Beneficial Plant-Microbe Interactions and Stress Tolerance in Maize

Beneficial microbes are crucial for improving crop adaptation and growth under various stresses. They enhance nutrient uptake, improve plant immune responses, and help plants tolerate stresses like drought, salinity, and heat. The yield potential of any crop is significantly influenced by its associated microbiomes and their potential to improve growth under different stressful environments. Therefore, it is crucial and exciting to understand the mechanisms of plant-microbe interactions. Maize (Zea mays L.) is one of the primary staple foods worldwide, in addition to wheat and rice. Maize is also an industrial crop globally, contributing 83% of its production for use in feed, starch, and biofuel industries. Maize requires significant nitrogen fertilization to achieve optimal growth and yield. Maize plants are highly susceptible to heat, salinity, and drought stresses and require innovative methods to mitigate the harmful effects of environmental stresses and reduce the use of chemical fertilizers. This review summarizes our current understanding of the beneficial interactions between maize plants and specific microbes. These beneficial microbes improve plant resilience to stress and increase productivity. For example, they regulate electron transport, downregulate catalase, and upregulate antioxidants. We also review the roles of plant growth-promoting rhizobacteria (PGPR) in enhancing stress tolerance in maize. Additionally, we explore the application of these microbes in maize production and identify major knowledge gaps that need to be addressed to utilize the potential of beneficial microbes fully

Expression of the High-Affinity K+ Transporter 1 (PpHKT1) Gene From Almond Rootstock ‘Nemaguard’ Improved Salt Tolerance of Transgenic Arabidopsis

Soil salinity affects plant growth and development, which directly impact yield. Plants deploy many mechanisms to cope with, or mitigate, salt stress. One of such mechanism is to control movement of ions from root to shoot by regulating the loading of Na+ in the transpiration stream. The high-affinity K+ transporter 1 (HKT1) is known to play a role in the removal of Na+from the xylem and bring it back to the root. As almond is a salt-sensitive crop, the rootstock plays an important role in successful almond cultivation in salt-affected regions. We currently lack knowledge on the molecular mechanisms involved in salt tolerance of almond rootstocks. In this study, we complemented the Arabidopsis athkt1 knockout mutant with HKT1 ortholog (PpHKT1) from the almond rootstock ‘Nemaguard’. Arabidopsis transgenic lines that were generated in athkt1 background with the constitutive promoter (PpHKT1OE2.2) and the native promoter (PpHKT1NP6) were subjected to different salt treatments. Both transgenic lines survived salt concentrations up to 120 mM NaCl, however, the mutant athkt1 died after 18 days under 120 mM NaCl. At 90 mM NaCl, the dry weight of athkt1 decreased significantly compared to the transgenic lines. Both transgenic lines showed significantly longer lateral roots compared to the athkt1 mutant at 80 mM NaCl treatment. The transgenic lines, PpHKT1OE2.2 and PpHKTNP6 had lower electrolyte leakage and higher relative water content compared to athkt1, suggesting that transgenic plants coped well with increased salt concentration by maintaining the integrity of the membranes. The expression analyses showed that PpHKT1 was induced in PpHKT1OE2.2 and PpHKTNP6 lines under salt treatment, which confirmed that both over-expression and native expression of PpHKT1 in the Arabidopsis mutant can complement salt tolerance function

Genetic Diversity of New Almond Accessions from Central Asian and Cold-adapted North American Germplasm

We evaluated the genetic diversity of a newly available collection of 94 almond [Prunus dulcis (Mill.) D.A. Webb] accessions from the former Improving Perennial Plants for Food and Bioenergy (IPPFBE) Foundation. Most of the collection (87 accessions) were collected as seeds from trees growing in the central Asian nations of Kyrgyzstan, Tajikistan, and Uzbekistan, and included several examples of Prunus bucharica (Korsh.) Hand.-Mazz, and related wild species. Of the remaining accessions, six were sourced from a nursery in northern Utah in the United States, and one was a seedling of ‘Nonpareil’, a major commercial cultivar. DNA fingerprints were generated from 10 simple sequence repeat markers. To evaluate the comparative diversity of these new accessions, 66 accessions from the US Department of Agriculture, National Plant Germplasm System (NPGS) almond germplasm collection near Davis, CA, USA, were also included. These NPGS accessions were chosen to represent those collected in similar regions of Central Asia and the Caucasus. The fingerprints were analyzed via hierarchical clustering, principal components analysis (PCA), and discriminant analysis of principal components (DAPC). Hierarchical clustering suggested that half of the Utah-sourced accessions are closely related to each other and to the ‘Nonpareil’ seedling. Additional close relationships were detected (including at least one duplication or mislabeling), and two P. bucharica accessions from the IPPFBE collection were separated from the rest of the collection. A plot of the first two principal components clearly separated wild almond relatives (P. bucharica and Prunus fenzliana Fritsch) from the remaining accessions. PCA after removal of the wild species separated the ‘Nonpareil’ seedling, the Utah-sourced accessions, and many of the IPPFBE accessions (mostly from Uzbekistan) from nearly all other individuals. The third principal component identified an additional population structure that separated groups of predominantly IPPFBE or NPGS accessions. DAPC showed a considerable admixture of accessions from Azerbaijan, and a little to no admixture of accessions from Georgia and Tajikistan. These results suggest that central Asian/Caucasian almond germplasm is generally distinct from ‘Nonpareil’ and its relatives, and that although there is overlap between the NPGS and IPPFBE collections from this region, the IPPFBE collection does enhance the diversity of available almond germplasm

Drought Stress Acclimation Imparts Tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested

Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction During Nonhost Resistance in Nicotiana benthamiana and Arabidopsis

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H2O2 accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response–causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H2O2 during both gene-for-gene and nonhost resistance responses

GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata

Formate Dehydrogenase (FDH1) Localizes to Both Mitochondria and Chloroplast to Play a Role in Host and Nonhost Disease Resistance

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In this study, the metabolic enzyme formate dehydrogenase (FDH1) was identified to be involved in nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. Arabidopsis Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that SA is most likely involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to only mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 localized to mitochondria and the amount of FDH1 localized to mitochondria increased upon infection with host or nonhost pathogens. Interestingly, the subcellular localization of FDH1 was observed in both mitochondria and chloroplasts after infection with a nonhost pathogen in Arabidopsis. We speculate that FDH1 plays a role in cellular signaling networks between mitochondria and chloroplasts to produce coordinated defense responses such as SA-induced reactive oxygen species (ROS) generation and hypersensitive response (HR)-induced cell death against nonhost bacterial pathogens

Glycolate Oxidase Activity Assay in Plants

Glycolate oxidase is located in the peroxisome and is involved in the photorespiratory cycle which recovers some of the carbon loss during photosynthesis. Glycolate oxidase converts glycolate to glyxoylate with the concomitant production of H2O2. In this assay, the H2O2 generated, in the presence of HRP, oxidizes O-dianisidine into a colored O-dianisidine radical cation that can be quantified spectrophotometrically. The amount of color produces is directly proportional to the glycolate oxidase activity

Measurement of NADPH Oxidase Activity in Plants

NADPH oxidase is a membrane-bound enxyme that generates (O2-) by transferring electrons from NADPH to molecular oxygen O2. O2- is spontaneously dismasted to the more stable form H2O2. Both O2- and H2O2 are forms of reactive oxygen species (ROS), which are involved in regulation of many cellular activities such as transcription, intracellular signaling, and host defense. The NADPH oxidase - dependent generation of O2- in total membrane fraction of plant tissue has been determined by the reduction of the tetrazolium salt XTT by O2-. In the presense of O2-, XTT generates a soluble yellow formazan that can be quantified spectrophotometrically

The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein

ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants