Ubiquitous presence of β-glucuronidase (GUS) in plants and its regulation in some model plants

The enzyme β-glucuronidase (GUS) is well characterized in animals and microbes. However, this enzyme is not well studied in plants and is widely assumed to be absent in them. In this study we document the ubiquitous presence of GUS in the model plants Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays and record its expression pattern. The pH of the assay buffer was found to be critical with pH 4.0 being optimum for detection in all the species. GUS in plants appears to be associated with growth. In general, younger regions of the organs showed more GUS activity than the older and more mature tissues. In Brassica juncea roots stained for GUS, intense blue color could be seen in the trichoblast cells and the growing root hair cells as compared to the non-root hair forming epidermal cells or the fully elongated root hairs. Cotton fibers showed high GUS activity during the initial phase of elongation while the seed coat, from which the fibers formed, did not stain for GUS activity. The activity in the fibers disappeared after they were fully elongated. The level of GUS activity increased 2.58 folds in leaf tissues of N. tabacum when cultured in MS medium supplemented with 6-benzylaminopurine, while gibberellic acid enhanced GUS activity 2.9 folds in the inter-nodal regions of rice in 12-h treatment. In addition, elongation of stem, root and root hairs in tobacco seedlings was strongly inhibited by the specific inhibitor of GUS, saccharo-1-4-lactone in a reversible manner. Taken together, these evidences suggest a probable association of plant GUS in cell growth.Charu Sudan and Shiva Prakash, the first two authors, have contributed equally.     

Biochemical features of maize tissues with different capacities to regenerate plants

Metabolic profiling using GC–MS and LC–MS analyses of soluble metabolites and cell wall bound phenolic compounds from maize calluses of different morphogenic competence revealed a number of biochemical characteristics that distinguish tissues with high plant regeneration ability from tissues that cannot efficiently regenerate plants in vitro. Maize cultures of different ages from H99 (compact type I callus) and HiII (friable type II callus) were divided into two different samples: regenerable (R) and non-regenerable (NR) based on known morphologies. Tissues from both genotypes with high morphogenic potential had higher asparagine and aspartate and indole-3-butenol concentrations, decreased sugar and DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) concentrations, low levels of 4-aminobutyric acid (GABA) and chlorogenic acid and lower levels of feruloyl- and sinapoyl glucosides compared to NR tissues. The ether bound cell wall phenolics of tissues with high regeneration potential had higher levels of the predominant G (guaiacyl) units and lower levels of H (p-hydroxyphenyl) and S (syringyl) units and higher ferulic acid/coumaric acid and ferulic acid/diferulic acid ratios. The same trends were found with the ester-bound phenolics of HiII, however, there were only small differences between the H99 R and NR tissues. Concentrations of the major sugars, organic acids, amino acids and soluble aromatic compounds tended to increase as the time after culture initiation increased. The results show that there are differences in general metabolism, phenolic secondary compounds and cell wall composition between R and NR cell types.     

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N₆BM and N₆K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.     

Genetic Analysis of Maize Root Characteristics in Response to Low Nitrogen Stress

Under low-input cropping systems, nitrogen (N) can be a limiting factor in plant growth and yield. Identifying genotypes that are more efficient at capturing limited N resources and the traits and mechanisms responsible for this ability is important. Root trait has a substantial influence on N acquisition from soils. Nevertheless, inconsistencies still exist as to the effect of low N on root length and its architecture in terms of lateral and axial roots. For maize, a crop utilizing heterosis, little is known about the relationship between parents and their crosses in the response of root architecture to N availability. Here 7 inbred maize lines and 21 of their crosses created by diallel mating were used to study the effect of N stress on root morphology as well as the relationship between the inbreds and their crosses. With large genotypic differences, low N generally suppresses shoot growth and increases the root to shoot ratio with or without increasing root biomass in maize. Maize plants responded to N deficiency by increasing total root length and altering root architecture by increasing the elongation of individual axial roots and enhancing lateral root growth, but with a reduction in the number of axial roots. Here, the inbreds showed weaker responses in root biomass and other root parameters than their crosses. Heterosis of root traits was significant at both N levels and was attributed to both the general combining ability (GCA) and special combining ability (SCA). Low N had substantial affects on the pattern of heterosis, GCA and SCA affects on root traits for each of the crosses suggesting that selection under N stress is necessary in generating low N-tolerant maize genotypes.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

The Effects of H<Superscript>+</Superscript>-ATPase Activator and Inhibitors on Cell Growth in the Maize Root

We studied the effects of H⁺-ATPase activator fusicoccin (FC) and its inhibitors, sodium orthovanadate (Na₃VO₄) and diethylstilbestrol (DES), on the rate of proton secretion by root regions located at various distances from the root tip, the rate of root growth, the length of the fully-elongated root cells, the sizes of growth zones, the relative growth rate of cells along the root length, and the number of fully-elongated cells in the root length increment. FC (10⁻⁶ M) stimulated proton secretion by root segments and enhanced root growth due to the greater length of fully-elongated cells. DES (10⁻⁴ M) suppressed proton secretion and retarded root growth, decreased the length of fully-elongated cells, inhibited cell division, and slowed down cell transition to elongation by prolonging the life-span of cells in the meristem. Na₃VO₄ (10⁻³ and 10⁻⁴ M) exerted similar effects. FC, DES, and orthovanadate did not affect the ratio of the relative rate of cell growth in the elongation zone to that in meristem.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 558–565.Original Russian Text Copyright © 2005 by Mesenko, Ivanov.     

Zea mays L. extracts modify glomerular function and potassium urinary excretion in conscious rats

Diuretic and uricosuric properties have traditionally been attributed to corn silk, stigma/style of Zea mays L. Although the diuretic effect was confirmed, studies of the plant's effects on renal function or solute excretion were lacking. Thus, we studied the effects of corn silk aqueous extract on the urinary excretion of water, Na+, K+, and uric acid. Glomerular and proximal tubular function and Na+ tubular handling were also studied. Conscious, unrestrained adult male rats were housed in individual metabolic cages (IMC) with continuous urine collection for 5 and 3 h, following two protocols. The effects of 25, 50, 200, 350, and 500 mg/kg body wt. corn silk extract on urine volume plus Na+ and K+ excretions were studied in water-loaded conscious rats (2.5 ml/100 g body wt.) in the IMC for 5 h (Protocol 1). Kaliuresis was observed with doses of 350 (100.42 +/- 22.32-120.28 +/- 19.70 microEq/5 h/100 g body wt.; n = 13) and 500 mg/kg body wt. (94.97+/- 29.30-134.32 +/- 39.98 microEq/5h/100 g body wt.; n = 12; p<0.01), and the latter dose resulted in diuresis as well (1.98 +/- 0.44-2.41 +/- 0.41 ml/5 h/100 g body wt.; n = 12; p<0.05). The effects of a 500 mg/kg body wt. dose of corn silk extract on urine volume, Na+, K+ and uric acid excretions, and glomerular and proximal tubular function, were measured respectively by creatinine (Cler) and Li+ (ClLi) clearances and Na+ tubular handling, in water-loaded rats (5 ml/100 g body wt.) in the IMC for 3 h (Protocol 2). Clcr (294.6 +/- 73.2, n = 12, to 241.7 +/- 48.0 microl/ min/100 g body wt.; n = 13; p<0.05) and the Na+ filtered load (41.9 +/- 10.3, n = 12, to 34.3 +/- .8, n = 13, p<0.05) decreased and ClLi and Na+ excretion were unchanged, while K+ excretion (0.1044 +/- 0.0458, n=12, to 0.2289 +/- 0.0583 microEq/min/100 body wt.; n = 13; p<0.001) increased. For Na+ tubular handling, the fractional proximal tubular reabsorption (91.5 +/- 3.5, n = 12, to 87.5 +/- 3.4%; n = 13; p<0.01) decreased, and both fractional distal reabsorptions--I and II--increased (96.5 +/- 1.5, n = 12, to 97.8 +/- 0.9%; n = 13; p<0.01; and 8.2 +/- 3.5, n = 12, to 12.2 +/- 3.4%, n = 13, p<0.01, respectively). To summarize, in water-loaded conscious rats (2.5 ml/100 body wt.), corn silk aqueous extract is diuretic at a dose of 500 mg/kg body wt. and kaliuretic at doses of 350 and 500 mg/kg body wt. In water-loaded conscious rats (5.0 ml/100 g body wt.), corn silk aqueous extract is kaliuretic at a dose of 500 mg/kg body wt., but glomerular filtration and filtered load decrease without affecting proximal tubular function, Na+, or uric acid excretion.     

Relationship between activities of key enzymes involved in starch synthesis and accumulation in maize inbred lines during grain filling

Time course of starch production and the key enzyme activities in the grains of four maize inbred lines (two high-starch and two low-starch lines) were studied. Accumulation of grain starch and its components in four maize inbred lines rose continuously after pollination and increased as a sigmoid curve during grain filling. The accumulation rates showed single-peak curves. The accumulation rates of starch and its components reached their peaks on 25–32 days after pollination (DAP), respectively. Activities of adenosine diphosphoglucose pyrophosphorylase (AGPPase) and starch synthase in the grains of four lines followed single-peak curves with the peaks on 24–31 DAP. The highest activity of the starch-branching enzyme (Q-enzyme) in the grains of both high-starch lines appeared on 23 DAP, but that of both low-starch inbred lines showed double-peak curves, the peaks being at 15–20 DAP and 30–35 DAP. There was significant positive correlation between AGPPase, soluble starch synthase (SSS), and starch granule-bound synthase (GBSS) activities. The results indicated the Q-enzyme had different expression patterns in the high-and the low-starch maize inbred lines, and that AGPPase, SSS, and GBSS activities were significantly and positively correlated with amylose, amylopectin, and starch accumulation rates in all lines. This text was submitted by the authors in English     

Photosynthesis and Dark Respiration in Leaves of Different Ages of Partly Flooded Maize Seedlings

Maize seedlings were flooded for periods from 1 to 15 days, and the leaves of different ages were then taken to examine photosynthesis, dark respiration, transpiration, chlorophyll content, and some morphometric parameters. The responses of leaves to root submergence essentially depended on the leaf layer and the treatment duration. A short-term flooding (1–24 h) induced primary stress responses in the first leaf. Photosynthesis and respiration in this leaf oscillated around the control levels with amplitudes of ±15–25% and ±40–60%, respectively. After a longer flooding, the CO₂ exchange in the second leaf was suppressed, while oxygen uptake was stimulated. In the third leaf, which was formed during submergence, the photosynthetic rate increased and the respiratory activity decreased. The transpiration rate did not change in these leaves for 15 days of flooding. The hypoxic treatment, at its early stages, retarded growth and disturbed the source–sink relations. At later stages the plants adapted to hypoxic environment: the seedling growth was restored, which elevated the demand for assimilates and stimulated photosynthesis. It is concluded that plants overcome negative impact of the root hypoxia at the systemic level.     

The Stimulatory Effect of the Antibiotic Cefotaxime on Plant Regeneration in Maize Tissue Culture

In order to elucidate the effects of the antibiotic cefotaxime on callus growth and morphogenesis, we incubated embryogenic maize calli (Zea mays L.) of A188 and R91 lines and of their F1 hybrid with 50–500 mg/l cefotaxime throughout several subcultures. Cefotaxime did not affect the induction frequency and growth of the embryogenic callus but enhanced its morphogenesis. In both tested lines and a hybrid, the highest increase in the number of regenerated plants was observed at the antibiotic concentration of 150 mg/l. The degree of morphogenesis stimulation and the range of cefotaxime concentrations effective in stimulation of plant regeneration depended on the properties of calli obtained from tested genotypes.