Physiological and Transcriptomic Analysis Reveals That Melatonin Alleviates Aluminum Toxicity in Alfalfa (Medicago sativa L.).

Aluminum (Al) toxicity is the most common factor limiting the growth of alfalfa in acidic soil conditions. Melatonin (MT), a significant pleiotropic molecule present in both plants and animals, has shown promise in mitigating Al toxicity in various plant species. This study aims to elucidate the underlying mechanism by which melatonin alleviates Al toxicity in alfalfa through a combined physiological and transcriptomic analysis. The results reveal that the addition of 5 µM melatonin significantly increased alfalfa root length by 48% and fresh weight by 45.4% compared to aluminum treatment alone. Moreover, the 5 µM melatonin application partially restored the enlarged and irregular cell shape induced by aluminum treatment, resulting in a relatively compact arrangement of alfalfa root cells. Moreover, MT application reduces Al accumulation in alfalfa roots and shoots by 28.6% and 27.6%, respectively. Additionally, MT plays a crucial role in scavenging Al-induced excess H2O2 by enhancing the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), consequently reducing malondialdehyde (MDA) levels. More interestingly, the RNA-seq results reveal that MT application significantly upregulates the expression of xyloglucan endotransglucosylase/hydrolase (XTH) and carbon metabolism-related genes, including those involved in the glycolysis process, as well as sucrose and starch metabolism, suggesting that MT application may mitigate Al toxicity by facilitating the binding of Al to the cell walls, thereby reducing intracellular Al accumulation, and improving respiration and the content of sucrose and trehalose. Taken together, our study demonstrates that MT alleviates Al toxicity in alfalfa by reducing Al accumulation and restoring redox homeostasis. These RNA-seq results suggest that the alleviation of Al toxicity by MT may occur through its influence on cell wall composition and carbon metabolism. This research advances our understanding of the mechanisms underlying MT's effectiveness in mitigating Al toxicity, providing a clear direction for our future investigations into the underlying mechanisms by which MT alleviates Al toxicity in alfalfa.

Mesophyll conductance exerts a significant limitation on photosynthesis during light induction.

Past studies have established mesophyll diffusion conductance to CO2 (gm ) as a variable and significant limitation to plant photosynthesis under steady-state conditions. However, the role of gm in influencing photosynthesis (A) during the transient period of light induction is largely unknown. We combined gas exchange measurements with laser-enabled carbon isotope discrimination measurements to assess gm during photosynthetic induction, using Arabidopsis as the measurement species. Our measurements revealed three key findings: (1) we found that the rate at which gm approached steady state during induction was not necessarily faster than the induction rate of the carboxylation process, contradictory to what has been suggested in previous studies; (2) gm displayed a strong and consistent coordination with A under both induction and steady-state settings, hinting that the mechanism driving gm -A coupling does not require physiological stability as a prerequisite; and (3) photosynthetic limitation analysis of our data revealed that when integrated over the entire induction period, the relative limitation of A imposed by gm can be as high as > 35%. The present study provides the first demonstration of the important role of gm in limiting CO2 assimilation during photosynthetic induction, thereby pointing to a need for more research attention to be devoted to gm in future induction studies.

Daily rhythms of phytomelatonin signaling modulate diurnal stomatal closure via regulating reactive oxygen species dynamics in Arabidopsis.

Melatonin is a well-studied neurohormone oscillating in a 24-h cycle in vertebrates. Phytomelatonin is widespread in plant kingdom, but it remains elusive whether this newly characterized putative hormone underlies the regulation by daily rhythms. Here, we report phytomelatonin signaling, as reflected by changes in endogenous concentrations of phytomelatonin and expression of genes associated with biosynthesis of phytomelatonin (AtSNAT1, AtCOMT1, and AtASMT) and its receptor (AtPMTR1), shows 24-h oscillations in Arabidopsis. The variation of reactive oxygen species (ROS) production and scavenging and expression of ROS-related genes significantly decrease in pmtr1 and snat and increase in PMTR1-OE seedlings, indicating the rhythmicity in phytomelatonin signaling is required for maintenance of ROS dynamics. Additionally, the ROS signaling feedback influences the expression of AtSNAT1, AtCOMT1, AtASMT, and AtPMTR1, suggesting the phytomelatonin and ROS signaling are coordinately interrelated. The pmtr1 mutant plants lose diurnal stomatal closure, with stomata remaining open during daytime as well as nighttime and mutants showing more water loss and drought sensitivity when compared with the wild-type Col-0 plants. Taken together, our results suggest that PMTR1-regulated ROS signaling peaks in the afternoon and may transmit the darkness signals to trigger stomatal closure, which might be essential for high water-use efficiency and drought tolerance.

Molecular character of a phosphatase 2C (PP2C) gene relation to stress tolerance in Arabidopsis thaliana.

Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. HAI-1 gene had been shown to affect both seed and vegetative responses to ABA, which is one of PP2Cs clade A in Arabidopsis thaliana. Transgenic plants containing pHAI-1::GUS (ß-glucuronidase) displayed GUS activity existing in the vascular system of leave veins, stems and petioles. Green fluorescent protein fused HAI-1 (HAI-1-GFP) was found in the nucleus through transient transformation assays with onion epidermal cells. The water-loss assays indicated the loss-of-function mutants did not show symptoms of wilting and they had still turgid green rosette leaves. The assays of seed germination by exogenous ABA and NaCl manifested that the loss-of-function mutants displayed higher insensitivity than wild-type plants. Taken together, the final results suggest that the HAI-1 (AT5G59220) encoded a nuclear protein and it can be highly induced by ABA and wound in Arabidposis, the stress-tolerance phenotype showed a slightly improvement when HAI-1 gene was disrupted.

Regulation and function of Arabidopsis AtGALK2 gene in abscisic acid response signaling.

AtGALK2 belongs to galactokinase of GHMP family in Arabidopsis thaliana. Two homozygous T-DNA insertion mutants (Atgalk2-1 and Atgalk2-2) of the AtGALK2 gene were identified. The AtGALK2 gene was highly expressed in flowers and roots, but less in stems, leaves and petioles. It was found that the expression of AtGALK2 gene was induced by NaCl and ABA. The two Atgalk2 mutants showed higher germination activity when treated with ABA and NaCl than wild type (Col-0). Through comparing the results of seed germination, root growth, stomatal aperture, water loss, and proline accumulation between the Atgalk2 mutants and Col-0, it was found that Atgalk2 mutants showed less sensitive to ABA than Col-0. The expression levels of ABI1, ABI2, RAB18, ABF3, RD22, RD29A, and RD29B in the Atgalk2 mutants were higher than in Col-0. However, the expression level of OST1 in the Atgalk2 mutants was lower than in Col-0. Taken together, these results suggested AtGALK2 was required for abscisic acid regulation of seed germination, root growth and gene expression, and was involved in salt and osmotic stress response in the early development stage. This study provides important clues to galactokinase activities of GHMP family in ABA signaling and plant development.

The Role of Light Quality in Regulating Early Seedling Development.

It is well-established that plants are sessile and photoautotrophic organisms that rely on light throughout their entire life cycle. Light quality (spectral composition) is especially important as it provides energy for photosynthesis and influences signaling pathways that regulate plant development in the complex process of photomorphogenesis. During previous years, significant progress has been made in light quality's physiological and biochemical effects on crops. However, understanding how light quality modulates plant growth and development remains a complex challenge. In this review, we provide an overview of the role of light quality in regulating the early development of plants, encompassing processes such as seed germination, seedling de-etiolation, and seedling establishment. These insights can be harnessed to improve production planning and crop quality by producing high-quality seedlings in plant factories and improving the theoretical framework for modern agriculture.

The genetic and physiological analysis of late-flowering phenotype of T-DNA insertion mutants of AtCAL1 and AtCAL2 in Arabidopsis.

The homozygous T-DNA mutants of AtCAL1 (Rat1) and AtCAL2 (Rat2) were obtained. The double mutant of Rat2/Rat1RNAi was constructed which showed obvious late-flowering phenotype from others. The expression of various flowering-related genes was studied among mutants and wild-type plants by quantitative RT-PCR. The double mutant plants showed the shortest root length compared with T-DNA insertion mutants and wild type plants under red light, blue light, and white light. The double mutants showed hypersensitivity to NaCl and ABA. However, these mutants had no effect on stomatal closure by ABA.

Mutational analysis of Arabidopsis PP2CA2 involved in abscisic acid signal transduction.

The homozygous T-DNA mutant of the PP2CA2 gene in Arabidopsis thaliana was identified at DNA and RNA levels. The semi-quantitative RT-PCR analysis showed expression of PP2CA2 was induced by NaCl and ABA. When grown in presence of increasing concentration of exogenous ABA the pp2ca2 mutant showed a significant loss of ABA sensitivity in terms of seed germination, efficiency of post-germination growth and root growth. In presence of all ABA and NaCl concentrations tested the germination percentage of wild-type seeds was lower than that of mutant ppca2 seeds. Furthermore, in the presence of exogenous ABA, the pp2ca2 seeds showed higher germination percentages than wild-type at different stages of development and the pp2ca2 seedlings showed a reduced inhibition of root growth compared with wild-type plants. The above results indicated that the pp2ca2 was an ABA-hyposensitive mutant.

[Preliminary studies on the function of Arabidopsis CK1A gene].

A casein kinase 1 protein gene, CK1A, was isolated from Arabidopsis seedlings by RT-PCR method. This gene contains an open reading frame of 2,112 bp, which encodes 703 amino acids. The plant expression vector of 35S: GFP: CK1A was constructed by the Gateway System. The 35S: GFP: CK1A fusion protein was localized to the nucleus in onion epidermal cell, indicating that the product of CK1A gene plays a role in the cell nucleus. The semi-quantitative RT-PCR analysis showed that CK1A was highly expressed in flowers, stems and roots, but less in leaves and leafstalks. The yeast two-hybrid analysis demonstrated that CK1A and CRY2 can interact in vivo under blue light, which indicates that CK1A may play an important role in blue light signal induction of Arabidopsis.

RPN1a negatively regulates ABA signaling in Arabidopsis.

The 26S proteasome selectively regulates key abscisic acid (ABA) signaling proteins, but the physiological functions and mechanisms of RPN1a (a subunit of the 26S proteasome) in ABA signaling remain largely unknown. In this study, we found that the mRNA expression of RPN1a was suppressed by ABA treatment, and that RPN1a protein was expressed abundantly in guard cells. In the presence of ABA, rpn1a mutants showed rapid stomatal closure, low water loss, delayed germination, and inhibited root elongation. In addition, the transcripts of key ABA signaling genes, including ABI5, RD22, RD29A, and RD29B, were upregulated in rpn1a mutant plants in response to ABA. Furthermore, the ABI5 protein level was higher in rpn1a mutants subjected to ABA treatment. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that RPN1a interacts with ABI1. Overall, these findings suggest that RPN1a negatively regulates ABA signaling in Arabidopsis.

RPN1a, a subunit of the 26S proteasome, controls trichome development in Arabidopsis.

The ubiquitin-mediated 26S proteasome pathway (UPS) is of great importance to plant growth and development. Previously research showed that a subunit of the 26S proteasome, named RPN1a, was involved in trichome's branching in Arabidopsis. Mutation in RPN1a give rise to more trichome branches on leaves. Here, we found that T-DNA insertion mutation in RPN1a resulted in increased trichome branches on main stem, and trichome number on rosette leaves and the main stem compared with the wild type plant. Expression analysis results showed that the transcription levels of ZFP6, ZFP5, GIS, GL1, GL2, GL3, TTG1 and MYB23, which promote trichome initiation, were up-regulated in the rpn1a mutant, and expression of FRC4, which is responsible for increased trichome branching, was also increased in the rpn1a mutant. Moreover, the mRNA expression level of RPN1a was significantly repressed by GA (gibberellin) and CK (cytokinin) treatment, which are two important phytohormones that play essential roles in trichome development. These results demonstrate that RPN1a may be involved in trichome development through the GA and CK signaling pathways.

Arabidopsis F-box gene FOA1 involved in ABA signaling.

The expression of FOA1 (F-box overexpressed/oppressed ABA signaling) in different organs of Arabidopsis, and in response to ABA and NaCl, was analyzed. The expression level of FOA1 is higher in the root and is lower in the stem, and is induced rapidly by ABA and NaCl. The phenotypes of T-DNA insertion mutant foa1 and FOA1 overexpression lines FOA1ox1 and FOA1ox2 were analyzed. The foa1 mutant exhibited a lower germination rate, shorter root length, more stomatal opening, increased proline accumulation and hypersensitivity to ABA compared with the wild type. In contrast, the overexpression lines showed lower sensitivity to ABA than the wild type. The expression levels of several ABA and stress-responsive transcription factors and genes were altered in the foa1 mutant in response to ABA. Compared with the wild type, the expression levels of ABA-responsive transcription factors were higher, but ABA and stress-responsive genes were lower in foa1 mutant. This study demonstrates that FOA1 is an ABA signaling-related gene, and may play a negative role in ABA signaling.