Recruitment of HAB1 and SnRK2.2 by C2-domain protein CAR1 in plasma membrane ABA signaling.

Plasma membrane (PM)-associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2-domain ABA-related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane-delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS-tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM-associated ABA signaling. Single-particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1-related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single-molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction.

SLPA-Net: a real-time recognition network for intelligent stomata localization and phenotypic analysis.

Plant stomatal phenotype traits play an important role in improving crop water use efficiency, stress resistance and yield. However, at present, the acquisition of phenotype traits mainly relies on manual measurement, which is time-consuming and laborious. In order to obtain high-throughput stomatal phenotype traits, we proposed a real-time recognition network SLPA-Net for stomata localization and phenotypic analysis. After locating and identifying stomatal density data, ellipse fitting is used to automatically obtain phenotype data such as apertures. Aiming at the problems of small stomata and high similarity to background, we introduced ECANet to improve the accuracy of stoma and aperture location. In order to effectively alleviate the unbalance problem in bounding box regression, we replaced the Loss function with a more effective Focal EIoU Loss. The experimental results show that SLPA-Net has excellent performance in the migration generalization and robustness of stomata and apertures detection and identification, as well as the correlation between stomata phenotype data obtained and artificial data. For convenience, we developed SLPA-Net into a freely available software, the software can be obtained at https://github.com/AITAhenu/SLPA.

Mapping of cytosol-facing organelle outer membrane proximity proteome by proximity-dependent biotinylation in living Arabidopsis cells.

The cytosol-facing outer membrane (OM) of organelles communicates with other cellular compartments to exchange proteins, metabolites, and signaling molecules. Cellular surveillance systems also target OM-resident proteins to control organellar homeostasis and ensure cell survival under stress. However, the OM proximity proteomes have never been mapped in plant cells since using traditional approaches to discover OM proteins and identify their dynamically interacting partners remains challenging. In this study, we developed an OM proximity labeling (OMPL) system using biotin ligase-mediated proximity biotinylation to identify the proximity proteins of the OMs of mitochondria, chloroplasts, and peroxisomes in living Arabidopsis (Arabidopsis thaliana) cells. Using this approach, we mapped the OM proximity proteome of these three organelles under normal conditions and examined the effects of the ultraviolet-B (UV-B) or high light (HL) stress on the abundances of OM proximity proteins. We demonstrate the power of this system with the discovery of cytosolic factors and OM receptor candidates potentially involved in local protein translation and translocation. The candidate proteins that are involved in mitochondrion-peroxisome, mitochondrion-chloroplast, or peroxisome-chloroplast contacts, and in the organellar quality control system are also proposed based on OMPL analysis. OMPL-generated OM proximity proteomes are valuable sources of candidates for functional validation and suggest directions for further investigation of important questions in cell biology.

Trade-offs between the accumulation of cuticular wax and jasmonic acid-mediated herbivory resistance in maize.

Plants have evolved complex physical and chemical defense systems that allow them to withstand herbivory infestation. Composed of a complex mixture of very-long-chain fatty acids (VLCFAs) and their derivatives, cuticular wax constitutes the first physical line of defense against herbivores. Here, we report the function of Glossy 8 (ZmGL8), which encodes a 3-ketoacyl reductase belonging to the fatty acid elongase complex, in orchestrating wax production and jasmonic acid (JA)-mediated defenses against herbivores in maize (Zea mays). The mutation of GL8 enhanced chemical defenses by activating the JA-dependent pathway. We observed a trade-off between wax accumulation and JA levels across maize glossy mutants and 24 globally collected maize inbred lines. In addition, we demonstrated that mutants defective in cuticular wax biosynthesis in Arabidopsis thaliana and maize exhibit enhanced chemical defenses. Comprehensive transcriptomic and lipidomic analyses indicated that the gl8 mutant confers chemical resistance to herbivores by remodeling VLCFA-related lipid metabolism and subsequent JA biosynthesis and signaling. These results suggest that VLCFA-related lipid metabolism has a critical role in regulating the trade-offs between cuticular wax and JA-mediated chemical defenses.

E3 ligases MAC3A and MAC3B ubiquitinate UBIQUITIN-SPECIFIC PROTEASE14 to regulate organ size in Arabidopsis.

The molecular mechanisms controlling organ size during plant development ultimately influence crop yield. However, a deep understanding of these mechanisms is still lacking. UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by DA3, is an essential factor determining organ size in Arabidopsis (Arabidopsis thaliana). Here, we identified two suppressors of the da3-1 mutant phenotype, namely SUPPRESSOR OF da3-1 1 and 2 (SUD1 and SUD2), which encode the E3 ligases MOS4-ASSOCIATED COMPLEX 3A (MAC3A) and MAC3B, respectively. The mac3a-1 and mac3b-1 mutations partially suppressed the high ploidy level and organ size phenotypes observed in the da3-1 mutant. Biochemical analysis showed that MAC3A and MAC3B physically interacted with and ubiquitinated UBP14/DA3 to modulate its stability. We previously reported that UBP14/DA3 acts upstream of the B-type cyclin-dependent kinase CDKB1;1 and maintains its stability to inhibit endoreduplication and cell growth. In this work, MAC3A and MAC3B were found to promote the degradation of CDKB1;1 by ubiquitinating UBP14/DA3. Genetic analysis suggests that MAC3A and MAC3B act in a common pathway with UBP14/DA3 to control endoreduplication and organ size. Thus, our findings define a regulatory module, MAC3A/MAC3B-UBP14-CDKB1;1, that plays a critical role in determining organ size and endoreduplication in Arabidopsis.

A platform for whole-genome speed introgression from Aegilops tauschii to wheat for breeding future crops.

Breeding new and sustainable crop cultivars of high yields and desirable traits has been a major challenge for ensuring food security for the growing global human population. For polyploid crops such as wheat, introducing genetic variation from wild relatives of its subgenomes is a key strategy to improve the quality of their breeding pools. Over the past decades, considerable progress has been made in speed breeding, genome sequencing, high-throughput phenotyping and genomics-assisted breeding, which now allows us to realize whole-genome introgression from wild relatives to modern crops. Here, we present a standardized protocol to rapidly introgress the entire genome of Aegilops tauschii, the progenitor of the D subgenome of bread wheat, into elite wheat backgrounds. This protocol integrates multiple modern high-throughput technologies and includes three major phases: development of synthetic octaploid wheat, generation of hexaploid A. tauschii-wheat introgression lines (A-WIs) and homozygosis of the generated A-WIs. Our approach readily generates stable introgression lines in 2 y, thus greatly accelerating the generation of A-WIs and the introduction of desirable genes from A. tauschii to wheat cultivars. These A-WIs are valuable for wheat-breeding programs and functional gene discovery. The current protocol can be easily modified and used for introgressing the genomes of wild relatives to other polyploid crops.

Reactive oxygen species: Multidimensional regulators of plant adaptation to abiotic stress and development.

Reactive oxygen species (ROS) are produced as undesirable by-products of metabolism in various cellular compartments, especially in response to unfavorable environmental conditions, throughout the life cycle of plants. Stress-induced ROS production disrupts normal cellular function and leads to oxidative damage. To cope with excessive ROS, plants are equipped with a sophisticated antioxidative defense system consisting of enzymatic and non-enzymatic components that scavenge ROS or inhibit their harmful effects on biomolecules. Nonetheless, when maintained at relatively low levels, ROS act as signaling molecules that regulate plant growth, development, and adaptation to adverse conditions. Here, we provide an overview of current approaches for detecting ROS. We also discuss recent advances in understanding ROS signaling, ROS metabolism, and the roles of ROS in plant growth and responses to various abiotic stresses.

The dual role of GoPGF reveals that the pigment glands are synthetic sites of gossypol in aerial parts of cotton.

Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites. Here, we showed that GoPGF, a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis. RNA-sequencing (RNA-seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP-seq and RNA-seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF-dependent and -independent regulation of gossypol production in cotton roots. Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species.

A maize epimerase modulates cell wall synthesis and glycosylation during stomatal morphogenesis.

The unique dumbbell-shape of grass guard cells (GCs) is controlled by their cell walls which enable their rapid responses to the environment. The molecular mechanisms regulating the synthesis and assembly of GC walls are as yet unknown. Here we have identified BZU3, a maize gene encoding UDP-glucose 4-epimerase that regulates the supply of UDP-glucose during GC wall synthesis. The BZU3 mutation leads to significant decreases in cellular UDP-glucose levels. Immunofluorescence intensities reporting levels of cellulose and mixed-linkage glucans are reduced in the GCs, resulting in impaired local wall thickening. BZU3 also catalyzes the epimerization of UDP-N-acetylgalactosamine to UDP-N-acetylglucosamine, and the BZU3 mutation affects N-glycosylation of proteins that may be involved in cell wall synthesis and signaling. Our results suggest that the spatiotemporal modulation of BZU3 plays a dual role in controlling cell wall synthesis and glycosylation via controlling UDP-glucose/N-acetylglucosamine homeostasis during stomatal morphogenesis. These findings provide insights into the mechanisms controlling formation of the unique morphology of grass stomata.

PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis.

Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.

FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions.

Reversible protein S-nitrosylation regulates a wide range of biological functions and physiological activities in plants. However, it is challenging to quantitively determine the S-nitrosylation targets and dynamics in vivo. In this study, we develop a highly sensitive and efficient fluorous affinity tag-switch (FAT-switch) chemical proteomics approach for S-nitrosylation peptide enrichment and detection. We quantitatively compare the global S-nitrosylation profiles in wild-type Arabidopsis and gsnor1/hot5/par2 mutant using this approach, and identify 2,121 S-nitrosylation peptides in 1,595 protein groups, including many previously unrevealed S-nitrosylated proteins. These are 408 S-nitrosylated sites in 360 protein groups showing an accumulation in hot5-4 mutant when compared to wild type. Biochemical and genetic validation reveal that S-nitrosylation at Cys337 in ER OXIDOREDUCTASE 1 (ERO1) causes the rearrangement of disulfide, resulting in enhanced ERO1 activity. This study offers a powerful and applicable tool for S-nitrosylation research, which provides valuable resources for studies on S-nitrosylation-regulated ER functions in plants.

Liverwort bHLH transcription factors and the origin of stomata in plants.

Stomata are distributed in nearly all major groups of land plants, with the only exception being liverworts. Instead of having stomata on sporophytes, many complex thalloid liverworts possess air pores in their gametophytes. At present, whether stomata in land plants are derived from a common origin remains under debate.1,2,3 In Arabidopsis thaliana, a core regulatory module for stomatal development comprises members of the bHLH transcription factor (TF) family, including AtSPCH, AtMUTE, and AtFAMA of subfamily Ia and AtSCRM1/2 of subfamily IIIb. Specifically, AtSPCH, AtMUTE, and AtFAMA each successively form heterodimers with AtSCRM1/2, which in turn regulate the entry, division, and differentiation of stomatal lineages.4,5,6,7 In the moss Physcomitrium patens, two SMF (SPCH, MUTE and FAMA) orthologs have been characterized, one of which is functionally conserved in regulating stomatal development.8,9 We here provide experimental evidence that orthologous bHLH TFs in the liverwort Marchantia polymorpha affect air pore spacing as well as the development of the epidermis and gametangiophores. We found that the bHLH Ia and IIIb heterodimeric module is highly conserved in plants. Genetic complementation experiments showed that liverwort SCRM and SMF genes weakly restored a stomata phenotype in atscrm1, atmute, and atfama mutant backgrounds in A. thaliana. In addition, homologs of stomatal development regulators FLP and MYB88 also exist in liverworts and weakly rescued the stomatal phenotype of atflp/myb88 double mutant. These results provide evidence not only for a common origin of all stomata in extant plants but also for relatively simple stomata in the ancestral plant.

Cell type-specific proteomics uncovers a RAF15-SnRK2.6/OST1 kinase cascade in guard cells.

Multicellular organisms such as plants contain various cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level. Guard cells (GCs) are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange, and provide a model genetic system for analyzing cell fate, signaling, and function. Several proteomics analyses of GC are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified approximately 3,000 proteins not previously found in the GC proteome and more than 600 proteins that may be specific to GC. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15 and Snf1-related kinase2.6 (SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)-induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. We also showed enrichment of enzymes and flavone metabolism in GC, and consistent, dramatic accumulation of flavone metabolites. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development, metabolism, structure, and function.

Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis.

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes. Serine palmitoyltransferase (SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway. The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT (ssSPTs). However, whether LCB1 is modified and how such modification might regulate SPT activity have to date been unclear. Here, we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 by upstream MKK9 and treatment with Flg22 (a pathogen-associated molecular pattern) increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana, with activated MPK3 and MPK6 phosphorylating AtLCB1. Phosphorylation of AtLCB1 strengthened its binding with AtLCB2b, promoted its binding with ssSPTs, and stimulated the formation of higher order oligomeric and active SPT complexes. Our findings therefore suggest a novel regulatory mechanism for SPT activity.

Direct balancing of lipid mobilization and reactive oxygen species production by the epoxidation of fatty acid catalyzed by a cytochrome P450 protein during seed germination.

Fatty acid (FA) ß-oxidation provides energy for oil seed germination but also produces massive byproduct reactive oxygen species (ROS), posing potential oxidative damage to plant cells. How plants overcome the contradiction between energy supply and ROS production during seed germination remains unclear. In this study, we identified an Arabidopsis mvs1 (methylviologen-sensitive) mutant that was hypersensitive to ROS and caused by a missense mutation (G1349 substituted as A) of a cytochrome P450 gene, CYP77A4. CYP77A4 was highly expressed in germinating seedling cotyledons, and its protein is localized in the endoplasmic reticulum. As CYP77A4 catalyzes the epoxidation of unsaturated FA, disruption of CYP77A4 resulted in increased unsaturated FA abundance and over accumulated ROS in the mvs1 mutant. Consistently, scavenging excess ROS or blocking FA ß-oxidation could repress the ROS overaccumulation and hypersensitivity in the mvs1 mutant. Furthermore, H2 O2 transcriptionally upregulated CYP77A4 expression and post-translationally modified CYP77A4 by sulfenylating its Cysteine-456, which is necessary for CYP77A4's role in modulating FA abundance and ROS production. Together, our study illustrates that CYP77A4 mediates direct balancing of lipid mobilization and ROS production by the epoxidation of FA during seed germination.

Deep Transfer Learning-Based Multi-Object Detection for Plant Stomata Phenotypic Traits Intelligent Recognition.

Plant stomata phenotypic traits can provide a basis for enhancing crop tolerance in adversity. Manually counting the number of stomata and measuring the height and width of stomata obviously cannot satisfy the high-throughput data. How to detect and recognize plant stomata quickly and accurately is the prerequisite and key for studying the physiological characteristics of stomata. In this research, we consider stomata recognition as a multi-object detection problem, and propose an end-to-end framework for intelligent detection and recognition of plant stomata based on feature weights transfer learning and YOLOv4 network. It is easy to operate and greatly facilitates the analysis of stomata phenotypic traits in high-throughput plant epidermal cell images. For different cultivars, multi-scales, rich background features, high density, and small stomata object images, the proposed method can precisely locate multiple stomata in microscope images and automatically give phenotypic traits of stomata. Users can also adjust the corresponding parameters to maximize the accuracy and scalability of automatic stomata detection and recognition. Experimental results on actual data provided by the National Maize Improvement Center show that the proposed method is superior to the existing methods in high stomata automatic detection and recognition accuracy, low training cost, strong generalization ability.