Early Leaf Development Research Articles

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development.

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice.

Remodelling of lace plant leaves: antioxidants and ROS are key regulators of programmed cell death

Main conclusionAntioxidants and reactive oxygen species are integral for programmed cell death signaling during perforation formation in the lace plant (Aponogeton madagascariensis).The lace plant is an excellent model system for studying developmentally regulated programmed cell death (PCD). During early lace plant leaf development, PCD systematically deletes cells resulting in a perforated leaf morphology that is unique in planta. A distinct feature in young lace plant leaves is an abundance of anthocyanins, which have antioxidant properties. The first sign of PCD induction is the loss of anthocyanin pigmentation in cells that are targeted for destruction, which results in a visible gradient of cell death. The cellular dynamics and time course of lace plant PCD are well documented; however, the signals involved in the pathway remain elusive. This study investigates the roles of antioxidants and ROS in developmental PCD signaling during lace plant perforation formation. The involvement of antioxidants and ROS in the pathway was determined using a variety of techniques including pharmacological whole plant experimentation, long-term live cell imaging, the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid anti-radical activity assay, and western blot analysis. Results indicate that antioxidants and ROS are key regulators of PCD during the remodelling of lace plant leaves.

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana.

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates.

AtLSG1-2 Regulates Leaf Growth by Affecting Cell Proliferation and the Onset of Endoreduplication and Synergistically Interacts with AtNMD3 during Cell Proliferation Process.

AtLSG1-2 is a circularly permuted GTPase required for ribosome biogenesis and recently shown to be involved in early leaf development, although it was unclear how AtLSG1-2 affects leaf growth. Here, we found that atlsg1-2 mutants had reduced leaf size as a result of decreased cell size and cell number. Leaf kinematic analysis and CYCB1;1::GUS expression pattern in atlsg1-2 mutant indicated that loss of function of AtLSG1-2 delays the transition from cell division to cell expansion. Decreases in ploidy levels and trichome branch number suggest that AtLSG1-2 deficiency suppresses endoreduplication. Real-time PCR analysis showed that genes specifically expressed in the proliferation stage were highly expressed and those involved in endoreduplication were differentially regulated. LSG1 is known to mediate the recruitment of nucleocytoplasmic shuttling protein NMD3 back to the nucleus in yeast, yet their relationship was unclear in plants. Our genetic analysis revealed that the atlsg1 atnmd3 double mutant displayed enhanced phenotypes as compared with the respective single mutant and that AtLSG1-2 and AtNMD3 synergistically affect the cell proliferation process.

Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice

BackgroundNADPH: protochlorophyllide oxidoreductase (POR) is an essential enzyme that catalyzes the photoreduction of protochlorophyllide to chlorophyllide, which is ultimately converted to chlorophyll in developing leaves. Rice has two POR isoforms, OsPORA and OsPORB. OsPORA is expressed in the dark during early leaf development; OsPORB is expressed throughout leaf development regardless of light conditions. The faded green leaf (fgl) is a loss-of-function osporB mutant that displays necrotic lesions and variegation in the leaves due to destabilized grana thylakoids, and has increased numbers of plastoglobules in the chloroplasts. To investigate whether the function of OsPORA can complement that of OsPORB, we constitutively overexpressed OsPORA in fgl mutant.ResultsIn the 35S:OsPORA/fgl (termed OPAO) transgenic plants, the necrotic lesions of the mutant disappeared and the levels of photosynthetic pigments and proteins, as well as plastid structure, were recovered in developing leaves under natural long days in the paddy field and under short days in an artificially controlled growth room. Under constant light conditions, however, total chlorophyll and carotenoid levels in the developing leaves of OPAO plants were lower than those of wild type. Moreover, the OPAO plants exhibited mild defects in mature leaves beginning at the early reproductive stage in the paddy field.ConclusionsThe physiological function of OsPORB in response to constant light or during reproductive growth cannot be completely replaced by constitutive activity of OsPORA, although the biochemical functions of OsPORA and OsPORB are redundant. Therefore, we suggest that the two OsPORs have differentiated over the course of evolution, playing distinct roles in the adaptation of rice to the environment.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis.

Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

SummaryCoordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN‐DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin‐dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK‐insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis.

Erratum to: Dynamics of Biologically Active Compound Contents from Betula pendula Leaves During Early Leaf Development

Erratum to: Dynamics of Biologically Active Compound Contents from Betula pendula Leaves During Early Leaf Development

Leaf ontogeny and meristem activity of Typha domingensis Pers. (Typhaceae) under different phosphate concentrations

Phosphorus eutrophication may promote increased growth, photosynthesis, and shoot investment in cattail (Typha domingensis). However, its influence in early leaf development and meristem traits remains unclear. Therefore, this work aimed to evaluate the effects of phosphate levels on meristem structure and activity as well as tissue differentiation during the leaf development of T. domingensis. Plants were subjected to modified nutrient solutions containing three phosphate concentrations. The leaves were collected daily for 7days, and were used to evaluate growth, meristem characteristics and tissue differentiation. In addition, phosphorus content was measured in roots and rhizomes exposed to phosphate treatments. The leaf primordia cultured in 0.4 and 0.8mM phosphate levels showed a larger proportion of ground meristem than those grown in 0.1mM. The procambium proportion was higher under 0.8mM phosphate when compared to other concentrations. However, the proportion of protodermis was greater in 0.1mM phosphate when compared to 0.4 and 0.8mM. In addition, leaf primordia submitted to 0.4 and 0.8mM phosphate increased meristem cell production. These primordia also showed fewer dividing cells and shorter cell cycle as compared to 0.1mM phosphate. The development of aerenchyma and palisade parenchyma was promoted by greater phosphate concentrations. Higher aerenchyma proportion was found in plants from 0.8mM phosphate treatment. Phosphorus content was higher in root than rhizomes for plants grown at 0.4 and 0.8mM of phosphate. Likewise, the higher phosphate concentrations increased P levels in both roots and rhizomes. Therefore, optimal phosphorus levels promote the development of leaves with a higher photosynthetic potential, which may contribute to the uncontrolled growth of cattail under phosphorus eutrophication.

Soil properties, seasonality and crop growth stage exert a stronger effect on rhizosphere prokaryotes than the fungal biocontrol agent Fusarium oxysporum f.sp. strigae

Fusarium oxysporum f.sp. strigae (Fos) is an effective biocontrol agent (BCA) against the parasitic weed Striga hermonthica. It acts in the rhizosphere of several tropical cereals, where it may interfere with indigenous microbial populations. To test this impact, we assessed in a 2-season field experiment at two contrasting tropical agro-ecological sites the response of nitrifying and total indigenous prokaryotic communities in the rhizosphere of maize to the exposure of the Fos-BCA “Foxy-2”. At early leaf development (EC30), flowering (EC60) and senescence (EC90) stage of maize, rhizosphere samples were obtained and subjected to community analysis of bacterial and archaeal amoA (ammonia monooxigenase) (AOB, AOA) and 16S rRNA genes. Abundance and community composition of all studied genes were predominantly influenced by soil type, crop growth stage and seasonality. No major effect of “Foxy-2” was found. Notably, total archaeal community relative to bacteria dominated in the clayey soil which was linked to its strong soil organic carbon (SOC) background. Compared to bacterial nitrifiers, domination of nitrifying archaea increased towards senescence stage which was explained by biochemical differences in organic resource availability between the crop growth stages. During the short rain season, the higher archaeal abundance was mainly driven by increased availability of organic substrates, i.e., extractable organic carbon. Our findings suggested that archaea had greater rhizosphere competence than “Foxy-2” in soils with higher clay and SOC contents. We verified that “Foxy-2” in maize rhizospheres is compatible with nitrifying prokaryotes under the given environments, in particular in clayey soils dominated by archaea.

Dynamics of Biologically Active Compound Contents from Betula pendula Leaves During Early Leaf Development

The individual group compositions of hydroxycinnamic acid derivatives, flavonoids, terpenoids, fatty acids, and hydrocarbons from Betula pendula leaves during early leaf development were studied using GC-MS and HPLC. The contents of biologically active compounds varied considerably during observation for 20 d after leaf emergence. It was shown that the dynamics of antifeedant triterpenoid contents correlated with the dynamics of L. dispar caterpillar deaths during the study period. The dynamics of the contents of total phenols, slightly oxidized phenols, and soluble proanthocyanidins were studied using spectrophotometry.

A comparison of the early developmental morphologies ofAponogeton madagascariensisandA.boivinianus

Aponogeton madagascariensis (Mirb.) H. Bruggen is an aquatic monocot that develops perforations in its leaves through developmentally regulated programmed cell death (PCD). Aponogeton boivinianus Baill. ex Jum. is a close relative found in comparable environments with leaves that have a similar shape with no perforations. Little is known about the early developmental morphology of the family. This study characterized and compared these two species via scanning electron microscopy (SEM) and confocal laser scanning microscopy using both fresh and fixed specimens. The shoot apical meristem (SAM) of A. madagascariensis was significantly larger than that of A. boivinianus, but there was no difference in phyllotaxy observed, as both exhibited a 2/5 spiral pattern. A novel technique using serial dissections and SEM of fresh, hydrated specimens revealed that there are 16 plastochrons before perforation formation in A. madagascariensis and 34 plastochrons until a similar developmental stage in A. boivinianus. The effects on early development of A. madagascariensis plants with supressed ethylene production were also analyzed. Ethylene inhibition alters leaf development by blocking PCD, but had no significant effect on the SAM morphology or early leaf development in A. madagascariensis.

Auxin influx importers modulate serration along the leaf margin.

SummaryLeaf shape in Arabidopsis is modulated by patterning events in the margin that utilize a PIN‐based auxin exporter/CUC2 transcription factor system to define regions of promotion and retardation of growth, leading to morphogenesis. In addition to auxin exporters, leaves also express auxin importers, notably members of the AUX1/LAX family. In contrast to their established roles in embryogenesis, lateral root and leaf initiation, the function of these transporters in leaf development is poorly understood. We report that three of these genes (AUX1, LAX1 and LAX2) show specific and dynamic patterns of expression during early leaf development in Arabidopsis, and that loss of expression of all three genes is required for observation of a phenotype in which morphogenesis (serration) is decreased. We used these expression patterns and mutant phenotypes to develop a margin‐patterning model that incorporates an AUX1/LAX1/LAX2 auxin import module that influences the extent of leaf serration. Testing of this model by margin‐localized expression of axr3–1 (AXR17) provides further insight into the role of auxin in leaf morphogenesis.

Foliar anti-herbivore defenses in Qualea multiflora Mart. (Vochysiaceae): Changing strategy according to leaf development

Knowledge about plant defense systems against herbivory is crucial to understanding trophic relationships in terrestrial ecosystems. Distinct defenses may act either alone or in combination during foliar sprouting. In our study, we explored the effect of the three most conspicuous defenses found in the leaves of Qualea multiflora (Vochysiaceae) during foliar sprouting, which are: (1) the presence of trichomes, (2) foliar toughness and (3) activity of extrafloral nectaries (EFNs). In addition, we quantified foliar herbivory at different leaf stages. We determined that the three defenses evaluated vary in effectiveness during leaf development. The number of trichomes was higher during early leaf development, toughness at the end, and the activity of EFNs (biotic defense) during the middle period. Foliar herbivory was similar at different leaf ages. The results indicate that Q. multiflora synchronizes its foliar defenses in order to optimize performance in anti-herbivory protection over time.

REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis.

As sessile organisms, plants have to continuously adjust growth and development to ever-changing environmental conditions. At the end of the growing season, annual plants induce leaf senescence to reallocate nutrients and energy-rich substances from the leaves to the maturing seeds. Thus, leaf senescence is a means with which to increase reproductive success and is therefore tightly coupled to the developmental age of the plant. However, senescence can also be induced in response to sub-optimal growth conditions as an exit strategy, which is accompanied by severely reduced yield. Here, we show that class III homeodomain leucine zipper (HD-ZIPIII) transcription factors, which are known to be involved in basic pattern formation, have an additional role in controlling the onset of leaf senescence in Arabidopsis. Several potential direct downstream genes of the HD-ZIPIII protein REVOLUTA (REV) have known roles in environment-controlled physiological processes. We report that REV acts as a redox-sensitive transcription factor, and directly and positively regulates the expression of WRKY53, a master regulator of age-induced leaf senescence. HD-ZIPIII proteins are required for the full induction of WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success.

The 60S associated ribosome biogenesis factor LSG1-2 is required for 40S maturation in Arabidopsis thaliana.

Ribosome biogenesis involves a large ensemble of trans-acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre-60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C-terminus, which is highly charged in atLSG1-2 but missing in atLSG1-1. This C-terminus of atLSG1-2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1-2 to the nucleolus. Furthermore, only atLSG1-2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T-DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1-2 18S rRNA precursors accumulate and a 20S pre-rRNA is detected, while the amount of pre-rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre-60S subunit maturation is important for the final steps of pre-40S maturation in plants. In addition, the lsg1-2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.

Isoprene emissions from downy oak under water limitation during an entire growing season: what cost for growth?

Increases in the production of terpene- and phenolic-like compounds in plant species under abiotic stress conditions have been interpreted in physiological studies as a supplementary defense system due to their capacity to limit cell oxidation. From an ecological perspective however, these increases are only expected to confer competitive advantages if they do not imply a significant cost for the plant, that is, growth reduction. We investigated shifts of isoprene emissions, and to a lesser extent phenolic compound concentration, of Quercus pubescens Willd. from early leaf development to leaf senescence under optimal watering (control: C), mild and severe water stress (MS, SS). The impact of water stress was concomitantly assessed on plant physiological (chlorophyll fluorescence, stomatal conductance, net photosynthesis, water potential) functional (relative leaf water content, leaf mass per area ratio) and growth (aerial and root biomass) traits. Growth changes allowed to estimate the eventual costs related to the production of isoprene and phenolics. The total phenolic content was not modified under water stress whereas isoprene emissions were promoted under MS over the entire growing cycle despite the decline of Pn by 35%. Under SS, isoprene emissions remained similar to C all over the study despite the decline of Pn by 47% and were thereby clearly uncoupled to Pn leading to an overestimation of the isoprene emission factor by 44%. Under SS, maintenance of isoprene emissions and phenolic compound concentration resulted in very significant costs for the plants as growth rates were very significantly reduced. Under MS, increases of isoprene emission and maintenance of phenolic compound concentration resulted in moderate growth reduction. Hence, it is likely that investment in isoprene emissions confers Q. pubescens an important competitive advantage during moderate but not severe periods of water scarcity. Consequences of this response for air quality in North Mediterranean areas are also discussed.

Switchgrass (Panicum virgatum L) flag leaf transcriptomes reveal molecular signatures of leaf development, senescence, and mineral dynamics.

Switchgrass flag leaves can be expected to be a source of carbon to the plant, and its senescence is likely to impact the remobilization of nutrients from the shoots to the rhizomes. However, many genes have not been assigned a function in specific stages of leaf development. Here, we characterized gene expression in flag leaves over their development. By merging changes in leaf chlorophyll and the expression of genes for chlorophyll biosynthesis and degradation, a four-phase molecular roadmap for switchgrass flag leaf ontogeny was developed. Genes associated with early leaf development were up-regulated in phase 1. Phase 2 leaves had increased expression of genes for chlorophyll biosynthesis and those needed for full leaf function. Phase 3 coincided with the most active phase for leaf C and N assimilation. Phase 4 was associated with the onset of senescence, as observed by declining leaf chlorophyll content, a significant up-regulation in transcripts coding for enzymes involved with chlorophyll degradation, and in a large number of senescence-associated genes. Of considerable interest were switchgrass NAC transcription factors with significantly higher expression in senescing flag leaves. Two of these transcription factors were closely related to a wheat NAC gene that impacts mineral remobilization. The third switchgrass NAC factor was orthologous to an Arabidopsis gene with a known role in leaf senescence. Other genes coding for nitrogen and mineral utilization, including ureide, ammonium, nitrate, and molybdenum transporters, shared expression profiles that were significantly co-regulated with the expression profiles of the three NAC transcription factors. These data provide a good starting point to link shoot senescence to the onset of dormancy in field-grown switchgrass.