Plant stem cells under low oxygen: metabolic rewiring by phytoglobin underlies stem cell functionality.

Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low-oxygen tensions. Overexpression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.

Preserving root stem cell functionality under low oxygen stress: the role of nitric oxide and phytoglobins.

MAIN CONCLUSION: The preservation of quiescent center stem cell integrity in hypoxic roots by phytoglobins is exercised through their ability to scavenge nitric oxide and attenuate its effects on auxin transport and cell degradation. Under low oxygen stress, the retention or induction of phytoglobin expression maintains cell viability while loss or lack of induction of phytoglobin leads to cell degradation. Plants have evolved unique attributes to ensure survival in the environment in which they must exist. Common among the attributes is the ability to maintain stem cells in a quiescent (or low proliferation) state in unfriendly environments. From the seed embryo to meristematic regions of the plant, quiescent stem cells exist to regenerate the organism when environmental conditions are suitable to allow plant survival. Frequently, plants dispose of mature cells or organs in the process of acclimating to the stresses to ensure survival of meristems, the stem cells of which are capable of regenerating cells and organs that have been sacrificed, a feature not generally available to mammals. Most of the research on plant stress responses has dealt with how mature cells respond because of the difficulty of specifically examining plant meristem responses to stress. This raises the question as to whether quiescent stem cells behave in a similar fashion to mature cells in their response to stress and what factors within these critical cells determine whether they survive or degrade when exposed to environmental stress. This review attempts to examine this question with respect to the quiescent center (QC) stem cells of the root apical meristem. Emphasis is put on how varying levels of nitric oxide, influenced by the expression of phytoglobins, affect QC response to hypoxic stress.

The inhibition of maize (Zea mays L.) root stem cell regeneration by low oxygen is attenuated by Phytoglobin 1 (Pgb1) through changes in auxin and jasmonic acid.

MAIN CONCLUSIONS: Over-expression of Phytoglobin1 increases the viability of maize root stem cells to low oxygen stress through changes in auxin and jasmonic acid responses. Hypoxia inhibits maize (Zea mays L.) root growth by deteriorating the quiescent center (QC) stem cells of the root apical meristem. Over-expression of the Phytoglobin1 ZmPgb1.1 alleviates these effects through the retention of the auxin flow along the root profile required for the specification of the QC stem cells. To identify QC-specific hypoxia responses and determine whether ZmPgb1.1 exercises a direct role on QC stem cells, we performed a QC functionality test. This was done by estimating the ability of QCs to regenerate a root in vitro in a hypoxic environment. Hypoxia decreased the functionality of the QCs by depressing the expression of several genes participating in the synthesis and response of auxin. This was accompanied by a decrease in DR5 signal, a suppression of PLETHORA and WOX5, two markers of QC cell identity, and a reduction in expression of genes participating in JA synthesis and signaling. Over-expression of ZmPgb1.1 was sufficient to mitigate all these responses. Through pharmacological alterations of auxin and JA, it is demonstrated that both hormones are required for QC functionality under hypoxia, and that JA acts downstream of auxin during QC regeneration. A model is proposed whereby the ZmPgb1.1 maintenance of auxin synthesis in hypoxic QCs is determinant for the retention of their functionality, with JA supporting the regeneration of roots from the QCs.

The Arabidopsis Phytoglobin 2 mediates phytochrome B (phyB) light signaling responses during somatic embryogenesis.

MAIN CONCLUSIONS: During the light induction of somatic embryogenesis, phyB-Pfr suppresses Phytoglobin 2, known to elevate nitric oxide (NO). NO depresses Phytochrome Interacting Factor 4 (PIF4) relieving its inhibition on embryogenesis through auxin. An obligatory step of many in vitro embryogenic systems is the somatic-embryogenic transition culminating with the formation of the embryogenic tissue. In Arabidopsis, this transition requires light and is facilitated by high levels of nitric oxide (NO) generated by either suppression of the NO scavenger Phytoglobin 2 (Pgb2), or its removal from the nucleus. Using a previously characterized induction system regulating the cellular localization of Pgb2, we demonstrated the interplay between phytochrome B (phyB) and Pgb2 during the formation of embryogenic tissue. The deactivation of phyB in the dark coincides with the induction of Pgb2 known to reduce the level of NO; consequently, embryogenesis is inhibited. Under light conditions, the active form of phyB depresses the levels of Pgb2 transcripts, thus expecting an increase in cellular NO. Induction of Pgb2 increases Phytochrome Interacting Factor 4 (PIF4) suggesting that high levels of NO repress PIF4. The PIF4 inhibition is sufficient to induce several auxin biosynthetic (CYP79B2, AMI1, and YUCCA 1, 2, and 6) and response (ARF5, 8, and 16) genes, conducive to the formation of the embryonic tissue and production of somatic embryos. Auxin responses mediated by ARF10 and 17 appear to be regulated by Pgb2, possibly through NO, in a PIF4-independent fashion. Overall, this work provides a new and preliminary model integrating Pgb2 (and NO) with phyB in the light regulation of in vitro embryogenesis.

Arabidopsis root apical meristem survival during waterlogging is determined by phytoglobin through nitric oxide and auxin.

MAIN CONCLUSION: Over-expression of phytoglobin mitigates the degradation of the root apical meristem (RAM) caused by waterlogging through changes in nitric oxide and auxin distribution at the root tip. Plant performance to waterlogging is ameliorated by the over-expression of the Arabidopsis Phytoglobin 1 (Pgb1) which also contributes to the maintenance of a functional RAM. Hypoxia induces accumulation of ROS and damage in roots of wild type plants; these events were preceded by the exhaustion of the RAM resulting from the loss of functionality of the WOX5-expressing quiescent cells (QCs). These phenotypic deviations were exacerbated by suppression of Pgb1 and attenuated when the same gene was up-regulated. Genetic and pharmacological studies demonstrated that degradation of the RAM in hypoxic roots is attributed to a reduction in the auxin maximum at the root tip, necessary for the specification of the QC. This reduction was primarily caused by alterations in PIN-mediated auxin flow but not auxin synthesis. The expression and localization patterns of several PINs, including PIN1, 2, 3 and 4, facilitating the basipetal translocation of auxin and its distribution at the root tip, were altered in hypoxic WT and Pgb1-suppressing roots but mostly unchanged in those over-expressing Pgb1. Disruption of PIN1 and PIN2 signal in hypoxic roots suppressing Pgb1 initiated in the transition zone at 12 h and was specifically associated to the absence of Pgb1 protein in the same region. Exogenous auxin restored a functional RAM, while inhibition of the directional auxin flow exacerbated the degradation of the RAM. The regulation of root behavior by Pgb1 was mediated by nitric oxide (NO) in a model consistent with the recognized function of Pgbs as NO scavengers. Collectively, this study contributes to our understanding of the role of Pgbs in preserving root meristem function and QC niche during conditions of stress, and suggests that the root transition zone is most vulnerable to hypoxia.

Specificity in root domain accumulation of Phytoglobin1 and nitric oxide (NO) determines meristematic viability in water-stressed Brassica napus roots.

BACKGROUND AND AIMS: Drought reduces plant productivity, especially in the susceptible species Brassica napus. Water stress, mimicked by applications of 10 % polyethylene glycol (PEG), elevates nitric oxide (NO) in root cells after a few hours, contributing to degradation of the root apical meristems (RAMs), the function of which relies on auxin and brassinosteroids (BRs). Phytoglobins (Pgbs) are effective NO scavengers induced by this stress. This study examines the effects of BnPgb1 dysregulation in dehydrating B. napus roots, and the spatiotemporal relationship between Pgb1 and activities of auxin and BRs in the regulation of the RAM. METHODS: Brassica napus lines over-expressing [BnPgb1(S)] or down-regulating [BnPgb1(RNAi)] BnPgb1 were exposed to PEG-induced water stress. The localization of BnPgb1, NO, auxin and PIN1 were analysed during the first 48 h, while the expression level of biosynthetic auxin and BR genes was measured during the first 24 h. Pharmacological treatments were conducted to assess the requirement of auxin and BR in dehydrating roots. KEY RESULTS: During PEG stress, BnPgb1 protein accumulated preferentially in the peripheral domains of the root elongation zone, exposing the meristem to NO, which inhibits polar auxin transport (PAT), probably by interfering with PIN1 localization and the synthesis of auxin. Diminished auxin at the root tip depressed the synthesis of BR and caused the degradation of the RAMs. The strength of BnPgb1 signal in the elongation zone was increased in BnPgb1(S) roots, where NO was confined to the most apical cells. Consequently, PAT and auxin synthesis were retained, and the definition of RAMs was maintained. Auxin preservation of the RAM required BRs, although BRs alone was not sufficient to fully rescue drought-damaged RAMs in auxin-depleted environments. CONCLUSIONS: The tissue-specific localization of BnPgb1 and NO determine B. napus root responses to water stress. A model is proposed in which auxin and BRs act as downstream components of BnPgb1 signalling in the preservation of RAMs in dehydrating roots.

Phytoglobin Expression Alters the Na+/K+ Balance and Antioxidant Responses in Soybean Plants Exposed to Na2SO4.

Soybean (Glycine max) is an economically important crop which is very susceptible to salt stress. Tolerance to Na2SO4 stress was evaluated in soybean plants overexpressing or suppressing the phytoglobin GmPgb1. Salt stress depressed several gas exchange parameters, including the photosynthetic rate, caused leaf damage, and reduced the water content and dry weights. Lower expression of respiratory burst oxidase homologs (RBOHB and D), as well as enhanced antioxidant activity, resulting from GmPgb1 overexpression, limited ROS-induced damage in salt-stressed leaf tissue. The leaves also exhibited higher activities of the H2O2-quenching enzymes, catalase (CAT) and ascorbate peroxidase (APX), as well as enhanced levels of ascorbic acid. Relative to WT and GmPgb1-suppressing plants, overexpression of GmPgb1 attenuated the accumulation of foliar Na+ and exhibited a lower Na+/K+ ratio. These changes were attributed to the induction of the Na+ efflux transporter SALT OVERLY SENSITIVE 1 (SOS1) limiting Na+ intake and transport and the inward rectifying K+ channel POTASSIUM TRANSPORTER 1 (AKT1) required for the maintenance of the Na+/K+ balance.

Stem cell fate in hypoxic root apical meristems is influenced by phytoglobin expression.

Root survival to flooding-induced hypoxic stress is dependent upon maintaining the functionality of the root apical meristem quiescent center (QC), a process that is governed by the basipetal flow of auxin leading to the formation of an auxin maximum, which is needed for the establishment of a highly oxidized environment specifying the QC niche. Perturbations in auxin flow and distribution along the root profile occurring during hypoxia can shift the redox state of the QC towards a more reduced environment, leading to the activation of the QC, degradation of the meristem, and root abortion. The maize phytoglobin gene ZmPgb1.1 is involved in minimizing these damaging effects during hypoxia in processes that result in sustaining the PIN-mediated auxin maximum and an oxidized environment in the QC. The oxidized environment is accomplished by maintaining the activity of redox enzymes oxidizing ascorbate and glutathione. These events, compromised in QCs suppressing ZmPgb1.1, ensure the functionality of the QC and root meristems under conditions of low oxygen, resulting in stable root performance.

Spatio-temporal expression of phytoglobin: a determining factor in the NO specification of cell fate.

Plant growth and development rely on the orchestration of cell proliferation, differentiation, and ultimately death. After varying rounds of divisions, cells respond to positional cues by acquiring a specific fate and embarking upon distinct developmental pathways which might differ significantly from those of adjacent cells exposed to diverse cues. Differential cell behavior is most apparent in response to stress, when some cells might be more vulnerable than others to the same stress condition. This appears to be the case for stem cells which show abnormal features of differentiation and ultimately signs of deterioration at the onset of specific types of stress such as hypoxia and water deficit. A determining factor influencing cell behavior during growth and development, and cell response during conditions of stress is nitric oxide (NO), the level of which can be regulated by phytoglobins (Pgbs), known scavengers of NO. The modulation of NO by Pgbs can be cell, tissue, and/or organ specific, as revealed by the expression patterns of Pgbs dictated by the presence of distinct cis-regulatory elements in their promoters. This review discusses how the temporal and spatial Pgb expression pattern influences NO-mediated responses and ultimately cell fate acquisition in plant developmental processes.

Elevation of cytosolic Ca2+ in response to energy deficiency in plants: the general mechanism of adaptation to low oxygen stress.

Ca2+ can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca2+ release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca2+ release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca2+ is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca2+ activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb-nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca2+- and low pH-activated external NADH and NADPH dehydrogenases participate. Ca2+ can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.

Phytoglobins regulate nitric oxide-dependent abscisic acid synthesis and ethylene-induced program cell death in developing maize somatic embryos.

MAIN CONCLUSION: During maize somatic embryogenesis, suppression of phytoglobins (Pgbs) reduced ABA levels leading to ethylene-induced programmed cell death in the developing embryos. These effects modulate embryonic yield depending on the cellular localization of specific phytoglobin gene expression. Suppression of Zea mays phytoglobins (ZmPgb1.1 or ZmPgb1.2) during somatic embryogenesis induces programmed cell death (PCD) by elevating nitric oxide (NO). While ZmPgb1.1 is expressed in many embryonic domains and its suppression results in embryo abortion, ZmPgb1.2 is expressed in the basal cells anchoring the embryos to the embryogenic tissue. Down-regulation of ZmPgb1.2 is required to induce PCD in these anchor cells allowing the embryos to develop further. Exogenous applications of ABA could reverse the effects caused by the suppression of either of the two ZmPgbs. A depletion of ABA, ascribed to a down-regulation of biosynthetic genes, was observed in those embryonic domains where the respective ZmPgbs were repressed. These effects were mediated by NO. Depletion in ABA content increased the transcription of genes participating in the synthesis and response of ethylene, as well as the accumulation of ethylene, which influenced embryogenesis. Somatic embryo number was reduced by high ethylene levels and increased with pharmacological treatments suppressing ethylene synthesis. The ethylene inhibition of embryogenesis was linked to the production of reactive oxygen species (ROS) and the execution of PCD. Integration of ABA and ethylene in the ZmPgb regulation of embryogenesis is proposed in a model where NO accumulates in ZmPgb-suppressing cells, decreasing the level of ABA. Abscisic acid inhibits ethylene biosynthesis and the NO-mediated depletion of ABA relieves this inhibition causing ethylene to accumulate. Elevated ethylene levels trigger production of ROS and induce PCD. Ethylene-induced PCD in the ZmPgb1.1-suppressing line [ZmPgb1.1 (A)] leads to embryo abortion, while PCD in the ZmPgb1.2-suppressing line [ZmPgb1.2 (A)] results in the elimination of the anchor cells and the successful development of the embryos.

Phytoglobins Improve Hypoxic Root Growth by Alleviating Apical Meristem Cell Death.

Hypoxic root growth in maize (Zea mays) is influenced by the expression of phytoglobins (ZmPgbs). Relative to the wild type, suppression of ZmPgb1.1 or ZmPgb1.2 inhibits the growth of roots exposed to 4% oxygen, causing structural abnormalities in the root apical meristems. These effects were accompanied by increasing levels of reactive oxygen species (ROS), possibly through the transcriptional induction of four Respiratory Burst Oxidase Homologs TUNEL-positive nuclei in meristematic cells indicated the involvement of programmed cell death (PCD) in the process. These cells also accumulated nitric oxide and stained heavily for ethylene biosynthetic transcripts. A sharp increase in the expression level of several 1-aminocyclopropane synthase (ZmAcs2, ZmAcs6, and ZmAcs7), 1-aminocyclopropane oxidase (Aco15, Aco20, Aco31, and Aco35), and ethylene-responsive (ZmErf2 and ZmEbf1) genes was observed in hypoxic ZmPgb-suppressing roots, which overproduced ethylene. Inhibiting ROS synthesis with diphenyleneiodonium or ethylene perception with 1-methylcyclopropene suppressed PCD, increased BAX inhibitor-1, an effective attenuator of the death programs in eukaryotes, and restored root growth. Hypoxic roots overexpressing ZmPgbs had the lowest level of ethylene and showed a reduction in ROS staining and TUNEL-positive nuclei in the meristematic cells. These roots retained functional meristems and exhibited the highest growth performance when subjected to hypoxic conditions. Collectively, these results suggest a novel function of Pgbs in protecting root apical meristems from hypoxia-induced PCD through mechanisms initiated by nitric oxide and mediated by ethylene via ROS.

Cellular localization of the Arabidopsis class 2 phytoglobin influences somatic embryogenesis.

Mutation of phytoglobin 2 (Pgb2) increases the number of somatic embryos in Arabidopsis. To assess the effects of the cellular localization of Pgb2 on embryo formation, an inducible system expressing a fusion protein consisting of Pgb2 linked to the steroid-binding domain of the rat glucocorticoid receptor (GR) was introduced in a pgb2 mutant line lacking the ability to express Pgb2. In this transgenic system, Pgb2 remains in the cytoplasm but migrates into the nucleus upon exposure to dexamethasone (DEX). Pgb2 retention in the cytoplasm, in the absence of DEX, increased the number of somatic embryos and reduced the expression of MYC2 - an inhibitor of the synthesis of auxin, which is the inductive signal for embryogenesis. Removal of DEX also induced the expression of several genes involved in the biosynthesis of tryptophan and the auxin, indole-3-acetic acid (IAA). These genes included: tryptophan synthase-α subunit (TSA1) and tryptophan synthase-ß subunit (TSB1), which are involved in the synthesis of tryptophan, cytochrome P450 CYP79B2 (CYP79B2) and amidase 1 (AMI1), which participate in the formation of IAA via indole-3-acetaldoxime, and several members of the YUCCA family, including YUC1 and 4, which are also required for IAA synthesis. Retention of Pgb2 in the cytoplasm by removal of DEX increased the staining pattern of IAA along the cotyledons of the explants generating embryogenic tissue. Staining for IAA decreased when Pgb2 translocated into the nucleus in response to the application of DEX. Collectively, these results suggest that the presence of Pgb2 in the cytoplasm, but not in the nucleus, phenocopies the effects of Pgb2 mutation in inducing somatic embryogenesis.

Expression of Arabidopsis class 1 phytoglobin (AtPgb1) delays death and degradation of the root apical meristem during severe PEG-induced water deficit.

Maintenance of a functional root is fundamental to plant survival in response to some abiotic stresses, such as water deficit. In this study, we found that overexpression of Arabidopsis class 1 phytoglobin (AtPgb1) alleviated the growth retardation of polyethylene glycol (PEG)-induced water stress by reducing programmed cell death (PCD) associated with protein folding in the endoplasmic reticulum (ER). This was in contrast to PEG-stressed roots down-regulating AtPgb1 that exhibited extensive PCD and reduced expression of several attenuators of ER stress, including BAX Inhibitor-1 (BI-1). The death program experienced by the suppression of AtPgb1 in stressed roots was mediated by reactive oxygen species (ROS) and ethylene. Suppression of ROS synthesis or ethylene perception reduced PCD and partially restored root growth. The PEG-induced cessation of root growth was preceded by structural changes in the root apical meristem (RAM), including the loss of cell and tissue specification, possibly as a result of alterations in PIN1- and PIN4-mediated auxin accumulation at the root pole. These events were attenuated by the overexpression of AtPgb1 and aggravated when AtPgb1 was suppressed. Specifically, suppression of AtPgb1 compromised the functionality of the WOX5-expressing quiescent cells (QCs), leading to the early and premature differentiation of the adjacent columella stem cells and to a rapid reduction in meristem size. The expression and localization of other root domain markers, such as SCARECROW (SCR), which demarks the endodermis and QCs, and WEREWOLF (WER), which specifies the lateral root cap, were also most affected in PEG-treated roots with suppressed AtPgb1. Collectively, the results demonstrate that AtPgb1 exercises a protective role in roots exposed to lethal levels of PEG, and suggest a novel function of this gene in ensuring meristem functionality through the retention of cell fate specification.

Regulation of programmed cell death by phytoglobins.

Programmed cell death (PCD) is a fundamental plant process in growth and development and in response to both biotic and abiotic stresses. Nitric oxide (NO) is a central component in determining whether a cell undergoes PCD, either as a direct elicitor of the response or as a factor in signal transduction from various hormones. Both NO and hormones that use NO as a signal transducer are mobile in the plant. Why do one set of cells die while adjacent cells remain alive, if this is the case? There is evidence to suggest that phytoglobins (Pgbs; previously termed non-symbiotic hemoglobins) may act as binary switches to determine plant cellular responses to perturbations. There are anywhere from one to five Pgb genes in plants that are expressed in response to growth and development and to stress. One of their main functions is to scavenge NO. This review will discuss how Pgb modulates cellular responses to auxin, cytokinin, and jasmonic acid during growth and development and in response to stress. The moderation in the production of reactive oxygen species (ROS) by Pgbs and the effects on PCD will also be discussed. An overall mechanism for Pgb involvement will be presented.

Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin.

Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos.

Phytoglobin expression influences soil flooding response of corn plants.

Background and Aims Excess water is a limiting factor for crop productivity. Under conditions of full submergence or flooding, plants can experience prolonged oxygen depletion which compromises basic physiological and biochemical processes. Severe perturbations of the photosynthetic machinery with a concomitant decline in photosynthetic potential as a result of elevated levels of reactive oxygen species (ROS) are the major consequences of water excess. Phytoglobins (Pgbs) are ubiquitous proteins induced by several types of stress which affect plant response by modulating nitric oxide. Methods Maize plants overexpressing or downregulating two Pgb genes were subjected to soil flooding for 10 d and their performance was estimated by measuring several gas exchange parameters including photosynthetic rate. Above-ground tissue was utilized to localize ROS and to measure the expression and activities of major antioxidant enzymes. Key Results Relative to the wild type, flooded plants overexpressing Pgb genes retained a greater photosynthetic rate and enhanced activity of several antioxidant enzymes. These plants also exhibited high levels of ascorbic acid and reduced ROS staining. This was in contrast to flooded plants downregulating Pgb genes and characterized by the lowest photosynthetic rates and reduced expression and activities of many antioxidant enzymes. Conclusions Induction of Pgb genes alleviates flooding stress by limiting ROS-induced damage and ensuring a sustained photosynthetic rate. This is achieved through improvements of the ascorbate antioxidant status including an enrichment of the ascorbate pool via de novo and recycling mechanisms, and increased activities of several ROS-scavenging enzymes.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis.

Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

Function of type-2 Arabidopsis hemoglobin in the auxin-mediated formation of embryogenic cells during morphogenesis.

Suppression of Arabidopsis GLB2, a type-2 nonsymbiotic hemoglobin, enhances somatic embryogenesis by increasing auxin production. In the glb2 knock-out line (GLB2-/-), polarization of PIN1 proteins and auxin maxima occurred at the base of the cotyledons of the zygotic explants, which are the sites of embryogenic tissue formation. These changes were also accompanied by a transcriptional upregulation of WUSCHEL (WUS) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK1), which are markers of embryogenic competence. The increased auxin levels in the GLB2-/- line were ascribed to the induction of several key enzymes of the tryptophan and IAA biosynthetic pathways, including ANTHRANILATE SYNTHASE (α subunit; ASA1), CYTOCHROME P79B2 (CYP79B2) and AMIDASE1 (AMI1). The effects of GLB2 suppression on somatic embryogenesis and IAA synthesis are mediated by increasing levels of nitric oxide (NO) within the embryogenic cells, which repress the expression of the transcription factor MYC2, a well-characterized repressor of the auxin biosynthetic pathway. A model is proposed in which the suppression of GLB2 reduces the degree of NO scavenging by oxyhemoglobin, thereby increasing the cellular NO concentration. The increased levels of NO repress the expression of MYC2, relieving the inhibition of IAA synthesis and increasing cellular IAA, which is the inductive signal promoting embryogenic competence. Besides providing a model for the induction phase of embryogenesis in vitro, these studies propose previously undescribed functions for plant hemoglobins.

The Brassica napus phytoglobin 1 (BnPgb1) mitigates the decrease in plant fertility resulting from high temperature stress.

High temperature stress during flowering adversely affects plant fertility, decreasing plant productivity. Daily cycles of heat stress (HS), imposed on Brassica napus L. plants by slowly ramping the temperature from 23 °C to 35 °C before lowering back to pre-stress conditions, inhibited flower and silique formation, with fewer seeds per silique during the stress period, as well as decreased pollen viability. Heat stress also elevated the transcripts and protein levels of class 1 phytoglobin BnPgb1, with the protein accumulating preferentially within the anther walls. Over-expression of BnPgb1 was sufficient to attenuate the reduction in plant fertility at high temperatures while its down-regulation exacerbated the effects of HS. Relative to WT anthers, the rise in ROS and ROS-induced damage caused by HS was limited when BnPgb1 was over-expressed, and this was linked to changes in antioxidant responses. High temperatures reduced the level of ascorbic acid (AsA) in anthers by favoring its oxidation via ascorbate oxidase (AOA) and limiting its regeneration through suppression of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR). Anthers of heat-stressed plants over-expressing BnPgb1 retained a higher AsA content with concomitant increased activities of DHAR, MDHAR, ascorbate peroxidase (APX) and superoxide dismutase (SOD). These changes suggest that BnPgb1 potentiates antioxidant responses during HS which mitigate the depression of fertility.