Different effects of gellan gum and agar on change in root elongation in Arabidopsis thaliana by polyploidization: the key role of aluminum.

Agar and gellan gum have been considered to have different effects on polyploidy-dependent growth in plants. We aim to demonstrate that agar and gellan gum differently affect the change in root elongation in Arabidopsis thaliana by polyploidization and examined the physico-chemical parameters in each gelling agent to elucidate key factors that caused the differences. Each polyploid strain was cultured vertically on agar and gellan gum solidified medium under fixed conditions. Root elongation rate was measured during 4-10 days after sowing. As a result, agar promoted root elongation of polyploids more than the gellan gum. Then water potential, gel hardness, and trace elements of each medium were quantified in each medium. Water potential and gel hardness of agar medium were significantly higher than those of gellan gum medium. The decrease in water potential and gel hardness in agar medium, however, did not affect the change in polyploidy-dependent growth. Elemental analysis showed that gellan gum contained more aluminum than agar. Subsequently, the polyploids were grown on agar media with additional aluminum, on which the root elongation in tetraploids and octoploids was significantly suppressed. These results revealed that agar and gellan gum affect the change in growth of root elongation in A. thaliana by polyploidization in different ways and the different effects on change in polyploidy-dependent growth is partially caused by aluminum in the gellan gum, which may be due to cell wall composition of polyploids.

Mitochondrial gene defects in Arabidopsis can broadly affect mitochondrial gene expression through copy number.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization.

Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a precise spatial profile of fluorescent signals in roots using conventional WM-FISH mainly because of the severe damage caused during the processing. To address this problem, we established a novel WM-FISH analysis for intact roots of Arabidopsis thaliana and successfully obtained a precise spatial profile of nuclear size and centromere signals. The two main improvements in the novel WM-FISH analysis are: (i) hybridization was performed directly on MAS-coated glass slides covered with silicon wells and (ii) conditions for enzyme treatment were optimized (37 °C, 45 s). After the WM-FISH using a centromere probe, we analyzed the results by 3D data processing to quantify the nuclear volume and number of centromere signals of the obtained cortical cell files and determined the position of each nucleus in intact roots. Then we plotted the nuclear volume and number of centromere signals versus distance from the quiescent center to evaluate the precise spatial profile of each parameter.

Mechanical Forces in Floral Development.

Mechanical forces acting within the plant body that can mold flower shape throughout development received little attention. The palette of action of these forces ranges from mechanical pressures on organ primordia at the microscopic level up to the twisting of a peduncle that promotes resupination of a flower at the macroscopic level. Here, we argue that without these forces acting during the ontogenetic process, the actual flower phenotype would not be achieved as it is. In this review, we concentrate on mechanical forces that occur at the microscopic level and determine the fate of the flower shape by the physical constraints on meristems at an early stage of development. We thus highlight the generative role of mechanical forces over the floral phenotype and underline our general view of flower development as the sum of interactions of known physiological and genetic processes, together with physical aspects and mechanical events that are entangled towards the shaping of the mature flower.

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri.

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration is mediated by the auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia), without intervening the callus mass formation in culture with cytokinin; yet, its molecular mechanisms remain unaddressed. Here, we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Mathematical model studies of the comprehensive generation of major and minor phyllotactic patterns in plants with a predominant focus on orixate phyllotaxis.

Plant leaves are arranged around the stem in a beautiful geometry that is called phyllotaxis. In the majority of plants, phyllotaxis exhibits a distichous, Fibonacci spiral, decussate, or tricussate pattern. To explain the regularity and limited variety of phyllotactic patterns, many theoretical models have been proposed, mostly based on the notion that a repulsive interaction between leaf primordia determines the position of primordium initiation. Among them, particularly notable are the two models of Douady and Couder (alternate-specific form, DC1; more generalized form, DC2), the key assumptions of which are that each leaf primordium emits a constant power that inhibits new primordium formation and that this inhibitory effect decreases with distance. It was previously demonstrated by computer simulations that any major type of phyllotaxis can occur as a self-organizing stable pattern in the framework of DC models. However, several phyllotactic types remain unaddressed. An interesting example is orixate phyllotaxis, which has a tetrastichous alternate pattern with periodic repetition of a sequence of different divergence angles: 180°, 90°, -180°, and -90°. Although the term orixate phyllotaxis was derived from Orixa japonica, this type is observed in several distant taxa, suggesting that it may reflect some aspects of a common mechanism of phyllotactic patterning. Here we examined DC models regarding the ability to produce orixate phyllotaxis and found that model expansion via the introduction of primordial age-dependent changes of the inhibitory power is absolutely necessary for the establishment of orixate phyllotaxis. The orixate patterns generated by the expanded version of DC2 (EDC2) were shown to share morphological details with real orixate phyllotaxis. Furthermore, the simulation results obtained using EDC2 fitted better the natural distribution of phyllotactic patterns than did those obtained using the previous models. Our findings imply that changing the inhibitory power is generally an important component of the phyllotactic patterning mechanism.

Correction to: Floral development of petaloid Alismatales as an insight into the origin of the trimerous Bauplan in monocot flowers.

The caption of Figure 5 was published incorrectly in the original publication of the article.

Floral development of petaloid Alismatales as an insight into the origin of the trimerous Bauplan in monocot flowers.

Monocots are remarkably homogeneous in sharing a common trimerous pentacyclic floral Bauplan. A major factor affecting monocot evolution is the unique origin of the clade from basal angiosperms. The origin of the floral Bauplan of monocots remains controversial, as no immediate sister groups with similar structure can be identified among basal angiosperms, and there are several possibilities for an ancestral floral structure, including more complex flowers with higher stamen and carpel numbers, or strongly reduced flowers. Additionally, a stable Bauplan is only established beyond the divergence of Alismatales. Here, we observed the floral development of five members of the three 'petaloid' Alismatales families Butomaceae, Hydrocharitaceae, and Alismataceae. Outer stamen pairs can be recognized in mature flowers of Alismataceae and Butomaceae. Paired stamens always arise independently, and are either shifted opposite the sepals or close to the petals. The position of stamen pairs is related to the early development of the petals. In Butomaceae, the perianth is not differentiated and the development of the inner tepals is not delayed; the larger inner tepals (petals) only permit the initiation of stamens in antesepalous pairs. Alismataceae has delayed petals and the stamens are shifted close to the petals, leading to an association of stamen pairs with petals in so-called stamen-petal complexes. In the studied Hydrocharitaceae species, which have the monocot floral Bauplan, paired stamens are replaced by larger single stamens and the petals are not delayed. These results indicate that the origin of the floral Bauplan, at least in petaloid Alismatales, is closely linked to the position of stamen pairs and the rate of petal development. Although the petaloid Alismatales are not immediately at the base of monocot divergence, the floral evolution inferred from the results should be a key to elucidate the origin of the floral Bauplan of monocots.

Floral anatomy and vegetative development in Ceratophyllum demersum: a morphological picture of an "unsolved" plant.

PREMISE OF THE STUDY: The phylogenetic position of Ceratophyllum is still controversial in recent molecular analyses of angiosperms, with various suggestions of a sister group relation to all other angiosperms, eudicots, monocots, eudicots + monocots, and magnoliids. Therefore, the morphological characters of Ceratophyllum are important for resolving the phylogeny of angiosperms. In this study, we observed the detailed developmental anatomy of all lateral organs and their configurations to elucidate the floral development and phyllotactic pattern of Ceratophyllum demersum. METHODS: We observed fixed shoots of C. demersum with scanning electron microscopy and serial sections of the samples with light microscopy. KEY RESULTS: Bract primordia arise first, followed by the stamen primordia in staminate flowers. Both bracts and stamens initiate unidirectionally, first on the abaxial side of the floral apex and later on the adaxial side, most likely due to the contact pressure imposed by the leaf primordium at the superior node. In pistillate flowers, bract primordia on the abaxial side were also initiated first. The configuration of buds at one node showed six patterns and each pattern included at least one vegetative bud, and flower buds were always accompanied by vegetative buds at the same node. CONCLUSIONS: The initiation pattern of organs in the outer whorls of C. demersum flowers is distorted by mechanical pressure, resulting in the phyllotactic variation of staminate flowers. Vegetative buds are the main axillary buds with floral buds as accessory buds, which suggests that the shoot of C. demersum has been modified from a decussate phyllotaxis.

Transcriptional repression by MYB3R proteins regulates plant organ growth.

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program.

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.

Insight into the basis of root growth in Arabidopsis thaliana provided by a simple mathematical model.

Plant organ growth changes under genetic and environmental influences can be observed as altered cell proliferation and volume growth. The two aspects are mutually dependent and intricately related. For comprehensive growth analysis, it is necessary to specify the relationship quantitatively. Here, we develop a simple mathematical model for this purpose. Our model assumes that the biological activity of a given organ is proportional to the cell number of the organ and is allocated into three aspects: cell proliferation, volume growth, and organ maintenance. We analyzed the growth of primary roots of Arabidopsis thaliana (L.) Heynh. in one tetraploid and four diploid strains using this model. The analysis determined various growth parameters, such as specific cost coefficients of cell proliferation and volume growth for each strain. The results provide insight into the basis of interstrain variations and ploidy effects in root growth.

Development and structure of trichotomous branching in Edgeworthia chrysantha (Thymelaeaceae).

We studied the development and structure of the unusual trichotomous branching of Edgeworthia chrysantha. Three "branch primordia" are formed sequentially on the shoot apex of a main axis and develop into trichotomous branching. The branch primordia are clearly distinguishable from the typical axillary buds of other angiosperms; they develop much more rapidly than axillary buds, and the borders between the branch primordia and shoot apex of the main axis are anatomically unclear. Furthermore, at a later stage, leaves subtending the branch primordia produce typical axillary buds. These results suggest that the trichotomous branching in this species involves the division of the shoot apical meristem. Expression analysis of genes involved in branching or maintenance of the shoot apical meristem in this species should clarify the control mechanism of this novel branching pattern in angiosperms. We also observed the phyllotactic patterns in trichotomous branching and have related these patterns to the shoot system as a whole.

Floral development and phyllotactic variation in Ceratophyllum demersum (Ceratophyllaceae).

The floral development of staminate and pistillate flowers of Ceratophyllum demersum was observed, with particular focus on the phyllotactic variation in staminate flowers, using scanning electronic microscopy (SEM). We discerned patterns of development of some important new morphological features, e.g., the difference and discontinuity between the organ initiation in stamens and that in bracts (or tepals) and the initial presence of a mucilaginous appendage on each pistil. Female flowers are considered to be very specialized through reduction. In male flowers stamen initiation changes between early and late floral development. The four or five stamens in the outermost whorl initiate first on the abaxial and lateral sides of the floral apex and only later on the adaxial side (unidirectional). Later the inner stamens initiate spirally, and this is the main pattern in the stamen initiation. Members of each whorl differ among themselves in time of initiation and in ultimate size. The phyllotactic variation in staminate flowers of Ceratophyllum, suggested by previous studies, is derived from the variation in stamen number and the difference of stamen initiation between the early and later stages. The development in Ceratophyllum has some similarities to those of ANITA plants except for Nymphaeales.