Quantitative determination of indole-3-acetic Acid in sugarbeet leaves using a double standard isotope dilution gas chromatographic assay

Quantitative levels of indole-3-acetic acid (IAA) were determined in leaf blades of two sugarbeet cultivars by a double standard isotope dilution assay using column chromatography followed by reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography with nitrogen thermionic detection. The double standard method was validated as a quantitative tool by gas chromatography/selected ion monitoring mass spectrometry using 2,′,4′,5′,6′,7′-d₅-IAA as the internal standard. Progenies of one breeding line that had been selected for a high taproot to leaf weight ratio were used to correlate IAA levels with varying leaf and plant size at day 31 from germination. In spite of size differences, no significant difference in IAA levels per unit leaf weight could be found. The possible relationship between day 31 leaves and IAA content at an earlier stage of development is discussed in the text. A second analysis used four developmental leaf stages, classified as expanding, recently mature, aging, and senescing leaves. Expanding leaves contained the most IAA, senescing leaves contained the least IAA, with recently mature leaves and aging leaves containing intermediate amounts. The DNA content of each of the four developmental leaf stages was determined and DNA levels per gram fresh weight were found to be constant at all developmental stages.     

Quantitative relationship between indole-3-acetic acid and abscisic acid during leaf growth in Coleus blumei

Quantitative determinations by gas chromatography-mass spectrometry ofindole-3-acetic acid (IAA) and abscisic acid (ABA) in growing leaves ofColeusblumei plants show parallel declines in leaf concentrations of bothhormones,except in leaf number 3 (about three-fourths of full size) where IAA level wasthe lowest of those measured. Expansion of the most recently unfurled leaf tofull size serves, in effect, to dilute both IAA and ABA about 1.7 to 1.Althoughabsolute levels of leaf IAA varied as much as an order of magnitude from onebatch of plants to another, ABA levels were proportional to the IAA level withan overall correlation coefficient of 0.91. Evidence, both correlative andcausal, for the determination of ABA status by IAA—and of IAA status byABA—in leaves and other developing organs is summarized.     

PRIMORDIAL LEAF AND PHYTOHORMONE EFFECTS ON EXCISED SHOOT APICAL MERISTEMS OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. apical meristems and apical meristems +1, +2, +3 primordial leaf pairs were cultured to examine phytohormone influences on development and correlative effects of developing primordial leaves on in vitro responses. The meristem with no phytohormones or low levels of IAA could not develop in vitro. At least 0.1 mg/l IAA and optimumly 1-2 mg/l IAA were required for development into complete plants. IAA from 0.1 to 3 mg/l also resulted in root development with no apparent leaf or shoot formation. Levels of IAA higher than 3 mg/l were inhibitory to development. Kinetin, as a substitute for naturally occurring cytokinins, alone (0.0003 to 3 mg/l) resulted in development of rosettes of leaves. In the presence of IAA (***1 mg/l) and kinetin (0.003 mg/l) plants, rosettes, individual leaves with roots, and roots developed from isolated meristems. Glutamine and adenine sulfate both appeared inhibitory to meristem development. With +1, +2, +3 developing primordial leaf pairs left attached to the apical dome, three pairs were required for plant formation in the absence of phytohormones. In the presence of IAA, two pairs of primordial leaves resulted in plant formation; whereas, with IAA and low levels of kinetin one pair of primordial leaves was enough. Higher levels of kinetin were inhibitory to plant development with primordial leaves present. ABA appeared to be inhibitory to development of meristems and meristems +1, +3 primordial leaves at low concentrations and resulted in death at ***1 mg/l. Developing primordial leaves appear to supply the apical meristem with a balance of phytohormones during growth. Meristem development into a plant first involved formation of leaf primordia. Establishment of a bipolar axis with root formation followed.     

Auxin-induced leaf blade expansion in Arabidopsis requires both wounding and detachment

Elevation of leaf auxin (indole-3-acetic acid; IAA) levels in intact plants has been consistently found to inhibit leaf expansion whereas excised leaf strips grow faster when treated with IAA. Here we test two hypothetical explanations for this difference in growth sensitivity to IAA by expanding leaf tissues in vivo versus in vitro. We asked if, in Arabidopsis, IAA-induced growth of excised leaf strips results from the wounding required to excise tissue and/or results from detachment from the plant and thus loss of some shoot or root derived growth controlling factors. We tested the effect of a range of exogenous IAA concentrations on the growth of intact attached, wounded attached, detached intact, detached wounded as well as excised leaf strips. After 24 h, the growth of intact attached, wounded attached, and detached intact leaves was inhibited by IAA concentrations as little as 1 µM in some experiments. Growth of detached wounded leaves and leaf strips was induced by IAA concentrations as low as 10 µM. Stress, in the form of high light, increased the growth response to IAA by leaf strips and reduced growth inhibition response by intact detached leaves. Endogenous free IAA content of intact attached leaves and excised leaf strips was found not to change over the course of 24 h. Together these results indicate growth induction of Arabidopsis leaf blade tissue by IAA requires both substantial wounding as well as detachment from the plant and suggests in vivo that IAA induces parallel pathways leading to growth inhibition.     

Light adaptation and the role of autotrophic epiphytes in primary production of the temperate seagrass, Zostera marina L.

Photosynthetic features of Zostera marina L. and its autotrophic epiphyte community were investigated in a population inhabiting a shallow (1.3 m depth) water meadow in Great Harbor, Woods Hole, MA (U.S.A.). Photosynthesis versus irradiance (P-I) relationships were measured with respect to leaf age determined by the leaf position in the shoot bundle and by location of the tissue along the leaf axis. Therefore both age and light intensity gradients along the leaf axis were considered. The maximum photosynthesis (P_max) per dm² typically increased nearly two-fold along the leaf axis from leaf bases to apices. Photosynthetic rate on a chlorophyll (Chl) basis did not increase as dramatically along the leaf axis, and rates were usually lowest in tissues with the highest Chl content. The P-I relationships of leaves of different ages did not reveal photoinhibition even at light intensities > 1400 μE • m⁻² • s ⁻¹. Furthermore, no photoinhibition was observed in tissues from leaf blade bases, which never experienced high light levels (> 500 μE • m ⁻² • s⁻¹) in situ in Great Harbor. The initial slopes of the P-I curves and light compensation and saturation values varied along the leaf axis in relation to in situ light intensity gradients and in relation to leaf or tissue age. It appeared that leaf and/or tissue age was more important than light environment in determining P-I responses. The contribution of the autotrophic epiphyte community on Z. marina leaves to total photosynthesis per dm² was between 27 and 50%, and between 10 and 44% per mg chlorophyll. These levels of epiphyte photosynthesis can double the primary production of Z. marina leaves. No detrimental effects of epiphyte cover were realized in leaf maximal photosynthesis or P-I relationships. Non-epiphytized leaves and leaves from which epiphytes were removed showed essentially identical photosynthetic features. Light intensity and age gradients along the leaf axis control both the photosynthetic performance of the leaves and epiphyte biomass and photosynthesis.     

Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth

The distribution and biosynthesis of indole-3-acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg(-1) of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred-fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten-fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue-dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue-specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.     

水分胁迫对棉花叶片中IAA从含量、IAA氧化酶和过氧化物酶活性的影响

整株干旱降低盐棉46号叶片中的IAA总量,叶龄愈小下降愈多。幼叶中IAA总量的下降主要是结合态IAA减少的结果。气干和-1.7MPa PEG溶液渗透胁迫处理也降低离体成熟叶片的IAA总量,其变化与叶片含水量呈直线相关(r=0.905)。整株干旱处理提高各叶片的过氧化物酶活性,叶龄愈小提高愈多,但IAA氧化酶活性无显著变化。离体和整株干旱时IAA总量的下降,可能是过氧化物酶活性增加所致。     

Indoleacetic acid, abscisic acid and phenolic substances during development of hazel leaves

The levels of indoleacetic acid (IAA), abscisic acid (ABA) and phenolic substances were measured monthly in leaves of hazel ( Corylus avellana L. cv. Negreta) in different stages of development from April till abscission in November. IAA and ABA were quantified by flame ionization detector gas-chromatography (GLC), and the total phenolic content by a colorimetric technique. During spring, IAA levels were the highest, coinciding with the greatest expansion of the leaves; after which IAA gradually decreased as the leaves became older, although minor increases were observed in July-August and November. The ABA level in hazel leaves was low throughout the period of leaf existence, with a maximum in July and a second increase in November, coinciding with the last month in which hazel bears leaves. The phenols of the neutral fraction showed a minimum in May and a pronounced increase in June, remaining at this level during summer and decreasing in November. The acid and alkaline fractions showed a maximum in August, which could be related to the decrease in the growth rate of leaves. The residual fraction remained at a relatively low level until September, increasing rapidly in October and November.     

Effects of pod removal on the transport and accumulation of abscisic Acid and indole-3-acetic Acid in soybean leaves

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the second most recently expanded trifoliolate leaf were determined during reproductive development of soybean (Glycine max [L.] Merr cv `Chippewa 64'). The concentration of ABA in leaves was constant during most of the seed filling period until the seeds began to dry. The concentration of IAA in the leaves decreased throughout development. Removal of pods 36 hours prior to sampling resulted in increased concentrations of ABA in leaves during the period of rapid pod filling but had little effect on the concentration of IAA in leaves. ABA appears to accumulate in leaves after fruit removal only when fruits represent the major sink for photosynthate.

ABA and IAA moving acropetally and basipetally in petioles of soybean were estimated using a phloem exudation technique. ABA was found to move mostly in the basipetal direction in petioles (away from laminae). IAA, primarily in the form of ester conjugate(s), was found to be moving acropetally (toward laminae) in petioles. The highest amount of IAA ester(s) was found in petiole exudate during the mid and late stages of seed filling. Removal of fruits 36 hours prior to exudation reduced the amount of IAA ester recovered in exudate, suggesting that fruits were a source of the IAA conjugate in petiole exudate.

     

Leaf water 18O and 2H maps show directional enrichment discrepancy in Colocasia esculenta

Spatial patterns of leaf water isotopes are challenging to predict because of the intricate link between vein and lamina water. Many models have attempted to predict these patterns, but to date, most have focused on monocots with parallel veins. These provide a simple system to study, but do not represent the majority of plant species. Here, a new protocol is developed using a Picarro induction module coupled to a cavity ringdown spectrometer to obtain maps of the leaf water isotopes (¹⁸O and ²H). The technique is applied to Colocasia esculenta leaves. The results are compared with isotope ratio mass spectrometry. In C. esculenta, a large enrichment in the radial direction is observed, but not in the longitudinal direction. The string‐of‐lakes model fails to predict the observed patterns, while the Farquhar–Gan model is more successful, especially when enrichment is accounted for along the radial direction. Our results show that reticulate‐veined leaves experience a larger enrichment along the axis of the secondary veins than along the midrib. We hypothesize that this is due to the lower major/minor vein ratio that leads to longer pathways between major veins and sites of evaporation.     

Leaf Expansion in the Potato, Solanum tuberosum

The effects of a number of manipulative treatments together with the application of the growth hormones IAA and GA on leaf expansion in the potato, variety Majestic, were investigated. It was found that removal of the apex and its surrounding non-expanded leaves resulted in an increase in both the rate and the extent of expansion of Ihe remaining upper leaves on the system. Application of IAA and GA to decapitated shoots showed that the rate of leaf expansion was decreased by IAA both alone and in combination with GA, whereas it was increased by GA treatment. Further investigations indicated that the response of a leaf to GA treatment was determined by the area and position of the leaf al the time of treatment and also by the number of expanding leaves on the system. It is postulated that this is due, at least in part, to competition for some factor or factors, which could be nutrients, within the system.     

DEVELOPMENTAL POTENTIAL OF EXCISED PRIMORDIAL AND EXPANDING LEAVES OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. primordial leaves 1 through 4 and expanding leaves 5 to 8 were isolated and cultured to examine the effects of auxin and kinetin on development. Without the plant growth regulators in the medium, expanding leaves 7 and 8 developed as leaves; younger leaf primordia did not develop. With 0.01 to 5.0 mg/1 IAA, 2–7% of the youngest pair of primordial leaves (1 and 2) developed as roots. Small leaf blade development occurred on IAA at 0.5 to 5.0 mg/1 with 10–12% of the explants, and shoots developed from 2% of the youngest primordia explants at 3 mg/1 IAA. With 2–28% of the second set of primordial leaves (3 and 4), a leaf with a root developed with 0.01 to 5.0 mg/1 IAA. At 3.0 mg/1 IAA, in addition to leaf formation, 2% of the explants formed a rosette of leaves and 1% formed a shoot. With the highest level of IAA (5 mg/1), 2% of the explants formed a root. Expanding leaves 5 through 8 developed mostly into leaves without petioles on IAA and kinetin. Plant development occurred from 2% of the youngest primordial leaves on 0.03 mg/1 kinetin; otherwise, these primordia on 0.003 to 2 mg/1 kinetin developed into abnormal leaves. Primordia 3 and 4 developed into normal appearing leaves at levels of kinetin between 0.03 and 2 mg/1. At lower levels the leaves were abnormal.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [¹⁴C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1° and 3° explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.     

Movement of 14C- and 11C-labelled Assimilate in Wheat Leaves: the Effect of IAA

The rate of translocation of naturally-loaded, radiolabelledassimilate has been studied in leaves of wheat treated withIAA. The velocity was measured directly by following the movementof ¹¹C-labelled material along the leaf. The kinetics of translocationwere also estimated by using a two-compartment model to calculatethe rate constant of disappearance of ¹⁴C from the fed area.IAA was applied at three different sites on the leaf and atvarious times up to 24 h before ¹⁴CO₂ feeding. No effects ofIAA were observed on: (1) direction and velocity of transport;(2) loading of assimilate into the phloem; or (3) the site andkinetics of unloading. These results are discussed with referenceto the movement of IAA along the transport path and the useof different tissues as experimental systems.     

Changes in the Levels of IAA and ABA in Cucumber Leaves under Progressive Soil Drought

Changes in the IAA and ABA contents in cucumber (Cucumis sativus L.) leaves during adaptation to drought were studied. An increase in the water-retaining capacity and heat resistance of leaves indicating the onset of adaptation occurred when the leaf growth has been already suppressed. There was a transient increase in the ABA content during the initial stage of adaptation. An increased IAA content was maintained for a longer period, throughout about two-third of the adaptation period. A second increase in the ABA content was observed before the onset of leaf permanent wilting, when IAA content already decreased. Our data suggest that not only ABA, but also IAA are involved in the development of defense responses during the adaptation to drought.     

Changes in the hydroxamic acid content of maize leaves with time and after artificial damage; implications for insect attack

The dynamics of hydroxamic acids (Hx) in maize plants were examined, along with the effects on this process of artificial leaf damage. The concentration of Hx in maize (cv. LGH) leaves declined rapidly with age, although young emerging tissue did have relatively high levels. The decline was not entirely due to a growth dilution effect, as the total Hx content in leaves also decreased with age. This effect was less pronounced in isolated leaves. The plant hormone abscisic acid (ABA) accelerated the decline in Hx in isolated maize leaves, while indole acetic acid (IAA) had no apparent effect. Artificial leaf damage on growing plants produced a significant increase in the concentration of Hx relative to undamaged controls. The difference was greatest (about 18%) two days after damage, but seemed to be largely due to a slower rate of decline relative to controls. Isolated maize leaves generally showed a much higher level of increase (20 - 40%). The results are generally consistent with the theory that Hx functions as a defence against insect attack.     

Effect of Salinity on the Composition, Number and Size of Epidermal Cells along the Mature Blade of Wheat Leaves

Salinity inhibits leaf growth in association with changes in cell size. The objective of this study was to determine the spatial distributions of the composition, number and dimensions of epidermal cells in the mature blades of leaf four of wheat seedlings under saline conditions. Plants were grown in loamy soil either with or without 120 mmol/L NaCl in a growth chamber, and harvested after leaf four was fully developed. The results of the spatial distribution analyses of width along the blade showed that salinity not only reduced the width of the leaf blade, but that it also altered the distribution pattern of blade width along the leaf axis. The reduction in the final size of the leaf blade was associated with a reduction in the total number of epidermal cells and in their widths and lengths. This study also revealed the spatial effects of salinity on the blade and epidermal cell dimensions along the leaf axis. In particular, salinity inhibited the total cell number for interstomatal, sister and elongated cells, implying that cell division in wheat leaves is inhibited by salinity. However, the lengths of interstomatal cells were not affected by salinity （unlike those for the sister and elongated cells）, suggesting the relative contributions of cell length and numbers to the reduction in the final length of the blade under salinity is dependent on cell type.