Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

MAIN CONCLUSION: phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

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.

Interplay between nitric oxide and inorganic nitrogen sources in root development and abiotic stress responses.

Nitrogen (N) is an essential nutrient for plants, and the sources from which it is obtained can differently affect their entire development as well as stress responses. Distinct inorganic N sources (nitrate and ammonium) can lead to fluctuations in the nitric oxide (NO) levels and thus interfere with nitric oxide (NO)-mediated responses. These could lead to changes in reactive oxygen species (ROS) homeostasis, hormone synthesis and signaling, and post-translational modifications of key proteins. As the consensus suggests that NO is primarily synthesized in the reductive pathways involving nitrate and nitrite reduction, it is expected that plants grown in a nitrate-enriched environment will produce more NO than those exposed to ammonium. Although the interplay between NO and different N sources in plants has been investigated, there are still many unanswered questions that require further elucidation. By building on previous knowledge regarding NO and N nutrition, this review expands the field by examining in more detail how NO responses are influenced by different N sources, focusing mainly on root development and abiotic stress responses.

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.

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.

Seed-specific expression of the class 2 Phytoglobin (Pgb2) increases seed oil in Brassica napus.

To examine the function of phytoglobin 2 (Pgb2) on seed oil level in the oil-producing crop Brassica napus L., we generated transgenic plants in which BnPgb2 was over-expressed in the seeds using the cruciferin1 promoter. Over-expression of BnPgb2 elevated the amount of oil, which showed a positive relationship with the level of BnPgb2, without altering the oil nutritional value, as evidenced by the lack of major changes in composition of fatty acids (FA), and key agronomic traits. Two key transcription factors, LEAFY COTYLEDON1 (LEC1) and WRINKLED1 (WRI1), known to promote the synthesis of fatty acids (FA) and potentiate oil accumulation, were induced in BnPgb2 over-expressing seeds. The concomitant induction of several enzymes of sucrose metabolism, SUCROSE SYNTHASE1 (SUS) 1 and 3, FRUCTOSE BISPHOSPHATE ALDOLASE (FPA), and PHOSPHOGLYCERATE KINASE (PGK), and starch synthesis, ADP-GLUCOSE PHOSPHORYLASE (AGPase) suggests that BnPgb2 favors sugar mobilization for FA production. The two plastid FA biosynthetic enzymes SUBUNIT A OF ACETYL-CoA CARBOXYLASE (ACCA2), and MALONYL-CoA:ACP TRANSACYLASE (MCAT) were also up-regulated by the over-expression of BnPgb2. The requirement of BnPgb2 for oil deposition was further evidenced in natural germplasm by the higher levels of BnPgb2 in seeds of high-oil genotypes relative to their low-oil counterparts.

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.

Interplay between the Brassica napus phytoglobin (BnPgb1), folic acid, and antioxidant responses enhances plant tolerance to waterlogging.

Oxygen deprivation by waterlogging reduces the productivity of several crop species, including the oil-producing crop Brassica napus L., which is highly sensitive to excess moisture. Among factors induced by oxygen deficiency are phytoglobins (Pgbs), heme-containing proteins known to ameliorate the response of plants to the stress. This study examined the early responses to waterlogging in B. napus plants over-expressing or down-regulating the class 1 (BnPgb1) and class 2 (BnPgb2) Pgbs. The depression of gas exchange parameters and plant biomass was exacerbated by the suppression of BnPgb1, while suppression of BnPgb2 did not evoke any changes. This suggests that natural occurring levels of BnPgb1 (but not BnPg2) are required for the response of the plants to waterlogging. Typical waterlogging symptoms, including the accumulation of reactive oxygen species (ROS) and the deterioration of the root apical meristem (RAM) were attenuated by over-expression of BnPgb1. These effects were associated with the activation of antioxidant system and the transcriptional induction of folic acid (FA). Pharmacological treatments revealed that high levels of FA were sufficient to revert the inhibitory effect of waterlogging, suggesting that the interplay between BnPgb1, antioxidant responses and FA might contribute to plant tolerance to waterlogging 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.

Over-expression of the barley Phytoglobin 1 (HvPgb1) evokes leaf-specific transcriptional responses during root waterlogging.

Oxygen deprivation (hypoxia) in the root due to waterlogging causes profound metabolic changes in the aerial organs depressing growth and limiting plant productivity in barley (Hordeum vulgare L.). Genome-wide analyses in waterlogged wild type (WT) barley (cv. Golden Promise) plants and plants over-expressing the phytoglobin 1 HvPgb1 [HvPgb1(OE)] were performed to determine leaf specific transcriptional responses during waterlogging. Normoxic WT plants outperformed their HvPgb1(OE) counterparts for dry weight biomass, chlorophyll content, photosynthetic rate, stomatal conductance, and transpiration. Root waterlogging severely depressed all these parameters in WT plants but not in HvPgb1(OE) plants, which exhibited an increase in photosynthetic rate. In leaftissue, root waterlogging repressed genes encoding photosynthetic components and chlorophyll biosynthetic enzymes, while induced those of reactive oxygen species (ROS)-generating enzymes. This repression was alleviated in HvPgb1(OE) leaves which also exhibited an induction of enzymes participating in antioxidant responses. In the same leaves, the transcript levels of several genes participating in nitrogen metabolism were also higher relative to WT leaves. Ethylene levels were diminished by root waterlogging in leaves of WT plants, but not in HvPgb1(OE), which were enriched in transcripts of ethylene biosynthetic enzymes and ethylene response factors. Pharmacological treatments increasing the level or action of ethylene further suggested the requirement of ethylene in plant response to root waterlogging. In natural germplasm an elevation in foliar HvPgb1 between 16h and 24h of waterlogging occurred in tolerant genotypes but not in susceptible ones. By integrating morpho-physiological parameters with transcriptome data, this study provides a framework defining leaf responses to root waterlogging and indicates that the induction of HvPgb1 may be used as a selection tool to enhance resilience to excess moisture.

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.

Light induction of somatic embryogenesis in Arabidopsis is regulated by PHYTOCHROME E.

The requirement of light on somatic embryogenesis (SE) has been documented in many species; however, no mechanism of action has been elucidated. Using Arabidopsis SE as a model, the effect of red light (660 nm) during the induction phase corresponding to the formation of the embryogenic tissue was examined. Analyses of several phytochrome mutants revealed that red light signaling, conducive to SE, was mediated by PHYTOCHROME E (PHYE). Both phyE and darkness were sufficient to repress the formation of somatic embryos and reduced the expression of CONSTITUTIVE PHOTOMORPHIC DWARF 3 (CPD3), a rate limiting step in brassinosteroid (BR) biosynthesis, as well as AGAMOUS LIKE 15 (AGL15), a key inducer of many SE genes. We further integrated BR signaling and nitric oxide (NO) with PHYE by demonstrating that applications of both compounds to phyE explants and WT explants cultured in the dark partially restored AGL15 expression. These results demonstrate that SE induction by red light operates via PHYE through BR signaling and NO required to induce AGL15.

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.

Anaerobiosis modulation of two phytoglobins in barley (Hordeum vulgare L.), and their regulation by gibberellin and abscisic acid in aleurone cells.

The transcript levels of the phytoglobin (Pgb) genes Pgb1 and Pgb3, and the protein content of Pgb1 were responsive to anaerobiosis in several tissues of barley (Hordeum vulgare L.). Oxygen deficiency induced the level of both Pgb transcripts and protein in aleurone layers and coleoptiles, as well as up-regulated both Pgb1 and Pgb3 in leaves, apexes and more strongly in roots of barley seedlings. In O2-depleted aleurone cells the induction of the Pgb transcript-protein pair was reversed by re-supplying O2. Based on this observation, it is suggested that Pgb1 and Pgb3 are inducible in all tissues. In aleurone cells, gibberellic acid (GA) induced Pgb1 and Pgb3 together with α-amylase, whereas abscisic acid (ABA) eliminated the GA stimulating effects on both α-amylase and Pgb1 and Pgb3 expression. While GA had no effects on alcohol dehydrogenase (Adh1, Adh2 and Adh3) transcripts, ABA induced all three Adh genes. It is concluded that Pgb and α-amylase in seeds are regulated reciprocally with the ethanolic fermentation pathway, and that Pgb induction is mediated by GA. Nitric oxide turnover and scavenging mediated by Pgb represents an important alternative to fermentation under anoxia.

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.

Transduction of Signals during Somatic Embryogenesis.

Somatic embryogenesis (SE) is an in vitro biological process in which bipolar structures (somatic embryos) can be induced to form from somatic cells and regenerate into whole plants. Acquisition of the embryogenic potential in culture is initiated when some competent cells within the explants respond to inductive signals (mostly plant growth regulators, PRGs), and de-differentiate into embryogenic cells. Such cells, "canalized" into the embryogenic developmental pathway, are able to generate embryos comparable in structure and physiology to their in vivo counterparts. Genomic and transcriptomic studies have identified several pathways governing the initial stages of the embryogenic process. In this review, the authors emphasize the importance of the developmental signals required for the progression of embryo development, starting with the de-differentiation of somatic cells and culminating with tissue patterning during the formation of the embryo body. The action and interaction of PGRs are highlighted, along with the participation of master regulators, mostly transcription factors (TFs), and proteins involved in stress responses and the signal transduction required for the initiation of the embryogenic process.

Synthetic Strigolactone GR24 Improves Arabidopsis Somatic Embryogenesis through Changes in Auxin Responses.

Somatic embryogenesis in Arabidopsis encompasses an induction phase requiring auxin as the inductive signal to promote cellular dedifferentiation and formation of the embryogenic tissue, and a developmental phase favoring the maturation of the embryos. Strigolactones (SLs) have been categorized as a novel group of plant hormones based on their ability to affect physiological phenomena in plants. The study analyzed the effects of synthetic strigolactone GR24, applied during the induction phase, on auxin response and formation of somatic embryos. The expression level of two SL biosynthetic genes, MOREAXILLARY GROWTH 3 and 4 (MAX3 and MAX4), which are responsible for the conversion of carotene to carotenal, increased during the induction phase of embryogenesis. Arabidopsis mutant studies indicated that the somatic embryo number was inhibited in max3 and max4 mutants, and this effect was reversed by applications of GR24, a synthetic strigolactone, and exacerbated by TIS108, a SL biosynthetic inhibitor. The transcriptional studies revealed that the regulation of GR24 and TIS108 on somatic embryogenesis correlated with changes in expression of AUXIN RESPONSIVE FACTORs 5, 8, 10, and 16, known to be required for the production of the embryogenic tissue, as well as the expression of WUSCHEL (WUS) and Somatic Embryogenesis Receptor-like Kinase 1 (SERK1), which are markers of cell dedifferentiation and embryogenic tissue formation. Collectively, this work demonstrated the novel role of SL in enhancing the embryogenic process in Arabidopsis and its requirement for inducing the expression of genes related to auxin signaling and production of embryogenic tissue.

The soybean Phytoglobin1 (GmPgb1) is involved in water deficit responses through changes in ABA metabolism.

Soybean (Glycine max), a major grain crop worldwide, is susceptible to severe yield loss due to drought. Soybean plants over-expressing and downregulating the soybean Phytoblobin1 (GmPgb1) were evaluated for their ability to cope with polyethylene glycol (PEG)-induced water deficit. Sense transformation of GmPgb1, which was more expressed in shoot tissue relative to roots, increased overall plant performance and tolerance to water stress by attenuating the PEG depression of photosynthetic gas exchange parameters and chlorophyll content, as well as reducing leaf injury and promoting root growth. The higher plant relative water content, as a result of GmPgb1 over-expression, was associated with higher transcript levels of three aquaporins: GmTIP1;5 and GmTIP2;5 GmPIP2;9, known to confer water stress tolerance. Opposite results were observed in plants suppressing GmPgb1, which were highly susceptible to PEG-induced stress. Transcriptional and metabolic analyses revealed higher ABA synthesis in dehydrating leaves of plants over-expressing GmPgb1 relative to those suppressing the same gene. The latter plants exhibited a transcriptional induction of ABA catabolic enzymes and higher accumulation of the ABA catabolite dehydrophaseic acid (DPA). Administration of 8'-acetylene ABA, an ABA agonist resistant to the ABA catabolic activity, was sufficient to restore tolerance in the GmPgb1 down-regulating plants suggesting that regulation of ABA catabolism is as important as ABA synthesis in conferring PEG-induced water stress tolerance. Screening of natural soybean germplasm also revealed a rapid and transient increase in foliar GmPgb1 in tolerant plants relative to their susceptible counterparts, thus confirming the key role exercised by this gene during water stress.

Cold stress in maize (Zea mays) is alleviated by the over-expression of Phytoglobin 1 (ZmPgb1.1).

Maize (Zea mays) plants over-expressing or suppressing the class 1 Phytoglobin (ZmPgb1.1) were evaluated for their ability to cope with low temperature stress. Cold treatment (10 °C day/4 °C night) depressed several gas exchange parameters including photosynthetic rate, stomatal conductance and transpiration, while elevated the levels of reactive oxygen species (ROS) and ROS-induced damage. These effects were attenuated by the over-expression of ZmPgb1.1, and aggravated when the level of the same gene was suppressed. Combination of transcriptomic and pharmacological studies revealed that over-expression of ZmPgb1.1 suppressed the level of nitric oxide (NO), which lowers the transcription of several Brassinosteroid (BR) biosynthetic and response genes. Cellular BR was required to induce the expression of ZmMPK5, a component of the mitogen-activated protein kinase (MAPK) cascade, which is known to be involved in the regulation of ROS-producing pathways. Experimental reduction of NO content, suppression of BR or inhibition of ZmMPK5 reverted the beneficial effects of ZmPgb1.1 over-expression, and increased plant susceptibility to cold stress through accumulation of ROS. Conversely, tolerance to cold was augmented in the ZmPgb1.1 down-regulating line when the levels of NO or BR were elevated. Together, this study demonstrates a novel role of ZmPgb1.1 in modulating plant performance to cold stress, and integrates the ZmPgb1.1 response in a model requiring NO and BR to alleviate oxidative stress through ZmMPK5.

Tolerance to excess moisture in soybean is enhanced by over-expression of the Glycine max Phytoglobin (GmPgb1).

Excess moisture in the form of waterlogging or full submergence can cause severe conditions of hypoxia or anoxia compromising several physiological and biochemical processes. A decline in photosynthetic rate due to accumulation of ROS and damage of leaf tissue are the main consequences of excess moisture. These effects compromise crop yield and quality, especially in sensitive species, such as soybean (Glycine max.). Phytoglobins (Pgbs) are expressed during hypoxia and through their ability to scavenge nitric oxide participate in several stress-related responses. Soybean plants over-expressing or suppressing the Pgb1 gene GmPgb1 were generated and their ability to cope with waterlogging and full submergence conditions was assessed. Plants over-expressing GmPgb1 exhibited a higher retention of photosynthetic rate during waterlogging and survival rate during submergence relative to wild type plants. The same plants also had lower levels of ROS due to a reduction in expression of Respiratory Burst Oxidase Homologs (RBOH), components of the NADPH oxidase enzyme, and enhanced antioxidant system characterized by higher expression of catalases (CAT) and superoxide dismutase (SOD), as well as elevated expression and activity of ascorbate peroxidase (APX). Plants over-expressing GmPgb1 also exhibited an expression pattern of aquaporins typical of excess moisture resilience. This was in contrast to plants downregulating GmPgb1 which were characterized by the lowest photosynthetic rates, higher ROS signal, and reduced expression and activities of many antioxidant enzymes. Results from these studies suggest that GmPgb1 exercises a protective role during conditions of excess moisture with similar mechanisms operating during waterlogging and submergence.