＋相对累积少的状况在叶中表现尤为明显。两者叶中Ｋ＋含量随盐浓度的增加显著下降,但突变体的含量均高于野生型。突变体根中Ｋ＋的含量也显著高于野生型,且这种差异随盐浓度的提高而增大。分别统计突变体和野生型根和叶中Ｎａ＋的含量以及每株幼苗的Ｎａ＋总量以分析二者Ｎａ＋在根和叶中的分布差异,结果表明３００ｍｍｏｌ／ＬＮａＣｌ胁迫９６ｈ后,突变体根中Ｎａ＋的累积量占其整株幼苗Ｎａ＋累积总量的４４４％,而野生型根中含量占其总量的２４３％。相对于野生型而言,在盐胁迫下突变体根中Ｎａ＋分布比例的提高可有效地减少根中Ｎａ＋向地上部分转运。     

Difference in Na+,K+ Accumulation in the Salt tolerant Mutant and the Wild Type of Wheat During Exposure to NaCl Stress

The difference in Na+, K+ accumulation between the mutant and the wild type of wheat ( Triticum aestivum L. ) has been investigated. The authors report here that the mutant accumulated less Na + in the root and leaf than the wild type in response to NaCI stress. This difference in Na + accumulation in leaf was more significant than that in the root. The mutant kept a lower net accumulation rate of Na + than that in the wild type during the stress. K+ content in the leaves and roots of beth species reduced severely when exposed to NaC1, but the contents in the leaf and root of the mutant was higher than those of the wild type. The Na + dis- tribution in the seedlings of the mutant and the wild type was significantly different. When exposed to salt stress for 96 h, the quantity of the accumulated Na + in root was 44.3 % of the total Na + per seedling of the mutant, whereas it was 24.3% in the wild type, which was likely resulted from the reduction of Na+ transfer from roots to shoots in the mutant.     

拟南芥ANAC055基因突变体对高盐和渗透胁迫的响应

高盐和渗透等非生物胁迫是影响农作物产量和品质的重要因素,非生物胁迫发生时,植物通过体内各类转录因子启动胁迫应答反应,进而降低非生物胁迫对植物的损伤。本研究筛选出植物特异性转录因子ANAC055编码基因的纯合T-DNA插入突变体SALK_152738,测序分析发现T-DNA插在ANAC055基因的3'UTR区域。实时荧光定量PCR结果表明叶中ANAC055基因表达量最高;与野生型相比,突变体叶、茎和花中ANAC055基因表达量分别下降了40%、50%和70%。高盐胁迫后,野生型和突变体叶中ANAC055基因表达量分别比对照上升了320%和55.4%;而渗透胁迫时,该基因叶中的表达量分别比对照下降了47.7%和56.3%;电子表达谱分析发现该基因根中的表达可受高盐和渗透等多种非生物胁迫的诱导表达。高盐和渗透胁迫时野生型和突变体幼根的生长均受到明显抑制,但高盐胁迫对突变体根生长的抑制作用比对野生型根生长的抑制作用更大。上述分析表明拟南芥ANAC055基因可受高盐和渗透等非生物胁迫的诱导表达,并且其在拟南芥幼根的生长发育过程中具有一定的作用,本研究有助于进一步明确其在非生物胁迫过程中的作用。     

Carbohydrate sources and sinks in woody plants

Each perennial woody plant is a highly integrated system of competing carbohydrate sinks (utilization sites). Internal competition for carbohydrates is shown by changes in rates of carbohydrate movement from sources to sinks and reversals in direction of carbohydrate transport as the relative sink strengths of various organs change. Most carbohydrates are produced in foliage leaves but some are synthesized in cotyledons, hypocotyls, buds, twigs, stems, flowers, fruits, and strobili. Although the bulk of the carbohydrate pool moves to sinks through the phloem, some carbohydrates are obtained by sinks from the xylem sap. Sugars are actively accumulated in the phloem and move passively to sinks along a concentration gradient. The dry weight of a mature woody plant represents only a small proportion of the photosynthate it produced. This discrepancy results not only from consumption of plant tissues by herbivores and shedding of plant parts, but also from depletion of carbohydrates by respiration, leaching, exudation, secretion, translocation to other plants through root grafts and mycorrhizae and losses to parasites. Large spatial and temporal variations occur in the use of reserve- and currently produced carbohydrates in metabolism and growth of shoots, stems, roots, and reproductive structures. A portion of the carbohydrate pool is diverted for production of chemicals involved in defense against fungi, herbivores, and competing plants. Woody plants accumulate carbohydrates during periods of excess production and deplete carbohydrates when the rate of utilization exceeds the rate of production. Stored carbohydrates play an important role in metabolism, growth, defense, cold hardiness, and postponement or prevention of plant mortality.