Role of ABA in the adaptive response of Arabidopsis plants to long-term boron toxicity treatment.

Boron (B) toxicity causes impairments in several plant metabolic and physiological processes. Under conditions of excessive B availability, this micronutrient is passively transported through the transpiration stream and accumulates in leaves, causing the development of necrotic regions in leaf tips. Some plants have developed adaptive mechanisms to minimize the toxic effects of excessive B accumulation in their tissues. Thus, for instance, in Arabidopsis it has been described an ABA-dependent decrease in the transpiration rate that would restrict B accumulation in aerial plant tissues in response to short-term B toxicity, this effect being mediated by AtNCED3 (which encodes a key enzyme for ABA biosynthesis). The present work aimed to study the possible involvement of ABA in the adjustment of plant water balance and B homeostasis during the adaptive response of Arabidopsis to prolonged B toxicity. For this purpose, Arabidopsis wild-type and the ABA-deficient nced3-2 mutant plants were subjected to B toxicity for 7 days. We show that ABA-dependent stomatal closure is determinant for the adjustment of plant water relations under conditions of prolonged B toxicity. Results suggest that, in addition to the AtNCED3 gene, the AtNCED5 gene could also be involved in this ABA-dependent stomatal closure. Finally, our results also indicate the possible role of endogenous root ABA content in the mechanism of active efflux of B via BOR4 (efflux-type B transporter) from the root to the external environment under excess B conditions.

Leaf Proteomic Analysis in Seedlings of Two Maize Landraces with Different Tolerance to Boron Toxicity.

Boron (B) toxicity is an important stressor that negatively affects maize yield and the quality of the produce. The excessive B content in agricultural lands is a growing problem due to the increase in arid and semi-arid areas because of climate change. Recently, two Peruvian maize landraces, Sama and Pachía, were physiologically characterized based on their tolerance to B toxicity, the former being more tolerant to B excess than Pachía. However, many aspects regarding the molecular mechanisms of these two maize landraces against B toxicity are still unknown. In this study, a leaf proteomic analysis of Sama and Pachía was performed. Out of a total of 2793 proteins identified, only 303 proteins were differentially accumulated. Functional analysis indicated that many of these proteins are involved in transcription and translation processes, amino acid metabolism, photosynthesis, carbohydrate metabolism, protein degradation, and protein stabilization and folding. Compared to Sama, Pachía had a higher number of differentially expressed proteins related to protein degradation, and transcription and translation processes under B toxicity conditions, which might reflect the greater protein damage caused by B toxicity in Pachía. Our results suggest that the higher tolerance to B toxicity of Sama can be attributed to more stable photosynthesis, which can prevent damage caused by stromal over-reduction under this stress condition.

Crosstalk of Cytokinin with Ethylene and Auxin for Cell Elongation Inhibition and Boron Transport in Arabidopsis Primary Root under Boron Deficiency.

Several studies have shown the role of phytohormones in the regulation of root growth of Arabidopsis plants under boron (B) deficiency. Ethylene and auxin play an important role in the control of Arabidopsis primary root cell elongation under short-term B deprivation, whereas cytokinins regulate root growth inhibition under B deficiency by controlling meristem cell proliferation. In this work, we study the possible interaction among cytokinin, ethylene, and auxin in the primary root response to B-deprivation treatment, as well as their possible role in B uptake and transport. Wild type (WT) and two mutants related to auxin and ethylene (aux1 and acs11) Arabidopsis plants were grown in control (10 µM B) or B starvation (0 µM B) treatment, in the absence or presence of trans-zeatin, and their primary root growth was analyzed. The possible interaction between these hormones was also studied by analyzing AUX1 gene expression in the acs11 mutant and ACS11 gene expression in the aux1 mutant. The GUS reporter lines ARR5::GUS, IAA2::GUS, and EBS::GUS were used to observe changes in cytokinin, auxin, and ethylene levels in the root, respectively. The results of this work suggest that cytokinin inhibits root cell elongation under B deficiency through two different mechanisms: (i) an ethylene-dependent mechanism through increased expression of the ACS11 gene, which would lead to increased ethylene in the root, and (ii) an ethylene-independent mechanism through decreased expression of the AUX1 gene, which alters auxin signaling in the meristematic and elongation zones and stele. We also report that changes in the expression of several B transporters occur in response to auxin, ethylene, and cytokinin that may affect the plant B content.

Characterization of two Peruvian maize landraces differing in boron toxicity tolerance.

Boron (B) toxicity is a major agricultural problem that causes a considerable decrease in crop yield and quality. The soil in arid and semi-arid areas is often subjected to excessive B content. Southwestern Perú (department of Tacna) is characterized by high B levels in its agricultural land and irrigation water. This work analyzes the response of two local maize (Zea mays) landraces (Pachía and Sama) from Tacna to B toxicity. Both landraces were, therefore, grown in hydroponic media under control and B toxicity conditions, and after 10 days, seedlings were harvested and B content, B-transporter gene expressions, and several morphological and physiological parameters were determined. The leaf and root soluble B content was lower in Sama than in Pachía when both landraces were subjected to high B concentrations, which could be explained by its higher expression levels of B-efflux transporters. The capacity of Sama to maintain reduced levels of soluble B in its leaves and roots led to decreased leaf damage and higher photosynthetic and growth parameters under B toxicity conditions. These results support the proposal that Sama would perform better than Pachía under excessive B conditions, thus making it a more suitable landrace to be used in soils with toxic levels of B.

Boron Deficiency Increases Cytosolic Ca2+ Levels Mainly via Ca2+ Influx from the Apoplast in Arabidopsis thaliana Roots.

Boron (B) is a micronutrient for plant development, and its deficiency alters many physiological processes. However, the current knowledge on how plants are able to sense the B-starvation signal is still very limited. Recently, it has been reported that B deprivation induces an increase in cytosolic calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana roots. The aim of this work was to research in Arabidopsis whether [Ca2+]cyt is restored to initial levels when B is resupplied and elucidate whether apoplastic Ca2+ is the major source for B-deficiency-induced rise in [Ca2+]cyt. The use of chemical compounds affecting Ca2+ homeostasis showed that the rise in root [Ca2+]cyt induced by B deficiency was predominantly owed to Ca2+ influx from the apoplast through plasma membrane Ca2+ channels in an IP3-independent manner. Furthermore, B resupply restored the root [Ca2+]cyt. Interestingly, expression levels of genes encoding Ca2+ transporters (ACA10, plasma membrane PIIB-type Ca2+-ATPase; and CAX3, vacuolar cation/proton exchanger) were upregulated by ethylene glycol tetraacetic acid (EGTA) and abscisic acid (ABA). The results pointed out that ACA10, and especially CAX3, would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.

Boron deficiency inhibits root cell elongation via an ethylene/auxin/ROS-dependent pathway in Arabidopsis seedlings.

One of the earliest symptoms of boron (B) deficiency is the inhibition of root elongation which can reasonably be attributed to the damaging effects of B deprivation on cell wall integrity. It is shown here that exposure of wild-type Arabidopsis thaliana seedlings to B deficiency for 4h led to a drastic inhibition of root cell length in the transition between the elongation and differentiation zones. To investigate the possible mediation of ethylene, auxin, and reactive oxygen species (ROS) in the effect of B deficiency on root cell elongation, B deficiency was applied together with aminoethoxyvinylglycine (AVG, a chemical inhibitor of ethylene biosynthesis), silver ions (Ag(+), an antagonist of ethylene perception), α-(phenylethyl-2-oxo)-indoleacetic acid (PEO-IAA, a synthetic antagonist of TIR1 receptor function), and diphenylene iodonium (DPI, an inhibitor of ROS production). Interestingly, all these chemicals partially or fully restored cell elongation in B-deficient roots. To further explore the possible role of ethylene and auxin in the inhibition of root cell elongation under B deficiency, a genetic approach was performed by using Arabidopsis mutants defective in the ethylene (ein2-1) or auxin (eir1-4 and aux1-22) response. Root cell elongation in these mutants was less sensitive to B-deficient treatment than that in wild-type plants. Altogether, these results demonstrated that a signalling pathway involving ethylene, auxin, and ROS participates in the reduction of root cell elongation when Arabidopsis seedlings are subjected to B deficiency. A similar signalling process has been described to reduce root elongation rapidly under various types of cell wall stress which supports the idea that this signalling pathway is triggered by the impaired cell wall integrity caused by B deficiency.

Root Responses to Boron Deficiency Mediated by Ethylene.

Low boron (B) supply alters the architecture of the root system in Arabidopsis thaliana seedlings, leading to a reduction in the primary root growth and an increase in the length and number of root hairs. At short-term (hours), B deficiency causes a decrease in the cell elongation of the primary root, resulting in a lower growth. Experimental approaches using ethylene insensitive Arabidopsis mutants, inhibitors of ethylene response, and GUS reporter lines suggest that ethylene is involved in these responses of the primary root to B deficiency. Furthermore, it has been shown that auxin participates in the inhibition of cell elongation under short-term B deprivation. These results support that an interaction between ethylene and auxin plays an important role in controlling the primary root elongation, in which a number of genes related to the synthesis, transport, and signaling of both phytohormones could modulate this effect. Evidence for a root cross-talk among both hormones and other possible intermediates (abscisic acid, calcium sensors, and reactive oxygen species) in response to B deficiency is provided and discussed.

Is Ca2+ involved in the signal transduction pathway of boron deficiency? New hypotheses for sensing boron deprivation.

Plants sense and transmit nutrient-deprivation signals to the nucleus. This increasingly interesting research field advances knowledge of signal transduction pathways for mineral deficiencies. The understanding of this topic for most micronutrients, especially boron (B), is more limited. Several hypotheses have been proposed to explain how a B deprivation signal would be conveyed to the nucleus, which are briefly summarized in this review. These hypotheses do not explain how so many metabolic and physiological processes quickly respond to B deficiency. Short-term B deficiency affects the cytosolic Ca(2+) levels as well as root expression of genes involved in Ca(2+) signaling. We propose and discuss that Ca(2+) and Ca(2+)-related proteins - channels/transporters, sensor relays, and sensor responders - might have major roles as intermediates in a transduction pathway triggered by B deprivation. This hypothesis may explain how plants sense and convey the B-deprivation signal to the nucleus and modulate physiological responses. The possible role of arabinogalactan-proteins in the B deficiency signaling pathway is also taken into account.

Transcription factors as potential participants in the signal transduction pathway of boron deficiency.

Boron (B) plays a well-known structural role in the cell wall, however the way of perceiving B deficiency by roots and transmitting this environmental signal to the nucleus to elicit a response is not well established. It is known that the direct interaction between Ca2+ sensors and transcription factors (TFs) is a necessary step to regulate the expression of downstream target genes in some signaling pathways. Interestingly, B deprivation affected gene expressions of several TFs belonging to MYB, WRKY, and bZIP families, as well as expressions of Ca2+ -related genes such as several CML (calmodulin-like protein) and CPK (Ca2+ -dependent protein kinase) genes. Taken together, these results suggest that B deficiency could affect the expression of downstream target genes by alteration of a calcium signaling pathway in which the interaction between CMLs and/or CPKs with TFs (activator or repressor) would be a crucial step, which would explain why some genes are upregulated whereas others are repressed upon B deprivation.

Boron deficiency increases the levels of cytosolic Ca(2+) and expression of Ca(2+)-related genes in Arabidopsis thaliana roots.

Boron (B) deficiency affects the expressions of genes involved in major physiological processes. However, signal transduction pathway through which plants are able to sense and transmit B-deprivation signal to the nucleus is unknown. The aim of this work was to research in Arabidopsis thaliana roots whether the short-term B deficiency affects cytosolic Ca(2+) levels ([Ca(2+)]cyt) as well as expression of genes involved in Ca(2+) signaling. To visualize in vivo changes in root [Ca(2+)]cyt, Arabidopsis seedlings expressing Yellow Cameleon (YC) 3.6 were grown in a nutrient solution supplemented with 2 µM B and then transferred to a B-free medium for 24 h. Root [Ca(2+)]cyt was clearly higher in B-deficient seedlings upon 6 and 24 h of B treatments when compared to controls. Transcriptome analyses showed that transcript levels of Ca(2+) signaling-related genes were affected by B deprivation. Interestingly, Ca(2+) channel (CNGC19, cyclic nucleotide-gated ion channel) gene was strongly upregulated as early as 6 h after B deficiency. Expression levels of Ca(2+) transporter (ACA, autoinhibited Ca(2+)-ATPase; CAX, cation exchanger) genes increased when seedlings were subjected to B deficiency. Gene expressions of calmodulin-like proteins (CMLs) and Ca(2+)-dependent protein kinases (CPKs) were also overexpressed upon exposure to B starvation. Our results suggest that B deficiency causes early responses in the expression of CNGC19 Ca(2+)-influx channel, ACA- and CAX-efflux, and Ca(2+) sensor genes to regulate Ca(2+) homeostasis. It is the first time that changes in the levels of in vivo cytosolic Ca(2+) and expression of Ca(2+) channel/transporter genes are related with short-term B deficiency in Arabidopsis roots.

Expression of root glutamate dehydrogenase genes in tobacco plants subjected to boron deprivation.

Recently it has been reported that boron (B) deficiency increases the expression of Nicotiana tabacum asparagine synthetase (AS) gene in roots, and that AS might play a main role as a detoxifying mechanism to convert ammonium into asparagine. Interestingly, glutamate dehydrogenase (GDH) genes, Ntgdh-NAD;A1 and Ntgdh-NAD;B2, were up-regulated when tobacco roots were subjected to B deprivation for 8 and 24 h. In addition, aminating and deaminating GDH (EC 1.4.1.2) activities were higher in B-deficient than in B-sufficient plants after 24 h of B deficiency. Ammonium concentrations were kept sufficiently low and with similar values in B-deficient roots when compared to control. Glucose and fructose contents decreased after 24 h of B deprivation. This drop in hexoses, which was corroborated by metabolomic analysis, correlated with higher GDH gene expression. Furthermore, metabolomic profiling showed that concentrations of several organic acids, phenolics, and amino acids increased after 24 h of B deficiency. Our results suggest that GDH enzyme plays an important role in metabolic acclimation of tobacco roots to B deprivation. A putative model to explain these results is proposed and discussed.

Boron deficiency and transcript level changes.

Boron (B) is an essential element for plant growth whose deficiency causes an alteration in the expression of a wide range of genes involved in several physiological processes. However, our understanding of the signal transduction pathways that trigger the B-deficiency responses in plants is still poor. The aims of this review are (i) to summarize the genes whose transcript levels are affected by B deficiency and (ii) to provide an update on recent findings that could help to understand how the signal(s) triggered by B deficiency is transferred to the nucleus to modulate gene expression. In this contribution we review the effects of B deficiency on the transcript level of genes related to B uptake and translocation, maintenance of cell wall and membrane function, nitrogen assimilation and stress response. In addition, we discuss the possible mediation of calcium, arabinogalactan-proteins and other cis-diol containing compounds in the signaling mechanisms that transfer the signal of B deficiency to nuclei. Finally, we conclude that the advance in the knowledge of the molecular basis of B deficiency response in plants will allow improving the tolerance of crops to B deficiency stress.

Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings.

Changes in root architecture are one of the adaptive strategies used by plants to compensate for nutrient deficiencies in soils. In this work, the temporal responses of Arabidopsis (Arabidopsis thaliana) root system architecture to low boron (B) supply were investigated. Arabidopsis Col-0 seedlings were grown in 10 µM B for 5 days and then transferred to a low B medium (0.4 µM) or control medium (10 µM) for a 4-day period. Low B supply caused an inhibition of primary root (PR) growth without altering either the growth or number of lateral roots (LRs). In addition, low B supply induced root hair formation and elongation in positions close to the PR meristem not observed under control conditions. The possible role of auxin and ethylene in the alteration of root system architecture elicited by low B supply was also studied by using two Arabidopsis reporter lines (DR5:GUS and EBS:GUS) and two Arabidopsis mutants with impaired auxin and ethylene signaling (aux1-22 and ein2-1). Low B supply increased auxin reporter DR5:GUS activity in PR tip, suggesting that low B alters the pattern of auxin distribution in PR tip. Moreover, PR elongation in aux1-22 mutant was less sensitive to low B treatment than in wild-type plants, which suggests that auxin resistant 1 (AUX1) participates in the inhibition of PR elongation under low B supply. From all these results, a hypothetical model to explain the effect of low B treatment on PR growth is proposed. We also show that ethylene, via ethylene-insensitive 2 (EIN2) protein, is involved in the induction of root hair formation and elongation under low B treatment.

Boron in plants: deficiency and toxicity.

Boron (B) is an essential nutrient for normal growth of higher plants, and B availability in soil and irrigation water is an important determinant of agricultural production. To date, a primordial function of B is undoubtedly its structural role in the cell wall; however, there is increasing evidence for a possible role of B in other processes such as the maintenance of plasma membrane function and several metabolic pathways. In recent years, the knowledge of the molecular basis of B deficiency and toxicity responses in plants has advanced greatly. The aim of this review is to provide an update on recent findings related to these topics, which can contribute to a better understanding of the role of B in plants.

PRD, an Arabidopsis AINTEGUMENTA-like gene, is involved in root architectural changes in response to phosphate starvation.

Changes in root architecture are one of the adaptive strategies used by plants to compensate for local phosphate (Pi) deficiency in soils. Root architecture variables triggered by Pi availability are well documented in Arabidopsis (Arabidopsis thaliana), but the molecular mechanisms behind these adaptive responses remain to be elucidated. By the use of transcriptomic and quantitative RT-PCR analysis, we observed that an AINTEGUMENTA-like gene, named PRD for Phosphate Root Development, was rapidly repressed in roots under low Pi conditions. The physiological function of the PRD gene was analyzed through the null allele mutant prd, which displayed less development of primary and lateral roots under Pi-starvation conditions than wild-type plants. Complementation of the prd mutant with the wild-type gene led to a similar response to Pi starvation as wild-type plants, indicating the complete rescue of the mutant phenotype. These results suggest that PRD gene is involved in the regulation of root architectural responses to Pi starvation by controlling primary and lateral root elongation. This model is in agreement with the tissue-specific pattern of PRD gene expression, which was observed to occur specifically in the apex in both the primary and lateral roots. However, Pi influx, anionic profiles and root expression of genes typically induced by Pi starvation, such as high affinity Pi transporters (PHT1;1 and PHT1;4) and an acid phosphatase (AtACP5), were similar in wild type and prd plants in response to Pi starvation. These results support the hypothesis that the PRD gene is not a checkpoint for Pi-starvation responses, but acts specifically as a regulator of root architectural responses to Pi starvation.

Is boron involved solely in structural roles in vascular plants?

It is very well proved that boron (B) plays a primary structural role in the plant cell wall. In addition, this micronutrient has been involved in a great variety of physiological processes in vascular plants. It has been reported that B deficiency induces stress-responsive genes and, in tobacco plants, it seems to decrease net nitrate uptake by repressing expression of root plasmalemma H(+)-ATPase gene. Moreover, root asparagine concentration is clearly increased under B deficiency, as also observed for other abiotic stresses. Accumulation of asparagine in response to abiotic stresses could be an ammonium detoxification mechanism when high amounts of ammonium are internally generated by deamination of soluble amino acids released from enhanced proteolysis under stress conditions. Nevertheless, the mechanisms underlying the several effects caused by B deficiency are unknown. Although a mechanism has been reported to explain B effects based on signals via the cell wall-plasma membrane-cytoskeleton continuum, we propose and discuss the possible role of B as a cellular signal through transcription factors. This hypothetical mechanism could explain not only its diverse effects on so many physiological processes, but also that a negligible amount of boron into the protoplast can be decisive for the normal development of such events.

The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii.

The expression of nitrite uptake activity and the induction of transcripts from several nitrate assimilation genes (Nii1, Nrt2;1, Nrt2;3, and Nar1) have been analysed in Chlamydomonas reinhardtii Dang. strains bearing several sets of genes encoding nitrate transport systems. Different nitrate concentrations resulted in a differential induction pattern of nitrite uptake activity depending on which particular nitrate transport system was present in the cells, and that was directly related to their relative efficiency for nitrate transport. The presence of the high-affinity nitrate transport system I (NRT2;1, NAR2) made cells able to sense the very low concentrations of nitrate present in culture medium with no added nitrate and to express optimally Nii1, Nrt2;1, Nrt2;3, and Nar1 genes involved in nitrate/nitrite assimilation. In addition, strains lacking nitrate reductase activity overexpressed these gene transcripts as a result of continuous signalling by nitrate, but only those bearing an active system I. This study supports the hypothesis that signalling of nitrate assimilation genes occurs intracellularly in a process dependent on the nitrate uptake activity. The bispecific nitrate/nitrite transport system I with the highest affinity for nitrate is the most efficient one for signalling the expression of nitrate/nitrite assimilation genes in C. reinhardtii.

Nitrite transport to the chloroplast in Chlamydomonas reinhardtii: molecular evidence for a regulated process.

Nitrite transport to the chloroplast is not a well documented process in spite of being a central step in the nitrate assimilation pathway. The lack of molecular evidence, as well as the easy diffusion of nitrite through biological membranes, have made this physiological process difficult to understand in plant nutrition. The aim of this review is to illustrate that nitrite transport to the chloroplast is a regulated step, intimately related to the efficiency of nitrate utilization. In Chlamydomonas reinhardtii, the Nar1;1 gene has been shown to have this role in nitrate assimilation. NAR1;1 corresponds to a plastidic membrane transporter protein related to the bacterial formate/nitrite transporters. At least four Nar1 genes might exist in Chlamydomonas. The existence of orthologous Nar1 genes in plants is discussed.