Palisade Mesophyll Cells Research Articles

Cutinsomes of Malus Mill. (Rosaceae) leaf and pericarp: genesis, localization, and transport

New data were obtained on specific bionanostructures, cutinsomes, which are involved in the formation of cuticles on the surface of leaf blades and pericarp of Malus domestica Borkh (Malus Mill., Rosaceae)introduced to the mountains at the altitudes of 1200 and 1700 m above sea level. Cutinsomes, which are electron-dense structures of spherical shape, have been identified by transmission electron microscopy. It was demonstrated that plastids can be involved in the synthesis of their constituent nanocomponents. The greatest number of nanoparticles was observed in the granal thylakoid lumen of the chloroplasts in palisade mesophyll cells and pericarp hypodermal cells. The transmembrane transport of cutinsomes into the cell wall cuticle proper by exocytosis has been visualized for the first time. The plasma membrane is directly involved in the excretion of nanostructures from the cell. Nanoparticles of cutinsomes in the form of necklace-like formations line up in a chain near cell walls, merge into larger conglomerates and are loaded into plasmalemma invaginations, and then, in membrane packing, they move into the cuticle, which covers both outer and inner cell walls of external tissues. The original materials obtained by us supplement the ideas about the non-enzymatic synthesis of cuticle components available in the literature and expand the cell compartment geography involved in this process.

Single-Cell Transcriptome Atlas of Leaves at Different Developmental Stages in Populus alba × Populus glandulosa Clone 84K

Leaves are crucial photosynthetic plant organs. The development of poplar leaves has spatio-temporal specificity and it is of great significance to study the single-cell transcription atlas of leaves to reveal the temporal regulation of gene expression in different cell types. Here, single-cell RNA sequencing was performed on 17,768 tender leaf and 5846 functional leaf cells of Poplar 84K to construct a transcriptome atlas and developmental trajectory. The results showed that there were five and six cell types in tender and functional leaves, respectively. According to a pseudo-time trajectory analysis and the clustering of expressed genes into different cell types, the development of tender and functional leaves was divided into two temporal stages. Tender leaf epidermal cells developed earliest and were enriched with genes related to cell division and growth, indicating that tender leaves were in the stage of cell expansion and functional differentiation. Functional leaf palisade mesophyll cells were enriched with genes related to photosynthesis and carbon metabolism and cell types performing different functions tended to mature, indicating that functional leaves were in the stage of leaf development and the initial formation of photosynthesis. Our in-depth analysis of the transcriptional regulation at the single-cell level during leaf development provides an important basis for studying the mechanisms involved in cell differentiation and leaf development in poplar as well as other plants.

Morphoanatomical Responses and Stomatal Conductance in Shaded and Sun-Exposed Broussonetia papyrifera Seedlings

Investigating invasive species’ morphoanatomical characteristics, such as leaf area and thickness of vascular tissues and storage cells, can reveal information about their adaptive ability and competitive advantage. Thus, we analyzed the variation in leaf, stem, and root morphoanatomical structure and stomatal conductance between shaded and sun-exposed seedlings of Broussonetia papyrifera to provide insights into management strategies to control its invasive ability. The variations in leaf area (LA), Leaf Mass per Area (LMA), stomatal density, thickness of palisade and spongy mesophyll tissues, thickness of xylem parenchyma cells, diameter of all xylem vessels in stem and roots and stomatal conductance were analyzed between shaded and sun-exposed seedlings. Results revealed that the LA and LMA of B. papyrifera were significantly larger in the sun-exposed than in the shaded condition. Seedlings exposed to sunlight also had thicker palisade mesophyll and xylem parenchyma cells. The diameter of xylem vessels in roots was also significantly higher in sun-exposed seedlings. Contrarily, a reverse pattern was observed in the diameter of stem xylem vessels (i.e., shaded > sun-exposed seedlings). Physiologically, a higher stomatal conductance was also observed in sun-exposed seedlings. In conclusion, we found significant variations in leaf, stem, and root morphoanatomy and stomatal conductance between shaded and sun-exposed seedlings of B. papyrifera. The findings of the present study may provide valuable implications for managing or controlling its invasibility in the introduced range. Keywords: freehand technique, invasive species, Moraceae, paper mulberry, stomatal conductance

Structural and functional characteristics of the leaves of economically valuable plants in arid environments ​

The difficult forest conditions of the Volgograd region determine the relevance of research in the field of agroforestry. For the selection and use of tree species in plantations during the arrangement of agricultural land, it is necessary to identify the structural and functional features of the leaves, which are reliable indicators of the mechanism of plant adaptation to stress factors. The experiment was set up during June to September 2022 under field and laboratory conditions with the participation of the dendrological collections of the Nizhnevolzhskaya station for tree species breeding, a branch of the Federal Scientific Center for Agroecology of the Russian Academy of Sciences (50°078ʹN, 45°370ʹE). The objects of research are shrubs of different geographical origin: Berberis vulgaris L., Berberis heteropoda Schrenk., Ligustrum vulgare L., Cotinus coggygria Scop., Mahonia aquifolium Nutt., Corylus avellana L. and Ribes aureum Pursh. An optical method was used to assess the permeability of the epidermis of native mature leaves of the middle tier of the crown. The dynamics and variation of the content of photosynthetic pigments has been established. In Ligustrum vulgare, Mahonia aquifolium and Cotinus coggygria, a higher content of chlorophylls and a smaller number of palisade mesophyll cells were found than in heliophytes. The species specificity of the structural features of the palisade mesophyll, taking into account the ecological groups of shrubs, was revealed, as well as a positive correlation between the number of cells and the amount of chlorophyll of the palisade mesophyll in Cotinus coggygria (0.87) and Ribes aureum (0.85), Corylus avellana (0.63). The conducted studies allow obtaining new data on the adaptive capabilities of shrubs under the action of stress factors for their selection and further practical application in the field of agroforestry.

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

Phytotoxic effects of chemically synthesized copper oxide nanoparticles induce physiological, biochemical, and ultrastructural changes in Cucumis melo.

Nanotechnology has achieved great attention due to its impressive performance especially engineered nanoparticles (ENPs). Copper-based nanoparticles offer favorable development in the fabrication of agrochemicals including fertilizers and pesticides in the field of agriculture. However, their toxic impact on melon plants (Cucumis melo) still needs to be investigated. Therefore, the aim of the current work was performed to focus on the toxic impact of Cu oxide nanoparticles (CuONPs) in hydroponically grown Cucumis melo. Our results demonstrated that CuONPs with 75, 150, and 225 mg/L significantly (P<0.005) suppressed the growth rate and badly affect physiological and biochemical activities in melon seedlings. Also, results revealed remarkable phenotypical changes besides significantly reduced fresh biomass and decreased levels of total chlorophyll contents in a dose-dependent manner. Atomic absorption spectroscopy (ASS) analysis exhibited that C. melo treated with CuONPs accumulates NPs in the shoot. Moreover, exposure to higher CuONPs (75-225mg/L) significantly increased the reactive oxygen species (ROS) accumulation, malondialdehyde (MDA), and hydrogen peroxide (H2O2) level in the shoot and induced toxicity in melon root with an increase in electrolyte leakage. Furthermore, antioxidant enzyme peroxidase (POD) and superoxide dismutase (SOD) activity in the shoot significantly increased under exposure to higher CuONPs. Exposure to higher concentrations of CuONPs (225 mg/L) significantly deformed the stomatal aperture. Furthermore, reducing the number and abnormal size of palisade mesophyll and spongy mesophyll cells were investigated especially at high doses of CuONPs. Overall, our current work demonstrates that CuONPs of 10-40 nm size provide direct evidence for a toxic effect in C. melo seedlings. Our findings were expected to inspire the safe production of NPs and agrifood security. Thus, CuONPs prepared from toxic route and its bioaccumulation into our food chain through crop plants possess a serious threat to the ecological system.

Variations in leaf anatomical characteristics drive the decrease of mesophyll conductance in poplar under elevated ozone.

Decline in mesophyll conductance (gm ) plays a key role in limiting photosynthesis in plants exposed to elevated ozone (O3 ). Leaf anatomical traits are known to influence gm , but the potential effects of O3 -induced changes in leaf anatomy on gm have not yet been clarified. Here, two poplar clones were exposed to elevated O3 . The effects of O3 on the photosynthetic capacity and anatomical characteristics were assessed to investigate the leaf anatomical properties that potentially affect gm . We also conducted global meta-analysis to explore the general response patterns of gm and leaf anatomy to O3 exposure. We found that the O3 -induced reduction in gm was critical in limiting leaf photosynthesis. Changes in liquid-phase conductance rather than gas-phase conductance drive the decline in gm under elevated O3, and this effect was associated with thicker cell walls and smaller chloroplast sizes. The effects of O3 on palisade and spongy mesophyll cell traits and their contributions to gm were highly genotype-dependent. Our results suggest that, while anatomical adjustments under elevated O3 may contribute to defense against O3 stress, they also cause declines in gm and photosynthesis. These results provide the first evidence of anatomical constraints on gm under elevated O3 .

Cellular-level distribution of manganese in Macadamia integrifolia, M. ternifolia, and M. tetraphylla from Australia.

Macadamia integrifolia and M. tetraphylla, unlike M. ternifolia, are known for their edible nuts. All three species over-accumulate the trace metal nutrient manganese (Mn) in their shoots. This study seeks to examine tissue- and cellular-level distribution of Mn and other plant nutrients in the three Macadamia species. The distribution of Mn, calcium, iron, and potassium were investigated in whole leaves and cross-sections of roots, petioles, and leaves using synchrotron-based X-ray fluorescence microscopy (XFM) in M. integrifolia, M. tetraphylla, and M. ternifolia. The results show Mn sequestration primarily in the leaf and midrib palisade mesophyll cells of all three species. Leaf interveinal regions, root cortical cells, and phloem cells were also found to be Mn loaded. The current study confirms earlier findings but further reveals that Mn is concentrated in the vacuoles of mesophyll cells owing to the exceptional resolution of the synchrotron XFM data, and the fact that fresh hydrated samples were used. New insights gained here into Mn compartmentalization in these highly Mn-tolerant Macadamias expand knowledge about potentially toxic over-accumulation of an essential micronutrient, which ultimately stands to inform strategies around farming edible species in particular.

Evolutionary history of CAM photosynthesis in NeotropicalClusia: insights from genomics, anatomy, physiology and climate

AbstractClusia is a remarkable genus of Neotropical woody plants as its members engage in either C3 photosynthesis or employ, to varying degrees, crassulacean acid metabolism (CAM) photosynthesis. Information about the evolutionary history of CAM in Clusia is scarce. Restriction site-associated sequencing of 64 species (20% of the genus) provided strong support for most of the previously recognized nine lineages. Ancestral reconstruction using maximum parsimony or maximum likelihood under a one-rate model suggested that CAM evolved at least four times independently from a most recent common ancestor (MRCA) with C3, whereas a maximum likelihood two-rate model suggested that CAM was already present in the MRCA followed by reversions to C3 in several lineages. Phylogenetic generalized least square analysis assessed variation in seven leaf anatomical traits and CAM activity measured as δ 13C. Results indicate that CAM is highly correlated with palisade mesophyll layer thickness and cell size. In addition, correlation between 19 bioclimatic variables and δ 13C was evaluated. It was found that CAM is positively correlated with habitats with a more severe dry season and greater precipitation seasonality. Since CAM is weakly and/or only periodically expressed in many Clusia spp., and thus not readily reflected in δ 13C, future analysis of phylogenetically-informed CAM expression in Clusia must include physiological measurements such as CO2 exchange and/or diel changes in leaf acidity for each species under investigation.

Singlet Oxygen Leads to Structural Changes to Chloroplasts during their Degradation in the Arabidopsis thaliana plastid ferrochelatase two Mutant.

During stress, chloroplasts produce large amounts of reactive oxygen species (ROS). Chloroplasts also contain many nutrients, including 80% of a leaf's nitrogen supply. Therefore, to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues, chloroplasts are prime targets for degradation. Multiple chloroplast degradation pathways are induced by photo-oxidative stress or nutrient starvation, but the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole and degraded are poorly defined. Here, we investigated the structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Under mild 1O2 stress, most fc2 chloroplasts appeared normal, but had reduced starch content. A subset of chloroplasts was degrading, and some protruded into the central vacuole via 'blebbing' structures. A 3D electron microscopy analysis demonstrated that up to 35% of degrading chloroplasts contained such structures. While the location of a chloroplast within a cell did not affect the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics, allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A correlation was observed among chloroplast swelling, 1O2 signaling and the state of degradation. Finally, plastoglobule (PG) enzymes involved in chloroplast disassembly were upregulated while PGs increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

Effects of polyploidization on photosynthetic characteristics in three Lilium species

Photosynthesis play an important role in plant growth. Generally, it will be increased with increased DNA content per cell. However, in organ levels, the changes of photosynthesis are species dependent. So, for target breeding materials, chromosome duplication how to influence photosynthetic performances need further investigation. In this study, three lily (Lilium) species, L. rosthornii (LRI), L. primulinum var. orchraceun (LPO) and L. regale (LRE), and their colchicine-induced autotetraploids were employed in comparing photosynthetic related characteristics. For anatomic traits, the number of palisade mesophyll cells was significantly increased in LRI and LPO but decreased in LRE after polyploidization. Moreover, except the intercellular space decreased, the mesophyll cell sizes and shapes presented different changes in the three species after polyploidization. For physical-biochemical traits, the chloroplast number, thylakoid grana thickness, and photosynthetic pigments and efficiency were significantly increased in LRI after polyploidization. For LPO, the chloroplast number, and weak light absorbed and CO2 assimilated rates were significantly increased, while others showed no significant changes after polyploidization. Out of expecting, for LRE, the chloroplast number and photosynthetic pigments were significantly decreased after polyploidization. Moreover, the structures of chloroplasts were abnormal with extremely thicker thylakoid grana and more tightly arranged thylakoid layer in tetraploids than in diploids. The photosynthetic rates of LRE were similar in diploid and tetraploid in natural conditions, while the maximum photosynthetic rate (Pmax) of light-/CO2-response curves were higher in diploids than in diploids. After plants withered, larger bulblet with more developed root systems were well observed from the three tetraploid species.

Identification of putative viroplasms within banana cells infected by banana streak MY virus.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

Accumulation of phosphorus and calcium in different cells protects the phosphorus-hyperaccumulator Ptilotus exaltatus from phosphorus toxicity in high-phosphorus soils

Ptilotus exaltatus accumulates phosphorus (P) to > 40 mg g-1 without toxicity symptoms, while Kennedia prostrata is intolerant of increased P supply. What physiological mechanisms underlie this difference and protect P. exaltatus from P toxicity? Ptilotus exaltatus and K. prostrata were grown in a sandy soil with low-P, high-P and P-pulse treatments. Both species hyperaccumulated P (>20 mg g−1) under high-P and P-pulse treatments; shoot dry weight was unchanged for P. exaltatus, but decreased by >50% for K. prostrata. Under high-P, in young fully-expanded leaves, both species accumulated P predominantly as inorganic P. However, P. exaltatus preferentially allocated P to mesophyll cells and stored calcium (Ca) as occasional crystals in specific lower mesophyll cells, separate from P, while K. prostrata preferentially allocated P to epidermal and spongy mesophyll cells, but co-located P and Ca in palisade mesophyll cells where granules with high [P] and [Ca] were evident. Mesophyll cellular [P] correlated positively with [potassium] for both species, and negatively with [sulfur] for P. exaltatus. Thus, P. exaltatus tolerated a very high leaf [inorganic P] (17 mg g-1), associated with P and Ca allocation to different cell types and formation of Ca crystals, thereby avoiding deleterious precipitation of Ca3(PO4)2. It also showed enhanced [potassium] and decreased [sulfur] to balance high cellular [P]. Phosphorus toxicity in K. prostrata arose from co-location of Ca and P in palisade mesophyll cells. This study advances understanding of leaf physiological mechanisms for high P tolerance in a P-hyperaccumulator and indicates P. exaltatus as a promising candidate for P-phytoextraction.

Distribution of aluminium in hydrated leaves of tea (Camellia sinensis) using synchrotron- and laboratory-based X-ray fluorescence microscopy.

Aluminium (Al) is highly toxic to plant growth, with soluble concentrations being elevated in the ∼40% of arable soils worldwide that are acidic. Determining the distribution of Al in plant tissues is important for understanding the mechanisms by which it is toxic and how some plants tolerate high concentrations. Synchrotron- and laboratory-based X-ray fluorescence microscopy (XFM) is a powerful technique to quantitatively analyse the distribution of elements, including in hydrated and living plants. However, analysis of light elements (z < phosphorus) is extremely challenging due to signal losses in air, and the unsuitability of vacuum environments for (fresh) hydrated plant tissues. This study uses XFM in a helium environment to avoid Al signal loss to reveal the distribution of Al in hydrated plant tissues of Tea (Camellia sinensis). The results show that Al occurs in localised areas across the foliar surface, whereas in cross-sections Al is almost exclusively concentrated in the apoplastic space above and in between adaxial epidermal cells. This distribution of Al is related to the Al tolerance of this species, and accumulation of phytotoxic elements in the apoplastic space, away from sensitive processes such as photosynthesis in the palisade mesophyll cells, is a common tolerance mechanism reported in many different plant species. This study develops an XFM method on both synchrotron and laboratory sources that overcomes the drawbacks of existing analytical techniques, permitting measurement of light elements down to Al in (fresh) hydrated plant tissues.

Morphological, physiological and genetic analyses of an upward-curling leaf mutant in Brassica napus L.

Leaf rolling is a common phenotypic trait and has recently gained more attention due to its involvement in photosynthetic efficiency and crop yield. An upward-curling leaf mutant (Bnucl1) was obtained from the rapeseed cultivar Zhongshuang 9 population (Brassica napus L.) via ethyl methanesulfonate mutagenesis. In this study, morphological and histological features, physiological characters, and inheritance of the Bnucl1 were investigated. The results indicated that leaf structure such as midvein, epidermal cells, and palisade mesophyll cells displayed remarkable differences in the mutant compared to wild type (WT). Pronounced variations in the chloroplast size and shape, starch grana between the mutant and WT leaves were observed, and the chloroplasts might be largely undifferentiated in the mutant. Moreover, the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were significantly decreased in the mutant. Likewise, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were also significantly decreased in the mutant compared to WT. Enzyme activity of SOD, POD, and CAT was reduced; however, the content of H2O2 and MDA was increased in the mutant. Among the tested agronomic traits and leaf area, all the traits except siliques on the terminal raceme decreased in the mutant plants. Hence, the mutation has imposed remarkable impacts on the structure and function of leaves and agronomic traits. Genetic study indicated that this trait was monogenic and the allele for curling leaf was dominant. These results provide inroads for further evaluation to understand the mechanism of leaf development in B. napus.

The Developmental Enhancement of a C4 System With Non-Typical C4 Physiological Characteristics in Salsola ferganica (Kranz Anatomy), an Annual Desert Halophyte.

Variations of photosynthetic structures in different tissues or cells are in coordination with changes in various aspects, e.g. physiology, biochemistry, gene expression, etc. Most C4 plant species undergo developmental enhancement of the photosynthetic system, which may present different modes of changes between anatomy and physiology/biochemistry. In the current study, we investigated a Kranz-type C4 species Salsola ferganica with the progressive development of photosynthetic (PS) structure, performance of PS physiology, induction of PS enzymes, and transcriptional and translational regulation of PS genes, results revealed that S. ferganica presented C3 type anatomy in cotyledons but C4 type in leaves (C3/L4), with the C4 system separation of initial carbon fixation in the palisade mesophyll (M) cells and the following incorporation into triosephosphates and sugars in the bundle sheath (BS) cells, respectively. The BS cells continuously surrounded the vascular bundles and water storage cells in leaf anatomic structure. Compared to the single-cell C4 species Suaeda aralocaspica, S. ferganica exhibited similar developmental enhancement of C4 syndrome temporally and spatially in anatomic structures, enzyme activities, and gene expression, which suggests that completion of differentiation of the photosynthetic system is necessary for a C4 assimilation pathway. Besides, S. ferganica also displayed some different characteristics compared to S. aralocaspica in photosynthetic physiology, e.g. a more flexible δ13C value, much lower phosphoenolpyruvate carboxylase (PEPC) activity, and an insensitive response to stimuli, etc., which were not typical C4 characteristics. We speculate that this may suggest a different status of these two species in the evolutionary process of the photosynthesis pathway. Our findings will contribute to further understanding of the diversity of photosynthesis systems in Kranz-type C4 species and the Salsola genus.

A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure.

TRNA Wobble Modification Affects Leaf Cell Development in Arabidopsis thaliana.

The tRNA modification at the wobble position of Lys, Glu and Gln (wobbleU* modification) is responsible for the fine-tuning of protein translation efficiency and translation rate. This modification influences organism function in accordance with growth and environmental changes. However, the effects of wobbleU* modification at the cellular, tissue, or individual level have not yet been elucidated. In this study, we show that sulfur modification of wobbleU* of the tRNAs affects leaf development in Arabidopsis thaliana. The sulfur modification was impaired in the two wobbleU*-modification mutants: the URM1-like protein-defective mutant and the Elongator complex-defective mutants. Analyses of the mutant phenotypes revealed that the deficiency in the wobbleU* modification increased the airspaces in the leaves and the leaf size without affecting the number and the area of palisade mesophyll cells. On the other hand, both mutants exhibited increased number of leaf epidermal pavement cells but with reduced cell size. The deficiency in the wobbleU* modification also delayed the initiation of the endoreduplication processes of mesophyll cells. The phenotype of ASYMMETRIC LEAVES2-defective mutant was enhanced in the Elongator-defective mutants, while it was unchanged in the URM1-like protein-defective mutant. Collectively, the findings of this study suggest that the tRNA wobbleU* modification plays an important role in leaf morphogenesis by balancing the development between epidermal and mesophyll tissues.

Genetic and Developmental Basis for Increased Leaf Thickness in the Arabidopsis Cvi Ecotype.

Leaf thickness is a quantitative trait that is associated with the ability of plants to occupy dry, high irradiance environments. Despite its importance, leaf thickness has been difficult to measure reproducibly, which has impeded progress in understanding its genetic basis, and the associated anatomical mechanisms that pattern it. Here, we used a custom-built dual confocal profilometer device to measure leaf thickness in the Arabidopsis Ler × Cvi recombinant inbred line population and found statistical support for four quantitative trait loci (QTL) associated with this trait. We used publically available data for a suite of traits relating to flowering time and growth responses to light quality and show that three of the four leaf thickness QTL coincide with QTL for at least one of these traits. Using time course photography, we quantified the relative growth rate and the pace of rosette leaf initiation in the Ler and Cvi ecotypes. We found that Cvi rosettes grow slower than Ler, both in terms of the rate of leaf initiation and the overall rate of biomass accumulation. Collectively, these data suggest that leaf thickness is tightly linked with physiological status and may present a tradeoff between the ability to withstand stress and rapid vegetative growth. To understand the anatomical basis of leaf thickness, we compared cross-sections of Cvi and Ler leaves and show that Cvi palisade mesophyll cells elongate anisotropically contributing to leaf thickness. Flow cytometry of whole leaves show that endopolyploidy accompanies thicker leaves in Cvi. Overall, our data suggest that mechanistically, an altered schedule of cellular events affecting endopolyploidy and increasing palisade mesophyll cell length contribute to increase of leaf thickness in Cvi. Ultimately, knowledge of the genetic basis and developmental trajectory leaf thickness will inform the mechanisms by which natural selection acts to produce variation in this adaptive trait.

Isolation of Arabidopsis Palisade and Spongy Mesophyll Cells.

Cell-type-specific transcription factors are key to deducing the distinct functions of specialized cells from gene expression profiles. Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. Palisade and spongy mesophyll cells are sub-tissues that have different morphologies and physiologies. Thus, determining the palisade and spongy mesophyll-specific transcription factors from the respective sub-tissue-specific transcriptomes is vital to understanding or verifying functions of major plant tissues. One way in which gene expression profiles can be addressed is through direct isolation. Here, we present rapid and simple methods to isolate palisade and spongy mesophyll cells mechanically and enzymatically. This method provides a good yield of each isolated cell type, and the isolated cells can be used for various downstream applications.