Modified Pseudo-Schiff Propidium Iodide for Staining the Shoot Apical Meristem in Arabidopsis.

Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.

Alternative Polyadenylation Is a Novel Strategy for the Regulation of Gene Expression in Response to Stresses in Plants.

Precursor message RNA requires processing to generate mature RNA. Cleavage and polyadenylation at the 3'-end in the maturation of mRNA is one of key processing steps in eukaryotes. The polyadenylation (poly(A)) tail of mRNA is an essential feature that is required to mediate its nuclear export, stability, translation efficiency, and subcellular localization. Most genes have at least two mRNA isoforms via alternative splicing (AS) or alternative polyadenylation (APA), which increases the diversity of transcriptome and proteome. However, most previous studies have focused on the role of alternative splicing on the regulation of gene expression. In this review, we summarize the recent advances concerning APA in the regulation of gene expression and in response to stresses in plants. We also discuss the mechanisms for the regulation of APA for plants in the adaptation to stress responses, and suggest that APA is a novel strategy for the adaptation to environmental changes and response to stresses in plants.

Comprehensive Insight into Tapetum-Mediated Pollen Development in Arabidopsis thaliana.

In flowering plants, pollen development is a key process that is essential for sexual reproduction and seed set. Molecular and genetic studies indicate that pollen development is coordinatedly regulated by both gametophytic and sporophytic factors. Tapetum, the somatic cell layer adjacent to the developing male meiocytes, plays an essential role during pollen development. In the early anther development stage, the tapetal cells secrete nutrients, proteins, lipids, and enzymes for microsporocytes and microspore development, while initiating programmed cell death to provide critical materials for pollen wall formation in the late stage. Therefore, disrupting tapetum specification, development, or function usually leads to serious defects in pollen development. In this review, we aim to summarize the current understanding of tapetum-mediated pollen development and illuminate the underlying molecular mechanism in Arabidopsis thaliana.

SKI-INTERACTING PROTEIN interacts with SHOOT MERISTEMLESS to regulate shoot apical meristem formation.

The shoot apical meristem (SAM), which is formed during embryogenesis, generates leaves, stems, and floral organs during the plant life cycle. SAM development is controlled by SHOOT MERISTEMLESS (STM), a conserved Class I KNOX transcription factor that interacts with another subclass homeodomain protein, BELL, to form a heterodimer, which regulates gene expression at the transcriptional level in Arabidopsis (Arabidopsis thaliana). Meanwhile, SKI-INTERACTING PROTEIN (SKIP), a conserved protein in eukaryotes, works as both a splicing factor and as a transcriptional regulator in plants to control gene expression at the transcriptional and posttranscriptional levels by interacting with distinct partners. Here, we show that, similar to plants with a loss of function of STM, a loss of function of SKIP or the specific knockout of SKIP in the SAM region resulted in failed SAM development and the inability of the mutants to complete their life cycle. In comparison, Arabidopsis mutants that expressed SKIP specifically in the SAM region formed a normal SAM and were able to generate a shoot system, including leaves and floral organs. Further analysis confirmed that SKIP interacts with STM in planta and that SKIP and STM regulate the expression of a similar set of genes by binding to their promoters. In addition, STM also interacts with EARLY FLOWERING 7 (ELF7), a component of Polymerase-Associated Factor 1 complex, and mutation in ELF7 exhibits similar SAM defects to that of STM and SKIP. This work identifies a component of the STM transcriptional complex and reveals the mechanism underlying SKIP-mediated SAM formation in Arabidopsis.

The N-terminal acetyltransferase Naa50 regulates tapetum degradation and pollen development in Arabidopsis.

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9-11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

A chloride efflux transporter, BIG RICE GRAIN 1, is involved in mediating grain size and salt tolerance in rice.

Grain size is determined by the size and number of cells in the grain. The regulation of grain size is crucial for improving crop yield; however, the genes and molecular mechanisms that control grain size remain elusive. Here, we report that a member of the detoxification efflux carrier /Multidrug and Toxic Compound Extrusion (DTX/MATE) family transporters, BIG RICE GRAIN 1 (BIRG1), negatively influences grain size in rice (Oryza sativa L.). BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the outer parenchyma layer cells of spikelet hulls are larger than in wild-type (WT) grains, but the cell number is unaltered. When expressed in Xenopus laevis oocytes, BIRG1 exhibits chloride efflux activity. Consistent with this role of BIRG1, the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level. Moreover, grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions, and the roots of birg1 accumulate more chloride than those of WT under saline conditions. Collectively, the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis.

Co-Transcriptional RNA Processing in Plants: Exploring from the Perspective of Polyadenylation.

Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and proteomic diversity. Following transcription, a nascent RNA usually undergoes capping, splicing, cleavage, and polyadenylation, resulting in a mature messenger RNA (mRNA); however, increasing evidence suggests that transcription and RNA processing are coupled. Plants, which must produce rapid responses to environmental changes because of their limited mobility, exhibit such coupling. In this review, we summarize recent advances in our understanding of the coupling of transcription with RNA processing in plants, and we describe the possible spatial environment and important proteins involved. Moreover, we describe how liquid-liquid phase separation, mediated by the C-terminal domain of RNA polymerase II and RNA processing factors with intrinsically disordered regions, enables efficient co-transcriptional mRNA processing in plants.

A transceptor-channel complex couples nitrate sensing to calcium signaling in Arabidopsis.

Nitrate-induced Ca2+ signaling is crucial for the primary nitrate response in plants. However, the molecular mechanism underlying the generation of the nitrate-specific calcium signature remains unknown. We report here that a cyclic nucleotide-gated channel (CNGC) protein, CNGC15, and the nitrate transceptor (NRT1.1) constitute a molecular switch that controls calcium influx depending on nitrate levels. The expression of CNGC15 is induced by nitrate, and its protein is localized at the plasma membrane after establishment of young seedlings. We found that disruption of CNGC15 results in the loss of the nitrate-induced Ca2+ signature (primary nitrate response) and retards root growth, reminiscent of the phenotype observed in the nrt1.1 mutant. We further showed that CNGC15 is an active Ca2+-permeable channel that physically interacts with the NRT1.1 protein in the plasma membrane. Importantly, we discovered that CNGC15-NRT1.1 interaction silences the channel activity of the heterocomplex, which dissociates upon a rise in nitrate levels, leading to reactivation of the CNGC15 channel. The dynamic interactions between CNGC15 and NRT1.1 therefore control the channel activity and Ca2+ influx in a nitrate-dependent manner. Our study reveals a new nutrient-sensing mechanism that utilizes a nutrient transceptor-channel complex assembly to couple nutrient status to a specific Ca2+ signature.

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding.

Common wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat.

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.

The N-Terminal Acetyltransferase Naa50 Regulates Arabidopsis Growth and Osmotic Stress Response.

N-terminal acetylation (Nt-acetylation) is one of the most common protein modifications in eukaryotes. The function of Naa50, the catalytic subunit of the evolutionarily conserved N-terminal acetyltransferase (Nat) E complex, has not been reported in Arabidopsis. In this study, we found that a loss of Naa50 resulted in a pleiotropic phenotype that included dwarfism and sterility, premature leaf senescence and a shortened primary root. Further analysis revealed that root cell patterning and various root cell properties were severely impaired in naa50 mutant plants. Moreover, defects in auxin distribution were observed due to the mislocalization of PIN auxin transporters. In contrast to its homologs in yeast and animals, Naa50 showed no co-immunoprecipitation with any subunit of the Nat A complex. Moreover, plants lacking Naa50 displayed hypersensitivity to abscisic acid and osmotic stress. Therefore, our results suggest that protein N-terminal acetylation catalyzed by Naa50 plays an essential role in Arabidopsis growth and osmotic stress responses.

To Splice or to Transcribe: SKIP-Mediated Environmental Fitness and Development in Plants.

Gene expression in eukaryotes is controlled at multiple levels, including transcriptional and post-transcriptional levels. The transcriptional regulation of gene expression is complex and includes the regulation of the initiation and elongation phases of transcription. Meanwhile, the post-transcriptional regulation of gene expression includes precursor messenger RNA (pre-mRNA) splicing, 5' capping, and 3' polyadenylation. Among these events, pre-mRNA splicing, conducted by the spliceosome, plays a key role in the regulation of gene expression, and the efficiency and precision of pre-mRNA splicing are critical for gene function. Ski-interacting protein (SKIP) is an evolutionarily conserved protein from yeast to humans. In plants, SKIP is a bifunctional regulator that works as a splicing factor as part of the spliceosome and as a transcriptional regulator via interactions with the transcriptional regulatory complex. Here, we review how the functions of SKIP as a splicing factor and a transcriptional regulator affect environmental fitness and development in plants.

SKIP regulates environmental fitness and floral transition by forming two distinct complexes in Arabidopsis.

Ski-interacting protein (SKIP) is a bifunctional regulator of gene expression that works as a splicing factor as part of the spliceosome and as a transcriptional activator by interacting with EARLY FLOWERING 7 (ELF7). MOS4-Associated Complex 3A (MAC3A) and MAC3B interact physically and genetically with SKIP, mediate the alternative splicing of c. 50% of the expressed genes in the Arabidopsis genome, and are required for the splicing of a similar set of genes to that of SKIP. SKIP interacts physically and genetically with splicing factors and Polymerase-Associated Factor 1 complex (Paf1c) components. However, these splicing factors do not interact either physically or genetically with Paf1c components. The SKIP-spliceosome complex mediates circadian clock function and abiotic stress responses by controlling the alternative splicing of pre-mRNAs encoded by clock- and stress tolerance-related genes. The SKIP-Paf1c complex regulates the floral transition by activating FLOWERING LOCUS C (FLC) transcription. Our data reveal that SKIP regulates floral transition and environmental fitness via its incorporation into two distinct complexes that regulate gene expression transcriptionally and post-transcriptionally, respectively. It will be interesting to discover in future studies whether SKIP is required for integration of environmental fitness and growth by control of the incorporation of SKIP into spliceosome or Paf1c in plants.

An expression atlas of miRNAs in Arabidopsis thaliana.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.

Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility in bread wheat.

Male sterility is an essential trait in hybrid seed production for monoclinous crops, including rice and wheat. However, compared with the high percentage of hybrid rice planted in the world, little commercial hybrid wheat is planted globally as a result of the lack of a suitable system for male sterility. Therefore, understanding the molecular nature of male fertility in wheat is critical for commercially viable hybrid wheat. Here, we report the cloning and characterization of Male Sterility 1 (Ms1) in bread wheat by using a combination of advanced genomic approaches. MS1 is a newly evolved gene in the Poaceae that is specifically expressed in microsporocytes, and is essential for microgametogenesis. Orthologs of Ms1 are expressed in diploid and allotetraploid ancestral species. Orthologs of Ms1 are epigenetically silenced in the A and D subgenomes of allohexaploid wheat; only Ms1 from the B subgenome is expressed. The encoded protein, Ms1, is localized to plastid and mitochondrial membranes, where it exhibits phospholipid-binding activity. These findings provide a foundation for the development of commercially viable hybrid wheat.

A Method for Characterizing Embryogenesis in Arabidopsis.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

Polycomb Group Proteins RING1A and RING1B Regulate the Vegetative Phase Transition in Arabidopsis.

Polycomb group (PcG) protein-mediated gene silencing is a major regulatory mechanism in higher eukaryotes that affects gene expression at the transcriptional level. Here, we report that two conserved homologous PcG proteins, RING1A and RING1B (RING1A/B), are required for global H2A monoubiquitination (H2Aub) in Arabidopsis. The mutation of RING1A/B increased the expression of members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family and caused an early vegetative phase transition. The early vegetative phase transition observed in ring1a ring1b double mutant plants was dependent on an SPL family gene, and the H2Aub status of the chromatin at SPL locus was dependent on RING1A/B. Moreover, mutation in RING1A/B affected the miRNA156a-mediated vegetative phase transition, and RING1A/B and the AGO7-miR390-TAS3 pathway were found to additively regulate this transition in Arabidopsis. Together, our results demonstrate that RING1A/B regulates the vegetative phase transition in Arabidopsis through the repression of SPL family genes.

Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants.

Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism's transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants.

NatA is required for suspensor development in Arabidopsis.

Suspensor development is essential for early embryogenesis. The filamentous suspensor plays vital roles in supporting the embryo proper and in exchanging nutrients and information between the embryo proper and embryo sac. In addition, at the globular stage, the uppermost suspensor cell differentiates into the hypophysis, which generates the progenitors of the quiescent center and columella stem cells. In naa10 and naa15 mutant plants, suspensor cell identity was found to be abnormal and embryo development was disturbed, leading to embryonic lethality. Therefore, the NatA complex is required for proper suspensor development in Arabidopsis.