CDC48A, an interactor of WOX2, is required for embryonic patterning in Arabidopsis thaliana.

KEY MESSAGE: Interactor of WOX2, CDC48A, is crucial for early embryo patterning and shoot meristem stem cell initiation, but is not required for WOX2 protein turnover or subcellular localization. During Arabidopsis embryo patterning, the WUSCHEL HOMEOBOX 2 (WOX2) transcription factor is a major regulator of protoderm and shoot stem cell initiation. Loss of WOX2 function results in aberrant protodermal cell divisions and, redundantly with its paralogs WOX1, WOX3, and WOX5, compromised shoot meristem formation. To elucidate the molecular basis for WOX2 function, we searched for protein interactors by IP-MS/MS from WOX2-overexpression roots displaying reprogramming toward shoot-like cell fates. Here, we report that WOX2 directly interacts with the type II AAA ATPase molecular chaperone CELL DIVISION CYCLE 48A (CDC48A). We confirmed this interaction with bimolecular fluorescence complementation and co-immunoprecipitation and found that both proteins co-localize in the nucleus. We show that CDC48A loss of function results in protoderm and shoot meristem stem cell initiation defects similar to WOX2 loss of function. We also provide evidence that CDC48A promotes WOX2 activity independently of proteolysis or the regulation of nuclear localization, common mechanisms of CDC48A function in other processes. Our results point to a new role of CDC48A in potentiating WOX2 function during early embryo patterning.

Empowering Psychiatric Inpatients to Vote: Perceptions of Voting and the Barriers Encountered.

Individuals with psychiatric illness believe that voting is important. However, these individuals have lower rates of voting when compared to the general population. A survey of psychiatrically hospitalized adult patients was conducted to assess perceptions of and barriers to voting in patients with psychiatric illness. Data from 113 surveys was analyzed. A majority of survey participants agreed that they cared about voting, that their vote made a difference, and that their vote was important. 74% of individuals reported previously experiencing at least one barrier when exercising their right to vote. The most commonly experienced barriers reported were not having enough information to make an informed choice, not knowing where to vote, not having transportation, and not being registered to vote. Individuals who encountered a higher number of barriers in the past had a higher chance of encountering barriers more often. In conclusion, a high percentage of individuals with mental illness severe enough to warrant hospitalization have experienced barriers to voting, with many experiencing multiple barriers. Reduction of these barriers is important, as voting and the resultant public policies can directly affect this population's mental health and access to both mental and physical healthcare services.

The RPT2a-MET1 axis regulates TERMINAL FLOWER1 to control inflorescence meristem indeterminacy in Arabidopsis.

Plant inflorescence architecture is determined by inflorescence meristem (IM) activity and controlled by genetic mechanisms associated with environmental factors. In Arabidopsis (Arabidopsis thaliana), TERMINAL FLOWER1 (TFL1) is expressed in the IM and is required to maintain indeterminate growth, whereas LEAFY (LFY) is expressed in the floral meristems (FMs) formed at the periphery of the IM and is required to activate determinate floral development. Here, we address how Arabidopsis indeterminate inflorescence growth is determined. We show that the 26S proteasome subunit REGULATORY PARTICLE AAA-ATPASE 2a (RPT2a) is required to maintain the indeterminate inflorescence architecture in Arabidopsis. rpt2a mutants display reduced TFL1 expression levels and ectopic LFY expression in the IM and develop a determinate zigzag-shaped inflorescence. We further found that RPT2a promotes DNA METHYLTRANSFERASE1 degradation, leading to DNA hypomethylation upstream of TFL1 and high TFL1 expression levels in the wild-type IM. Overall, our work reveals that proteolytic input into the epigenetic regulation of TFL1 expression directs inflorescence architecture in Arabidopsis, adding an additional layer to stem cell regulation.

miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana.

Many plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro-RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared with Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.

Promoting Health Equity Through Voter Support Activities for the Inpatient Psychiatric Population.

Individuals with mental illness often face barriers to voting. One of the primary barriers is not being registered to vote. This paper describes voter support activities (VSAs) provided to hospitalized adults on the acute inpatient psychiatric units at Pennsylvania Psychiatric Institute. During the six weeks preceding the 2020 general election, adult inpatients were offered six VSAs and an optional survey examining previous voting behaviors and barriers encountered to voting. VSAs included checking voter registration status and polling location, completing a paper or electronic voter registration application, and requesting a mail-in ballot. Of 189 patients approached, 119 individuals participated in the survey and 60 individuals utilized at least one VSA. This project demonstrates that VSAs are a welcome and feasible resource for psychiatrically hospitalized adults. Psychiatric providers can serve an important role in promoting access to voting-related activities for their patients.

A root phloem pole cell atlas reveals common transcriptional states in protophloem-adjacent cells.

Single-cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here we use a second-generation single-cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks common to protophloem sieve element (PSE)-adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.

Integration of pluripotency pathways regulates stem cell maintenance in the Arabidopsis shoot meristem.

In the shoot meristem, both WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM), two transcription factors with overlapping spatiotemporal expression patterns, are essential for maintaining stem cells in an undifferentiated state. Despite their importance, it remains unclear how these two pathways are integrated to coordinate stem cell development. Here, we show that the WUS and STM pathways in Arabidopsis thaliana converge through direct interaction between the WUS and STM proteins. STM binds to the promoter of CLAVATA3 (CLV3) and enhances the binding of WUS to the same promoter through the WUS-STM interaction. Both the heterodimerization and simultaneous binding of WUS and STM at two sites on the CLV3 promoter are required to regulate CLV3 expression, which in turn maintains a constant number of stem cells. Furthermore, the expression of STM depends on WUS, and this WUS-activated STM expression enhances the WUS-mediated stem cell activity. Our data provide a framework for understanding how spatial expression patterns within the shoot meristem are translated into regulatory units of stem cell homeostasis.

Stem cell ageing of the root apical meristem of Arabidopsis thaliana.

Plants form new organs from pluripotent stem cells throughout their lives and under changing environmental conditions. In the Arabidopsis root meristem, a pool of stem cells surrounding a stem cell organizer, named Quiescent Center (QC), gives rise to the specific root tissues. Among them, the columella stem cell niche that gives rise to the gravity-sensing columella cells has been used as a model system to study stem cell regulation at the young seedling stage. However, little is known about the changes of the stem cell niche during later development. Here, we report that the columella stem cell niche undergoes pronounced histological and molecular reorganization as the plant progresses towards the adult stage. Commonly-used reporters for cellular states undergo re-patterning after an initial juvenile meristem phase. Furthermore, the responsiveness to the plant hormone abscisic acid, an integrator of stress response, strongly decreases. Many ageing effects are reminiscent of the loss-of-function phenotype of the central stem cell regulator WOX5 and can be explained by gradually decreasing WOX5 expression levels during ageing. Our results show that the architecture and central regulatory components of the root stem cell niche are already highly dynamic within the first weeks of development.

Dose-Dependent AGO1-Mediated Inhibition of the miRNA165/166 Pathway Modulates Stem Cell Maintenance in Arabidopsis Shoot Apical Meristem.

Pluripotent stem cells localized in proliferating growth centers, the meristems, are the origin of life-long organ formation and growth in higher plants. In the shoot apical meristem of Arabidopsis thaliana, the closely related ARGONAUTE proteins AGO1 and ZLL/AGO10 bind miR165/166 species to regulate mRNAs of HD-ZIP III transcription factors that are essential to maintaining stem cells. Several genetic studies showed that AGO1 and ZLL/AGO10 act redundantly to maintain stem cells. By contrast, the reported biochemical data suggested antagonistic functions: AGO1 utilizes miR165/166 to slice HD-ZIP III mRNAs, whereas ZLL/AGO10 promotes degradation of miR165/166 and thus stabilizes HD-ZIP III mRNAs. How these different functions are balanced in stem cell regulation has remained enigmatic. Here, we show that autorepression of AGO1 through miR168-mediated slicing of its own RNA is required to maintain the ability of AGO1 to suppress HD-ZIP III mRNAs. Increased AGO1 expression, either in the miR168a-2 mutant or by transgenic expression, inhibits this ability despite the presence of high levels of miR165/166, effectively uncoupling HD-ZIP III and miR165/166 expression. AGO1 activity can be restored, however, by increasing the levels of chaperones SQN and HSP90, which promote assembly of RNA-induced silencing complex (RISC). This suggests that cellular abundance of SQN and HSP chaperones limits AGO1-mediated RNA interference in shoot meristem stem cell regulation. Localized misexpression of AGO1 indicates that the cells surrounding the shoot meristem primordium play a crucial role in stem cell development. Taken together, our study provides a framework that reconciles biochemical and genetic data, showing that restriction of AGO1 levels by miR168-mediated autorepression is key to RISC homeostasis and the function of AGO1 in stem cell regulation.

Author Correction: Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

In the Supplementary Information file originally published with this Letter, the authors mistakenly omitted Supplementary Table 14; this has now been amended.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30° N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.

Plant Development: Adding HAM to Stem Cell Control.

The stem cell niche of the shoot meristem is stably maintained despite a rapidly changing cellular context. Recent papers reveal a mechanism controlling the spatial patterning of the stem cell niche that prevents its self-termination.

Maternal auxin supply contributes to early embryo patterning in Arabidopsis.

The angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically, producing a small apical embryonic cell and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes, suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5-7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8-10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules after fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally produced auxin is required for correct embryo development.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook.

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses.

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis.

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.

Transcriptional integration of paternal and maternal factors in the Arabidopsis zygote.

In many plants, the asymmetric division of the zygote sets up the apical-basal axis of the embryo. Unlike animals, plant zygotes are transcriptionally active, implying that plants have evolved specific mechanisms to control transcriptional activation of patterning genes in the zygote. In Arabidopsis, two pathways have been found to regulate zygote asymmetry: YODA (YDA) mitogen-activated protein kinase (MAPK) signaling, which is potentiated by sperm-delivered mRNA of the SHORT SUSPENSOR (SSP) membrane protein, and up-regulation of the patterning gene WOX8 by the WRKY2 transcription factor. How SSP/YDA signaling is transduced into the nucleus and how these pathways are integrated have remained elusive. Here we show that paternal SSP/YDA signaling directly phosphorylates WRKY2, which in turn leads to the up-regulation of WOX8 transcription in the zygote. We further discovered the transcription factors HOMEODOMAIN GLABROUS11/12 (HDG11/12) as maternal regulators of zygote asymmetry that also directly regulate WOX8 transcription. Our results reveal a framework of how maternal and paternal factors are integrated in the zygote to regulate embryo patterning.

A Molecular Framework for the Embryonic Initiation of Shoot Meristem Stem Cells.

The establishment of pluripotent stem cells is a key event during plant and animal embryogenesis, but the underlying mechanisms remain enigmatic. We show that in the flowering plant Arabidopsis thaliana, expression of the shoot meristem stem cell marker CLV3 becomes detectable in transition stage embryos. Surprisingly, the key regulator of stem cell homeostasis WUSCHEL (WUS) is expressed but dispensable for stem cell initiation. Rather, the WUS paralog WOX2, a regulator of embryo patterning initiated in the zygote, functions in this process by shielding stem cell progenitors from differentiation. WOX2 upregulates HD-ZIP III transcription factors required for shoot identity and balances cytokinin versus auxin hormone pathways, revealing that classical plantlet regeneration procedures recapitulate the natural induction mechanism. Our findings link transcriptional regulation of early embryo patterning to hormonal control of stem cell initiation and suggest that similar strategies have evolved in plant and animal stem cell formation.