Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.     

Auxin-induced leaf blade expansion in Arabidopsis requires both wounding and detachment

Elevation of leaf auxin (indole-3-acetic acid; IAA) levels in intact plants has been consistently found to inhibit leaf expansion whereas excised leaf strips grow faster when treated with IAA. Here we test two hypothetical explanations for this difference in growth sensitivity to IAA by expanding leaf tissues in vivo versus in vitro. We asked if, in Arabidopsis, IAA-induced growth of excised leaf strips results from the wounding required to excise tissue and/or results from detachment from the plant and thus loss of some shoot or root derived growth controlling factors. We tested the effect of a range of exogenous IAA concentrations on the growth of intact attached, wounded attached, detached intact, detached wounded as well as excised leaf strips. After 24 h, the growth of intact attached, wounded attached, and detached intact leaves was inhibited by IAA concentrations as little as 1 µM in some experiments. Growth of detached wounded leaves and leaf strips was induced by IAA concentrations as low as 10 µM. Stress, in the form of high light, increased the growth response to IAA by leaf strips and reduced growth inhibition response by intact detached leaves. Endogenous free IAA content of intact attached leaves and excised leaf strips was found not to change over the course of 24 h. Together these results indicate growth induction of Arabidopsis leaf blade tissue by IAA requires both substantial wounding as well as detachment from the plant and suggests in vivo that IAA induces parallel pathways leading to growth inhibition.     

Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development

Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.     

Key proliferative activity in the junction between the leaf blade and leaf petiole of Arabidopsis

Leaves are the most important, fundamental units of organogenesis in plants. Although the basic form of a leaf is clearly divided into the leaf blade and leaf petiole, no study has yet revealed how these are differentiated from a leaf primordium. We analyzed the spatiotemporal pattern of mitotic activity in leaf primordia of Arabidopsis (Arabidopsis thaliana) in detail using molecular markers in combination with clonal analysis. We found that the proliferative zone is established after a short interval following the occurrence of a rod-shaped early leaf primordium; it is separated spatially from the shoot apical meristem and seen at the junction region between the leaf blade and leaf petiole and produces both leaf-blade and leaf-petiole cells. This proliferative region in leaf primordia is marked by activity of the ANGUSTIFOLIA3 (AN3) promoter as a whole and seems to be differentiated into several spatial compartments: activities of the CYCLIN D4;2 promoter and SPATULA enhancer mark parts of it specifically. Detailed analyses of the an3 and blade-on-petiole mutations further support the idea that organogenesis of the leaf blade and leaf petiole is critically dependent on the correct spatial regulation of the proliferative region of leaf primordia. Thus, the proliferative zone of leaf primordia is spatially differentiated and supplies both the leaf-blade and leaf-petiole cells.     

Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes

BACKGROUND: Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem. RESULTS: We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth. CONCLUSIONS: Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar

During the shade-avoidance response, leaf blade expansion is inhibited and petiole elongation is enhanced. In this study, we examined the roles of photoreceptors and sugar on the differential growth of the leaf blade and petiole in shade conditions. Under the conditions examined, cell expansion, not cell division, played a major role in the differential leaf growth. The enhanced cell expansion in the leaf blade is associated with an increase in the ploidy level, whereas cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. Analysis of phytochrome, cryptochrome and phototropin mutants revealed that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade. Examination of the effects of photo-assimilated sucrose on the growth of the leaf blade and petiole revealed growth-promotional effects of sucrose that are highly dependent on the light conditions. The leaf blades of abscisic acid-deficient and sugar-insensitive mutants did not expand in blue light, but expanded normally in red light. These results suggest that both the regulation of light signals and the modulation of responses to sugar are important in the control of the differential photomorphogenesis of the leaf blade and petiole.     

Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition

Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of (15)N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early- and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.     

Signals derived from YABBY gene activities in organ primordia regulate growth and partitioning of Arabidopsis shoot apical meristems

Shoot apical meristems (SAMs) are self-sustaining groups of cells responsible for the ordered initiation of all aerial plant tissues, including stems and lateral organs. The precise coordination of these processes argues for crosstalk between the different SAM domains. The products of YABBY (YAB) genes are limited to the organ primordium domains, which are situated at the periphery of all SAMs and which are separated by a margin of three to seven cells from the central meristem zone marked by WUSCHEL and CLAVATA3 expression. Mutations in the two related YAB1 genes, FILAMENTOUS FLOWER and YABBY3 (YAB3), cause an array of defects, including aberrant phyllotaxis. We show that peripheral YAB1 activity nonautonomously and sequentially affects the phyllotaxis and growth of subsequent primordia and coordinates the expression of SAM central zone markers. These effects support a role for YAB1 genes in short-range signaling. However, no evidence was found that YAB1 gene products are themselves mobile. A screen for suppression of a floral YAB1 overexpression phenotype revealed that the YAB1-born signals are mediated in part by the activity of LATERAL SUPPRESSOR. This GRAS protein is expressed at the boundary of organ primordia and the SAM central zone, distinct from the YAB1 expression domain. Together, these results suggest that YAB1 activity stimulates signals from the organs to the meristem via a secondary message or signal cascade, a process essential for organized growth of the SAM.     

Day length affects the dynamics of leaf expansion and cellular development in Arabidopsis thaliana partially through floral transition timing

Background and Aims: Plant aerial development is well known to be affected by daylength in terms of the timing and developmental stage of floraltransition. Arabidopsis thaliana is a ‘long day’plant in which the time to flower is delayed by short days andleaf number is increased. The aim of the work presented herewas to determine the effects of different day lengths on individualleaf area expansion. The effect of flower emergence per se onthe regulation of leaf expansion was also tested in this study. Methods: Care was taken to ensure that day length was the only sourceof micro-meteorological variation. The dynamics of individualleaf expansion were analysed in Ler and Col-0 plants grown underfive day lengths in five independent experiments. Responsesat cellular level were analysed in Ler plants grown under variousday lengths and treatments to alter the onset of flowering. Key Results: When the same leaf position was compared, the final leaf areaand both the relative and absolute rates of leaf expansion weredecreased by short days, whereas the duration of leaf expansionwas increased. Epidermal cell number and cell area were alsoaltered by day-length treatments and some of these responsescould be mimicked by manipulating the date of flowering. Conclusions: Both the dynamics and cellular bases of leaf development arealtered by differences in day length even when visible phenotypesare absent. To some extent, cell area and its response to daylength are controlled by whole plant control mechanisms associatedwith the onset of flowering.     

Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development

In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and cell differentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a component of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf development. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1 promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of the protein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development when it promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it promotes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts this developmental program by promoting unscheduled cell expansion during the proliferation phase.