Maize stem tissues: ferulate deposition in developing internode cell walls

It has been hypothesized that ferulates are only deposited in the primary cell wall of grasses. To test this hypothesis, the fourth elongating, above-ground internode of maize (Zea mays l.) was sampled from three maize hybrids throughout development. Cell wall composition was determined by the Uppsala Dietary Fibre method. Ester- and ether-linked ferulates were determined by HPLC analysis of ferulic acid released from the internodes by low and high temperature alkaline treatments. Internode length increased from 9 to 152 mm over 96 days of growth, with elongation being complete in the first 12 days. More than half of the cell wall material in the maize internodes accumulated after elongation had ended. Deposition of cell wall material appeared to reach its maximum extent 40 days after sampling began, well before physiological maturity of the maize plants. Galactose and arabinose began to accumulate early in cell wall development which was presumed to be associated with primary wall growth during internode elongation. The major secondary wall constituents (analyzed as glucose, xylose, and Klason lignin) did not begin to accumulate rapidly until shortly before internode elongation ended. Ferulate ester deposition began before ferulate ethers were observed in the cell wall, but both forms of ferulate continued to accumulate in secondary cell walls, long after internode elongation had ceased. These data clearly show that contrary to the hypothesis, ferulate deposition was not restricted to the primary wall and that active lignin/polysaccharide cross-linking mediated by ferulates occurs in the secondary wall.     

Proteomic Analysis Demonstrates that Elongation of Below-Ear Internodes in Maize is Related to Three Different Hormones

In many grain crops, the length of internodes below ears is related to lodging resistance in the field. To clarify the relationship between internode morphological differentiation and internode proteins during primary elongation stages in maize (Zea mays L.), we used proteomics analysis to explore factors regulating internodes in eight elite inbred maize lines: Zong3, Yu87-1, Xun9058, Xun928, Chang7-2, Zheng58, P2, and A50—the parents of four commercial hybrids in China (Yuyu22, Xundan20, Zhengdan958, and Jinsai6850). A total of 66 protein spots corresponding to 48 non-redundant proteins were identified in developing seventh to ninth leaf internodes. Of these spots, seven spots corresponding to six non-redundant proteins were related to the gibberellin (GA) pathway. Nineteen protein spots corresponding to 13 non-redundant proteins were related to the auxin (IAA) pathway, and 31 protein spots corresponding to 20 non-redundant proteins were associated with ethylene biosynthesis. A correlation analysis revealed that GA and IAA contents are negatively correlated with internode length, with the first hormone more strongly length-correlated than the second.     

Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field‐grown sugarcane

Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole‐targeted trehalulose synthase modified from the gene in ‘Pseudomonas mesoacidophila MX‐45’ and controlled by the maize ubiquitin (Ubi‐1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mm , with near‐complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.     

Interaction among planting dates,transgenic maize,seed treatment,corn rootworm damage and grain yield

A study was conducted from 2006 to 2008 at South Charleston and Wooster, Ohio, USA, to evaluate the potential use of planting dates in combination with Bacillus thuringiensis transgenic maize and insecticidal seed treatments to manage the root feeding of western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae). Three planting dates (early, middle and late), targeting late April/early, mid‐May and early June, respectively, were used. We planted six hybrid treatments consisting of two seed‐treated hybrids with seed treatment, two transgenic hybrids and two untreated hybrids, each set represented by one short and one full season maturity hybrid. When root injury was high, significant lodging and stunted growth were observed on untreated maize and declined when planting was delayed. Root injury by rootworm larval feeding was significantly reduced by later planting maize, that is, early June. The use of transgenic maize and seed treatment also significantly reduced root injury by rootworm larvae. The influence of planting date on grain yield was inconsistent from year to year. Grain yields from short season hybrids were comparable to full season hybrids especially on later plantings. These results showed that the use of a seed treatment and transgenic maize might be beneficial only when rootworm population is high and planting is early.     

Influence of soil temperature during root appearance on the trajectory of nodal roots of field grown maize

Trajectories of maize nodal roots were studied to test the hypothesis that roots which appear on a common internode have similar geometrical characteristics, and to assess the effect of soil temperature on root trajectory. Treatments consisted of three sowing dates, a comparison between mulched and non-mulched soil, a replication of one sowing date for two years in two locations, and a comparison between two cultivars at one sowing date. All these sources of variation, except the cultivar, had an appreciable effect on the trajectories of roots which appeared on the first four internodes. The horizontal component of the trajectory differed significantly between treatments, ranging from 93 to 700 mm in roots which appeared on the second internode, and from 71 to 569 for those on the third internode. The original hypothesis had, therefore, to be rejected. Mean soil temperature during the 100°C.days after root appearance accounted for the differences in trajectory between location, year, sowing date and mulch treatments, and for the differences between internodes within each location. The critical period during which temperature affected root trajectory probably began at root appearance, and ended between 50 and 100°C days after root appearance, i.e. when the root was less than 100 mm long.     

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.     

Transformation through agroinfection on decapitated shoot apex of field-growing <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A₄M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A₄M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A₄M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.     

Gibberellic acid improved shoot multiplication in Cephaelis ipecacuanha

The internodes of Cephaelis ipecacuanha elongated when cultured on Gamborg B5 solid medium supplemented with 0.5 or 1 mg/L gibberellic acid (GA₃). The size of the elongated internode doubled in length compared to the untreated shoots, and the adventitious shoots formed on the elongated internodes. The shoots grew easily into plantlets without the use of auxin for rooting. The ex vitro regenerates cultivated in the greenhouse showed normal characteristics. Emetic alkaloids were detected in the leaves of in vitro shoots and the roots of regenerates cultivated in the greenhouse. This method using GA₃ propagated numerous plants at a rate of more than 100 times compared to the method without GA₃.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Effect of Sowing Time on Toxigenic Fungal Infection and Mycotoxin Contamination of Maize Kernels

Field experiments were set up from 2000 to 2002 in northwest Italy to determine the effects of sowing date on the susceptibility of maize hybrids to ear rot and mycotoxin contamination in natural infection conditions. Three sowing dates (March, April and May) were compared using two hybrids with different maturity classes. The ears were rated for the incidence and severity of disease symptoms at harvest and the harvested kernels were analysed for mycotoxin fumonisin B₁, zearalenone, deoxynivalenol, aflatoxin B₁ and ochratoxin A. The last sowing date resulted in higher values of European corn borer incidence (+39%), kernel Fusarium infection and fungal ear rot severity (increased respectively by a factor of 4 and 3) than the plots sowed in March. The sowing date did not influence the type of mycotoxin found in the kernel, which only depended on the climatic conditions during the season and on their influence on the infection and the development of the fungal species. The natural occurrence of fumonisin B₁ and deoxynivalenol was always significantly higher in late‐sowed plots. A late sowing, after May 10, multiplies the risk of the occurrence of fumonisins and deoxynivalenol in the grains at harvest by a factor of 11.2 and 1.9, respectively. No significant difference was observed for the contamination of zearalenone or aflatoxin B₁ for the different sowing dates. An earlier sowing time than April, resulted in a significant reduction in mycotoxin contamination only in the years in which the meteorological trend protracted the kernel dry down to the autumn months. The use of a late‐maturity hybrid with late sowing, compared with a medium‐maturity hybrid, could lead to a serious risk of mycotoxin contamination. To the authors’ knowledge, this is the first study to report on the effects of sowing time on maize kernel contamination of mycotoxins other than aflatoxins in non‐inoculated conditions.     

Carbon allocation to the insoluble fraction,respiration and triose-phosphate cycling in the sugarcane culm

The changes in carbon allocation to non-sucrose metabolic pathways were investigated in developing internodes of sugarcane. Radiolabelling studies were done on internode 3, 6 and 9 tissues, representing three stages of increasing maturity. Carbon partitioning into sucrose increased from 34% of total ¹⁴C uptake in internode 3, to 66% in internodes 9. In immature tissue, the protein and fibre components were the dominant competing sinks with sucrose for incoming carbon, to which 14 and 16% of carbon was allocated. Increased carbon allocation to sucrose with tissue maturity coincided with a decrease in partitioning to fibre and total respiration. Between internodes 3 and 9 carbon allocation to total respiration decreased by 9%, and to fibre by 14%. Carbon cycling between the triose- and hexose phosphate pools was evident in all internodes. More than 90% of carbon entering triose-phosphates was returned to hexose in internode 3 tissue, and this flux decreased with tissue maturity.     

Adventitious root induction in Ophiorrhiza prostrata: a tool for the production of camptothecin (an anticancer drug) and rapid propagation

Roots of Ophiorrhiza prostrata D. Don serve as a rich source of camptothecin (CPT), an anticancer drug. Because of the large-scale collection of its roots, the plant has become a threatened species. The present study accomplishes the induction of adventitious roots as a means for the production of CPT as well as for the large-scale propagation of this anticancer drug plant using leaf and internode explants. The biomass yield and CPT content of adventitious roots induced from different explants were compared to roots developed on ex vitro rooted stem cuttings. Adventitious roots were produced on half-strength Murashige and Skoog (MS) medium supplemented with 10.74 μM α-naphthaleneacetic acid and 2.32 μM kinetin at mean fresh weights of 0.753, 0.739 and 0.748 g roots from leaf, internode and shoot, respectively. CPT yield from in vitro derived roots after 50, 80 and 120 days of incubation (0.028, 0.06 and 0.1% dry weight, respectively) was not significantly different from those harvested at the same age from ex vitro rooted (0.03, 0.06 and 0.13%, respectively) stem cuttings. CPT from subcultured roots derived from solid (0.08%) medium was lower than from suspension culture medium (0.12%). Subsequent cultures of the adventitious roots showed a stable production of CPT (0.16%). The yield of CPT from 360-day-old plant-derived roots was 0.19%. Elicitation using methyl jasmonate and acetyl salicylic acid exhibited no enhancement in CPT yield. In vitro propagation through direct shoot regeneration was achieved from the adventitious roots upon transfer to MS medium with 8.87 μM N ⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) with a mean of 21.2 shoots per culture in 50 days. The shoots upon subculture on medium having the same level of BA and IBA underwent rapid proliferation. The shoots transferred to field conditions after in vitro rooting exhibited 95% survival. Adventitious root induction, from leaf and internode explants, enables the feasible production of CPT as well as the large-scale rapid propagation of this species which can safeguard it from extinction.     

Phytosiderophore release from nodal,primary, and complete root systems in maize

Some maize (Zea mays L.) hybrids grown in high pH soil in Nebraska suffer from severely reduced yields caused by iron (Fe) deficiency chlorosis. Hybrids which recover from early season Fe-deficiency chlorosis and yield well are termed Fe-efficient or tolerant. Most Fe-efficient gramineous species respond to Fe-deficiency stress by releasing phytosiderophores (mugineic acid and its derivatives) into the rhizosphere, thereby increasing Fe availability and uptake of the Fe³⁺-phytosiderophore complex via a high affinity uptake system. Field-grown Fe-efficient maize recovers from Fe-deficiency chlorosis at a stage when nodal roots have become the dominant root system. Quantifying phytosiderophore release from hydroponically grown plants has been proposed as a viable alternative to time-consuming and variable field trials and has been used successfully to delineate among Fe-efficient and Fe-inefficient lines of oat (Avena sativa L.) and wheat (Triticum aestivum L.). Our objectives were (1) to determine if phytosiderophore release differed between nodal- and primary-root systems of maize, and (2) to compare phytosiderophore release from 12 hybrids. Root exudates secreted during daily 4-h collections were analyzed for their Fe-solubilizing ability, which was equated to phytosiderophore release. Nodal root systems released significantly more phytosiderophore than primary- or complete-root systems. In early experiments, an Fe-efficient hybrid (P3279) released more phytosiderophore from nodal roots than an Fe-inefficient hybrid (P3489). Tests of an additional 10 hybrids showed that phytosiderophore release varied significantly among the cultivars but did not clearly distinguish between hybrids classified as Fe-efficient or Fe-inefficient in individual company trials. We recommend using nodal roots when studying Fe-stress response mechanisms in maize.     

Population parameters for resistance to Fusarium graminearum and Fusarium verticillioides ear rot among large sets of early, mid-late and late maturing European maize (Zea mays L.) inbred lines

Infection of maize ears with Fusarium graminearum (FG) and Fusarium verticillioides (FV) reduces yield and quality by mycotoxin contamination. Breeding and growing varieties resistant to both Fusarium spp. is the best alternative to minimize problems. The objectives of our study were to draw conclusions on breeding for ear rot resistance by estimating variance components, heritabilities and correlations between resistances to FV and FG severity and to investigate different inoculation methods. In 2007 and 2008, three maturity groups (early, mid-late, late) each comprising about 150 inbred lines were tested in Germany, France, Italy, and Hungary according to their maturity group. They were silk channel inoculated by FG (early) and FV (all groups). In the late maturity group, additionally kernel inoculation was applied in a separate trial. The percentage of mycelium coverage on the ear was rated at harvest (0–100%). Significant (P < 0.01) genotypic variances of ear rot severity were found in all groups. Inoculation was superior to natural infection because of higher disease severities and heritabilities. In early maturing flints and dents, FG caused significantly (P < 0.01) higher ear rot severity than FV (61.7 and 55.1% FG vs. 18.2 and 11.1% FV ear rot severity, respectively). FV inoculation in Southern Europe (mid-late, late) resulted in similar means between 10.3 and 14.0%. Selection is complicated by significant (P < 0.01) genotype × environment interactions. Correlation between FG and FV severity was moderate in flints and dents (r = 0.59 and 0.49, respectively) but lines resistant to both fungi exist. We conclude that chances for selecting improved European elite maize material within the existing germplasms is promising by multi-environmental inoculation trials.     

Ontogenetic development in architecture and biomass allocation of 13 rattan species in Indonesia

Rattans are climbing, nonbranching palms with diverse growth forms ranging from stems that remain at the forest floor to stems that reach the canopy. We analyzed changes in architecture and biomass allocation during ontogenetic development of 13 Indonesian species in the genera Calamus, Ceratolobus, Daemonorops, Korthalsia, and Plectocomiopsis. Species included both nonclimbers (<5 m tall maximum) and climbers (10 to 50 m tall). Nonclimbers retain a rosette form—that is, thick, short internodes with length/internode diameter ratio <10 but with long petioles—throughout their lives. Leaf and internode shapes of climbers at the early stage resemble those of nonclimbers, but internodes later become longer (internode length/diameter ratio ≥10) and petioles become shorter and developed one of two kinds of climbing organs at the adult stage. These developmental changes reduce self-shading within the crown. Some climbers have dwarf blades at the early stage and skip the rosette form. Principal component analysis of biomass allocation indicated that growth strategies to attain the adult stage are diverse. These results suggest that rattans reach maturity at different phases along a series of ontogenetic development stages and generate diverse growth forms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vertical and radial profiles in tracheid characteristics along the trunk of Douglas-fir trees with implications for water transport

The main stems of three young Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirbel) Franco) trees were dissected to obtain samples of secondary xylem from internodes axially along the trunk and radially within each internode. From these samples, measurements were obtained of tracheid diameter, length, the number of inter-tracheid pits per tracheid, and the diameter of the pit membranes. In addition, samples were obtained along the trunks of three old growth trees and also a small sample of roots for measurement of tracheid diameter. A gradient was apparent in all measured anatomical characters vertically along a sequence among the outer growth rings. These gradients arose not because of a gradient vertically along the internodes, but because of the strong gradients present at each internode among growth rings out from the pith. Tracheid characteristics were correlated: wider and longer tracheids had more numerous pits and wider pits, such that total pit area was about 6% of tracheid wall area independent of tracheid size. A stem model combining growth rings in parallel and internodes in series allowed for estimates of whole trunk conductance as a function of tree age. Conductance of the stem (xylem area specific conductivity) declined during the early growth of the trees, but appeared to approach a stable value as the trees aged.     

Thigmomorphogenesis: Evidence for a translocatable thigmomorphogenetic factor induced by mechanical perturbation of beans (Phaseolus vulgaris)

Mechanical perturbation of bean (Phaseolus vulgaris L.) internodes results in reduced elongation and increased diameter of the internodes (thigmomorphogenesis). Perturbation of a single lower internode results in thigmorphogenesis in that internode and all of those internodes above it, the degree of which depends on the age (size) of the internodes and the frequency of perturbation. Application of ethephon to the internodes mimics mechanical perturbation. Early removal of the shoot tip or the cotyledons does not effect thigmomorphogenesis, indicating that those organs do not exert control over the response. Mechanical perturbation of one plant of a pair grafted together at the first internodes results in thigmomorphogenesis in both plants. This indicates the transport of some factor from the mechanically perturbed donor to the non-treated receiver. Evidence is presented to support the contention that ethylene is not this transportable factor.     

A simple shoot multiplication procedure using internode explants,and its application for particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in watercress

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 μM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1–2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 μM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.     

Influence of arbuscular mycorrhiza on lipid peroxidation and antioxidant enzyme activity of maize plants under temperature stress

The influence of the arbuscular mycorrhizal (AM) fungus, Glomus etunicatum, on characteristics of growth, membrane lipid peroxidation, osmotic adjustment, and activity of antioxidant enzymes in leaves and roots of maize (Zea mays L.) plants was studied in pot culture under temperature stress. The maize plants were placed in a sand and soil mixture under normal temperature for 6 weeks and then exposed to five different temperature treatments (5oC, 15oC, 25oC, 35oC, and 40oC) for 1 week. AM symbiosis decreased membrane relative permeability and malondialdehyde content in leaves and roots. The contents of soluble sugar content and proline in roots were higher, but leaf proline content was lower in mycorrhizal than nonmycorrhizal plants. AM colonization increased the activities of superoxide dismutase, catalase, and peroxidase in leaves and roots. The results indicate that the AM fungus is capable of alleviating the damage caused by temperature stress on maize plants by reducing membrane lipid peroxidation and membrane permeability and increasing the accumulation of osmotic adjustment compounds and antioxidant enzyme activity. Consequently, arbuscular mycorrhiza formation highly enhanced the extreme temperature tolerance of maize plant, which increased host biomass and promoted plant growth.