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Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the Arabidopsis root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica , primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica -induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting a transcriptional control mechanism that facilitates the accommodation of beneficial microbes in the roots. As plant roots grow deeper into the soil, they encounter various fungi and bacteria. Some of these microbes attempt to infect the roots, but certain interactions can be mutually beneficial, promoting microbe survival while protecting plants from harmful infections. However, microbes rarely invade the root tip, which is protected by a special type of tissue called the root cap. As roots grow, root cap cells are constantly removed and replaced. In the model plant Arabidopsis thaliana , root cap turnover occurs via a combination of cellular shedding and developmental programmed cell death (dPCD). Proteins known as SMB and BFN1 regulate this process by triggering cell death and ensuring dead cells are removed. To investigate whether the rate of dPCD and the degree of post-mortem corpse clearance affect how fungi accumulate in plant roots, Charura et al. studied Arabidopsis plants with a non-functional SMB protein. Staining techniques revealed an accumulation of dead cells remaining in the root cap, as well as increased growth of the fungus Serendipita indica in the root tip. These changes also disrupted the growth-promoting effects typically initiated by the fungus. Taken together, the findings suggest that under normal conditions, SMB drives the continuous clearance of cells through dPCD, which limits fungal growth in the root tip that could otherwise harm the plant. Charura et al. next looked at how S. indica infection affects the expression of genes that drive dPCD. This revealed reduced expression of the gene for BFN1 in Arabidopsis plants infected with the fungus. Staining the roots of plants containing a non-functional form of BFN1 also revealed increased dead cell remnants and greater fungal growth further up the root, suggesting that S. indica may exploit host cell clearance pathways to colonize the roots. In conclusion, the findings show that the rate of dPCD in plant roots is key to limiting fungal invasion. The decreased BFN1 gene expression observed with S. indica infection suggests that fungi may manipulate BFN1 to help them form more beneficial partnerships. Understanding the interplay between root cap turnover and fungal invasion could lead to more sustainable agricultural practices and may help researchers to improve plant nutrition and tolerance without relying on chemical fertilizers or pesticides.

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. $smt1^{orc}$ mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the $smt1^{orc}$ root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-β-glucuronidase were aberrant in $smt1^{orc}$. Patterning defects in $smt1^{orc}$ resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in $smt1^{orc}$, whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.

Acidification of intracellular compartments by the vacuolar-type H+-ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the $\\text{V}_{1}\\text{V}_{\\text{o}}$-ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane Vo sector was investigated. Expression of VHA-c1, monitored by promoter-driven β-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wild-type seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the Vo sector and $\\text{V}_{1}\\text{V}_{\\text{o}}$-ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley α-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles ($\\text{GA}_{1}$ by 11-fold and GA4 by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

• Background and Aims Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes. • Methods The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max). • Key Results Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0-to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean. • Conclusions These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps.

For most plants survival depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Because land plants cannot escape environmental stress they use developmental solutions to remodel themselves in order to better adapt to the new conditions. The primary site for perception of underground signals is the root cap (RC). Plant roots have positive hydrotropic response and modify their growth direction in search of water. Using a screening system with a water potential gradient, we isolated a no hydrotropic response (nhr) semi-dominant mutant of Arabidopsis that continued to grow downwardly into the medium with the lowest water potential contrary to the positive hydrotropic and negative gravitropic response seen in wild type-roots. The lack of hydrotropic response of nhr1 roots was confirmed in a system with a gradient in air moisture. The root gravitropic response of nhr1 seedlings was significantly faster in comparison with those of wild type. The frequency of the waving pattern in nhr1 roots was increased compared to those of wild type. nhr1 seedlings had abnormal root cap morphogenesis and reduced root growth sensitivity to abscisic acid (ABA) and the polar auxin transport inhibitor N-(1-naphtyl)phtalamic acid (NPA). These results showed that hydrotropism is amenable to genetic analysis and that an ABA signaling pathway participates in sensing water potential gradients through the root cap.

The rice Eui （ELONGATED UPPERMOST INTERNODE） gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins （GAs）. In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmylA and GAmyb were highly upregulated in eui aleurone ceils in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3oxl and GA20ox2 were downregulated and GA2oxl was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 （GIBBERELLIN INSENSITIVE DWARF 1）, GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA2Oox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.