CRISPR-mediated promoter editing of a cis-regulatory element of OsNAS2 increases Zn uptake/translocation and plant yield in rice.

Developing nutritious rice with a higher yield is one approach to alleviating the problem of micronutrient deficiency in developing countries, especially human malnutrition involving zinc and iron (Fe) deficiency, and achieving better adoption. The transport of micronutrients such as Fe and Zn is mainly regulated via the nicotianamine synthase (OsNAS) gene family, whereas yield is a complex trait that involves multiple loci. Genome editing via CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, focusing on the OsNAS2 promoter, particularly the deletion of the cis-regulatory element ARR1AT at position -933, was conducted for an enhanced accumulation of Zn in the grain and per plant. The results showed that our promoter editing increased Zn concentration per plant. Evidence also showed that an improved spikelet number per main panicle led to increased grain per plant. The traits were inherited in "transgene-free" and homozygous plant progenies. Further investigation needs to be conducted to validate trait performance under field conditions and elucidate the cause of the spikelet increase.

Genome editing reagent delivery in plants.

Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.

Genetic Biofortification to Enrich Rice and Wheat Grain Iron: From Genes to Product.

The micronutrient iron (Fe) is not only essential for plant survival and proliferation but also crucial for healthy human growth and development. Rice and wheat are the two leading staples globally; unfortunately, popular rice and wheat cultivars only have a minuscule amount of Fe content and mainly present in the outer bran layers. Unavailability of considerable Fe-rich rice and wheat germplasms limits the potential of conventional breeding to develop this micronutrient trait in both staples. Agronomic biofortification, defined as soil and foliar fertilizer application, has potential but remains quite challenging to improve grain Fe to the significant level. In contrast, recent accomplishments in genetic biofortification can help to develop Fe-enriched cereal grains to sustainably address the problem of "hidden hunger" when the roadmap from proof of concept to product and adoption can be achieved. Here, we highlight the different genetic biofortification strategies for rice and wheat and path to develop a product.

An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture microdissection.

BACKGROUND: Cyst nematodes are biotrophs that form specialized feeding structures in the roots of host plants, which consist of a syncytial fusion of hypertrophied cells. The formation of syncytium is accompanied by profound transcriptional changes and active metabolism in infected tissues. The challenge in gene expression studies for syncytium has always been the isolation of pure syncytial material and subsequent extraction of intact RNA. Root fragments containing syncytium had been used for microarray analyses. However, the inclusion of neighbouring cells dilutes the syncytium-specific mRNA population. Micro-sectioning coupled with laser capture microdissection (LCM) offers an opportunity for the isolation of feeding sites from heterogeneous cell populations. But recovery of intact RNA from syncytium dissected by LCM is complicated due to extended steps of fixation, tissue preparation, embedding and sectioning. RESULTS: In the present study, we have optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies. We investigated the effect of various sucrose concentrations as cryoprotectant on RNA quality and morphology of syncytial sections. We also compared various types of microscopic slides for strong adherence of sections while removing embedding material. CONCLUSION: The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections. Treatment with higher sucrose concentrations minimizes the risk of RNA degradation, whereas longer incubation times help maintaining the morphology of tissue sections. Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments.

Conserved and unique features of the homeologous maize Aux/IAA proteins ROOTLESS WITH UNDETECTABLE MERISTEM 1 and RUM1-like 1.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) is a key regulator of lateral and seminal root formation. An ancient maize genome duplication resulted in the emergence of its homeolog rum1-like1 (rul1), which displays 92% amino acid sequence identity with RUM1. Both, RUL1 and RUM1 exhibit the canonical four domain structure of Aux/IAA proteins. Moreover, both are localized to the nucleus, are instable and have similar short half-lives of ~23min. Moreover, RUL1 and RUM1 can be stabilized by specific mutations in the five amino acid degron sequence of domain II. In addition, proteins encoded by both genes interact in vivo with auxin response factors (ARFs) such as ZmARF25 and ZmARF34 in protoplasts. Although it was demonstrated that RUL1 and RUM1 can homo and heterodimerize in vivo, rul1 expression is independent of rum1. Moreover, on average rul1 expression is ~84-fold higher than rum1 in the 12 tested tissues and developmental stages, although the relative expression levels in different root tissues are very similar. While RUM1 and RUL1 display conserved biochemical properties, yeast-two-hybrid in combination with BiFC experiments identified a RUM1-associated protein 1 (RAP1) that specifically interacts with RUM1 but not with RUL1. This suggests that RUM1 and RUL1 are at least in part interwoven into different molecular networks.

LATERAL ROOT PRIMORDIA 1 of maize acts as a transcriptional activator in auxin signalling downstream of the Aux/IAA gene rootless with undetectable meristem 1.

Only little is known about target genes of auxin signalling downstream of the Aux/IAA-ARF module. In the present study, it has been demonstrated that maize lateral root primordia 1 (lrp1) encodes a transcriptional activator that is directly regulated by the Aux/IAA protein ROOTLESS WITH UNDETECTABLE MERISTEM 1 (RUM1). Expression of lrp1 is confined to early root primordia and meristems and is auxin-inducible. Based on its primary protein structure, LRP1 is predicted to be a transcription factor. This notion is supported by exclusive LRP1 localization in the nucleus and its ability to activate downstream gene activity. Based on the observation that lrp1 transcription is completely repressed in the semi-dominant gain of function mutant rum1, it was demonstrated that the lrp1 promoter is a direct target of RUM1 proteins. Subsequently, promoter activation assays indicated that RUM1 represses the expression of a GFP reporter fused to the native promoter of lrp1. Constitutive repression of lrp1 in rum1 mutants is a consequence of the stability of mutated rum1 proteins which cannot be degraded by the proteasome and thus constitutively bind to the lrp1 promoter and repress transcription. Taken together, the repression of the transcriptional activator lrp1 by direct binding of RUM1 to its promoter, together with specific expression of lrp1 in root meristems, suggests a function in maize root development via the RUM1-dependent auxin signalling pathway.

Cooperative action of the paralogous maize lateral organ boundaries (LOB) domain proteins RTCS and RTCL in shoot-borne root formation.

The paralogous maize (Zea mays) LBD (Lateral Organ Boundaries Domain) genes rtcs (rootless concerning crown and seminal roots) and rtcl (rtcs-like) emerged from an ancient whole-genome duplication. RTCS is a key regulator of crown root initiation. The diversity of expression, molecular interaction and phenotype of rtcs and rtcl were investigated. The rtcs and rtcl genes display highly correlated spatio-temporal expression patterns in roots, despite the significantly higher expression of rtcs. Both RTCS and RTCL proteins bind to LBD downstream promoters and act as transcription factors. In line with its auxin inducibility and binding to auxin response elements of rtcs and rtcl promoters, ARF34 (AUXIN RESPONSE FACTOR 34) acts as transcriptional activator. Yeast two-hybrid screening combined with bimolecular fluorescence complementation (BiFC) experiments revealed conserved and unique interaction partners of RTCS and RTCL. The rtcl mutation leads to defective shoot-borne root elongation early in development. Cooperative action of RTCS and RTCL during shoot-borne root formation was demonstrated by rtcs-dependent repression of rtcl transcription in coleoptilar nodes. Although RTCS is instrumental in shoot-borne root initiation, RTCL controls shoot-borne root elongation early in development. Their conserved role in auxin signaling, but diverse function in shoot-borne root formation, is underscored by their conserved and unique interaction partners.

Diversity of stability, localization, interaction and control of downstream gene activity in the Maize Aux/IAA protein family.

AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are central regulators of auxin signal transduction. They control many aspects of plant development, share a conserved domain structure and are localized in the nucleus. In the present study, five maize Aux/IAA proteins (ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 and ZmIAA33) representing the evolutionary, phylogenetic and expression diversity of this gene family were characterized. Subcellular localization studies revealed that ZmIAA2, ZmIAA11 and ZmIAA15 are confined to the nucleus while ZmIAA20 and ZmIAA33 are localized in both the nucleus and the cytoplasm. Introduction of specific point mutations in the degron sequence (VGWPPV) of domain II by substituting the first proline by serine or the second proline by leucine stabilized the Aux/IAA proteins. While protein half-life times between ∼11 min (ZmIAA2) to ∼120 min (ZmIAA15) were observed in wild-type proteins, the mutated forms of all five proteins were almost as stable as GFP control proteins. Moreover, all five maize Aux/IAA proteins repressed downstream gene expression in luciferase assays to different degrees. In addition, bimolecular fluorescence complementation (BiFC) analyses demonstrated interaction of all five Aux/IAA proteins with RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1, ZmIAA10) while only ZmIAA15 and ZmIAA33 interacted with the RUM1 paralog RUL1 (RUM-LIKE 1, ZmIAA29). Moreover, ZmIAA11, ZmIAA15 ZmIAA33 displayed homotypic interaction. Hence, despite their conserved domain structure, maize Aux/IAA proteins display a significant variability in their molecular characteristics which is likely associated with the wide spectrum of their developmental functions.

Laser microdissection of plant cells.

Different plant cell types express unique transcriptomes, proteomes, and metabolomes. Therefore, the isolation of specific cell types prior to molecular analyses is important to understand the specification, differentiation, and function of these cells. Isolation of specific plant cell types from composite organs can be achieved by laser microdissection (LMD). A wide variety of methods to fix and embed tissues prior to LMD and downstream molecular analyses have been developed for different plant species and tissues. The present review summarizes and highlights the most recently applied LMD approaches in plant science.

The maize (Zea mays L.) AUXIN/INDOLE-3-ACETIC ACID gene family: phylogeny, synteny, and unique root-type and tissue-specific expression patterns during development.

The plant hormone auxin plays a key role in the coordination of many aspects of growth and development. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode instable primary auxin responsive regulators of plant development that display a protein structure with four characteristic domains. In the present study, a comprehensive analysis of the 34 members of the maize Aux/IAA gene family was performed. Phylogenetic reconstructions revealed two classes of Aux/IAA proteins that can be distinguished by alterations in their domain III. Seven pairs of paralogous maize Aux/IAA proteins were discovered. Comprehensive root-type and tissue-specific expression profiling revealed unique expression patterns of the diverse members of the gene family. Remarkably, five of seven pairs of paralogous genes displayed highly correlated expression patterns in roots. All but one (ZmIAA23) tested maize Aux/IAA genes were auxin inducible, displaying two types of auxin induction within three hours of treatment. Moreover, 51 of 55 (93%) differential Aux/IAA expression patterns between different root-types followed the expression tendency: crown roots > seminal roots > primary roots > lateral roots. This pattern might imply root-type-specific regulation of Aux/IAA transcript abundance. In summary, the detailed analysis of the maize Aux/IAA gene family provides novel insights in the evolution and developmental regulation and thus the function of these genes in different root-types and tissues.

Investigation of nuclear envelope structure and passive permeability.

We present an experimental approach by the help of which structure and passive permeability of the nuclear envelope (NE) can be investigated thoroughly, by combining imaging, fluorescent, and electrophysiological techniques. A mature Xenopus laevis oocyte features a large nucleus offering an excellent system for these investigations. Using the emerging technique of atomic force microscopy, NE structure and the conformational state of nuclear pore complexes (NPCs), known to rule NPC and inevitably NE permeability, can be visualised at high resolution and under near physiological conditions. Passive NE permeability to macromolecules can be determined by the long-established confocal laser scanning microscopy, applying fluorescent macromolecules (dextran). Passive NE permeability to small molecules, which has long remained confounded by the lack of an appropriate technique, can finally be investigated following development of a proper technique designated the "nuclear hourglass" technique. The experimental approach presented here thus opens unique perspectives towards understanding the correlation between NE structure and passive permeability. This chapter describes in detail the protocols for performing such investigations.

Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore.

Nuclear pore complexes (NPCs) mediate and control the transport of virtually all material between the cytosol and the nucleus. It is, therefore, unsurprising that they have long taken centre stage in physiology. A precise understanding of the NPC structure and function that remain to be thoroughly investigated yet is, thus, of crucial importance. The NPC can mediate transport both actively and passively. It remains to be clarified, however, whether transport of small molecules and macromolecules proceeds through the same route in the NPC. Furthermore, it has been shown that surface hydrophobicity represents a major sorting criterion for the active transport through NPCs. Transport factors like importin beta, which exhibit a rather large surface hydrophobicity, bind to their cargo and are believed to interact with a supposedly hydrophobic meshwork that is assumed to reside in the central channel of the NPC but has not yet been visualised. This interaction is presumed to lead to a partial breakdown of the meshwork, thereby, permitting the transport-cargo complexes to pass through. In this study, by using the nano-imaging approach, atomic force microscopy, we visualised under near-physiological conditions, for the first time, the presence of a hydrophobic meshwork in the NPC central channel. Furthermore, our data lend strong support for the existence of two segregated transport routes in the NPC.

A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules.

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.

Ethanol alters access to the cell nucleus.

Ethanol is the most frequently used drug among humans. We tested the hypothesis whether ethanol, at clinically relevant concentrations modifies, signaling across the nuclear envelope (NE). In cell nuclei isolated from Xenopus oocytes, we measured NE electrical resistance and NE macromolecule permeability 1 to 20 h after addition of ethanol (0.05 to 0.2%). Furthermore, with atomic force microscopy, nuclear pores of the NE were imaged after exposure to ethanol. We found that NE permeability decreased within hours of ethanol exposure. In parallel, nuclei swell and nuclear pores form clusters in the NE. Force measurements on individual nuclear pores indicate that pores found in clusters are stiffer than those found randomly distributed in the NE. Application of a transcription blocker (actinomycin D) or RNase treatment of isolated nuclei in vitro after ethanol exposure prevents the permeability changes. In conclusion, ethanol, at commonly used concentrations, changes NE structure by transcriptional processes in the cell nucleus. Within hours, the NE becomes less permeable for diffusible ions and macromolecules. This could explain altered signaling to and communication with the cell nucleus in the pathophysiology of alcohol abuse.

Direct aldosterone action on mouse cardiomyocytes detected with atomic force microscopy.

There is growing evidence that aldosterone acts on heart where it causes cellular remodeling and hypertrophy. Since it is still unclear whether aldosterone directly acts on cardiomyocytes or indirectly, by an altered electrolyte balance in the organism, we applied atomic force microscopy (AFM) in primary cultures of neonatal mouse cardiomyocytes to measure hormone-induced changes in cell volume and plasma membrane surface. AFM measures cell volume and, at the same time, provides quantitative information on cell surface properties. Neonatal mouse cardiomyocytes were cultured for 28 hours in absence or presence of 100 nM aldosterone. Spironolactone was applied as a selective aldosterone receptor antagonist. At the microscopic level, single cell volume and single cell surface were found unchanged by aldosterone. However, nanoscopy of the cell surface, i.e. analysis of the plasma membrane at the nanometer level, revealed a specific increase in plasma membrane nano-enfoldings (roughness). This aldosterone-mediated increase in cell surface roughness was completely prevented by spironolactone. We conclude: (i) Aldosterone directly acts upon cardiomyocytes. (ii) At the microscopic level, no changes of cell volume and cell surface are detectable. (iii) At the nanoscopic level, aldosterone increases plasma membrane roughness. These nanometer changes, detectable only with AFM in cells scanned in fluid after fixation under physiological conditions, indicate plasma membrane remodeling of cardiomyocytes by mineralocorticoids.

Hot spot formation in the nuclear envelope of oocytes in response to steroids.

A Glucocorticoid-sensitive cell rapidly responds to hormone stimulation with bidirectional exchange of specific macromolecules between cytosol and nucleus. Glucocorticoid-initiated macromolecules (GIMs) must overcome the nuclear envelope (NE) to enter or leave the nucleus. GIM translocation occurs through nuclear pore complexes (NPCs) that span the NE. We investigated the question whether transport of GIMs through NPCs occurs random or involves selected groups of NPCs (hot spots). Glucocorticoid receptors were expressed in Xenopus laevis oocytes and GIM transport was activated by triamcinolone acetonide, a potent synthetic glucocorticoid analogon. Glucocorticoid receptors associated with the NE and the chromatin were identified using western blot analysis and, at single molecule level, atomic force microscopy. Fluorescence-labeled dextran was used to describe passive NE permeability. We observed that after hormone injection (i) small GIMs, most likely GRs, localize within seconds on both sides of the NE. (ii) large GIMs, most likely ribonucleoproteins, localize within minutes on NPCs at the nucleoplasmic side (iii) both small and large GIMs accumulate on selected NPC clusters (iv) NE permeability transiently decreases when GIMs attach to NPCs. We conclude that GIM transport across the nuclear barrier does not randomly take place but is carried out by a selected population of NPCs.

Nuclear envelope barrier leak induced by dexamethasone.

Nuclear pore complexes (NPCs) are multiprotein channels that span the nuclear envelope. They strongly limit the efficiency of gene transfection by restriction of nuclear delivery of exogenously applied therapeutic macromolecules. NPC dilation could significantly increase this efficiency. Recently, it was shown in oocytes of Xenopus laevis that NPCs dilate from about 82 to 110 nm within min after injection of the glucocorticoid analog dexamethasone (dex). In the present paper we analyzed by means of atomic force microscopy the structural details of NPC dilation and correlated them with functional changes in nuclear envelope permeability. 5-11 min after Dex injection NPC dilation was found at its maximum (approximately 140 nm). In addition, a yet unknown configuration, so-called giant pore, up to 300 nm in diameter, was visualized. Giant pore formation was paralleled by an increase in nuclear envelope permeability tested by electrophysiology and confocal fluorescence microscopy. Even large macromolecules lacking any nuclear localization signal (77 kDa FITC-dextran, molecule diameter up to 36 nm) could gain access to the nucleus. We conclude that dex transiently opens unspecific pathways for large macromolecules. Dex treatment could be potentially useful for improving the efficiency of nuclear gene transfection.

The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure.

Incoming herpes simplex virus type-1 (HSV-1) capsids are known to dock to the nuclear pore complex (NPC) and release their genome. It has remained elusive, however, how the huge viral DNA translocates through the comparatively small NPC channel. In the present study, the interaction of HSV-1 with NPCs was analyzed by atomic force microscopy. In addition to capsids, smaller subviral structures--most with a diameter of 35-40 nm and a length of 130-160 nm--were visualized at the cytoplasmic side of the NPC. These components differed from capsids in their adhesion and stiffness properties, and were the sole subviral structures translocated through dilated NPCs towards the nucleus. It is presumed that they are the HSV-1 genome, and that a change in NPC conformation allows translocation of this genome as a densely packaged, rodlike structure.

Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells.

During thymic T cell development, immature CD4+CD8+ double-positive (DP) thymocytes develop either into CD4+CD8- Th cells or CD4-CD8+ CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The transcription factor Runx3/AML-2 Runx, runt [corrected] dominant factor; AML, acute myeloid leukemia is expressed specifically during the development of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction of CD8 SP T cells and concomitant accumulation of CD4+CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identified integrin alpha(E)/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only to repress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression of Runx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and an increased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8 lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics of regulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8 SP T cells during thymocyte development.

Glucocorticoids remodel nuclear envelope structure and permeability.

The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.