Bioengineering plant resistance to abiotic stresses by the global calcium signal system

Considerable progresses have taken place both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades. It is generally accepted that the global calcium signal system functions importantly in coping with plant abiotic stresses, especially drought stress, which has been proved further by the recent transgenic and molecular breeding reports under soil water deficits. In plant cells, calcium plays roles as a universal transducer coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin (CAM) is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of CAM targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plant cells and remain characterized, calling for a concerted effort from plant and animal scientists to elucidate their functions. To date, three major classes of plant calcium signals encoding elements in the calcium signal system, including calcium-permeable ion channels,Ca(2)+/ H(+) antiporters and Ca(2)+-ATPases, are responsible for drought stress signal transduction directly or indirectly. This review summarizes the current knowledge of calcium signals involved in plant abiotic stresses and presents suggestions for future focus areas of study.     

Advances of calcium signals involved in plant anti-drought

Considerable progresses have taken place, both in the methodology available to study changes in intracellular cytosolic calcium and in our understanding of calcium signaling cascades, but how calcium signals function in plant drought resistance is questionable. In plant cells, calcium plays roles as a second messenger coupling a wide range of extracellular stimuli with intracellular responses. Different extracellular stimuli trigger specific calcium signatures: dynamics, amplitude and duration of calcium transients specify the nature, implication and intensity of stimuli. Calcium-binding proteins (sensors) play a critical role in decoding calcium signatures and transducing signals by activating specific targets and corresponding metabolic pathways. Calmodulin is a calcium sensor known to regulate the activity of many mammalian proteins, whose targets in plants are now being identified. Higher plants possess a rapidly growing list of calmodulin targets with a variety of cellular functions. Nevertheless, many targets appear to be unique to higher plants and remain characterized, calling for a concerted effort to elucidate their functions. To date, three major classes of plant calcium signals, including calcium permeable ion channels, Ca(2+)/H(+) antiporters and Ca(2+)-ATPases, have been responsible for drought-stress signal transduction. This review summarizes the current knowledge of calcium signals involved in plant anti-drought and plant water use efficiency (WUE) and presents suggestions for future focus of study.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.     

非生物逆境胁迫下植物钙信号转导的分子机制

Ca2+作为植物细胞中最重要的第二信使, 参与植物对许多逆境信号的转导。在非生物逆境条件下, 植物细胞质内的钙离子在时间、空间及浓度上会出现特异性变化, 即诱发产生钙信号。钙信号再通过其下游的钙结合蛋白进行感受和转导, 进而在细胞内引起一系列的生物化学反应以适应或抵制各种逆境胁迫。目前在植物细胞中发现Ca2+/CDPK、Ca2+/CaM和Ca2+/CBL 3类钙信号系统, 研究表明它们与非生物逆境胁迫信号转导密切相关。本文通过从植物在非生物逆境条件下钙信号的感受、转导到产生适应性和抗性等方面, 介绍钙信号转导分子机制的一些研究进展。     

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

Modelling and experimental analysis of the role of interacting cytosolic and vacuolar pools in shaping low temperature calcium signatures in plant cells

A major challenge to understanding low temperature calcium signatures in plants is defining how these signatures emerge from the interactions of different molecular components that are stored in different subcellular pools of a plant cell. Here we develop an integrative model that incorporates the interactions of Ca2?, H?, K?, Cl? and ATP in both cytosolic and vacuolar pools. Our analysis reveals how these four major ions along with ATP forms a complex network to relate the emergence of calcium signatures to other responses (e.g. pH response). Modelling results are in agreement with experimental observations for both cytosolic free calcium concentration ([Ca2?](c)) and pH. The model is further validated by experimentally measuring the response of [Ca2?](c) to six fluctuating (rather than constant) temperature profiles. We found that modelling results are in reasonable agreement with experimental observations, in particular, if the rate of reducing temperature is relatively high. In addition, we show that both calcium-induced calcium release (CICR) at the vacuolar membrane and transport of ions from the cytosolic pool to the vacuolar membrane play important roles in the interaction between cytosolic and vacuolar pools. In combination they control the amount and timing of calcium release from the vacuolar to cytosolic pool, shaping the specific calcium signature. The methodology and principles developed here establish an integrative view on the role of cytosolic and vacuolar pools in shaping calcium signatures in general, and they are universally applicable to study of the interactions of multiple subcellular pools.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.     

Multiple calmodulin-like proteins in Arabidopsis are induced by insect-derived (Spodoptera littoralis) oral secretion

In plant cells, diverse environmental changes often induce transient elevation in the intracellular calcium concentrations, which are involved in signaling pathways leading to the respective cellular reactions. Therefore, these calcium elevations need to be deciphered into specific downstream responses. Calmodulin-like-proteins (CMLs) are calcium-sensing proteins present only in higher plants. They are involved in signaling processes induced by both abiotic as well as biotic stress factors. However, the role of CMLs in the interaction of plants with herbivorous insects is almost unknown. Here we show that in Arabidopsis thaliana a number of CMLs genes (CML9, 11,12,16,17 and 23) are upregulated due to treatments with oral secretion of larvae of the herbivorous insect Spodoptera littoralis. We identified that these genes belong to two groups that respond with different kinetics to the treatment with oral secretion. Our data indicate that signaling networks involving multiple CMLs very likely have important functions in plant defense against insect herbivores, in addition to their involvement in many other stress-induced processes in plants.     

Insects feeding on plants: rapid signals and responses preceding the induction of phytochemical release

The ability of plants to withstand herbivores relies on direct and indirect chemical defense. By using toxic phytochemicals, plants can deter and/or poison herbivores, while by releasing volatile organic compounds (VOCs) into the atmosphere plants can attract predators of the herbivores. The activation of specific responses requires recognition and appropriate response towards the attacking enemy and most of the events which finally lead to gene activation (the signaling pathway) occur within a few minutes. Among the several signaling molecules involved, reactive oxygen species (ROS) and intracellular calcium signatures belong to early events, which are responsible for most of the ensuing cascades of chemical and molecular reactions. In this review, we will focus on rapid early events following insect feeding on plants that eventually lead to the production and release of phytochemicals. Furthermore, we compare and discuss the impact of mechanical and biotroph wounding.     

Apoptotic-like programmed cell death in plants

Programmed cell death (PCD) is now accepted as a fundamental cellular process in plants. It is involved in defence, development and response to stress, and our understanding of these processes would be greatly improved through a greater knowledge of the regulation of plant PCD. However, there may be several types of PCD that operate in plants, and PCD research findings can be confusing if they are not assigned to a specific type of PCD. The various cell-death mechanisms need therefore to be carefully described and defined. This review describes one of these plant cell death processes, namely the apoptotic-like PCD (AL-PCD). We begin by examining the hallmark 'apoptotic-like' features (protoplast condensation, DNA degradation) of the cell's destruction that are characteristic of AL-PCD, and include examples of AL-PCD during the plant life cycle. The review explores the possible cellular 'executioners' (caspase-like molecules; mitochondria; de novo protein synthesis) that are responsible for the hallmark features of the cellular destruction. Finally, senescence is used as a case study to show that a rigorous definition of cell-death processes in plant cells can help to resolve arguments that occur in the scientific literature regarding the timing and control of plant cell death.     

Cytosolic calcium and pH signaling in plants under salinity stress

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH_cyt, either individually, or in coordination with changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca²⁺ and protons. The variety and complexity of Ca²⁺ and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca²⁺]_cyt concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca²⁺]_cyt and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca²⁺]_cyt and pH_cyt at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.Key words: cytosolic calcium, ionic toxicity, osmotic stress, pH, salinity stress, salt tolerance, signaling     

Reactive oxygen species (ROS) as defenses against a broad range of plant fungal infections and case study on ROS employed by crops against Verticillium dahliae wilts

Reactive oxygen species (ROS) are natural by products of cellular metabolism that were initially considered only deleterious towards the cellular macromolecules. Research advances have broadened the scope and now numerous studies are available rendering ROS molecules essential for plants to combat several biotic and abiotic stresses after being involved in essential defense mechanisms such as hypersensitivity reactions (HR) that lead to programmed cell death (PCD), cell wall reinforcement by cross-linking of cellular glycoproteins with other entities and salicylic acid mediated signal transduction pathways. During fungal attack, the fungal components like chitin and other elicitors activates the plant immune responses that employ ROS with other molecules like nitric oxide (NO), calcium ions to fight back the pathogen attack and restrict its spread to further plant parts. Here, several defense mechanisms mediated by ROS are discussed. Verticillium dahliae is one of the dreadful fungal pathogen to plants that cause wilts in many important plant species causing huge economic burden in food sector. The major constraint in its scenario being the deficit of field management systems based on chemicals or agronomics. It is evident by studying their interactions with the variety of hosts that in most cases, ROS mediated defenses play a key central role via cross-talk with other mechanisms making them a potential target for transgenics as well as resistant genotype selection.     

Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells

Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.     

荒漠植物不同功能群性状特征及其与土壤环境的关系

研究不同功能群植物性状差异及其与土壤环境关系对于充分掌握植物的环境适应策略至关重要。以艾比湖流域为研究区,利用荒漠植物的植物高度、叶片碳、氮、磷、硫、钾、钙、钠、镁含量等9个性状,将高、低土壤水盐环境下的植物划分为5个功能群,分析不同功能群的植物组成、性状差异及其与土壤环境的关系。结果表明：(1)不同土壤水盐环境下,其植物功能群组成不同;其中白刺、胡杨和罗布麻在两个土壤水盐环境下的功能群中均存在。(2)植物的功能性状在不同土壤水盐环境下也发生了适应性的变化。高土壤水盐环境下3个功能群的植物高度、叶片碳、氮、磷和钙含量显著高于低土壤水盐环境功能群(P<0.05);低土壤水盐环境下2个功能群的植物叶片硫、钠和镁含量高于高土壤水盐环境功能群。(3)土壤含水量(SVWC)、电导率(EC)、pH以及土壤磷含量对荒漠植物功能性状影响较大。在高土壤水盐环境下,EC、pH与植物高度,叶片钾、钙含量正相关,与叶片硫含量负相关;在低土壤水盐环境下,SVWC、EC与植物高度呈显著正相关(P<0.05)。研究有助于理解荒漠植物对极端环境的适应对策,为保护荒漠地区生物多样性提供理论依据。     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

喀斯特地区植物钙含量特征与高钙适应方式分析

喀斯特地区土壤的高钙含量是影响该地区植物生理特征的最重要环境因素之一。高钙影响植物的光合作用、生长速率及磷代谢, 从而限制了许多物种在该地区的分布。选取贵州4个石漠化程度不同的地区, 测定采集地内45种优势种或常见种的地上部分和地下部分的全钙含量以及土壤的交换性钙含量。通过分析喀斯特地区植物与土壤钙含量的特征发现: 喀斯特地区植物具有较高的钙含量平均值; 土壤交换性钙含量对植物地上部分钙含量的影响总体上不显著, 对植物地下部分钙含量的影响显著; 不同类别植物的钙含量存在显著差异, 蕨类植物地上部分钙含量平均值明显低于被子植物; 不同类别植物钙的分布部位也存在显著差异, 在蕨类植物和单子叶植物中地上部分和地下部分的钙含量相近, 而双子叶植物的地上部分钙含量明显高于地下部分。分析了喀斯特地区14种优势灌木和草本植物地上部分与地下部分钙含量的差异性以及与土壤交换性钙含量的相关关系, 以此为根据将14种优势植物对土壤高钙的适应方式分为3种类型: 随遇型、高钙型和低钙型。随遇型植物的钙含量主要受土壤交换性钙含量影响, 其地上部分和地下部分的钙含量均与土壤交换性钙含量成显著正相关关系; 高钙型植物具有较强的钙富集能力, 其地上部分即使在低钙含量的土壤中也可维持较高的钙含量; 低钙型植物的地上部分即使在高钙含量的土壤中亦可维持较低的钙含量。对植物适应钙的不同方式的研究可用于筛选退化生态系统恢复所需的植物资源。     

Isotopic equilibration of calcium-45 with labile soil calcium

Summary When soils are suspended in solutions of labelled calcium chloride isotopic exchange with the labile soil calcium occurs rapidly. This may be followed by a slow secondary exchange reaction, but its magnitude is not great and equilibrium is nearly, if not completely, attained within 7 days.When, however, plants are grown in soil throughout which carrier-free calcium-45 has been thoroughly mixed, it is found that the calcium-45 absorbed by the plants has equilibrated with a quantity of soil calcium larger than that which undergoes isotopic exchange when soils are suspended in solutions of labelled calcium chloride. The analysis of plants grown for varying periods shows that equilibration can continue for several weeks, and that the quantity of soil calcium with which the calcium-45 is associated can be increased both by the addition of electrolytes to the soil and by growing plants under exhaustion conditions. In 5 soils the extra calcium which equilibrated with calcium-45 in this way never exceeded 3.5 per cent of the total soil calcium, and was usually considerably lower.The continued equilibration of calcium-45 with soil calcium causes the specific activity (Ca⁴⁵/stable Ca) of the calcium entering plants to decrease. Because the calcium in plant roots has, on average, been absorbed more recently than that in shoots, the latter show higher specific activities.The causes of these effects are discussed and consideration is given to their significance in the interpretation of results of experiments involving the use of calcium-45 as a tracer in soils.     

Provenance- and life-history stage-specific responses of the dwarf shrub Calluna vulgaris to elevated vapour pressure deficit

Climate change may alter microscale-effective ecosystem properties such as atmospheric water vapour pressure, but consequences for plant growth are insufficiently understood. Within a northwest German heathland an open-top chamber experiment was established to analyse the effects of elevated vapour pressure deficit (eVPD) on growth responses of Calluna vulgaris considering both plant origin (Atlantic (AP), sub-Atlantic (SAP), sub-Continental (SCP)) and life-history stage (1-year vs. 10-year old plants). We hypothesised that the plants’ sensitivity to eVPD decreases (i) from AP to SCP and (ii) with progressing life-history stage. Elevated VPD caused a provenance-specific decrease of shoot increment whilst aboveground biomass productivity remained unaffected. AP and SAP responded with increasing belowground biomass δ¹³C signatures to eVPD, whereas δ¹³C values decreased for SCP. Moreover, eVPD increased and decreased belowground biomass δ¹³C signatures of 1- and 10-year old plants, respectively. These responses to eVPD were related to differences in morphological-chemical traits and the plants’ trait plasticity in response to eVPD. SCP showed the highest aboveground tissue mass density and significantly increased tissue C:N ratios under eVPD. One-year old plants had a tenfold higher shoot:root ratio than 10-year old plants, making young plants more sensitive to eVPD. Our findings demonstrate that the atmospheric water status affects the morphology and physiology of Calluna independent of the soil water status. The results have implications for the conservation of heathlands under climate change: (i) SCP may constitute an appropriate ecotype for assisted migration-approaches, and (ii) management needs to weigh different options for heathland rejuvenation.