Kenneth D. Birnbaum

Cell-specific nitrogen responses mediate developmental plasticity

The organs of multicellular species consist of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change. Some cell-specific responses to changes in environmental conditions are known, but the scale of cell-specific responses within an entire organ as it perceives an environmental flux has not been well characterized in plants or any other multicellular organism. Here, we use cellular profiling of five Arabidopsis root cell types in response to an influx of a critical resource, nitrogen, to uncover a vast and predominantly cell-specific response. We show that cell-specific profiling increases sensitivity several-fold, revealing highly localized regulation of transcripts that were largely hidden from previous global analyses. The cell-specific data revealed responses that suggested a coordinated developmental response in distinct cell types or tissues. One example is the cell-specific regulation of a transcriptional circuit that we showed mediates lateral root outgrowth in response to nitrogen via microRNA167, linking small RNAs to nitrogen responses. Together, these results reveal a previously cryptic component of cell-specific responses to nitrogen. Thus, the results make an important advance in our understanding of how multicellular organisms cope with environmental change at the cell level.

Transcriptional Profile of the Arabidopsis Root Quiescent Center

The self-renewal characteristics of stem cells render them vital engines of development. To better understand the molecular mechanisms that determine the properties of stem cells, transcript profiling was conducted on quiescent center (QC) cells from the Arabidopsis thaliana root meristem. The AGAMOUS-LIKE 42 (AGL42) gene, which encodes a MADS box transcription factor whose expression is enriched in the QC, was used to mark these cells. RNA was isolated from sorted cells, labeled, and hybridized to Affymetrix microarrays. Comparisons with digital in situ expression profiles of surrounding tissues identified a set of genes enriched in the QC. Promoter regions from a subset of transcription factors identified as enriched in the QC conferred expression in the QC. These studies demonstrated that it is possible to successfully isolate and profile a rare cell type in the plant. Mutations in all enriched transcription factor genes including AGL42 exhibited no detectable root phenotype, raising the possibility of a high degree of functional redundancy in the QC.

Slicing across kingdoms: regeneration in plants and animals.

Multicellular organisms possessing relatively long life spans are subjected to diverse, constant, and often intense intrinsic and extrinsic challenges to their survival. Animal and plant tissues wear out as part of normal physiological functions and can be lost to predators, disease, and injury. Both kingdoms survive this wide variety of insults by strategies that include the maintenance of adult stem cells or the induction of stem cell potential in differentiated cells. Repatterning mechanisms often deploy embryonic genes, but the question remains in both plants and animals whether regeneration invokes embryogenesis, generic patterning mechanisms, or unique circuitry comprised of well-established patterning genes.

Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines

Cell type–specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines

Nitrogen economics of root foraging: Transitive closure of the nitrate–cytokinin relay and distinct systemic signaling for N supply vs. demand

As sessile organisms, root plasticity enables plants to forage for and acquire nutrients in a fluctuating underground environment. Here, we use genetic and genomic approaches in a “split-root” framework—in which physically isolated root systems of the same plant are challenged with different nitrogen (N) environments—to investigate how systemic signaling affects genome-wide reprogramming and root development. The integration of transcriptome and root phenotypes enables us to identify distinct mechanisms underlying “N economy” (i.e., N supply and demand) of plants as a system. Under nitrate-limited conditions, plant roots adopt an “active-foraging strategy”, characterized by lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate deprivation. By contrast, in nitrate-replete conditions, plant roots adopt a “dormant strategy”, characterized by a repression of lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate supply. Sentinel genes responding to systemic N signaling identified by genome-wide comparisons of heterogeneous vs. homogeneous split-root N treatments were used to probe systemic N responses in Arabidopsis mutants impaired in nitrate reduction and hormone synthesis and also in decapitated plants. This combined analysis identified genetically distinct systemic signaling underlying plant N economy: (i) N supply, corresponding to a long-distance systemic signaling triggered by nitrate sensing; and (ii) N demand, experimental support for the transitive closure of a previously inferred nitrate–cytokinin shoot–root relay system that reports the nitrate demand of the whole plant, promoting a compensatory root growth in nitrate-rich patches of heterogeneous soil.

Organ regeneration does not require a functional stem cell niche in plants.

Plants rely on the maintenance of stem cell niches at their apices for the continuous growth of roots and shoots. However, although the developmental plasticity of plant cells has been demonstrated, it is not known whether the stem cell niche is required for organogenesis. Here we explore the capacity of a broad range of differentiating cells to regenerate an organ without the activity of a stem cell niche. Using a root-tip regeneration system in Arabidopsis thaliana to track the molecular and functional recovery of cell fates, we show that re-specification of lost cell identities begins within hours of excision and that the function of specialized cells is restored within one day. Critically, regeneration proceeds in plants with mutations that fail to maintain the stem cell niche. These results show that stem-cell-like properties that mediate complete organ regeneration are dispersed in plant meristems and are not restricted to niches, which nonetheless seem to be necessary for indeterminate growth. This regenerative reprogramming of an entire organ without transition to a stereotypical stem cell environment has intriguing parallels to recent reports of induced transdifferentiation of specific cell types in the adult organs of animals.

Regulation of Leaf Maturation by Chromatin-Mediated Modulation of Cytokinin Responses

Plant shoots display indeterminate growth, while their evolutionary decedents, the leaves, are determinate. Determinate leaf growth is conditioned by the CIN-TCP transcription factors, which promote leaf maturation and are negatively regulated by miR319 in leaf primordia. Here we show that CIN-TCPs reduce leaf sensitivity to cytokinin (CK), a phytohormone implicated in inhibition of differentiation in the shoot. We identify the SWI/SNF chromatin remodeling ATPase BRAHMA (BRM) as a genetic mediator of CIN-TCP activities and CK responses. An interactome screen further revealed that SWI/SNF complex components including BRM preferentially interacted with basic-helix-loop-helix (bHLH) transcription factors and the bHLH-related CIN-TCPs. Indeed, TCP4 and BRM interacted in planta. Both TCP4 and BRM bound the promoter of an inhibitor of CK responses, ARR16, and induced its expression. Reconstituting ARR16 levels in leaves with reduced CIN-TCP activity restored normal growth. Thus, CIN-TCP and BRM together promote determinate leaf growth by stage-specific modification of CK responses.

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis

Significance Cellular signals evoke rapid and broad changes in gene regulatory networks. To uncover these network dynamics, we developed an approach able to monitor primary targets of a transcription factor (TF) based solely on gene regulation, in the absence of detectable binding. This enabled us to follow the transient propagation of a nitrogen (N) nutrient signal as a direct impact of the master TF Basic Leucine Zipper 1 (bZIP1). Unexpectedly, the largest class of primary targets that exhibit transient associations with bZIP1 is uniquely relevant to the rapid and dynamic propagation of the N signal. Our ability to uncover this transient network architecture has revealed the “dark matter” of dynamic N nutrient signaling in plants that has previously eluded detection. The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF–target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1–5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, “hit-and-run” transcription model, which enables a “catalyst TF” to activate a large set of targets within minutes of signal perturbation.

A map of cell type‐specific auxin responses

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin‐responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue‐specific transcriptional regulation of cell‐identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin‐response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome‐level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development.

Integration of responses within and across Arabidopsis natural accessions uncovers loci controlling root systems architecture

Significance Species display a range of plastic phenotypes that presumably have evolved as a result of adaptation to heterogeneous environments. We asked whether the genetic mechanisms that underlie adaptation across populations also determine the response of an individual plant to environmental cues in Arabidopsis. Using an integrative root phenotyping approach, genes that underlie natural variation in root architecture across populations were shown to control plasticity responses within an individual. Together, our results uncover a genetic mechanism underlying the phenotypic plasticity of an individual and phenotypic diversity across natural variants. Phenotypic plasticity is presumed to be involved in adaptive change toward species diversification. We thus examined how candidate genes underlying natural variation across populations might also mediate plasticity within an individual. Our implementation of an integrative “plasticity space” approach revealed that the root plasticity of a single Arabidopsis accession exposed to distinct environments broadly recapitulates the natural variation “space.” Genome-wide association mapping identified the known gene PHOSPHATE 1 (PHO1) and other genes such as Root System Architecture 1 (RSA1) associated with differences in root allometry, a highly plastic trait capturing the distribution of lateral roots along the primary axis. The response of mutants in the Columbia-0 background suggests their involvement in signaling key modulators of root development including auxin, abscisic acid, and nitrate. Moreover, genotype-by-environment interactions for the PHO1 and RSA1 genes in Columbia-0 phenocopy the root allometry of other natural variants. This finding supports a role for plasticity responses in phenotypic evolution in natural environments.