Kranz Anatomy Research Articles

Jack of all trades - C4 photosynthesis, CAM and HCO3- use in the same tissue. A commentary on: 'Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides'.

Jack of all trades - C4 photosynthesis, CAM and HCO3- use in the same tissue. A commentary on: 'Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides'.

The Pyruvate-Phosphate Dikinase (C4-SmPPDK) Gene From Suaeda monoica Enhances Photosynthesis, Carbon Assimilation, and Abiotic Stress Tolerance in a C3 Plant Under Elevated CO2 Conditions.

A pyruvate-phosphate dikinase (C4-PPDK) gene was cloned from Suaeda monoica, which had a single-cell C4 photosynthesis pathway without Kranz anatomy and was functionally validated in a C3 model plant under different abiotic stress conditions in an ambient and elevated CO2 environment. Overexpression of SmPPDK promoted growth of C3 transgenic plants, enhancing their photosynthesis (CO2 assimilation) by lowering photorespiration under stress conditions. Transgenic plants also showed an improved physiological status, with higher relative water content (RWC), membrane integrity, concentration of glycine betaine, total soluble sugars, free amino acids, polyphenols and antioxidant activity, and lower electrolyte leakage, lipid peroxidation, free radical accumulation, and generation of reactive oxygen species (ROS), compared to control plants. Moreover, SmPPDK transgenic plants exhibited earlier flowering and higher dry biomass compared to controls. These results suggested that the C4-PPDK gene was appropriate for improvement of carbon assimilation, and it also played an important role in adaption to salinity and severe drought-induced stress. More intriguingly, an elevated CO2 environment alleviated the adverse effects of abiotic stress, particularly caused by drought through coordination of osmoprotectants and antioxidant defense systems. The molecular, physiological, metabolic, and biochemical indicators ameliorated the overall performance of model C3 plants overexpressing the C4-PPDK gene in an elevated CO2 environment, by lowering photorespiration metabolic processes, however, further studies are needed to confirm its precise role in C3 plants as protection against future climate change.

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.

Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides.

Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

Cytogenetic analysis of Bienertia sinuspersici Akhani as the first step in genome sequencing.

C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani is one of the few species in the family Amaranthaceae that can perform C4 photosynthesis within individual chlorenchyma cells, without the conventional Kranz anatomy in its leaf. This plant is salt-tolerant and is well-adapted to thrive in hot and humid climates. To date, there have been no reported cytogenetic analyses yet on this species. This study aims to provide a cytogenetic analysis of B. sinuspersici as the first step in genome sequencing. Fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici probed with 5S and 45S rDNA and Arabidopsis-type telomeric repeats. Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n = 2x = 18 consisting of nine pairs of metacentric chromosomes. Two loci of 45S rDNA were found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA were found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also colocalized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous. The FISH karyotype analysis, based on the combination of rDNAs, telomeric tandem repeat markers and C0t DNA chromosome landmarks, allowed efficient chromosome identification and provided useful information in characterizing the genome of B. sinuspersici.

Zulustylis (Abildgaardieae, Cyperaceae) – a new genus from sub-Saharan Africa

The generic placement of two African species currently included in the genus Fimbristylis, F. hygrophila and F. variegata, has been contentious. These two species, which have been known for the past half century, are glabrous tufted perennials whose spikelets have semi-distichous to spiral glume arrangement, bisexual flowers, and a pistil with an enlarged and deciduous style base. While the two species fit the gross morphology of Fimbristylis, they differ in lacking the characteristic Kranz anatomy. We present phylogenetic inference of the Abildgaardieae based on DNA sequence data and map photosynthetic type. C4 photosynthesis has arisen twice in Abildgaardieae, in the Bulbostylis and the Fimbistylis clades, with a C3 clade (including Arthrostylis) sister to rest of tribe and a C3 grade (the two African Fimbristylis species, Trachystylis, Actinoschoenus) separating the two C4 clades. The new genus Zulustylis is described here, to segregate the two aberrant African Fimbristylis species, and its taxonomy presented.

Comparative analysis of leaf vein density in Sri Lankan traditional rice varieties

The C3 photosynthetic pathway of rice is less efficient than the C4 pathway. The possibility of conversion from C3 to C4 pathways in rice is currently under experiment. In plants undergoing such a transition, higher leaf-vein densities (LVD), and development of Kranz anatomy is expected to be present at an initial stage. High LVD could exist among traditional rice and those could be used as a starting point towards developing C4 rice. In the present study, LVD (estimated by average vein length between, two large longitudinal veins (LLV), two small longitudinal veins (SLV), and the vein length per unit area (TLV)) of 23 Sri Lankan rice accessions were analysed by performing a one-way ANOVA, with mean separation using Duncan’s multiple range test. Among the tested varieties LLV, SLV, and TLV were found to be significantly (p<0.05) different among accessions. Based on the mean separation, the highest LLV was shown by Thanthiribalan, and the lowest by Dewareddhiri, Mahasuduwee, Suwadel and five other accessions. The highest SLV was reported in three accessions and the lowest was in Mahasuduwee, Suwadel and Mahakuruwee. The highest TLV was reported in Mahasuduwee, Suwadel and Mahakuruwee and the lowest in IR 64. Unlike TLV estimation, SLV can be measured directly. Given that TLV and SLV are negatively correlated (r=-0.9183) a low SLV will be a suitable parameter to select for high TLV. The accessions, Mahasuduwee, Suwadel and Mahakuruwee carried a higher LVD (lower SLV and LLV leading to higher TLV) and can be recommended as candidates for studying C3 to C4 photosynthetic pathway conversion in rice.

Activities of key enzymes in the C4 pathway and anatomy of sugarcane infected by Leifsonia xyli subsp. xyli.

Ratoon stunting disease caused by Leifsonia xyli subsp. xyli (Lxx) is a bacterial disease that has plagued sugarcane-planting countries for a long time. This study mainly analysed Lxx localization and its effects on sugarcane leaf. Badila were inocultated by bacteria of Lxx. It was noted that the number of Lxx cells were rapidly enriched in sugarcane leaves from the 150th to the 210th days of post inoculation (dpi). Lxx infection disrupted the integrity of vascular bundle sheath cells (BSC) in the 'Kranz anatomy' of leaves, resulting in irregular accumulation of starch in vascular BSC of leaves. In situ PCR showed that the Lxx localized in the xylem vessels, mesophyll cell (MC) and BSC as described before in sugarcane leaf, a new niche within the host tissues in the phloem of sugarcane stem. The gene expression and activities of phosphoenolpyruvate carboxylase (PEPC), pyruvate, orthophosphate dikinase (PPDK) and NADP-malic enzyme (NADP-ME) enzymes were lower in Lxx-inoculated sugarcane plants as compared to the MI group. Lxx infection not only disrupted the structure of vascular BSC in the C4 'Kranz anatomy' of sugarcane leaves, but also affected the activities and gene expression of the key enzymes PEPC, PPDK and NADP-ME in the C4 cycle of sugarcane suggesting a reduction in CO2 fixation. The effect of Leifsonia xyli subsp. xyli (Lxx) infection on the photosynthetic physiology of sugarcane is currently limited to the evaluation of photosynthetic parameters. This study assessed the impact of Lxx infection on the mechanism of C4 cycle CO2 fixation and to accompanying plant anatomy.

English

The aim of this work was to determine the anatomical adaptations of leaves and stems of four species of Poaceae (Cynodon dactylon L. Pers., Chloris barbata SW. (Chloridoideae), Setaria verticillata L. P. Beauv., and Panicum coloratum L. (Panicoideae)) growing naturally at Al-Ha&#39;ir region near Riyadh, Saudi Arabia. Cross-sections of the leaves revealed that the epidermis cells were spherical to oval and formed one layer with a thick cuticle as well as numerous bulliform cells in a fan shape and prickles. Ground tissue (mesophyll) consists mostly of chlorenchyma cells. A large vascular bundle surrounded by two bundle sheaths, outer sheath parenchyma and inner sheath sclerenchyma were observed in C. barbata and C. dactylon, while one bundle sheath of sclerenchyma surrounds the vascular bundle in S. verticillata and P. coloratum, while one bundle sheath with chlorenchymatous cells surrounded the small vascular bundles. Our results showed that all species contained a Kranz anatomy indicative of a C4 photosynthetic pathway despite belonging to two different subfamilies, Panicoideae and Chloridoideae. Oil droplets appeared in the mesophyll tissue of P. coloratum and S. verticillata. Cross-sections of stems revealed an epidermis which consists of one layer of cells with spherical to oval shape and had a thick cuticle. Ground tissue contains strands of chlorenchyma cells followed by sclerenchyma tissue surrounding vascular bundles, thereby making a continuous cylinder. The vascular bundles were scattered in the ground tissue, with each vascular bundle surrounded by a single sclerenchymatous bundle sheath. Our results indicate that these plants were characterized by anatomical adaptations that enhance drought-tolerance capabilities, facilitating survival in arid and semi-arid regions such as Al-Ha&#39;ir and thus these plants can be used to increase vegetation cover and pasture area in dry environments. &nbsp; Key words: Anatomy, adaptations, Poaceae, Cynodon dactylon, Chloris barbata, Setaria verticillata, Panicum coloratum.

What is the fate of xylem-transported CO2 in Kranz-type C4 plants?

High concentrations of dissolved inorganic carbon in stems of herbaceous and woody C3 plants exit leaves in the dark. In the light, C3 species use a small portion of xylem-transported CO2 for leaf photosynthesis. However, it is not known if xylem-transported CO2 will exit leaves in the dark or be used for photosynthesis in the light in Kranz-type C4 plants. Cut leaves of Amaranthus hypochondriacus were placed in one of three solutions of [NaH13 CO3 ] dissolved in KCl water to measure the efflux of xylem-transported CO2 exiting the leaf in the dark or rates of assimilation of xylem-transported CO2 * in the light, in real-time, using a tunable diode laser absorption spectroscope. In the dark, the efflux of xylem-transported CO2 increased with increasing rates of transpiration and [13 CO2 *]; however, rates of 13 Cefflux in A.hypochondriacus were lower compared to C3 species. In the light, A.hypochondriacus fixed nearly 75% of the xylem-transported CO2 supplied to the leaf. Kranz anatomy and biochemistry likely influence the efflux of xylem-transported CO2 out of cut leaves of A.hypochondriacus in the dark, as well as the use of xylem-transported CO2 * for photosynthesis in the light. Thus increasing the carbon use efficiency of Kranz-type C4 species over C3 species.

Structural and photosynthetic re-acclimation to low light in C4 maize leaves that developed under high light.

C4 plants have higher photosynthetic capacity than C3 plants, but this advantage comes at an energetic cost that is problematic under low light. In the crop canopy, lower leaves first develop under high light but later experience low light because of mutual shading. To explore the re-acclimation of C4 leaves to low light, we investigated the structural and physiological changes of the leaves of maize plants grown in shaded pots. Plants were first grown under high light, and then some of them were shaded (20 % of sunlight) for 3 weeks. Four types of leaves were examined: new leaves that developed under low light during shading (L), new leaves that developed under high light (H), mature leaves that developed under high light before shading and were then subjected to low light (H-L) and mature leaves that always experienced high light (H-H). The leaf mass per area, nitrogen and chlorophyll contents per unit leaf area, chlorophyll a/b ratio and activities of C3 and C4 photosynthetic enzymes were lower in H-L than in H-H leaves and in L than in H leaves. Unlike L leaves, H-L leaves maintained the thickness and framework of the Kranz anatomy of H leaves, but chloroplast contents in H-L leaves were reduced. This reduction of chloroplast contents was achieved mainly by reducing the size of chloroplasts. Although grana of mesophyll chloroplasts were more developed in L leaves than in H leaves, there were no differences between H-L and H-H leaves. The light curves of photosynthesis in H-L and L leaves were very similar and showed traits of shade leaves. Mature maize leaves that developed under high light re-acclimate to low-light environments by adjusting their biochemical traits and chloroplast contents to resemble shade leaves but maintain the anatomical framework of sun leaves.

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Screening of Mutants Related to the C4 Photosynthetic Kranz Structure in Foxtail Millet

C4 plants exhibit significantly higher photosynthetic, water and nutrient use efficiency compared with C3 plants. Kranz anatomy is associated with many C4 plants in which bundle sheath cells surround the veins and are themselves surrounded by mesophyll cells. This specialized Kranz anatomy is elucidated as an important contributor to C4 photosynthetic activities in C4 plant. Characterizing the molecular basis of Kranz structure formation has become a key objective for studies of C4 photosynthesis. However, severe mutants that specifically disrupt Kranz anatomy have not been identified. In this study, we detected 549 stable ethyl methane sulfonate-induced foxtail millet (cultivar Yugu1) mutants related to leaf development and photosynthesis among 2,709 mutants screened (M3/M4 generation). The identified mutants included 52 that had abnormal leaf veins (with abnormal starch accumulation based on iodine staining). Each of the 52 mutants was characterized through an analysis of leaf morphology, and through microscopic observations of leaf tissue sections embedded in resin and paraffin. In total, 14 mutants were identified with abnormal Kranz structures exemplified by small bundle sheath cell size. Additional phenotypes of the mutants included poorly differentiated mesophyll and bundle sheath cells, increased vein density and the absence of chloroplasts in the bundle sheath cells. Kranz structure mutations were accompanied by varying leaf thickness, implying these mutations induced complex effects. We identified mutations related to Kranz structure development in this trial, which may be useful for the mapping and cloning of genes responsible for mediating Kranz structure development.

Understanding the Genetic Basis of C4 Kranz Anatomy with a View to Engineering C3 Crops.

One of the most remarkable examples of convergent evolution is the transition from C3 to C4 photosynthesis, an event that occurred on over 60 independent occasions. The evolution of C4 is particularly noteworthy because of the complexity of the developmental and metabolic changes that took place. In most cases, compartmentalized metabolic reactions were facilitated by the development of a distinct leaf anatomy known as Kranz. C4 Kranz anatomy differs from ancestral C3 anatomy with respect to vein spacing patterns across the leaf, cell-type specification around veins, and cell-specific organelle function. Here we review our current understanding of how Kranz anatomy evolved and how it develops, with a focus on studies that are dissecting the underlying genetic mechanisms. This research field has gained prominence in recent years because understanding the genetic regulation of Kranz may enable the C3-to-C4 transition to be engineered, an endeavor that would significantly enhance crop productivity.

Aspects of ecological anatomy of Traganum nudatum Del. (Amaranthaceae) from the Northeast of the Algerian Sahara

This study focuses on the anatomical strategies developed by the Traganum nudatum Del., prevalent in the Algerian Sahara, particularly in the region of Oued Righ, which allows to this species to survive in a harsh environment (aridity and salinity). The anatomical structure of this species was studied using fresh materials (roots, stems and leaves). These materials have been collected from several individuals in different saline habitats. Some interesting features such as successive cambia phenomenon, calcium oxalate crystals, Kranz anatomy (salsoloid subtype), succulence, low stomata density, low stomata index, the presence of the papillae, paracytic stomata and other structures have been noticed. We can conclude that the ecological significance of evidenced adaptations by T. nudatum is supported in this article by the analysis of adaptations of other species belonging either to the Amaranthaceae or to other botanical families; and that this adaptation has no link with botanical families. In these species, the key adaptation is the ability to maintain growth processes and water saving under difficult living conditions (high summer temperatures or salty soils), regardless of the evolutionary level of the taxon.

Cross-sectional anatomy of leaf blade and leaf sheath of cogon grass (Imperata cylindrica L.)

Histology of leaf blade and sheath of cogon grass (Imperata cylindrica L.) Beauv., indicated typical C4 Kranz anatomy. Cells of adaxial epidermis were smaller and bulliform cells were present on the adaxial epidermis. The shape of bulliform cells was bulbous; 3-7 cells were present in a group and 3-5 folds larger than epidermal cells. Three types of vascular bundles in respect of size and structure were extra large, large and small and they were part of leaf blade histology. These three sizes of vascular bundles were arranged in successive manner from midrib to leaf margin. Leaf sheath bundles were of two types: large and small. Extra large bundles were flanked by five small and four large bundles but small bundles were alternate found to be with large typed bundles. Extra large bundles were of typical monocotyledonous type but the large type had reduced xylem elements and the small typed was found to be transformed into treachery elements. Small be bundles occupied half the thickness of the flat portion of leaf blade topped by large bulliform cells of the adaxial epidermis. Extra large and large bundle had been extended to upper and lower epidermis. Kranz mesophyll completely encircled the bundle sheath and radiated out into ground tissue. Midrib was projected in abaxial direction and had a central vascular bundle with large and small bundles on either side of it along the abaxial regions. The midrib vascular bundle was devoid of chlorenchymatous bundle sheath and was of non-Kranz type. Continuous sub-epidermal sclerenchyma girders were noted as adaxial hypodermis. Anatomical traits exhibited an important adaptive defense against draught and saline stress of the plant. Quantitative measurement of various anatomical traits indicated strong variations among them.J. bio-sci. 25: 17-26, 2017

Characterization of maize leaf pyruvate orthophosphate dikinase using high throughput sequencing.

In C4 photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss-of-function mutants are seedling lethal, even when plants were grown under 2% CO2 , and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

C4 photosynthesis and transition of Kranz anatomy in cotyledons and leaves of Tetraena simplex.

Tetraena simplex is an independently evolved C4 species in the Zygophylloideae (Zygophyllaceae) and a characteristic forb of saline flats in hot and sandy desert habitats. During early ontogeny, the species had a morphological shift from planar cotyledons (dorsiventral symmetry) to terete, succulent leaves (radial symmetry). We tested whether this shift had a corresponding change in internal Kranz anatomy and tissue patterning. For a comprehensive characterization of C4 photosynthesis across early ontogeny in T. simplex, structural and ultrastructural anatomical properties and localization patterns, activities, and immunoblotting of key C4 photosynthetic enzymes were compared in mesophyll and bundle sheath tissues in cotyledons and leaves. Cotyledons and leaves possessed different types of Kranz anatomy (atriplicoid type and a "Tetraena" variant of the kochioid type, respectively), reflecting the change in leaf morphology. In bundle sheath cells, key differences in ultrastructural features included increased organelle numbers and chloroplast thylakoid stacking. C4 enzymes had strict tissue-specific localization patterns within bundle sheath and mesophyll cells in both cotyledons and leaves. The decarboxylase NAD-ME maintained the highest activity, increasing from cotyledons to leaves. This classified T. simplex as fully C4 across ontogeny and a strictly NAD-ME biochemical subtype. Tetraena simplex cotyledons and leaves showed differences in Kranz type, with associated progression in ultrastructural features, and differing activities/expression levels of C4 enzymes. Furthermore, leaves characterized a new "Tetraena" variation of the kochioid Kranz anatomy.

Anatomy and ultrastructure of embryonic leaves of the C4 species Setaria viridis.

Setaria viridis is being promoted as a model C4 photosynthetic plant because it has a small genome (~515 Mb), a short life cycle (~60 d) and it can be transformed. Unlike other C4 grasses such as maize, however, there is very little information about how C4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves. Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy. Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1-3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis. The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C4 grasses.

Comparative Survey of Lignification in Aerial Organs during Development of Sugarcane (Saccharum officinarum L.)

Cell-wall lignification is among the new features that appeared as early plants began to adapt to land, possibly aiding in the development of more complex bodies. Saccharum officinarum L., a typical C4 plant with unique structure and anatomy, is one of the most important energy crops in the world with a very high yield of biomass. We analyzed lignification in the anatomy of leaf, sheath and stem, in sugarcane seedlings at four distinct stages of growth: germination stage, tillering stage, ground growth stage and maturation stage. Kranz anatomy with well-developed bundle sheath associated with Kranz mesophyll was observed but Kranz system was absent in large vascular bundles. Lignification of tissues and cells in sheath during early developmental stages is expected confirmed because during this stage, when stem tissue is absent in sugarcane, may be critical for the expansion of lignification and development of the stem and increasing the mechanical rigidity of the young plant. The lignified tissues and the intensity of lignification increased during later stages of development, especially in the stem. The most lignified tissues of the stem can be observed during the maturation stage and a little before it, which provides the stem with mechanical strength. These differentiation features may be linked to stem development, while the relevant changes in metabolites may be related to sucrose accumulation.