Argonaute Proteins: Why Are They So Important for the Legume-Rhizobia Symbiosis?

Unlike most other land plants, legumes can fulfill their nitrogen needs through the establishment of symbioses with nitrogen-fixing soil bacteria (rhizobia). Through this symbiosis, fixed nitrogen is incorporated into the food chain. Because of this ecological relevance, the genetic mechanisms underlying the establishment of the legume-rhizobia symbiosis (LRS) have been extensively studied over the past decades. During this time, different types of regulators of this symbiosis have been discovered and characterized. A growing number of studies have demonstrated the participation of different types of small RNAs, including microRNAs, in the different stages of this symbiosis. The involvement of small RNAs also indicates that Argonaute (AGO) proteins participate in the regulation of the LRS. However, despite this obvious role, the relevance of AGO proteins in the LRS has been overlooked and understudied. Here, we discuss and hypothesize the likely participation of AGO proteins in the regulation of the different steps that enable the establishment of the LRS. We also briefly review and discuss whether rhizobial symbiosis induces DNA damages in the legume host. Understanding the different levels of LRS regulation could lead to the development of improved nitrogen fixation efficiency to enhance sustainable agriculture, thereby reducing dependence on inorganic fertilizers.

Redox Systemic Signaling and Induced Tolerance Responses During Soybean-Bradyrhizobium japonicum Interaction: Involvement of Nod Factor Receptor and Autoregulation of Nodulation.

The symbiotic relationship between legumes and nitrogen-fixing rhizobia induces local and systemic responses, which ultimately lead to nodule formation. The autoregulation of nodulation (AON) is a systemic mechanism related to innate immunity that controls nodule development and involves different components ranging from hormones, peptides, receptors to small RNAs. Here, we characterized a rapid systemic redox changes induced during soybean-Bradyrhizobium japonicum symbiotic interaction. A transient peak of reactive oxygen species (ROS) generation was found in soybean leaves after 30 min of root inoculation with B. japonicum. The ROS response was accompanied by changes in the redox state of glutathione and by activation of antioxidant enzymes. Moreover, the ROS peak and antioxidant enzyme activation were abolished in leaves by the addition, in either root or leaf, of DPI, an NADPH oxidase inhibitor. Likewise, these systemic redox changes primed the plant increasing its tolerance to photooxidative stress. With the use of non-nodulating nfr5-mutant and hyper-nodulating nark-mutant soybean plants, we subsequently studied the systemic redox changes. The nfr5-mutant lacked the systemic redox changes after inoculation, whereas the nark-mutant showed a similar redox systemic signaling than the wild type plants. However, neither nfr5- nor nark-mutant exhibited tolerance to photooxidative stress condition. Altogether, these results demonstrated that (i) the early redox systemic signaling during symbiotic interaction depends on a Nod factor receptor, and that (ii) the induced tolerance response depends on the AON mechanisms.

Learning control for batch thermal sterilization of canned foods.

A control technique based on Reinforcement Learning is proposed for the thermal sterilization of canned foods. The proposed controller has the objective of ensuring a given degree of sterilization during Heating (by providing a minimum temperature inside the cans during a given time) and then a smooth Cooling, avoiding sudden pressure variations. For this, three automatic control valves are manipulated by the controller: a valve that regulates the admission of steam during Heating, and a valve that regulate the admission of air, together with a bleeder valve, during Cooling. As dynamical models of this kind of processes are too complex and involve many uncertainties, controllers based on learning are proposed. Thus, based on the control objectives and the constraints on input and output variables, the proposed controllers learn the most adequate control actions by looking up a certain matrix that contains the state-action mapping, starting from a preselected state-action space. This state-action matrix is constantly updated based on the performance obtained with the applied control actions. Experimental results at laboratory scale show the advantages of the proposed technique for this kind of processes.

Takagi-Sugeno control of nocturnal temperature in greenhouses using air heating.

A solution to the problem of controlling the minimum temperature in greenhouses using controllers developed from nonlinear models of the system is discussed and applied on a real greenhouse. More precisely, the controllers designed are Takagi-Sugeno type controllers, and the proposed design method is an iterative method based on solving a set of Linear Matrix Inequalities, which ensures stability and performance in closed-loop. The tests in a real greenhouse show that it is possible to design controllers for control of nocturnal temperature that give good performance, and guarantee stability in a wide range of working conditions.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus.

The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A1 (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions.

Pollination Increases Gibberellin Levels in Developing Ovaries of Seeded Varieties of Citrus.

Reproductive and vegetative tissues of the seeded Pineapple cultivars of sweet orange (Citrus sinensis L.) contained the following C-13 hydroxylated gibberellins (GAs): GA53, GA17, GA19, GA20, GA1, GA29, and GA8, as well as GA97, 3-epi-GA1, and several uncharacterized GAs. The inclusion of 3-epi-GA1 as an endogenous substance was based on measurements of the isomerization rates of previously added [2H2]GA1. Pollination enhanced amounts of GA19, GA20, GA29, and GA8 in developing ovaries. Levels of GA1 increased from 5.0 to 9.5 ng/g dry weight during anthesis and were reduced thereafter. The amount of GA in mature pollen was very low. Emasculation reduced GA levels and caused a rapid 100% ovary abscission. This effect was partially counteracted by either pollination or application of GA3. In pollinated ovaries, repeated paclobutrazol applications decreased the amount of GA and increased ovary abscission, although the pattern of continuous decline was different from the sudden abscission induced by emasculation. The above results indicate that, in citrus, pollination increases GA levels and reduces ovary abscission and that the presence of exogenous GA3 in unpollinated ovaries also suppresses abscission. Evidence is also presented that pollination and GAs do not, as is generally assumed, suppress ovary abscission through the reactivation of cell division.

Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots.

The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

Cellular changes induced by exogenous and endogenous gibberellins in shoot tips of the long-day plant Silene armeria.

Stem elongation and flowering are two processes induced by long-day (LD) treatment in Silene armeria L. Whereas photoperiodic control of stem growth is mediated by gibberellins (GAs), the flowering response cannot be obtained by GA applications. Microscopic observations on early cellular changes in the shoot meristem following LD induction or GA treatment in short days (SD) were combined with GA analyses of stem sections at various distances below the shoot apex. The earliest effects of both LD and GA induction on the subapical meristem were an increase in the number of cells per cell file and a reduction of cell length in the meristematic tissue approx. 1.0-3.0 mm below the shoot apex. Within 8 d after the beginning of LD induction or after GA application, the cells in the subapical meristem were oriented in long files. In induced tips, cellulose deposition occurred mostly in longitudinal walls, indicating that many transverse cell divisions had taken place which, in turn, increased the length of the stem. In contrast to LD induction, GA treatments did not promote the transition from the vegetative to the floral stage. Endogenous GAs were analyzed by selected ion monitoring (SIM), using labeled internal standards, in extracts from transverse sections of the tip at various distances below the apical meristem. In control plants, the levels of the six 13-hydroxy GAs studied (GA53, GA44, GA19, GA20, GA1, and GA8) decreased as the distance from the apical meristem increased. Except for GA53, GA levels were higher in tips of LD-induced plants, particularly in the meristematic zone approx. 0.5-1.5 mm below the apical meristem. In comparison with SD, the highest increase observed was for GA1, the content of which increased 30-fold in the zone 0.5-3.5 mm below the shoot apex. These data indicate a spatial correlation between the accumulation of GA1 and its precursors, and the enhanced mitotic activity which occurs in the subapical meristem of elongating Silene apices.