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.

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination.

Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and well-characterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase.

The ARC1 E3 ligase promotes a strong and stable self-incompatibility response in Arabidopsis species: response to the Nasrallah and Nasrallah commentary.

Following the identification of the male (S-locus Cysteine Rich/S-locus Protein 11) and female (S Receptor kinase [SRK]) factors controlling self-incompatibility in the Brassicaceae, research in this field has focused on understanding the nature of the cellular responses activated by these regulators. We previously identified the ARM Repeat Containing1 (ARC1) E3 ligase as a component of the SRK signaling pathway and demonstrated ARC1's requirement in the stigma for self-incompatible pollen rejection in Brassica napus, Arabidopsis lyrata, and Arabidopsis thaliana. Here, we discuss our findings on the role of ARC1 in reconstructing a strong and stable A. thaliana self-incompatibility phenotype, in the context of the putative issues outlined in a commentary by Nasrallah and Nasrallah. Additionally, with their proposed standardized strategy for studying self-incompatibility in A. thaliana, we offer our perspective on what constitutes a strong and stable self-incompatibility phenotype in A. thaliana and how this should be investigated and reported to the greater community.

Glyoxalase Goes Green: The Expanding Roles of Glyoxalase in Plants.

The ubiquitous glyoxalase enzymatic pathway is involved in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis. The glyoxalase system has been more extensively studied in animals versus plants. Plant glyoxalases have been primarily associated with stress responses and their overexpression is known to impart tolerance to various abiotic stresses. In plants, glyoxalases exist as multigene families, and new roles for glyoxalases in various developmental and signaling pathways have started to emerge. Glyoxalase-based MG detoxification has now been shown to be important for pollination responses. During self-incompatibility response in Brassicaceae, MG is required to target compatibility factors for proteasomal degradation, while accumulation of glyoxalase leads to MG detoxification and efficient pollination. In this review, we discuss the importance of glyoxalase systems and their emerging biological roles in plants.

ABI3 controls embryo degreening through Mendel's I locus.

Chlorophyll (chl) is essential for light capture and is the starting point that provides the energy for photosynthesis and thus plant growth. Obviously, for this reason, retention of the green chlorophyll pigment is considered a desirable crop trait. However, the presence of chlorophyll in mature seeds can be an undesirable trait that can affect seed maturation, seed oil quality, and meal quality. Occurrence of mature green seeds in oil crops such as canola and soybean due to unfavorable weather conditions during seed maturity is known to cause severe losses in revenue. One recently identified candidate that controls the chlorophyll degradation machinery is the stay-green gene, SGR1 that was mapped to Mendel's I locus responsible for cotyledon color (yellow versus green) in peas. A defect in SGR1 leads to leaf stay-green phenotypes in Arabidopsis and rice, but the role of SGR1 in seed degreening and the signaling machinery that converges on SGR1 have remained elusive. To decipher the gene regulatory network that controls degreening in Arabidopsis, we have used an embryo stay-green mutant to demonstrate that embryo degreening is achieved by the SGR family and that this whole process is regulated by the phytohormone abscisic acid (ABA) through ABSCISIC ACID INSENSITIVE 3 (ABI3); a B3 domain transcription factor that has a highly conserved and essential role in seed maturation, conferring desiccation tolerance. Misexpression of ABI3 was sufficient to rescue cold-induced green seed phenotype in Arabidopsis. This finding reveals a mechanistic role for ABI3 during seed degreening and thus targeting of this pathway could provide a solution to the green seed problem in various oil-seed crops.

The S-Domain Receptor Kinase Arabidopsis Receptor Kinase2 and the U Box/Armadillo Repeat-Containing E3 Ubiquitin Ligase9 Module Mediates Lateral Root Development under Phosphate Starvation in Arabidopsis.

When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots (LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients, reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate for synthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. In our screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, the Arabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this module in regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plants lacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grown under phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin in their primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin was sufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for this process. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interaction between ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plants resulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role for autophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions.

Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development.

The Arabidopsis protein AtTTM3 belongs to the CYTH superfamily named after its two founding members, the CyaB adenylate cyclase from Aeromonas hydrophila and the mammalian thiamine triphosphatase. In this study we report the three-dimensional structure of a plant CYTH domain protein, AtTTM3, determined at 1.9 Å resolution. The crystal structure revealed the characteristic tunnel architecture of CYTH proteins, which specialize in the binding of nucleotides and other organic phosphates and in phosphoryl transfer reactions. The ß barrel is composed of eight antiparallel ß strands with a cluster of conserved inwardly facing acidic and basic amino acid residues. Mutagenesis of these residues in the catalytic core led to an almost complete loss of enzymatic activity. We established that AtTTM3 is not an adenylate cyclase. Instead, the enzyme displayed weak NTP phosphatase as well as strong tripolyphosphatase activities similar to the triphosphate tunnel metalloenzyme proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM). AtTTM3 is most highly expressed in the proximal meristematic zone of the plant root. Furthermore, an AtTTM3 T-DNA insertion knockout line displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development.

Proteomics approaches advance our understanding of plant self-incompatibility response.

Self-incompatibility (SI) in plants is a genetic mechanism that prevents self-fertilization and promotes out-crossing needed to maintain genetic diversity. SI has been classified into two broad categories: the gametophytic self-incompatibility (GSI) and the sporophytic self-incompatibility (SSI) based on the genetic mechanisms involved in 'self' pollen rejection. Recent proteomic approaches to identify potential candidates involved in SI have shed light onto a number of previously unidentified mechanisms required for SI response. SI proteome research has progressed from the use of isoelectric focusing in early days to the latest third-generation technique of comparative isobaric tag for relative and absolute quantitation (iTRAQ) used in recent times. We will focus on the proteome-based approaches used to study self-incompatibility (GSI and SSI), recent developments in the field of incompatibility research with emphasis on SSI and future prospects of using proteomic approaches to study self-incompatibility.

Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1.

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK.

Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during pollen responses.

Mate selection and maintenance of genetic diversity is crucial to successful reproduction and species survival. Plants utilize self-incompatibility system as a genetic barrier to prevent self pollen from developing on the pistil, leading to hybrid vigor and diversity. In Brassica (canola, kale, and broccoli), an allele-specific interaction between the pollen SCR/SP11 (S-locus cysteine rich protein/S locus protein 11) and the pistil S Receptor Kinase, results in the activation of SRK which recruits the Arm Repeat Containing 1 (ARC1) E3 ligase to the proteasome. The targets of Arm Repeat Containing 1 are proposed to be compatibility factors, which when targeted for degradation by Arm Repeat Containing 1 results in pollen rejection. Despite the fact that protein degradation is predicted to be important for successful self-pollen rejection, the identity of the various proteins whose abundance is altered by the SI pathway has remained unknown. To identify potential candidate proteins regulated by the SI response, we have used the two-dimensional difference gel electrophoresis analysis, coupled with matrix-assisted laser desorption ionization/time of flight/MS. We identified 56 differential protein spots with 19 unique candidate proteins whose abundance is down-regulated following self-incompatible pollinations. The identified differentials are predicted to function in various pathways including biosynthetic pathways, signaling, cytoskeletal organization, and exocytosis. From the 19 unique proteins identified, we investigated the role of tubulin and the microtubule network during both self-incompatible and compatible pollen responses. Moderate changes in the microtubule network were observed with self-incompatible pollinations; however, a more distinct localized break-down of the microtubule network was observed during compatible pollinations, that is likely mediated by EXO70A1, leading to successful pollination.

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.

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.

Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex.

In the Brassicaceae, compatible pollen-pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat-containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.

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.

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.

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.