Genome-Wide Identification and Analysis of the Valine-Glutamine Motif-Containing Gene Family in Brassica napus and Functional Characterization of BnMKS1 in Response to Leptosphaeria maculans.

Proteins containing valine-glutamine (VQ) motifs play important roles in plant growth and development as well as in defense responses to both abiotic and biotic stresses. Blackleg disease, which is caused by Leptosphaeria maculans, is the most important disease in canola (Brassica napus) worldwide; however, the identification of Brassica napus VQs and their functions in response to blackleg disease have not yet been reported. In this study, we conducted a genome-wide identification and characterization of the VQ gene family in Brassica napus, including chromosome location, phylogenetic relations, gene structure, motif domain, synteny analysis, and cis-elements categorization of their promoter regions. To understand Brassica napus VQ gene function in response to blackleg disease, we overexpressed BnVQ7 (BnaA01g36880D, also known as the mitogen-activated protein kinase 4 substrate 1 [MKS1] gene) in a blackleg-susceptible canola variety, Westar. Overexpression of BnMKS1 in canola did not improve its resistance to blackleg disease at the seedling stage; however, transgenic canola plants overexpressing BnMKS1 displayed an enhanced resistance to L. maculans infection at the adult plant stage. Expression levels of downstream and defense marker genes in cotyledons increased significantly at the necrotrophic stage of L. maculans infection in the overexpression line of BnMKS1, suggesting that the salicylic acid- and jasmonic acid-mediated signaling pathways were both involved in the defense responses. Together, these results suggest that BnMKS1 might play an important role in defense against L. maculans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.

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.

A Six-Year Investigation of the Dynamics of Avirulence Allele Profiles, Blackleg Incidence, and Mating Type Alleles of Leptosphaeria maculans Populations Associated with Canola Crops in Manitoba, Canada.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is one of the most economically important diseases of canola (Brassica napus, oilseed rape) worldwide. This study assessed incidence of blackleg, the avirulence allele, and mating type distributions of L. maculans isolates collected in commercial canola fields in Manitoba, Canada, from 2010 to 2015. A total of 956 L. maculans isolates were collected from 2010 to 2015 to determine the presence of 12 avirulence alleles using differential canola cultivars and/or PCR assays specific for each avirulence allele. AvrLm2, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm11, and AvrLmS were detected at frequencies ranging from 97 to 33%, where the AvrLm1, AvrLm3, AvrLm9, AvrLepR1, and AvrLepR2 alleles were the least abundant. When the race structure was examined, a total of 170 races were identified among the 956 isolates, with three major races, AvrLm-2-4-5-6-7-11, AvrLm-2-4-5-6-7-11-S, and Avr-1-4-5-6-7-11-(S) accounting for 15, 10, and 6% of the total fungal population, respectively. The distribution of the mating type alleles (MAT1-1 and MAT1-2) indicated that sexual reproduction was not inhibited in any of the nine Manitoba regions in any of the years L. maculans isolates were collected.

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.

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.

Amino-functionalization enhanced CO2 reduction reaction in pure water.

The electrochemical reduction of carbon dioxide (CO2RR) to carbon monoxide represents a cost-effective pathway towards realizing carbon neutrality. To suppress the hydrogen evolution reaction (HER), the presence of alkali cations is critical, which can however lead to precipitate formation on the electrode, adversely impacting the device stability. Employing pure water as the electrolyte in zero-gap CO2 electrolyzers can address this challenge, albeit at the cost of diminished catalyst performance due to the absence of alkali cations. In this study, we introduce a novel approach by implementing amino modifications on the catalyst surface to mimic the function of alkali metal cations, while simultaneously working in pure water. This modification enhances the adsorption of carbon dioxide and protons, thereby facilitating the CO2RR while concurrently suppressing the HER. Utilizing this strategy in a zero-gap CO2 electrolyzer with pure water as the anolyte resulted in an impressive carbon monoxide faradaic efficiency (FECO) of 95.5% at a current density of 250 mA cm-2, while maintaining stability for over 180 hours without any maintenance.

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.

Validating the Strategic Deployment of Blackleg Resistance Gene Groups in Commercial Canola Fields on the Canadian Prairies.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus L.) production in western Canada. Crop scouting and extended crop rotation, along with the use of effective genetic resistance, have been key management practices available to mitigate the impact of the disease. In recent years, new pathogen races have reduced the effectiveness of some of the resistant cultivars deployed. Strategic deployment and rotation of major resistance (R) genes in cultivars have been used in France and Australia to help increase the longevity of blackleg resistance. Canada also introduced a grouping system in 2017 to identify blackleg R genes in canola cultivars. The main objective of this study was to examine and validate the concept of R gene deployment through monitoring the avirulence (Avr) profile of L. maculans population and disease levels in commercial canola fields within the Canadian prairies. Blackleg disease incidence and severity was collected from 146 cultivars from 53 sites across Manitoba, Saskatchewan, and Alberta in 2018 and 2019, and the results varied significantly between gene groups, which is likely influenced by the pathogen population. Isolates collected from spring and fall stubble residues were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm9, AvrLm10, AvrLm11, AvrLepR1, AvrLepR2, AvrLep3, and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR-based markers. The Simpson's evenness index was very low, due to two dominant L. maculans races (AvrLm2-4-5-6-7-10-11 and AvrLm2-5-6-7-10-11) representing 49% of the population, but diversity of the population was high from the 35 L. maculans races isolated in Manitoba. AvrLm6 and AvrLm11 were found in all 254 L. maculans isolates collected in Manitoba. Knowledge of the blackleg disease levels in relation to the R genes deployed, along with the L. maculans Avr profile, helps to measure the effectiveness of genetic resistance.

Directing Trophic Divergence in Plant-Pathogen Interactions: Antagonistic Phytohormones With NO Doubt?

A fundamental process culminating in the mechanisms of plant-pathogen interactions is the regulation of trophic divergence into biotrophic, hemibiotrophic, and necrotrophic interactions. Plant hormones, of almost all types, play significant roles in this regulatory apparatus. In plant-pathogen interactions, two classical mechanisms underlying hormone-dependent trophic divergence are long recognized. While salicylic acid dominates in the execution of host defense response against biotrophic and early-stage hemibiotrophic pathogens, jasmonic acid, and ethylene are key players facilitating host defense response against necrotrophic and later-stage hemibiotrophic pathogens. Evidence increasingly suggests that trophic divergence appears to be modulated by more complex signaling networks. Acting antagonistically or agonistically, other hormones such as auxins, cytokinins, abscisic acid, gibberellins, brassinosteroids, and strigolactones, as well as nitric oxide, are emerging candidates in the regulation of trophic divergence. In this review, the latest advances in the dynamic regulation of trophic divergence are summarized, emphasizing common and contrasting hormonal and nitric oxide signaling strategies deployed in plant-pathogen interactions.

Protection of root apex meristem during stress responses.

By regulating the levels of nitric oxide (NO) in a cell and tissue specific fashion, Phytoglobins (Pgbs), plant hemoglobin-like proteins, interfere with many NO-mediated pathways participating in developmental and stress-related responses. Recent evidence reveals that one of the functions of Pgbs is to protect the root apical meristem (RAM) from stress conditions by retaining the viability and function of the quiescent center (QC), required to maintain the stem cells in an undifferentiated state and ensure proper tissue patterning and root viability. Based on this and other evidence, it is suggested that Pgbs regulate cell fate by modulating NO homeostasis.

Dying with Style: Death Decision in Plant Embryogenesis.

Embryogenesis is a fascinating event during the plant life cycle encompassing several steps whereby the zygote develops into a fully developed embryo which, in angiosperms, is composed of an axis separating the apical meristems, and two cotyledons. Recapitulation of embryogenesis can also occur in vitro through somatic embryogenesis, where somatic cells are induced to form embryos, and androgenesis, in which embryos originate from immature male gametophytes. Besides cell division and differentiation, embryo patterning in vivo and in vitro requires the dismantling and selective elimination of cells and tissues via programmed cell death (PCD). While the manifestation of the death program has long been acknowledged in vivo, especially in relation to the elimination of the suspensor during the late phases of embryo development, PCD during in vitro embryogenesis has only been described in more recent years. Independent studies using the gymnosperm Norway spruce and the angiosperm maize have shown that the death program is crucial for the proper formation and further development of immature somatic embryos. This chapter summarizes the recent advances in the field of PCD during embryogenesis and proposes novel regulatory mechanisms activating the death program in plants.

Plant hemoglobin participation in cell fate determination.

Plant hemoglobins (Hbs) have been identified as master regulators in determining the developmental fate of specific cells during maize embryogenesis. Whether an embryogenic cell lives or undergoes programmed cell death (PCD) is modulated by Hbs, through their tight interactions with nitric oxide (NO) and auxin. During maize embryogenesis, Hb-suppressing cells accumulate NO, are depleted of auxin, and are committed to die. We propose that Hbs control cell fate by regulating NO and auxin homeostasis, and that this type of mechanism may influence other hormonal responses modulating plant behavior during development and stress conditions.

Hemoglobins, programmed cell death and somatic embryogenesis.

Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival.