Search

Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing. [Copyright &y& Elsevier]

Plants have an amazing ability to adapt to their environment, and this extends beyond biochemical responses and includes developmental changes that help them better exploit resources and survive. The plasticity observed in individual plant morphology is associated with robust developmental pathways that are influenced by environmental factors. However, there is still much to learn about the mechanisms behind the formation of the root system. In Arabidopsis thaliana, the root system displays a hierarchical structure with primary and secondary roots. The process of lateral root (LR) organogenesis involves multiple steps, including LR pre-patterning, LR initiation, LR outgrowth, and LR emergence. The study of root developmental plasticity in Arabidopsis has led to significant progress in understanding the mechanisms governing lateral root formation. The importance of root system architecture lies in its ability to shape the distribution of roots in the soil, which affects the plant's ability to acquire nutrients and water. In Arabidopsis, lateral roots originate from pericycle cells adjacent to the xylem poles known as the xylem-pole-pericycle (XPP). The positioning of LRs along the primary root is underpinned by a repetitive pre-patterning mechanism that establishes primed sites for future lateral root formation. In a subset of primed cells, the memory of a transient priming stimulus leads to the formation of stable pre-branch sites and the establishment of founder cell identity. These founder cells undergo a series of highly organized periclinal and anticlinal cell divisions and expansion to form lateral root primordia. Subsequently, LRP emerges through three overlying cell layers of the primary root, giving rise to fully developed LRs. In addition to LRs Arabidopsis can also develop adventitious lateral roots from the primary root in response to specific stress signals such as wounding or environmental cues. Overall, this review creates an overview of the mechanisms governing root lateral root formation which can be a stepping stone to improved crop yields and a better understanding of plant adaptation to changing environments. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana is a suitable host for phytoparasitic nematodes of the genus Meloidogyne. Successful nematode infection leads to the formation of root galls. We tested for natural genetic variation and inoculation density effects on nematode reproductive success in the interaction between A. thaliana and Meloidogyne javanica. We inoculated different Arabidopsis genotypes with two sources of nematodes at two different doses, using a mild protocol for inoculum preparation. We counted root galls and egg masses 2 months after inoculation. We obtained a high number of successful nematode infections. Infection success differed among Arabidopsis genotypes in interaction with the nematode source. Overall, infection success and reproductive success of nematodes were lower at a higher inoculum dose of nematodes. Our results indicate that natural genetic variation in both host plants and nematodes, as well as short‐ and long‐term negative density effects, shape nematode reproductive success. [ABSTRACT FROM AUTHOR]

Cdc6 is a key regulator of DNA replication in eukaryotes. In this work, the expression pattern of an Arabidopsis cdc6 homologue is characterized by RT‐PCR and in situ hybridization. The data suggest that cdc6At expression is cell cycle regulated. During development, high cdc6At mRNA levels are found in regular cycling cells. In addition, cdc6At expression is also observed in cells that are probably undergoing endoreduplication, suggesting a possible role of Cdc6At in this process in plants. [ABSTRACT FROM PUBLISHER]

Nematode migration, feeding site formation, withdrawal of plant assimilates, and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognized as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana , since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode–plant interactions. Through imaging of tolerance-related parameters, the green canopy area was identified as an accessible and robust measure for assessing damage due to cyst nematode infection. Subsequently, a high-throughput phenotyping platform simultaneously measuring the green canopy area growth of 960 A. thaliana plants was developed. This platform can accurately measure cyst nematode and root-knot nematode tolerance limits in A. thaliana through classical modelling approaches. Furthermore, real-time monitoring provided data for a novel view of tolerance, identifying a compensatory growth response. These findings show that our phenotyping platform will enable a new mechanistic understanding of tolerance to below-ground biotic stress. [ABSTRACT FROM AUTHOR]

The root-knot nematode Meloidogyne incognita is a pathogenic pest that causes severe economic loss to agricultural production by forming a parasitic relationship with its hosts. During the development of M. incognita in the host plant roots, giant cells are formed as a nutrient sink. However, the roles of sugar transporters during the giant cells gain sugar from the plant cells are needed to improve. Meanwhile, the eventual function of sugars will eventually be exported transporters (SWEETs) in nematode-plant interactions remains unclear. In this study, the expression patterns of Arabidopsis thaliana SWEETs were examined by inoculation with M. incognita at 3 days post inoculation (dpi) (penetration stage) and 18 dpi (developing stage). We found that few AtSWEETs responded sensitively to M. incognita inoculation, with the highest induction of AtSWEET1 (AT1G21460), a glucose transporter gene. Histological analyses indicated that the b-glucuronidase (GUS) and green fluorescent protein (GFP) signals were observed specifically in the galls of AtSWEET1-GUS and AtSWEET1-GFP transgenic plant roots, suggesting that AtSWEET1 was induced specifically in the galls. Genetic studies have shown that parasitism of M. incognita was significantly affected in atsweet1 compared to wildtype and complementation plants. In addition, parasitism of M. incognita was significantly affected in atsweet10 but not in atsweet13 and atsweet14, expression of which was induced by inoculation with M. incognita. Taken together, these data prove that SWEETs play important roles in plant and nematode interactions. [ABSTRACT FROM AUTHOR]

The synergy between drought-responsive traits across different organs is crucial in the whole-plant mechanism influencing drought resilience. These organ interactions, however, are poorly understood, limiting our understanding of drought response strategies at the whole-plant level. Therefore, we need more integrative studies, especially on herbaceous species that represent many important food crops but remain underexplored in their drought response. We investigated inflorescence stems and rosette leaves of six Arabidopsis thaliana genotypes with contrasting drought tolerance, and combined anatomical observations with hydraulic measurements and gene expression studies to assess differences in drought response. The soc1ful double mutant was the most drought-tolerant genotype based on its synergistic combination of low stomatal conductance, largest stomatal safety margin, more stable leaf water potential during non-watering, reduced transcript levels of drought stress marker genes, and reduced loss of chlorophyll content in leaves, in combination with stems showing the highest embolism resistance, most pronounced lignification, and thickest intervessel pit membranes. In contrast, the most sensitive Cvi ecotype shows the opposite extreme of the same set of traits. The remaining four genotypes show variations in this drought syndrome. Our results reveal that anatomical, ecophysiological, and molecular adaptations across organs are intertwined, and multiple (differentially combined) strategies can be applied to acquire a certain level of drought tolerance. [ABSTRACT FROM AUTHOR]

MonocotJRLs are Poaceae-specific two-domain proteins that consist of a jacalin-related lectin (JRL) and a dirigent (DIR) domain which participate in multiple developmental processes, including disease resistance. For OsJAC1, a monocotJRL from rice, it has been confirmed that constitutive expression in transgenic rice or barley plants facilitates broad-spectrum disease resistance. In this process, both domains of OsJAC1 act cooperatively, as evidenced from experiments with artificially separated JRL- or DIR-domain-containing proteins. Interestingly, these chimeric proteins did not evolve in dicotyledonous plants. Instead, proteins with a single JRL domain, multiple JRL domains or JRL domains fused to domains other than DIR domains are present. In this study, we wanted to test if the cooperative function of JRL and DIR proteins leading to pathogen resistance was conserved in the dicotyledonous plant Arabidopsis thaliana. In Arabidopsis, we identified 50 JRL and 24 DIR proteins, respectively, from which seven single-domain JRL and two single-domain DIR candidates were selected. A single-cell transient gene expression assay in barley revealed that specific combinations of the Arabidopsis JRL and DIR candidates reduced the penetration success of barley powdery mildew. Strikingly, one of these pairs, AtJAX1 and AtDIR19, is encoded by genes located next to each other on chromosome one. However, when using natural variation and analyzing Arabidopsis ecotypes that express full-length or truncated versions of AtJAX1, the presence/absence of the full-length AtJAX1 protein could not be correlated with resistance to the powdery mildew fungus Golovinomyces orontii. Furthermore, an analysis of the additional JRL and DIR candidates in a bi-fluorescence complementation assay in Nicotiana benthamiana revealed no direct interaction of these JRL/DIR pairs. Since transgenic Arabidopsis plants expressing OsJAC1-GFP also did not show increased resistance to G. orontii, it was concluded that the resistance mediated by the synergistic activities of DIR and JRL proteins is specific for members of the Poaceae, at least regarding the resistance against powdery mildew. Arabidopsis lacks the essential components of the DIR-JRL-dependent resistance pathway. [ABSTRACT FROM AUTHOR]

Some of the hormone crosstalk and transcription factors (TFs) involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. In previous work, we established Solanum lycopersicum "Micro-Tom" explants without the addition of exogenous hormones as a model to investigate wound-induced de novo organ formation. The current working model indicates that cell reprogramming and founder cell activation requires spatial and temporal regulation of auxin-to-cytokinin (CK) gradients in the apical and basal regions of the hypocotyl combined with extensive metabolic reprogramming of some cells in the apical region. In this work, we extended our transcriptomic analysis to identify some of the gene regulatory networks involved in wound-induced organ regeneration in tomato. Our results highlight a functional conservation of key TF modules whose function is conserved during de novo organ formation in plants, which will serve as a valuable resource for future studies. [ABSTRACT FROM AUTHOR]

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes. [ABSTRACT FROM AUTHOR]

Root meristem is a reserve of undifferentiated cells which guide root development. To maintain root meristem identity and therefore continuous root growth, the rate of cell differentiation must coordinate with the rate of generation of new cells. The E2 promoter-binding factor a (E2Fa) has been shown to regulate root growth through controlling G1/S cell cycle transitions in Arabidopsis thaliana. Here, we found that NAC1, a member of the NAM/ATAF/CUC family of transcription factors, regulated root growth by directly repressing the transcription of E2Fa. Loss of NAC1 triggers an up-regulation of the E2Fa expression and causes a reduced meristem size and short-root phenotype, which are largely rescued by mutation of E2Fa. Further analysis showed that NAC1 was shown to regulate root meristem by controlling endopolyploidy levels in an E2Fa-dependent manner. This study provides evidence to show that NAC1 maintains root meristem size and root growth by directly repressing the transcription of E2Fa in Arabidopsis. [ABSTRACT FROM AUTHOR]

Summary: Magnesium (Mg2+) serves as a cofactor for a number of photosynthetic enzymes in the chloroplast, and is the central atom of the Chl molecule. However, little is known about the molecular mechanism of Mg2+ transport across the chloroplast envelope.Here, we report the functional characterization of two transport proteins in Arabidopsis: Magnesium Release 8 (MGR8) and MGR9, of the ACDP/CNNM family, which is evolutionarily conserved across all lineages of living organisms.Both MGR8 and MGR9 genes were expressed ubiquitously, and their encoded proteins were localized in the inner envelope of chloroplasts. Mutations of MGR8 and MGR9 together, but neither of them alone, resulted in albino ovules and chlorotic seedlings. Further analysis revealed severe defects in thylakoid biogenesis and assembly of photosynthetic complexes in the double mutant. Both MGR8 and MGR9 functionally complemented the growth of the Salmonella typhimurium mutant strain MM281, which lacks Mg2+ uptake capacity. The embryonic and early seedling defects of the mgr8/mgr9 double mutant were rescued by the expression of MGR9 under the embryo‐specific ABI3 promoter. The partially rescued mutant plants were hypersensitive to Mg2+ deficient conditions and contained less Mg2+ in their chloroplasts than wild‐type plants.Taken together, we conclude that MGR8 and MGR9 serve as Mg2+ transporters and are responsible for chloroplast Mg2+ uptake. [ABSTRACT FROM AUTHOR]

Summary: Root‐knot nematodes (RKNs) are root endoparasites that induce the dedifferentiation of a few root cells and the reprogramming of their gene expression to generate giant hypermetabolic feeding cells.We identified two microRNA families, miR408 and miR398, as upregulated in Arabidopsis thaliana and Solanum lycopersicum roots infected by RKNs. In plants, the expression of these two conserved microRNA families is known to be activated by the SPL7 transcription factor in response to copper starvation.By combining functional approaches, we deciphered the network involving these microRNAs, their regulator and their targets. MIR408 expression was located within nematode‐induced feeding cells like its regulator SPL7 and was regulated by copper. Moreover, infection assays with mir408 and spl7 knockout mutants or lines expressing targets rendered resistant to cleavage by miR398 demonstrated the essential role of the SPL7/MIR408/MIR398 module in the formation of giant feeding cells.Our findings reveal how perturbation of plant copper homeostasis, via the SPL7/MIR408/MIR398 module, modulates the development of nematode‐induced feeding cells. [ABSTRACT FROM AUTHOR]

Lateral root organogenesis is a key process in the development of a plant's root system and its adaptation to the environment. During lateral root formation, an early phase of cell proliferation first produces a four-cell-layered primordium, and only from this stage onwards is a root meristem-like structure, expressing root stem cell niche marker genes, being established in the developing organ. Previous studies reported that the gene regulatory network controlling lateral root formation is organized into two subnetworks whose mutual inhibition may contribute to organ patterning. PUCHI encodes an AP2/ERF transcription factor expressed early during lateral root primordium development and required for correct lateral root formation. To dissect the molecular events occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral root development in puchi-1 mutants and wild types. Transcriptomic and reporter analyses revealed that meristem-related genes were expressed ectopically at early stages of lateral root formation in puchi-1 mutants. We conclude that, consistent with the inhibition of genetic modules contributing to lateral root development, PUCHI represses ectopic establishment of meristematic cell identities at early stages of organ development. These findings shed light on gene network properties that orchestrate correct timing and patterning during lateral root formation. [ABSTRACT FROM AUTHOR]

Sedentary plant-parasitic nematodes, such as cyst and root-knot nematodes, induce feeding structures in the host root that undergo extensive changes in the gene expression. This phenomenon has previously been studied by gene chip analysis and qRT-PCR. Housekeeping genes are often used routinely as internal references for relative qRT-PCR analyses. However, due to the strong influence of nematode infection on host cell metabolism and physiology, expression of housekeeping genes may be altered considerably, thus limiting reliability of qRT-PCR analyses. Therefore, in the present work we tested UBQ10, ACT2, EF1a, UBP22 and 18S rRNA as potential candidates for relative qRT-PCR studies of gene expression in nematode infection sites in roots of Arabidopsis thaliana. Among the tested candidates only UBP22 and 18S rRNA were stably expressed and, therefore, are reliable reference genes for studying cyst and root-knot nematode infections. [ABSTRACT FROM AUTHOR]

Association of phenotypes or exposures with genomic and epigenomic data faces important statistical challenges. One of these challenges is to account for variation due to unobserved confounding factors, such as individual ancestry or cell-type composition in tissues. This issue can be addressed with penalized latent factor regression models, where penalties are introduced to cope with high dimension in the data. If a relatively small proportion of genomic or epigenomic markers correlate with the variable of interest, sparsity penalties may help to capture the relevant associations, but the improvement over non-sparse approaches has not been fully evaluated yet. Here, we present least-squares algorithms that jointly estimate effect sizes and confounding factors in sparse latent factor regression models. In simulated data, sparse latent factor regression models generally achieved higher statistical performance than other sparse methods, including the least absolute shrinkage and selection operator and a Bayesian sparse linear mixed model. In generative model simulations, statistical performance was slightly lower (while being comparable) to non-sparse methods, but in simulations based on empirical data, sparse latent factor regression models were more robust to departure from the model than the non-sparse approaches. We applied sparse latent factor regression models to a genome-wide association study of a flowering trait for the plant Arabidopsis thaliana and to an epigenome-wide association study of smoking status in pregnant women. For both applications, sparse latent factor regression models facilitated the estimation of non-null effect sizes while overcoming multiple testing issues. The results were not only consistent with previous discoveries, but they also pinpointed new genes with functional annotations relevant to each application. [ABSTRACT FROM AUTHOR]

Parasitic root-knot nematodes transform the host's vascular cells into permanent feeding giant cells (GCs) to withdraw nutrients from the host plants. GCs are multinucleated metabolically active cells with distinctive cell wall structures; however, the genetic regulation of GC formation is largely unknown. In this study, the functions of the Arabidopsis thaliana transcription factor PUCHI during GC development were investigated. PUCHI expression was shown to be induced in early developing galls, suggesting the importance of the PUCHI gene in gall formation. Despite the puchi mutant not differing significantly from the wild type in nematode invasion and reproduction rates, puchi GC cell walls appeared to be thicker and lobate when compared to the wild type, while the cell membrane sometimes formed invaginations. In three-dimensional (3D) reconstructions of puchi GCs, they appeared to be more irregularly shaped than those in the wild type, with noticeable cell-surface protrusions and folds. Interestingly, the loss-of-function mutant of 3-KETOACYL-COA SYNTHASE 1 showed GC morphology and cell wall defects similar to those of the puchi mutant, suggesting that PUCHI may regulate GC development via very long chain fatty acid synthesis. [ABSTRACT FROM AUTHOR]

SUMMARY: The butenolide molecule, karrikin (KAR), emerging in smoke of burned plant material, enhances light responses such as germination, inhibition of hypocotyl elongation, and anthocyanin accumulation in Arabidopsis. The KAR signaling pathway consists of KARRIKIN INSENSITIVE 2 (KAI2) and MORE AXILLARY GROWTH 2 (MAX2), which, upon activation, act in an SCF E3 ubiquitin ligase complex to target the downstream signaling components SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1‐LIKE 2 (SMXL2) for degradation. How degradation of SMAX1 and SMXL2 is translated into growth responses remains unknown. Although light clearly influences the activity of KAR, the molecular connection between the two pathways is still poorly understood. Here, we demonstrate that the KAR signaling pathway promotes the activity of a transcriptional module consisting of ELONGATED HYPOCOTYL 5 (HY5), B‐BOX DOMAIN PROTEIN 20 (BBX20), and BBX21. The bbx20 bbx21 mutant is largely insensitive to treatment with KAR2, similar to a hy5 mutant, with regards to inhibition of hypocotyl elongation and anthocyanin accumulation. Detailed analysis of higher order mutants in combination with RNA‐sequencing analysis revealed that anthocyanin accumulation downstream of SMAX1 and SMXL2 is fully dependent on the HY5‐BBX module. However, the promotion of hypocotyl elongation by SMAX1 and SMXL2 is, in contrast to KAR2 treatment, only partially dependent on BBX20, BBX21, and HY5. Taken together, these results suggest that light‐ and KAR‐dependent signaling intersect at the HY5‐BBX transcriptional module. Significance Statement: Although the core mechanism of karrikin signaling has been largely resolved, it has remained unclear how different growth responses to karrikins are carried out. Many of the responses to karrikin that are induced through the KAI2‐MAX2‐dependent degradation of SMAX1 and SMXL2 relate to light signaling. Here, we show that these two pathways intersect at the HY5‐BBX transcriptional module downstream of SMAX1 and SMXL2 to regulate gene expression, hypocotyl elongation, and accumulation of anthocyanins. [ABSTRACT FROM AUTHOR]

The biogenesis of root-knot nematode (Meloidogyne spp.)-induced galls requires the hyperactivation of the cell cycle with controlled balance of mitotic and endocycle programs to keep its homeostasis. To better understand gall functioning and to develop new control strategies for this pest, it is essential to find out how the plant host cell cycle programs are responding and integrated during the nematode-induced gall formation. This work investigated the spatial localization of a number of gene transcripts involved in the pre-replication complex during DNA replication in galls and report their akin colocation with the cell cycle S-phase regulator Armadillo BTB Arabidopsis Protein 1 (ABAP1). ABAP1 is a negative regulator of pre-replication complex controlling DNA replication of genes involved in control of cell division and proliferation; therefore, its function has been investigated during gall ontogenesis. Functional analysis was performed upon ABAP1 knockdown and overexpression in Arabidopsis thaliana. We detected ABAP1 promoter activity and localized ABAP1 protein in galls during development, and its overexpression displayed significantly reduced gall sizes containing atypical giant cells. Profuse ABAP1 expression also impaired gall induction and hindered nematode reproduction. Remarkably, ABAP1 knockdown likewise negatively affected gall and nematode development, suggesting its involvement in the feeding site homeostasis. Microscopy analysis of cleared and nuclei-stained whole galls revealed that ABAP1 accumulation resulted in aberrant giant cells displaying interconnected nuclei filled with enlarged heterochromatic regions. Also, imbalanced ABAP1 expression caused changes in expression patterns of genes involved in the cell division control as demonstrated by qRT-PCR. CDT1a , CDT1b , CDKA;1 , and CYCB1;1 mRNA levels were significantly increased in galls upon ABAP1 overexpression, possibly contributing to the structural changes in galls during nematode infection. Overall, data obtained in galls reinforced the role of ABAP1 controlling DNA replication and mitosis and, consequently, cell proliferation. ABAP1 expression might likely take part of a highly ordered mechanism balancing of cell cycle control to prevent gall expansion. ABAP1 expression might prevent galls to further expand, limiting excessive mitotic activity. Our data strongly suggest that ABAP1 as a unique plant gene is an essential component for cell cycle regulation throughout gall development during nematode infection and is required for feeding site homeostasis. [ABSTRACT FROM AUTHOR]

Summary: The root‐knot nematode Meloidogyne incognita secretes specific effectors (MiEFF) and induces the redifferentiation of plant root cells into enlarged multinucleate feeding 'giant cells' essential for nematode development.Immunolocalizations revealed the presence of the MiEFF18 protein in the salivary glands of M. incognita juveniles. In planta, MiEFF18 localizes to the nuclei of giant cells demonstrating its secretion during plant–nematode interactions. A yeast two‐hybrid approach identified the nuclear ribonucleoprotein SmD1 as a MiEFF18 partner in tomato and Arabidopsis. SmD1 is an essential component of the spliceosome, a complex involved in pre‐mRNA splicing and alternative splicing.RNA‐seq analyses of Arabidopsis roots ectopically expressing MiEFF18 or partially impaired in SmD1 function (smd1b mutant) revealed the contribution of the effector and its target to alternative splicing and proteome diversity. The comparison with Arabidopsis galls data showed that MiEFF18 modifies the expression of genes important for giant cell ontogenesis, indicating that MiEFF18 modulates SmD1 functions to facilitate giant cell formation.Finally, Arabidopsis smd1b mutants exhibited less susceptibility to M. incognita infection, and the giant cells formed on these mutants displayed developmental defects, suggesting that SmD1 plays an important role in the formation of giant cells and is required for successful nematode infection. [ABSTRACT FROM AUTHOR]

The root stem cell niche (SCN) of Arabidopsis thaliana consists of the quiescent center (QC) cells and the surrounding initial stem cells that produce progeny to replenish all the tissues of the root. The QC cells divide rather slowly relative to the initials, yet most root tissues can be formed from these cells, depending on the requirements of the plant. Hormones are fundamental cues that link such needs with the cell proliferation and differentiation dynamics at the root SCN. Nonetheless, the crosstalk between hormone signaling and the mechanisms that regulate developmental adjustments is still not fully understood. Developmental transcriptional regulatory networks modulate hormone biosynthesis, metabolism, and signaling, and conversely, hormonal responses can affect the expression of transcription factors involved in the spatiotemporal patterning at the root SCN. Hence, a complex genetic–hormonal regulatory network underlies root patterning, growth, and plasticity in response to changing environmental conditions. In this review, we summarize the scientific literature regarding the role of hormones in the regulation of QC cell proliferation and discuss how hormonal signaling pathways may be integrated with the gene regulatory network that underlies cell fate in the root SCN. The conceptual framework we present aims to contribute to the understanding of the mechanisms by which hormonal pathways act as integrators of environmental cues to impact on SCN activity. [ABSTRACT FROM AUTHOR]

Under environmental stress, plants are believed to actively repress their growth to save resource and alter its allocation to acquire tolerance against the stress. Although a lot of studies have uncovered precise mechanisms for responding to stress and acquiring tolerance, the mechanisms for regulating growth repression under stress are not as well understood. It is especially unclear which particular genes related to cell cycle control are involved in active growth repression. Here, we showed that decreased growth in plants exposed to moderate salt stress is mediated by MYB3R transcription factors that have been known to positively and negatively regulate the transcription of G2/M-specific genes. Our genome-wide gene expression analysis revealed occurrences of general downregulation of G2/M-specific genes in Arabidopsis under salt stress. Importantly, this downregulation is significantly and universally mitigated by the loss of MYB3R repressors by mutations. Accordingly, the growth performance of Arabidopsis plants under salt stress is significantly recovered in mutants lacking MYB3R repressors. This growth recovery involves improved cell proliferation that is possibly due to prolonging and accelerating cell proliferation, which were partly suggested by enlarged root meristem and increased number of cells positive for CYCB1;1-GUS. Our ploidy analysis further suggested that cell cycle progression at the G2 phase was delayed under salt stress, and this delay was recovered by loss of MYB3R repressors. Under salt stress, the changes in expression of MYB3R activators and repressors at both the mRNA and protein levels were not significant. This observation suggests novel mechanisms underlying MYB3R-mediated growth repression under salt stress that are different from the mechanisms operating under other stress conditions such as DNA damage and high temperature. [ABSTRACT FROM AUTHOR]

The correct development of a diploid sporophyte body and a haploid gametophyte relies on a strict coordination between cell divisions in space and time. During plant reproduction, these divisions have to be temporally and spatially coordinated with cell differentiation processes, to ensure a successful fertilization. Armadillo BTB Arabidopsis protein 1 (ABAP1) is a plant exclusive protein that has been previously reported to control proliferative cell divisions during leaf growth in Arabidopsis. Here, we show that ABAP1 binds to different transcription factors that regulate male and female gametophyte differentiation, repressing their target genes expression. During male gametogenesis, the ABAP1-TCP16 complex represses CDT1b transcription, and consequently regulates microspore first asymmetric mitosis. In the female gametogenesis, the ABAP1-ADAP complex represses EDA24-like transcription, regulating polar nuclei fusion to form the central cell. Therefore, besides its function during vegetative development, this work shows that ABAP1 is also involved in differentiation processes during plant reproduction, by having a dual role in regulating both the first asymmetric cell division of male gametophyte and the cell differentiation (or cell fusion) of female gametophyte. [ABSTRACT FROM AUTHOR]

Summary: Large amounts of effectors are secreted by the oesophageal glands of plant‐parasitic nematodes, but their molecular mode of action remains largely unknown. We characterized a Meloidogyne incognita protein disulphide isomerase (PDI)‐like effector protein (MiPDI1) that facilitates nematode parasitism.In situ hybridization showed that MiPDI1 was expressed specifically in the subventral glands of M. incognita. It was significantly upregulated during parasitic stages. Immunolocalization demonstrated MiPDI1 secretion in planta during nematode migration and within the feeding cells. Host‐induced silencing of the MiPDI1 gene affected the ability of the nematode to infect the host, whereas MiPDI1 expression in Arabidopsis increased susceptibility to M. incognita, providing evidence for a key role of MiPDI1 in M. incognita parasitism.Yeast two‐hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assays showed that MiPDI1 interacted with a tomato stress‐associated protein (SlSAP12) orthologous to the redox‐regulated AtSAP12, which plays an important role in plant responses to abiotic and biotic stresses. SAP12 silencing or knocking out in Nicotiana benthamiana and Arabidopsis increased susceptibility to M. incognita.Our results suggest that MiPDI1 acts as a pathogenicity factor promoting disease by fine‐tuning SAP‐mediated responses at the interface of redox signalling, defence and stress acclimation in Solanaceae and Arabidopsis. [ABSTRACT FROM AUTHOR]

Copper deficiency reduces plant growth, male fertility, and seed set. The contribution of copper to female fertility and the underlying molecular aspects of copper deficiency‐caused phenotypes are not well known. We show that among copper deficiency‐caused defects in Arabidopsis thaliana were also the increased shoot branching, delayed flowering and senescence, and entirely abolished gynoecium fertility. The increased shoot branching of copper‐deficient plants was rescued by the exogenous application of auxin or copper. The delayed flowering was associated with the decreased expression of the floral activator, FT. Copper deficiency also decreased the expression of senescence‐associated genes, WRKY53 and SAG13, but increased the expression of SAG12. The reduced fertility of copper‐deficient plants stemmed from multiple factors including the abnormal stigma papillae development, the abolished gynoecium fertility, and the failure of anthers to dehisce. The latter defect was associated with reduced lignification, the upregulation of copper microRNAs and the downregulation of their targets, laccases, implicated in lignin synthesis. Copper‐deficient plants accumulated ROS in pollen and had reduced cytochrome c oxidase activity in both leaves and floral buds. This study opens new avenues for the investigation into the relationship between copper homeostasis, hormone‐mediated shoot architecture, gynoecium fertility, and copper deficiency‐derived nutritional signals leading to the delay in flowering and senescence. [ABSTRACT FROM AUTHOR]

Cell division is precisely regulated and highly tissue‐specific; studies have suggested that diverse signals in the epidermis, especially the epidermal brassinosteroids (BRs), can regulate root growth. However, the underlying molecular mechanisms that integrate hormonal cues such as BR signaling with other endogenous, tissue‐specific developmental programs to regulate epidermal cell proliferation remain unclear. In this study, we used molecular and biochemical approaches, microscopic imaging and genetic analysis to investigate the function and mechanisms of a P‐type cyclin in root growth regulation. We found that CYCP3;1, specifically expressed in the root meristem epidermis and lateral root cap, can regulate meristem cell division. Mitotic analyses and biochemical studies demonstrated that CYCP3;1 promotes cell division at the G2‐M duration by associating and activating cyclin‐dependent kinase B2‐1 (CDKB2;1). Furthermore, we found that CYCP3;1 expression was inhibited by BR signaling through BRI1‐EMS‐SUPPRESSOR1 (BES1), a positive downstream transcription factor in the BR signaling pathway. These findings not only provide a mechanism of how root epidermal‐specific regulators modulate root growth, but also reveal why the excess of BRs or enhanced BR signaling inhibits cell division in the meristem to negatively regulate root growth. [ABSTRACT FROM AUTHOR]

Intracellular levels of Mg2+ are tightly regulated, as Mg2+ deficiency or excess affects normal plant growth and development. In Arabidopsis, we determined that phospholipase Dα1 (PLDα1) is involved in the stress response to high‐magnesium conditions. The T‐DNA insertion mutant pldα1 is hypersensitive to increased concentrations of magnesium, exhibiting reduced primary root length and fresh weight. PLDα1 activity increases rapidly after high‐Mg2+ treatment, and this increase was found to be dose dependent. Two lines harbouring mutations in the HKD motif, which is essential for PLDα1 activity, displayed the same high‐Mg2+ hypersensitivity of pldα1 plants. Moreover, we show that high concentrations of Mg2+ disrupt K+ homeostasis, and that transcription of K+ homeostasis‐related genes CIPK9 and HAK5 is impaired in pldα1. Additionally, we found that the akt1, hak5 double mutant is hypersensitive to high‐Mg2+. We conclude that in Arabidopsis, the enzyme activity of PLDα1 is vital in the response to high‐Mg2+ conditions, and that PLDα1 mediates this response partially through regulation of K+ homeostasis. Intracellular levels of Mg2+ are tightly regulated. The present study shows that in Arabidopsis, the enzyme activity of phospholipase Dα1 (PLDα1) is vital in the response to high‐Mg2+ conditions, and that PLDα1 mediates this response partially through regulation of K+ homeostasis. [ABSTRACT FROM AUTHOR]

Summary: The use of Arabidopsis thaliana as a model plant increased the rate of molecular discoveries of plant-pathogen interactions. Although Meloidogyne graminicola has a relatively broad host range, it is not known whether it can infect A. thaliana. In this study, we showed that M. graminicola is able to invade A. thaliana and complete its life cycle 12-14 days after invasion. No significant difference in the total number of nematodes inside roots of A. thaliana and rice, Oryza sativa , was found at 14 day after inoculation (dai). Significantly more galls were formed in A. thaliana roots compared to the numbers in O. sativa roots at 14 dai. Females laid egg masses on the A. thaliana root surface and a large number of hatched juveniles of the next generation were obtained from infected A. thaliana roots. In addition, the infection of M. graminicola can induce expression of A. thaliana basal defence genes, such as AtMYB51 , AtWRKY11 , AtPR1 and AtFRK1 , at 24 h after inoculation. Therefore, A. thaliana can be considered as a suitable host to study host- M. graminicola interactions and to understand the molecular mechanisms developed by M. graminicola to infect its dicotyledonous host plants. In addition, our results also showed that a delayed development of M. graminicola occurred in A. thaliana compared to O. sativa , and a higher proportion of empty galls appeared in A. thaliana roots than in O. sativa roots, suggesting A. thaliana is a less optimal host than rice. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a second messenger that regulates a broad range of physiological processes in plants. NO-derived molecules called reactive nitrogen species (RNS) can react with unsaturated fatty acids generating nitrated fatty acids (NO2-FA). NO2-FA work as signaling molecules in mammals where production and targets have been described under different stress conditions. Recently, NO2-FAs were detected in plants, however their role(s) on plant physiological processes is still poorly known. Although in this work NO2-OA has not been detected in any Arabidopsis seedling tissue, here we show that exogenous application of nitro-oleic acid (NO2-OA) inhibits Arabidopsis primary root growth; this inhibition is not likely due to nitric oxide (NO) production or impaired auxin or cytokinin root responses. Deep analyses showed that roots incubated with NO2-OA had a lower cell number in the division area. Although this NO2-FA did not affect the hormonal signaling mechanisms maintaining the stem cell niche, plants incubated with NO2-OA showed a reduction of cell division in the meristematic area. Therefore, this work shows that the exogenous application of NO2-OA inhibits mitotic processes subsequently reducing primary root growth. [ABSTRACT FROM AUTHOR]

Summary: We investigated the interaction between osmotic stress and auxin signaling in leaf growth regulation. Therefore, we grew Arabidopsis thaliana seedlings on agar media supplemented with mannitol to impose osmotic stress and 1‐naphthaleneacetic acid (NAA), a synthetic auxin.We performed kinematic analysis and flow‐cytometry to quantify the effects on cell division and expansion in the first leaf pair, determined the effects on auxin homeostasis and response (DR5::β‐glucuronidase), performed a next‐generation sequencing transcriptome analysis and investigated the response of auxin‐related mutants.Mannitol inhibited cell division and expansion. NAA increased the effect of mannitol on cell division, but ameliorated its effect on expansion. In proliferating cells, NAA and mannitol increased free IAA concentrations at the cost of conjugated IAA and stimulated DR5 promotor activity. Transcriptome analysis shows a large overlap between NAA and osmotic stress‐induced changes, including upregulation of auxin synthesis, conjugation, transport and TRANSPORT INHIBITOR RESPONSE1 (TIR1) and AUXIN RESPONSE FACTOR (ARF) response genes, but downregulation of Aux/IAA response inhibitors. Consistently, arf7/19 double mutant lack the growth response to auxin and show a significantly reduced sensitivity to osmotic stress.Our results show that osmotic stress inhibits cell division during leaf growth of A. thaliana at least partly by inducing the auxin transcriptional response. [ABSTRACT FROM AUTHOR]

The development of lateral roots in Arabidopsis thaliana is strongly dependent on signaling directed by the AUXIN RESPONSE FACTOR7 (ARF7), which in turn activates LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors (LBD16, LBD18 and LBD29). Here, the product of PRH1, a PR-1 homolog annotated previously as encoding a pathogen-responsive protein, was identified as a target of ARF7-mediated auxin signaling and also as participating in the development of lateral roots. PRH1 was shown to be strongly induced by auxin treatment, and plants lacking a functional copy of PRH1 formed fewer lateral roots. The transcription of PRH1 was controlled by the binding of both ARF7 and LBDs to its promoter region. Author summary: In Arabidopsis thaliana AUXIN RESPONSE FACTOR7 (ARF7)-mediated auxin signaling plays a key role in lateral roots (LRs) development. The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors (LBD16, LBD18 and LBD29) act downstream of ARF7-mediated auxin signaling to control LRs formation. Here, the PR-1 homolog PRH1 was identified as a novel target of both ARF7 and LBDs (especially the LBD29) during auxin induced LRs formation, as both ARF7 and LBDs were able to bind to the PRH1 promoter. This study provides new insights about how auxin regulates lateral root development. [ABSTRACT FROM AUTHOR]

Summary: Leaf growth is a complex trait for which many similarities exist in different plant species, suggesting functional conservation of the underlying pathways. However, a global view of orthologous genes involved in leaf growth showing conserved expression in dicots and monocots is currently missing. Here, we present a genome‐wide comparative transcriptome analysis between Arabidopsis and maize, identifying conserved biological processes and gene functions active during leaf growth. Despite the orthology complexity between these distantly related plants, 926 orthologous gene groups including 2829 Arabidopsis and 2974 maize genes with similar expression during leaf growth were found, indicating conservation of the underlying molecular networks. We found 65% of these genes to be involved in one‐to‐one orthology, whereas only 28.7% of the groups with divergent expression had one‐to‐one orthology. Within the pool of genes with conserved expression, 19 transcription factor families were identified, demonstrating expression conservation of regulators active during leaf growth. Additionally, 25 Arabidopsis and 25 maize putative targets of the TCP transcription factors with conserved expression were determined based on the presence of enriched transcription factor binding sites. Based on large‐scale phenotypic data, we observed that genes with conserved expression have a higher probability to be involved in leaf growth and that leaf‐related phenotypes are more frequently present for genes having orthologues between dicots and monocots than clade‐specific genes. This study shows the power of integrating transcriptomic with orthology data to identify or select candidates for functional studies during leaf development in flowering plants. [ABSTRACT FROM AUTHOR]

PIN-FORMED (PIN) auxin efflux carriers with a long central hydrophilic loop (long PINs) have been implicated in organogenesis. However, the role of short hydrophilic loop PINs (short PINs) in organogenesis is largely unknown. In this study, we investigated the role of a short PIN, PIN8, in lateral root (LR) development in Arabidopsis thaliana. The loss-of-function mutation in PIN8 significantly decreased LR density, mostly by affecting the emergence stage. PIN8 showed a sporadic expression pattern along the root vascular cells in the phloem, where the PIN8 protein predominantly localized to intracellular compartments. During LR primordium development, PIN8 was expressed at the late stage. Plasma membrane (PM)-localized long PINs suppressed LR formation when expressed in the PIN8 domain. Conversely, an auxin influx carrier, AUX1, restored the wild-type (WT) LR density when expressed in the PIN8 domain of the pin8 mutant root. Moreover, LR emergence was considerably inhibited when AXR2-1, the dominant negative form of Aux/IAA7, compromised auxin signaling in the PIN8 domain. Consistent with these observations, the expression of many genes implicated in late LR development was suppressed in the pin8 mutant compared with the WT. Our results suggest that the intracellularly localized PIN8 affects LR development most likely by modulating intracellular auxin translocation. Thus, the function of PIN8 is distinctive from that of PM-localized long PINs, where they generate local auxin gradients for organogenesis by conducting cell-to-cell auxin reflux. [ABSTRACT FROM AUTHOR]

Summary: Galls induced by plant‐parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident.We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode‐induced galls.Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1‐knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls.Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1‐knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root‐knot nematodes. Together with data on DNA‐damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls. [ABSTRACT FROM AUTHOR]

Key message: Agrobacterium-mediated transient overexpression of E2Fb triggers new cell divisions in pavement cells of Nicotiana benthamiana leaves. Transient transformation in Nicotiana benthamiana enables the study of multiple biological processes in a simple and fast manner. Here, we describe that, upon A. tumefaciens-mediated transient overexpression of the cell cycle regulator E2Fb from either Arabidopsis thaliana or N. benthamiana, cell divisions occur in epidermal pavement cells in N. benthamiana leaves, following a sequence of events that encompasses the nucleus taking a central position and being surrounded by chloroplasts, nuclear division, and formation of a new wall that divides the initial cell in two. Our results indicate that transient expression in N. benthamiana can be used to study cell division in plants, from DNA replication to cell wall formation, in a simple, controlled, and rapid manner. [ABSTRACT FROM AUTHOR]

Many Arabidopsis thaliana genes have been reported to affect plant cell size by regulating the level of endoreduplication, which is a modified cell cycle. However, the role of endoreduplication on the altered cell size in these reports must be reconsidered based on a number of findings. First, not all plant species exhibit endoreduplication, which indicates that endoreduplication-driven cell size regulation is not universal among plants. Second, while ploidy level and cell size are correlated in the epidermal pavement cells of Arabidopsis leaves, the size of mesophyll cells appears to be comparatively uniform regardless of whether there is heterogeneity in the ploidy level. Third, changes in the cell sizes reported in mutant and transgenic Arabidopsis seem to be too large to be solely the result of altered endoreduplication level. Fourth, compensated cell enlargement, which is triggered by a severe decrease in cell proliferation in Arabidopsis leaves, is usually independent of altered endoreduplication. We re-examined the role of endoreduplication on cell-size regulation in Arabidopsis, mainly in leaves, and revealed biases in the previous studies. This paper provides an overview of the work carried out in the past decade, and presents rationale to correct the previous assumptions. Based on the considerations provided in this report, a re-examination of previous reports regarding the roles of mutations and/or transgenes in the regulation of cell size is recommended. [ABSTRACT FROM AUTHOR]

Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell–cell junctions. [ABSTRACT FROM AUTHOR]

Main conclusion: Gymnosperms respond differently to light intensity and R:FR; although some aspects of shade response appear conserved, yet underlying mechanisms seem to be diverse in gymnosperms as compared to angiosperms. Shade avoidance syndrome (SAS) is well-characterized in the shade intolerant model species Arabidopsis thaliana whereas much less is known about shade tolerance response (STR), yet regulation of SAS and STR with reference to conifers remains poorly understood. We conducted a comparative study of two conifer species with contrasting responses to shade, Scots pine (shade-intolerant) and Norway spruce (shade-tolerant), with the aim to understand mechanisms behind SAS and STR in conifers. Pine and spruce seedlings were grown under controlled light and shade conditions, and hypocotyl and seedling elongation following different light treatments were determined in both species as indicators of shade responses. Red to far-red light ratio (R:FR) was shown to trigger the shade response in Norway spruce. In Scots pine, we observed an interaction between R:FR and light intensity. RNA sequencing (RNA-Seq) data revealed that SAS and STR responses included changes in expression of genes involved primarily in hormone signalling and pigment biosynthesis. From the RNA-Seq analysis, we propose that although some aspects of shade response appear to be conserved in angiosperms and gymnosperms, yet the underlying mechanisms may be different in gymnosperms that warrants further research. [ABSTRACT FROM AUTHOR]

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting. [ABSTRACT FROM AUTHOR]

Summary: Plasmodiophora brassicae is a soil‐borne biotroph whose life cycle involves reprogramming host developmental processes leading to the formation of galls on its underground parts. Formation of such structures involves modification of the host cell cycle leading initially to hyperplasia, increasing the number of cells to be invaded, followed by overgrowth of cells colonised by the pathogen. Here we show that P. brassicae infection stimulates formation of the E2Fa/RBR1 complex and upregulation of MYB3R1,MYB3R4 and A‐ and B‐type cyclin expression. These factors were previously described as important regulators of the G2−M cell cycle checkpoint. As a consequence of this manipulation, a large population of host hypocotyl cells are delayed in cell cycle exit and maintained in the proliferative state. We also report that, during further maturation of galls, enlargement of host cells invaded by the pathogen involves endoreduplication leading to increased ploidy levels. This study characterises two aspects of the cell cycle reprogramming efforts of P. brassicae: systemic, related to the disturbance of host hypocotyl developmental programs by preventing cell cycle exit; and local, related to the stimulation of cell enlargement via increased endocycle activity. Significance Statement: We describe the changes in host cell cycle gene expression in response to P. brassicae infection, and characterise the impact of key cell cycle regulators (MYB3R4, E2Fa, CCS52A1), establishing that cell proliferation during gall development stems from repression of cell cycle exit, whereas hypertrophy in later disease stages depends on stimulation of endoreduplication. This provides a molecular framework for understanding the progress of clubroot disease and gall formation. [ABSTRACT FROM AUTHOR]

Summary: Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development.We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle.Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large‐leaf mutants.Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general. [ABSTRACT FROM AUTHOR]

Stomata have a critical function in the exchange of gases and water vapor between plants and their environment. Stomatal development is under the rigorous control of many regulators. The last step of development is the terminal division of guard mother cells (GMC) into two guard cells (GC). It is still unclear how the symmetric division of GMCs is regulated. Here, we show that the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) is required for the symmetric division of GMCs into GCs in Arabidopsis. Exogenous application of the ACC biosynthesis inhibitor aminoethoxyvinylglycine (AVG) induced the formation of single guard cells (SGCs). Correspondingly, an acs octuple-mutant with extremely low endogenous ACC also developed SGCs, and exogenous ACC dramatically decreased the number of SGCs in this mutant whereas exogenous ethephon (which is gradually converted into ethylene) had no effect. Furthermore, neither blocking of endogenous ethylene synthesis nor disruption of ethylene signaling transduction could induce the production of SGCs. Further investigation indicated that ACC promoted the division of GMCs in fama-1 and flp-1myb88 mutants whereas AVG inhibited it. Moreover, ACC positively regulated the expression of CDKB1;1 and CYCA2;3 in the fama-1 and flp-1myb88 mutants. The SGC number was not affected by ACC or AVG in cdkb1;11;2 and cyca2;234 mutants. Taken together, the results demonstrate that ACC itself, but not ethylene, positively modulates the symmetric division of GMCs in a manner that is dependent on CDKB1s and CYCA2s. [ABSTRACT FROM AUTHOR]

Summary: Root‐knot nematodes transform vascular host cells into permanent feeding structures to selectively withdraw their nutrients from host plants during the course of several weeks. The susceptibility of host plants to root‐knot nematode infections is thought to be a complex trait involving many genetic loci. However, genome‐wide association (GWA) analysis has so far revealed only four quantitative trait loci (QTLs) linked to the reproductive success of the root‐knot nematode Meloidogyne incognita in Arabidopsis thaliana, which suggests that the genetic architecture underlying host susceptibility could be much simpler than previously thought. Here, we report that, by using a relaxed stringency approach in a GWA analysis, we could identify 15 additional loci linked to quantitative variation in the reproductive success of M. incognita in Arabidopsis. To test the robustness of our analysis, we functionally characterized six genes located in a QTL with the lowest acceptable statistical support and smallest effect size. This led us to identify ETHYLENE RESPONSE FACTOR 6 (ERF6) as a novel susceptibility gene for M. incognita in Arabidopsis. ERF6 functions as a transcriptional activator and suppressor of genes in response to various abiotic stresses independent of ethylene signalling. However, whole‐transcriptome analysis of nematode‐infected roots of the Arabidopsis erf6‐1 knockout mutant line showed that allelic variation at this locus may regulate the conversion of aminocyclopropane‐1‐carboxylate (ACC) into ethylene by altering the expression of 1‐aminocyclopropane‐1‐carboxylate oxidase 3 (ACO3). Our data further suggest that tolerance to abiotic stress mediated by ERF6 forms a novel layer of control in the susceptibility of Arabidopsis to M. incognita. [ABSTRACT FROM AUTHOR]

Metal trace elements, such as Fe, Zn, and Mn, are necessary micronutrients required by all plants. In this study, the MxNAS3 gene was cloned from Malus xiaojinensis and MxNAS3 was localized in the cytoplasmic membrane. The expression level of MxNAS3 in root and new leaf was higher than in mature leaf and phloem, which was greatly influenced by high and low Fe stresses, IAA and ABA treatments in M. xiaojinensis. Over-expression of MxNAS3 in transgenic Arabidopsis thaliana contributed to enhanced Fe stress tolerance, as well as higher levels of root length, fresh weight, concentrations of chlorophyll, nicotianamine, Fe, Zn, and Mn, especially under high and low Fe stresses. More importantly, it was the first time for us to find that higher expression of MxNAS3 in transgenic A. thaliana contributed to misshappen flowers. Moreover, the MxNAS5-OE A. thaliana had increased expression levels of flowering-related genes (AtYSL1, AtYSL3, AtAFDL, AtAP1, ATMYB21, and AtSAP). [ABSTRACT FROM AUTHOR]

A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips. [ABSTRACT FROM AUTHOR]

SUMMARY: Root‐knot nematodes (RKNs, Meloidogyne spp.) are sedentary biotrophic pathogens that establish within the vascular cylinder of plant roots, forming a gall and inducing several feeding cells, giant cells (GCs), essential for completion of their life cycle. GCs suffer gene expression changes, repeated mitosis and endoreduplication events. Transcriptomics has revealed that an extensive down‐regulation of transcripts, a molecular signature of early‐developing galls and GCs that is conserved in tomato and Arabidopsis, may be achieved through small RNA (sRNA) gene silencing pathways. The role of some microRNAs (miRNAs) in plant–RKN interactions has recently been addressed, but little is known about the regulatory roles of other sRNA types. Here, we perform a differential accumulation analysis to show which repeat‐associated small interfering RNAs (rasiRNAs) are distinctive or enriched in early Arabidopsis galls vs. uninfected roots. Those distinctive from galls are preferentially located in pericentromeric regions with predominant sizes of 24 and 22 nucleotides. Gall‐distinctive rasiRNAs target primarily GYPSY and COPIA retrotransposons, which show a marked repression in galls vs. uninfected roots. Infection tests and phenotypic studies of galls from Meloidogyne javanica in Arabidopsis mutants impaired in post‐transcriptional gene silencing and/or canonical RNA‐directed DNA methylation (RdDM) pathways, as well as quantitative polymerase chain reaction analysis, suggest the implication of canonical and non‐canonical RdDM pathways during gall formation, possibly through the regulation of retrotransposons. This process may be crucial for the maintenance of genome integrity during the reprogramming process of galls/GCs from their vascular precursor cells, and/or to ensure a faithful DNA replication during the repeated mitosis/endoreduplication that concurs with feeding site formation. [ABSTRACT FROM AUTHOR]

In the post-embryonic stage of Arabidopsis thaliana, roots can be initiated from the vascular region of the existing roots or non-root organs; they are designated as lateral roots (LRs) and adventitious roots (ARs), respectively. Some root-like organs can also be initiated from the vasculature. In tissue culture, auxin-induced callus, which is a group of pluripotent root-primordium-like cells, is formed via the rooting pathway. The formation of feeding structures from the vasculature induced by root-knot nematodes also borrows the rooting pathway. In this review, we summarize and discuss recent progress on the role of LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16; also known as ASYMMETRIC LEAVES2-LIKE18, ASL18), a member of the LBD/ASL gene family encoding plant-specific transcription factors, in roots and root-like organ initiation. Different root and root-like organ initiation processes have distinct priming mechanisms to specify founder cells. All these priming mechanisms converge to activate LBD16 expression in the primed founder cells. The activation of LBD16 expression leads to organ initiation via promotion of cell division and establishment of root-primordium identity. Therefore, LBD16 might play a common and pivotal role in root and root-like organ initiation. [ABSTRACT FROM AUTHOR]

Summary: Leaves are essential organs for plants. We previously identified a functional gene possibly encoding a component of the SWI/SNF complex named Leaf and Flower Related (LFR) in Arabidopsis thaliana. Loss‐of‐function mutants of LFR displayed obvious defects in leaf morphogenesis, indicating its vital role in leaf development. Here an allelic null mutant of ASYMMETRIC LEAVES2 (AS2), as2‐6, was isolated as an enhancer of lfr‐1 in petiole length, vasculature pattern and leaf margin development. The lfr as2 double‐mutants showed enhanced ectopic expression of BREVIPEDICELLUS (BP) compared with each of the single‐mutants, which is consistent with their synergistic genetic enhancement in multiple BP‐dependent development processes. Moreover, LFR and several putative subunits of the SWI/SNF complex interacted physically with AS2. LFR associated with BP chromatin in an AS1–AS2‐dependent manner to promote the nucleosome occupancy for appropriate BP repression in leaves. Taken together, our findings reveal that LFR and the SWI/SNF complex play roles in leaf development at least partly by repressing BP transcription as interacting factors of AS2, which expounds our understanding of BP repression at the chromatin structure level in leaf development. [ABSTRACT FROM AUTHOR]

Tetranychus urticae (two-spotted spider mite) is a striking example of polyphagy among herbivores with an extreme record of pesticide resistance and one of the most significant pests in agriculture. The T. urticae genome contains a large number of cysteine- and serine-proteases indicating their importance in the spider mite physiology. This work is focused on the potential role of the Kunitz trypsin inhibitor (KTI) family on plant defense responses against spider mites. The molecular characterization of two of these genes, AtKTI4 and AtKTI5, combined with feeding bioassays using T-DNA insertion lines for both genes was carried out. Spider mite performance assays showed that independent KTI silencing Arabidopsis lines conferred higher susceptibility to T. urticae than WT plants. Additionally, transient overexpression of these inhibitors in Nicotiana benthamiana demonstrated their ability to inhibit not only serine- but also cysteine-proteases, indicating the bifunctional inhibitory role against both types of enzymes. These inhibitory properties could be involved in the modulation of the proteases that participate in the hydrolysis of dietary proteins in the spider mite gut, as well as in other proteolytic processes. [ABSTRACT FROM AUTHOR]