José Sánchez-Bravo

Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants

Following exposure to salinity, the root/shoot ratio is increased (an important adaptive response) due to the rapid inhibition of shoot growth (which limits plant productivity) while root growth is maintained. Both processes may be regulated by changes in plant hormone concentrations. Tomato plants (Solanum lycopersicum L. cv Moneymaker) were cultivated hydroponically for 3 weeks under high salinity (100 mM NaCl) and five major plant hormones (abscisic acid, ABA; the cytokinins zeatin, Z, and zeatin-riboside, ZR; the auxin indole-3-acetic acid, IAA; and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, ACC) were determined weekly in roots, xylem sap, and leaves. Salinity reduced shoot biomass by 50–60% and photosynthetic area by 20–25% both by decreasing leaf expansion and delaying leaf appearance, while root growth was less affected, thus increasing the root/shoot ratio. ABA and ACC concentrations strongly increased in roots, xylem sap, and leaves after 1 d (ABA) and 15 d (ACC) of salinization. By contrast, cytokinins and IAA were differentially affected in roots and shoots. Salinity dramatically decreased the Z+ZR content of the plant, and induced the conversion of ZR into Z, especially in the roots, which accounted for the relative increase of cytokinins in the roots compared to the leaf. IAA concentration was also strongly decreased in the leaves while it accumulated in the roots. Decreased cytokinin content and its transport from the root to the shoot were probably induced by the basipetal transport of auxin from the shoot to the root. The auxin/cytokinin ratio in the leaves and roots may explain both the salinity-induced decrease in shoot vigour (leaf growth and leaf number) and the shift in biomass allocation to the roots, in agreement with changes in the activity of the sink-related enzyme cell wall invertase.

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K+ (20%) and decreases in the toxic ion Na+ (by 30%) and abscisic acid (by 20–40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.

Rootstock-mediated changes in xylem ionic and hormonal status are correlated with delayed leaf senescence, and increased leaf area and crop productivity in salinized tomato

Tomato crop productivity under salinity can be improved by grafting cultivars onto salt-tolerant wild relatives, thus mediating the supply of root-derived ionic and hormonal factors that regulate leaf area and senescence. A tomato cultivar was grafted onto rootstocks from a population of recombinant inbred lines (RILs) derived from a Solanum lycopersicum x Solanum cheesmaniae cross and cultivated under moderate salinity (75 mM NaCl). Concentrations of Na(+), K(+) and several phytohormones [abscisic acid (ABA); the cytokinins (CKs) zeatin, Z; zeatin riboside, ZR; and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)] were analysed in leaf xylem sap in graft combinations of contrasting vigour. Scion leaf area correlated with photosystem II (PSII) efficiency (F(v)/F(m)) and determined fruit productivity. Xylem K(+) (but not Na(+)), K(+)/Na(+), the active CK Z, the ratio with its storage form Z/ZR and especially the ratio between CKs and ACC (Z/ACC and Z + ZR/ACC) were positively loaded into the first principal component (PC) determining both leaf growth and PSII efficiency. In contrast, the ratio ACC/ABA was negatively correlated with leaf biomass. Although the underlying physiological mechanisms by which rootstocks mediate leaf area or chlorophyll fluorescence (and thus influence tomato salt tolerance) seem complex, a putative potassium-CK interaction involved in regulating both processes merits further attention.

INDOLE-3-CARBINOL AS A SCAVENGER OF FREE RADICALS

The ability of indole‐3‐carbinol (indole‐3‐methanol) to trap a metastable synthetic‐free radical is presented. Indole‐3‐carbinol is capable of acting as a scavenger of free radicals in an in vitro system. The presence of indole‐3‐carbinol determines the disappearance of the free radicals, the reaction being time‐ and concentration‐dependent. The scavenging activity of different indoles is compared. Indole‐3‐carbinol and indole‐3‐acetic acid are both able to scavenge free radicals, but indole‐3‐carbinol is more effective. Other indoles such as indole‐3‐aldehyde and indole‐3‐carboxylic acid do not show the ability to trap free radicals. Indole‐3‐aldehyde appears as a product of indole‐3‐carbinol reaction with free radicals. The formation of an adduct between the free radical generated in vitro and indole‐3‐carbinol has also been detected. Stability of indole‐3‐carbinol in buffered media at different pH values and formation of 3,3′‐diindolylmethane from indole‐3‐carbinol is also studied. The scavenging activity of indole‐3‐carbinol and its implications on the anti‐carcinogenesis process is discussed.

Influence of 2,3,5-Triiodobenzoic Acid and 1-N-Naphthylphthalamic Acid on Indoleacetic Acid Transport in Carnation Cuttings: Relationship with Rooting.

Abstract.3H-IAA transport in excised sections of carnation cuttings was studied by using two receiver systems for recovery of transported radioactivity: agar blocks (A) and wells containing a buffer solution (B). When receivers were periodically renewed, transport continued for up to 8 h and ceased before 24 h. If receivers were not renewed, IAA transport decreased drastically due to immobilization in the base of the sections. TIBA was as effective as NPA in inhibiting the basipetal transport irrespective of the application site (the basal or the apical side of sections). The polarity of IAA transport was determined by measuring the polar ratio (basipetal/acropetal) and the inhibition caused by TIBA or NPA. The polar ratio varied with receiver, whereas the inhibition by TIBA or NPA was similar. Distribution of immobilized radioactivity along the sections after a transport period of 24 h showed that the application of TIBA to the apical side or NPA to the basal side of sections, increased the radioactivity in zones further from the application site, which agrees with a basipetal and acropetal movement of TIBA and NPA, respectively. The existence of a slow acropetal movement of the inhibitor was confirmed by using 3H-NPA. From the results obtained, a methodological approach is proposed to measure the variations in polar auxin transport. This method was used to investigate whether the variations in rooting observed during the cold storage of cuttings might be related to changes in polar auxin transport. As the storage period increased, a decrease in intensity and polarity of auxin transport occurred, which was accompanied by a delay in the formation and growth of adventitious roots, confirming the involvement of polar auxin transport in supplying the auxin for rooting.

Early steps of adventitious rooting: morphology, hormonal profiling and carbohydrate turnover in carnation stem cuttings

The rooting of stem cuttings is a common vegetative propagation practice in many ornamental species. A detailed analysis of the morphological changes occurring in the basal region of cultivated carnation cuttings during the early stages of adventitious rooting was carried out and the physiological modifications induced by exogenous auxin application were studied. To this end, the endogenous concentrations of five major classes of plant hormones [auxin, cytokinin (CK), abscisic acid, salicylic acid (SA) and jasmonic acid] and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid were analyzed at the base of stem cuttings and at different stages of adventitious root formation. We found that the stimulus triggering the initiation of adventitious root formation occurred during the first hours after their excision from the donor plant, due to the breakdown of the vascular continuum that induces auxin accumulation near the wounding. Although this stimulus was independent of exogenously applied auxin, it was observed that the auxin treatment accelerated cell division in the cambium and increased the sucrolytic activities at the base of the stem, both of which contributed to the establishment of the new root primordia at the stem base. Further, several genes involved in auxin transport were upregulated in the stem base either with or without auxin application, while endogenous CK and SA concentrations were specially affected by exogenous auxin application. Taken together our results indicate significant crosstalk between auxin levels, stress hormone homeostasis and sugar availability in the base of the stem cuttings in carnation during the initial steps of adventitious rooting.

Lateral diffusion of polarly transported indoleacetic acid and its role in the growth of Lupinus albus L. hypocotyls.

The transport and metabolism of indole-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L, cv. Multolupa) hypocotyls, following application of dual-isotope-labelled indole-3-acetic acid, [5-3H]IAA plus [1-14C]IAA, to decapitated plants. To study the radial distribution of the transported and metabolized IAA, experiments were carried out with plants in which the stele was separated from the cortex by a glass capillary. After local application of labelled IAA to the cortex, radioactivity remained immobilized in the cortex, near the application point, showing that polar transport cannot occur in the outer tissues. However, following application of IAA to the stele, radioactivity appeared in the cortex in those hypocotyl sections below the first 1 cm (in which the capillary was inserted), and the basipetal IAA movement was similar to that observed after application of IAA to the complete cut surface. In both assays, longitudinal distribution of 14C and 3H in the stele outside the first 1 cm was positively correlated with that of cortex, indicating that there was a lateral migration of IAA from the transport pathway (in the stele) to the outer tissues and that this migration depended on the amount of IAA in the stele. Both tissues (stele and cortex) exhibited intensive IAA metabolism, decarboxylation being higher in the stele than in the cortex while IAA conjugation was the opposite. Decapitation of the seedlings caused a drastic reduction of hypocotyl growth in the 24 h following decapitation, unless the hypocotyls were treated apically with IAA. Thus, exogenous IAA, polarly transported, was able to substitute the endogenous source of auxin (cotyledons plus meristem) to permit hypocotyl growth. It is proposed that IAA escapes from the transporting cells (in the stele) to the outer tissues in order to reach the growth-responsive cells. The IAA metabolism in the outer tissues could generate the IAA gradient necessary for the maintenance of its lateral flow, and consequently the auxin-induced cell elongation.

Influence of cold storage period and auxin treatment on the subsequent rooting of carnation cuttings

Abstract The influence of the duration of cold storage and a subsequent auxin treatment on the rooting of cuttings from three carnation cultivars (‘Oriana’, ‘Elsy’ and ‘Virginie’) was studied. When cuttings were stored for short periods (2 weeks), auxin (indole-3-butyric acid plus naphthale-neacetic acid) treatment stimulated the rooting in ‘Oriana’ and ‘Elsy’ but produced no effect in ‘Virginie’. However, no significant auxin effect was observed in any variety when cuttings were stored for long periods (12 weeks). Increasing the storage period from 2 to 10 weeks (in ‘Oriana’) or to 8 weeks (in ‘Elsy’) was as effective as the auxin treatment in stimulating the rooting. The onset of rooting in untreated ‘Virginie’ cuttings was delayed as the storage period increased from 2 to 8 weeks. The stimulation of the rooting by auxin treatment or storage in ‘Oriana’ and ‘Elsy’ meant a reduction of about 4 days in the time required to reach optimal rooting. Neither the percentage of rooting nor the quality of rooted plants was modified at the end of the rooting period, irrespective of auxin treatment or storage. Changes in the endogenous auxin levels and auxin sensitivity during storage might account for the results obtained.

Oxygen consumption and enzyme inactivation in the indolyl-3-acetic acid oxidation catalyzed by peroxidase

Abstract Oxidation of indolyl-3-acetic acid (IAA) catalyzed by peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) can be brought about alternatively by two different pathways that correspond to the oxidase or peroxidase activities of the enzyme. The relative participation of the enzyme in the two pathways depends on the enzyme/substrate ratio and/or the oxygen concentration. Low levels of oxygen favour the oxidase pathway due to the high affinity for oxygen of the ferroperoxidase, the enzymatic form that initiates this pathway. The inactivation of the enzyme estimated either by appearance of P-670 or by measurements of the IAA degraded when the reaction has finished ( P ∞ ) is a good indication that the peroxidase pathway operates. Therefore, the conditions that reduce the enzyme inactivation (for example, high IAA concentrations), also favour the oxidase pathway.

QUANTITATION OF INDOLE-3-ACETIC ACID BY LC WITH ELECTROCHEMICAL DETECTION IN ETIOLATED HYPOCOTYLS OF LUPINUS ALBUS

The objective of this work was to develop a simple procedure to determine the level of the plant hormone indole-3-acetic acid (IAA) in etiolated lupin hypocotyls using liquid chromatography with amperometric detection. A C18 reversed-phased column was used with a potential of +0.85V applied to the carbon electrode. We used two different auxin extraction procedures from lupin hypocotyl and similar quantities of indole-3-acetic were obtained. The method was capable of determining indole-3-acetic acid with a limit of quantification of 2.2 pg. The identity of the indole-3-acetic acid in lupin was confirmed by GC-MS. The content of this hormone in three zones of hypocotyl (apical, central, and basal) was estimated, and a concentration gradient: apical > central > basal, was observed with mean values of 657 ng IAA/g FW, 261 ng IAA/g FW, and 145 ng IAA/g FW, respectively.