小麦根尖细胞分化过程中DNA,RNA和蛋白质含量变化的研究

小麦(Triticum aestivum L.)种子在25℃条件下萌发3d,根生长至1～2cm长时,于双筒解剖镜下严格切取根分生区、伸长区和成熟区。用专一性荧光染料Hochest33258、Pyronin G和FITC分别染细胞核DNA、RNA和蛋白质,并用自动图像分析技术和细胞荧光测定术分别测定三个区中各125个细胞核DNA的相对含量和各100个细胞中RNA和蛋白质的相对含量。核DNA相对含量随着根尖细胞分化的进程,DNA含量递增,成熟区细胞中含量最高。RNA的相对含量则与之相反,在分生区细胞中含量最高,成熟区细胞中含量最低。蛋白质的相对含量则在伸长区细胞中最高,分生区细胞中最低。讨论了根尖细胞分化过程中DNA、RNA和蛋白质三者之间变化的一些内在联系。     

小麦根尖细胞分化过程中超微结构变化的研究

本文研究了小麦根尖分生区、伸长区和成熟区中细胞的超微结构变化。发现细胞核从分生区到成熟区,其大小、形态及其内核仁和异染色质结构均发生一些有规律的变化;内质网、液泡、线粒体、质体、细胞壁和胞间隙也存在着一系列有规律的变化;并讨论了这些动态变化与根尖细胞分化的内在联系。     

Inhibition of mitosis in pisum root meristems by continuous gamma radiation: the influence of temperature on the synthesis of DNA, RNA and protein during inhibition

Tritiated precursors of DNA, RNA and protein were used to measure synthesis at 10 and 20C in root meristem cells of Pisum after they were mitotically arrested by continuous irradiation with gamma rays. The experiments were designed to determine if the arrested cells accumulated in a certain part of interphase, to determine the effect on DNA, RNA and protein synthesis, to find out if the effects were temperature dependent, and finally to reveal possible relationships between growth inhibition and altered synthesis. The results showed that the incorporation of DNA and RNA precursors was impaired by irradiation and that decreased temperature further increased radiation impairment of DNA synthesis. Protein synthesis on the other hand was not impaired by irradiation at either temperature. Irradiation at 20C reduced the number of DNA-synthesizing cells; at 10C this number was reduced to near zero. Although irradiated cells synthesizing RNA showed a reduction in grain counts when compared to the controls, they still retained the ability to incorporate tritiated uridine at 10C. It was hypothesized that the combination of reduced DNA and RNA synthesis and unaffected protein synthesis resulted in precocious maturation of the arrested meristem cells. Growth which occurred in the absence of cell division was attributed to meristematic cells which precociously matured and cells which were in the region of elongation.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.     

Restoration of cell growth and proliferation in the wheat roots after their inhibition by nickel sulfate

The dynamics of cell growth and proliferation restoration in different tissues and quiescent center (QC) in the wheat (Triticum aestivum L.) seedling roots and also the differentiation of rhizodermal cells and lateral root initiation after 48-h treatment with 100 μM NiSO₄ were studied. Within 24 h after nickel removal from medium, root growth was resumed due to the increase in the rate of cell growth in the meristem and the region where cell elongation started in control roots. Stimulation of cell proliferation was restored in the main part of the meristem and later in the initial cells of the files and QC. Cell proliferation was not observed in the QC. The time of cell proliferation resumption in the roots and in tested tissues depended on the degree of their injury by nickel treatment. In most tested roots, DNA synthesis and cell division were restored in 32 h. In the cells leaving the meristem due to the resumption of their growth and proliferation, growth of root hairs started. In 48 h, the number of roots with perished cells in the rhizodermis in the meristem was sharply increased and the regeneration of the damaged region by the cells of outer cortex was observed. Only after the appearance of root hairs, the cells coming from the meristem started to elongate. In most roots, the formation of the new elongation zone occurred in 56 h. During its formation, the initiation of lateral root primordia was shifted in the basipetal direction. It was concluded that the cessation of cell growth and proliferation under the influence of high concentration of heavy metal (HM) ions is not lethal for the root. At the action of toxic HM concentrations, the plant strategy is the maintenance of meristematic cell capacity for cell growth and proliferation resumption. The cellular mechanism of this capacity maintenance is the transition of meristematic cells from G₁ phase to dormancy due to growth inhibition and the inhibition of the transition to DNA synthesis.     

NUCLEOLAR VARIATION DURING DIFFERENTIATION OF PHLEUM ROOT EPIDERMIS

Striking differences in nucleolar volume were found between trichoblasts and hairless initials all along the differentiation gradient of the tissue. The larger nucleoli of the trichoblasts were evident from the onset of their differentiation in the meristem and remained so throughout the growing 1000μ of epidermis. At the same time, nucleoli of the alternating, hairless initials rapidly became reduced in volume and virtually disappeared in maturing cells. Nuclear volume was relatively constant throughout the epidermis, so that nuclear:nucleolar volume ratios clearly indicated the nucleolar differences between the shorter trichoblasts and longer hairless initials. Along with the first signs of nucleolar volume difference in the meristem, there were higher concentrations of RNA and ribonucleoprotein in nucleoli and cytoplasm of trichoblasts compared with hairless initials. Although these chemical differences occurred principally in meristem cells, protein content was much higher in trichoblasts than in the alternating hairless initials of the enlargement zone, 300–500μ from the root tip apex. There was no essential difference in protein content between the 2 cell types in the meristem or in older enlarging cells. The data suggested that the initial increase in nucleolar volume and content of the meristem trichoblasts led to their increased protein content and enzyme activities during the enlargement phase of their differentiation. The sharp reduction in nucleoli of the hairless initials, at the same time, led to their generally lower metabolism during tissue differentiation.     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

EARLY HISTOGENESIS AND SEMIQUANTITATIVE HISTOCHEMISTRY OF LEAF INITIATION IN TRITICUM AESTIVUM

The shoot apex of Triticum aestivum cv. Ramona 50 was investigated histologically to describe cell lineages and events during leaf initiation. During histogenesis three periclinal divisions occurred in the first apical layer, with one or two divisions in the second apical layer. This sequence of cell divisions initially occurred in one region and spread laterally in both directions to encircle the meristem. Cells of the third apical layer were not involved in leaf histogenesis. Initially, young leaf primordia were produced from daughter cells of periclinal divisions in the two outer apical layers. Nuclear contents of protein, histone, and RNA in the shoot apex were evaluated as ratios to DNA by means of semiquantitative histochemistry. Daughter cells of periclinal divisions in the outer apical layer which produced the leaf primordia had higher histone/DNA ratios than cells of the remaining meristem. However, protein/DNA and RNA/DNA ratios were similar in both regions. Leaf initial cells had a higher ³H-thymidine labeling index, a higher RNA synthesis rate, and smaller nuclear volumes than cells of the residual apical meristem.     

Dependence of Root Cell Growth and Division on Root Diameter

Primary roots of 98 species from different families of monocotyledonous and dicotyledonous plants and adventitious roots obtained from bulbs and rhizomes of 24 monocot species were studied. Root growth rate, root diameter, length of the meristem and elongation zones, number of meristematic cells in a file of cortical cells, and length of fully elongated cells were evaluated in each species after the onset of steady growth. The mitotic cycle duration and relative cell elongation rate were calculated. In all species, the meristem length was approximately equal to two root diameters. When comparing different species, the rate of root growth increased with a larger root diameter. This was due to an increase in the number of meristematic cells in a row and, to a lesser degree, to a greater length of fully elongated cells. The duration of the mitotic cycle and the relative cell elongation rate did not correlate with the root diameter. It is suggested that the meristem size depends on the level of nutrient inflow from upper tissues, and is thereby controlled during further growth.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

Changes in the Nucleus during Cellular Development in the Pea Seedling

Nuclei were isolated from three regions of the root and fromthe epicotyls of growing pea seedlings. As the dry mass of thenuclei was the same before and after isolation, and as the DNAcontent of the nuclei accounted for the DNA of the cells, theisolated nuclei are assumed to be a random sample of the nucleiin the intact tissues. The DNA, RNA, and protein contents percell were much greater in the epicotyl than in the root, butthe RNA and protein contents of the nucleus were similar inboth epicotyl and root. Although the amounts of DNA, RNA, andprotein per cell increased with increasing distance from theroot tip, increase in the protein content of the nucleus occurredmainly between the meristematic and expanding regions, and atthis same point nuclear RNA decreased. Nuclear volume increasedwith increasing distance from the root tip, whereas nucleardry mass remained virtually unchanged. The nucleus thereforebecomes increasingly hydrated as the cells mature. The datasuggest a changing interaction between the nucleus and the cytoplasmduring cellular development.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

ORIGIN,DEVELOPMENT, AND GROWTH OF DIFFERENTIATING TRICHOBLASTS IN ELODEA CANADENSIS

Root hairs of Elodea canadensis develop only from cells which undergo a particular series of developmental steps. These cells, the trichoblasts, are formed as the smaller, proximal product of an asymmetric division, and immediately enter a prolonged phase of synthesis. Histochemical tests show that large amounts of RNA and protein accumulate in the vastly enlarged nucleolus and cytoplasm, while histone increases in the enlarging nucleus. Cytophotometry shows that DNA in the nucleus reaches polyploid levels. Throughout the synthetic phase, almost to the point of root hair initiation at 9.5 mm proximal to the meristem initials, vacuolation is delayed and the trichoblasts elongate less extensively. All results suggest that this synthesis is the type which normally follows cell division, but is greatly enhanced in the trichoblast. In contrast, the initially larger atrichoblasts only accumulate RNA, DNA, and protein in the region from 1 mm to 2 mm proximal to the meristem tip, and they then enter a phase of extensive vacuolation and elongation.     

Changes in cell length and mitotic index in vascular pattern-related pericycle cell types along the apical meristem and elongation zone of the onion root

Summary Three pericycle cell types (opposite xylem, opposite phloem and intervening) distinguished by their location in relation to different elements of the vascular system were studied in the adventitious root ofAllium cepa L. Changes in cell length and mitotic index were analysed in these cells along the apical meristem and elongation zone of the root. The opposite phloem and intervening pericycle cells are significantly shorter than the opposite xylem pericycle cells in the apical half of the meristem. Between 1,200 and 1,400 m behind the tip, length became similar in all three pericycle cell types, while in more proximal zones the opposite phloem cells were significantly longer. These results suggest that the number of transverse divisions is different in the three types of pericycle cells. In the apical half of the meristem, mitotic index increased in intervening and opposite xylem cells but remained unchanged in opposite phloem cells, a fact likely to account for the relative lengthening of the latter. In the proximal half of the meristem, mitotic index fell in all three cell types until cell division had ceased. However, mitotic index in opposite xylem cells remained high for longer than in the other two cell types, implying that increase of the mean cell length in the former was slower. These results suggest that differences in mean cell length between the three pericycle cell types are due to different rates of proliferation.     

Cytochemical determination of changes in nuclear histone content in differentiating root cells of barley and garlic

Summary Changes in nuclear histone content in differentiating root cells of barley and garlic have been studied by cytochemical methods. The results obtained indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclear histones of cell nuclei found in meristem contain histones rich in lysine. Intermediate or transitional types of nuclear histones were also observed in cell nuclei which were undergoing differentiation or elongation and in chromosomes of mitotic cells.Contribution from the Division of Genetics and Cytology, Department of Botany, University of Tokyo, No. 395.     

DNA, RNA, and Protein in the Pea Shoot Apex in Relation to Leaf Initiation

The average amounts of DNA, RNA, and protein per cell measuredhistochemically in each region of the shoot apical meristemremained unchanged during the course of a plastochron and duringthe early development of the leaf primordium. The average contentof DNA, RNA, and protein per cell was the same in all regionsof the shoot apical meristem.