Expansins and Internodal Growth of Deepwater Rice

The distribution and activity of the cell wall-loosening protein expansin is correlated with internodal growth in deepwater rice (Oryza sativa L.). Acid-induced extension of native cell walls and reconstituted extension of boiled cell walls were confined to the growing region of the internode, i.e. to the intercalary meristem (IM) and the elongation zone. Immunolocalization by tissue printing and immunoblot analysis, using antibody against cucumber expansin 29 as a probe, confirmed that rice expansin occurred primarily in the IM and elongation zone. Rice expansin was localized mainly around the vascular bundles at the base of the IM and along the inner epidermal cell layer surrounding the internodal cavity. Submergence greatly promoted the growth of rice internodes, and cell walls of submerged internodes extended much more in response to acidification than did the cell walls of air-grown internodes. Susceptibility of cell walls to added expansin was also increased in submerged internodes, and analysis by immunoblotting showed that cell walls of submerged internodes contained more expansin than did cell walls of air-grown internodes. Based on these data, we propose that expansin is involved in mediating rapid internodal elongation in submerged deepwater rice internodes.     

Clonal analysis of corn plant development

The formation of the lower nodes and internodes in maize (Zea mays L.) and the progression of their differentiation was investigated by generating clonal sectors from cells of the apical meristem. Marked clones were induced by irradiating dry seeds (kernels) and 2-, 8- and 13-day-old seedlings heterozygous for anthocyanin markers (b, pl) and a chlorophyll factor (wd). The extent and apparent number of cells generating the internodes 2–6, which normally remain condensed, were traced by promoting the elongation of these internodes with gibberellic acid. At the mature seed stage, internodes 2 and 3 are undergoing longitudinal expansion and each is represented by two or three circumferential populations of cells. Internodes 4 and 5 are in the process of radial expansion and each is represented by a single circumferential population of cells. At nodes 2–4, the cells for leaves and internodes have separated but such a separation has not occurred for nodes 5 and 6. The formation and expansion of basal six internodes progressed acropetally, i.e. from the base toward distal nodes. Analysis of sectors induced at the seedling stage shows that the formation of middle and top internodes also progress acropetally. The basal, middle and top internodes were found to develop at different apparent cell numbers in the apical meristem.     

Floral-stimulus activity in tobacco stem pieces

The growth patterns of axillary buds of dayneutral tobacco (Nicotiana tabacum L. cv. Wisconsin 38) plants were assessed by using expiants of single buds attached to leafless stem cuttings and allowing the buds to grow to flowering without additional manipulation. Buds located 5, 10 and 15 nodes below the inflorescence were employed. For a given bud position, when a cutting had few internodes the growth pattern of a bud tended to fall into one of two groups: buds that produced few-noded shoots and buds that produced many-noded shoots. For example, in a group of 13 cuttings composed of bud 5 with 2 associated internodes, 11 buds produced 14.2 nodes (range, 11–17) and 2 buds produced 32.0 nodes (range, 30–34). As the number of internodes on the cutting increased, the number of buds producing few-noded shoots increased and the number of nodes produced decreased (e.g. in contrast to the data above, all 5th buds with 6 internodes produced 12.8 nodes; range 11–15). When cuttings from the 3 positions had the same number of internodes, the more apical cuttings had buds that produced fewer nodes (e.g. for cuttings with 6 internodes all 5th buds produced 12.8 nodes, all 10th buds produced 15.5 nodes and 85% of 15th buds produced few-noded shoots with 19.3 nodes). The number of nodes produced by a bud was a function of the original position of the stem piece and not the original position of the bud. That is, bud 5 associated with the 6 internodes below it produced 12.8 nodes and bud 10 associated with essentially the same 6 internodes (i.e. the 6 above it) produced 12.9 nodes while bud 10 associated with the 6 internodes below it produced 15.5 nodes. Thus, the number of nodes produced by a bud was dependent upon the original main-axis position of the cutting as well as the number of internodes on the cutting. Buds forced to grow out in situ on main axes devoid of leaves produced substantially more nodes than similar buds on cuttings. Buds isolated without associated internodes produced many-noded plants with a number of nodes similar to that of plants grown from seed. The simplest interpretation of these data is that stem pieces contain floral-stimulus activity and that this activity is present in a gradient with the highest activity being located in the apical part of the stem.We thank Susan Smith and Harry Roy (Rensselaer) for comments, and the National Science Foundation for financial support (IBN-9003739 to C.N.M.).     

水稻上部节间长度等数量性状基因的定位及其遗传效应分析

对水稻籼粳杂交（窄叶青８号×京系１７）Ｆ＿１的花药进行离体培养,建立一个含１３３个ＤＨ系的作图群体,通过构建分子连锁图谱,对水稻上部节间长度、株高和抽穗期的ＱＴＬ进行区间作图,定位了影响上部节间长度的１２个ＱＴＬ、株高的４个ＱＴＬ和抽穗期的１个ＱＴＬ。对这些ＱＴＬ的遗传效应分析的结果表明,控制抽穗期的１个ＱＴＬ即Ｈｄ８ａ为主效基因,其余的１６个ＱＴＬ为微效基因。控制上部节间长度单个的ＱＴＬ对表型的贡献率介于８－１８％,其加性效应可使所控制的节间长度增加大约１．６－３．６ｃｍ。值得注意的是,一些控制相关性状的、作用方向相同的ＱＴＬ定位于同一染色体的相同或邻近区段上。这一结果揭示了这些性状相关的遗传基础,在水稻育种中运用这些ＱＴＬ将有助于对株高进行精细的遗传调控。     

Expression analysis of genes associated with sucrose accumulation in sugarcane (Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage

Sucrose synthesis/accumulation in sugarcane is a complex process involving many genes and regulatory sequences that control biochemical events in source–sink tissues. Among these, sucrose synthase (SuSy), sucrose phosphate synthase (SPS), soluble acid (SAI) and cell wall (CWI) invertases are important. Expression of these enzymes was compared in an early (CoJ64) and late (BO91) maturing sugarcane variety using end‐point and qRT‐PCR. Quantitative RT‐PCR at four crop stages revealed high CWI expression in upper internodes of CoJ64, which declined significantly in both top and bottom internodes with maturity. In BO91, CWI expression was high in top and bottom internodes and declined significantly only in top internodes as the crop matured. Overall, CWI expression was higher in CoJ64 than in BO91. During crop growth, there was no significant change in SPS expression in bottom internodes in CoJ64, whereas in BO91 it decreased significantly. Apart from a significant decrease in expression of SuSy in mature bottom internodes of BO91, there was no significant change. Similar SAI expression was observed with both end‐point and RT‐PCR, except for significantly increased expression in top internodes of CoJ64 with maturity. SAI, being a major sucrose hydrolysing enzyme, was also monitored with end‐point PCR expression in internode tissues of CoJ64 and BO91, with higher expression of SAI in BO91 at early crop stages. Enzyme inhibitors, e.g. manganese chloride (Mn⁺⁺), significantly suppressed expression of SAI in both early‐ and late‐maturing varieties. Present findings enhance understanding of critical sucrose metabolic gene expression in sugarcane varieties differing in content and time of peak sucrose storage. Thus, through employing these genes, improvement of sugarcane sucrose content is possible.     

Production of polyhydroxybutyrate in sugarcane

We report here the production of the bacterial polyester, polyhydroxybutyrate (PHB), in the crop species sugarcane ( Saccharum spp. hybrids). The PHB biosynthesis enzymes of Ralstonia eutropha [β-ketothiolase (PHAA), acetoacetyl-reductase (PHAB) and PHB synthase (PHAC)] were expressed in the cytosol or targeted to mitochondria or plastids. PHB accumulated in cytosolic lines at trace amounts, but was not detected in mitochondrial lines. In plastidic lines, PHB accumulated in leaves to a maximum of 1.88% of dry weight without obvious deleterious effects. Epifluorescence and electron microscopy of leaf sections from these lines revealed that PHB granules were visible in plastids of most cell types, except mesophyll cells. The concentration of PHB in culm internodes of plastidic lines was substantially lower than in leaves. Western blot analysis of these lines indicated that expression of the PHB biosynthesis proteins was not limiting in culm internodes. Epifluorescence microscopy of culm internode sections from plastidic lines showed that PHB granules were visible in most cell types, except photosynthetic cortical cells in the rind, and that the lower PHB concentration in culm internodes was probably a result of dilution of PHB-containing cells by the large number of cells with little or no PHB. We discuss strategies for producing PHB in mitochondria and mesophyll cell plastids, and for increasing PHB yields in culms.     

乙烯利对甘蔗分蘖期茎三种酶活性的影响及其与分蘖的关系

在分蘖前期喷施适宜浓度(100mg/L)乙烯利提高了蔗茎的过氧化物酶活性和IAA氧化酶的活性。用100mg/L乙烯利处理后,两个品种根部的过氧化物酶和IAA氧化酶活性明显高于上部的活性,并且比对照和400mg/L乙烯利处理的效果明显。乙烯利处理后新台糖16号上部节间的酸性转化酶活性始终高于下部节间的酶活性,其中100mg/L乙烯利处理下部节间的明显低于对照的;乙烯利处理后新台糖22号茎内的酸性转化酶活性也低于对照的,但与对照的差异相对比新台糖16号的小。     

Developmental Changes in the Anatomy of the Sugarcane Stem in Relation to Phloem Unloading and Sucrose Storage

Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo-chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick-walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso-dium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.     

The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia

The differentiation of phloem anastomoses linking the longitudinal vascular bundles has been studied in stem internodes of Cucurbita maxima Duchesne, C. pepo L. and Dahlia pinnata Cav. These anastomoses comprise naturally occurring regenerative sieve tubes which redifferentiate from interfascicular parenchyma cells in the young internodes. In all three species, severing a vascular bundle in a young internode resulted in regeneration of xylem to form a curved by-pass immediately around the wound. The numerous phloem anastomoses in these young internodes were not involved in this process, the regenerated vessels originating from interfascicular parenchyma alone. Conversely, in mature internodes of Dahlia, the regenerated vessels originated from initials of the interfascicular cambia, and their phloem anastomoses did not influence the pattern of xylogenesis. On the other hand, in old internodes of Cucurbita, in which an interfascicular cambium was not yet developed, the parenchyma cells between the bundles had lost the ability to redifferentiate into vessel elements, and instead, regenerated vessels were produced in the phloem anastomoses. Thus, the wounded region of the vascular bundle was not bypassed via the shortest, curved pathway, but by more circuitous routes further away from the wound. Some of the regenerated vessels produced in the phloem anastomoses were extremely wide, and presumably efficient conductors of water. It is proposed that the dense network of phloem anastomoses developed during evolution as a mechanism of adaptation to possible damage in mature internodes by providing flexible alternative pathways for efficient xylem regeneration in plants with limited or no interfascicular cambium.This paper is dedicated to the memory of the late Isaac Blachmann (deceased 19 November 1995), father-in-law of the senior author, for encouragement and advice throughout the yearsThis research was supported by an International Scientific Exchange Award to R.A. from the Israel Academy of Sciences and The Royal Society.     

Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Regulatory Mechanisms in Anthocyanin Biosynthesis in First Internodes of Sorghum vulgare: Effect of Presumed Inhibitors of Protein Synthesis

There was a 6 to 24-hour lag in the production of anthocyanins in the light after excision of 4-day-old etiolated internodes of Sorghum vulgare variety Wheatland milo. In internodes infiltrated with water, apigeninidin was formed first at 12 to 24 hours and continued to be produced slowly. Luteolinidin was formed slightly later, but its formation rapidly exceeded that of apigeninidin. Cyanidin was the last type to be produced, but equaled the amounts of luteolinidin by 4 days. In noninfiltrated internodes, the production of cyanidin was greatly accelerated, beginning at about 6 hours.     

Gibberellins in Diffusates from Shoots of Apple Trees

Excised shoot apices, leaves and internodes from shoots of apple trees (Malus×domestica) give off gibberellins by diffusion on agar. A methanol extract of the agar was prepared, the extract separated on thin layer plates, and the gibberellin activity estimated by means of Rumex and lettuce hypocotyl bioassays. The largest amounts of gibberellin are found in diffusates from the shoot apex, the two upper leaves and the two upper internodes. Several promotive fractions are found on the chromatograms as well as growth inhibitors. Removal of young leaves retards elongation of the internodes. Probably gibberellins produced in young leaves exercise some control over this process. The growth regulators Alar and CCC also retard internode elongation. Diffusates from shoots treated with these substances were also examined. Preliminary results suggest that the amount of diffusible gibberellins from treated shoots is not reduced.     

Changes in total nitrogen, soluble protein, and peroxidases in the expanding leaf zone of eastern cottonwood

Nitrogen content and soluble protein and anodal peroxidase banding in acrylamide gel changed with leaf and internode development in the expanding leaf zone of eastern cottonwood (Populus deltoides Bartr.). Nitrogen per unit leaf area was high near the apex and decreased to a constant value at the sixth node below it. Soluble protein banding was qualitatively similar for leaves and internodes in this zone, but anodal peroxidases differed between leaves and internodes. The major leaf peroxidase band was absent from the second leaf below the apex but present in the fourth and sixth leaves; its appearance and intensification seemed to parallel the development of photosynthetic activity. The major internode peroxidase band was present in the apex, second, fourth, and sixth internodes, and intensified during internodal development. It is suggested that these two “isoenzymes” may have different functions in vivo.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Flower formation in vitro in a quantitative short-day tobacco: interrelation between photoperiod and infructescence development

The flowering response of thin layers excised from branch internodes of Nicotiana tabacum cv. Maryland Catterton (quantitative short-day plant for induction) was studied under three photoperiodic treatments. The explants were excised from inflorescences bearing flowers only, flowers and green fruits, or from infructescences with green fruits only. The aim of the study was to investigate the post-inductive photoperiodic effects on in vitro flower bud formation in a quantitative short-day tobacco and the relation with infructescence development. Short days quantitatively enhanced the flower bud regeneration capacities of explants in all stages of development, both as number of explants induced to produce flowers and as mean number of flowers per explant. There was no significant difference in flower bud formation on explants of the first two stages, which produced much more flowers than those of the third stage. Observations in planta showed that, during the 20 days separating the second stage from the first stage, there was no significant difference in the number of floral buds and flowers present on the inflorescence; however, the branch internodes lengthened, as did the floral buds and flowers. During the 10 days leading to the third stage, the number of capsules did not change significantly, but a high rate of floral abscission occurred. The present results show that in Nicotiana tabacum cv. Maryland Catterton short day quantitatively controls not only the inductive step of the flowering process, but also affects the capacity to regenerate flower buds during the late post-inductive phases. The responsiveness to the photoperiodic signal decreases only when the plant exhibits only fruits.     

Interconversion of free sugars in relation to activities of enzymes catalyzing synthesis and cleavage of sucrose in growing stem tissues of sorghum

Free sugar interconversion and activities of soluble acidic (pH 4.8) and neutral (pH 7.5) invertases, sucrose synthase (synthesis) and sucrose phosphate synthase were investigated in the growing nodes and internodes of sorghum (Sorghum vulgare). The results were substantiated with incorporation of 14C from supplied sucrose and hexoses into endogenous sugars of these stem tissues. With the advancement in plant growth, the content of total free sugars in apical nodes and internodes increased till 70 DAS (flowering stage) followed by a decline. In the corresponding basal tissues, the sugar build-up continued even beyond this stage of plant growth. Compared with basal stem tissues, the apical ones contained high activities of soluble invertases and a low proportion amongst free sugars of sucrose. The activities of sucrose-hydrolyzing enzymes were higher as compared with those of sucrose-synthesizing ones in both nodes and internodes and with the growth of plant, the activity of neutral invertase increased in these tissues. More 14C from supplied sucrose and hexoses appeared in extracted sugars from cut discs of apical nodes and internodes in comparison with their basal counterparts. 14C from supplied sucrose appeared in glucose, fructose and from supplied hexoses appeared in sucrose. The results suggest that in apical nodes and internodes, where a rapid cell division and cell expansion occur, sucrose is obligatorily inverted to meet the increased requirement of hexoses and there is a compartmentalized synthesis and cleavage of sucrose in the nodes and internodes of growing sorghum plant.     

An endogenous growth inhibitor, 3-hydroxy-β-ionone. III. Its longitudinal gradients in the first internode of Phaseolus vulgaris

Dark-grown, 10-day-old bean seedlings (Phaseolus vulgaris L. cv. Morocco) were transferred to continuous light, and the resulting changes in growth rate and concentration of 3-hydroxy-β-ionone, an endogenous growth inhibitor, were monitored. The growth rate of the first internodes of the seedlings decreased rapidly and leveled off 20 h after onset of light. This plateau value was about 25% of the growth rate of the non-irradiated control. The concentration of 3-hydroxy-β-ionone in the internodes of the irradiated seedlings increased rapidly and reached a plateau value after 16 h. This increased level of the inhibitor was about 5 times the level in the non-irradiated control. The changes in the levels of the inhibitor in the internodes reflected the light-induced growth inhibition of the internodes. The longitudinal distribution of the growth inhibitor along the first internodes of the seedlings was closely correlated with the light-induced growth inhibition of the corresponding region of the internodes. The present results suggest that the endogenous growth inhibitor 3-hydroxy-β-ionone may play an important role in the inhibition by light of internode growth of bean seedlings.     

Carbon Partitioning during Sucrose Accumulation in Sugarcane Internodal Tissue

The temporal relationship between sucrose (Suc) accumulation and carbon partitioning was investigated in developing sugarcane internodes. Radiolabeling studies on tissue slices, which contained Suc concentrations ranging from 14 to 42% of the dry mass, indicated that maturation coincided with a redirection of carbon from water-insoluble matter, respiration, amino acids, organic acids, and phosphorylated intermediates into Suc. It is evident that a cycle of Suc synthesis and degradation exists in all of the internodes. The decreased allocation of carbon to respiration coincides with a decreased flux from the hexose pool. Both the glucose and fructose (Fru) concentrations significantly decrease during maturation. The phosphoenolpyruvate, Fru-6-phosphate (Fru-6-P), and Fru-2,6-bisphosphate (Fru-2, 6-P2) concentrations decrease between the young and older internodal tissue, whereas the inorganic phosphate concentration increases. The calculated mass-action ratios indicate that the ATP-dependent phosphofructokinase, pyruvate kinase, and Fru-1,6-bisphosphatase reactions are tightly regulated in all of the internodes, and no evidence was found that major changes in the regulation of any of these enzymes occur. The pyrophosphate-dependent phosphofructokinase reaction is in apparent equilibrium in all the internodes. Substrate availability might be one of the prime factors contributing to the observed decrease in respiration.     

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of -cellulose, whereas the relative amounts of -cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.     

Phyllotaxis in two species ofRubia,R. akane andR. sikkimensis

Phyllotaxis and vascular course in the vegetative shoots ofRubia akane andR. sikkimensis were studied. Each node of both species has a whorl of four leafy members among which two are true leaves. Arrangement of the true leaves is not decussate but bijugate, i.e., opposite leaves are arranged spirally. Bijugy was ascertained not only by gross morphology but also by arrangement of primordia around the shoot apex and vascular course through several internodes. Divergence angle differed widely with internodes even within a single shoot and with shoots even in the internodes which are separated by a same number of nodes from the apex. Mean divergence angles obtained for five youngest internodes of some shoots were between 49.4° and 61.8° inR. akane and between 53.6° and 59.4° inR. sikkimensis. Young seedlings ofR. akane showed decussate phyllotaxis in the lowermost several internodes. In the internodes near the lower end of the bijugate part, the divergence angle was wider than in the upper internodes. The directions of the phyllotactic spirals in the main axis and the lateral branches were either homodromous or antidromous, and those in the oppositely paired branches also were either homo- or antidromous.