Hormonal and cell division analyses in Watsonia lepida seedlings

The regeneration ability, cell division activity, auxin and cytokinin content of seedling regions and hypocotyl subsections of Watsonia lepida were studied. A total of 21 different cytokinins or conjugates were found in seedlings, with the highest cytokinin content in meristematic regions (root and shoot apical meristems). The greatest contribution to the cytokinin pool came from the biologically inactive cZRMP, suggesting that significant de novo synthesis was occurring. Five different auxins or conjugates were detected, being concentrated largely in the shoot apical meristem and leaves, IAA being the most abundant. Analysis of hypocotyl subsections (C1–C4) revealed that cell division was highest in subsection C2, although regeneration in vitro was significantly lower than in subsection C1. Anatomically, subsection C1 contains the apical meristem, and hence has meristematic cells that are developmentally plastic. In contrast, subsection C2 has cells that have recently exited the meristem and are differentiating. Despite high rates of cell division, cells in subsection C2 appear no longer able to respond to cues that promote proliferation in vitro. Auxin and cytokinin analyses of these subsections were conducted. Possibly, a lower overall cytokinin content, and in particular the free-base cytokinins, could account for this observed difference.     

Studies of aberrant phyllotaxy1 Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.     

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.     

Emerging role of cytokinin as a regulator of cellular differentiation

Perhaps the most amazing feature of plants is their ability to grow and regenerate for years, sometimes even centuries. This fascinating characteristic is achieved thanks to the activity of stem cells, which reside in the shoot and root apical meristems. Stem cells function as a reserve of undifferentiated cells to replace organs and sustain postembryonic plant growth. To maintain meristem function, stem cells have to generate new cells at a rate similar to that of cells leaving the meristem and differentiating, thus achieving a balance between cell division and cell differentiation. Recent findings have improved our knowledge on the molecular mechanisms necessary to establish this balance and reveal a fundamental signaling role for the plant hormone cytokinin. Evidence has been provided to show that in the root meristem cytokinin acts in defined developmental domains to control cell differentiation rate, thus controlling root meristem size.     

Synergistic action of auxin and cytokinin mediates aluminum‐induced root growth inhibition in Arabidopsis

Auxin acts synergistically with cytokinin to control the shoot stem‐cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress‐induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al‐induced auxin signaling. Al stress triggers a local cytokinin response in the root‐apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up‐regulated specifically in the root‐apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum‐induced root growth inhibition in Arabidopsis.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

Hormone signaling in plant development

Hormone signaling plays diverse and critical roles during plant development. In particular, hormone interactions regulate meristem function and therefore control formation of all organs in the plant. Recent advances have dissected commonalities and differences in the interaction of auxin and cytokinin in the regulation of shoot and root apical meristem function. In addition, brassinosteroid hormones have recently been discovered to regulate root apical meristem size. Further insights have also been made into our understanding of the mechanism of crosstalk among auxin, cytokinin, and strigolactone in axillary meristems.     

The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus

Legume–rhizobium symbiosis contributes large quantities of fixed nitrogen to both agricultural and natural ecosystems. This global impact and the selective interaction between rhizobia and legumes culminating in development of functional root nodules have prompted detailed studies of the underlying mechanisms. We performed a screen for aberrant nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here, we describe the identification of amsh1 mutants that only develop small nodule primordia and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by LORE1 insertions in the Amsh1 gene may be separated from the shoot phenotype. In amsh1 mutants, rhizobia initially became entrapped in infection threads with thickened cells walls. Some rhizobia were released into plant cells much later than observed for the wild‐type; however, no typical symbiosome structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating enzyme, and that AMSH1 specifically cleaves K63‐linked ubiquitin chains. Post‐translational ubiquitination and deubiquitination processes involving the AMSH1 deubiquitinating enzyme are thus involved in both infection and organogenesis in Lotus japonicus.     

Cytokinin biosynthesis and transport for systemic nitrogen signaling

The plasticity of growth and development in response to environmental changes is one of the essential aspects of plant behavior. Cytokinins play an important role as signaling molecules in the long-distance communication between organs in systemic growth regulation in response to nitrogen. The spatial distribution of the expression sites of cytokinin biosynthesis genes leads to structural differences in the molecular species transported through the xylem and phloem, giving root-borne trans-hydroxylated cytokinins, namely trans-zeatin (tZ) type, a specialized efficacy in regulating shoot growth. Furthermore, root-to-shoot translocation via the xylem, tZ, and its precursor, the tZ riboside, controls different sets of shoot growth traits to fine-tune shoot growth in response to nitrogen availability. In addition to nitrogen, photosynthetically generated sugars positively regulate de novo cytokinin biosynthesis in the roots, and contribute to plant growth under elevated CO₂ conditions. In shoot-to-root signaling, cytokinins also play a role in the regulation of nutrient acquisition and root system growth in cooperation with other types of signaling molecules, such as C-TERMINALLY ENCODED PEPTIDE DOWNSTREAMs. As cytokinin is a key regulator for the maintenance of shoot apical meristem, deepening our understanding of the regulatory mechanisms of cytokinin biosynthesis and transport in response to nitrogen is important not only for basic comprehension of plant growth, but also to ensure the stability of agricultural production.     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex,affects development through cell division in Arabidopsis thaliana

Kinetochore, a protein super‐complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured‐1 (mun‐1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun‐1 is a weak allele because of the insertion of T‐DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9‐mediated mutagenesis showed zygotic embryonic lethality similar to nuf2‐1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two‐hybrid assay and in planta co‐immunoprecipitation. MUN is co‐localized at the centromere with HTR12/CENH3, which is a centromere‐specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.     

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.     

MOL1 is required for cambium homeostasis in Arabidopsis

Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine‐rich repeat receptor‐like kinase (LRR‐RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR‐RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR‐RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis‐expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress‐related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.     

Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem

Cytokinin phytohormones regulate a variety of developmental processes in the root such as meristem size, vascular pattern, and root architecture [1-3]. Long-distance transport of cytokinin is supported by the discovery of cytokinins in xylem and phloem sap [4] and by grafting experiments between wild-type and cytokinin biosynthesis mutants [5]. Acropetal transport of cytokinin (toward the shoot apex) has also been implicated in the control of shoot branching [6]. However, neither the mode of transport nor a developmental role has been shown for basipetal transport of cytokinin (toward the root apex). In this paper, we combine the use of a new technology that blocks symplastic connections in the phloem with a novel approach to visualize radiolabeled hormones in planta to examine the basipetal transport of cytokinin. We show that this occurs through symplastic connections in the phloem. The reduction of cytokinin levels in the phloem leads to a destabilization of the root vascular pattern in a manner similar to mutants affected in auxin transport or cytokinin signaling [7]. Together, our results demonstrate a role for long-distance basipetal transport of cytokinin in controlling polar auxin transport and maintaining the vascular pattern in the root meristem.     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.