Serotonin, a tryptophan-derived signal conserved in plants and animals, regulates root system architecture probably acting as a natural auxin inhibitor in Arabidopsis thaliana

Serotonin (5-hydroxytryptamine) is a well-known neurotransmitter in mammals and is widely distributed in plants. This compound is synthesized from tryptophan and shares structural similarity with IAA. To date, little is known about the morphological, physiological and molecular responses of plants to serotonin. In this study, we characterized the effects of serotonin on growth and development in Arabidopsis thaliana seedlings. Gas chromatography-mass spectrometry (GC-MS) analysis showed that plants are able to take up serotonin from the growth medium, which coincided with greatly stimulated lateral root development at concentrations from 10 to 160 μM. In contrast, higher doses of serotonin repressed lateral root growth, primary root growth and root hair development, but stimulated adventitious root formation. To investigate the role of serotonin in modulating auxin responses, we performed experiments using transgenic Arabidopsis lines expressing the auxin-responsive marker constructs DR5:uidA, BA3:uidA and HS::AXR3NT-GUS, as well as a variety of Arabidopsis mutants defective at the AUX1, AXR1, AXR2 and AXR4 auxin-related loci. We found that serotonin strongly inhibited both DR5:uidA and BA3:uidA gene expression in primary and adventitious roots and in lateral root primordia. This compound also abolished the effects of IAA or naphthaleneacetic acid on auxin-regulated developmental and genetic responses, indicating an anti-auxin activity in the plant. Mutant analysis further showed that lateral root induction elicited by serotonin was independent of the AUX1 and AXR4 loci but required AXR1 and AXR2. Our results show that serotonin regulates root development probably by acting as a natural auxin inhibitor.     

AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling

Arabidopsis root architecture is regulated by shoot-derived signals such as nitrate and auxin. We report that mutations in the putative auxin influx carrier AUX1 modify root architecture as a result of the disruption in hormone transport between indole-3-acetic acid (IAA) source and sink tissues. Gas chromatography-selected reaction monitoring-mass spectrometry measurements revealed that the aux1 mutant exhibited altered IAA distribution in young leaf and root tissues, the major IAA source and sink organs, respectively, in the developing seedling. Expression studies using the auxin-inducible reporter IAA2::uidA revealed that AUX1 facilitates IAA loading into the leaf vascular transport system. AUX1 also facilitates IAA unloading in the primary root apex and developing lateral root primordium. Exogenous application of the synthetic auxin 1-naphthylacetic acid is able to rescue the aux1 lateral root phenotype, implying that root auxin levels are suboptimal for lateral root primordium initiation in the mutant.     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana

When growing under limiting phosphate (P) conditions, Arabidopsis thaliana plants show dramatic changes in root architecture, including a reduction in primary root length, increased formation of lateral roots and greater formation of root hairs. Here we report that primary root growth inhibition by low P is caused by a shift from an indeterminate to a determinate developmental program. In the primary root, the low P-induced determinate growth program initiates with a reduction of cell elongation followed by the progressive loss of meristematic cells. At later stages, cell proliferation ceases and cell differentiation takes place at the former cell elongation and meristematic regions of the primary root. In low P, not only the primary but also almost all mature lateral roots enter the determinate developmental program. Kinetic studies of expression of the cell cycle marker CycB1;1:uidA and the quiescent center (QC) identity marker QC46:GUS showed that in low P conditions, reduction in proliferation in the primary root was preceded by alterations in the QC. These results suggest that in Arabidopsis, P limitation can induce a determinate root developmental program that plays an important role in altering root system architecture and that the QC could act as a sensor of environmental signals.     

Cytokinin receptors are involved in alkamide regulation of root and shoot development in Arabidopsis

Alkamides and N-acilethanolamides are a class of lipid compounds related to animal endocannabinoids of wide distribution in plants. We investigated the structural features required for alkamides to regulate plant development by comparing the root responses of Arabidopsis (Arabidopsis thaliana) seedlings to a range of natural and synthetic compounds. The length of the acyl chain and the amide moiety were found to play a crucial role in their biological activity. From the different compounds tested, N-isobutyl decanamide, a small saturated alkamide, was found to be the most active in regulating primary root growth and lateral root formation. Proliferative-promoting activity of alkamide treatment was evidenced by formation of callus-like structures in primary roots, ectopic blades along petioles of rosette leaves, and disorganized tumorous tissue originating from the leaf lamina. Ectopic organ formation by N-isobutyl decanamide treatment was related to altered expression of the cell division marker CycB1:uidA and an enhanced expression of the cytokinin-inducible marker ARR5:uidA both in roots and in shoots. The involvement of cytokinins in mediating the observed activity of alkamides was tested using Arabidopsis mutants lacking one, two, or three of the putative cytokinin receptors CRE1, AHK2, and AHK3. The triple cytokinin receptor mutant was insensitive to N-isobutyl decanamide treatment, showing absence of callus-like structures in roots, the lack of lateral root proliferation, and absence of ectopic outgrowths in leaves under elevated levels of this alkamide. Taken together our results suggest that alkamides and N-acylethanolamides may belong to a class of endogenous signaling compounds that interact with a cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development.     

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.     

GCR1和GCR2在N-酰基高丝氨酸内酯调节拟南芥根生长中的作用

N-酰基高丝氨酸内酯（AHLs）是革兰氏阴性细菌群体感应的信号分子。培养基中添加1μmol·L-13OC6-HSL和10μmol·L-13OC8-HSL可显著促进野生型拟南芥主根生长,但拟南芥G蛋白偶联受体GCR1和GCR2基因缺失突变体gcr1-1和gcr2-2对AHLs处理不敏感;实时荧光定量PCR分析显示,这2种AHLs的处理可以使拟南芥GCR1和GCR2基因表达量上调2～4倍。结果表明,G蛋白偶联受体GCR1和GCR2可能参与植物感应细菌信号进而做出根生长响应的信号转导。     

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.     

Plant growth-promoting rhizobacteria modulate root-system architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> through volatile organic compound emission

Extensive communication occurs between plants and microorganisms during different stages of plant development in which signaling molecules from the two partners play an important role. Volatile organic compounds (VOCs) emission by certain plant-growth promoting rhizobacteria (PGPR) has been found to be involved in plant growth. However, little is known about the role of bacterial VOCs in plant developmental processes. In this work, we investigated the effects of inoculation with twelve bacterial strains isolated from the rhizosphere of lemon plants (Citrus aurantifolia) on growth and development of Arabidopsis thaliana seedlings. Several bacterial strains showed a plant growth promoting effect stimulating biomass production, which was related to differential modulation of root-system architecture. The isolates L263, L266, and L272a stimulated primary root growth and lateral root development, while L254, L265a and L265b did not significantly alter primary root growth but strongly promoted lateral root formation. VOC emission analysis by SPME-GC-MS identified aldehydes, ketones and alcohols as the most abundant compounds common to most rhizobacteria. Other VOCs, including 1-octen-3-ol and butyrolactone were strain specific. Characterization of L254, L266 and L272a bacterial isolates by 16S rDNA analysis revealed the identity of these strains as Bacillus cereus, Bacillus simplex and Bacillus sp, respectively. Taken together, our data suggest that rhizospheric bacterial strains can modulate both plant growth promotion and root-system architecture by differential VOC emission.     

Aberrant temporal growth pattern and morphology of root and shoot caused by a defective circadian clock in Arabidopsis thaliana

Circadian clocks synchronized with the environment allow plants to anticipate recurring daily changes and give a fitness advantage. Here, we mapped the dynamic growth phenotype of leaves and roots in two lines of Arabidopsis thaliana with a disrupted circadian clock: the CCA1 over‐expressing line (CCA1ox) and the prr9 prr7 prr5 (prr975) mutant. We demonstrate leaf growth defects due to a disrupted circadian clock over a 24 h time scale. Both lines showed enhanced leaf growth compared with the wild‐type during the diurnal period, suggesting increased partitioning of photosynthates for leaf growth. Nocturnal leaf growth was reduced and growth inhibition occurred by dawn, which may be explained by ineffective starch degradation in the leaves of the mutants. However, this growth inhibition was not caused by starch exhaustion. Overall, these results are consistent with the notion that the defective clock affects carbon and energy allocation, thereby reducing growth capacity during the night. Furthermore, rosette morphology and size as well as root architecture were strikingly altered by the defective clock control. Separate analysis of the primary root and lateral roots revealed strong suppression of lateral root formation in both CCA1ox and prr975, accompanied by unusual changes in lateral root growth direction under light–dark cycles and increased lateral extension of the root system. We conclude that growth of the whole plant is severely affected by improper clock regulation in A. thaliana, resulting not only in altered timing and capacity for growth but also aberrant development of shoot and root architecture.     

Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis

Phosphorus, one of the essential elements for plants, is often a limiting nutrient in soils. Low phosphate (Pi) availability induces sugar-dependent systemic expression of genes and modulates the root system architecture (RSA). Here, we present the differential effects of sucrose (Suc) and auxin on the Pi deficiency responses of the primary and lateral roots of Arabidopsis (Arabidopsis thaliana). Inhibition of primary root growth and loss of meristematic activity were evident in seedlings grown under Pi deficiency with or without Suc. Although auxin supplementation also inhibited primary root growth, loss of meristematic activity was observed specifically under Pi deficiency with or without Suc. The results suggested that Suc and auxin do not influence the mechanism involved in localized Pi sensing that regulates growth of the primary root and therefore delineates it from sugar-dependent systemic Pi starvation responses. However, the interaction between Pi and Suc was evident on the development of the lateral roots and root hairs in the seedlings grown under varying levels of Pi and Suc. Although the Pi+ Suc- condition suppressed lateral root development, induction of few laterals under the Pi- Suc- condition point to increased sensitivity of the roots to auxin during Pi deprivation. This was supported by expression analyses of DR5uidA, root basipetal transport assay of auxin, and RSA of the pgp19 mutant exhibiting reduced auxin transport. A significant increase in the number of lateral roots under the Pi- Suc- condition in the chalcone synthase mutant (tt4-2) indicated a potential role for flavonoids in auxin-mediated Pi deficiency-induced modulation of RSA. The study thus demonstrated differential roles of Suc and auxin in the developmental responses of ontogenetically distinct root traits during Pi deprivation. In addition, lack of cross talk between local and systemic Pi sensing as revealed by the seedlings grown under either the Pi- Suc- condition or in the heterogeneous Pi environment highlighted the coexistence of Suc-independent and Suc-dependent regulatory mechanisms that constitute Pi starvation responses.     

N-3-oxo-decanoyl-L-homoserine-lactone activates auxin-induced adventitious root formation via hydrogen peroxide- and nitric oxide-dependent cyclic GMP signaling in mung bean

N-Acyl-homoserine-lactones (AHLs) are bacterial quorum-sensing signaling molecules that regulate population density. Recent evidence demonstrates their roles in plant defense responses and root development. Hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and cyclic GMP (cGMP) are essential messengers that participate in various plant physiological processes, but how these messengers modulate the plant response to N-acyl-homoserine-lactone signals remains poorly understood. Here, we show that the N-3-oxo-decanoyl-homoserine-lactone (3-O-C10-HL), in contrast to its analog with an unsubstituted branch chain at the C3 position, efficiently stimulated the formation of adventitious roots and the expression of auxin-response genes in explants of mung bean (Vigna radiata) seedlings. This response was mimicked by the exogenous application of auxin, H(2)O(2), NO, or cGMP homologs but suppressed by treatment with scavengers or inhibitors of H(2)O(2), NO, or cGMP metabolism. The 3-O-C10-HL treatment enhanced auxin basipetal transport; this effect could be reversed by treatment with H(2)O(2) or NO scavengers but not by inhibitors of cGMP synthesis. Inhibiting 3-O-C10-HL-induced H(2)O(2) or NO accumulation impaired auxin- or 3-O-C10-HL-induced cGMP synthesis; however, blocking cGMP synthesis did not affect auxin- or 3-O-C10-HL-induced H(2)O(2) or NO generation. Additionally, cGMP partially rescued the inhibitory effect of H(2)O(2) or NO scavengers on 3-O-C10-HL-induced adventitious root development and auxin-response gene expression. These results suggest that 3-O-C10-HL, unlike its analog with an unmodified branch chain at the C3 position, can accelerate auxin-dependent adventitious root formation, possibly via H(2)O(2)- and NO-dependent cGMP signaling in mung bean seedlings.     

Two G-protein-coupled-receptor candidates, Cand2 and Cand7, are involved in Arabidopsis root growth mediated by the bacterial quorum-sensing signals N-acyl-homoserine lactones

Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules to coordinate their group behavior. Recently, it was shown that plants can perceive and respond to these bacterial AHLs. However, little is known about the molecular mechanism underlying the response of plants to bacterial QS signals. In this study, we show that the promotion of root elongation in wild type Arabidopsis thaliana induced by the AHLs N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) or N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) was completely abolished in plants with loss-of-function mutations in two candidate G-protein Coupled Receptors (GPCRs), Cand2 and Cand7. Furthermore, real-time PCR analysis revealed that the expression levels of Cand2 and Cand7 were elevated in plants treated with 3OC6-HSL or 3OC8-HSL. These results suggest that Cand2 and Cand7 are involved in the regulation of root growth by bacterial AHLs and that GPCRs play a role in mediating interactions between plants and microbes.     

Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings

Changes in root architecture are one of the adaptive strategies used by plants to compensate for nutrient deficiencies in soils. In this work, the temporal responses of Arabidopsis (Arabidopsis thaliana) root system architecture to low boron (B) supply were investigated. Arabidopsis Col-0 seedlings were grown in 10 μM B for 5 days and then transferred to a low B medium (0.4 μM) or control medium (10 μM) for a 4-day period. Low B supply caused an inhibition of primary root (PR) growth without altering either the growth or number of lateral roots (LRs). In addition, low B supply induced root hair formation and elongation in positions close to the PR meristem not observed under control conditions. The possible role of auxin and ethylene in the alteration of root system architecture elicited by low B supply was also studied by using two Arabidopsis reporter lines (DR5:GUS and EBS:GUS) and two Arabidopsis mutants with impaired auxin and ethylene signaling (aux1-22 and ein2-1). Low B supply increased auxin reporter DR5:GUS activity in PR tip, suggesting that low B alters the pattern of auxin distribution in PR tip. Moreover, PR elongation in aux1-22 mutant was less sensitive to low B treatment than in wild-type plants, which suggests that auxin resistant 1 (AUX1) participates in the inhibition of PR elongation under low B supply. From all these results, a hypothetical model to explain the effect of low B treatment on PR growth is proposed. We also show that ethylene, via ethylene-insensitive 2 (EIN2) protein, is involved in the induction of root hair formation and elongation under low B treatment.     

3-羰基辛酰基高丝氨酸内酯诱导拟南芥根细胞Ca~（2＋）内流

N-酰基高丝氨酸内酯（AHLs）是革兰氏阴性细菌群体感应系统（QS）中的胞间通讯信号分子。近年的研究表明AHLs可以调控植物生长发育及防卫反应,但其调控机制尚不清楚。本研究以拟南芥为材料,采用3-羰基辛酰基高丝氨酸内酯（3OC8-HSL）处理转水母发光蛋白基因的拟南芥幼根细胞,利用冷光仪检测3OC8-HSL对拟南芥根细胞中胞质游离Ca2＋浓度（[Ca2＋]cyt）变化的影响,同时采用Ca2＋专一性螯合剂EGTA和Ca2＋通道抑制剂预处理转基因拟南芥根细胞,用全细胞膜片钳技术分析3OC8-HSL诱导拟南芥根细胞中[Ca2＋]cyt升高的Ca2＋来源。结果表明,3OC8-HSL可诱导拟南芥根细胞中[Ca2＋]cyt瞬时升高。这种诱导效应可被EGTA、异搏定（verapamil）、LaCl3所抑制,但LiCl预处理对这种诱导效应无影响。膜片钳分析结果显示,3OC8-HSL可激活质膜Ca2＋通道,增加胞外Ca2＋内流。说明细菌AHLs可诱导植物Ca2＋信号产生,且这种Ca2＋信号主要源于胞外Ca2＋内流,暗示Ca2＋信使系统参与植物对细菌QS信号的响应。     

Inhibition of primary roots and stimulation of lateral root development in Arabidopsis thaliana by the rhizobacterium Serratia marcescens 90–166 is through both auxin-dependent and -independent signaling pathways

The rhizobacterium Serratia marcescens strain 90–166 was previously reported to promote plant growth and induce resistance in Arabidopsis thaliana. In this study, the influence of strain 90-166 on root development was studied in vitro. We observed inhibition of primary root elongation, enhanced lateral root emergence, and early emergence of second order lateral roots after inoculation with strain 90–166 at a certain distance from the root. Using the DR5::GUS transgenic A. thaliana plant and an auxin transport inhibitor, N-1-naphthylphthalamic acid, the altered root development was still elicited by strain 90–166, indicating that this was not a result of changes in plant auxin levels. Intriguingly, indole-3-acetic acid, a major auxin chemical, was only identified just above the detection limit in liquid culture of strain 90–166 using liquid chromatography-mass spectrometry. Focusing on bacterial determinants of the root alterations, we found that primary root elongation was inhibited in seedlings treated with cell supernatant (secreted compounds), while lateral root formation was induced in seedlings treated with lysate supernatant (intracellular compounds). Further study revealed that the alteration of root development elicited by strain 90–166 involved the jasmonate, ethylene, and salicylic acid signaling pathways. Collectively, our results suggest that strain 90–166 can contribute to plant root development via multiple signaling pathways.     

Phosphate availability regulates root system architecture in Arabidopsis

Plant root systems are highly plastic in their development and can adapt their architecture in response to the prevailing environmental conditions. One important parameter is the availability of phosphate, which is highly immobile in soil such that the arrangement of roots within the soil will profoundly affect the ability of the plant to acquire this essential nutrient. Consistent with this, the availability of phosphate was found to have a marked effect on the root system architecture of Arabidopsis. Low phosphate availability favored lateral root growth over primary root growth, through increased lateral root density and length, and reduced primary root growth mediated by reduced cell elongation. The ability of the root system to respond to phosphate availability was found to be independent of sucrose supply and auxin signaling. In contrast, shoot phosphate status was found to influence the root system architecture response to phosphate availability.     

The xipotl mutant of Arabidopsis reveals a critical role for phospholipid metabolism in root system development and epidermal cell integrity

Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity.