Carmen Pérez-Rontomé

Stem xylem features in three Quercus (Fagaceae) species along a climatic gradient in NE Spain

Abstract Stem xylem features in two evergreen Quercus species (Q. coccifera and Q. ilex) and a deciduous one (Q. faginea) were analysed along an Atlantic-Mediterranean climatic gradient in which rainfall and winter cold experience strong variation. Mean maximum vessel diameter, vessel density, vessel element length, xylem transverse sectional area, Huber value (xylem transverse sectional area per leaf area unit), theoretical leaf specific conductivity (estimated hydraulic conductance per leaf area unit) and total leaf area were determined in 3-year-old branches. Q. faginea presented the widest vessels and the highest theoretical leaf specific conductivity while Q. coccifera showed the lowest total leaf area and the highest Huber value. Studied features did not exhibit significant correlations with mean minimum January temperature in any species but did show significant relationships with rainfall. In Q. coccifera, mean maximum vessel diameter, vessel element length and theoretical leaf specific conductivity increased with higher rainfall while vessel density decreased. Mean maximum vessel diameter and total leaf area in Q. ilex increased with precipitation whereas variables of Q. faginea did not show any significant trend. Results suggest that aridity, rather than minimum winter temperature, controls stem xylem responses in the studied evergreen species. Q. faginea traits did not show any response to precipitation, probably because this species develops deep roots, which in turn makes edaphic and topographic factors more important in the control of soil water availability. In response to aridity Q. coccifera only exhibits adjustment at a xylem level by reducing its water transport capacity through a reduction of vessel diameter without changing the amount of xylem tissue or foliage, whereas Q. ilex adjusts its water transport capacity in parallel to the foliage area.

Leaf morphology and leaf chemical composition in three Quercus (Fagaceae) species along a rainfall gradient in NE Spain

Abstract Leaf features were examined in three Quercus species (Q. coccifera, Q. ilex and Q. faginea) along a steep rainfall gradient in NE Spain. The analyzed leaf traits were area, thickness, density, specific mass, leaf concentration of nitrogen, phosphorous, lignin, cellulose and hemicellulose, both on a dry weight basis (Nw, Pw, Lw, Cw, Hw) and on an area basis (Na, Pa, La, Ca, Ha). These traits were regressed against annual precipitation and correlated with each other, revealing different response patterns in the three species. Q. faginea, a deciduous tree, did not show any significant correlation with rainfall. In Q. coccifera, an evergreen shrub, Nw, Na, Lw, La and Ca increased with higher annual rainfall, while Hw decreased. In Q. ilex, an evergreen tree, leaf area, Pw and Lw increased with precipitation, whereas specific leaf mass, thickness and Ha showed the reverse response. Correlations between the leaf features revealed that specific mass variation in Q. faginea and Q. coccifera could be explained by changes in leaf density, while in Q. ilex specific leaf mass was correlated with thickness. Specific leaf mass in the three species appeared positively correlated with all the chemical components on a leaf area basis except with lignin in Q. ilex and with P in Q. ilex and Q. faginea. In these two tree species Pw showed a negative correlation with specific leaf mass. It is suggested that each species has a different mechanism to cope with water shortage which is to a great extent related to its structure as a whole, and to its habit.

Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants

The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.

Leaf morphology, leaf chemical composition and stem xylem characteristics in two Pistacia (Anacardiaceae) species along a climatic gradient

Summary Leaf and stem xylem features in Pistacia lentiscus (an evergreen shrub) and P. terebinthus (a deciduous shrub) were analysed along an Atlantic-Mediterranean gradient, which is characterised by a variation of rainfall and winter temperature. Leaf features were foliage area of 3-year-old shoots (FA), leaflet area (LA), specific mass (SLM), thickness (LTh), density (LD) and chemical composition on a dry weight basis (N, P, cellulose Cel, hemicellulose Hcel, and lignin Lig). Stem traits referred to xylem anatomy: mean vessel diameter of the 25 largest vessels (MxD), vessel element length (VEL), number of vessels per xylem cross section area unit (VD) and the ratio xylem/foliage surface of shoots (HV). Comparisons of traits have been made both at an interspecific and at an intraspecific level, this latter by analysing how these traits respond to variations of precipitation and winter temperature. P. terebinthus showed higher FA, LA, MxD, Nand Cel, but lower SLM, LTh, Hcel, VD and HV than P. lentiscus. Both species showed opposite responses of LTh to the reduction of mean minimum January temperature, decreasing in the former and increasing in the latter. In P. lentiscus VD increased and VEL decreased when winter temperature became colder, while YD decreased and N increased towards the arid part of the gradient. It is suggested that leaf habit plays an important role in determining the function of species and their response to the climatic gradient.

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus

• Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. • Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. • The exposure of plants to 10 μM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 μM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. • We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 μM Al.

Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O2 transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O2 and facilitate its diffusion to the N2-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lbam, Lbcm, and Lbdm, of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O2 binding.

Seasonal variability of dry matter content and its relationship with shoot growth and nonstructural carbohydrates

This study assesses how different phases of shoot growth underlie seasonal change in leaf and stem dry matter content (LDMC and SDMC, respectively) of 12 woody Mediterranean species. The relationship between LDMC and nonstructural carbohydrate (NSC) concentrations is also explored and the seasonal vs interspecies variability of LDMC compared. LDMC, SDMC and shoot elongation rate (SER) were measured on a monthly basis for a minimum of 12 months. Bud growth rate (BGR) and NSC concentrations were also assessed in several of the study species. LDMC and SDMC decreased during shoot elongation in spring and increased in summer, showing a significant negative correlation with SER, but were unrelated to BGR. Half of the species analysed showed a positive relationship between LDMC and NSC. Seasonal fluctuations of LDMC within species were higher than interspecies differences, and species ranking was significantly affected by the month of sampling, except during winter months. Seasonal changes in LDMC and SDMC are mainly related to shoot elongation phenology, and NSC sink-source relationships between old and growing organs can explain this relationship in some species. Owing to the high seasonal variability in LDMC, it is recommended that samples for comparative purposes should be collected as close to the winter as possible.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Fruit growth dynamics and their effects on the phenological pattern of native Pistacia populations in NE Spain

Summary The phenological pattern of Pistacia terebinthus, P. lentiscus and their hybrid P. x saportae, all native to the NE of the Iberian Peninsula, has been studied to explore in natural populations the patterns of phenophase overlapping between vegetative and reproductive growth. We aimed to test the hypothesis that the existence of a long period of arrested fruit growth in P. lentiscus determines a sequential arrangement of vegetative and reproductive phenophases, while the lack of arrested fruit growth in P. terebinthus leads to a high superposition of these phenophases. Two natural populations of P. lentiscus and P. terebinthus and one of P. x saportae were studied on a monthly basis by the method of Orshan (1989c), modified to take into account three frequency levels of phenophase manifestation in the population. The phenophases considered were dolichoblast vegetative growth (DVG), flower bud formation (FBF), flowering (F), fruit setting (FS), fruit growth (FG), seed dispersal (SD), and leaf shedding of dolichoblasts (LSD). It was necessary to define the phenophase FG to differentiate the phase of arrested fruit growth in P. lentiscus. The phenological pattern of P. x saportae presented most of its features as being intermediate with those of the parental species, although the pattern of fruit development was similar to that of P. terebinthus, without producing full fruits. P. terebinthus clearly superposed DVG with FBF and F, while P. lentiscus tended to develop these phenophases sequentially. P. terebinthus presented a partial overlapping between DVG and FG, while P. lentiscus tended to reduce this overlap to a minimum level. Both species avoid DVG overlapping with FG by means of two different mechanisms: P. lentiscus presented a long period of arrested fruit growth necessary to delay fruit growth towards the end of its long DVG, while P. terebinthus lacked the phase of arrested fruit growth but presented a shorter DVG, mostly developed before the onset of FG. The initial hypothesis proved to be valid for P. lentiscus but not totally correct for P. terebinthus, as it also tended to avoid DVG superposition with FG.

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes

The combined use of EPR spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate uncovers sites of nitric oxide production in legume nodules and warns of potential artifacts.