Month: January 2023

For instance, the pre-side dress soil nitrate test is used widely in conventional systems to indicate plant available N just before the exponential growth phase of the crop. However, low soil NO3 – pools can occur even when N availability is high if soil NO3 – turns over rapidly, such as when high input and high output fluxes occur simultaneously. The higher soil carbon availability often resulting from organic management can increase both microbial N demand and gross soil N transformation rates, thereby increasing plant-soil-microbe soil N cycling and turnover of inorganic N. Thus, new indicators of N availability are needed that take into account active C and N processes in organic systems. Good candidates are labile soil organic matter fractions, which will benefit from more on-farm validation and standardization. Expression levels of genes involved in root N uptake and assimilation may also indirectly indicate plant available N in soil and provide a complement to bio-geochemical indicators of N availability, especially when soil NO3 – turnover is high. Plant N uptake and assimilation systems respond to wide variation in external N availability and internal N metabolites that reflect plant N status through regulatory mechanisms that optimize capture of limiting nutrients. Recent work has expanded knowledge of plant root transcriptional responses to N availability from laboratory-based systems into natural soil conditions, thus providing a basis for selecting candidate genes as indicators of soil N processes. These genes include high-affinity transporters of NH4 + and NO3 – ; nitrite reductase, responsible for reduction of NO3 – to nitrite; and glutamine synthetase and glutamate synthase,vertical agriculture which are involved in NH4 + assimilation into amino acids. Analyzing expression of these genes in roots may provide a “plant’s eye view” of soil N availability, and show how root Nassimilation is high even when soil inorganic N pools are low, i.e. in situations of tightly coupled and rapid N cycling.

If working organic farms can achieve both tightly-coupled N cycling and high crop yields, then how do farmers do it? Are there indeed bio-geochemical or plant-based indicator measures that will help organic farmers learn about their systems and provide the basis for adaptive management? Tomato , a model species for plant N metabolism and plant genetics, is widely grown on organic farms in California, where organic farmers use a variety of management practices. This provides a unique opportunity for a landscape study on how variability in SOM and management relate to yield and N cycling on working organic farms and how root expression of N metabolism genes could indicate rapid plant-soil-microbe N cycling . The overall hypothesis of this study is that tightly-coupled N cycling will be associated with higher levels of total and labile soil C and N and more diverse nutrient inputs . In turn, expression of root N metabolism genes will be elevated and more closely related to soil bio-assays for N availability than inorganic N pools in such fields. A landscape approach was used to assess crop yields, plant-soil N cycling, root gene expression, and the potential for soil N retention across a representative set of organic fields growing Roma-type tomatoes in one county in California, USA.The study took place during the tomato growing season to focus on the synchrony between soil N availability and crop N demand. The participatory framework in concert with GIS-based evaluation of land in organic tomato production was designed to provide real world context for evaluating novel combinations of indicators for N cycling in organic systems. The specific objectives were to: 1) identify different N cycling patterns in organic fields representative of the local landscape based on a suite of plant, soil, and soil microbial variables; 2) examine how root expression of key N metabolism genes relates to biogeochemical indicators of plant-microbe-soil N cycling; and 3) evaluate trade offs among ecosystem functions in N cycling scenarios.The organically-managed fields in this study were on similar parent material in Yolo County, California, which is situated along the western side of the Sacramento Valley.

Annual precipitation in 2011 was 403 mm, and the mean maximum and minimum temperatures were 21.7 and 7.3°C, respectively, compared to 462 mm, 23.1°C, and 8.4°C for the previous 20 years . From 1989–2011, certified organic acreage in Yolo County, California increased 15-fold while production value increased nearly 30-fold to >$30M.Farms growing organic Roma-type tomatoes in 2011 in Yolo County were identified using the California Certified Organic Farmers directory and farmers were contacted during the winter of 2010–11. CCOF is the primary organic certifier in this region of California. Widespread interest among organic farmers in this region to improve N cycling and increasing concerns about N loss due to state-level policy initiatives related to greenhouse gas emissions and water quality provided an entry point to engage a variety of farmers in this study. Eight growers expressed interest in the project and identified the fields in which they expected to transplant tomatoes in early April 2011 . Through multiple one-on-one meetings with these farmers we learned management practices and following the study, we discussed biophysical and management data from their field relative to data from other fields in the study and potential reasons for differences.GIS analysis of the land in organic tomato production was performed in order to ascertain how well the 13 fields that were sampled compared to the range of variability in organic tomato fields in Yolo County. Soil, landscape, and management attributes of all fields in organic tomato production in Yolo County were characterized with a landscape regionalization approach. A set of 103 points were randomly assigned to all such fields based on a 2008 field-scale county survey, representing one point every 4 hectares. For each of these points, the values of 12 variables were compiled from several sources. Categorical variables included soil great group and soil drainage class from the SSURGO database, the number of crop rotation types in a one mile surrounding square, and an agricultural sub-region classification. Continuous variables from the SSURGO database included percent sand, silt, and clay, organic matter, elevation, and the Storie index .GIS data were subjected to a clustering algorithm, partitioning around medoids , based on a distance matrix derived from Gower’s dissimilarity algorithm. PAM analysis with five clusters returned the best defined clusters yielding an average silhouette width of 0.499. The proportion of the landscape in organic tomato production represented by each cluster was calculated by performing a Voronoi tessellation of the 103 points, assigning eachpolygon of the tessellation to a cluster type,vertical farming aeroponics and then intersecting the tessellation with the field boundaries to allow determination of cluster areas.

Based on a lack of grower interest, cluster 2 was not represented.Soil and plant sampling was designed to capture indicators of ecosystem functions related to plant-soil N cycling at times corresponding to key agronomic and phenological events, including immediately prior to tomato transplanting , peak tomato growth period , and tomato harvest . In each field, plots were established at six random locations within a 0.25 ha area. Pre-transplant measurements took place several days prior to tomato transplanting but after other field operations, such as tillage, incorporation of organic amendments and/or vetch cover crops, and bed formation. Tomatoes were transplanted in all fields between 6 April and 20 April, 2011. In each of the six plots, three soil cores for each depth were removed from tomato beds and composited in the field, separately for each plot.For mid-season measurements, fields were all sampled within two weeks of one another, an average of 68 days after transplanting.A soil core was removed in each plot, situated between two tomato plants 15 cm from the planting row. Three 50–150 mg sub-samples of roots were promptly removed from the soil core in the field under minimized/indirect light, rinsed, patted dry, and flash frozen in liquid nitrogen for subsequent RNA extraction .The two plants adjacent to this core were cut at the base and petiole samples from recently matured leaves were removed. Plants were rinsed and dried at 60°C for two weeks before grinding and analyzing for C and N . Tomato yields were sampled just before the farmer’s harvest. In each plot , two 1m × 2m sub-plots were established. At each of these subplots, individual tomato plants were cut at the base and ripe fruit was separated by hand from green and decayed fruit . This process uses criteria similar to that of machine harvested tomatoes as well as those harvested by hand for fresh market sales. Biomass of fruits and shoots were weighed in the field then sub-sampled and dried at 60°C for 2 weeks, before grinding and analyzing for C and N . Soil cores were also taken from each subplot and composited in the field for measurements described below.Soil samples were kept on ice and processed within several hours of field extraction by thoroughly homogenizing by hand. Soils from the 0–15 cm depth were analyzed for a variety of soil C and N fractions, bio-assays for N availability, and soil properties, while deeper depths were analyzed for inorganic N and gravimetric water content only. Inorganic N was extracted from moist soils with 2M KCl and analyzed colorimetrically for NH4 + and NO3 -. Potentially-mineralizable N was measured as NH4 + liberated during a seven-day anaerobic incubation at 37°C. Chloroform fumigation-extraction followed by UV-persulfate oxidation and alkaline persulfate oxidation was used to measure microbial biomass C and N , respectively. K2SO4 extractable organic C and N were quantified in non-fumigated samples. Permanganate oxidizable C , which reflects a processed soil fraction that is sensitive to management was measured according to standard procedures. Gravimetric water content was determined by drying at 105°C for 48 h. Air dried soil samples were sieved to 2 mm, ground, and analyzed for total C and N at the UC Davis Stable Isotope Facility. Shoots and fruit were analyzed for total C and N, δ13C, and δ15N at the UC Davis Stable Isotope Facility. Petiole NO3 – , an indicator of recent N status in conventionally-produced vegetables, was measured in the most recently-matured leaves. Petiole NO3 – changes rapidly with growth stage, so the data are graphed by post-transplanting growing degree day to account for phenological differences among fields as a result of slightly different sampling times relative to transplanting.Root RNA was extracted using Trizol reagent according to the manufacturer’s guidelines followed by DNase digestion using RQ1 RNase-free DNase . Total RNA was purified using the RNeasy Plant Mini Kit . RNA concentrations and quality were assessed using the Agilent Nano drop and the RNA 6000 Nano Assay . Only RNA samples with RNA integrity numbers of at least 7.0 were used for subsequent analyses. These RNA were used for cDNA synthesis for qRT-PCR analysis. cDNA was synthesized from 0.5 μg DNase-treated total RNA using the Superscript III kit .Expression of cytosolic glutamine synthetase GS1 in roots was more strongly related to indicators of plant-soil N cycling than were the other six key genes involved in root N metabolism . Of the soil variables, GS1 was more strongly related to soil bio-assays for N availability than to inorganic N pools . Microbial biomass N and PMN were most strongly associated with expression of GS1 in roots, followed by soil NO3 – . Permanganate oxidizable C and MBC, both indicators of labile soil C pools, also had significant associations with GS1 expression in roots, but soil NH4 + did not. Expression of GS1 also was positively associated with shoot N and petiole NO3 – , as was glutamate synthase NADH-GOGAT. Inclusion of GWC as a covariate in multiple linear regression models improved the proportion of explained variation in GS1 expression .PCA of 28 indicators of yield and plant nutrient status, root N metabolism, and soil C and N cycling showed strong relationships among suites of variables, which clearly differentiated fields along the first two principal components . The first principal component explained 28.3% of the variation; on the left side of the biplot are higher values of most variables, including yield, soil bio-assays, expression of root GS1 and NADH-GOGAT, and labile and total soil C and N pools . Soil NH4 + and NO3 – concentrations from all three sampling times as well as AMT1.2 were associated with one another and with positive values along principal component 2, which explained 19.4% of the variation.

Using weighted Principal Coordinates Analysis , the differences between the soil microbial communities of the two soils can be seen by the two distinct clusters, regardless of surfactant amendment . Due to their broad occurrence in numerous contaminated sites and diverse metabolic pathways for xenobiotic degradation, genera of the Sphingomonadaceae family such as Sphingomonas are considered effective PAH-degrading soil microorganisms . Bastida et al. evaluated PAH biodegradation in a semiarid petroleum-contaminated soil amended with compost and concluded that Sphingomonadales played a dominant role in the initial steps of PAH biodegradation, suggesting that Sphingomonadales were primarily responsible for the conversion of the aromatic hydrocarbons into cis-dihdyrodiol via dioxygenases as well as in the metacleavage pathway to catechol. Kaistobacter has only recently been linked with PAH degradation and their role in PAH biodegradation is still unclear; however, Li et al. utilized 13C-phenanthrene and stable isotope probing in activated sludge and suggested that Kaistobacter was among the primary native microorganisms responsible for phenanthrene degradation. Wang et al. utilized KEGG functional prediction and PICRUSt analysis of PAH-contaminated sediment and concluded that Kaistobacter contributed to the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, specifically, the process of metabolizing pyrene to 3,4-dihydroxyphenanthrene. Although Firmicutes and Proteobacteria phyla comprised a substantial proportion of the soil microbial community, the effects of Brij-35 and rhamnolipid surfactant application, particularly at the high rates, on soil microbial dynamics was apparent. In the pyrene-contaminated clay and sandy loam soils, the OTU numbers and Shannon diversity index were not different from the surfactant-amended treatments in both native and bio-augmented soil treatments,hydroponic bucket except for the addition of rhamnolipid at the high rate, which resulted in a dramatic decrease in OTU number and Shannon diversity index .

The Shannon diversity index of the Native+Pyrene clay and sandy loam soils decreased from 6.53 and 7.74 to 2.54 and 4.18, respectively, in the soils amended with rhamnolipid at the high rate. Notably, the abundance of Bacillus present after the 50-d incubation of the pyrene-contaminated clay soil, with or without bio-augmentation, was less than 2% when rhamnolipid bio-surfactant was amended at the high rate . In the native clay soil amended with rhamnolipid at the high rate, the most dominant genus was Mycoplana . The ability of Mycoplana to effectively use the rhamnolipid bio-surfactant as a carbon source likely resulted in a substantial decrease in the abundance of known PAH degraders, such as Bacillus, Sphingomonas, Kaistobacter, Mycobacterium, and Rhodococcus that were present in other soil treatments . Although it has been shown that some species of Mycoplana such as Mycoplana sp. MWVMB2 were capable of effective PAH biodegradation in soils contaminated with phenanthrene up to 200 mg kg-1 with or without the use of surfactants such as Span 80, Tween 20, cetyl trimethyl ammonium bromide, sodium dodecyl sulfate, and Triton X-100, the Mycoplana sp. that was the dominant genus identified in this study was not able to mineralize pyrene after 50 d . In contrast, the native sandy loam soil amended with rhamnolipid at the high rate did not follow this trend and Bacillus comprised approximately 58% of the genera relative abundance . It should be noted that at the end of the 50-d mineralization study, the native sandy loam amended with rhamnolipid at the medium or high rate was just commencing pyrene mineralization, suggesting that the rhamnolipid biosurfactant was potentially exhausted as a preferential carbon source by the soil microbes . A study by Wang et al. considered the influence of rhamnolipid biosurfactant, Tween 80, and sodium dodecyl benzenesulfonate at 5, 10, 50, 100, and 1,000 mg kg-1 on soil microbial dynamics and PAH biodegradation in aged PAH-contaminated soil. The researchers reapplied the surfactants after 42 d due to surfactant adsorption onto solid matrices as well as partial surfactant biodegradation based upon surfactant degradation results by Cserháti et al. . Wang et al. observed similar results to the sandy loam soil amended with rhamnolipid at the high rate in this study, with Bacillus abundance being three to five times as high as that of the other surfactant-amended PAH-contaminated soils.Additionally, the native sandy loam soil amended with rhamnolipid biosurfactant at the medium rate as well as the bioaugmented sandy loam soil amended with rhamnolipid at the high rate contained a substantially greater relative abundance of Pseudomonas compared to the unamended and bioaugmented sandy loam soil .

Pseudomonas are known PAH-degrading soil microorganisms and have been shown to effectively degrade PAHs such as naphthalene, phenanthrene, pyrene, and anthracene in crude-oil contaminated soils. The PAH biodegradation by Pseudomonas was also shown to be enhanced in the presence of surfactants such as Tween 80, Triton 100, and rhamnolipid biosurfactant . Cébron et al. used DNA stable isotope probing in 13C-phenanthrene-contaminated soil to assess the effects of ryegrass root exudates on PAH biodegradation and concluded that Pseudomonas sp. was one of the few soil microorganisms activated by the root exudates because the easily degradable carbon source addition provided by the root exudates favored the development of fast-growing rstrategists and copiotrophic soil microorganisms belonging to Gammaproteobacteria . Rhamnolipid biosurfactant, which is composed of a β-hydroxy fatty acid connected by the carboxyl moiety to a rhamnose sugar molecule, has the potential to also be utilized by Pseudomonas as an easily degradable carbon source similar to root exudates . Colores et al. investigated the effect of Witconol SN70 nonionic surfactant on the soil microbial community as well as the biodegradation of hexadecane and concluded that Pseudomonas populations in the soil could utilize both the surfactant and hexadecane for growth, which could have important implications on remediation efforts. The effect of rhamnolipid at the high rate can also be seen using weighted PCoA, where the treatments in both soils clustered separate of the other unamended and surfactant-amended treatments . Additionally, Brij-35 surfactant at the high rate resulted in a cluster separate from the unamended and surfactants amended at the low and medium rates, which were clustered together, indicating no substantial difference in the soil microbial communities . The amendment of either surfactant at various rates, except rhamnolipid at the high rate, to the sandy loam soil resulted in an increase in Brevibacillus abundance compared to the unamended native or bioaugmented sandy loam soil . Wei et al. evaluated Brevibacillus in liquid culture spiked with pyrene and showed that Brevibacillus was able to degrade 57% of pyrene as the sole energy and carbon source; however, these findings have yet to be repeated in a soil system and warrant future research,stackable planters as the increased abundance of Brevibacillus may be attributable to growth due to surfactant degradation in the sandy loam soil and may have important implications for surfactant-enhanced  bio-remediation.

The addition of Brij-35 at the low rate to the native or bio-augmented sandy loam soil resulted in a dramatic increase in Bacillus compared to the unamended native or bio-augmented sandy loam . The greater Bacillus abundance in the native sandy loam soil amended with Brij-35 at the low rate may have contributed to the increased pyrene mineralization compared to the native unamended sandy loam soil . The inoculation of PAH-degrading bacteria in a wide range of contaminated soils has been successfully implemented for the removal of priority PAH pollutants and continues to be a promising remediation method due to its low cost, lack of secondary pollution, and environmental safety . The bio-augmentation of M. vanbaalenii PYR-1, an isolate from an oil-contaminated estuary of the Gulf of Mexico, Redfish Bay, near Aransas Pass, has previously been shown to significantly enhance the initiation and rate of PAH mineralization in both PAH-contaminated soils compared to the native soils.The effectiveness of the bioaugmentation of M. vanbaalenii PYR-1 on pyrene mineralization was evident in the unamended, Brij-35 amended at all rates, and rhamnolipid biosurfactant amendment at the low rate, in both soil systems . Mycobacterium vanbaalenii PYR-1 has been studied in detail with respect to the molecular genetics of PAH degradation and has been shown to encode PAH ringhydroxylating oxygenases nidAB/nidA3B3, which are utilized in the oxidation of HMW PAHs such as pyrene . Additionally, M. vanbaalenii PYR-1 has a complex and very hydrophobic rigid cell envelope that is enriched in mycolic acids and the mycolic acid wall monolayer acts as a biosurfactant to enhance PAH solubility and bio-degradation . Because of these characteristics, M. vanbaalenii PYR-1 is considered an excellent candidate for bio-augmentation in PAH-contaminated soils. The 16S rRNA gene analysis was used in this study to determine if M. vanbaalenii PYR-1 was capable of successfully acclimating after the introduction in both soil systems with or without the addition of the surfactants at different rates. As shown in Figs. 3.2 and 3.3, the bio-augmentation of M. vanbaalenii PYR-1 in all clay and sandy loam soil treatments, except for rhamnolipid at the high ratein both soils and rhamnolipid at the medium rate in the sandy loam soil, resulted in an increase in Mycobacterium compared to the native treatments. This increase in Mycobacterium was especially evident when comparing the Native+Pyrene and PYR- 1+Pyrene treatments . Additionally, LEfSE software was used to determine which soil microorganisms were differentially abundant between the bioaugmented and native soil systems. The abundance of Mycobacterium was found to be significantly greater in the bio-augmented soil treatments compared to the native soil treatments . Functions of different OTUs and prediction of the functional composition of the metagenome in both soils was accomplished using the 16S rRNA gene data, Greengenes reference database, KEGG pathways, and PICRUSt to evaluate the effectiveness of surfactant addition as well as the bio-augmentation of M. vanbaalenii PYR-1 on pyrene mineralization.

By analyzing soil functional genes, contributions of different bacteria involved in the biodegradation of PAHs were assessed. For instance, according to the “Xenobiotics biodegradation and metabolism” list on the KEGG website , pyrene and phenanthrene can be degraded to 3,4-dihydroxyphenanthrene via “Polycyclic aromatic hydrocarbon degradation”, which can then be further metabolized into the TCA cycle via “Naphthalene degradation” and “Benzoate degradation” . These KEGG pathways include numerous predicted PAH-degradation-related KOs, such as PAH oxygenase large subunit , PAH oxygenase small subunit , and extradiol dioxygenase and determine whether bioaugmentation or surfactant addition had any significant effect on these genes and thus, PAH biodegradation in the two soils. Upon analysis of the “Polycyclic aromatic hydrocarbon degradation” KEGG pathway, M. vanbaalenii PYR-1 bioaugmentation in both soils significantly increased the KOs associated with the PAH biodegradation pathway compared to the native soils . The same trend of bioaugmentation of M. vanbaalenii PYR-1 resulted in increased PAH-biodegradation-related KOs in the “Naphthalene degradation” and “Benzoate degradation” compared to the native soil systems . Additionally, PICRUSt was utilized to assess which taxa contributed to the PAH-related KOs. For example, M. vanbaalenii PYR-1 substantially contributed to PAH oxygenase large and small unit; however, other soil microbes in addition to M. vanbaalenii PYR-1 contributed to the increased extradiol dioxygenase in the bioaugmented soils compared to the native soils . These results were in agreement with Niepceron et al. who evaluated phenanthrene biodegradation potential by assessing the PAH-ring hydroxylating dioxygenase sequences in PAH-contaminated soil and showed that PAH-RHDα was closely related to either Burkholderia or Mycobacterium. Wang et al. also used PICRUSt to investigate the successions of bacterial communities in PAH-contaminated soils undergoing  bio-remediation and concluded that bacteria in the Mycobacterium genus contributed substantially to functional genes in all PAH-degradation pathways for metabolizing pyrene to the TCA cycle.Target shooting is an increasingly popular recreational sport with over approximately 100,000 shooting ranges worldwide . In the United States , there are an estimated 12,000 shooting ranges, consisting of 9,000 civilian and 3,000 military shooting ranges . Over 18 million adults participated in any type of clay target shooting on these civilian shooting ranges in 2014, which was a 3.6% increase compared to participants in 2012 . Accordingly, there have been vast numbers of clay targets used for these outdoor clay target shooting activities. Baer et al. determined that clay target use in the U.S. since 1970 has averaged approximately 560 million targets/year. Until recently, clay targets were composed of approximately 67- 70% clay or dolomitic limestone, 30-32% coal tar or petroleum pitch used as a binding agent, and fluorescent paint . The coal tar or petroleum pitch binding agent is a large source of PAHs with concentrations up to 3,000-40,000 mg/kg clay target . Each clay target used in these outdoor shooting activities weighs approximately 100 g each and spread into fragments of various sizes when shot.

In addition, most of the Cu was sequestered in root tissues. Therefore, the likelihood of Cu over-accumulation in fruit is low. According to US Department of Agriculture, annual per capita consumption of fresh cucumbers in the United States is 3.0 kg in 2013, which means average daily cucumber consumption is ∼8.2 g per person-day . The average cucumber water content is 95%, so the daily consumption is 0.41 g dry weight per person-day. Thus, daily personal Cu intake from cucumber used in this study would be 10.0, 11.2, 11.8, and 12.9 μg from control, low, medium and high treatments. According to the Food and Nutrition Board at the U.S. Institute of Medicine of the National Academies, the recommended average requirement for Cu is 700 μg per person-day, with a tolerable upper intake level of 10 mg per person-day.Therefore, Cu intake from consumption of cucumber exposed to nCu enriched soil would be within the recommended Cu levels, even at the higher application level. Hence, consumption of nCu treated cucumbers, even at the high level, represents no significant added risk to consumers. Fruit quality is affected by, among others, sugars and fatty, amino, and carboxylic acids. The profile alteration of these nutrients may result in flavor and nutritional supply changes induced by exposure to nCu.Leaves growing in sunny locations have comparatively high photosynthetic capacities, Rubisco activity, rates of electron transport, and rates of dark respiration . Some species are restricted to sunny or shady locations, and the leaves of these plants are often genetically adapted to their characteristic light environment. The leaves of other species, including those that are naturally exposed to particularly variable light environments,fodder system acclimate to local conditions . Acclimation to extended changes in light enhances net assimilation and nitrogen use efficiency while decreasing vulnerability to high light stress . Either anatomical or biochemical mechanisms may be involved in acclimation .

The local light environment influences the morphological development of leaves in many species, resulting in comparatively thick leaves in bright locations . Fully expanded leaves have a limited capacity for morphological change , and acclimation by these leaves requires biochemical changes in carboxylation, electron transport, and light harvesting, as well as modifications to chloroplast structure and orientation . Monocotyledons with basal meristems, long leaves, and dense canopies may represent a case where photosynthetic acclimation by biochemical change is particularly advantageous. The grass Lolium multiflorum exhibits a strong capacity for local photosynthetic acclimation along the length of a leaf . The leaves of plants like Lolium are produced in dark or dim conditions at the base of plants, and, over time, are pushed to the upper part of the canopy. Typha latifolia , atall monocot that forms dense and highly productive monospecific stands in wetlands , may provide an even more extreme example. T. latifolia ramets originate from rhizomes that are buried in sediment, submerged under water, and often shaded by a dense layer of litter and existing plants. Initial leaf growth is supported by carbohydrates that are either mobilized from rhizomes or translocated from older leaves. Depending on sediment thickness and water depth, and the density of the litter layer and existing canopy, the lower 50–100 cm of a Typha leaf may experience almost total darkness . These characteristics make Typha a useful experimental system for investigating the acclimation capacity of morphologically mature leaves. Basal growth in Typha allows the separation of leaf age from light environment; the oldest segments of Typha leaves are exposed to the brightest light, as opposed to plants with apical meristems, where the youngest leaves are in bright conditions. We investigated the photosynthetic capacity of T. latifolia leaves over time following step changes in shading at different locations along leaves. We hypothesized that morphologically mature Typha leaves have a strong ability for local acclimation, and that individual leaf segments acclimate to the local light level autonomously from the rest of the leaf.T. latifolia rhizomes and crowns were collected in April 2004 at the San Joaquin Freshwater Marsh , located in Orange County, California . Rhizomes with an average weight of 49.7 ± 2.5 g were planted in 20-l pots filled with sand. Plants were grown in a greenhouse under either low light with a mean PPFD of 2.7 mol m−2 d−1 and a maximum of 159 mol m−2 s−1, which was created with 80% neutral shade cloth, or high light with a mean PPFD of 19.4 mol m−2 d−1 and a maximum of 990 mol m−2 s−1.

Each pot had one plant, and the pots were widely spaced. The lower segments of leaves were unshaded by either neighboring plants or upper leaf segments, and the light levels were approximately constant along the length of leaves. The water level in the pots was maintained 5 cm above the sand surface with daily additions of deionized water. The pots were fertilized every 2 weeks with Flora Grow and Flora Micro following the manufacturer’s instructions . The pots were drained before fertilization to avoid salt buildup.Two-month-old sun and shade grown plants with several fully expanded leaves were placed on a bench under high light, and a pair of fully expanded leaves from each plant were selected for experimentation. Individual leaf segments between 20 and 45 cm from the tip were exposed to either sun or shade during the 15-day transfer experiment using cylinders of 80% neutral shade cloth, creating the full combination of segments exposed to constant low light , constant high light , low to high light , or high to low light . Additionally, a set of segments on the same leaves were exposed to either constant high light or low to high light . All treatment combinations and locations were replicated six times. The photosynthesis rate under bright light , stomatal conductance and dark respiration rate were measured every two or three days for two weeks in the middle of the sun and shade segments , on six replicate plants using a portable gas exchange system . Afull sun was measured at a PPFD of 2000 mol m−2 s−1 and Rd was measured in darkness after allowing 3–5 min for equilibration. Leaf temperature was controlled at 25 ◦C and reference CO2 concentration at 370 mol mol−1. The leaf to air vapor pressure deficit ranged from 0.6 to 1.5 kPa. Photosynthetic light response curves were measured after leaves had fully acclimated to a change in light . The light response curves were fit using a non-rectangular hyperbola . Afull sun was calculated as the photosynthetic rate at 2000 mol m−2 s−1; Amax was calculated by extrapolating the regression to infinite light; Rd was calculated as the y-intercept; the apparent quantum yield was calculated as the slope extrapolated to darkness. The light response curves were started at high light , and assimilation was measured in response to stepwise PPFD decreases until full darkness.

Stomatal conductance decreased gradually in response to light decreases, and increased gradually in response to light increases. This sluggish stomatal response either led to lower rates of photosynthesis for light curves run from dim to bright conditions relative to curves run from bright to dim conditions, or forced unreasonably long equilibration times. Moreover,fodder system for sale midday field and greenhouse observations showed that leaves exposed to a continuous PPFD of 2000 mol m−2 s−1 for ∼15 min exhibited a steady CO2 assimilation. We therefore opted to carry out light curves from bright to dark conditions, but acknowledge that lags in stomatal adjustment may have resulted in somewhat higher Ci for the light curves than would have been observed for fully equilibrated leaves. Nonetheless, we emphasize that our study is comparative, and the key is consistency across treatments; we executed the light curves the same way for all treatments and leaf segments. Nitrogen concentration , and leaf mass per area , were measured on the leaf segments used for gas exchange. Nitrogen was determined using the micro Kjeldahl technique; samples were oven dried, ground in a Wiley mill, weighed, digested, and nitrogen concentration was determined with an auto analyzer .We characterized the vertical gradients of light and photosynthetic characteristics during midday sunny conditions in August 2004. The PPFD profile was measured through the canopy at 48 different locations in the SJFM using a horizontal quantum sensor mounted on a 2 m handheld pole. Each profile consisted of ten individual measurements recorded with a data logger at 0.0, 0.6, 1.2, and 3.0 m above the sediment surface. The 3.0 m measurement was above the canopy. LAI was measured at the base of the canopy with a LI-COR LAI-2000, assuming non-clumped leaves and without distinguishing between live leaves and litter. Photosynthetic light response curves were measured on three segments of fully expanded leaves from 5 different plants. The cross section of leaves changed from flat at the tip to triangular at the base, and it was not possible to seal the chamber on leaf segments further than 100 cm from the tip.The parameters derived from the light response curves, the nitrogen content, and the leaf mass per area, were compared between treatments using Univariate ANOVA or t tests. The effect of light treatment was analyzed by Student’s t-test. Univariate ANOVAs and Tukey tests were used to compare Afull sun, Amax, gs and Rd between the light treatments within each sampling period. The effects and interactions of treatment and time following transfer were analyzed with multivariate analysis of variance ; this analysis corrected F values due to temporal auto correlation. MANOVA does not require the response variables to be equally correlated, assuming an unstructured variance–covariance matrix . The effect of leaf position on the photosynthetic parameters of leaves growing in natural conditions was analyzed with three paired t-tests, because of the high variation among leaves.

Statistical analyses were performed with JMP software version 7.0 and Minitab statistical software version 15.Fully expanded T. latifolia leaf segments exhibited strong acclimation to a change in light. The light response curves of individual segments of shade grown leaves that were transferred to high light were similar to those of segments that remained in high light throughout the experiment . This acclimation was highly localized; the response curves of SH-SH segments did not change, even as adjacent SH-SU segments acclimated to high light. Likewise, the response curves of SU-SU segments did not change, even as adjacent SU-SH segments acclimated to shade . Photosynthesis in bright light , maximum photosynthetic capacity , dark respiration , and stomatal conductance differed significantly between sun and shade segments following acclimation, regardless of initial growth conditions . In contrast, intercellular CO2 concentration and apparent quantum yield showed no significant differences. When compared within plants that started in sun, Afull sun and Amax of SU-SU segments differed significantly from SU-SH segments. The same pattern was found for Rd and gs, but not ˚y and Ci, where no significant differences among treatments were found. When light treatments were compared within plants that started in the shade, SH-SU segments and SH-SH segments were significantly different for most parameters except ˚y and Ci .Acclimation to a change in light occurred over a 10 to 15 day period. The MANOVA showed significant effects of light treatment and time, as well as an interaction, on leaf gas exchange . The rate of Afull sun by SH-SU segments increased over time, and was significantly greater than that of adjacent SH-SH segments beginning on day. The Afull sun observed for the SH-SU segments after ∼10 days was comparable to that of SU-SU segments . Broadly similar, or somewhat faster, responses were observed for the SU-SH treatments; Afull sun decreased, reaching a rate that was comparable to that of SH-SH segments . Sun grown segments exposed continuously to high light alsoshowed a decrease in Afull sun over time , though this trend was smaller than that observed for the SU-SH segments, and Afull sun by the SU-SH segments was significantly less than that by the SU-SU segments beginning on day 8. Stomatal conductance paralleled the changes in Afull sun; the gs of SH-SU segments increased over time, becoming significantly different from that of SH-SH segments on day 4, and reaching a maximum after ∼10 days that was comparable to that of SU-SU segments . Likewise, the gs of SU-SH segments decreased significantly, reaching a rate that was comparable to that of SH-SH segments .