Early Stages Of Leaf Development Research Articles

Mineral Content Differs among Microgreen, Baby Leaf, and Adult Stages in Three Cultivars of Kale

Kale (Brassica oleracea L. and other species) is considered a rich source of important minerals. Kale at the early stage of leaf development is assumed to contain higher levels of minerals than at maturity. However, literature supporting this assumption is scarce. In this study, the concentrations of macronutrients [potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P)] and micronutrients [sodium (Na), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu)] either essential to plant growth and development, or important to human health, were determined. Three kale cultivars (green leaf ‘Dwarf Blue Curled’ and red leaf ‘Scarlet’ in B. oleracea, and green leaf with purple midvein ‘Red Russian’ in Brassica napus) were evaluated at five different leaf developmental stages; cotyledon [microgreen 1 (MG1)], two true leaf [microgreen 2 (MG2)], four true leaf [baby leaf 1 (BL1)], six true leaf [baby leaf 2 (BL2)], and adult. As kale matured, total mineral (ash) decreased from 14.6–19.1% at the microgreen stages to 3.9–6.4% at the adult stage, on a dry weight (DW) basis. Microgreen kale contained higher concentrations of most minerals than adult kale, on a DW basis, in all cultivars. On a fresh weight (FW) (as consumed) basis, the highest level of total mineral concentration was detected at baby leaf stage 1 (1.3–1.7%) and there was no difference between microgreen and adult stages. Fresh microgreens generally contained lower K, Ca, Mg, Fe, and Zn than fresh baby leaves, and lower concentrations of Ca and Mg and higher Na compared with fresh adult kale. Overall, water content deceased from 95.1% at MG1 stage to 80.0% at adult stage. The variation in water content and mineral accumulation during leaf development might contribute to the discrepancy. In addition, fresh leaves of ‘Scarlet’ contained higher concentration of total minerals than that of ‘Dwarf Blue Curled’ or ‘Red Russian’. Although ‘Dwarf Blue Curled’ and ‘Red Russian’ are different species, their mineral content profile during leaf development was similar. Together, cultivar and leaf developmental stage influenced mineral content in kale.

Trichome patterning control involves TTG1 interaction with SPL transcription factors.

Epidermal cell differentiation is a paramount and conserved process among plants. In Arabidopsis, a ternary complex formed by MYB, bHLH transcription factors and TTG1 modulates unicellular trichome morphogenesis. The formation of multicellular glandular trichomes of the xerophytic shrub Cistus creticus that accumulate labdane-type diterpenes, has attained much attention renowned for its medicinal properties. Here, we show that C. creticus TTG1 (CcTTG1) interacts with the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPLA/B) proteins, putative homologs of AtSPL4/5 that in turn interact with AtTTG1. These interactions occur between proteins from evolutionarily distant species supporting the conserved function of TTG1-SPL complex. Overexpression of AtSPL4 and AtSPL5 decreased the expression of GLABRA2 (AtGL2), the major regulator of trichome morphogenesis, resulting in trichome reduction on the adaxial surface of cauline leaves, thereby illuminating the significance of TTG1-SPLs interactions in trichome formation control. AtGL2 and AtSPL4 have opposite expression patterns during early stages of leaf development. We postulate an antagonistic effect between SPLs and the heterogeneous MYB-bHLH factors binding to TTG1. Hence, the SPLs potentially rearrange the complex, attenuating its transcriptional activity to control trichome distribution.

Foliar colleters in Anacardiaceae: first report for the family

Colleters are secretory structures widely distributed in eudicots and with taxonomic value in many families. Although glandular trichomes have been described in some Anacardiaceae species, the chemical characterization of their secretions is scarce and to date there are no reports on colleters. Light microscopy and scanning electron microscopy were used to study the distribution and structure of colleters on the vegetative buds of Anacardium humile A.St.-Hil., Lithraea molleoides (Vell.) Engl., Spondias dulcis Parkinson, and Tapirira guianensis Aubl., and to characterize their secretory products histochemically. In all of these Anacardiaceae species, colleters are multicellular and multiseriate ovoid or club-shaped glandular trichomes of protodermic origin, present on both surfaces of leaf primordia. They reach the secretory phase at early stages of leaf development, after which they gradually degenerate, become brown, and fall off. Histochemical tests indicate that the secretion within the glandular cells and outside the trichomes is a complex mixture containing mucilage, fatty acids, and phenolic compounds, which are secretory products that can play an important role in the protection of meristems against desiccation and attack by pathogens. Therefore, the distribution of these glandular trichomes, their short-life, the chemical nature of their secretions and their presumed functions support their being classified as colleters.

Herbivory-induced stress: Leaf developmental instability is caused by herbivore damage in early stages of leaf development

Herbivory is a major source of plant stress and its effects can be severe, decreasing plant fitness, or subtle, affecting the development of leaves by influencing the normal pattern of growth and expansion of leaf blades. Fluctuating asymmetry (FA) analysis is recognized as a measure of plant stress, and can be used to evaluate subtle effects of herbivory on the imperfect growth of bilaterally symmetrical traits, such as leaves. One general issue is that authors usually consider FA as an indicator of stress, which can attract herbivores (plant stress hypothesis), and studies showing that herbivores themselves affect leaf symmetry (herbivory-induced stress hypothesis) are scarce, with mixed results. Here, we investigated the relationship between herbivory by thrips and leaf FA in Banisteriopsis malifolia and Heteropterys escallonifolia (Malpighiaceae). Pseudophilothrips obscuricornis is a free-living, non-pest, sucking species that feeds mainly on leaf buds. We hypothesized that herbivory by thrips in the early stages of leaf development would provoke increased FA levels in mature leaves. The results showed that thrips herbivory rate was low, affecting barely more than 1% of the leaf blade. Nonetheless, thrips-attacked leaves of B. malifolia and H. escallonifolia presented increases of 15 and 27% in leaf asymmetry, respectively, compared to uninjured leaves, corroborating the herbivory-induced stress hypothesis. Since herbivory by thrips in leaf buds was related to significant increases in the stress of mature leaves, we assume that under these circumstances, FA can be used as a biomarker for plant stress following herbivory damage. To be useful as a biomarker of stress, FA in plants must be investigated with caution, taking into account the natural history of the herbivore species and timing of leaf damage.

Visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging

In comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific β-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a β-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L.japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.

The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants. Map-based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non-photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short-day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate-dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light-dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.

The TIE1 Transcriptional Repressor Links TCP Transcription Factors with TOPLESS/TOPLESS-RELATED Corepressors and Modulates Leaf Development inArabidopsis

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development.

Compositional analysis of leaf cuticular membranes isolated from tea plants (Camellia sinensis L.)

Chemical constituents of cuticular membranes (CMs) isolated from three tea cultivars (Camellia sinensis (L.) O. Kuntze cvs. Yabukita, Samidori and Gokou) were compared. All CMs from the adaxial side of the leaves showed higher accumulation of wax, cutin and polysaccharide, while those from the abaxial side were abundant in cutan, showing the adaptation of the adaxial side to abiotic stresses, such as wind and rain, in contrast to the abaxial side, which provides defence against pathogens. Yabukita, a major tea cultivar in Japan, developed thick CMs while Samidori and Gokou, shade-cultivated tea cultivars, had lighter CMs, reflecting their thin and soft leaves. CMs rapidly accumulated 9,10-epoxy-18-hydroxyoctadecanoic acid at a very early stage of leaf development. Additionally, shade treatment did not influence cutin biosynthesis in CMs, reflecting high adaptation of tea leaves under low light levels.

Elevated temperature altered the reaction norms of stomatal conductance in field-grown grapevine

We measured the effect of elevated temperature, and its interaction with fruit load (exp. 1) and water supply (exp. 2), on the stomatal conductance (gs) of Vitis vinifera, cv. Shiraz. Thermal regimes (control vs. elevated temperature using open-top chambers) were initiated in spring, thus affecting leaf development, and were maintained during the whole growing season. We used a top-down approach where reaction norms were derived that relate gs for each treatment and the mean gs across treatments; the slopes of reaction norms quantify phenotypic plasticity.Stomatal conductance responded to neither the interaction between temperature and fruit load (P=0.37) nor the interaction between temperature and water regime (P=0.33). We therefore dealt with each factor separately. Reaction norms of gs under elevated temperature and control treatments diverged, i.e. elevated temperature had no effect on gs under conditions conducive to low conductance (below ∼100mmolm−2s−1) but increased gs in relation to controls under conditions favouring high conductance. Stomata density was 159±6.7mm−2, and was unaffected by temperature. Stomata length increased from 20.6μm in controls to 23.2μm in the heated treatment (P<0.0001) and width increased from 14.4μm in controls to 16.8μm in the heated treatment (P<0.0001). Thus, longer and wider stomata contributed to the enhanced plasticity of stomatal conductance under elevated temperature. Consistently, reaction norms of light-saturated photosynthesis were divergent, i.e. elevated temperature did not affect photosynthesis under conditions conducive to low gs and photosynthesis, but increased photosynthesis in relation to controls under more favourable conditions. A high source:sink ratio reduced the plasticity of stomatal conductance. Water regime had a minor effect on the plasticity of stomatal conductance, but the reaction norms for irrigated and water deficit treatments were offset by ∼60mmolm−2s−1 thus indicating a consistent effect of water deficit across environmental conditions.We conclude that the responses of stomata derived from transient temperature treatments in fully expanded leaves are unsuitable to model the dynamics of gas exchange in response to projected warming. Our experiments support a conceptual model where current stomatal conductance is a function of (i) the prevailing environmental conditions during early stages of leaf development that set the upper limit of conductance through the modulation of stomatal density and size, (ii) the current environmental conditions and (iii) the source:sink ratio.

Overexpression of stress-related genes, BrERF4 and AtMYB44, in Arabidopsis thaliana alters cell expansion but not cell proliferation during leaf growth

We have previously shown that overexpression of BrERF4 (BrassicarapaETHYLENE-RESPONSIVEFACTOR4) increases salt and drought tolerance in Arabidopsis plants, and also retarded organ growth. In the present study, we investigated in detail the leaf growth retardation phenotype at the cellular level. Results showed that BrERF4-overexpressing Arabidopsis plants developed small leaves by reducing their cell size but not the cell number. Detailed kinematic analysis revealed that changes in cell size appeared from the very early stages of leaf development, directly affecting the size of leaf organs. RT-PCR analysis showed that expression of expansin genes was reduced in the overexpressors, whereas expression of the cell cycle gene, CYCB1;1, was not altered at all. In addition, overexpression of AtMYB44, another stress-related transcription factor gene, reduced leaf growth, which also resulted from reduction in cell size but not in cell number. These results suggest that overexpression of those transcription factors negatively affects cell expansion during leaf growth without altering cell number. We discuss about the advantages that the BrERF4- or AtMYB44-induced cell expansion retardation confers on plants under natural environmental adversity.

Air-Propelled Abrasive Grit for Postemergence In-Row Weed Control in Field Corn

Organic growers need additional tools for weed control. A new technique using abrasive grit propelled by compressed air was tested in field plots. Grit derived from corncobs was directed at seedlings of summer annual weeds growing at the bases of corn plants when the corn was at differing early stages of leaf development. Season-long, in-row weed control exceeded 90% when two or three abrasion events were coupled with between-row cultivation. Timing of weed abrasion was critical, with highest levels of control corresponding to the one- and five-leaf stages or the one-, three-, and five-leaf stages of corn development. Corn yields associated with these treatments were equivalent to those of hand-weeded controls in which no abrasive grit was applied. Thus, air-propelled abrasive grit applications at the one-, three-, and five-leaf stages of corn controlled weeds sufficiently to prevent weed-induced reductions in corn grain. Additionally, these applications were not harmful to corn plants. This new concept for weed control may be of interest to organic crop managers.

Enhanced antioxidant protection at the early stages of leaf expansion in ginkgo under natural environmental conditions

Photosynthetic pigments, gas exchange, chlorophyll (Chl) a fluorescence kinetics, antioxidant enzymes and chloroplast ultrastructure were investigated in ginkgo (Ginkgo biloba L.) leaves from emergence to full size. Under natural conditions, the net photosynthetic rate (PN), contents of Chl a, Chl b and total soluble proteins and fresh and dry leaf mass gradually increased during leaf expansion. The maximum photochemical efficiency of photosystem (PS) 2 (variable to maximum fluorescence ratio, Fv/Fm) was considerably higher at the early stages of leaf development than in fully expanded leaves. During daily course, only reversible decrease in Fv/Fm was distinguished at various stages, implying that no photo-damage occurred. Absorption flux per cross section (CS) and trapped energy flux per CS were significantly lower in newly expanding leaves compared with fully expanded ones, however, dissipated energy flux per CS was only slightly lower in expanding leaves. The ratio of carotenoids (Car)/Chl decreased gradually during leaf expansion due to increasing Chl content. Moreover, activities of the antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase and peroxidase, increased at the early stages of leaf expansion. The appearance of osmiophilic granules in fully expanded leaves further proves that photo-protection is significantly strengthened at the early stages of leaf expansion.

Enhanced antioxidant protection at the early stages of leaf expansion in ginkgo under natural environmental conditions

Photosynthetic pigments, gas exchange, chlorophyll (Chl) a fluorescence kinetics, antioxidant enzymes and chloroplast ultrastructure were investigated in ginkgo (Ginkgo biloba L.) leaves from emergence to full size. Under natural conditions, the net photosynthetic rate (PN), contents of Chl a, Chl b and total soluble proteins and fresh and dry leaf mass gradually increased during leaf expansion. The maximum photochemical efficiency of photosystem (PS) 2 (variable to maximum fluorescence ratio, Fv/Fm) was considerably higher at the early stages of leaf development than in fully expanded leaves. During daily course, only reversible decrease in Fv/Fm was distinguished at various stages, implying that no photo-damage occurred. Absorption flux per cross section (CS) and trapped energy flux per CS were significantly lower in newly expanding leaves compared with fully expanded ones, however, dissipated energy flux per CS was only slightly lower in expanding leaves. The ratio of carotenoids (Car)/Chl decreased gradually during leaf expansion due to increasing Chl content. Moreover, activities of the antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase and peroxidase, increased at the early stages of leaf expansion. The appearance of osmiophilic granules in fully expanded leaves further proves that photo-protection is significantly strengthened at the early stages of leaf expansion.

Early stages of leaf development in has mutant of Arabidopsis thaliana

The elucidation of molecular mechanisms underlying the leaf development can be facilitated by the detailed anatomical study of leaf development mutants. We present an analysis of leaf anatomy and morphogenesis during early developmental stages in has mutant of Arabidopsis thaliana. The recessive has mutation affects a number of aspects in plant development, including the shape and size of both cotyledons and leaves. The earliest developmental observations suggest almost synchronous growth of the first two leaf primordia of has mutant. No significant disruption of the cell division pattern in the internal tissue is observed at the earliest stages of development, with the major anatomical difference compared to wild type primordia being the untimely maturation of mesophyll tissue cells in has mutant. At the stage of leaf blade formation, structure disruption becomes clearly evident, by irregular arrangement of the cell layers and the lack of polarity in juvenile has leaves. One distinguishing feature of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. Altered has mutant leaf morphology could be at least partially accounted for by the ectopic STM activity that was found at the base of leaf primordia during early stages of leaf development in has plants.

Gibberellin partly mediates LANCEOLATE activity in tomato

Elaboration of a compound leaf shape depends on extended morphogenetic activity in developing leaves. In tomato (Solanum lycopersicum), the CIN-TCP transcription factor LANCEOLATE (LA) promotes leaf differentiation. LA is negatively regulated by miR319 during the early stages of leaf development, and decreased sensitivity of LA mRNA to miR319 recognition in the semi-dominant mutant La leads to prematurely increased LA expression, precocious leaf differentiation and a simpler and smaller leaf. Increased levels or responses of the plant hormone gibberellin (GA) in tomato leaves also led to a simplified leaf form. Here, we show that LA activity is mediated in part by GA. Expression of the SlGA20 oxidase1 (SlGA20ox1) gene, which encodes an enzyme in the GA biosynthesis pathway, is increased in gain-of-function La mutants and reduced in plants that over-express miR319. Conversely, the transcript levels of the GA deactivation gene SlGA2 oxidase4 (SlGA2ox4) are increased in plants over-expressing miR319. The miR319 over-expression phenotype is suppressed by exogenous GA application and by a mutation in the PROCERA (PRO) gene, which encodes an inhibitor of the GA response. SlGA2ox4 is expressed in initiating leaflets during early leaf development. Its expression expands as a result of miR319 over-expression, and its over-expression leads to increased leaf complexity. These results suggest that LA activity is partly mediated by positive regulation of the GA response, probably by regulation of GA levels.

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.

Positive and negative peptide signals control stomatal density.

The stoma is a micro valve found on aerial plant organs that promotes gas exchange between the atmosphere and the plant body. Each stoma is formed by a strict cell lineage during the early stages of leaf development. Molecular genetics research using the model plant Arabidopsis has revealed the genes involved in stomatal differentiation. Cysteine-rich secretory peptides of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play crucial roles as extracellular signaling factors. Stomatal development is orchestrated by the positive factor STOMAGEN/EPFL9 and the negative factors EPF1, EPF2, and CHALLAH/EPFL6 in combination with multiple receptors. EPF1 and EPF2 are produced in the stomatal lineage cells of the epidermis, whereas STOMAGEN and CHALLAH are derived from the inner tissues. These findings highlight the complex cell-to-cell and intertissue communications that regulate stomatal development. To optimize gas exchange, particularly the balance between the uptake of carbon dioxide (CO(2)) and loss of water, plants control stomatal activity in response to environmental conditions. The CO(2) level and light intensity influence stomatal density. Plants sense environmental cues in mature leaves and adjust the stomatal density of newly forming leaves, indicating the involvement of long-distance systemic signaling. This review summarizes recent research progress in the peptide signaling of stomatal development and discusses the evolutionary model of the signaling machinery.

An investigation of the glandular and non-glandular foliar trichomes of Orthosiphon labiatus N.E.Br. [Lamiaceae

Using a combination of light (stereo and compound) and scaning electron microscopy, this study investigates the distribution and morphology of foliar trichomes of Orthosiphon labiatus (Lamiaceae) at different stages of leaf development. Three types of trichomes were observed: two glandular types (large peltate and small capitate) and one non-glandular type. Results revealed that the development of peltate trichomes was initiated in the early stages of leaf development. However, a higher number of developed peltate trichomes was observed in mature leaves, indicating an increase with age. Capitate trichomes are, by contrast, rare and sparsely distributed on both leaf surfaces. The highest density of non-glandular trichomes was observed on the abaxial leaf surface, especially along the veins, with the density decreasing with leaf maturity. This paper contributes towards the body of knowledge being assembled on the family Lamiaceae as it manifests in southern Africa.

Chloroplast biogenesis during the early stage of leaf development in rice

In rice, the developmental process in leaf formation can be divided into 7 stages (stages P0 to P6). We investigated chloroplast biogenesis and physiological changes in the developing leaves at the stage P4, during which leaf blade elongation and establishment of basic leaf blade structure occur. Chlorophyll content was negligible in the leaves early in the P4 stage and increased rapidly as they enters the late P4 phase. Chlorophyll fluorescence ratio (Fv/Fm) also increased markedly and the final value was comparable with that of mature leaves. Gene expression analysis showed that during the P4 stage, chloroplasts in the leaf cell undergo all three steps of differentiation: (i) plastid division and DNA replication (ii) establishment of plastid genetic system (iii) activation of photosynthetic apparatus. These observations suggest that the P4 is key in the development of a leaf, during which leaf rapidly differentiated both morphologically and physiologically, and that the P4 leaf is suitable for investigation of physiological relationships between chloroplast and leaf development.

KNOX overexpression in transgenic Kohleria (Gesneriaceae) prolongs the activity of proximal leaf blastozones and drastically alters segment fate

KNOX (knotted1-like homeobox) genes have a widely conserved role in the generation of dissected leaves. Ectopic KNOX activity in leaves in various angiosperm lineages causes leaf form changes that can elucidate how the configuration of leaf development evolved. We present an analysis of leaf morphology and morphogenesis in transgenic Kohleria lines overexpressing a heterologous KNOX gene. Kohleria, like many members of Gesneriaceae, has simple-serrated leaves with pinnate venation. KNOX overexpression causes prolonged segment proliferation in proximal, but not distal, parts of leaf blades. Elaborate dissected segments reiterate the zonation of the whole leaf, with organogenic activity persisting between a distal maturation zone and a proximal intercalary elongation zone. The architecture of vascular bundles is severely altered, with a reduced midvein and a more palmate venation. The initial establishment of organogenically competent primordial margins (marginal blastozones) and the onset of tissue differentiation in early stages of leaf development were similar in wild-type and KNOX overexpressing lines. However, leaves overexpressing KNOX often failed to fully mature, and persistent marginal blastozones were found at the base of blades in mature portions of the shoot. We conclude that KNOX-mediated perpetuation of marginal blastozones in Kohleria is sufficient to induce a set of processes that result in highly dissected leaflets, which are unusual in this plant family. Spatial confinement of blastozones between an early maturing tip and a late elongating petiole zone reflects the presence of distinct maturation processes that limit the ability of the leaf margins to respond to ectopic KNOX gene expression.