Properties and applications of α-galactosidase in agricultural waste processing and secondary agricultural process industries.

Agriculture products form the foundation building blocks of our daily lives. Although they have been claimed to be renewable resources with a low carbon footprint, the agricultural community is constantly challenged to overcome two post-harvest bottlenecks: first, farm bio-waste, a substantial economic and environmental burden to the farming sector, and second, an inefficient agricultural processing sector, plagued by the need for significant energy input to generate the products. Both these sectors require extensive processing technologies that are demanding in their energy requirements and expensive. To address these issues, an enzyme(s)-based green chemistry is available to break down complex structures into bio-degradable compounds that source alternate energy with valuable by-products and co-products. α-Galactosidase is a widespread class of glycoside hydroxylases that hydrolyzes α-galactosyl moieties in simple and complex oligo and polysaccharides, glycolipids, and glycoproteins. As a result of its growing importance, in this review we discuss the source of the enzyme, production and purification systems, and enzyme properties. We also elaborate on the enzyme's potential in agricultural bio-waste management, secondary agricultural industries like sugar refining, soymilk derivatives, food and confectionery, and animal feed processing. Insight into this vital enzyme will provide new avenues for less expensive green chemistry-based secondary agricultural processing and agricultural sustainability. © 2023 Society of Chemical Industry.

Plant growth regulator extracts from seaweeds promote plant growth and confer drought tolerance in canola (Brassica napus).

Brassica napus, commonly known as canola, is an important oilseed crop in Canada contributing over $29.9 billion CAD to the Canadian economy annually. A major challenge facing Canadian canola is drought, which has become increasingly prevalent in recent years due to the changing climate. Research investigating novel agronomic techniques in mitigating drought is key to securing yields and sustainability in canola and other crops. One such technique is the use of bio-stimulant sprays to help offset biotic and abiotic stresses in plants through promoting stand establishment. Previous studies have shown that the application of seaweed extracts as bio-stimulant sprays to Brassicaceae has been successful in improving plant growth and development along with stress tolerance. However, this method has yet to be tested on canola. The organic nutrients that are waste products from processed seaweed help stimulate plant growth, yielding higher quality plants as a result. In association with Le Groupe Roullier, this study demonstrates that the Roullier extracts (RE) help increase plant growth characteristics and drought tolerance in canola when sprayed 3 times over a 3-week period. A high yielding but drought sensitive mutant of canola, d14 (developed through gene editing) was used for drought assays after 8 weeks of growth and where water was withheld for 6 days. Application of the REs prior to drought resulted in plants having enhanced survival rate and improved biomass retention indicating high drought tolerance. Subsequent RNA sequencing and gene ontological term analysis performed using RE treated plants in triplicates, revealed substantial levels of differential expression of growth-related genes along with stress-related genes. These REs elicited responses in plants that had previously only been achieved through gene editing and transgenic methodologies. Using bio-stimulant sprays provides a novel platform to promote beneficial agronomic traits, independent of genetic manipulation.

Plant reproduction: Stigma receptors regulate reactive oxygen species to establish pollination barriers.

Exciting new research highlights how stigmatic receptors purposed for recognizing self-incompatible pollen interact with the FERONIA pathway to regulate stigmatic reactive oxygen species production to enforce a barrier against self-, intra- and interspecific pollen.

Peroxisomal protein phosphatase PP2A-B' theta interacts with and piggybacks SINA-like 10 E3 ligase into peroxisomes.

Protein phosphatase 2A (PP2A) is targeted to the plant peroxisome via a C-terminal SSL sequence on its regulatory B' theta (θ) subunit. To date the substrates of peroxisomal PP2A are unknown but are thought to be recruited by the regulatory B'θ subunit. Employing yeast two hybrid screening, we have identified Arabidopsis E3 ligase SINA-like 10 as a B'θ binding partner. The E3 ligase SINA-like 10 was found to harbor the PP2A B'-binding Short Linear interaction Motif or SLiM, LxxIxE. This interaction was further verified both in vitro and in vivo using direct pulldown assays and bimolecular fluorescence complementation. Utilizing peroxisomal targeted and a cytosolic version of B'θ (lacking its C-terminal peroxisomal targeting sequence SSL>) bimolecular fluorescence complementation suggests an interaction to occur in the cytosol followed by piggybacking E3 ligase SINA-like 10 into peroxisomes. These results identify a first peroxisomal PP2A interactor, which also obtains a PP2A B'-binding SLiM.

Identification of Arabidopsis Protein Kinases That Harbor Functional Type 1 Peroxisomal Targeting Signals.

Peroxisomes are eukaryotic specific organelles that perform diverse metabolic functions including fatty acid ß-oxidation, reactive species metabolism, photorespiration, and responses to stress. However, the potential regulation of these functions by post-translational modifications, including protein phosphorylation, has had limited study. Recently, we identified and catalogued a large number of peroxisomal phosphorylated proteins, implicating the presence of protein kinases in this organelle. Here, we employed available prediction models coupled with sequence conservation analysis to identify 31 protein kinases from the Arabidopsis kinome (all protein kinases) that contain a putative, non-canonical peroxisomal targeting signal type 1 (PTS1). From this, twelve C-terminal domain-PTS1s were demonstrated to be functional in vivo, targeting enhanced yellow fluorescent protein to peroxisomes, increasing the list of presumptive peroxisomal protein kinases to nineteen. Of the twelve protein kinases with functional PTS1s, we obtained full length clones for eight and demonstrated that seven target to peroxisomes in vivo. Screening homozygous mutants of the presumptive nineteen protein kinases revealed one candidate (GPK1) that harbors a sugar-dependence phenotype, suggesting it is involved in regulating peroxisomal fatty acid ß-oxidation. These results present new opportunities for investigating the regulation of peroxisome functions.

Stylar steroids: Brassinosteroids regulate pistil development and self-incompatibility in Primula.

Manipulation of active brassinosteroid content in the developing flower of Primula dictates style length and female incompatibility type. A new study reveals the dual effects of brassinosteroids on establishing both the morphology of the pistil and mate recognition in self-incompatible heterostylous Primula forbseii.

Cell-cell signaling during the Brassicaceae self-incompatibility response.

Self-incompatibility (SI) is a mechanism that many plant families employ to prevent self-fertilization. In the Brassicaceae, the S-haplotype-specific interaction of the pollen-borne ligand, and a stigma-specific receptor protein kinase triggers a signaling cascade that culminates in the rejection of self-pollen. While the upstream molecular components at the receptor level of the signaling pathway have been extensively studied, the intracellular responses beyond receptor activation were not as well understood. Recent research has uncovered several key molecules and signaling events that operate in concert for the manifestation of the self-incompatible responses in Brassicaceae stigmas. Here, we review the recent discoveries in both the compatible and self-incompatible pathways and provide new perspectives on the early stages of Brassicaceae pollen-pistil interactions.

Stigma Receptivity Is Controlled by Functionally Redundant MAPK Pathway Components in Arabidopsis.

In angiosperms, the process of pollination relies on species-specific interaction and signaling between the male (pollen) and female (pistil) counterparts where the interplay between several pollen and stigma proteins decides the fate of the pollen. In Brassicaceae, the dry stigmatic papillary cells control pollen germination by releasing resources only to compatible pollen thereby allowing pollen to hydrate and germinate. Despite the identification of a number of stigmatic proteins that facilitate pollination responses, the signaling mechanisms that regulate functions of these proteins have remained unknown. Here, we show that, in Arabidopsis, an extremely functionally redundant mitogen-activated protein kinase (MAPK) cascade is required for maintaining stigma receptivity to accept compatible pollen. Our genetic analyses demonstrate that in stigmas, five MAPK kinases (MKKs), MKK1/2/3/7/9 are required to transmit upstream signals to two MPKs, MPK3/4, to mediate compatible pollination. Compromised functions of these five MKKs in the quintuple mutant (mkk1/2/3RNAi/mkk7/9) phenocopied pollination defects observed in the mpk4RNAi/mpk3 double mutant. We further show that this MAPK nexus converges on Exo70A1, a previously identified stigma receptivity factor essential for pollination. Given that pollination is the crucial initial step during plant reproduction, understanding the mechanisms that govern successful pollination could lead to development of strategies to improve crop yield.

Identification and characterization of a female gametophyte defect in sdk1-7 +/- abi3-6 +/- heterozygotes of Arabidopsis thaliana.

Successful reproduction in angiosperms is dependent on the highly synchronous development of their male and female gametophytes and the ensuing fusion of the gametes from these reproductive tissue types. When crossing a T-DNA insertion line sdk1-7-/-(Salk_024564), one of the S-domain receptor kinases involved in ABA responses with a fast neutron deletion line abi3-6-/-, the F1 heterozygotes (sdk1-7+/-abi3-6 +/-) displayed 50% ovule abortion suggesting a likely gametophytic defects. We identified and characterized an early stage female gametophyte developmental defect in the heterozygous mutant ovules. Recombination frequency analysis of the F2 progenies from selfed heterozygotes revealed a possible pseudo-linkage of sdk1-7 and abi3-6 suggesting a reciprocal translocation event in the heterozygote. Our study emphasizes the importance of robust analysis to distinguish gametophytic defect phenotypes caused by genetic interactions and that resulting from possible chromosomal translocation events.

Spatial regulation of alpha-galactosidase activity and its influence on raffinose family oligosaccharides during seed maturation and germination in Cicer arietinum.

Alpha-galactosides or Raffinose Family Oligosaccharides (RFOs) are enriched in legumes and are considered as anti-nutritional factors responsible for inducing flatulence. Due to a lack of alpha-galactosidases in the stomachs of humans and other monogastric animals, these RFOs are not metabolized and are passed to the intestines to be processed by gut bacteria leading to distressing flatulence. In plants, alpha(α)-galactosides are involved in desiccation tolerance during seed maturation and act as a source of stored energy utilized by germinating seeds. The hydrolytic enzyme alpha-galactosidase (α-GAL) can break down RFOs into sucrose and galactose releasing the monosaccharide α-galactose back into the system. Through characterization of RFOs, sucrose, reducing sugars, and α-GAL activity in maturing and germinating chickpeas, we show that stored RFOs are likely required to maintain a steady-state level of reducing sugars. These reducing sugars can then be readily converted to generate energy required for the high energy-demanding germination process. Our observations indicate that RFO levels are lowest in imbibed seeds and rapidly increase post-imbibition. Both RFOs and the α-GAL activity are possibly required to maintain a steady-state level of the reducing monosaccharide sugars, starting from dry seeds all the way through post-germination, to provide the energy for increased germination vigor.

A Flower-Specific Phospholipase D Is a Stigmatic Compatibility Factor Targeted by the Self-Incompatibility Response in Brassica napus.

Self-incompatibility (SI) is a genetic mechanism in hermaphroditic flowers that prevents inbreeding by rejection of self-pollen, while allowing cross- or genetically diverse pollen to germinate on the stigma to successfully fertilize the ovules. In Brassica, SI is initiated by the allele-specific recognition of pollen-encoded, secreted ligand (SCR/SP11) by the stigmatic receptor kinase S-locus receptor kinase (SRK), resulting in activation of SRK through phosphorylation [1-3]. Once activated, this phospho-relay converges on intracellular compatibility factors, which are immediately targeted for degradation by the E3 ligase, ARC1, resulting in the pollen rejection response [4, 5]. Through proteomics approach using proteins from SI activated stigmas of Brassica napus, we identified phospholipase D α1 (PLDα1) as one of the candidates that is most likely targeted for degradation after SI [6]. PLDα1 is enriched in the stigmas and functions as a stigmatic compatibility factor as loss of PLDα1 compromised compatible pollination, while overexpression of PLDα1 in self-incompatible stigmas led to breakdown of SI response. PLDα1 can be ubiquitinated by ARC1 and accumulate in ARC1-suppressed lines, confirming PLDα1 as a target of ARC1 during SI response. Addition of phosphatidic acid (PA) to PLDα1-deficient stigmas was sufficient to rescue compatibility, suggesting an essential role for PA generated by PLDα1 for compatible interactions. We propose that PA produced by PLDα1 activity during compatible pollination promotes vesicle fusion at the membrane to facilitate exocytosis necessary for pollen germination to occur, while SI response could abrogate this process by targeting PLDα1 for degradation.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

Abiotic Stress Signaling in Wheat - An Inclusive Overview of Hormonal Interactions During Abiotic Stress Responses in Wheat.

Rapid global warming directly impacts agricultural productivity and poses a major challenge to the present-day agriculture. Recent climate change models predict severe losses in crop production worldwide due to the changing environment, and in wheat, this can be as large as 42 Mt/°C rise in temperature. Although wheat occupies the largest total harvested area (38.8%) among the cereals including rice and maize, its total productivity remains the lowest. The major production losses in wheat are caused more by abiotic stresses such as drought, salinity, and high temperature than by biotic insults. Thus, understanding the effects of these stresses becomes indispensable for wheat improvement programs which have depended mainly on the genetic variations present in the wheat genome through conventional breeding. Notably, recent biotechnological breakthroughs in the understanding of gene functions and access to whole genome sequences have opened new avenues for crop improvement. Despite the availability of such resources in wheat, progress is still limited to the understanding of the stress signaling mechanisms using model plants such as Arabidopsis, rice and Brachypodium and not directly using wheat as the model organism. This review presents an inclusive overview of the phenotypic and physiological changes in wheat due to various abiotic stresses followed by the current state of knowledge on the identified mechanisms of perception and signal transduction in wheat. Specifically, this review provides an in-depth analysis of different hormonal interactions and signaling observed during abiotic stress signaling in wheat.

VEGFR2 promotes central endothelial activation and the spread of pain in inflammatory arthritis.

Chronic pain can develop in response to conditions such as inflammatory arthritis. The central mechanisms underlying the development and maintenance of chronic pain in humans are not well elucidated although there is evidence for a role of microglia and astrocytes. However in pre-clinical models of pain, including models of inflammatory arthritis, there is a wealth of evidence indicating roles for pathological glial reactivity within the CNS. In the spinal dorsal horn of rats with painful inflammatory arthritis we found both a significant increase in CD11b+ microglia-like cells and GFAP+ astrocytes associated with blood vessels, and the number of activated blood vessels expressing the adhesion molecule ICAM-1, indicating potential glio-vascular activation. Using pharmacological interventions targeting VEGFR2 in arthritic rats, to inhibit endothelial cell activation, the number of dorsal horn ICAM-1+ blood vessels, CD11b+ microglia and the development of secondary mechanical allodynia, an indicator of central sensitization, were all prevented. Targeting endothelial VEGFR2 by inducible Tie2-specific VEGFR2 knock-out also prevented secondary allodynia in mice and glio-vascular activation in the dorsal horn in response to inflammatory arthritis. Inhibition of VEGFR2 in vitro significantly blocked ICAM-1-dependent monocyte adhesion to brain microvascular endothelial cells, when stimulated with inflammatory mediators TNF-α and VEGF-A165a. Taken together our findings suggest that a novel VEGFR2-mediated spinal cord glio-vascular mechanism may promote peripheral CD11b+ circulating cell transmigration into the CNS parenchyma and contribute to the development of chronic pain in inflammatory arthritis. We hypothesise that preventing this glio-vascular activation and circulating cell translocation into the spinal cord could be a new therapeutic strategy for pain caused by rheumatoid arthritis.

Integration of amplification efficiency in qPCR analysis allows precise and relative quantification of transcript abundance of genes from large gene families using RNA isolated from difficult tissues.

The performance of the quantitative polymerase chain reaction (qPCR) assay in the analysis of gene expression belonging to multigene families in tissues rich in secondary metabolites is technically complicated. Here, we present the qPCR analysis of PMT2 gene, a predominant member of a multigene family from tobacco, expressed in the root tissues and is involved in the biosynthesis of nicotine. Consequently, we provide insight into the effect of polymerase chain reaction (PCR) amplification efficiency (AE) of reference and target genes of calibrator and test samples on the qPCR assay performance. Obviously, we found PCR AE as a critical indicator of qPCR assay performance involving multigene families and secondary metabolite-rich root tissues of tobacco. The integration of consistent and uniform PCR amplification efficiencies of reference and target genes of the samples into the relative quantification analysis is emphasized.

Farnesylation-mediated subcellular localization is required for CYP85A2 function.

Protein farnesylation refers to the addition of a 15-carbon farnesyl isoprenoid to the cysteine residue of the CaaX motif at the carboxy terminus of target proteins. In spite of its known roles in plant development and abiotic stress tolerance, how these processes are precisely regulated by farnesylation had remained elusive. We recently showed that CYP85A2, the cytochrome P450, which converts castasterone to brassinolide in the last step of brassinosteroid synthesis must be farnesylated in order to function in this pathway. Lack of either CYP85A2 or the farnesylation motif of CYP85A2 resulted in reduced brassinolide accumulation, hypersensitivity to ABA, and increased plant drought tolerance. In this study, we have assessed the influence of the N-terminal secretory signal and the C-terminal CaaX motif of CYP85A2 in mediating CYP85A2 function and targeting to endomembrane compartments. We show that CaaX motif could still target CYPA85A2 in the absence of an intact N-terminal secretory signal to the respective membrane compartments and partially rescue cyp85a2-2 phenotypes. However, in the absence of both the CaaX motif and the secretory signal, CYP85A2 is not targeted to the membranes and becomes unstable.