CRISPR/Cas9 Based Cell-Type Specific Gene Knock-Out in Arabidopsis Roots.

CRISPR/Cas9 (hereafter Cas9)-mediated gene knockout is one of the most important tools for studying gene function. However, many genes in plants play distinct roles in different cell types. Engineering the currently used Cas9 system to achieve cell-type-specific knockout of functional genes is useful for addressing the cell-specific functions of genes. Here we employed the cell-specific promoters of the WUSCHEL RELATED HOMEOBOX 5 (WOX5), CYCLIND6;1 (CYCD6;1), and ENDODERMIS7 (EN7) genes to drive the Cas9 element, allowing tissue-specific targeting of the genes of interest. We designed the reporters to verify the tissue-specific gene knockout in vivo. Our observation of the developmental phenotypes provides strong evidence for the involvement of SCARECROW (SCR) and GIBBERELLIC ACID INSENSITIVE (GAI) in the development of quiescent center (QC) and endodermal cells. This system overcomes the limitations of traditional plant mutagenesis techniques, which often result in embryonic lethality or pleiotropic phenotypes. By allowing cell-type-specific manipulation, this system has great potential to help us better understand the spatiotemporal functions of genes during plant development.

Transcriptional networks regulating suberin and lignin in endodermis link development and ABA response.

Vascular tissues are surrounded by an apoplastic barrier formed by endodermis that is vital for selective absorption of water and nutrients. Lignification and suberization of endodermal cell walls are fundamental processes in establishing the apoplastic barrier. Endodermal suberization in Arabidopsis (Arabidopsis thaliana) roots is presumed to be the integration of developmental regulation and stress responses. In root endodermis, the suberization level is enhanced when the Casparian strip, the lignified structure, is defective. However, it is not entirely clear how lignification and suberization interplay and how they interact with stress signaling. Here, in Arabidopsis, we constructed a hierarchical network mediated by SHORT-ROOT (SHR), a master regulator of endodermal development, and identified 13 key MYB transcription factors (TFs) that form multiple sub-networks. Combined with functional analyses, we further uncovered MYB TFs that mediate feedback or feed-forward loops, thus balancing lignification and suberization in Arabidopsis roots. In addition, sub-networks comprising nine MYB TFs were identified that interact with abscisic acid signaling to integrate stress response and root development. Our data provide insights into the mechanisms that enhance plant adaptation to changing environments.

Symplastic communication in the root cap directs auxin distribution to modulate root development.

Root cap not only protects root meristem, but also detects and transduces the signals of environmental changes to affect root development. The symplastic communication is an important way for plants to transduce signals to coordinate the development and physiology in response to the changing enviroments. However, it is unclear how the symplastic communication between root cap cells affects root growth. Here we exploit an inducible system to specifically block the symplastic communication in the root cap. Transient blockage of plasmodesmata (PD) in differentiated collumella cells severely impairs the root development in Arabidopsis, in particular in the stem cell niche and the proximal meristem. The neighboring stem cell niche is the region that is most sensitive to the disrupted symplastic communication and responds rapidly via the alteration of auxin distribution. In the later stage, the cell division in proximal meristem is inhibited, presumably due to the reduced auxin level in the root cap. Our results reveal the essential role of the differentiated collumella cells in the root cap mediated signaling system that directs root development.

Time-course observation of the reconstruction of stem cell niche in the intact root.

The stem cell niche (SCN) is critical in maintaining continuous postembryonic growth of the plant root. During their growth in soil, plant roots are often challenged by various biotic or abiotic stresses, resulting in damage to the SCN. This can be repaired by the reconstruction of a functional SCN. Previous studies examining the SCN's reconstruction often introduce physical damage including laser ablation or surgical excision. In this study, we performed a time-course observation of the SCN reconstruction in pWOX5:icals3m roots, an inducible system that causes non-invasive SCN differentiation upon induction of estradiol on Arabidopsis (Arabidopsis thaliana) root. We found a stage-dependent reconstruction of SCN in pWOX5:icals3m roots, with division-driven anatomic reorganization in the early stage of the SCN recovery, and cell fate specification of new SCN in later stages. During the recovery of the SCN, the local accumulation of auxin was coincident with the cell division pattern, exhibiting a spatial shift in the root tip. In the early stage, division mostly occurred in the neighboring stele to the SCN position, while division in endodermal layers seemed to contribute more in the later stages, when the SCN was specified. The precise re-positioning of SCN seemed to be determined by mutual antagonism between auxin and cytokinin, a conserved mechanism that also regulates damage-induced root regeneration. Our results thus provide time-course information about the reconstruction of SCN in intact Arabidopsis roots, which highlights the stage-dependent re-patterning in response to differentiated quiescent center.

Intersected functional zone of transcriptional regulators patterns stemness within stem cell niche of root apical meristem.

Root stem cell niche (SCN) consists of a quiescent center (QC) and surrounding stem cells. Disrupted symplastic communication leads to loss of stemness in the whole SCN. Several SCN regulators were reported to move between cells for SCN maintenance. However, single mutant of these regulators is insufficient to abolish QC stemness despite the high differentiation rate in surrounding stem cells. To dissect the mechanism behind such distinct stemness in SCN, we combined the mis-expression strategy with pWOX5:icals3m system in which QC is symplastically isolated. We found the starch accumulation in QC could be synergistically repressed by WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT). Like PLTs, other core regulators also exhibited dimorphic functions by inhibiting differentiation at a higher dose while promoting cell division at a low protein level. Being located in the center of the intersected expression zones, QC cells receive the highest level of core regulators, forming the most robust stemness within SCN. WUSCHEL-RELATED HOMEOBOX 5 was sufficient to activate PLT1/2 expression, contributing to the QC-enriched PLTs. Our results provide experimental evidence supporting the long-standing hypothesis that the combination of spatial expression, synergistic function and dosage effect of core regulators result in spatially distinct stemness in SCN.

Loss or duplication of key regulatory genes coincides with environmental adaptation of the stomatal complex in Nymphaea colorata and Kalanchoe laxiflora.

The stomatal complex is critical for gas and water exchange between plants and the atmosphere. Originating over 400 million years ago, the structure of the stomata has evolved to facilitate the adaptation of plants to various environments. Although the molecular mechanism of stomatal development in Arabidopsis has been widely studied, the evolution of stomatal structure and its molecular regulators in different species remains to be answered. In this study, we examined stomatal development and the orthologues of Arabidopsis stomatal genes in a basal angiosperm plant, Nymphaea colorata, and a member of the eudicot CAM family, Kalanchoe laxiflora, which represent the adaptation to aquatic and drought environments, respectively. Our results showed that despite the conservation of core stomatal regulators, a number of critical genes were lost in the N. colorata genome, including EPF2, MPK6, and AP2C3 and the polarity regulators BASL and POLAR. Interestingly, this is coincident with the loss of asymmetric divisions during the stomatal development of N. colorata. In addition, we found that the guard cell in K. laxiflora is surrounded by three or four small subsidiary cells in adaxial leaf surfaces. This type of stomatal complex is formed via repeated asymmetric cell divisions and cell state transitions. This may result from the doubled or quadrupled key genes controlling stomatal development in K. laxiflora. Our results show that loss or duplication of key regulatory genes is associated with environmental adaptation of the stomatal complex.

Cell-Fate Specification in Arabidopsis Roots Requires Coordinative Action of Lineage Instruction and Positional Reprogramming.

Tissue organization and pattern formation within a multicellular organism rely on coordinated cell division and cell-fate determination. In animals, cell fates are mainly determined by a cell lineage-dependent mechanism, whereas in plants, positional information is thought to be the primary determinant of cell fates. However, our understanding of cell-fate regulation in plants mostly relies on the histological and anatomical studies on Arabidopsis (Arabidopsis thaliana) roots, which contain a single layer of each cell type in nonvascular tissues. Here, we investigate the dynamic cell-fate acquisition in modified Arabidopsis roots with additional cell layers that are artificially generated by the misexpression of SHORT-ROOT (SHR). We found that cell-fate determination in Arabidopsis roots is a dimorphic cascade with lineage inheritance dominant in the early stage of pattern formation. The inherited cell identity can subsequently be removed or modified by positional information. The instruction of cell-fate conversion is not a fast readout during root development. The final identity of a cell type is determined by the synergistic contribution from multiple layers of regulation, including symplastic communication across tissues. Our findings underline the collaborative inputs during cell-fate instruction.

Symplastic communication spatially directs local auxin biosynthesis to maintain root stem cell niche in Arabidopsis.

Stem cells serve as the source of new cells for plant development. A group of stem cells form a stem cell niche (SCN) at the root tip and in the center of the SCN are slowly dividing cells called the quiescent center (QC). QC is thought to function as a signaling hub that inhibits differentiation of surrounding stem cells. Although it has been generally assumed that cell-to-cell communication provides positional information for QC and SCN maintenance, the tools for testing this hypothesis have long been lacking. Here we exploit a system that effectively blocks plasmodesmata (PD)-mediated signaling to explore how cell-to-cell communication functions in the SCN. We showed that the symplastic signaling between the QC and adjacent cells directs the formation of local auxin maxima and establishment of AP2-domain transcription factors, PLETHORA gradients. Interestingly we found symplastic signaling is essential for local auxin biosynthesis, which acts together with auxin polar transport to provide the guidance for local auxin enrichment. Therefore, we demonstrate the crucial role of cell-to-cell communication in the SCN maintenance and further uncover a mechanism by which symplastic signaling initiates and reinforces the positional information during stem cell maintenance via auxin regulation.

Symplastic signaling instructs cell division, cell expansion, and cell polarity in the ground tissue of Arabidopsis thaliana roots.

Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However, the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icals3m) that inducibly disrupts cell-to-cell communication specifically at PD. Our results show that loss of symplastic signaling to and from the endodermis has very significant effects on the root, including an increase in the number of cell layers in the root and a misspecification of stele cells, as well as ground tissue. Surprisingly, loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root.