Elimination of Leaf Angle Impacts on Plant Reflectance Spectra Using Fusion of Hyperspectral Images and 3D Point Clouds.

During recent years, hyperspectral imaging technologies have been widely applied in agriculture to evaluate complex plant physiological traits such as leaf moisture content, nutrient level, and disease stress. A critical component of this technique is white referencing used to remove the effect of non-uniform lighting intensity in different wavelengths on raw hyperspectral images. However, a flat white tile cannot accurately reflect the lighting intensity variance on plant leaves, since the leaf geometry (e.g., tilt angles) and its interaction with the illumination severely impact plant reflectance spectra and vegetation indices such as the normalized difference vegetation index (NDVI). In this research, the impacts of leaf angles on plant reflectance spectra were summarized, and an improved image calibration model using the fusion of leaf hyperspectral images and 3D point clouds was built. Corn and soybean leaf samples were imaged at different tilt angles and orientations using an indoor desktop hyperspectral imaging system and analyzed for differences in the NDVI values. The results showed that the leaf's NDVI largely changed with angles. The changing trends with angles differed between the two species. Using measurements of leaf tilt angle and orientation obtained from the 3D point cloud data taken simultaneously with the hyperspectral images, a support vector regression (SVR) model was successfully developed to calibrate the NDVI values of pixels at different angles on a leaf to a same standard as if the leaf was laid flat on a horizontal surface. The R-squared values between the measured and predicted leaf angle impacts were 0.76 and 0.94 for corn and soybean, respectively. This method has a potential to be used in any general plant imaging systems to improve the phenotyping quality.

MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples.

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the 'Fuji' apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.

PP2A and microtubules function in 5-aminolevulinic acid-mediated H2 O2 signaling in Arabidopsis guard cells.

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H2 O2 . However, the mechanisms behind ALA-decreased H2 O2 in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H2 O2 in guard cell signaling. Finally, we demonstrate the role of H2 O2 -mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H2 O2 decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.

Exogenous 5-aminolevulinic acid improves strawberry tolerance to osmotic stress and its possible mechanisms.

Cultivated strawberry, one of the major fruit crops worldwide, is an evergreen plant with shallow root system, and thus sensitive to environmental changes, including drought stress. To investigate the effect of 5-aminolevulinic acid (ALA), a new environment-friendly plant growth regulator, on strawberry drought tolerance and its possible mechanisms, we treated strawberry (Fragaria × annanasa Duch. cv. 'Benihoppe') with 15% polyethylene glycol 6000 to simulate osmotic stress with or without 10 mg l-1 ALA. We found that ALA significantly alleviated PEG-inhibited plant growth and improved water absorption and xylem sap flux, indicating ALA mitigates the adverse effect of osmotic stress on strawberry plants. Gas exchange and chlorophyll fluorescence analysis showed that ALA mitigated PEG-induced decreases of Pn , Gs , Tr , Pn /Ci , photosystem I and II reaction center activities, electron transport activity, and photosynthetic performance indexes. Equally important, ALA promoted PEG-increased antioxidant enzyme activities and repressed PEG-increased malondialdehyde and superoxide anion in both leaves and roots. Specially, ALA repressed H2 O2 increase in leaves, but stimulated it in roots. Furthermore, ALA repressed abscisic acid (ABA) biosynthesis and signaling gene expressions in leaves, but promoted those in roots. In addition, ALA blocked PEG-downregulated expressions of plasmalemma and tonoplast aquaporin genes PIP and TIP in both leaves and roots. Taken together, ALA effectively enhances strawberry drought tolerance and the mechanism is related to the improvement of water absorption and conductivity. The tissue-specific responses of ABA biosynthesis, ABA signaling, and H2 O2 accumulation to ALA in leaves and roots play key roles in ALA-improved strawberry tolerance to osmotic stress.

LeafScope: A Portable High-Resolution Multispectral Imager for In Vivo Imaging Soybean Leaf.

Portable devices for measuring plant physiological features with their isolated measuring chamber are playing an increasingly important role in plant phenotyping. However, currently available commercial devices of this type, such as soil plant analysis development (SPAD) meter and spectrometer, are dot meters that only measure a small region of the leaf, which does not perfectly represent the highly varied leaf surface. This study developed a portable and high-resolution multispectral imager (named LeafScope) to in-vivo image a whole leaf of dicotyledon plants while blocking the ambient light. The hardware system is comprised of a monochrome camera, an imaging chamber, a lightbox with different bands of light-emitting diodes (LEDs) array, and a microcontroller. During measuring, the device presses the leaf to lay it flat in the imaging chamber and acquires multiple images while alternating the LED bands within seconds in a certain order. The results of an experiment with soybean plants clearly showed the effect of nitrogen and water treatments as well as the genotype differences by the color and morphological features from image processing. We conclude that the low cost and easy to use LeafScope can provide promising imaging quality for dicotyledon plants, so it has great potential to be used in plant phenotyping.

Precise Estimation of NDVI with a Simple NIR Sensitive RGB Camera and Machine Learning Methods for Corn Plants.

The normalized difference vegetation index (NDVI) is widely used in remote sensing to monitor plant growth and chlorophyll levels. Usually, a multispectral camera (MSC) or hyperspectral camera (HSC) is required to obtain the near-infrared (NIR) and red bands for calculating NDVI. However, these cameras are expensive, heavy, difficult to geo-reference, and require professional training in imaging and data processing. On the other hand, the RGBN camera (NIR sensitive RGB camera, simply modified from standard RGB cameras by removing the NIR rejection filter) have also been explored to measure NDVI, but the results did not exactly match the NDVI from the MSC or HSC solutions. This study demonstrates an improved NDVI estimation method with an RGBN camera-based imaging system (Ncam) and machine learning algorithms. The Ncam consisted of an RGBN camera, a filter, and a microcontroller with a total cost of only $70 ~ 85. This new NDVI estimation solution was compared with a high-end hyperspectral camera in an experiment with corn plants under different nitrogen and water treatments. The results showed that the Ncam with two-band-pass filter achieved high performance (R2 = 0.96, RMSE = 0.0079) at estimating NDVI with the machine learning model. Additional tests showed that besides NDVI, this low-cost Ncam was also capable of predicting corn plant nitrogen contents precisely. Thus, Ncam is a potential option for MSC and HSC in plant phenotyping projects.

Stress Distribution Analysis on Hyperspectral Corn Leaf Images for Improved Phenotyping Quality.

High-throughput imaging technologies have been developing rapidly for agricultural plant phenotyping purposes. With most of the current crop plant image processing algorithms, the plant canopy pixels are segmented from the images, and the averaged spectrum across the whole canopy is calculated in order to predict the plant's physiological features. However, the nutrients and stress levels vary significantly across the canopy. For example, it is common to have several times of difference among Soil Plant Analysis Development (SPAD) chlorophyll meter readings of chlorophyll content at different positions on the same leaf. The current plant image processing algorithms cannot provide satisfactory plant measurement quality, as the averaged color cannot characterize the different leaf parts. Meanwhile, the nutrients and stress distribution patterns contain unique features which might provide valuable signals for phenotyping. There is great potential to develop a finer level of image processing algorithm which analyzes the nutrients and stress distributions across the leaf for improved quality of phenotyping measurements. In this paper, a new leaf image processing algorithm based on Random Forest and leaf region rescaling was developed in order to analyze the distribution patterns on the corn leaf. The normalized difference vegetation index (NDVI) was used as an example to demonstrate the improvements of the new algorithm in differentiating between different nitrogen stress levels. With the Random Forest method integrated into the algorithm, the distribution patterns along the corn leaf's mid-rib direction were successfully modeled and utilized for improved phenotyping quality. The algorithm was tested in a field corn plant phenotyping assay with different genotypes and nitrogen treatments. Compared with the traditional image processing algorithms which average the NDVI (for example) throughout the whole leaf, the new algorithm more clearly differentiates the leaves from different nitrogen treatments and genotypes. We expect that, besides NDVI, the new distribution analysis algorithm could improve the quality of other plant feature measurements in similar ways.

Transcriptomic Analysis of Ficus carica Peels with a Focus on the Key Genes for Anthocyanin Biosynthesis.

Fig (Ficus carica L.), a deciduous fruit tree of the Moraceae, provides ingredients for human health such as anthocyanins. However, little information is available on its molecular structure. In this study, the fig peels in the yellow (Y) and red (R) stages were used for transcriptomic analyses. Comparing the R with the Y stage, we obtained 6224 differentially expressed genes, specifically, anthocyanin-related genes including five CHS, three CHI, three DFR, three ANS, two UFGT and seven R2R3-MYB genes. Furthermore, three anthocyanin biosynthetic genes, i.e., FcCHS1, FcCHI1 and FcDFR1, and two R2R3-MYB genes, i.e., FcMYB21 and FcMYB123, were cloned; sequences analysis and their molecular characteristics indicated their important roles in fig anthocyanin biosynthesis. Heterologous expression of FcMYB21 and FcMYB123 significantly promoted anthocyanin accumulation in both apple fruits and calli, further suggesting their regulatory roles in fig coloration. These findings provide novel insights into the molecular mechanisms behind fig anthocyanin biosynthesis and coloration, facilitating the genetic improvement of high-anthocyanin cultivars and other horticultural traits in fig fruits.

Hydrogen peroxide as a mediator of 5-aminolevulinic acid-induced Na+ retention in roots for improving salt tolerance of strawberries.

To explore the mechanisms of 5-aminolevulinic acid (ALA)-improved plant salt tolerance, strawberries (Fragaria × ananassa Duch. cv. 'Benihoppe') were treated with 10 mg l-1 ALA under 100 mmol l-1 NaCl stress. We found that the amount of Na+ increased in the roots but decreased in the leaves. Laser scanning confocal microscopy (LSCM) observations showed that ALA-induced roots had more Na+ accumulation than NaCl alone. Measurement of the xylem sap revealed that ALA repressed Na+ concentrations to a large extent. The electron microprobe X-ray assay also confirmed ALA-induced Na+ retention in roots. qRT-PCR showed that ALA upregulated the gene expressions of SOS1 (encoding a plasma membrane Na+ /H+ antiporter), NHX1 (encoding a vacuolar Na+ /H+ antiporter) and HKT1 (encoding a protein of high-affinity K+ uptake), which are associated with Na+ exclusion in the roots, Na+ sequestration in vacuoles and Na+ unloading from the xylem vessels to the parenchyma cells, respectively. Furthermore, we found that ALA treatment reduced the H2 O2 content in the leaves but increased it in the roots. The exogenous H2 O2 promoted plant growth, increased root Na+ retention and stimulated the gene expressions of NHX1, SOS1 and HKT1. Diphenyleneiodonium (DPI), an inhibitor of H2 O2 generation, suppressed the effects of ALA or H2 O2 on Na+ retention, gene expressions and salt tolerance. Therefore, we propose that ALA induces H2 O2 accumulation in roots, which mediates Na+ transporter gene expression and more Na+ retention in roots, thereby improving plant salt tolerance.

Improving High-Throughput Phenotyping Using Fusion of Close-Range Hyperspectral Camera and Low-Cost Depth Sensor.

Hyperspectral sensors, especially the close-range hyperspectral camera, have been widely introduced to detect biological processes of plants in the high-throughput phenotyping platform, to support the identification of biotic and abiotic stress reactions at an early stage. However, the complex geometry of plants and their interaction with the illumination, severely affects the spectral information obtained. Furthermore, plant structure, leaf area, and leaf inclination distribution are critical indexes which have been widely used in multiple plant models. Therefore, the process of combination between hyperspectral images and 3D point clouds is a promising approach to solve these problems and improve the high-throughput phenotyping technique. We proposed a novel approach fusing a low-cost depth sensor and a close-range hyperspectral camera, which extended hyperspectral camera ability with 3D information as a potential tool for high-throughput phenotyping. An exemplary new calibration and analysis method was shown in soybean leaf experiments. The results showed that a 0.99 pixel resolution for the hyperspectral camera and a 3.3 millimeter accuracy for the depth sensor, could be achieved in a controlled environment using the method proposed in this paper. We also discussed the new capabilities gained using this new method, to quantify and model the effects of plant geometry and sensor configuration. The possibility of 3D reflectance models can be used to minimize the geometry-related effects in hyperspectral images, and to significantly improve high-throughput phenotyping. Overall results of this research, indicated that the proposed method provided more accurate spatial and spectral plant information, which helped to enhance the precision of biological processes in high-throughput phenotyping.

Dynamic Changes in Phenolics and Antioxidant Capacity during Pecan (Carya illinoinensis) Kernel Ripening and Its Phenolics Profiles.

Pecan (Carya illinoinensis) kernels have a high phenolics content and a high antioxidant capacity compared to other nuts-traits that have attracted great interest of late. Changes in the total phenolic content (TPC), condensed tannins (CT), total flavonoid content (TFC), five individual phenolics, and antioxidant capacity of five pecan cultivars were investigated during the process of kernel ripening. Ultra-performance liquid chromatography coupled with quadruple time-of-flight mass (UPLC-Q/TOF-MS) was also used to analyze the phenolics profiles in mixed pecan kernels. TPC, CT, TFC, individual phenolics, and antioxidant capacity were changed in similar patterns, with values highest at the water or milk stages, lowest at milk or dough stages, and slightly varied at kernel stages. Forty phenolics were tentatively identified in pecan kernels, of which two were first reported in the genus Carya, six were first reported in Carya illinoinensis, and one was first reported in its kernel. The findings on these new phenolic compounds provide proof of the high antioxidant capacity of pecan kernels.

Study on salt tolerance with YHem1 transgenic canola (Brassica napus).

5-Aminolevulinic acid (5-ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5-ALA, to study salt stress responses. In a long-term pot experiment, the transgenic plants produced higher yield under 200 mmol L(-1) NaCl treatment than the wild type (WT). In a short-term experiment, the YHem1 transformation accelerated endogenous 5-ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O2 ·(-) and malondialdehyde were lower than the latter. Additionally, the Na(+) content was higher in the transgenic leaves than that in the WT under salinity, but K(+) and Cl(-) were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.

Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples.

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.

Incorporation of ascorbic acid in chitosan-based coating combined with plasma-activated water: A technology for quality preservation of red grapes after simulated transportation.

Red grapes possess multiple bioactivities but are highly susceptible to spoilage due to the lack of efficient preservation techniques. Plasma-activated water (PAW) treatment and the incorporation of antioxidants in bio-based coatings are promising methods for preserving produce. In this study, we tested a novel combination by incorporating ascorbic acid (AA) into a chitosan-based edible coating (CH) and combining it with plasma-activated water (PAW) treatment (CA-PAW) before simulating transport vibrations to extend the shelf-life of red grapes. The results from storage at 4 °C for 20 d indicated that the CA-PAW treatment reduced microbial counts by 2.62 log10 CFU/g for bacteria, 1.72 log10 CFU/g for yeasts and molds, and 1.1 log10 CFU/g for coliforms, in comparison to the control group treated with sterile deionized water. Total phenols and total flavonoid content were the highest observed, at 111.2 mg GAE/100 g and 262.67 mg RE/100 g, respectively. This treatment also inhibited water migration and erosion, and reduced damage to cell structure. Microstructural observations revealed that the CH coating on the surface of red grapes diminished the degradation of bioactive components. In conclusion, the CA-PAW treatment effectively inhibited the adverse physiological changes caused by vibration and mechanical damage to red grapes, maintained their nutritional and sensory qualities, and extended the shelf life by at least 8 d.

Effects of plasma-activated water combined with ultrasonic treatment of corn starch on structural, thermal, physicochemical, functional, and pasting properties.

In this study, corn starch was used as the raw material, and modified starch was prepared using a method combining plasma-activated water and ultrasound treatment (PUL). This method was compared with treatments using plasma-activated water (PAW) and ultrasound (UL) alone. The structure, thermal, physicochemical, pasting, and functional properties of the native and treated starches were evaluated. The results indicated that PAW and UL treatments did not alter the shape of the starch granules but caused some surface damage. The PUL treatment increased the starch gelatinization temperature and enthalpy (from 11.22 J/g to 13.13 J/g), as well as its relative crystallinity (increased by 0.51 %), gel hardness (increased by 16.19 %) compared to untreated starch, without inducing a crystalline transition. The PUL treatment resulted in a whitening of the samples. The dual treatment enhanced the thermal stability of the starch paste, which can be attributed to the synergistic effect between PAW and ultrasound (PAW can modify the starch structure at a molecular level, while ultrasound can further disrupt the granule weak crystalline structures, leading to improved thermal properties). Furthermore, FTIR results suggested significant changes in the functional groups related to the water-binding capacity of starch, and the order of the double-helical structure was disrupted. The findings of this study suggest that PUL treatment is a promising new green modification technique for improving the starch structure and enhancing starch properties. However, further research is needed to tailor the approach based on the specific properties of the raw material.

Comparative proteomic and transcriptomic approaches to address the active role of GA4 in Japanese apricot flower bud dormancy release.

Hormones are closely associated with dormancy in deciduous fruit trees, and gibberellins (GAs) are known to be particularly important. In this study, we observed that GA4 treatment led to earlier bud break in Japanese apricot. To understand better the promoting effect of GA4 on the dormancy release of Japanese apricot flower buds, proteomic and transcriptomic approaches were used to analyse the mechanisms of dormancy release following GA4 treatment, based on two-dimensional gel electrophoresis (2-DE) and digital gene expression (DGE) profiling, respectively. More than 600 highly reproducible protein spots (P<0.05) were detected and, following GA4 treatment, 38 protein spots showed more than a 2-fold difference in expression, and 32 protein spots were confidently identified according to the databases. Compared with water treatment, many proteins that were associated with energy metabolism and oxidation-reduction showed significant changes after GA4 treatment, which might promote dormancy release. We observed that genes at the mRNA level associated with energy metabolism and oxidation-reduction also played an important role in this process. Analysis of the functions of the identified proteins and genes and the related metabolic pathways would provide a comprehensive proteomic and transcriptomic view of the coordination of dormancy release after GA4 treatment in Japanese apricot flower buds.

Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

ALA induces stomatal opening through regulation among PTPA, PP2AC, and SnRK2.6.

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has been proved to regulate protein phosphatase 2A (PP2A) activity to promote stomatal opening in apple (Malus domestica) leaves. However, the molecular mechanisms underlying remain unclear. Here, we cloned and transformed MdPTPA, MdPP2AC, and MdSnRK2.6 of apple into tobaccos (Nicotiana tabacum) and found that over-expression (OE)-MdPTPA or OE-MdPP2AC promoted stomatal aperture while OE-MdSnRK2.6 induced stomatal closure under normal or drought condition. The Ca2+ and H2O2 levels in the guard cells of OE-MdPTPA and OE-MdPP2AC was decreased but flavonols increased, and the results in OE-SnRK2.6 was contrary. Exogenous ALA stimulated PP2A activity but depressed SnRK2.6 activity in transgenic tobaccos, leading to less Ca2+, H2O2 and more flavonols in guard cells, and consequently stomatal opening. OE-MdPTPA improved stomatal opening and plant growth but impaired drought tolerance, while OE-MdSnRK2.6 improved drought tolerance but depressed the leaf P n. Only OE-MdPP2AC improved stomatal opening, leaf P n, plant growth, as well as drought tolerance. These suggest that the three genes involved in ALA-regulating stomatal movement have their respective unique biological functions. Yeast two-hybrid (Y2H) assays showed that MdPP2AC interacted with MdPTPA or MdSnRK2.6, respectively, but no interaction of MdPTPA with MdSnRK2.6 was found. Yeast three-hybrid (Y3H) assay showed that MdPTPA promoted the interactions between MdPP2AC and MdSnRK2.6. Therefore, we propose a regulatory module of PTPA-PP2AC-SnRK2.6 that may be involved in mediating the ALA-inducing stomatal aperture in green plants.

ALA reverses ABA-induced stomatal closure by modulating PP2AC and SnRK2.6 activity in apple leaves.

5-Aminolevulinic acid (ALA), known as a new natural plant growth regulator, can reverse abscisic acid (ABA)-induced stomatal closure. The protein phosphatase 2A (PP2A) played an important role in regulation of stomatal movement by ALA and ABA; however, the underlying molecular mechanisms remain unclear. Here, we report that ALA promotes MdPP2A activity and gene expression in the leaf epidermis of apple (Malus × domestica Borkh.), and expression of the catalytic subunit MdPP2AC was most significantly correlated with stomatal aperture. Western blotting showed that ALA enhanced MdPP2AC protein abundance and phosphorylation. Y2H (yeast two hybrid), FLC (firefly luciferase complementation imaging) and BiFC (Bimolecular fluorescence complementation) assays showed that MdPP2AC interacted with several other MdPP2A subunits as well as MdSnRK2.6 (Sucrose non-fermenting 1-related protein kinase 2.6), and the latter interaction was further verified by pull-down and MST (microscale thermophoresis) assays. ALA downregulated ABA-induced MdSnRK2.6 gene expression, kinase activity, and protein phosphorylation. In transiently transgenic apple leaves, OE-MdPP2AC promoted stomatal aperture by reducing Ca2+ and H2O2 levels but increasing flavonol levels in guard cells. Conversely, OE-MdSnRK2.6 induced stomatal closure by increasing Ca2+ and H2O2 but reducing flavonols. Partial silencing of these genes had opposite effects on Ca2+, H2O2, flavonols, and stomatal movement. Application of exogenous ALA stimulated PP2A activity, which promoted SnRK2.6 dephosphorylation and lower kinase activity in wild-type and transgenic apple leaves. We therefore propose that PP2AC, which dephosphorylates SnRK2.6 and represses its enzyme activity, mediates ALA signaling to inhibit ABA-induced stomatal closure in apple leaves.

MdMYB110a, directly and indirectly, activates the structural genes for the ALA-induced accumulation of anthocyanin in apple.

5-Aminolevulinic acid (ALA), an essential biosynthetic precursor of tetrapyrrole compounds, promotes the anthocyanin accumulation in many plant species. However, the underlying mechanism of ALA-induced accumulation is not yet fully understood. In this study, we identified an important regulator of the anthocyanin accumulation, MdMYB110a, which plays an important role in the ALA-induced anthocyanin accumulation. MdMYB110a activated the expression of MdGSTF12 by binding to its promoter. Additionally, two interacting MdMYB110a proteins, MdWD40-280 and MdHsfB3a, were isolated and confirmed as positive regulators of the ALA-induced anthocyanin accumulation. Both MdWD40-280 and MdHsfB3a enhanced the ability of MdMYB110a to transcribe MdGSTF12. A yeast one-hybrid assay revealed that MdWD40-280 did not bind to most structural genes in the anthocyanin biosynthetic and transport pathways, thus promoting anthocyanin accumulation by MdWD40-280 to depend on MdMYB110a. However, MdHsfB3a could bind to both the MdDFR and MdANS promoters, thereby directly regulating anthocyanin biosynthesis. Collectively, these results provide new insight into the mechanism of ALA-induced anthocyanin accumulation.