Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems

RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Correspondingly, plant viruses encode suppressors of silencing. In the positive-strand RNA virus Tobacco rattle virus (TRV), the suppressor of silencing is a 16-kDa (16K) protein encoded by RNA1. The suppressor action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by tomato bushy stunt virus. Mutant TRV that does not produce its suppressor, unlike other suppressor-defective viruses, is competent to accumulate and spread systemically in the infected plant. However, this mutant virus does not exhibit the transient invasion of the meristem that is characteristic of the wild-type virus. Based on this analysis, we propose that the 16K suppressor of silencing allows TRV to transiently invade the meristem. Our data are consistent with a mechanism of long-term meristem virus exclusion that is dependent on a transient invasion of the meristem early in the infection cycle. This novel mechanism of meristem exclusion may be associated with the phenomenon of recovery in virus-infected plants in which upper leaves have little or no virus and are immune to secondary infection by the same virus.     

ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression

Auxin and cytokinin direct cell proliferation and differentiation during the in vitro culture of plant cells, but the molecular basis of these processes, especially de novo shoot regeneration, has not been fully elucidated. Here, we describe the regulatory control of shoot regeneration in Arabidopsis thaliana (L.) Heynh, based on the interaction of ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and WUSCHEL (WUS). The major site of ARR12 expression coincided with the location where the shoot apical meristem (SAM) initiated. The arr12 mutants showed severely impaired shoot regeneration and reduced responsiveness to cytokinin; consistent with this, the overexpression of ARR12 enhanced shoot regeneration. Certain shoot meristem specification genes, notably WUSCHEL (WUS) and CLAVATA3, were significantly downregulated in the arr12 explants. Chromatin immunoprecipitation (ChIP) and transient activation assays demonstrated that ARR12 binds to the promoter of WUS. These observations indicate that during shoot regeneration, in vitro, ARR12 functions as a molecular link between cytokinin signaling and the expression of shoot meristem specification genes.     

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca2+ as a secondary cytosolic messenger

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non‐cell‐autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca²⁺ elevations, cyclic nucleotide (cGMP)‐activated Ca²⁺ channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca²⁺ elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca²⁺ and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP‐activated Ca²⁺ channel. In wild‐type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca²⁺ channel blocker or a guanylyl cyclase inhibitor. When CLV3‐dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca²⁺ channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca²⁺, and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.     

A viral RNA silencing suppressor interferes with abscisic acid‐mediated signalling and induces drought tolerance in Arabidopsis thaliana

Cucumber mosaic virus (CMV) encodes the 2b protein, which plays a role in local and systemic virus movement, symptom induction and suppression of RNA silencing. It also disrupts signalling regulated by salicylic acid and jasmonic acid. CMV induced an increase in tolerance to drought in Arabidopsis thaliana. This was caused by the 2b protein, as transgenic plants expressing this viral factor showed increased drought tolerance, but plants infected with CMVΔ2b, a viral mutant lacking the 2b gene, did not. The silencing effector ARGONAUTE1 (AGO1) controls a microRNA‐mediated drought tolerance mechanism and, in this study, we noted that plants (dcl2/3/4 triple mutants) lacking functional short‐interfering RNA‐mediated silencing were also drought tolerant. However, drought tolerance engendered by CMV may be independent of the silencing suppressor activity of the 2b protein. Although CMV infection did not alter the accumulation of the drought response hormone abscisic acid (ABA), 2b‐transgenic and ago1‐mutant seeds were hypersensitive to ABA‐mediated inhibition of germination. However, the induction of ABA‐regulated genes in 2b‐transgenic and CMV‐infected plants was inhibited more strongly than in ago1‐mutant plants. The virus engenders drought tolerance by altering the characteristics of the roots and not of the aerial tissues as, compared with the leaves of silencing mutants, leaves excised from CMV‐infected or 2b‐transgenic plants showed greater stomatal permeability and lost water more rapidly. This further indicates that CMV‐induced drought tolerance is not mediated via a change in the silencing‐regulated drought response mechanism. Under natural conditions, virus‐induced drought tolerance may serve viruses by aiding susceptible hosts to survive periods of environmental stress.     

<Emphasis Type="Italic">MIR166</Emphasis>/<Emphasis Type="Italic">165</Emphasis> genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The miR166/165 group and its target genes regulate diverse aspects of plant development, including apical and lateral meristem formation, leaf polarity, and vascular development. We demonstrate here that MIR166/165 genes are dynamically controlled in regulating shoot apical meristem (SAM) and floral development in parallel to the WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR166 and miR165 cleave same target mRNAs, individual MIR166/165 genes exhibit distinct expression domains in different plant tissues. The MIR166/165 expression is also temporarily regulated. Consistent with the dynamic expression patterns, an array of alterations in SAM activities and floral architectures was observed in the miR166/165-overproducing plants. In addition, when a MIR166a-overexpressing mutant was genetically crossed with mutants defective in the WUS-CLV pathway, the resultant crosses exhibited additive phenotypic effects, suggesting that the miR166/165-mediated signal exerts its role via a distinct signaling pathway.     

Phenotypic alterations in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> plants caused by<Emphasis Type="Italic"> Rhodococcus fascians</Emphasis> infection

Arabidopsis thaliana (L.) Heynh. plants were challenged with Rhodococcus fascians at several developmental stages and using different inoculation procedures. A variety of morphological alterations was scored on the infected plants; some of them resembled phenotypes of A. thaliana mutants in their shoot apical meristem (SAM) organization. Infection with R. fascians did not affect SAM organization in wild type nor in SAM mutants. Anatomical studies on the new organs formed after infection with R. fascians demonstrated extensive bacterial colonization. Colonization and concomitant production of specific signals are the likely cause of malformations.     

Recovery from Cucumber Mosaic Virus Infection for ‘Calwonder’ Bell Pepper Plants Does not Counter Negative Impacts on Plant Growth

Cucumber mosaic virus (CMV) accumulation in leaves and stems of infected bell pepper plants at specific symptom stages was evaluated with an emphasis on the transition from full infection to recovery from Cucumber mosaic disease. Four symptom phases occurred in successive order, designated chlorosis (leaves 6–8), mosaic (leaves 9–11/12), leaf distortion (first series of leaves on secondary and tertiary branches) and recovery (progressive recovery with newly emerging leaves in tertiary and younger branches). In situ detection of CMV in leaf tissues revealed widespread occurrence in leaves expressing chlorosis and mosaic symptoms but reduced, localized occurrence in leaves in the recovery phase. Similarly, CMV accumulated to high levels throughout stems expressing chlorosis and mosaic symptoms but with dramatically reduced levels for plants in the recovery symptom phase. Stunting of internodes occurred at all locations above the inoculated leaves by the first expression of systemic symptoms, suggesting an impact on stem growth in response to initial virus invasion of young developing tissues of the stem. Despite the recovery from CMV infection, plant growth was negatively impacted early in the infection process and remained so through the course of the experiment.     

Expression of SHOOT MERISTEMLESS, WUSCHEL, and ASYMMETRIC LEAVES1 Homologs in the Shoots of Podostemaceae: Implications for the Evolution of Novel Shoot Organogenesis

Podostemaceae (the river weeds) are ecologically and morphologically unusual angiosperms. The subfamily Tristichoideae has typical shoot apical meristems (SAMs) that produce leaves, but Podostemoideae is devoid of SAMs and new leaves arise below the base of older leaves. To reveal the genetic basis for the evolution of novel shoot organogenesis in Podostemaceae, we examined the expression patterns of key regulatory genes for shoot development (i.e., SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) orthologs) in Tristichoideae and Podostemoideae. In the SAM-mediated shoots of Tristichoideae, like in model plants, STM and WUS orthologs were expressed in the SAM. In the SAM-less shoots of Podostemoideae, STM and WUS orthologs were expressed in the initiating leaf/bract primordium. In older leaf/bract primordia, WUS expression disappeared and STM expression became restricted to the basal part, whereas ARP was expressed in the distal part in a complementary pattern to STM expression. In the reproductive shoots of Podostemoideae with a normal mode of flower development, STM and WUS were expressed in the floral meristem, but not in the floral organs, similar to the pattern in model plants. These results suggest that the leaf/bract of Podostemoideae is initiated as a SAM and differentiates into a single apical leaf/bract, resulting in the evolution of novel shoot-leaf mixed organs in Podostemaceae.     

The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE

The function of the SHOOT MERISTEMLESS (STM) gene in shoot and floral meristems throughout Arabidopsis development has been analyzed. The results show that STM plays a major role in maintaining shoot and floral meristems. In an allelic series of stm mutants the shoot meristem was either reduced or completely absent in mature embryos and mutant seedling cotyledons showed partial fusion, indicating that the STM gene affects embryonic shoot meristem development and spacing of cotyledons. Postembryonically, stm mutants initiated adventitious shoot development at a position corresponding to the shoot meristem in wild-type. Repetitively initiated defective mutant shoot and floral meristems were consumed during primordia formation and typically terminated prematurely in fused ectopic primordia, indicating that STM is required for continuous shoot and floral meristem function. Analogous defects were observed in stm embryonic and postembryonic development suggesting that similar mechanisms are employed in embryonic and postembryonic organ primordia initiation. Allelic combinations suggest different thresholds for STM requirement during plant development. STM requirement could not be bypassed by standard growth factor regimes or by shoot regeneration from calli. The results suggest that STM functions by preventing incorporation of cells in the meristem center into differentiating organ primordia and that this role can completely account for all defects observed in stm mutants. Mutations in the WUSCHEL (WUS) and ZWILLE (ZLL) genes result in defective organization and premature termination of shoot meristems. Genetic interactions between STM, WUS and ZLL were analyzed and the results indicate that STM acts upstream of WUS and ZLL. Therefore, while STM appears to function in keeping central meristem cells undifferentiated, WUS and ZLL seem to be subsequently required for proper function of these cells.     

Characterization of Synergy between Cucumber mosaic virus and Tobacco necrosis virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Cucumber mosaic virus (CMV) and Tobacco necrosis virus (TNV) exhibit a synergistic interaction and result in symptom enhancement. Accumulation of CMV(+) RNA as well as capsid protein (CP) in mixed infection was considerably higher than that of singly‐infected plants. There was also a slight increase in TNV(+) RNA and CP levels in doubly infected plants. Synergistic infection by CMV‐ and TNV‐induced higher increase in the levels of malonyldialdehyde, hydrogen peroxide (H₂O₂) and more decline in the activities of catalase than singly infected ones. Both peroxidase and superoxide dismutase activities increased rapidly for the first 10 days post inoculation (dpi) in doubly‐infected plants and then declined, whereas the enzyme activities continued to increase after 10 dpi in singly infected plants and had higher enzyme activities in the late stages than that of co‐infected plants. These results suggest that synergistic infection by CMV and TNV produced severes oxidative stress in N. benthamiana plants and the synergy between the two viruses was mutual.     

A systems approach to morphogenesis in Arabidopsis thaliana: II. Modeling the regulation of shoot apical meristem structure

Two models of the mechanism maintaining a zonal structure in the shoot apical meristem (SAM) were built based on the analysis of experimental data on the interactions between CLV1, CLV2, CLV3, and WUS genes and the concepts of their role in this mechanism. The first model, a simple one-dimensional model with two morphogens, which is a variant of Wolpert’s French flag model [1], describes the regulation of zone distribution along the SAM vertical axis. Despite a number of simplifications, this model has stationary solutions with biologically meaningful interpretation. The simplifying assumptions were successively abandoned in constructing a two-dimensional model of the mechanism underlying the regulation of SAM structure. This model provides a better understanding of the distributed system that regulates the SAM structure, and allows more detailed formalization of the modern concepts and experimental data concerning this mechanism.

     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamical modeling of the cholesterol regulatory pathway with Boolean networks

Background

RNA silencing has been implicated in virus symptom development in plants. One common infection symptom in plants is the formation of chlorotic tissue in leaves. Chlorotic and healthy tissue co-occur on a single leaf and form patterns. It has been shown that virus levels in chlorotic tissue are high, while they are low in healthy tissue. Additionally, the presence of siRNAs is confined to the chlorotic spots and the boundaries between healthy and infected tissue. These results strongly indicate that the interaction between virus growth and RNA silencing plays a role in the formation of infection patterns on leaves. However, how RNA silencing leads to the intricate patterns is not known.

Results

Here we elucidate the mechanisms leading to infection patterns and the conditions which lead to the various patterns observed. We present a modeling approach in which we combine intra- and inter-cellular dynamics of RNA silencing and viral growth. We observe that, due to the spread of viruses and the RNA silencing response, parts of the tissue become infected while other parts remain healthy. As is observed in experiments high virus levels coincide with high levels of siRNAs, and siRNAs are also present in the boundaries between infected and healthy tissue. We study how single- and double-stranded cleavage by Dicer and amplification by RNA-dependent RNA polymerase can affect the patterns formed.

Conclusion

This work shows that RNA silencing and virus growth within a cell, and the local spread of virions and siRNAs between cells can explain the heterogeneous spread of virus in leaf tissue, and therewith the observed infection patterns in plants.