Leaf Blade Development Research Articles

Quantitative MRI imaging of parenchyma and venation networks in Brassica napus leaves: effects of development and dehydration

BackgroundCharacterisation of the structure and water status of leaf tissues is essential to the understanding of leaf hydraulic functioning under optimal and stressed conditions. Magnetic Resonance Imaging is unique in its capacity to access this information in a spatially resolved, non-invasive and non-destructive way. The purpose of this study was to develop an original approach based on transverse relaxation mapping by Magnetic Resonance Imaging for the detection of changes in water status and distribution at cell and tissue levels in Brassica napus leaves during blade development and dehydration.ResultsBy combining transverse relaxation maps with a classification scheme, we were able to distinguish specific zones of areoles and veins. The tissue heterogeneity observed in young leaves still occurred in mature and senescent leaves, but with different distributions of T2 values in accordance with the basipetal progression of leaf blade development, revealing changes in tissue structure. When subjected to severe water stress, all blade zones showed similar behaviours.ConclusionThis study demonstrates the great potential of Magnetic Resonance Imaging in assessing information on the structure and water status of leaves. The feasibility of in planta leaf measurements was demonstrated, opening up many opportunities for the investigation of leaf structure and hydraulic functioning during development and/or in response to abiotic stresses.

Arabidopsis TGA256 Transcription Factors Suppress Salicylic-Acid-Induced Sucrose Starvation.

Salicylic acid (SA) is produced by plants in response to pathogen infection. SA binds the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) family of receptors to regulate both positive (NPR1) and negative (NPR3/4) plant immune responses by interacting with the clade II TGACG (TGA) motif-binding transcription factors (TGA2, TGA5, and TGA6). Here, we report that the principal metabolome-level response to SA treatment in Arabidopsis is a reduction in sucrose and other free sugars. We observed nearly identical effects in the tga256 triple mutant, which lacks all clade II TGA transcription factors. The tga256 mutant presents reduced leaf blade development and elongated hypocotyls, roots, and petioles consistent with sucrose starvation. No changes were detected in auxin levels, and mutant seedling growth could be restored to that of wild-type by sucrose supplementation. Although the retrograde signal 2-C-methyl-D-erythritol-2,4-cyclodiphosphate is known to stimulate SA biosynthesis and defense signaling, we detected no negative feedback by SA on this or any other intermediate of the 2-C-methyl-D-erythritol-4-phosphate pathway. Trehalose, a proxy for the sucrose regulator trehalose-6-phosphate (T6P), was highly reduced in tga256, suggesting that defense-related reductions in sugar availability may be controlled by changes in T6P levels. We conclude that the negative regulatory roles of TGA2/5/6 include maintaining sucrose levels in healthy plants. Disruption of TGA2/5/6-NPR3/4 inhibitory complexes by mutation or SA triggers sucrose reductions in Arabidopsis leaves, consistent with the 'pathogen starvation' hypothesis. These findings highlight sucrose availability as a mechanism by which TGA2/5/6 balance defense and development.

WOX family transcriptional regulators modulate cytokinin homeostasis during leaf blade development in Medicago truncatula and Nicotiana sylvestris.

The plant-specific family of WUSCHEL (WUS)-related homeobox (WOX) transcription factors is key regulators of embryogenesis, meristem maintenance, and lateral organ development in flowering plants. The modern/WUS clade transcriptional repressor STENOFOLIA/LAMINA1(LAM1), and the intermediate/WOX9 clade transcriptional activator MtWOX9/NsWOX9 antagonistically regulate leaf blade expansion, but the molecular mechanism is unknown. Using transcriptome profiling and biochemical methods, we determined that NsCKX3 is the common target of LAM1 and NsWOX9 in Nicotiana sylvestris. LAM1 and NsWOX9 directly recognize and bind to the same cis-elements in the NsCKX3 promoter to repress and activate its expression, respectively, thus controlling the levels of active cytokinins in vivo. Disruption of NsCKX3 in the lam1 background yielded a phenotype similar to the knockdown of NsWOX9 in lam1, while overexpressing NsCKX3 resulted in narrower and shorter lam1 leaf blades reminiscent of NsWOX9 overexpression in the lam1 mutant. Moreover, we established that LAM1 physically interacts with NsWOX9, and this interaction is required to regulate NsCKX3 transcription. Taken together, our results indicate that repressor and activator WOX members oppositely regulate a common downstream target to function in leaf blade outgrowth, offering a novel insight into the role of local cytokinins in balancing cell proliferation and differentiation during lateral organ development.

LFR Physically and Genetically Interacts With SWI/SNF Component SWI3B to Regulate Leaf Blade Development in Arabidopsis.

Leaves start to develop at the peripheral zone of the shoot apical meristem. Thereafter, symmetric and flattened leaf laminae are formed. These events are simultaneously regulated by auxin, transcription factors, and epigenetic regulatory factors. However, the relationships among these factors are not well known. In this study, we conducted protein-protein interaction assays to show that our previously reported Leaf and Flower Related (LFR) physically interacted with SWI3B, a component of the ATP-dependent chromatin remodeling SWI/SNF complex in Arabidopsis. The results of truncated analysis and transgenic complementation showed that the N-terminal domain (25–60 amino acids) of LFR was necessary for its interaction with SWI3B and was crucial for LFR functions in Arabidopsis leaf development. Genetic results showed that the artificial microRNA knockdown lines of SWI3B (SWI3B-amic) had a similar upward-curling leaf phenotype with that of LFR loss-of-function mutants. ChIP-qPCR assay was conducted to show that LFR and SWI3B co-targeted the promoters of YABBY1/FILAMENTOUS FLOWER (YAB1/FIL) and IAA carboxyl methyltransferase 1 (IAMT1), which were misexpressed in lfr and SWI3B-amic mutants. In addition, the association between LFR and the FIL and IAMT1 loci was partly hampered by the knockdown of SWI3B. These data suggest that LFR interacts with the chromatin-remodeling complex component, SWI3B, and influences the transcriptional expression of the important transcription factor, FIL, and the auxin metabolism enzyme, IAMT1, in flattened leaf lamina development.

Transcriptional Changes of Cell Wall Organization Genes and Soluble Carbohydrate Alteration during Leaf Blade Development of Rice Seedlings.

Plant cell walls have two constituent parts with different components and developmental stages. Much of the mystery concerning the mechanisms of synthesis, decomposition, modification, and so forth, has been resolved using omics and microscopic techniques. However, it still remains to be determined how cell wall development progresses over time after leaf emergence. Our focus in the present study was to expand our knowledge of the molecular mechanisms associated with cell wall synthesis in rice leaf blade during three distinct stages (sink, sink-to-source transition, and source). The RNA-seq, quantitative reverse transcription PCR (qRT-PCR) and carbohydrate concentrations were evaluated using developing fifth leaf blades harvested at different time points. The results revealed that some of the essential genes for the primary cell wall (PCW) were highly upregulated in the sink-to-source transition compared to the sink stage, whereas those essential to the secondary cell wall (SCW) displayed relatively higher levels (p < 0.05) during the source stage. The concentrations of soluble carbohydrates differed via type rather than stage; we observed higher monosaccharides during the sink stage and higher di- and oligo-saccharides during the sink-to-source transition and source stages. In conclusion, our findings suggest that the transcriptional regulation of plant cell wall biosynthesis genes are both synchronistic with and independent of, and directly and indirectly governed by, the abundance of soluble carbohydrates in the developing leaf blade, and, finally, raffinose is likely to play a transport role comparable to sucrose.

YABBY Genes in the Development and Evolution of Land Plants.

Mounting evidence from genomic and transcriptomic studies suggests that most genetic networks regulating the morphogenesis of land plant sporophytes were co-opted and modified from those already present in streptophyte algae and gametophytes of bryophytes sensu lato. However, thus far, no candidate genes have been identified that could be responsible for “planation”, a conversion from a three-dimensional to a two-dimensional growth pattern. According to the telome theory, “planation” was required for the genesis of the leaf blade in the course of leaf evolution. The key transcription factors responsible for leaf blade development in angiosperms are YABBY proteins, which until recently were thought to be unique for seed plants. Yet, identification of a YABBY homologue in a green alga and the recent findings of YABBY homologues in lycophytes and hornworts suggest that YABBY proteins were already present in the last common ancestor of land plants. Thus, these transcriptional factors could have been involved in “planation”, which fosters our understanding of the origin of leaves. Here, we summarise the current data on functions of YABBY proteins in the vegetative and reproductive development of diverse angiosperms and gymnosperms as well as in the development of lycophytes. Furthermore, we discuss a putative role of YABBY proteins in the genesis of multicellular shoot apical meristems and in the evolution of leaves in early divergent terrestrial plants.

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris.

WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that controlling transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in flowering plants.

Suppression of Leaf Blade Development by BLADE-ON-PETIOLE Orthologs Is a Common Strategy for Underground Rhizome Growth.

Rhizomes are modified stems that grow horizontally underground in various perennial species, a growth habit that is advantageous for vigorous asexual proliferation. In Oryza longistaminata, a rhizomatous wild relative of cultivated rice (Oryza sativa), leaves in the aerial shoots consist of a distal leaf blade and a proximal leaf sheath [1]. Leaf blade formation is, however, suppressed in rhizome leaves. In O.sativa, BLADE-ON-PETIOLE (BOP) genes are the main regulators of proximal-distal leaf patterning [2]. During the juvenile phase of O.sativa, BOP expression is maintained at high levels by the small regulatory RNA microRNA156 (miR156), leading to formation of leaves consisting predominantly of the sheath. Here, we show that in O.longistaminata, high expression of BOPs caused by miR156 was responsible for suppression of the blade in rhizomes and that bop loss-of-function mutants produced leaves consisting of the leaf blade only. Rhizome growth in soil was also hampered in the mutants due to a severe reduction in rhizome tip stiffness. Leaf blade formation is also suppressed in the stolons of Zoysia matrella, a monocot species, and in the rhizomes of Houttuynia cordata, a dicot species, indicating that leaf blade suppression is widely conserved. We also show that strong expression of BOP homologs in both rhizome and stolon leaves rather than in aerial leaves is another conserved feature. We propose that suppression of the leaf blade by BOP is an evolutionary strategy that has been commonly recruited by both rhizomatous and stoloniferous species to establish their unique growth habit.

Control of leaf blade outgrowth and floral organ development by LEUNIG, ANGUSTIFOLIA3 and WOX transcriptional regulators.

Plant lateral organ development is a complex process involving both transcriptional activation and repression mechanisms. The WOX transcriptional repressor WOX1/STF, the LEUNIG (LUG) transcriptional corepressor and the ANGUSTIFOLIA3 (AN3) transcriptional coactivator play important roles in leaf blade outgrowth and flower development, but how these factors coordinate their activities remains unclear. Here we report physical and genetic interactions among these key regulators of leaf and flower development. We developed a novel in planta transcriptional activation/repression assay and suggest that LUG could function as a transcriptional coactivator during leaf blade development. MtLUG physically interacts with MtAN3, and this interaction appears to be required for leaf and flower development. A single amino acid substitution at position 61 in the SNH domain of MtAN3 protein abolishes its interaction with MtLUG, and its transactivation activity and biological function. Mutations in lug and an3 enhanced each other's mutant phenotypes. Both the lug and the an3 mutations enhanced the wox1 prs leaf and flower phenotypes in Arabidopsis. Our findings together suggest that transcriptional repression and activation mediated by the WOX, LUG and AN3 regulators function in concert to promote leaf and flower development, providing novel mechanistic insights into the complex regulation of plant lateral organ development.

A new anatomical interpretation for abaxialization in unifacial leaf blade of stone plants (Aizoaceae, Caryophyllales)

Unifacial leaves are described in different angiosperm groups, especially in monocotyledons, which show anatomical and molecular evidence of an abaxialization process. Among the structural characters, the anatomy of epidermis and growth tissues has been reported. Special focus has been given to vascularization patterns, such as the presence and organization of peripheral vascular bundles. In the Aizoaceae, peripheral vascular bundles exhibit two patterns: exoscopic and endoscopic bundles; however, studies including an ontogenetic approach with evidence for a unifacial condition in the group are lacking. We investigated eleven Ruschioideae species, including members of two tribes (Ruschieae and Dorotheantheae), which present flat and terete leaves. Our results show that all leaves present a bifacial leaf base, with xylem cells oriented toward the adaxial face present in collateral vascular bundles. However, the occurrence of peripheral endoscopic vascular bundles in the leaf blade in all terete leaves demonstrates a unifacial condition which is present only at the leaf blades. Based on ontogenetics studies, we demonstrate that the peripheral endoscopic bundles begin their differentiation in the abaxial side of the leaf primordia, suggesting the occurrence of an abaxialization process during leaf blade development.

Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis.

Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

WOX3 in the scene: intimacy with hormones.

How do WOX genes function to regulate key developmental programs in plants? We’re aware of their love affair with hormones, but lack good evidence of direct cause-and-effect relationships linking transcriptional activity and hormonal changes. In this issue of Journal of Experimental Botany (pages 1677–1687), Cho et al. provide that evidence for rice OsWOX3A and gibberellic acid. The name WOX stands for WUSCHEL-related homeobox, named after the founding member of the group, Arabidopsis WUSCHEL (WUS) (Mayer et al., 1998). WOX transcription factors are plant-specific, homeodomain-containing transcriptional regulators known to function in several key plant developmental programs including embryo development, root and shoot apical meristem maintenance, lateral root and crown root development, tillering and vegetative growth, leaf blade development, vascular patterning, inflorescence development, floral organ development and seed development. A major outstanding question is how WOX genes function to regulate these important processes. WOX genes do appear to have a love affair with hormones since auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) have been implicated in their actions. However, most of these associations were inferred based on gene expression changes and fluorescent markers signaling changes in the activity of the corresponding hormones. Direct cause-and-effect relationships that demonstrate transcriptional activity of WOX genes linked to measured steady-state level hormonal changes, with classical reversal of effects with hormone applications, have not been well established. Now, Cho et al. (2016) have demonstrated that rice OsWOX3A is induced by GA and directly binds to the promoter of ent-kaurenoic acid oxidase (KAO), an enzyme involved in GA biosynthesis, repressing its activity. Transgenic rice plants overexpressing OsWOX3A became dwarf (Fig. 1), suggesting a defect in GA biosynthesis or signaling. By quantifying endogenous GA intermediates, Cho and colleagues were able to show that GA20 and GA1 levels decrease in these plants. Although in vivo data were not provided for the binding of OsWOX3A to the KAO promoter and other potential OsWOX3A binding sites in the GA pathway are still unclear, the induction of OsWOX3A by GA3 treatment, reversion of the dwarf phenotype in OsWOX3A transgenic plants by GA application, the measurement of GA intermediates and the interaction of OsWOX3A with the KAO promoter using yeast one-hybrid and EMSA assays are powerful pieces of evidence that together speak loudly for the involvement of OsWOX3A in gibberellin negative-feedback regulation. But then what is the contribution of GA to the Oswox3a (nal2/3) mutant phenotypes? What other hormones are involved in generating the pleiotropic defects? Fig. 1. OsWOX3A-OX transgenic plants in the field. Courtesy of Prof. Nam-Chon Paek.

Microsatellite mapping of the mutant gene conferring interrupted development of leaf blade in Triticum aestivum L.

The growth of the leaf blade is one of the limiting factors of the life cycle of a plant. The leaf elongation rate in early plant growth stages is important for plant establishment. The rlb (reduction of leaf blade) mutation in Triticum aestivum L. (2n = 6x = 42, BBAADD genome) caused a severe interruption of leaf blade development, a reduction of spike length and spikelet number, and decreased plant height. The heterozygous plants of the hybrid ‘ANK-31’ (rlb near-isogenic line of T. aestivum ‘Novosibirskaya 67’)/Novosibirskaya 67 was used to conserve the rlb trait, since the heterozygous plants were fertile and were characterized by incisions on the leaf edge. The F2 of ANK-31/T. aestivum ‘Chinese Spring’ segregated 559 normal leaf: 162 reduction of leaf blade, fitting a 3:1 ratio. This results confirmed that the recessive allele at a single locus controlled the rlb phenotype. Using microsatellite probes, the rlb gene was mapped between Xgwm133 and Xgwm582 in chromosome arm 6DL. We discussed possible use of ANK-31 to find the negative determinants of leaf development.

Repression of AS2 by WOX family transcription factors is required for leaf development in Medicago and Arabidopsis

WOX transcription factors are key regulators of meristematic activity in plants. The Medicago WOX gene, STF, functions in maintenance of leaf marginal meristem, analogous to the function of WUS in the shoot apical meristem. Both STF and WUS directly repress AS2 expression in their respective domains. Ectopic expression of AS2 with WUS promoter leads to a narrow leaf phenotype and other phenotypes similar to the wus mutant. We also found that a wox1 prs wus triple mutant produces much narrower leaf blades than the wox1 prs double mutant, indicating that WUS genetically interacts with WOX1 and PRS in Arabidopsis leaf blade development. Our data points to a general requirement for AS2 repression in meristematic regions to allow cell proliferation.

LOOSE FLOWER, a WUSCHEL-like Homeobox gene, is required for lateral fusion of floral organs in Medicago truncatula.

The Medicago truncatula WOX gene, STENOFOLIA (STF), and its orthologs in Petunia, pea, and Nicotiana sylvestris are required for leaf blade outgrowth and floral organ development as demonstrated by severe phenotypes in single mutants. But the Arabidopsis wox1 mutant displays a narrow leaf phenotype only when combined with the prs/wox3 mutant. In maize and rice, WOX3 homologs are major regulators of leaf blade development. Here we investigated the role of WOX3 in M. truncatula development by isolating the lfl/wox3 loss-of-function mutant and performing genetic crosses with the stf mutant. Lack of WOX3 function in M. truncatula leads to a loose-flower (lfl) phenotype, where defects are observed in sepal and petal development, but leaf blades are apparently normal. The stf lfl double mutant analysis revealed that STF and LFL act mainly independently with minor redundant functions in flower development, but LFL has no obvious role in leaf blade outgrowth in M. truncatula on its own or in combination with STF. Interestingly, LFL acts as a transcriptional repressor by recruiting TOPLESS in the same manner as STF does, and can substitute for STF function in leaf blade and flower development if expressed under the STF promoter. STF also complements the lfl mutant phenotype in the flower if expressed under the LFL promoter. Our data suggest that the STF/WOX1 and LFL/WOX3 genes of M. truncatula employ a similar mechanism of action in organizing cell proliferation for lateral outgrowth but may have evolved different cis elements to acquire distinct functions.

Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved.

Correlation of mRNA and protein abundance in the developing maize leaf

To help understand regulation of maize leaf blade development, including sink-source transitions and induction of C4 photosynthesis, we compared large-scale quantitative proteome and transcriptomes collected at specific stages along the developmental maize leaf blade gradient. Proteome data were based on label-free shotgun proteomics (spectral counting) and transcript data were based on RNA-seq using the same source materials, and had been published previously (Nat Genet, 42, 2010, 1060-1067; The Plant Cell, 22, 2010, 3509-3542). Transcript and protein abundance followed near normal distributions, in contrast with several studies with other organisms. Protein observability correlated with transcript abundance following a 'lazy step function' similar to that in bacteria and yeast. mRNA and protein abundance showed significant positive correlations (up to 0.8) for log-transformed length-weighted normalized spectral abundance factor (NSAF) and reads per kilobase of exon model per million mapped reads (RPKM) and non-weighted abundances (NadjSPC and COV) in dependence of function and development. Correlations were much weaker in the leaf 'sink-source' transition zone, i.e. the zone with massive investments in leaf chloroplast biogenesis and build-up of photosynthetic capacity. Clustering analyses of gene-specific protein-mRNA ratios revealed co-ordinated shifts in control points in gene expression along the leaf blade developmental gradient. The highest protein-mRNA ratio for each gene generally corresponded to leaf developmental stages in which the protein function was most important, with the exception of the 80S ribosome. Specific examples are discussed in the context of C4 photosynthesis, leaf development and sink-source transitions. This large-scale mRNA-protein correlation analysis in plants (maize) using label-free spectral counting for protein quantification and RNA-seq for mRNA abundance will provide a template for future mRNA-protein correlation studies.

Validation of leaf undulation traits in the taxonomy of Epipactis muelleri Godfery, 1921 (Orchidaceae, Neottieae)

Leaf undulation is used as a diagnostic feature in the taxonomy of different taxa of the Epipactis (Orchidaceae) genus. It is considered a specifically important trait, apart from the column (gynostemium) structure, in the identification of E. muelleri. However, leaf undulation is a common developmental phenomenon in various plants, including orchids, thus the main goal of the research presented was to validate the usefulness of this feature in Epipactis taxonomy. In the course of this study, the leaf structure of six Epipactis species was analyzed, i.e., E. albensis, E. atrorubens, E. helleborine, E. muelleri, E. palustris and E. purpurata. The leaf margin outline and the cellular pattern of the leaf surface, as well as the presence of papillae, were examined in detail. The results obtained showed that in all taxa analyzed, representing different groups of the Epipactis genus, leaf undulation was present. The phenomenon was related to leaf blade development and the local differential growth of leaf sectors rather than to the taxonomic position of the species. Therefore, leaf undulation studied in Helleborines does not have a diagnostic value as a non-programmed intrinsic feature and should not be applied to taxa identification. The valid trait in Epipactis classification (including E. muelleri) is the column structure.

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.

A Maize Thiamine Auxotroph Is Defective in Shoot Meristem Maintenance

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.