Diverging cell wall strategies for drought adaptation in two maize inbreds with contrasting lodging resistance.

The plant cell wall is a plastic structure of variable composition that constitutes the first line of defence against environmental challenges. Lodging and drought are two stressful conditions that severely impact maize yield. In a previous work, we characterised the cell walls of two maize inbreds, EA2024 (susceptible) and B73 (resistant) to stalk lodging. Here, we show that drought induces distinct phenotypical, physiological, cell wall, and transcriptional changes in the two inbreds, with B73 exhibiting lower tolerance to this stress than EA2024. In control conditions, EA2024 stalks had higher levels of cellulose, uronic acids and p-coumarate than B73. However, upon drought EA2024 displayed increased levels of arabinose-enriched polymers, such as pectin-arabinans and arabinogalactan proteins, and a decreased lignin content. By contrast, B73 displayed a deeper rearrangement of cell walls upon drought, including modifications in lignin composition (increased S subunits and S/G ratio; decreased H subunits) and an increase of uronic acids. Drought induced more substantial changes in gene expression in B73 compared to EA2024, particularly in cell wall-related genes, that were modulated in an inbred-specific manner. Transcription factor enrichment assays unveiled inbred-specific regulatory networks coordinating cell wall genes expression. Altogether, these findings reveal that B73 and EA2024 inbreds, with opposite stalk-lodging phenotypes, undertake different cell wall modification strategies in response to drought. We propose that the specific cell wall composition conferring lodging resistance to B73, compromises its cell wall plasticity, and renders this inbred more susceptible to drought.

A novel pectin methylesterase inhibitor, PMEI3, in common bean suggests a key role of pectin methylesterification in Pseudomonas resistance.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.

Immune Priming Triggers Cell Wall Remodeling and Increased Resistance to Halo Blight Disease in Common Bean.

The cell wall (CW) is a dynamic structure extensively remodeled during plant growth and under stress conditions, however little is known about its roles during the immune system priming, especially in crops. In order to shed light on such a process, we used the Phaseolus vulgaris-Pseudomonas syringae (Pph) pathosystem and the immune priming capacity of 2,6-dichloroisonicotinic acid (INA). In the first instance we confirmed that INA-pretreated plants were more resistant to Pph, which was in line with the enhanced production of H2O2 of the primed plants after elicitation with the peptide flg22. Thereafter, CWs from plants subjected to the different treatments (non- or Pph-inoculated on non- or INA-pretreated plants) were isolated to study their composition and properties. As a result, the Pph inoculation modified the bean CW to some extent, mostly the pectic component, but the CW was as vulnerable to enzymatic hydrolysis as in the case of non-inoculated plants. By contrast, the INA priming triggered a pronounced CW remodeling, both on the cellulosic and non-cellulosic polysaccharides, and CW proteins, which resulted in a CW that was more resistant to enzymatic hydrolysis. In conclusion, the increased bean resistance against Pph produced by INA priming can be explained, at least partially, by a drastic CW remodeling.

Perception and First Defense Responses Against Pseudomonas syringae pv. phaseolicola in Phaseolus vulgaris: Identification of Wall-Associated Kinase Receptors.

Common bean (Phaseolus vulgaris) is attacked by several pathogens such as the biotrophic gamma-proteobacterium Pseudomonas syringae pv. phaseolicola. To study the P. syringae pv. phaseolicola-bean interaction during the first stages of infection, leaf discs of a susceptible bean cultivar Riñón were infected with pathogenic P. syringae pv. phaseolicola. Using this experimental system, we tested six new putative wall-associated kinase (WAK) receptors, previously identified in silico. These six P. vulgaris WAKs (PvWAKs) showed high protein sequence homology to the well-described Arabidopsis thaliana WAK1 (AtWAK1) receptor and, by phylogenetic analysis, clustered together with AtWAKs. The expression of PvWAK1 increased at very early stages after the P. syringae pv. phaseolicola infection. Time course experiments were performed to evaluate the accumulation of apoplastic H2O2, Ca2+ influx, total H2O2, antioxidant enzymatic activities, lipid peroxidation, and the concentrations of abscisic acid and salicylic acid (SA), as well as the expression of six defense-related genes: MEKK-1, MAPKK, WRKY33, RIN4, PR1, and NPR1. The results showed that overexpression of PR1 occurred 2 h after P. syringae pv. phaseolicola infection without a concomitant increase in SA levels. Although apoplastic H2O2 increased after infection, the oxidative burst was neither intense nor rapid, and an efficient antioxidant response did not occur, suggesting that the observed cellular damage was caused by the initial increase in total H2O2 early after infection. In conclusion, Riñón can perceive the presence of P. syringae pv. phaseolicola, but this recognition results in only a modest and slow activation of host defenses, leading to high susceptibility to P. syringae pv. phaseolicola.

Elucidating compositional factors of maize cell walls contributing to stalk strength and lodging resistance.

Lodging is one of the causes of maize (Zea mays L.) production losses worldwide and, at least, the resistance to stalk lodging has been positively correlated with stalk strength. In order to elucidate the putative relationship between cell wall, stalk strength and lodging resistance, twelve maize inbreds varying in rind penetration strength and lodging resistance were characterized for cell wall composition and structure. Stepwise multiple regression indicates that H lignin subunits confer a greater rind penetration strength. Besides, the predictive model for lodging showed that a high ferulic acid content increases the resistance to lodging, whereas those of diferulates decrease it. These outcomes highlight that the strength and lodging susceptibility of maize stems may be conditioned by structural features of cell wall rather than by the net amount of cellulose, hemicelluloses and lignin. The results presented here provide biotechnological targets in breeding programs aimed at improving lodging in maize.

In Vitro Plant Regeneration in Conifers: The Role of WOX and KNOX Gene Families.

Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster.

The role of cell wall phenolics during the early remodelling of cellulose-deficient maize cells.

The habituation of cultured cells to cellulose biosynthesis inhibitors such as dichlobenil (dichlorobenzonitrile, DCB) has proven a valuable tool to elucidate the mechanisms involved in plant cell wall structural plasticity. Our group has demonstrated that maize cells cope with DCB through a modified cell wall in which cellulose is replaced by a more extensive network of highly cross-linked feruloylated arabinoxylans. In order to gain further insight into the contribution of phenolics to the early remodelling of cellulose-deficient cell walls, a comparative HPLC-PAD analysis was carried out of hydroxycinnamates esterified into nascent and cell wall polysaccharides obtained from non-habituated (NH) and habituated to low DCB concentrations (1.5 µM; H) maize suspension-cultured cells. Incipient DCB-habituated cell walls showed significantly higher levels of esterified ferulic acid and p-coumaric acid throughout the culture cycle. In terms of cell wall fortification, ferulic acid is associated to arabinoxylan crosslinking whereas the increase of p-coumaric suggests an early lignification response. As expected, the level of hydroxycinnamates esterified into nascent polysaccharides was also higher in DCB-habituated cells indicating an overexpression of phenylpropanoid pathway. Due to their key role in cell wall strengthening, special attention was paid into the dimerization pattern of ferulic acid. A quantitative comparison of diferulate dehydrodimers (DFAs) between cell lines and cell compartments revealed that an extra dimerization took place in H cells when both nascent and mature cell wall polysaccharides were analysed. In addition, qualitative differences in the ferulic acid coupling pattern were detected in H cells, allowing us to suggest that 8-O-4'-DFA and 8-5'-DFA featured the ferulic acid dimerization when it occurred in the protoplasmic and cell wall fractions respectively. Both qualitative and quantitative differences in the phenolic profile between NH and H cells point to a regioselectivity in the ferulate dehydrodimerization.

Determination of abscisic acid and its glucosyl ester in embryogenic callus cultures of Vitis vinifera in relation to the maturation of somatic embryos using a new liquid chromatography-ELISA analysis method.

The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4'-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC-ESI-MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation.

Rapid responses of C14 clone of Eucalyptus globulus to root drought stress: Time-course of hormonal and physiological signaling.

The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.

Caulogenic induction in cotyledons of stone pine (Pinus pinea): relationship between organogenic response and benzyladenine trends in selected families.

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.

Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro.

Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium.

Hormonal changes throughout maturation and ageing in Pinus pinea.

Phytohormones, which are responsible for certain age-related changes in plants, play a major role throughout maturation and ageing. Previous results dealing with this topic allowed us to describe an ageing and vigour index in Pinus radiata based on a ratio between different forms of cytokinins (Cks). The aim of the present study was to extend the studies on the changes in the hormonal status throughout maturation and ageing to Stone pine (Pinus pinea L.). With this aim in mind, a number of Cks were analysed in addition to indole-3-acetic acid (IAA) and abscisic acid (ABA) in terminal buds, axillary buds and in the apical portion of needles collected from trees at different stages of development. The results showed an increasing pattern in the levels of various Cks similar to that found in previous studies on P. radiata. Although the maintenance of the same ratio as an ageing and vigour index was not ratified, these results seem to point to Cks as major hormones throughout maturation and related processes in conifers. The distribution of hormones between the two parts of the needle is also discussed.

Alterations in endogenous levels of cytokinins following grafting of Pinus radiata support ratio of cytokinins as an index of ageing and vigour.

Markers for ageing or maturation in woody plants provides the possibility of identifying genotypes with a prolonged juvenile phase, or to evaluate reinvigoration procedures, such as grafting of adult material. This study focuses specifically on cytokinins (Cks) as markers, due to their role in the transition process from juvenile to adult states. Previous reports from our group disclosed that the ratio of isopentenyladenine-type (iP-type) to zeatin-type (Z-type) Cks decreases during tree maturation, a maturation and ageing index being likely in Pinus radiata. Based on these results, Cks were tested in terminal buds of adult and grafted adult P. radiata material to corroborate the importance of the aforementioned balance as an index in the reinvigoration process of adult trees associated with grafting procedures. Results revealed the reversion of this index pattern parallel to the reactivation of some juvenile traits, namely, increasing values of the ratio throughout the reinvigoration process. According to previous results, however, the opposite was the case during the reverse process. This fact allowed us to validate the iP-type/Z-type Ck ratio as an ageing and vigour index.