Evaluation of physiological traits for improving drought tolerance in faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

Among grain legumes, faba bean is becoming increasingly popular in European agriculture due to recent economic and environmental interests. Faba bean can be a highly productive crop, but it is sensitive to drought stress and yields can vary considerably from season to season. Understanding the physiological basis of drought tolerance would indicate traits that can be used as indirect selection criteria for the development of cultivars adapted to drought conditions. To assess genotypic variation in physiological traits associated with drought tolerance in faba bean and to determine relationships among these attributes, two pot experiments were established in a growth chamber using genetic materials that had previously been screened for drought response in the field. Nine inbred lines of diverse genetic backgrounds were tested under adequate water supply and limited water conditions. The genotypes showed substantial variation in shoot dry matter, water use, stomatal conductance, leaf temperature, transpiration efficiency, carbon isotope discrimination (Δ¹³C), relative water content (RWC) and osmotic potential, determined at pre-flowering vegetative stage. Moisture deficits decreased water usage and consequently shoot dry matter production. RWC, osmotic potential, stomatal conductance and Δ¹³C were lower, whereas leaf temperature and transpiration efficiency were higher in stressed plants, probably due to restricted transpirational cooling induced by stomatal closure. Furthermore, differences in stomatal conductance, leaf temperature, Δ¹³C and transpiration efficiency characterized genotypes that were physiologically more adapted to water deficit conditions. Correlation analysis also showed relatively strong relationships among these variables under well watered conditions. The drought tolerant genotypes, ILB-938/2 and Melodie showed lower stomatal conductance associated with warmer leaves, whereas higher stomatal conductance and cooler leaves were observed in sensitive lines (332/2/91/015/1 and Aurora/1). The lower value of Δ¹³C coupled with higher transpiration efficiency in ILB-938/2, relative to sensitive lines (Aurora/1 and Condor/3), is indeed a desirable characteristic for water-limited environments. Finally, the results showed that stomatal conductance, leaf temperature and Δ¹³C are promising physiological indicators for drought tolerance in faba bean. These variables could be measured in pot-grown plants at adequate water supply and may serve as indirect selection criteria to pre-screen genotypes.     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

Host physiological condition regulates parasitic plant performance: <Emphasis Type="Italic">Arceuthobium vaginatum</Emphasis> subsp. <Emphasis Type="Italic">cryptopodum</Emphasis> on <Emphasis Type="Italic">Pinus ponderosa</Emphasis>

Much research has focused on effects of plant parasites on host-plant physiology and growth, but little is known about effects of host physiological condition on parasite growth. Using the parasitic dwarf mistletoe Arceuthobium vaginatum subsp. cryptopodum (Viscaceae) and its host Pinus ponderosa, we investigated whether changes in host physiological condition influenced mistletoe shoot development in northern Arizona forests. We conducted two studies in two consecutive years and used forest thinning (i.e., competitive release) to manipulate host physiological condition. We removed dwarf mistletoe shoots in April, before the onset of the growing season, and measured the amount of regrowth in the first season after forest thinning (Study I: n=38 trees; Study II: n=35 trees). Thinning increased tree uptake of water and carbon in both studies, but had no effect on leaf N concentration or δ¹³C. Mistletoe shoot growth was greater on trees with high uptake of water and carbon in thinned stands than trees with low uptake in unthinned stands. These findings show that increased resource uptake by host trees increases resources to these heterotrophic dwarf mistletoes, and links mistletoe performance to changes in host physiological condition.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Xylem embolism and stomatal regulation in two rubber clones (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> Muell. Arg.)

Vulnerability to water-stress-induced embolism of stems, petioles, and leaf midribs was evaluated for two rubber clones (RRIM600 and RRIT251). The xylem conduits were relatively vulnerable to cavitation with 50% of embolism measured for xylem pressures between –1 and –2 MPa. This feature can be related to the tropical-humid origin of the species. A distinct basipetal gradient of vulnerability was found, leaf midribs being the least vulnerable. Substantial variation in vulnerability to cavitation was found between the two clones only at the petiole level. A correlation was found between the stomatal behavior and the development of cavitation. Stomata were nearly closed when the xylem pressure reached the point of xylem dysfunction. Stomata may thus contribute to controlling the risk of cavitation. However, for one clone a poor correlation was found between stomatal regulation and petiole vulnerability. This was consistent with a high degree of embolism measured in the petioles after a soil drought event. Therefore, xylem cavitation might represent a promising criterion to evaluate the performance of rubber clones under drought conditions.     

Gas exchange in the salt marsh species <Emphasis Type="Italic">Atriplex portulacoides</Emphasis> L. and <Emphasis Type="Italic">Limoniastrum monopetalum</Emphasis> L. in Southern Portugal

Salt marshes are ecosystems subjected to a variety of environmental stresses like high salinity, water deficit, intense radiation or high temperatures. Field measurements were conduced in two halophyte species, Atriplex portulacoides L. and Limoniastrum monopetalum L., in the Reserva Natural do Sapal de Castro Marim, to compare their physiological response, i.e., water potential (ψ), net photosynthetic rate (A), stomatal conductance (gs) under natural conditions. Both species demonstrated marked variations in ψ throughout the year, with very low values in the summer, the period of higher salinity, drought and temperature. Deficit water potential (Δψ = ψ_midday − ψ_predawn) was lower in the summer than in other seasons in A. portulacoides but not in L. monopetalum. The highest values for A and gs in L. monopetalum were observed in autumn and for A. portulacoides in winter, presenting both lowest values in spring and summer. A_max was particularly high for L. monopetalum than for A. portulacoides in summer and autumn, despite gs_max was similar in both species. Diurnal pattern of A and gs were similar in both species, with higher values in the morning, decreasing throughout the day.     

Effect of high temperature on photosynthesis and transpiration of sweet corn (<Emphasis Type="Italic">Zea mays</Emphasis> L. var. <Emphasis Type="Italic">rugosa</Emphasis>)

Four temperature treatments were studied in the climate controlled growth chambers of the Georgia Envirotron: 25/20, 30/25, 35/30, and 40/35 °C during 14/10 h light/dark cycle. For the first growth stage (V3-5), the highest net photosynthetic rate (P _N) of sweet corn was found for the lowest temperature of 28–34 μmol m⁻² s⁻¹ while the P _N for the highest temperature treatment was 50–60 % lower. We detected a gradual decline of about 1 P _N unit per 1 °C increase in temperature. Maximum transpiration rate (E) fluctuated between 0.36 and 0.54 mm h⁻¹ (≈5.0–6.5 mm d⁻¹) for the high temperature treatment and the minimum E fluctuated between 0.25 and 0.36 mm h⁻¹ (≈3.5–5.0 mm d⁻¹) for the low temperature treatment. Cumulative CO₂ fixation of the 40/35 °C treatment was 33.7 g m⁻² d⁻¹ and it increased by about 50 % as temperature declined. The corresponding water use efficiency (WUE) decreased from 14 to 5 g(CO₂) kg⁻¹(H₂O) for the lowest and highest temperature treatments, respectively. Three main factors affected WUE, P _N, and E of Zea: the high temperature which reduced P _N, vapor pressure deficit (VPD) that was directly related to E but did not affect P _N, and quasi stem conductance (QC) that was directly related to P _N but did not affect E. As a result, WUE of the 25/20 °C temperature treatment was almost three times larger than that of 40/35 °C temperature treatment.     

Irradiance influences tea leaf (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.) photosynthesis and transpiration

Rates of net photosynthesis (P _N) and transpiration (E), and leaf temperature (T_L) of maintenance leaves of tea under plucking were affected by photosynthetic photon flux densities (PPFD) of 200–2 200 μmol m⁻² s⁻¹. P _N gradually increased with the increase of PPFD from 200 to 1 200 μmol m⁻² s⁻¹ and thereafter sharply declined. Maximum P _N was 13.95 μmol m⁻² s⁻¹ at 1 200 μmol m⁻² s⁻¹ PPFD. There was no significant variation of P _N among PPFD at 1 400–1 800 μmol m⁻² s⁻¹. Significant drop of P _N occurred at 2 000 μmol m⁻² s⁻¹. PPFD at 2 200 μmol m⁻² s⁻¹ reduced photosynthesis to 6.92 μmol m⁻² s⁻¹. PPFD had a strong correlation with T_L and E. Both T_L and E linearly increased from 200 to 2 200 μmol m⁻² s⁻¹ PPFD. T_L and E were highly correlated. The optimum T_L for maximum P _N was 26.0 °C after which P _N declined significantly. E had a positive correlation with P _N.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.     

Transient expression of <Emphasis Type="Italic">AtNCED3</Emphasis> and <Emphasis Type="Italic">AAO3</Emphasis> genes in guard cells causes stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis>

Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore, stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native ABA-biosynthesis enzymes are active in guard cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. V. Melhorn and K. Matsumi contributed equally to this work.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Effects of long-term experimental night-time warming and drought on photosynthesis, <Emphasis Type="Italic">F</Emphasis>v/<Emphasis Type="Italic">F</Emphasis>m and stomatal conductance in the dominant species of a Mediterranean shrubland

We conducted a night-time warming and drought field experiment for 7 years (1999–2005) in a Mediterranean shrubland. We focused on the two dominant shrub species, Erica multiflora L. and Globularia alypum L. and the tree Pinus halepensis L. and the final years to study the effects of the experimental night-time warming and drought on Fv/Fm, photosynthesis, and stomatal conductance. Warming treatment increased mean air temperature and mean soil temperature through the years by an average of 0.7 and 0.9°C respectively, and drought treatment reduced soil moisture through the years by an average of 19%. Warming tended to increase photosynthetic rates in E. multiflora, G. alypum and P. halepensis mostly in the cold seasons, when plants were more limited by temperature, as shown by the lowest values of Fv/Fm being detected in winter in the three studied species. A negative effect of warming was only detected for E. multiflora in summer 2003. Drought treatment generated different responses of net photosynthetic rates depending on the species, season and year. Stomatal conductance showed the same pattern as photosynthesis for the three studied species, displaying seasonal and inter-annual variability, although with an overall negative effect of drought for P. halepensis. Photosynthetic rates decreased significantly in the dry winter 2005 and spring 2005 in comparison to the same seasons of 2003 and 2004. There were positive correlations between the photosynthetic rates in different seasons for E. multiflora, G. alypum and P. halepensis and the soil moisture of the week prior to measurements. The great variation in the photosynthetic rates was thus explained in a significant part by soil moisture levels. The lowest Fv/Fm values usually corresponded with lowest stomatal conductances suggesting that drought stress could be associated to stress by low temperatures in winter.     

Growth,biomass allocation,morphology, and photosynthesis of invasive <Emphasis Type="Italic">Eupatorium</Emphasis><Emphasis Type="Italic">adenophorum</Emphasis> and its native congeners grown at four irradiances

Eupatorium adenophorum is one of the more noxious invasive plants worldwide. However, the mechanisms underlying its invasiveness are still not well elucidated. In this study, we compared the invader with its two native congeners (E. heterophyllum and E. japonicum) at four irradiances in terms of growth, biomass allocation, morphology, and photosynthesis. The higher light-saturated photosynthetic rate (P _max) and total leaf area of the invader may contribute to its higher relative growth rate (RGR) and total biomass compared with its native congeners. Total biomass and RGR increased significantly with the increase of P _max and total leaf area. The higher support organ mass fraction and the lower root mass fraction of the invader may also contribute to its higher RGR and biomass through increasing carbon assimilation and reducing respiratory carbon loss, respectively. The higher growth rate of the invader increased its total leaf area, ramet number, and crown area. These traits may help the invader to form dense monoculture, outshading native plant species. However, consistently higher leaf area ratio, specific leaf area, and leaf mass fraction were not found across irradiances for the invader compared with its native congeners. Higher plasticity in response to irradiance was also not found for the invader. The invader retained advantages over the natives across irradiances, while its performance decreased with lower irradiance. The results indicate that the invader may be one of the few super invaders. Reducing irradiance may inhibit its invasions.     

Environmental induced variations in leaf dark respiration and net photosynthesis of <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The relationships between dark respiration rate (R _D) and net photosynthetic rate (P _N) in Quercus ilex L. shrubs growing at the Botanical Garden in Rome were analysed. Correlation analysis of the data sets collected in the year 2006 confirmed the dependence among the considered leaf traits, in particular, R _D was significantly (p<0.05) correlated with P _N (r = 0.40). R _D and P _N increased from March to May [1.40±0.10 and 10.1±1.8 μmol(CO₂) m⁻² s⁻¹ mean values of the period, respectively], when air temperature was in the range 14.8–25.2 °C, underlining the highest metabolic activity in the period of the maximum vegetative activity that favoured biomass accumulation. On the contrary, the highest R _D [1.60±0.02 μmol(CO₂) m⁻² s⁻¹], associated to the lowest P _N rates (44 % of the maximum) and carbon use efficiency (CUE) in July underlined the mobilization of stored material during drought stress by a higher air temperature (32.7 °C).     

Promotive effect of 5-aminolevulinic acid on chlorophyll,antioxidative enzymes and photosynthesis of Pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp<Emphasis Type="Italic">.</Emphasis><Emphasis Type="Italic">chinensis</Emphasis> var. communis Tsen et Lee)

The objective of this article is to study the effect of 5-aminolevulinic acid (ALA) and enhanced chlorophyll content, antioxidative enzymes and photosynthesis rate by foliar application of ALA. We evaluated three concentrations (control-distilled water, T1-50 mg l⁻¹, T2-150 mg l⁻¹, T3-250 mg l⁻¹) of ALA and seven cultivars, “Sanchidaye” (Sa-1), “Lichuandasuomian” (Li-1), “Aijiaohuang” (Ai-1), “Qingyou” No. 4 (Qi-1), “Aikang” No. 5 (Ak-1), “Hanxiao” (Ha-1) and “Shulv” (Sl-1). “Ak-1” showed strongest response of POD (peroxidase) enzyme activity (0.4 U g⁻¹ min⁻¹) in 250 mg l⁻¹ ALA solution. The highest CAT (catalase) activity (0.8 U g⁻¹ min⁻¹) after administration of 250 mg l⁻¹ ALA was observed in “Li-1”. Meanwhile, highest (1.42 mg l⁻¹) total chlorophyll content was also observed in “Ak-1”, when leaves were treated in 50 mg l⁻¹ ALA, “Li-1” and “Ai-1” showed strongest response of specific activity of superoxide dismutase (SOD) in 50 mg l⁻¹ and 50 mg l⁻¹ ALA. Two hundred and fifty milligram per milliliter of ALA-treatment significantly improved the net photosynthetic rate.     

<Emphasis Type="Italic">Withania somnifera</Emphasis> (Linn.) Dunal: a review of chemical and pharmacological diversity

Withania somnifera Dunal, is a commonly used herb in Indian Ayurvedic medicine system. Due to its pharmacological value and an inexhaustible source of novel biologically active compounds, it has been a great interest for researchers. The plant is known to possess anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory and hemopoetic properties. Various withanolides, steroidal lactones, have been isolated from W. somnifera and were known to have high therapeutic value. Based on the differences in the substitution patterns of withanolides the species has been classified into various chemotypes. So far, three different chemotypes have been identified, which have been further classified into ecotypes based on the contents of withanolides. Present review summarizes the phytochemical variability and pharmacological advances reported in literature.