Studying plant vascular development using single-cell approaches.

Vascular cells form a highly complex and heterogeneous tissue. Its composition, function, shape, and arrangement vary with the developmental stage and between organs and species. Understanding the transcriptional regulation underpinning this complexity thus requires a high-resolution technique that is capable of capturing rapid events during vascular cell formation. Single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) approaches provide powerful tools to extract transcriptional information from these lowly abundant and dynamically changing cell types, which allows the reconstruction of developmental trajectories. Here, we summarize and reflect on recent studies using single-cell transcriptomics to study vascular cell types and discuss current and future implementations of sc/snRNA-seq approaches in the field of vascular development.

The primary root procambium contributes to lateral root formation through its impact on xylem connection.

The postembryonic formation of lateral roots (LRs) starts in internal root tissue, the pericycle. An important question of LR development is how the connection of the primary root vasculature with that of the emerging LR is established and whether the pericycle and/or other cell types direct this process. Here, using clonal analysis and time-lapse experiments, we show that both the procambium and pericycle of the primary root (PR) affect the LR vascular connectivity in a coordinated manner. We show that during LR formation, procambial derivates switch their identity and become precursors of xylem cells. These cells, together with the pericycle-origin xylem, participate in the formation of what we call a "xylem bridge" (XB), which establishes the xylem connection between the PR and the nascent LR. If the parental protoxylem cell fails to differentiate, XB is still sometimes formed but via a connection with metaxylem cells, highlighting that this process has some plasticity. Using mutant analyses, we show that the early specification of XB cells is determined by CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors (TFs). Subsequent XB cell differentiation is marked by the deposition of secondary cell walls (SCWs) in spiral and reticulate/scalariform patterns, which is dependent on the VASCULAR-RELATED NAC-DOMAIN (VND) TFs. XB elements were also observed in Solanum lycopersicum, suggesting that this mechanism may be more widely conserved in plants. Together, our results suggest that plants maintain vascular procambium activity, which safeguards the functionality of newly established lateral organs by assuring the continuity of the xylem strands throughout the root system.

Bipartite phosphoinositide-dependent modulation of auxin signaling during xylem differentiation in Arabidopsis thaliana roots.

Efficient root-to-shoot delivery of water and nutrients in plants relies on the correct differentiation of xylem cells into hollow elements. While auxin is integral to the formation of xylem cells, it remains poorly characterized how each subcellular pool of this hormone regulates this process. Combining genetic and cell biological approaches, we investigated the bipartite activity of nucleoplasmic vs plasma membrane-associated phosphatidylinositol 4-phosphate kinases PIP5K1 and its homolog PIP5K2 in Arabidopsis thaliana roots and uncovered a novel mechanism by which phosphoinositides integrate distinct aspects of the auxin signaling cascade and, in turn, regulate the onset of xylem differentiation. The appearance of undifferentiated cells in protoxylem strands of pip5k1 pip5k2 is phenomimicked in auxin transport and perception mutants and can be partially restored by the nuclear residence of PIP5K1. By contrast, exclusion of PIP5K1 from the nucleus hinders the auxin-mediated induction of the xylem master regulator VASCULAR RELATED NAC DOMAIN (VND) 7. A xylem-specific increase of auxin levels abolishes pip5k1 pip5k2 vascular defects, indicating that the establishment of auxin maxima is required to activate VND7-mediated xylem differentiation. Our results describe a new mechanism by which distinct subcellular pools of phosphoinositides integrate auxin transport and perception to initiate xylem differentiation in a spatiotemporal manner.

Fe acquisition at the crossroad of calcium and reactive oxygen species signaling.

Due to its redox properties, iron is both essential and toxic. Therefore, soil iron availability variations pose a significant problem for plants. Recent evidence suggests that calcium and reactive oxygen species coordinate signaling events related to soil iron acquisition. Calcium was found to affect directly IRT1-mediated iron import through the lipid-binding protein EHB1 and to trigger a CBL-CIPK-mediated signaling influencing the activity of the key iron-acquisition transcription factor FIT. In parallel, under prolonged iron deficiency, reactive oxygen species both inhibit FIT function and depend on FIT through the function of the catalase CAT2. We discuss the role of calcium and reactive oxygen species signaling in iron acquisition, with post-translational mechanisms influencing the localization and activity of iron-acquisition regulators and effectors.

Reactive oxygen species coordinate the transcriptional responses to iron availability in Arabidopsis.

Reactive oxygen species play a central role in the regulation of plant responses to environmental stress. Under prolonged iron (Fe) deficiency, increased levels of hydrogen peroxide (H2O2) initiate signaling events, resulting in the attenuation of Fe acquisition through the inhibition of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). As this H2O2 increase occurs in a FIT-dependent manner, our aim was to understand the processes involved in maintaining H2O2 levels under prolonged Fe deficiency and the role of FIT. We identified the CAT2 gene, encoding one of the three Arabidopsis catalase isoforms, as regulated by FIT. CAT2 loss-of-function plants displayed severe susceptibility to Fe deficiency and greatly increased H2O2 levels in roots. Analysis of the Fe homeostasis transcription cascade revealed that H2O2 influences the gene expression of downstream regulators FIT, BHLH genes of group Ib, and POPEYE (PYE); however, H2O2 did not affect their upstream regulators, such as BHLH104 and ILR3. Our data shows that FIT and CAT2 participate in a regulatory loop between H2O2 and prolonged Fe deficiency.