Tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is a model plant widely studied for its diverse physiological and genetic traits. Understanding the intricate hormonal mechanisms governing its response to abiotic stresses, such as drought and waterlogging, is crucial to a world harshly affected by climate change. Therefore, we aimed at understanding how other hormones affect drought and waterlogging tolerance in MT, wild type (WT) with no hormonal variation and three variants with a genetic background for reduced cytokinin (−CK, 35S::CYTOKININ OXYDASE 2) and abscisic acid (−ABA, notabilis mutant) and increased gibberellin (+ GA, 35S::GA2ox-gibberellin). Thirty days after germination plants were subjected to drought, field capacity, and waterlogging for 7 days. Root-stem transition zone cross sections were analysed by microscopy. All treatments showed different responses. All plants showed decreased diameter in xylem vessel elements under field capacity. WT showed decreased epidermis cell area (ECa) under field capacity, decreased average intercellular space area (ISA), and epidermis thickness under drought, and increased epidermis cell wall thickness (Ecwt) under waterlogging. −CK showed increased Ecwt under waterlogging, −ABA showed decreased ECa and average ISA under drought. −CK and −ABA showed anomalies in vascular bundle morphology such as the position of tissues. + GA was unaffected by water stresses, while −ABA was harmed by drought and waterlogging. Increased ISA was found in −ABA under waterlogging, and in 35S::GA2ox under drought, indicating that ABA and GA regulate the initial signalling for tissues formed by programmed cell death such as adventitious root and aerenchyma in tomato at contrasting conditions of water saturation.

The spatial pattern distribution of plant hormones in response to aluminum (Al) toxicity in roots remains to be shown. This study was performed to assess the root hormonal accumulation and gene expression in response to Al toxicity in five transgenic miniature dwarf tomatoes cv. Micro-Tom (MT). MT and MT transgenics to acid indole acetic, cytokinin, gibberellin, abscisic acid and ethylene were cultivated in nutrient solutions containing different Al concentrations. Root growth elongation was measured and cellular damage was visualized by staining Evans's blue. The GUS reporter gene staining technique was used to visualize hormonal changes in MT apex root tissues. Data indicated that the MT is sensitive to Al that induced significant growth inhibition and cellular damage. Al concentration of 27 μM was significantly toxic, inducing root apex darkening and inhibition of root development. The qualitative evaluation of GUS reporter gene expression showed intense crosstalk among all hormones studied, underscoring the complexity of signaling induced by Al in apex roots. Results point out to a major understanding of the hormonal signaling in response to Al toxicity, which may induce a change of root growth and architecture with growth inhibition and cell constraints modulated by all different hormones evaluated.

SummaryIron (Fe) is essential for virtually all organisms, being irreplaceable because of its electrochemical properties that enable many biochemical processes, including photosynthesis. Besides its abundance, Fe is generally found in the poorly soluble form of ferric iron (Fe3+), while most plants uptake the soluble form Fe2+. The model angiosperm Arabidopsis thaliana, for example, captures Fe through a mechanism that lowers rhizosphere pH through proton pumping that increases Fe3+ solubility, which is then reduced by a membrane‐bound reductase and transported into the cell by the zinc‐regulated, iron‐regulated transporter‐like protein (ZIP) family protein AtIRT1. ZIP proteins are transmembrane transporters of divalent metals such as Fe2+, Zn2+, Mn2+, and Cd2+. In this work, we investigated the evolution of functional homologs of IRON‐REGULATED TRANSPORTER 1/ZIP in the supergroup Archaeplastida (Viridiplantae + Rhodophyta + Glaucophyta) using 51 genomes of diverse lineages. Our analyses suggest that Fe is acquired through deeply divergent ZIP proteins in land plants and chlorophyte green algae, indicating that Fe2+ uptake by ZIP proteins evolved independently at least twice throughout green plant evolution. Our results indicate that the archetypical IRON‐REGULATED TRANSPORTER (IRT) proteins from angiosperms likely emerged before the origin of land plants during early streptophyte algae terrestrialization, a process that required the evolution of Fe acquisition in terrestrial subaerial settings.

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4 +), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4 + and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N-NH4 + in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4 + transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g-1 root DW h-1). We were able to infer that ScAMT3;3 plays a presumed role in NH4 + source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

AMMONIUM TRANSPORTER/METHYLAMMONIUM PERMEASE/RHESUS (AMT) family members transport ammonium across membranes in all life domains. Plant AMTs can be categorized into AMT1 and AMT2 subfamilies. Functional studies of AMTs, particularly AMT1-type, have been conducted using model plants but little is known about the function of AMTs from crops. Sugarcane (Saccharum spp.) is a major bioenergy crop that requires heavy nitrogen fertilization but depends on a low carbon-footprint for competitive sustainability. Here, we identified and functionally characterized sugarcane ScAMT2;1 by complementing ammonium uptake-defective mutants of Saccharomyces cerevisiae and Arabidopsis thaliana. Reporter gene driven by the ScAMT2;1 promoter in A. thaliana revealed preferential expression in the shoot vasculature and root endodermis/pericycle according to nitrogen availability and source. Arabidopsis quadruple mutant plants expressing ScAMT2;1 driven by the CaMV35S promoter or by a sugarcane endogenous promoter produced significantly more biomass than mutant plants when grown in NH4+ and showed more 15N-ammonium uptake by roots and nitrogen translocation to shoots. In A. thaliana, ScAMT2;1 displayed a Km of 90.17 µM and Vmax of 338.99 µmoles h-1 g-1 root DW. Altogether, our results suggest that ScAMT2;1 is a functional high-affinity ammonium transporter that might contribute to ammonium uptake and presumably to root-to-shoot translocation under high NH4+ conditions.

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.

"Acid soil syndrome" is a worldwide phenomenon characterized by low pH (pH < 5.5), scarce nutrient avail-ability (K+, Ca2+, Mg2+, P), and mineral toxicity such as those caused by soluble aluminium (Al) forms. Regardless of the mineral toxicity, the low pH by itself is detrimental to crop development causing striking sensitivity responses such as root growth arrest. However, low pH-induced responses are still poorly understood and underrated. Here, we review and discuss the core evidence about the action of low pH upon specific root zones, distinct cell types, and possible cellular targets (cell wall, plasma membrane, and alternative oxidase). The role of different players in signaling processes leading to low pH-induced responses, such as the STOP tran-scription factors, the reactive oxygen species (ROS), auxin, ethylene, and components of the antioxidant system, is also addressed. Information at the molecular level is still lacking to link the low pH targets and the subsequent actors that trigger the observed sensitivity responses. Future studies will have to combine genetic tools to identify the signaling processes triggered by low pH, unraveling not only the mechanisms by which low pH affects root cells but also finding new ways to engineer the tolerance of domesticated plants to acidic stress.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato ( Solanum lycopersicum ) obscuravenosa ( obv ) mutant lacks BSEs. Here we identify the obv gene and the causative mutation, a non-synonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a rare polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection. One sentence summary distribution of tomato heterobaric and homobaric leaves is controlled by a single-nucleotide polymorphism in an auxin-related transcription factor

Nitrate uptake in sugarcane roots is regulated at the transcriptional and posttranscriptional levels based on the physiological status of the plant and is likely a determinant mechanism for discrimination against nitrate. Sugarcane (Saccharum spp.) is one of the most suitable energy crops for biofuel feedstock, but the reduced recovery of nitrogen (N) fertilizer by sugarcane roots increases the crop carbon footprint. The low nitrogen use efficiency (NUE) of sugarcane has been associated with the significantly low nitrate uptake, which limits the utilization of the large amount of nitrate available in agricultural soils. To understand the regulation of nitrate uptake in sugarcane roots, we identified the major canonical nitrate transporter genes (NRTs—NITRATE TRANSPORTERS) and then determined their expression profiles in roots under contrasting N conditions. Correlation of gene expression with 15N-nitrate uptake revealed that under N deprivation or inorganic N (ammonium or nitrate) supply in N-sufficient roots, the regulation of ScNRT2.1 and ScNRT3.1 expression is the predominant mechanism for the modulation of the activity of the nitrate high-affinity transport system. Conversely, in N-deficient roots, the induction of ScNRT2.1 and ScNRT3.1 transcription is not correlated with the marked repression of nitrate uptake in response to nitrate resupply or high N provision, which suggested the existence of a posttranscriptional regulatory mechanism. Our findings suggested that high-affinity nitrate uptake is regulated at the transcriptional and presumably at the posttranscriptional levels based on the physiological N status and that the regulation of NRT2.1 and NRT3.1 activity is likely a determinant mechanism for the discrimination against nitrate uptake observed in sugarcane roots, which contributes to the low NUE in this crop species.

Raphidiopsis (=Cylindrospermopsis) raciborskii was described as a subtropical-tropical cyanobacterium, later reported expanding into temperate regions. Heterocyte presence used to distinguish Cylindrospermopsis from the very similar Raphidiopsis, but recently the two genera were recognized as one and unified. This study aimed to investigate how heterocyte production is related to nitrogen (N) limitation in heterocytous and non-heterocytous strains of R.raciborskii. High N-concentrations did not inhibit heterocyte development in some strains, while prolonged N-starvation periods never stimulated production in others. RT-qPCR was used to examine the genetic background, through the expression patterns of nifH, ntcA and hetR. While gene expression increased under N-restriction, N-sufficiency did not suppress nifH transcripts as previously observed in other diazotrophyc cyanobacteria, suggesting that heterocyte production in R. raciborskii is not regulated by N-availability. Heterocytous and non-heterocytous strains were genotypically characterized to assess their phylogenetic relationships. In the phylogenetic tree, clusters were intermixed and confirmed Raphidiopsis and Cylindrospermopsis as the same genus. The tree supported previous findings of earlier splitting of American strains, while contesting the African origin hypothesis. The existence of two lines of Chinese strains, with distinct evolutionary patterns, is a significant addition that could lead to new hypotheses of the species biogeography.

Jatropha curcas plants were cultivated during 4 months in pots containing soil at 90% of field capacity (FC), in permanent drought at 30% of FC and in extreme drought without any irrigation. The permanent drought clearly resulted in significant reductions of 43% on spongy parenchyma thickness and increase of 170% on stomatal index and 80% in photosynthetic pigments content. The measurements of leaf gas showed that growing in permanent drought condition resulted in decreases of 38% on net CO2 assimilation rate, 81% on stomatal conductance, 69% on transpiration, 30% on intercellular carbon concentration (Ci) and reductions on the capacity to refresh leaves. The plants subjected to extreme drought took a period of 12 days of re-irrigation to recover values of CO2 assimilation and stomatal conductance similar to well-irrigated plants. Some of these morphophysiological changes look as though not to directly result from drought, but from secondary variations unleashed by stomatal closure and intensified by long duration of stressful conditions. This seems to result in plants photosynthetically more efficient with competitive advantage in arid situations. Besides these variations, it was possible to show and evaluate the resilience and extreme tolerance of these plants to survive in harsh environment.

Low pH stress (pH < 5.5) triggers sensitivity responses in roots such as cell death (CD) or arrest in elongation which are detrimental to the development of many plant species. The involvement of the cell wall (CW) with these sensitive responses and their spatiotemporal dynamics in the distinct root zones remains poorly understood. The spatio-temporal analysis of primary roots of Arabidopsis thaliana upon low pH (pH 4.6) revealed that the CD repeatedly started in cells of the transition zone (TZ). Then, CD dynamically moved downward to the meristematic zone (MZ) and upward to the early elongation zone (EZ). The dead cells exhibited coordinated in situ DNA fragmentation, highlighted by the deformity in the nuclei of dead cells and positive reaction for Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL). This DNA fragmentation strongly suggests that the low pH-driven CD occurred through a programmed cell death (PCD) pathway. In addition, a decreased CW stiffness in TZ epidermal cells before the onset of CD suggests that low pH first triggers cell wall disturbances (CWDs) in these root cells. Disruption in CW integrity caused by incorrect cellulose deposition in cobra (cob-1) mutant enhanced CD in TZ and early EZ. Supporting this notion, an increase in calcium concentration upon low pH alleviated the CD in roots, probably due to its role in stabilization of the pectin crosslinking in CWs and likely counteracting the low pH-induced CWDs. Moreover, the CD was significantly decreased when roots were exposed to low pH under reduced CW tension, however, a sudden increase in turgor pressure and CW tension (hypoosmotic treatment) combined with low pH accelerated CD in TZ and early EZ. The CD was not observed in the wild type late EZ trichoblasts upon low pH but rather an orchestrated arrest in elongation. An increase in calcium concentration inhibited this elongation arrest upon low pH, suggesting that the onset of this response also required CWDs. Altogether, these results indicate that low pH-induced CWDs triggered sensitive responses within defined root zones. The low pH-activated PCD and an orchestrated elongation arrest occurring in different root zones suggest the occurrence of yet to be identified signaling cascades.

One irreversible consequence of acidic pH for roots is cell death. Growing evidence suggests the role of hormones and cell wall-related enzymes in response to acidic pH that could possibly avoid cell mortality. Here, we have investigated the role of ethylene and class III peroxidases (CIII Prxs) activity on sensitivity to further low pH treatment. Seedlings of Arabidopsis thaliana were pretreated with ethylene, at various concentrations for various times, and then exposed to low pH. In contrast to non-treated roots, roots pretreated with ethylene for 3 h became tolerant to subsequent low pH, with negligible cell mortality in meristematic (MZ), transition (TZ), and early elongation (EZ) zones. This effect of ethylene was time dependent since it was achieved only when seedlings were pre-incubated with ethylene for at least 3 h. This tolerance induced by ethylene was not observed in the gain-of-function mutation etr1-1 (insensitive to ethylene 1–1). Besides, it was prevented by salicylhydroxamic acid (SHAM) which is an inhibitor of CIII Prxs activity. In late EZ, the decrease in cell expansion due to low pH was dependent on both ethylene signaling and a SHAM-sensitive process. The responses mediated by ethylene signaling might involve CIII Prxs-dependent cell wall modifications, leading to tolerance to low pH and arrest in cell expansion during stress.

Background: Coronary artery calcium (CAC) scanning has evolved into an important subclinical prediction method for cardiovascular diseases in asymptomatic subjects. However, the prognostic implication of CAC scanning in symptomatic individuals is less clear. Objectives: To assess the prognostic utility of CAC in predicting risk of major adverse cardiac events (MACE) in stable patients with suspected CAD. Methods: We did a systematic electronic literature search for studies presenting original data in CAC score, and reporting cardiovascular events in stable, symptomatic patients as primary outcome. Primary outcome of the meta-analysis was the occurrence of MACE, a composite of late coronary revascularization, hospitalization for unstable angina or heart failure, nonfatal myocardial infarction, and cardiac death or all-cause mortality. Using random effects models, we pooled relative risk ratios of CAC for MACE, and adjusted hazard ratios (HR) of the associations between different CAC strata (CAC 0–100,100–400, and ≥ 400, versus CAC = 0) and incident MACE. Results: We included 19 observational studies (n = 34,041). In total, 1601 events were analyzed, of which 158 in patients with CAC = 0. The pooled relative risk ratio was 5.71 (95%-CI: 3.98;8.19) for subjects with CAC > 0. The pooled estimate of adjusted HRs demonstrated increasing, positive associations, with the strongest association for CAC > 400 (HR: 4.88; 95%-CI: 2.44;9.27). Conclusions: This meta-analysis demonstrated that increased levels of CAC are strongly and independently associated with increased risk for MACE in stable, symptomatic patients with suspected CAD, showing increasing risk with greater CAC scores. Application of CAC scanning as a prediction method could be useful for a considerable number of such patients.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

Plants are sessile organisms that must perceive and respond to various environmental constraints throughout their life cycle. Among these constraints, drought stress has become the main limiting factor to crop production around the world. Water deprivation is perceived primarily by the roots, which efficiently signal the shoot to trigger drought responses in order to maximize a plant's ability to survive. In this study, the tomato (Solanum lycopersicum L.) mutant procera (pro), with a constitutive response to gibberellin (GA), and its near isogenic line cv. Micro-Tom (MT), were used in reciprocal grafting under well-watered and water stress conditions to evaluate the role of GA signaling in root-to-shoot communication during drought stress. Growth, oxidative stress, gene expression, water relations and hormonal content were measured in order to provide insights into GA-mediated adjustments to water stress. All graft combinations with pro (i.e. pro/pro, MT/pro and pro/MT) prevented the reduction of growth under stress conditions without a reduction in oxidative stress. The increase of oxidative stress was followed by upregulation of SlDREB2, a drought-tolerance related gene, in all drought-stressed plants. Scions harboring the pro mutation tended to increase the abscisic acid (ABA) content, independent of the rootstock. Moreover, the GA sensitivity of the rootstock modulated stomatal conductance and water use efficiency under drought stress, indicating GA and ABA crosstalk in the adjustment of growth and water economy.

Nitrogen (N) is quantitatively the main nutrient required by coffee plants, with acquisition mainly by the roots and mostly exported to coffee beans. Nitrate (NO3-) and ammonium (NH4+) are the most important inorganic sources for N uptake. Several N transporters encoded by different gene families mediate the uptake of these compounds. They have an important role in source preference for N uptake in the root system. In this study, we performed a genome-wide analysis, including in silico expression and phylogenetic analyses of AMT1, AMT2, NRT1/PTR, and NRT2 transporters in the recently sequenced Coffea canephora genome. We analyzed the expression of six selected transporters in Coffea arabica roots submitted to N deficiency. N source preference was also analyzed in C. arabica using isotopes. C. canephora N transporters follow the patterns observed for most eudicots, where each member of the AMT and NRT families has a particular role in N mobilization, and where some of these are modulated by N deficiency. Despite the prevalence of putative nitrate transporters in the Coffea genome, ammonium was the preferential inorganic N source for N-starved C. arabica roots. This data provides an important basis for fundamental and applied studies to depict molecular mechanisms involved in N uptake in coffee trees.

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer ( Tuta absoluta ), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on "in planta-induced transient gene silencing" (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic 'Micro-Tom' tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.