The course of root differentiation from root primordia in poplar stems

Upon rooting of poplar stem cuttiags the total inductive stimulation does not take place, but the individual root primordia maintain a relative independence. This becomes evident by various time parameters of their differentiation. Under experimental conditions, in dormant cuttings from one year individuals of the hybrid I 214 the activation of the first root primordia occurs after 24 h, the pre-emergent development of the roots formed was completed after 72 h. The activated root primordium is divided into two regions. In the distal region with the predominating cell division the root apex with histogens is formed by the action of initials. From peripheral cell layers of the distal region the so-called “Wurzeltasche” develops which covers the root cap. Due to cell elongation in the proximal region the root apex is pushed up towards the stem surface. The beginning differentiation of the connective vascular tissue is a preparative step for the connection of the vascular system of the developing root with the secondary vascular system of the maternal stem. Following the penetration of the root through the peripheral stem tissues this connection is realized with progressing development. In the developed root the protoxylem elements differentiate continuously and acropetally in direct continuity with tracheids of the basal connective region.     

Preformation of root primordia in shoots and root morphogenesis in Salix viminalis

Root primordia are formed in the stems of Salix viminalis L. during normal growth. Some of these primordia are produced at definite sites in the nodes. The initiation and early structural and ultrastructural development of the nodal primordia were studied in young shoots. In the fourth node below the terminal leaf cluster some parenchyma cells situated at the lateral leaf gaps formed a small group of initial cells. Derivatives of the newly formed interfascicular cambium added cells to that group, in which later on cell divisions in various directions occurred resulting in the formation of a root primordium. Root morphogenesis was studied in cuttings from one-season-old stems. The cells in the dormant primordia contained many lipid bodies but only a small amount of starch. After the cuttings had been 24 hours in water starch was accumulating in the plastids and lipid bodies were seen in the vacuoles. 48 hours after activation cell divisions occurred throughout the primordia and a layered apical mer-istem was organized. After 96 hours a root cap with amyloplasts was formed and the procambium was well developed. The amyloplasts were sedimented in response to gravity. After six days the first roots were ready to emerge from the stems. Their root caps had a well developed columella and endodermal and pericyclic cells were recognizable.     

Cellular localization of aquaporin mRNA in hybrid poplar stems

? Premise of the study: Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes. ? Methods: Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought. Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize spatial expression of three AQP genes in stem cross sections. ? Key results: The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable. High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization (control plants) and plants subjected to drought. ? Conclusions: Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.     

The seminal root primordia in barley and the participation of their non-meristematic cells in root construction

In addition to the primary seminal primordium, the so-called secondary seminal root primordia are also initiated in a barley embryo. The primary root primordium is developmentally most advanced. It is formed by root meristem covered with the root cap, and by a histologically determined region with completed cell division. On germination, the restoration of growth processes begins in this non-meristematic region of root primordium by cell elongation, with the exception of the zone adjacent to the scutellar node, the cells of which do not elongate but continue differentiating. In the root primordia initiated later, the zone with completed cell division is relatively shorter, in the youngest primordia the non-meristematic cells may be lacking. The root meristem is reactivated after the primary root primordium has broken through the sheath-like coleorrhiza and emerges from the caryopsis as the primary root. The character of root meristem indicates a reduced water content at the embryonic development of root primordium. With progressing growth the root apex becomes thinner, the meristematic region becomes longer, and the differences in the extent of cell division between individual cell types increase. — The primary root base is formed of cells pre-existing in the seminal root primordium. Upon desiccation of caryopsis in maturation, and subsequent quiescent period, their development was temporarily broken, proceeding with the onset of germination. The length of this postembryonically non-dividing basal zone is different in individual cell types. The column of central metaxylem characteristic of the smallest number of cell cycles, has, under the given conditions, a mean length of about 22 mm, whereas the pericycle, as the tissue with most prolonged cell division, has a mean length of about 6 mm. In the seminal root primordia initiated later the non-dividing areas are relatively shorter. The basal region of seminal roots thus differs in its ontogenesis from the increase which is formed “de novo” by the action of root meristem upon seed germination.     

Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia

Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Shoot meristem differentiation in shoot primordia culture (SPC) obtained from root primordia culture (RPC) of Solanum lycopersicoides Dun.

We present a unique example of conversion of the morphogenesis type (from rhisogenesis to shoot organogenesis) in in vitro cultures of Solanum lycopersicoides. Liquid shoot primordia cultures (SPC) were obtained from root primordia culture (RPC) on two kinds of MS-based media with BA. The first SMS₈, contained a full set of organic compounds; the second, ²SMS₈, was devoid of organic compounds except sucrose and edamine. Two weeks after passage of RPC onto both the media, disintegration of root primordia and cell aggregates began. After 8 weeks of cultivation on SMS₈ and ²SMS₈ media, SPC aggregates developed from meristematic cells located near the vascular tissue of disintegrating RPC aggregates. Initiation of shoot meristems started from meristematic cells centers which were localised under the surface of the newly formed SPC aggregates. The change in the type of morphogenesis occurred faster on medium SMS₈, but the SPC on medium ²SMS₈ was characterized by more frequent formation of shoots and plants (45% of aggregates, in the case of SPC on medium SMS₈, and 90% in the case of SPC on medium ²SMS₈). This fact was correlated with the structural organization of the meristematic centers. Our results indicate that an RPC system has high morphogenetic potential due to the continual presence of meristematic cells. The change in the type of morphogenesis was followed by a rebuilding of the aggregate structure on the basis of the meristematic cells already existing in RPC, which gave rise to SPC aggregates from which shoot meristems developed.     

Vesicular-arbuscular mycorrhizal fungi induced alteration in poplar root system morphology

Poplar was grown in a soil either inoculated with Scutellispora calospora, Glomus sp E3 or Glomus caledonium or to which a nutrient solution had been added, in order to determine effects on root morphology. Plants were harvested after 115 days. The lengths of individual roots were measured using image analysis and percentage colonisation was determined for different root orders. Colonisation did not affect plant size but induced large changes in root morphology, with lengths of individual secondary and tertiary roots increased in some cases by up to 100%. Root branching was also increased with number of laterals per unit length of colonised roots being up to 6 times greater than in non-colonised roots. These results clearly show that colonisation of roots by Vesicular-arbuscular mycorrhizal fungi can result in significant alteration to poplar root system morphology. They also suggest that the mechanisms of alteration are not entirely due to improved host plant nutrition.     

杨树人工林根际古菌群落随细根生长的演变

研究细根不同生长时期根际土壤古菌群落组成结构差异,对深入了解林木细根与土壤微生物互作关系具有重要理论意义.依据细根表面颜色,采集杨树一级细根不同生长时期(白色新生根、黄色成熟根、褐色衰老根)根际土壤并提取微生物总DNA,采用特异性引物对古菌16S rDNA V4-V5区进行扩增,利用Illumina MiSeq平台进行古菌高通量测序分析.结果表明: 新生根和衰老根根际土壤古菌群落操作分类单元(OTU)丰富度相似,而成熟根根际土壤古菌群落OTU数量较少.新生根和成熟根根际土壤共同含有134个OTU;成熟根和衰老根根际土壤共同含有87个OTU,新生根和衰老根根际土壤共同拥有90个OTU.α多样性分析表明,成熟根根际土壤古菌群落Chao1指数和ACE指数显著低于新生根和衰老根根际土壤,而衰老根根际土壤古菌群落Simpson指数和Shannon指数显著低于新生根和成熟根根际土壤.PERMANOVA分析表明,新生根和衰老根根际土壤古菌群落组成有显著差异.物种注释显示,杨树根际土壤共包含12个古菌属,其中新生根5个、成熟根10个、衰老根6个.β多样性指数表明,杨树根际土壤古菌群落相似度随着细根的生长逐渐下降,不同生长阶段细根根际土壤的古菌群落结构有较大差异.其中,占绝对优势的古菌为氨氧化古菌Candidatus_Nitrososphaera,其相对丰度超过70%.且随细根生长发育,该类古菌在根际土壤中的丰度呈现上升趋势,表明其可能与细根的生长发育关系密切.     

Proteome analysis of apical and basal regions of poplar stems under gravitropic stimulation

Gravity is a constant force guiding the direction of plant growth. In young poplar stem, reorientation of the apical region is mainly obtained by differential growth of elongating primary tissues. At the base, where elongation is achieved but where the cambium is active, reorientation is due to asymmetrical formation of reaction wood. After 45 min of gravistimulation, the stem showed no reorientation, but 1 week later, reaction wood was observed at the base of the stem. To determine the molecular mechanisms taking place at the top and base of the stem, after 45 min or 1 week of inclination, the changes induced in protein accumulation were studied by two-dimensional polyacrylamide gel electrophoresis and quantitatively analyzed using image analysis software. Around 300 protein spots were reproducibly detected and analyzed. Forty percent of these proteins showed significant changes after inclination. Mass spectrometry analysis of 135 spots led to the identification of 60 proteins involved in a wide range of activities and metabolisms. Very different patterns of protein expression were obtained according to conditions tested, highlighting the complexity of gravitropic responses. Our results suggest that primary and secondary tissues present specific mechanisms to sense reorientation and to respond to inclination. Some selected proteins are discussed.     

Isoenzymes of hydroxycinnamate: CoA ligase from poplar stems properties and tissue distribution

Three different forms of hydroxycinnamate: CoA ligase (EC 6.2.1-) have been separated by chromatofocusing from poplar stems. These three forms exhibit different substrate specificities and tissue distribution. A correlation was established between the monomeric composition of lignins isolated from xylem and sclerenchyma and the pattern of hydroxycinnamate: CoA ligase isoforms in these tissues. The results obtained indicate that, in poplar, the hydroxycinnamate: CoA ligase isoenzymes could play an important role in the control of the monomeric composition of lignins.     

Concerning variability in the delimitation of the vascular system in root primordia of maize embryos

1. The transfer of immature embryos from maternal plants to artificial media influenced the radial arrangement of vascular bundles in developing root primordia. The variability in the number of poles of the prospective protoxylem and protophloem, observed as a rule during embryogenesis under natural conditions, could not be suppressed even under the conditions ofin vitro cultivation. The possibility is admitted that when using agar medium the nutrient supply need not necessarily be equivalent for all embryos.
2. Using excised embryos of various ages the period of delimination of the vascular system in the root primordium was determined. It is relatively short and occurs in the first half of embryogenesis. The results obtained revealed no relationship between vascular system arrangement in root primordium and mature grain and mature embryo size.
3. Maize ear represents a type of inflorescence of which the apical part is delayed in development. Histogenically this uneven development becomes evident with the formation of a significantly lower mean number of poles in root primordia from the grains originating from the apical region of the cob. This is further evidence of the adaptibility of the vascular system development to environmental conditions.
4. As further causes of the variability in pole number those differences are considered which occur during sex cell formation, pollination and fertilization.

     

Influence of indole-3-acetic acid on adventitious root primordia of brittle willow

Summary Removal of the stem apex and certain leaves and axillary buds of brittle willows (Salix fragilis) was employed to limit the supply of endogenous auxin to adventitious root primordia during their formation, which occurs at predetermined sites. Limiting endogenous auxin by this surgical treatment resulted in reduced primordium initiation and, to a lesser degree, primordium growth in cell number. Root primordium cells in surgically treated plants differentiated into mature parenchyma after losing their meristematic character. Application of indole-3-acetic acid (IAA) to surgically treated plants partially overcame the effects of the surgical tretament, increasing root primordium initiation and growth by cell division. When IAA-2-¹⁴C was applied to surgically treated plants, label was detected in root primordium cells by means of autoradiography. Root primordium cells took up more label during the earliest stage of initiation than during a later stage of growth. The data indicate that the initiation of these primordia is more dependent on a supply of auxin than is their subsequent development. Further, the auxin apparently acts directly in the cells which initiate primordia.This investigation was supported in part by Public Health Service Research Grant No. UI 00110-07 (now 5R01 FD 00074-09) from the National Center for Urban and Industrial Health. Paper No. 7138, Min nesota Agricultural Experiment Station.     

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l^?1 of exogenous 6‐benzyl adenine (6‐BA) was administered to 3 and 7‐day‐old apple rootstocks ‘M.26’ cuttings (3 and 7 days 6‐BA) by transferring them from a rooting medium containing indole‐3‐butanoic acid to the medium containing 6‐BA. Anatomical and morphological observations revealed that the exogenous application of 6‐BA inhibited primordia formation in the 3 days 6‐BA but not the 7 days 6‐BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6‐BA than in 7 days 6‐BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6‐BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6‐BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3–7 days is the time period in which AR primordia formation occurs in cuttings of ‘M.26’ and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3–7 days period after culturing in rooting medium.     

The onset of cell proliferation is stimulated by ascorbate free radical in onion root primordia

Summary— Ascorbate free radical (AFR) accelerates the quiescency-proliferation shift in onion root primordia. The acceleration was detected by the increase of [³H]thymidine incorporation and by the anticipated kinetics of the increase in the labeling index. The shortening of the onset of cell proliferation is attributted to the role of AFR in the energization of the plasma membrane.     

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.Abbreviations PVS2 Vitrification solution - LN liquid nitrogen - BA 6-benzyladenine - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium