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Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.

RNA turnover is necessary for controlling proper mRNA levels posttranscriptionally. In general, RNA degradation is via exoribonucleases that degrade RNA either from the 5′ end to the 3′ end, such as XRN4, or in the opposite direction by the multisubunit exosome complex. Here, we use genome-wide mapping of uncapped and cleaved transcripts to reveal the global landscape of cotranslational mRNA decay in the Arabidopsis thaliana transcriptome. We found that this process leaves a clear three nucleotide periodicity in open reading frames. This pattern of cotranslational degradation is especially evident near the ends of open reading frames, where we observe accumulation of cleavage events focused 16 to 17 nucleotides upstream of the stop codon because of ribosomal pausing during translation termination. Following treatment of Arabidopsis plants with the translation inhibitor cycloheximide, cleavage events accumulate 13 to 14 nucleotides upstream of the start codon where initiating ribosomes have been stalled with these sequences in their P site. Further analysis in xrn4 mutant plants indicates that cotranslational RNA decay is XRN4 dependent. Additionally, studies in plants lacking CAP BINDING PROTEIN80/ABA HYPERSENSITIVE1, the largest subunit of the nuclear mRNA cap binding complex, reveal a role for this protein in cotranslational decay. In total, our results demonstrate the global prevalence and features of cotranslational RNA decay in a plant transcriptome.

The seed is a key evolutionary adaptation of land plants that facilitates dispersal and allows for germination when the environmental conditions are adequate. Mature seeds are dormant and desiccated, with accumulated storage products that are to be used by the seedling after germination. These properties are imposed on the developing embryo by a maturation program, which operates during the later part of embryogenesis. A number of \"master regulators\" (the \"LEC genes\") required for the induction of the maturation program have been described, but it is not known what prevents this program from being expressed during early embryogenesis. Here, we report that Arabidopsis (Arabidopsis thaliana) embryos mutant for strong alíeles of DICER-LIKE1, the enzyme responsible for the biosynthesis of microRNAs (miRNAs), mature earlier than their wildtype counterparts. This heterochronic phenotype indicates that miRNAs are key regulators of the timing of the maturation program. We demonstrate that miRNAs operate in part by repressing the master regulators LEAFY COTYLEDON2 and FUSCA3 and identify the trihelix transcription factors ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1) and ASIL2 and the histone deacetylase HDA6/SIL1 as components that act downstream of miRNAs to repress the maturation program early in embryogenesis. Both ASIL1 and HDA6/SIL1 are known to act to prevent the expression of embryonic maturation genes after germination, but to our knowledge, this is the first time they have been shown to have a role during embryogenesis. Our data point to a common negative regulatory module of maturation during early embryogenesis and seedling development.

In plants, numerous Ca2+-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.

Loss-of-function mutations of SQUINT (SQN)--which encodes the Arabidopsis orthologue of cyclophilin 40 (CyP40)--cause the precocious expression of adult vegetative traits, an increase in carpel number, and produce abnormal spacing of flowers in the inflorescence. Here we show that the vegetative phenotype of sqn is attributable to the elevated expression of miR156-regulated members of the SPL family of transcription factors and provide evidence that this defect is a consequence of a reduction in the activity of ARGONAUTE1 (AGO1). Support for this latter conclusion was provided by the phenotypic similarity between hypomorphic alleles of AGO1 and null alleles of SQN and by the genetic interaction between sqn and these alleles. Our results suggest that AGO1, or an AGO1-interacting protein, is a major client of CyP40 and that miR156 and its targets play a central role in the regulation of vegetative phase change in ARABIDOPSIS:

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana , rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

Innate immunity represents the first line of inducible defence against microbial infection in plants and animals. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity. Although Ca(2+) has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca(2+) signals are sensed and relayed into early MAMP signalling is unknown. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca(2+)-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence, the present study reveals Ca(2+) signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

Calcium sensing pivotal in innate immunity Plants and animals sense the invasion of pathogens using pattern recognition receptors that recognize diverse microbe-associated molecular patterns (MAMPs) and initiate innate immune responses. Early signalling events in the host include calcium influx, activation of mitogen-activated protein kinases (MAPKs), oxidative burst and transcriptional reprogramming. Boudsocq et al . use a functional genomic screen and genome-wide gene expression profiling in Arabidopsis to show that four calcium-dependent protein kinases (CDPKs) acting as Ca 2+ sensors are the convergence points for diverse MAMPs and are critical for transcriptional reprogramming and oxidative burst in plants. Plants and animals sense intruding pathogens by using proteins that recognize diverse microbe-associated molecular patterns (MAMPs) and initiate innate immune responses. Early signalling responses in the host include calcium influx, an oxidative burst and transcriptional reprogramming. Here, four calcium-dependent protein kinases are described that function as calcium sensors, act as convergence points for various MAMPs, and are crucial for transcriptional reprogramming and oxidative burst in plants. Innate immunity represents the first line of inducible defence against microbial infection in plants and animals 1 , 2 , 3 . In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity 1 , 2 , 3 , 4 . Although Ca 2+ has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca 2+ signals are sensed and relayed into early MAMP signalling is unknown 5 , 6 . Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca 2+ -sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence 7 , 8 , the present study reveals Ca 2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

Part 1 of this volume reviews advances in gene editing techniques such as insertion-based genome edits, base editing, guide RNAs and CRISPR/Cas off targeting. Part 2 surveys applications of gene editing in key cereal and vegetable crops.

RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20–24 nucleotide (nt) small RNAs (smRNAs). In Arabidopsis thaliana, smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3–5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.