Advantages of a novel in situ pH measurement for soilless media.

Rhizosphere pH determines nutrient bioavailability, but this pH is difficult to measure. Standard pH tests require adding water to growth media. This dilutes hydrogen ion activity and increases pH. We used a novel, in situ, pointed-tip electrode to estimate rhizosphere pH without dilution. Measurements from this electrode matched a research-grade pH meter in hydroponic nutrient solutions. We then compared measurements from this electrode to saturated paste and pour-through methods in peat moss, coconut coir, and pine bark. The pointed-tip electrode was unable to accurately measure pH in the highly-porous pine bark media. Adding deionized water to the other media at container capacity using the saturated paste method resulted in a pH that was 0.59 ± 0.30 units higher than the initial in situ measurement at the top of the container. This increase aligns with established solution chemistry principles. Measurements of pH using the pour-through method were 0.38 ± 0.24 pH units higher than in situ measurements at the bottom of the container. We conclude that in situ pH measurements are not subject to dilution and are thus more representative of the rhizosphere pH than the saturated paste and pour-through techniques.

An improved digestion and analysis procedure for silicon in plant tissue.

Silicon (Si) in plant tissues reduces abiotic and biotic stress, but it is incorporated as silica (SiO2), which is difficult to solubilize for analysis. We modified an oven-induced tissue-digestion and analysis method to improve Si solubilization and validated its accuracy by quantifying the mass-balance recovery of Si from the hydroponic solution and plant tissues of cucumber (Cucumis sativus). Leaf, stem, and root tissues were dried, finely-ground, and digested in 12.5 molar sodium hydroxide at 95°C for 4 hours. Solutions were then acidified with 6 molar hydrochloric acid to achieve a pH below 2 for measurement of Si using the molybdate blue colorimetric method. Interference of phosphorus in the analysis was minimized by increasing the addition of oxalic acid from 0.6 to 1.1 molar. We recovered 101% ± 13% of the expected Si, calculated using mass-balance recovery, in leaf, stem, and root tissues across 15 digestions. This Si recovery was fourteen-fold higher than the standard acid-extraction method and similar to a USDA-ARS alkaline-extraction method. Our procedure offers a low-cost, accurate method for extraction and analysis of Si in plant tissues.

Assessing water stress in a high-density apple orchard using trunk circumference variation, sap flow index and stem water potential.

Introduction: Automated plant-based measurements of water stress have the potential to advance precision irrigation in orchard crops. Previous studies have shown correlations between sap flow, line variable differential transform (LVDT) dendrometers and fruit tree drought response. Here we report season-long automated measurement of maximum daily change in trunk diameter using band dendrometers and heated needles to measure a simplified sap flow index (SFI). Methods: Measurements were made on two apple cultivars that were stressed at 7 to 12 day intervals by withholding irrigation until the average stem water potential (ΨStem) dropped below -1.5 MPa, after which irrigation was restored and the drought cycle repeated. Results: Dendrometer measurements of maximum daily trunk shrinkage (MDS) were highly correlated (r² = 0.85) with pressure chamber measurements of stem water potential. The SFI measurements were less correlated with stem water potential but were highly correlated with evaporative demand (r² = 0.82) as determined by the Penman-Monteith equation (ETr). Discussion: The high correlation of SFI to ETr suggests that high-density orchards resemble a continuous surface, unlike orchards with widely spaced trees. The correlations of MDS and SFI to ΨStem were higher during the early season than the late season growth. Band dendrometers are less labor intensive to install than LVDT dendrometers and are non-invasive so are well suited to commercialization.

Elevated UV photon fluxes minimally affected cannabinoid concentration in a high-CBD cultivar.

Ultraviolet photons (UV) can damage critical biochemical processes. Plants synthesize photo-protective pigments that absorb UV to minimize damage. Cannabinoids absorb UV, so increased UV has the potential to increase cannabinoid synthesis. Studies in the 1980's provided some evidence for this hypothesis in low-cannabinoid cultivars, but recent studies did not find an increase in cannabinoid synthesis with increasing UV in high-cannabinoid cultivars. These studies used low UV photon fluxes, so we examined the effect of higher UV photon fluxes. We used fluorescent UV lights with 55% UV-B (280 to 314 nm) and 45% UV-A (315 to 399 nm). Treatments began three weeks after the start of short days and continued for five weeks until harvest. Established weighting factors were used to calculate the daily biologically effective UV photon flux (UV-PFDBE; 280 to 399 nm). Daily UV-PFDBE levels were 0, 0.02, 0.05, and 0.11 mol m-2 d-1 with a background daily light integral (DLI) of 30 mol m-2 d-1. This provided a ratio of daily UV-PFDBE to DLI of 41 to 218% of summer sunlight in the field. Cannabinoid concentration was 3 to 13% higher than the control in UV treated plants, but the effect was not statistically significant. Fv/Fm and flower yield were reduced only in the highest UV treatment. These data support recent literature and lead us to conclude that an elevated flux of UV photons is not an effective approach to increase cannabinoid concentration in high-cannabinoid cultivars.

On the contrasting morphological response to far-red at high and low photon fluxes.

Plants compete for sunlight and have evolved to perceive shade through both relative increases in the flux of far-red photons (FR; 700 to 750 nm) and decreases in the flux of all photons (intensity). These two signals interact to control stem elongation and leaf expansion. Although the interacting effects on stem elongation are well quantified, responses for leaf expansion are poorly characterized. Here we report a significant interaction between far-red fraction and total photon flux. Extended photosynthetic photon flux density (ePPFD; 400 to 750 nm) was maintained at three levels (50/100, 200 and 500 µmol m-2 s-1), each with a range of 2 to 33% FR. Increasing FR increased leaf expansion in three cultivars of lettuce at the highest ePPFD but decreased expansion at the lowest ePPFD. This interaction was attributed to differences in biomass partitioning between leaves and stems. Increased FR favored stem elongation and biomass partitioning to stems at low ePPFD and favored leaf expansion at high ePPFD. In cucumber, leaf expansion was increased with increasing percent FR under all ePPFD levels showing minimal interaction. The interactions (and lack thereof) have important implications for horticulture and warrant further study for plant ecology.

Photosynthesis in rice is increased by CRISPR/Cas9-mediated transformation of two truncated light-harvesting antenna.

Plants compete for light partly by over-producing chlorophyll in leaves. The resulting high light absorption is an effective strategy for out competing neighbors in mixed communities, but it prevents light transmission to lower leaves and limits photosynthesis in dense agricultural canopies. We used a CRISPR/Cas9-mediated approach to engineer rice plants with truncated light-harvesting antenna (TLA) via knockout mutations to individual antenna assembly component genes CpSRP43, CpSRP54a, and its paralog, CpSRP54b. We compared the photosynthetic contributions of these components in rice by studying the growth rates of whole plants, quantum yield of photosynthesis, chlorophyll density and distribution, and phenotypic abnormalities. Additionally, we investigated a Poales-specific duplication of CpSRP54. The Poales are an important family that includes staple crops such as rice, wheat, corn, millet, and sorghum. Mutations in any of these three genes involved in antenna assembly decreased chlorophyll content and light absorption and increased photosynthesis per photon absorbed (quantum yield). These results have significant implications for the improvement of high leaf-area-index crop monocultures.

Sustainable Cannabis Nutrition: Elevated root-zone phosphorus significantly increases leachate P and does not improve yield or quality.

Phosphorus (P) is an essential but often over-applied nutrient in agricultural systems. Because of its detrimental environmental effects, P fertilization is well studied in crop production. Controlled environment agriculture allows for precise control of root-zone P and has the potential to improve sustainability over field agriculture. Medical Cannabis is uniquely cultivated for the unfertilized female inflorescence and mineral nutrition can affect the yield and chemical composition of these flowers. P typically accumulates in seeds, but its partitioning in unfertilized Cannabis flowers is not well studied. Here we report the effect of increasing P (25, 50, and 75 mg P per L) in continuous liquid fertilizer on flower yield, cannabinoid concentration, leachate P, nutrient partitioning, and phosphorus use efficiency (PUE) of a high-CBD Cannabis variety. There was no significant effect of P concentration on flower yield or cannabinoid concentration, but there were significant differences in leachate P, nutrient partitioning, and PUE. Leachate P increased 12-fold in response to the 3-fold increase in P input. The P concentration in the unfertilized flowers increased to more than 1%, but this did not increase yield or quality. The fraction of P in the flowers increased from 25 to 65% and PUE increased from 31 to 80% as the as the P input decreased from 75 to 25 mg per L. Avoiding excessive P fertilization can decrease the environmental impact of Cannabis cultivation.

Germination and seedling establishment for hydroponics: The benefit of slant boards.

Germination and seedling establishment for transplanting into hydroponics often uses porous substrates, but fine roots grow into these substrates, and they cannot be removed without damaging these roots. Seedlings transplanted without removal of substrates can cause interactions with solution chemistry or addition of particulates to the nutrient solution. Germination of seeds on slant boards is clean, uniform, and reduces the time to transplanting. Slant boards facilitate development of long roots, which maximize exposure of the primary root to the nutrient solution after transplanting. The "boards" are made from thin acrylic or polycarbonate sheets with germination paper on top. Seeds are held in place by covering with thin paper before vertical placement of the boards in the container. Four to twelve days later, the seedlings with long roots can be removed from the paper without damage and transplanted into the hydroponic system. Here we describe slant board construction and procedures for rapid germination and transplanting in hydroponics.

Photosynthesis in sun and shade: the surprising importance of far-red photons.

The current definition of photosynthetically active radiation includes only photons from 400 up to 700 nm, despite evidence of the synergistic interaction between far-red photons and shorter-wavelength photons. The synergy between far-red and shorter-wavelength photons has not been studied in sunlight under natural conditions. We used a filter to remove photons above 700 nm to quantify the effects on photosynthesis in diverse species under full sun, medium light intensity and vegetation shade. Far-red photons (701 to 750 nm) in sunlight are used efficiently for photosynthesis. This is especially important for leaves in vegetation shade, where far-red photons can be > 50% of the total incident photons between 400 and 750 nm. Far-red photons accounted for 24-25% of leaf gross photosynthesis (Pgross ) in a C3 and a C4 species when sunlight was filtered through a leaf, and 10-14% of leaf Pgross in a tree and an understory species in deep shade. Accounting for the photosynthetic activity of far-red photons is critical for accurate measurement and modeling of photosynthesis at single leaf, canopy and ecosystem scales. This, in turn, is crucial in understanding crop productivity, the global carbon cycle and climate change impacts on agriculture and ecosystems.

Colorimetric determination of urea using diacetyl monoxime with strong acids.

Urea is a byproduct of the urea cycle in metabolism and is excreted through urine and sweat. Ammonia, which is toxic at low levels, is converted to the safe storage form of urea, which represents the largest efflux of nitrogen from many organisms. Urea is an important nitrogen source in agriculture, is added to many industrial products, and is a large component in wastewater. The enzyme urease hydrolyzes urea to ammonia and bicarbonate. This reaction is microbially mediated in soils, hydroponic solutions, and wastewater recycling and is catalyzed in vivo in plants using native urease, making measurement of urea environmentally important. Both direct and indirect methods to measure urea exist. This protocol uses diacetyl monoxime to directly determine the concentration of urea in solution. The protocol provides repeatable results and stable reagents with good color stability and simple measurement techniques for use in any lab with a spectrophotometer. The reaction between diacetyl monoxime and urea in the presence of sulfuric acid, phosphoric acid, thiosemicarbazide, and ferric chloride produces a chromophore with a peak absorbance at 520 nm and a linear relationship between concentration and absorbance from 0.4 to 5.0 mM urea in this protocol. The lack of detectable interferences makes this protocol suitable for the determination of millimolar levels of urea in wastewater streams and hydroponic solutions.

Photons from NIR LEDs can delay flowering in short-day soybean and Cannabis: Implications for phytochrome activity.

Photons during the dark period delay flowering in short-day plants (SDP). Red photons applied at night convert phytochromes to the active far-red absorbing form (Pfr), leading to inhibition of flowering. Far-red photons (greater than 700 nm) re-induce flowering when applied after a pulse of red photons during the dark period. However, far-red photons at sufficiently high intensity and duration delay flowering in sensitive species. Mechanistically, this response occurs because phytochrome-red (Pr) absorbance is not zero beyond 700 nm. We applied nighttime photons from near infrared (NIR) LEDs (peak 850 nm) over a 12 h dark period. Flowering was delayed in Glycine max and Cannabis sativa (two photosensitive species) by 3 and 12 days, respectively, as the flux of photons from NIR LEDs was increased up to 83 and 116 µmol m-2 s-1. This suggests that long wavelength photons from NIR LEDs can activate phytochromes (convert Pr to Pfr) and thus alter plant development.

Improving the Predictive Value of Phytochrome Photoequilibrium: Consideration of Spectral Distortion Within a Leaf.

The ratio of active phytochrome (Pfr) to total phytochrome (Pr + Pfr), called phytochrome photo-equilibrium (PPE; also called phytochrome photostationary state, PSS) has been used to explain shade avoidance responses in both natural and controlled environments. PPE is commonly estimated using measurements of the spectral photon distribution (SPD) above the canopy and photoconversion coefficients. This approach has effectively predicted morphological responses when only red and far-red (FR) photon fluxes have varied, but controlled environment research often utilizes unique ratios of wavelengths so a more rigorous evaluation of the predictive ability of PPE on morphology is warranted. Estimations of PPE have rarely incorporated the optical effects of spectral distortion within a leaf caused by pigment absorbance and photon scattering. We studied stem elongation rate in the model plant cucumber under diverse spectral backgrounds over a range of one to 45% FR (total photon flux density, 400-750 nm, of 400 µmol m-2 s-1) and found that PPE was not predictive when blue and green varied. Preferential absorption of red and blue photons by chlorophyll results in an SPD that is relatively enriched in green and FR at the phytochrome molecule within a cell. This can be described by spectral distortion functions for specific layers of a leaf. Multiplying the photoconversion coefficients by these distortion functions yields photoconversion weighting factors that predict phytochrome conversion at the site of photon perception within leaf tissue. Incorporating spectral distortion improved the predictive value of PPE when phytochrome was assumed to be homogeneously distributed within the whole leaf. In a supporting study, the herbicide norflurazon was used to remove chlorophyll in seedlings. Using distortion functions unique to either green or white cotyledons, we came to the same conclusions as with whole plants in the longer-term study. Leaves of most species have similar spectral absorbance so this approach for predicting PPE should be broadly applicable. We provide a table of the photoconversion weighting factors. Our analysis indicates that the simple, intuitive ratio of FR (700-750 nm) to total photon flux (far-red fraction) is also a reliable predictor of morphological responses like stem length.

Does Green Really Mean Go? Increasing the Fraction of Green Photons Promotes Growth of Tomato but Not Lettuce or Cucumber.

The photon flux in the green wavelength region is relatively enriched in shade and the photon flux in the blue region is selectively filtered. In sole source lighting environments, increasing the fraction of blue typically decreases stem elongation and leaf expansion, and smaller leaves reduce photon capture and yield. Photons in the green region reverse these blue reductions through the photoreceptor cryptochrome in Arabidopsis thaliana, but studies in other species have not consistently shown the benefits of photons in the green region on leaf expansion and growth. Spectral effects can interact with total photon flux. Here, we report the effect of the fraction of photons in the blue (10 to 30%) and green (0 to 50%) regions at photosynthetic photon flux densities of 200 and 500 µmol m-2 s-1 in lettuce, cucumber and tomato. As expected, increasing the fraction of photons in the blue region consistently decreased leaf area and dry mass. By contrast, large changes in the fraction of photons in the green region had minimal effects on leaf area and dry mass in lettuce and cucumber. Photons in the green region were more potent at a lower fraction of photons in the blue region. Photons in the green region increased stem and petiole length in cucumber and tomato, which is a classic shade avoidance response. These results suggest that high-light crop species might respond to the fraction of photons in the green region with either shade tolerance (leaf expansion) or shade avoidance (stem elongation).

Cannabis lighting: Decreasing blue photon fraction increases yield but efficacy is more important for cost effective production of cannabinoids.

LED technology facilitates a range of spectral quality, which can be used to optimize photosynthesis, plant shape and secondary metabolism. We conducted three studies to investigate the effect of blue photon fraction on yield and quality of medical hemp. Conditions were varied among studies to evaluate potential interactions with environment, but all environmental conditions other than the blue photon fraction were maintained constant among the five-chambers in each study. The photosynthetic photon flux density (PPFD, 400 to 700 nm) was rigorously maintained at the set point among treatments in each study by raising the fixtures. The lowest fraction of blue photons was 4% from HPS, and increased to 9.8, 10.4, 16, and 20% from LEDs. There was a consistent, linear, 12% decrease in yield in each study as the fraction of blue photons increased from 4 to 20%. Dry flower yield ranged from 500 to 750 g m-2. This resulted in a photon conversion efficacy of 0.22 to 0.36 grams dry flower mass yield per mole of photons. Yield was higher at a PPFD of 900 than at 750 µmol m-2 s-1. There was no effect of spectral quality on CBD or THC concentration. CBD and THC were 8% and 0.3% at harvest in trials one and two, and 12% and 0.5% in trial three. The CBD/THC ratio was about 25 to 1 in all treatments and studies. The efficacy of the fixtures ranged from 1.7 (HPS) to 2.5 µmol per joule (white+red LED). Yield under the white+red LED fixture (10.4% blue) was 4.6% lower than the HPS on a per unit area basis, but was 27% higher on a per dollar of electricity basis. These findings suggest that fixture efficacy and initial cost of the fixture are more important for return on investment than spectral distribution at high photon flux.

Substituting Far-Red for Traditionally Defined Photosynthetic Photons Results in Equal Canopy Quantum Yield for CO2 Fixation and Increased Photon Capture During Long-Term Studies: Implications for Re-Defining PAR.

Far-red photons regulate shade avoidance responses and can have powerful effects on plant morphology and radiation capture. Recent studies have shown that far-red photons (700 to 750 nm) efficiently drive photosynthesis when added to traditionally defined photosynthetic photons (400-700 nm). But the long-term effects of far-red photons on canopy quantum yield have not yet been determined. We grew lettuce in a four-chamber, steady-state canopy gas-exchange system to separately quantify canopy photon capture, quantum yield for CO2 fixation, and carbon use efficiency. These measurements facilitate a mechanistic understanding of the effect of far-red photons on the components of plant growth. Day-time photosynthesis and night-time respiration of lettuce canopies were continuously monitored from seedling to harvest in five replicate studies. Plants were grown under a background of either red/blue or white light, each background with or without 15% (50 µmol m-2 s-1) of far-red photons substituting for photons between 400 and 700 nm. All four treatments contained 31.5% blue photons, and an equal total photon flux from 400 to 750 nm of 350 µmol m-2 s-1. Both treatments with far-red photons had higher canopy photon capture, increased daily carbon gain (net photosynthesis minus respiration at night), and 29 to 31% more biomass than control treatments. Canopy quantum yield was similar among treatments (0.057 ± 0.002 mol of CO2 fixed in gross photosynthesis per mole of absorbed photons integrated over 400 to 750 nm). Carbon use efficiency (daily carbon gain/gross photosynthesis) was also similar for mature plants (0.61 ± 0.02). Photosynthesis increased linearly with increasing photon capture and had a common slope among all four treatments, which demonstrates that the faster growth with far-red photon substitution was caused by enhanced photon capture through increased leaf expansion. The equivalent canopy quantum yield among treatments indicates that the absorbed far-red photons were equally efficient for photosynthesis when acting synergistically with the 400-700 nm photons.

Steady-state stomatal responses of C3 and C4 species to blue light fraction: Interactions with CO2 concentration.

Blue light induced stomatal opening has been studied by applying a short pulse (~5 to 60 s) of blue light to a background of saturating photosynthetic red photons, but little is known about steady-state stomatal responses. Here we report stomatal responses to blue light at high and low CO2 concentrations. Steady-state stomatal conductance (gs ) of C3 plants increased asymptotically with increasing blue light to a maximum at 20% blue (120 µmol m-2 s-1 ). This response was consistent from 200 to 800 µmol mol-1 atmospheric CO2 (Ca ). In contrast, blue light induced only a transient stomatal opening (~5 min) in C4 species above a Ca of 400 µmol mol-1 . Steady-state gs of C4 plants generally decreased with increasing blue intensity. The net photosynthetic rate of all species decreased above 20% blue because blue photons have lower quantum yield (moles carbon fixed per mole photons absorbed) than red photons. Our findings indicate that photosynthesis, rather than a blue light signal, plays a dominant role in stomatal regulation in C4 species. Additionally, we found that blue light affected only stomata on the illuminated side of the leaf. Contrary to widely held belief, the blue light-induced stomatal opening minimally enhanced photosynthesis and consistently decreased water use efficiency.

Far-red photons have equivalent efficiency to traditional photosynthetic photons: Implications for redefining photosynthetically active radiation.

Far-red photons (701-750 nm) are abundant in sunlight but are considered inactive for photosynthesis and are thus excluded from the definition of photosynthetically active radiation (PAR; 400-700 nm). Several recent studies have shown that far-red photons synergistically interact with shorter wavelength photons to increase leaf photochemical efficiency. The value of far-red photons in canopy photosynthesis has not been studied. Here, we report the effects of far-red photons on single leaf and canopy photosynthesis in 14 diverse crop species. Adding far-red photons (up to 40%) to a background of shorter wavelength photons caused an increase in canopy photosynthesis equal to adding 400-700 nm photons. Far-red alone minimally increased photosynthesis. This indicates that far-red photons are equally efficient at driving canopy photosynthesis when acting synergistically with traditionally defined photosynthetic photons. Measurements made using LEDs with peak wavelength of 711, 723, or 746 nm showed that the magnitude of the effect was less at longer wavelengths. The consistent response among diverse species indicates that the mechanism is common in higher plants. These results suggest that far-red photons (701-750 nm) should be included in the definition of PAR.

Phototrophic N2 and CO2 Fixation Using a Rhodopseudomonas palustris-H2 Mediated Electrochemical System With Infrared Photons.

A promising approach for the synthesis of high value reduced compounds is to couple bacteria to the cathode of an electrochemical cell, with delivery of electrons from the electrode driving reductive biosynthesis in the bacteria. Such systems have been used to reduce CO2 to acetate and other C-based compounds. Here, we report an electrosynthetic system that couples a diazotrophic, photoautotrophic bacterium, Rhodopseudomonas palustris TIE-1, to the cathode of an electrochemical cell through the mediator H2 that allows reductive capture of both CO2 and N2 with all of the energy coming from the electrode and infrared (IR) photons. R. palustris TIE-1 was shown to utilize a narrow band of IR radiation centered around 850 nm to support growth under both photoheterotrophic, non-diazotrophic and photoautotrophic, diazotrophic conditions with growth rates similar to those achieved using broad spectrum incandescent light. The bacteria were also successfully cultured in the cathodic compartment of an electrochemical cell with the sole source of electrons coming from electrochemically generated H2, supporting reduction of both CO2 and N2 using 850 nm photons as an energy source. Growth rates were similar to non-electrochemical conditions, revealing that the electrochemical system can fully support bacterial growth. Faradaic efficiencies for N2 and CO2 reduction were 8.5 and 47%, respectively. These results demonstrate that a microbial-electrode hybrid system can be used to achieve reduction and capture of both CO2 and N2 using low energy IR radiation and electrons provided by an electrode.