Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation

The plant hormone cytokinin is a key morphogenic factor controlling cell division and differentiation, and thus the formation and growth rate of organs during a plant's life cycle. In order to explore the relevance of cytokinin during the initial phase of leaf primordia formation and its impact on subsequent leaf development, we increased cytokinin degradation in young shoot organ primordia of Arabidopsis thaliana by expressing a cytokinin oxidase/dehydrogenase (CKX) gene under control of the AINTEGUMENTA (ANT) promoter. The final leaf size in ANT:CKX3 plants was reduced to ∼27% of the wild-type size and the number of epidermal cells was reduced to ∼12% of the wild type. Kinematic analysis revealed that cell proliferation ceased earlier and cell expansion was accelerated in ANT:CKX3 leaves, demonstrating that cytokinin controls the duration of the proliferation phase by delaying the onset of cell differentiation. The reduction of the cell number was partially compensated by an increased cell expansion. Interestingly, ANT:CKX3 leaf cells became about 60% larger than those of 35S:CKX3 leaves, indicating that cytokinin has an important function during cell expansion as well. Furthermore, ANT:CKX3 expression significantly reduced the capacity of both the vegetative as well as the generative shoot apical meristem to initiate the formation of new leaves and flowers, respectively. We therefore hypothesize that the cytokinin content in organ primordia is important for regulating the activity of the shoot meristem in a non-autonomous fashion.     

Regulation of CLV3 expression by two homeobox genes in Arabidopsis

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. The WUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3 (CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes in CLV3 expression levels, and adjustment of WUS expression via the CLV signal transduction pathway. We have analyzed whether expression of CLV3 is controlled by the activity of WUS or another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends on WUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level of CLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.     

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.     

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.     

CORONA, a member of the class III homeodomain leucine zipper gene family in Arabidopsis, regulates stem cell specification and organogenesis

Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.     

THE COST OF MERISTEM LIMITATION IN POLYGONUM ARENASTRUM: NEGATIVE GENETIC CORRELATIONS BETWEEN FECUNDITY AND GROWTH

Growth and reproduction in higher plants depend on meristems, which have three developmental fates. A meristem can become reproductive, but doing so terminates its activity, it can differentiate vegetatively, or it can remain quiescent for extended periods. The first two fates are mutually exclusive, and only the second leads to the production of additional meristems for subsequent growth and reproduction. In Polygonum arenastrum (frequently referred to as P. aviculare in North American Floras), an annual species lacking quiescent meristems, a quantitative genetic analysis of inbred full-sibling families revealed genetic variation in the developmental pattern of axillary meristem commitment to vegetative growth versus reproduction. Developmental variation resulted in family differences in the age of first reproduction, in age-specific fecundity and growth, and in final plant size and reproductive output. Furthermore, there were strong negative genetic correlations between age-specific growth and fecundity. Early commitment of meristems to reproduction favors high early fecundity, but reduces the number of meristems available for vegetative differentiation, and leads to lowered growth rates and fecundity later in life, when meristems are limiting. Conversely, meristem commitment to vegetative growth early in life results in low early fecundity but high late fecundity and growth. Meristem limitation, like resource limitation, is a proximate mechanism that generates trade-offs between life history traits. Differences between meristem limitation and resource limitation are discussed. Meristem limitation leads automatically to a senescent life history because of the determinate fate of reproductive meristems. Developmental characters were also found to be genetically correlated with metamer characters (leaf size, internode length) and seed size in this selfing species. The pattern of correlation is suggestive of selection for particular suites of life history and morphological characters.     

The Stem Cell Niche in Leaf Axils Is Established by Auxin and Cytokinin in Arabidopsis

Plants differ from most animals in their ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems harboring new stem cell niches. In seed plants, this is achieved by axillary meristems, which are established in the axil of each leaf base and develop into lateral branches. Here, we describe the initial processes of Arabidopsis thaliana axillary meristem initiation. Using reporter gene expression analysis, we find that axillary meristems initiate from leaf axil cells with low auxin through stereotypical stages. Consistent with this, ectopic overproduction of auxin in the leaf axil efficiently inhibits axillary meristem initiation. Furthermore, our results demonstrate that auxin efflux is required for the leaf axil auxin minimum and axillary meristem initiation. After lowering of auxin levels, a subsequent cytokinin signaling pulse is observed prior to axillary meristem initiation. Genetic analysis suggests that cytokinin perception and signaling are both required for axillary meristem initiation. Finally, we show that cytokinin overproduction in the leaf axil partially rescue axillary meristem initiation-deficient mutants. These results define a mechanistic framework for understanding axillary meristem initiation.     

Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation

Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7–GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7‐expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis‐zeatin (cZ) and N‐glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ‐deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone.     

Arabidopsis KNOXI proteins activate cytokinin biosynthesis

Plant architecture is shaped through the continuous formation of organs by meristems. Class I KNOTTED1-like homeobox (KNOXI) genes are expressed in the shoot apical meristem (SAM) and are required for SAM maintenance. KNOXI proteins and cytokinin, a plant hormone intimately associated with the regulation of cell division, share overlapping roles, such as meristem maintenance and repression of senescence, but their mechanistic and hierarchical relationship have yet to be defined. Here, we show that activation of three different KNOXI proteins using an inducible system resulted in a rapid increase in mRNA levels of the cytokinin biosynthesis gene isopentenyl transferase 7 (AtIPT7) and in the activation of ARR5, a cytokinin response factor. We further demonstrate a rapid and dramatic increase in cytokinin levels following activation of the KNOXI protein SHOOT MERISTEMLESS (STM). Application of exogenous cytokinin or expression of a cytokinin biosynthesis gene through the STM promoter partially rescued the stm mutant. We conclude that activation of cytokinin biosynthesis mediates KNOXI function in meristem maintenance. KNOXI proteins emerge as central regulators of hormone levels in plant meristems.     

The dynamic plant stem cell niches

Stem cells exist in specific locations called niches, where extracellular signals maintain stem cell division and prevent differentiation. In plants, the best characterised niches are within the shoot and root meristems. Networks of regulatory genes and intercellular signals maintain meristem structure in spite of constant cell displacement by division. Recent works have improved our understanding of how these networks function at the cellular and molecular levels, particularly in the control of the stem cell population in the shoot meristem. The meristem regulatory genes have been found to function partly through localised control of widely used signals such as cytokinin and auxin. The retinoblastoma protein has also emerged as a key regulator of cell differentiation in the meristems.     

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.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.