Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns ...about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The regulation of stem cell proliferation in plants is controlled by intercellular signaling pathways driven by the diffusible CLAVATA3 (CLV3p) peptide. CLV3p perception is thought to be mediated by ...an overlapping array of receptors in the stem cell niche including the transmembrane receptor kinase CLV1, Receptor-Like Protein Kinase 2 (RPK2), and a dimer of the receptor-like protein CLV2 and the CORYNE (CRN) pseudokinase. Mutations in these receptors have qualitatively similar effects on stem cell function but it is unclear if this represents common or divergent signaling outputs. Previous work in heterologous systems has suggested that CLV1, RPK2 and CLV2/CRN could form higher order complexes but it is also unclear what relevance these putative complexes have to in vivo receptor functions. Here I use the in vivo regulation of a specific transcriptional target of CLV1 signaling in Arabidopsis to demonstrate that, despite the phenotypic similarities between the different receptor mutants, CLV1 controls distinct signaling outputs in living stem cell niches independent of other receptors. This regulation is separable from stem cell proliferation driven by WUSCHEL, a proposed common transcriptional target of CLV3p signaling. In addition, in the absence of CLV1, CLV1-related receptor kinases are ectopically expressed but also buffer stem cell proliferation through the auto-repression of their own expression. Collectively these data reveal a unique in vivo role for CLV1 separable from other stem cell receptors and provides a framework for dissecting the signaling outputs in stem cell regulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Precise control of plant stem cell proliferation is necessary for the continuous and reproducible development of plant organs
. The peptide ligand CLAVATA3 (CLV3) and its receptor protein kinase ...CLAVATA1 (CLV1) maintain stem cell homeostasis within a deeply conserved negative feedback circuit
. In Arabidopsis, CLV1 paralogs also contribute to homeostasis, by compensating for the loss of CLV1 through transcriptional upregulation
. Here, we show that compensation
operates in diverse lineages for both ligands and receptors, but while the core CLV signaling module is conserved, compensation mechanisms have diversified. Transcriptional compensation between ligand paralogs operates in tomato, facilitated by an ancient gene duplication that impacted the domestication of fruit size. In contrast, we found little evidence for transcriptional compensation between ligands in Arabidopsis and maize, and receptor compensation differs between tomato and Arabidopsis. Our findings show that compensation among ligand and receptor paralogs is critical for stem cell homeostasis, but that diverse genetic mechanisms buffer conserved developmental programs.
Abstract
Robust agricultural yields require consistent flower production throughout fluctuating environmental conditions. Floral primordia are produced in the inflorescence meristem, which contains a ...pool of continuously dividing stem cells. Daughter cells of these divisions either retain stem cell identity or are pushed to the SAM periphery, where they become competent to develop into floral primordia after receiving the appropriate signal. Thus, flower production is inherently linked to regulation of the stem cell pool. The plant hormone auxin promotes flower development throughout its early phases and has been shown to interact with the molecular pathways regulating stem cell maintenance. Here, we will summarize how auxin signaling contributes to stem cell maintenance and promotes flower development through the early phases of initiation, outgrowth, and floral fate establishment. Recent advances in this area suggest that auxin may serve as a signal that integrates stem cell maintenance and new flower production.
The development of plant tissues requires cell–cell communication facilitated by chemical and peptide hormones, including small signaling peptides in the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) ...family. The paradigmatic CLE signaling peptide CLAVATA3 regulates the size of the shoot apical meristem and the expression of the stem cell–promoting WUSCHEL transcription factor through an unknown mechanism. This review discusses recent advances in CLE signaling, showing that CLE pathways are conserved in bryophytes, that CLE peptides in Arabidopsis thaliana regulate stem cell identity and cell division in root tissues, and connections to auxin biosynthesis in regulating flower and leaf development. These advances shed light on potential WUSCHEL family–independent aspects of CLE signaling and the overlap between CLE and auxin signaling
The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides ...by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (
). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between
and
suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.
Arabidopsis thaliana seed development requires the concomitant development of two zygotic compartments, the embryo and the endosperm. Following fertilization, the endosperm expands and the embryo ...grows invasively through the endosperm, which breaks down. Here, we describe a structure we refer to as the embryo sheath that forms on the surface of the embryo as it starts to elongate. The sheath is deposited outside the embryonic cuticle and incorporates endosperm-derived material rich in extensin-like molecules. Sheath production is dependent upon the activity of ZHOUPI, an endosperm-specific transcription factor necessary for endosperm degradation, embryo growth, embryo-endosperm separation, and normal embryo cuticle formation. We show that the peptide KERBEROS, whose expression is ZHOUPI dependent, is necessary both for the formation of a normal embryo sheath and for embryo-endosperm separation. Finally, we show that the receptor-like kinases GSO1 and GSO2 are required for sheath deposition at the embryo surface but not for production of sheath material in the endosperm. We present a model in which sheath formation depends on the coordinated production of material in the endosperm and signaling within the embryo, highlighting the complex molecular interaction between these two tissues during early seed development.
The CLAVATA3 (CLV3)-CLAVATA1 (CLV1) ligand-receptor kinase pair negatively regulates shoot stem cell proliferation in plants. clv1 null mutants are weaker in phenotype than clv3 mutants, but the clv1 ...null phenotype is enhanced by mutations in the related receptor kinases BARELY ANY MERISTEM 1, 2 and 3 (BAM1, 2 and 3). The basis of this genetic redundancy is unknown. Here, we demonstrate that the apparent redundancy in the CLV1 clade is in fact due to the transcriptional repression of BAM genes by CLV1 signaling. CLV1 signaling in the rib meristem (RM) of the shoot apical meristem is necessary and sufficient for stem cell regulation. CLV3-CLV1 signaling in the RM represses BAM expression in wild-type Arabidopsis plants. In clv1 mutants, ectopic BAM expression in the RM partially complements the loss of CLV1. BAM regulation by CLV1 is distinct from CLV1 regulation of WUSCHEL, a proposed CLV1 target gene. In addition, quadruple receptor mutants are stronger in phenotype than clv3, pointing to the existence of additional CLV1/BAM ligands. These data provide an explanation for the genetic redundancy seen in the CLV1 clade and reveal a novel feedback operating in the control of plant stem cells.
Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem ...cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
