Tala Awada

Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169

Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and carbohydrates, and the environmental factors influencing their accumulation is necessary to realize the full potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accumulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress in medium containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under strictly photoautotrophic conditions. Interestingly, nutrient depletion triggered a similar pattern of early synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green microalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in ribosomal polypeptides and some key enzymes for CO₂ assimilation like ribulose-1,5-bisphosphate carboxylase/oxygenase. These results suggest that turnover of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, several genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engineering of algal strains with improved capacity to convert their biomass into useful biofuel precursors.

Seasonal changes in depth of water uptake for encroaching trees Juniperus virginiana and Pinus ponderosa and two dominant C4 grasses in a semiarid grassland

We used the natural abundance of stable isotopic ratios of hydrogen and oxygen in soil (0.05-3 m depth), plant xylem and precipitation to determine the seasonal changes in sources of soil water uptake by two native encroaching woody species (Pinus ponderosa P. & C. Lawson, Juniperus virginiana L.), and two C(4) grasses (Schizachyrium scoparium (Michx.) Nash, Panicum virgatum L.), in the semiarid Sandhills grasslands of Nebraska. Grass species extracted most of their water from the upper soil profile (0.05-0.5 m). Soil water uptake from below 0.5 m depth increased under drought, but appeared to be minimal in relation to the total water use of these species. The grasses senesced in late August in response to drought conditions. In contrast to grasses, P. ponderosa and J. virginiana trees exhibited significant plasticity in sources of water uptake. In winter, tree species extracted a large fraction of their soil water from below 0.9 m depth. In spring when shallow soil water was available, tree species used water from the upper soil profile (0.05-0.5 m) and relied little on water from below 0.5 m depth. During the growing season (May-August) significant differences between the patterns of tree species water uptake emerged. Pinus ponderosa acquired a large fraction of its water from the 0.05-0.5 and 0.5-0.9 m soil profiles. Compared with P. ponderosa, J. virginiana acquired water from the 0.05-0.5 m profile during the early growing season but the amount extracted from this profile progressively declined between May and August and was mirrored by a progressive increase in the fraction taken up from 0.5-0.9 m depth, showing plasticity in tracking the general increase in soil water content within the 0.5-0.9 m profile, and being less responsive to growing season precipitation events. In September, soil water content declined to its minimum, and both tree species shifted soil water uptake to below 0.9 m. Tree transpiration rates (E) and water potentials (Psi) indicated that deep water sources did not maintain E which sharply declined in September, but played an important role in the recovery of tree Psi. Differences in sources of water uptake among these species and their ecological implications on tree-grass dynamics and soil water in semiarid environments are discussed.

Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes

Abiotic stresses have adverse effects on plant growth and productivity. The homologous RD29A and RD29B genes are exquisitely sensitive to various abiotic stressors. Therefore, RD29A and RD29B gene sequences have potential to confer abiotic stress resistance in crop species grown in arid and semi-arid regions. To our knowledge, no information on the physiological roles of the proteins encoded by RD29A and RD29B are available in the literature. To understand how these proteins function, we used reverse genetic approaches, including identifying rd29a and rd29b T-DNA knockout mutants, and examining the effects of complementing transgenes with the genes under control of their native promoters and chimeric genes with the native promoters swapped. Four binary vectors with the RD29A and RD29B promoters upstream of the cognate RD29A and RD29B cDNAs and as chimeric genes with noncognate promoters were used to transform rd29a and rd29b plants. Cold, drought, and salt induced both genes; the promoter of RD29A was found to be more responsive to drought and cold stresses, whereas the promoter of RD29B was highly responsive to salt stress. Morphological and physiological responses of rd29a and rd29b plants to salt stress were further investigated. Root growth, and photosynthetic properties declined significantly, while solute concentration (Ψπ), water use efficiency (WUE) and δ13C ratio increased under salt stress. Unexpectedly, the rd29a and rd29b knockout mutant lines maintained greater root growth, photosynthesis, and WUE under salt stress relative to control. We conclude that the RD29A and RD29B proteins are unlikely to serve directly as protective molecules.

Ecophysiology of Two Native Invasive Woody Species and Two Dominant Warm‐Season Grasses in the Semiarid Grasslands of the Nebraska Sandhills

Populations of Pinus ponderosa and Juniperus virginiana are expanding into semiarid Sandhills grasslands in Nebraska. To evaluate the physiological basis of their success, we measured the seasonal course of leaf gas exchange, plant water status, and carbon isotope discrimination in these two native trees and two native C4 grasses (Schizachyrium scoparium and Panicum virgatum). Compared to the trees, grasses had higher net photosynthetic rates (Anet) and water use efficiency (WUE) and more negative predawn and midday water potentials (Ψ) in June and July. While leaf Ψ and rates of leaf gas exchange declined for all four species during August, the Ψmid of the grasses were significantly more negative than those of the two trees. The deeply rooted trees maintained water status during summer, in contrast to the grasses, which senesced. Juniperus virginiana in particular was well adapted to xeric conditions, with low stomatal conductance, high WUE, and positive Anet at low Ψ. The highest values of Anet were observed in May for J. virginiana and in May and September for P. ponderosa. Both species maintained low but positive Anet throughout the winter at temperatures above 0°C. Leaf carbon isotopic signature differed between tree and grass species but did not exhibit significant within‐species seasonal variability. The semiarid grassland climate of Nebraska does not appear to limit P. ponderosa and J. virginiana, which use growth during the nongrowing season and access to deep soil moisture to compensate for growing‐season drought.

Comparative Transcriptome Analysis of Two Olive Cultivars in Response to NaCl-Stress

Background Olive (Olea europaea L.) cultivation is rapidly expanding and low quality saline water is often used for irrigation. The molecular basis of salt tolerance in olive, though, has not yet been investigated at a system level. In this study a comparative transcriptomics approach was used as a tool to unravel gene regulatory networks underlying salinity response in olive trees by simulating as much as possible olive growing conditions in the field. Specifically, we investigated the genotype-dependent differences in the transcriptome response of two olive cultivars, a salt-tolerant and a salt-sensitive one. Methodology/Principal Findings A 135-day long salinity experiment was conducted using one-year old trees exposed to NaCl stress for 90 days followed by 45 days of post-stress period during the summer. A cDNA library made of olive seedling mRNAs was sequenced and an olive microarray was constructed. Total RNA was extracted from root samples after 15, 45 and 90 days of NaCl-treatment as well as after 15 and 45 days of post-treatment period and used for microarray hybridizations. SAM analysis between the NaCl-stress and the post-stress time course resulted in the identification of 209 and 36 differentially expressed transcripts in the salt–tolerant and salt–sensitive cultivar, respectively. Hierarchical clustering revealed two major, distinct clusters for each cultivar. Despite the limited number of probe sets, transcriptional regulatory networks were constructed for both cultivars while several hierarchically-clustered interacting transcription factor regulators such as JERF and bZIP homologues were identified. Conclusions/Significance A systems biology approach was used and differentially expressed transcripts as well as regulatory interactions were identified. The comparison of the interactions among transcription factors in olive with those reported for Arabidopsis might indicate similarities in the response of a tree species with Arabidopsis at the transcriptional level under salinity stress.

Photosynthetic performance of invasive Pinus ponderosa and Juniperus virginiana seedlings under gradual soil water depletion

Changes in climate, land management and fire regime have contributed to woody species expansion into grasslands and savannas worldwide. In the USA, Pinus ponderosa P.&C. Lawson and Juniperus virginiana L. are expanding into semiarid grasslands of Nebraska and other regions of the Great Plains. We examined P. ponderosa and J. virginiana seedling response to soil water content, one of the most important limiting factors in semiarid grasslands, to provide insight into their success in the region. Photosynthesis, stomatal conductance, maximum photochemical efficiency of PSII, maximum carboxylation velocity, maximum rate of electron transport, stomatal limitation to photosynthesis, water potential, root-to-shoot ratio, and needle nitrogen content were followed under gradual soil water depletion for 40 days. J. virginiana maintained lower L(s), higher A, g(s), and initial F(v)/F(m), and displayed a more gradual decline in V(cmax) and J(max) with increasing water deficit compared to P. ponderosa. J. virginiana also invested more in roots relative to shoots compared to P. ponderosa. F(v)/F(m) showed high PSII resistance to dehydration in both species. Photoinhibition was observed at approximately 30% of field capacity. Soil water content was a better predictor of A and g(s) than Psi, indicating that there are other growth factors controlling physiological processes under increased water stress. The two species followed different strategies to succeed in semiarid grasslands. P. ponderosa seedlings behaved like a drought-avoidant species with strong stomatal control, while J. virginiana was more of a drought-tolerant species, maintaining physiological activity at lower soil water content. Differences between the studied species and the ecological implications are discussed.

Stomatal variability of native warm-season grasses from the Nebraska Sandhills

Soil moisture deficit is usually the major limiting factor for herbage production in the Sandhills of Nebraska. We examined inter-population and interspecific variability in stomatal characteristics and drought tolerance in sand bluestem (Andropogon hallii Vitman), little bluestem [Schizachyrium scoparium (Michx.) Nash], prairie sandreed [Calamovilfa longifolia (Hook) Scribn.], and switchgrass (Panicum virgatum L.). Ramets were collected during the dormant season across an aridity gradient from east to west (ranging from 560 mm to 340 mm average annual precipitation) in the Sandhills of Nebraska. Plants were grown in individual pots under greenhouse conditions. Once plants were well established, stomatal characteristics were determined and stomatal conductance (gs) was measured through a dry-down period of no watering. Populations did not differ in stomatal characteristics across the gradient, except for stomatal density on the adaxial leaf surface of prairie sandreed and the abaxial leaf surface of sand ...

Animal anti-apoptotic genes ameliorate the loss of turgor in water-stressed transgenic tobacco

Nicotiana tabacum L. ‘Glurk’ plants were transformed with antiapoptotic animal genes [chicken Bcl-xl; nematode CED-9; chicken Bcl-xl(GA) a mutant of Bcl-xl ; and a 3’ non-coding region of human Bcl-2, referred to as 161-1]. Our objectives were to determine whether plant transformation with anti-apoptotic genes ameliorates drought tolerance in tobacco plants by subjecting the plants to a dry-down period. The non-transformed Glurk and the transgenic Glurk harboring G115, which expresses β-glucuronidase, served as controls. Transformation of tobacco plants with animal anti-apoptotic genes significantly impacted the rates of photosynthesis (A) and stomatal conductance (gs), but not to the same extent in every line. Controls generally exhibited higher A and gs than the transformed plants under well-watered conditions. Photosynthesis and stomatal conductance declined significantly on the 3rd day, and approached zero on the 11th day of water deprivation. Differences among controls and transformed tobacco plants ...

Carbon sequestration in the total and light fraction soil organic matter along a chronosequence in grazing exclosures in a semiarid degraded sandy site in China

Horqin Sandy Land is a fragile, seriously desertified region located in Inner Mongolia of China. Over- grazing is one of the primary drivers of desertification in this region. We investigated whether the establishment of grazing exclosures in areas with active sand dunes enhances soil carbon (C) sequestration and benefits soil re- covery. The results showed that soil organic C storage was 1.4, 1.9, and 3.5 times, and light fraction C storage was 2.3, 3.2, and 4.4 times in the 100-cm topsoil after 7, 12, and 25 years of grazing exclusion, respectively, compared to the case in active sand dunes. The light fraction of soil played an important role in soil C sequestration, although it might not change rapidly to provide an early indication of how soil C is increasing in response to grazing exclusion. The results indicated that soils could potentially sequester up to 13.8 Mt C in 25 years if active sand dunes in the study area were to be protected by exclosures. This corresponds to 12.8% of the estimated carbon loss (107.53 Mt) that has been associated with desertification over the past century in the Horqin Sandy Land. Our results suggested that exclosures have the capacity to increase soil C sequestration; however, decades will be required for soil C to recover to historical grassland levels observed prior to desertification.

Accumulation of soil organic carbon during natural restoration of desertified grassland in China’s Horqin Sandy Land

China’s Horqin Sandy Land, a formerly lush grassland, has experienced extensive desertification that caused considerable carbon (C) losses from the plant-soil system. Natural restoration through grazing exclusion is a widely suggested option to sequester C and to restore degraded land. In a desertified grassland, we investigated the C accumulation in the total and light fractions of the soil organic matter from 2005 to 2013 during natural restoration. To a depth of 20 cm, the light fraction organic carbon (LFOC) storage increased by 221 g C/m2 (84%) and the total soil organic carbon (SOC) storage increased by 435 g C/m2 (55%). The light fraction dry matter content represented a small proportion of the total soil mass (ranging from 0.74% in 2005 to 1.39% in 2013), but the proportion of total SOC storage accounted for by LFOC was remarkable (ranging from 33% to 40%). The C sequestration averaged 28 g C/(m2·a) for LFOC and 54 g C/(m2·a) for total SOC. The total SOC was strongly and significantly positively linearly related to the light fraction dry matter content and the proportions of fine sand and silt+clay. The light fraction organic matter played a major role in total SOC sequestration. Our results suggest that grazing exclusion can restore desertified grassland and has a high potential for sequestering SOC in the semiarid Horqin Sandy Land.