Overexpression of three related root-cap outermost-cell-specific C2H2-type zinc-finger protein genes suppresses the growth of Arabidopsis in an EAR-motif-dependent manner.

The root meristem of Arabidopsis thaliana is protected by the root cap, the size of which is tightly regulated by the balance between the formative cell divisions and the dispersal of the outermost cells. We isolated an enhancer-tagged dominant mutant displaying the short and twisted root by the overexpression of ZINC-FINGER OF ARABIDOPSIS THALIANA1 (ZAT1) encoding an EAR motif-containing zinc-finger protein. The growth inhibition by ZAT1 was shared by ZAT4 and ZAT9, the ZAT1 homologues. The ZAT1 promoter was specifically active in the outermost cells of the root cap, in which ZAT1-GFP was localized when expressed by the ZAT1 promoter. The outermost cell-specific expression pattern of ZAT1 was not altered in the sombrero (smb) or smb bearskin1 (brn1) brn2 accumulating additional root-cap layers. In contrast, ZAT4-GFP and ZAT9- GFP fusion proteins were distributed to the inner root-cap cells in addition to the outermost cells where ZAT4 and ZAT9 promoters were active. Overexpression of ZAT1 induced the ectopic expression of PUTATIVE ASPARTIC PROTEASE3 involved in the programmed cell death. The EAR motif was essential for the growth inhibition by ZAT1. These results suggest that the three related ZATs might regulate the maturation of the outermost cells of the root cap. [BMB Reports 2020; 53(3): 160-165].

POLTERGEIST and POLTERGEIST-LIKE1 are essential for the maintenance of post-embryonic shoot and root apical meristems as revealed by a partial loss-of-function mutant allele of pll1 in Arabidopsis.

BACKGROUND: POLTERGEIST (POL) and POL-LIKE1 (PLL1) encoding related protein phosphatase 2Cs are essential for the establishment of both shoot and root meristems during embryogenesis. As the strong pol pll1 are seedling-lethal due to the lack of hypocotyl vasculature, the roles of POL/PLL1 for the post-embryonic development is difficult to be assessed. OBJECTIVE: To prepare a weak pol pll1 double mutant that are able to produce post-embryonic organs. METHODS: Several T-DNA insertion mutants of pll1 were crossed to pol-6 for the preparation of weak pol pll1. To understand the epistatic interactions between POL/PLL1 and CLAVATAs, the phenotypes of clvs pol pll1 were assessed and the expression patterns of stem cell markers were examined in pol pll1. POLpro:PLL1-GFP expression was examined during the embryogenesis with confocal microscopy. RESULTS: We isolated a pll1-3 (S544N) allele and prepared a weak pol-6 pll1-3. About 5% of pol-6 pll1-3 seedlings continued the post-embryonic development displaying short roots with reduced root meristem, wuschel-like adventitious phyllotaxis, and defective flowers lacking carpel. The clv1, clv2, and clv3 phenotypes led by enlarged shoot meristems were almost completely suppressed in the pol-6 pll1-3. POL/PLL1 were required for the indeterminate floral organ development displayed by agamous. PLL1-GFP asymmetrically localized in the shootward sides of columella cells and increased the size of distal root meristem region by enhancing the WUS-RELATED HOMEOBOX 5 expression suggesting that PLL1 might provide the stem cells and progenies with proper positional information for the asymmetric cell divisions. CONCLUSION: Together, POL/PLL1 are required for the maintenance of stem cell pools for the post-embryonic development in Arabidopsis.

Misexpression of AtTX12 encoding a Toll/interleukin-1 receptor domain induces growth defects and expression of defense-related genes partially independently of EDS1 in Arabidopsis.

In this study, a tissue-specific GAL4/UAS activation tagging system was used for the characterization of genes which could induce lethality when ubiquitously expressed. A dominant mutant exhibiting stunted growth was isolated and named defective root development 1-D (drd1-D). The T-DNA tag was located within the promoter region of AtTX12, which is predicted to encode a truncated nucleotide-binding leucinerich repeat (NLR) protein, containing a Toll/interleukin-1 receptor (TIR) domain. The transcript levels of AtTX12 and defense-related genes were elevated in drd1-D, and the misexpression of AtTX12 recapitulated the drd1-D phenotypes. In the presence of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), a key transducer of signals triggered by TIR-type NLRs, a low-level of AtTX12 misexpression induced strong defective phenotypes including seedling lethality whereas, in the absence of EDS1, a high-level of AtTX12 misexpression induced weak growth defects like dwarfism, suggesting that AtTX12 might function mainly in an EDS1-dependent and partially in an EDS1-independent manner. [BMB Reports 2016; 49(12): 693-698].

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

WEREWOLF and ENHANCER of GLABRA3 are interdependent regulators of the spatial expression pattern of GLABRA2 in Arabidopsis.

In multicellular organisms, cell fates are specified through differential regulation of transcription. Epidermal cell fates in the Arabidopsis thaliana root are precisely specified by several transcription factors, with the GLABRA2 (GL2) homeodomain protein acting at the farthest downstream in this process. To better understand the regulation of GL2 expression, we ectopically expressed WEREWOLF (WER) and ENHANCER OF GLABRA3 (EGL3) in various tissues and examined GL2 expression. Here we show that WER expressed ubiquitously in the root induced GL2 expression only in the root epidermis, whereas co-expression of WER and EGL3 induced GL2 expression in the corresponding tissues. We also found that GL3 accumulated in the nucleus at the early meristematic region and EGL3 accumulated later in the nucleus of epidermal cells. We further found that ectopic expression of WER and EGL3 in ground tissues inhibited GL2 expression in the epidermis. Our results suggest that the co-expression of WER and EGL3 is sufficient for driving GL2 and CPC expression.

ANGUSTIFOLIA mediates one of the multiple SCRAMBLED signaling pathways regulating cell growth pattern in Arabidopsis thaliana.

In Arabidopsis thaliana, an atypical leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), is required for multiple developmental processes including root epidermal cell fate determination, silique dehiscence, inflorescence growth, ovule morphogenesis, and tissue morphology. Previous work suggested that SCM regulates these multiple pathways using distinct mechanisms via interactions with specific downstream factors. ANGUSTIFOLIA (AN) is known to regulate cell and tissue morphogenesis by influencing cortical microtubule arrangement, and recently, the AN protein was reported to interact with the SCM protein. Therefore, we examined whether AN might be responsible for mediating some of the SCM-dependent phenotypes. We discovered that both scm and an mutant lines cause an abnormal spiral or twisting growth of roots, but only the scm mutant affected root epidermal patterning. The siliques of the an and scm mutants also exhibited spiral growth, as previously reported, but only the scm mutant altered silique dehiscence. Interestingly, we discovered that the spiral growth of roots and siliques of the scm mutant is rescued by a truncated SCM protein that lacks its kinase domain, and that a juxtamembrane domain of SCM was sufficient for AN binding in the yeast two-hybrid analysis. These results suggest that the AN protein is one of the critical downstream factors of SCM pathways specifically responsible for mediating its effects on cell/tissue morphogenesis through cortical microtubule arrangement.

Nuclear trapping controls the position-dependent localization of CAPRICE in the root epidermis of Arabidopsis.

Cell fate determination and differentiation are central processes in the development of multicellular organisms, and the Arabidopsis (Arabidopsis thaliana) root epidermis provides a model system to study the molecular basis of these processes. A lateral inhibition mechanism mediated by an R3 single-repeat MYB protein, CAPRICE (CPC), has been proposed to explain the specification of the two types of root epidermal cells (hair cells and nonhair cells). However, it is not clear how CPC acts preferentially in the H-position cells, rather than the N-position cells, where its gene is expressed. To explore this issue, we examined the effect of misexpressed CPC on cell fate specification and CPC localization in the root epidermis. We show that CPC is able to move readily within the root epidermis when its expression level is high and that CPC can induce the hair cell fate in a cell-autonomous manner. We provide evidence that CPC is capable of moving from the stele tissue in the center of the root to the outermost epidermal layer, where it can induce the hair cell fate. In addition, we show that CPC protein accumulates primarily in the nuclei of H-position cells in the early meristematic region, and this localization requires the H-cell-expressed ENHANCER OF GLABRA3 (EGL3) basic helix-loop-helix transcription factor. These results suggest that cell-cell movement of CPC occurs readily within the meristematic region of the root and that EGL3 preferentially traps the CPC protein in the H-position cells of the epidermis.

Cell fate in the Arabidopsis root epidermis is determined by competition between WEREWOLF and CAPRICE.

The root hair and nonhair cells in the Arabidopsis (Arabidopsis thaliana) root epidermis are specified by a suite of transcriptional regulators. Two of these are WEREWOLF (WER) and CAPRICE (CPC), which encode MYB transcription factors that are required for promoting the nonhair cell fate and the hair cell fate, respectively. However, the precise function and relationship between these transcriptional regulators have not been fully defined experimentally. Here, we examine these issues by misexpressing the WER gene using the GAL4-upstream activation sequence transactivation system. We find that WER overexpression in the Arabidopsis root tip is sufficient to cause epidermal cells to adopt the nonhair cell fate through direct induction of GLABRA2 (GL2) gene expression. We also show that GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), two closely related bHLH proteins, are required for the action of the overexpressed WER and that WER interacts with these bHLHs in plant cells. Furthermore, we find that CPC suppresses the WER overexpression phenotype quantitatively. These results show that WER acts together with GL3/EGL3 to induce GL2 expression and that WER and CPC compete with one another to define cell fates in the Arabidopsis root epidermis.

Funneling of gibberellin signaling by the GRAS transcription regulator scarecrow-like 3 in the Arabidopsis root.

During plant development, because no cell movement takes place, control of the timing and extent of cell division and coordination of the direction and extent of cell expansion are particularly important for growth and development. The plant hormone gibberellins (GAs) play key roles in the control of these developmental processes. However, little is known about the molecular components that integrate the generic GA signaling into a specific cell/tissue to coordinate cell division and cell expansion. Here we report that scarecrow-like 3 (SCL3), a GRAS protein, acts as a positive regulator to integrate and maintain a functional GA pathway by attenuating the DELLA repressors in the root endodermis. The tissue-specific maintenance of GA signaling in the root endodermis plays distinct roles along the longitudinal root axis. While in the elongation/differentiation zone (EDZ), the endodermis-confined GA pathway by SCL3 controls primarily coordination of root cell elongation; in the meristem zone (MZ) SCL3 in conjunction with the short-root/scarecrow (SHR/SCR) pathway controls GA-modulated ground tissue maturation. Our findings highlight the regulatory network of the GRAS transcription regulators (SCL3, DELLAs, and SHR/SCR) in the root endodermis, shedding light on how GA homeostasis is achieved and how the maintenance of GA signaling controls developmental processes in roots.

Developmental and environmental regulation of soybean SE60 gene expression during embryogenesis and germination.

Soybean SE60 belongs to the gamma-thionin family of proteins. We recently demonstrated that SE60 plays a role in defense during soybean development. Here, we show that SE60 is expressed in a tissue-specific and developmentally regulated manner. The expression of SE60 is distinct from that of the glycinin (Gy2) and extensin (SbHRGP3) genes of soybean during embryogenesis and germination. A SE60::GUS(-809) transgene, comprising -809 bp of the 5'-flanking region of SE60 fused to the GUS reporter gene, was expressed specifically in developing embryos, but not in the endosperms, from the globular stage of transgenic tobacco and Arabidopsis seeds. Furthermore, light affected the SE60::GUS(-809) expression pattern in germinating seedlings. Electrophoretic mobility shift assay (EMSA) revealed that soybean nuclear proteins as well as E. coli-expressed SB16, a high mobility group protein (HMG), were bound sequence-specifically to the fragment containing AT-rich motifs identified in the SE60 promoter. Interestingly, the soybean nuclear proteins binding to the two G-boxes and RY repeat were prevalent in seeds of 2-4 mm in size. In contrast, the nuclear proteins binding to the AT-rich motif and SE60 RNA expression were more prominent in seeds of 4-6 mm in size. Therefore, we propose that factors binding to the G-boxes or RY repeat initiate SE60 expression during embryogenesis.

Key divisions in the early Arabidopsis embryo require POL and PLL1 phosphatases to establish the root stem cell organizer and vascular axis.

Arabidopsis development proceeds from three stem cell populations located at the shoot, flower, and root meristems. The relationship between the highly related shoot and flower stem cells and the very divergent root stem cells has been unclear. We show that the related phosphatases POL and PLL1 are required for all three stem cell populations. pol pll1 mutant embryos lack key asymmetric divisions that give rise to the root stem cell organizer and the central vascular axis. Instead, these cells divide in a superficially symmetric fashion in pol pll1 embryos, leading to a loss of embryonic and postembryonic root stem cells and vascular specification. We present data that show that POL/PLL1 drive root stem cell specification by promoting expression of the WUS homolog WOX5. We propose that POL and PLL1 are required for the proper divisions of shoot, flower, and root stem cell organizers, WUS/WOX5 gene expression, and stem cell maintenance.

Large-scale analysis of the GRAS gene family in Arabidopsis thaliana.

GRAS proteins belong to a plant-specific transcription factor family. Currently, 33 GRAS members including a putative expressed pseudogene have been identified in the Arabidopsis genome. With a reverse genetic approach, we have constructed a "phenome-ready unimutant collection" of the GRAS genes in Arabidopsis thaliana. Of this collection, we focused on loss-of-function mutations in 23 novel GRAS members. Under standard conditions, homozygous mutants have no obvious morphological phenotypes compared with those of wild-type plants. Expression analysis of GRAS genes using quantitative real-time RT-PCR (qRT-PCR), microarray data mining, and promoter::GUS reporter fusions revealed their tissue-specific expression patterns. Our analysis of protein-protein interaction and subcellular localization of individual GRAS members indicated their roles as transcription regulators. In our yeast two-hybrid (Y2H) assay, we confirmed the protein-protein interaction between SHORT-ROOT (SHR) and SCARECROW (SCR). Furthermore, we identified a new SHR-interacting protein, SCARECROW-LIKE23 (SCL23), which is the most closely related to SCR. Our large-scale analysis provides a comprehensive evaluation on the Arabidopsis GRAS members, and also our phenome-ready unimutant collection will be a useful resource to better understand individual GRAS proteins that play diverse roles in plant growth and development.

POLTERGEIST and PLL1 are required for stem cell function with potential roles in cell asymmetry and auxin signaling.

Stem cell establishment and maintenance are essential for the largely post-embryonic developmental patterning that occurs in higher plants. Plant embryos establish two stem cell populations at the shoot and root meristems which then function to continuously generate shoot and root tissues, respectively. Research has uncovered entirely separate sets of regulators for the shoot and root stem cells, raising questions about the origin of the later-evolving root meristem and the relationship between the two meristems. We have recently demonstrated that the related Arabidopsis phosphatases POL and PLL1 are essential for both shoot and root stem cell maintenance and that they act in each population by promoting expression of related WOX transcription factors. Furthermore, pol pll1 mutant embryos exhibit defects in key asymmetric divisions in the early embryo. We hypothesize that the primary functions of POL and PLL1 are to establish and/or maintain stem cell polarity. Here we show additional data linking POL/PLL1 function to the proper polarization and localization of auxin signaling during embryogenesis.

POL and PLL1 phosphatases are CLAVATA1 signaling intermediates required for Arabidopsis shoot and floral stem cells.

The post-embryonic development of above-ground tissues in plants is dependent upon the maintenance and differentiation of stem cells at the shoot meristem. The Arabidopsis WUSCHEL (WUS) transcription factor establishes an organizing center within the shoot meristem that is essential for specification of stem-cell identity in overlying cells. The CLAVATA (CLV) signaling pathway, including the CLV1 receptor-kinase, promotes the differentiation of stem cells by limiting the WUS expression domain, yet the mechanism of CLV signaling is largely unknown. Previously, we have shown that mutations in two protein phosphatases, POLTERGEIST (POL) and PLL1, partially suppress clv mutant phenotypes. Here, we demonstrate that POL and PLL1 are integral components of the CLV1 signaling pathway. POL and PLL1 are essential for stem-cell specification, and can also block stem-cell differentiation when overexpressed. We provide extensive evidence that POL and PLL1 act downstream of CLV signaling to maintain WUS expression and that they regulate WUS at a transcriptional level. Our findings suggest that POL and PLL1 are central players in regulating the balance between stem-cell maintenance and differentiation, and are the closest known factors to WUS regulation in the shoot meristem.

POL and related phosphatases are dosage-sensitive regulators of meristem and organ development in Arabidopsis.

CLAVATA1 (CLV1) regulates stem cell accumulation at Arabidopsis shoot and flower meristems. CLV1 encodes a receptor-like kinase, but very little is known about downstream signaling components of receptor-kinase signaling in plants. poltergeist (pol) mutants suppress the accumulation of stem cells that occur in clv mutants, and POL has been hypothesized to modulate CLV1 signaling. The POL gene, which encodes a functional protein phosphatase type 2C, is a member of a six-gene family in Arabidopsis. We have isolated loss-of-function alleles for each of the five POL-like genes (PLL1-PLL5). All gene family members, with the exception of PLL3, are expressed broadly within the plant, albeit at differing levels. We show that PLL1 regulates meristem development in parallel with POL. We observe a strong dosage sensitivity at the meristem for POL and PLL1 function in both loss- and gain-of-function analyses, suggesting that these proteins are rate-limiting modulators of stem cell specification. PLL genes also function outside of the meristem: POL and PLL1 regulate pedicel length in interaction with ERECTA, while PLL4 and PLL5 regulate leaf development. We observed no developmental role for either PLL2 or PLL3 based on single and double mutant analysis.

Expression of an expansin gene is correlated with root elongation in soybean.

Expansin is a family of proteins that catalyze long-term expansion of cell walls and has been considered a principal protein that affects cell expansion in plants. We have identified the first root-specific expansin gene in soybean (Glycine max), GmEXP1, which may be responsible for root elongation. Expression levels of GmEXP1 were very high in the roots of 1- to 5-d-old seedlings, in which rapid root elongation takes place. Furthermore, GmEXP1 mRNA was most abundant in the root tip region, where cell elongation occurs, but scarce in the region of maturation, where cell elongation ceases, implying that its expression is closely related to root development processes. In situ hybridization showed that GmEXP1 transcripts were preferentially present in the epidermal cells and underlying cell layers in the root tip of the primary and secondary roots. Ectopic expression of GmEXP1 accelerated the root growth of transgenic tobacco (Nicotiana tabacum) seedlings, and the roots showed insensitivity to obstacle-touching stress. These results imply that the GmEXP1 gene plays an important role in root development in soybean, especially in the elongation and/or initiation of the primary and secondary roots.