CPK10 protein kinase regulates Arabidopsis tolerance to boron deficiency through phosphorylation and activation of BOR1 transporter.

Boron (B) is crucial for plant growth and development. B deficiency can impair numerous physiological and metabolic processes, particularly in root development and pollen germination, seriously impeding crop growth and yield. However, the molecular mechanism underlying boron signal perception and signal transduction is rather limited. In this study, we discovered that CPK10, a calcium-dependent protein kinase in the CPK family, has the strongest interaction with the boron transporter BOR1. Mutations in CPK10 led to growth and root development defects under B-deficiency conditions, while constitutively active CPK10 enhanced plant tolerance to B deficiency. Furthermore, we found that CPK10 interacted with and phosphorylated BOR1 at the Ser689 residue. Through various biochemical analyses and complementation of B transport in yeast and plants, we revealed that Ser689 of BOR1 is important for its transport activity. In summary, these findings highlight the significance of the CPK10-BOR1 signaling pathway in maintaining B homeostasis in plants and provide targets for the genetic improvement of crop tolerance to B-deficiency stress.

TaNRAMP3 is essential for manganese transport in Triticum aestivum.

Manganese (Mn) is an essential trace element for almost all living organisms. In plants, Mn deficiency, which is occurs in calcareous soils or alkaline soils, severely limiting crop yields. However, the potential mechanism of Mn transport in Triticum aestivum is still obscure. Here, we found that TaNRAMP3, a member of the naturally resistant macrophage protein (NRAMP) family in Triticum aestivum, is located in the plasma membrane of protoplasts and functions as an influx transporter for Mn in yeast (Δsmf1). The expression of TaNRAMP3 was induced under Mn-deficiency conditions. Furthermore, TaNRAMP3-RNAi plants exhibited a sensitive phenotype, while transgenic plants overexpressing TaNRAMP3 showed a tolerant phenotype. In addition, TaNRAMP3 rescued the sensitive phenotype of Arabidopsis nramp1 mutant under Mn deficiency condition. In summary, our study reveals the key role of TaNRAMP3 in Mn transport in Triticum aestivum, allowing it to adapt to Mn-deficiency stress. These findings provide new insights for the cultivation of Mn-deficiency tolerant wheat varieties.

Calcium-dependent protein kinases CPK21 and CPK23 phosphorylate and activate the iron-regulated transporter IRT1 to regulate iron deficiency in Arabidopsis.

Iron (Fe) is an essential micronutrient for all organisms. Fe availability in the soil is usually much lower than that required for plant growth, and Fe deficiencies seriously restrict crop growth and yield. Calcium (Ca2+) is a second messenger in all eukaryotes; however, it remains largely unknown how Ca2+ regulates Fe deficiency. In this study, mutations in CPK21 and CPK23, which are two highly homologous calcium-dependent protein kinases, conferredimpaired growth and rootdevelopment under Fe-deficient conditions, whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions. Furthermore, we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRON-REGULATED TRANSPORTER1 (IRT1) at the Ser149 residue. Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity. Taken together, these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.

CBL1/9-CIPK23-NRAMP1 axis regulates manganese toxicity.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

Plasma membrane-associated calcium signaling regulates arsenate tolerance in Arabidopsis.

Arsenate [As(V)] is a metalloid with heavy metal properties and is widespread in many environments. Dietary intake of food derived from arsenate-contaminated plants constitutes a major fraction of the potentially health-threatening human exposure to arsenic. However, the mechanisms underlying how plants respond to arsenate stress and regulate the function of relevant transporters are poorly understood. Here, we observed that As(V) stress induces a significant Ca2+ signal in Arabidopsis (Arabidopsis thaliana) roots. We then identified a calcium-dependent protein kinase, CALCIUM-DEPENDENT PROTEIN KINASE 23 (CPK23), that interacts with the plasma membrane As(V)/Pi transporter PHOSPHATE TRANSPORTER 1;1 (PHT1;1) in vitro and in vivo. cpk23 mutants displayed a sensitive phenotype under As(V) stress, while transgenic Arabidopsis plants with constitutively active CPK23 showed a tolerant phenotype. Furthermore, CPK23 phosphorylated the C-terminal domain of PHT1;1, primarily at Ser514 and Ser520. Multiple experiments on PHT1;1 variants demonstrated that PHT1;1S514 phosphorylation is essential for PHT1;1 function and localization under As(V) stress. In summary, we revealed that plasma-membrane-associated calcium signaling regulates As(V) tolerance. These results provide insight for crop bioengineering to specifically address arsenate pollution in soils.

Diurnal transcriptome dynamics reveal the photoperiod response of Pyrus.

Photoperiod provides a key environmental signal that controls plant growth. Plants have evolved an integrated mechanism for sensing photoperiods with internal clocks to orchestrate physiological events. This mechanism has been identified to enable timely plant growth and improve fitness. Although the components and pathways underlying photoperiod regulation have been described in many species, diurnal patterns of gene expression at the genome-wide level under different photoperiods are rarely reported in perennial fruit trees. To explore the global gene expression in response to photoperiod, pear plants were cultured under long-day (LD) and short-day (SD) conditions. A time-series transcriptomic study was implemented using LD and SD samples collected at 4 h intervals over 2 days. We identified 13,677 rhythmic genes, of which 7639 were identified under LD and 10,557 under SD conditions. Additionally, 4674 genes were differentially expressed in response to photoperiod change. We also characterized the candidate homologs of clock-associated genes in pear. Clock genes were involved in the regulation of many processes throughout the day, including photosynthesis, stress response, hormone dynamics, and secondary metabolism. Strikingly, genes within photosynthesis-related pathways were enriched in both the rhythmic and differential expression analyses. Several key candidate genes were identified to be associated with regulating photosynthesis and improving productivity under different photoperiods. The results suggest that temporal variation in gene expression should not be ignored in pear gene function research. Overall, our work expands the understanding of photoperiod regulation of plant growth, particularly by extending the research to non-model trees.

Plasma membrane-associated calcium signaling modulates cadmium transport.

Cadmium (Cd) is a toxic heavy element for plant growth and development, and plants have evolved many strategies to cope with Cd stress. However, the mechanisms how plants sense Cd stress and regulate the function of transporters remain very rudimentary. Here, we found that Cd stress induces obvious Ca2+ signals in Arabidopsis roots. Furthermore, we identified the calcium-dependent protein kinases CPK21 and CPK23 that interacted with the Cd transporter NRAMP6 through a variety of protein interaction techniques. Then, we confirmed that the cpk21 23 double mutants significantly enhanced the sensitive phenotype of cpk23 single mutant under Cd stress, while the overexpression and continuous activation of CPK21 and CPK23 enhanced plants tolerance to Cd stress. Multiple biochemical and physiological analyses in yeast and plants demonstrated that CPK21/23 phosphorylate NRAMP6 primarily at Ser489 and Thr505 to inhibit the Cd transport activity of NRAMP6, thereby improving the Cd tolerance of plants. Taken together, we found a plasma membrane-associated calcium signaling that modulates Cd tolerance. These results provide new insights into the molecular breeding of crop tolerance to Cd stress.

Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Ca2+-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis.

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families have been characterized, we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants. Here, we demonstrate that two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, are key regulators of plant Mn homeostasis. Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we show that sequential phosphorylation of MTP8, initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26, provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport. Collectively, our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.

Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity. Despite their essential roles in Mn homeostasis, transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood. Here, we demonstrated that high-Mn stress induces an obvious Ca2+ signature in Arabidopsis. We identified four calcium-dependent protein kinases, CPK4/5/6/11, that interact with the tonoplast-localized Mn and iron (Fe) transporter MTP8 in vitro and in vivo. The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant. CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues. Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8S31/32A and phospho-mimetic variant MTP8S31/32D plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function. In addition, genetic analysis showed that CPKs functioned upstream of MTP8. In summary, we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity, Ca2+ signaling, and Mn transporters. These findings provide new insight into Mn homeostasis mechanisms and Ca2+ signaling pathways in plants, providing potential targets for engineering heavy metal toxicity-tolerant plants.

Water quality induced corrosion of stainless steel valves during long-term service in a reverse osmosis system.

The current study analyzes the contribution of 10 water quality parameters (including pH, turbidity, conductivity, total dissolved solids (TDS), hardness, total organic carbon (TOC), alkalinity, calcium ions, chlorides and sulfates) to corrosion extent of stainless steel valves taken from different locations in a reverse osmosis system of a reclaimed water plant. The valves were in service for 5 years. Raman spectroscopy and X-ray photoelectron spectroscopy analyses are conducted to quantify corrosion products on different valves under various water quality conditions. On that basis, bivariate and multivariate regression analyses between the 10 water quality parameters and the corrosion extent of valve specimens (represented by metal loss percentage (MLP) values) are carried out to check the contribution of those water quality parameters to MLP. The results indicate that the proportions of metal oxides as corrosion products vary according to the corrosion extent of the valves. Although no linear correlation is found, all 10 water quality parameters except for pH show a significant positive correlation with the MLP values of the valve specimens. Moreover, results of multivariate regression suggest that the variation of MLP can be explained by turbidity, TDS, TOC and sulfates. A positive contribution of turbidity, TDS and TOC to MLP is observed, whereas the contribution of sulfates is negative. The results from the current work help to identify the reasons for water quality-induced failure of stainless steel equipment in RO systems.