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.

Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads.

Co-immunoprecipitation (Co-IP) is a widely used and powerful approach for studying protein-protein interactions in vivo. Here, we describe a protocol for antibody purification and immobilization followed by immunoprecipitation from plant tissue extracts using magnetic beads. The protocol has been used to detect regulators in the Zea mays phenylpropanoid pathway. The protocol is amenable to a variety of downstream assays, including western blotting and mass spectrometry. For complete details on the use and execution of this protocol, please refer to Vélez-Bermúdez et al. (2015).

Expression Characterization of Flavonoid Biosynthetic Pathway Genes and Transcription Factors in Peanut Under Water Deficit Conditions.

Drought is one of the hostile environmental stresses that limit the yield production of crop plants by modulating their growth and development. Peanut (Arachis hypogaea) has a wide range of adaptations to arid and semi-arid climates, but its yield is prone to loss due to drought. Other than beneficial fatty acids and micronutrients, peanut harbors various bioactive compounds including flavonoids that hold a prominent position as antioxidants in plants and protect them from oxidative stress. In this study, understanding of the biosynthesis of flavonoids in peanut under water deficit conditions was developed through expression analysis and correlational analysis and determining the accumulation pattern of phenols, flavonols, and anthocyanins. Six peanut varieties (BARD479, BARI2011, BARI2000, GOLDEN, PG1102, and PG1265) having variable responses against drought stress have been selected. Higher water retention and flavonoid accumulation have been observed in BARI2011 but downregulation has been observed in the expression of genes and transcription factors (TFs) which indicated the maintenance of normal homeostasis. ANOVA revealed that the expression of flavonoid genes and TFs is highly dependent upon the genotype of peanut in a spatiotemporal manner. Correlation analysis between expression of flavonoid biosynthetic genes and TFs indicated the role of AhMYB111 and AhMYB7 as an inhibitor for AhF3H and AhFLS, respectively, and AhMYB7, AhTTG1, and AhCSU2 as a positive regulator for the expression of Ah4CL, AhCHS, and AhF3H, respectively. However, AhbHLH and AhGL3 revealed nil-to-little relation with the expression of flavonoid biosynthetic pathway genes. Correlational analysis between the expression of TFs related to the biosynthesis of flavonoids and the accumulation of phenolics, flavonols, and anthocyanins indicated coregulation of flavonoid synthesis by TFs under water deficit conditions in peanut. This study would provide insight into the role of flavonoid biosynthetic pathway in drought response in peanut and would aid to develop drought-tolerant varieties of peanut.

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 seedlings of Pinus radiata, jasmonic acid and auxin are differentially distributed on opposite sides of tilted stems affecting lignin monomer biosynthesis and composition.

Plants respond to the loss of vertical growth re-orientating their affected organs. In trees, this phenomenon has received the scientific attention due to its importance for the forestry industry. Nowadays it is accepted that auxin distribution is involved in the modulation of the tilting response, but how this distribution is controlled is not fully clear. Auxin transporters that determine the spatio-temporal auxin distribution in radiate pine seedlings exposed to 45° of tilting were identified. Additionally, based on indications for an intimate plant hormone crosstalk in this process, IAA and JA contents were evaluated. The experiments revealed that expression of the auxin transporters was down-regulated in the upper half of the tilted stem, while being induced in the lower half. Moreover, transporter-coding genes were first induced at the apical zone of the stem. IAA was consistently redistributed toward the lower half, which is in accordance with the expression profile of the auxin transporters. In contrast, JA was mainly accumulated in the upper half of tilted stems. Finally, lignin content and monomeric composition were analyzed in both sides of stem and along the time course of tilting. As expected, lignin accumulation was higher at the lower half of stem at longer times of tilting. However, the most marked difference was the accumulation of the H-lignin monomer in the lower half, while the G-lignin unit was more dominant in the upper half. Here, we provide detailed insight in the distribution of IAA and JA, affecting the lignin composition during the tilting response in Pinus radiata seedlings.

Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock.

Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail.

Changes in Cell Wall Polymers and Degradability in Maize Mutants Lacking 3'- and 5'-O-Methyltransferases Involved in Lignin Biosynthesis.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) and caffeic acid-O-methyltransferase (COMT) are key enzymes in the biosynthesis of coniferyl and sinapyl alcohols, the precursors of guaiacyl (G) and syringyl (S) lignin subunits. The function of these enzymes was characterized in single and double mutant maize plants. In this work, we determined that the comt (brown-midrib 3) mutant plants display a reduction of the flavonolignin unit derived from tricin (a dimethylated flavone), demonstrating that COMT is a key enzyme involved in the synthesis of this compound. In contrast, the ccoaomt1 mutants display a wild-type amount of tricin, suggesting that CCoAOMT1 is not essential for the synthesis of this compound. Based on our data, we suggest that CCoAOMT1 is involved in lignin biosynthesis at least in midribs. The phenotype of ccoaomt1 mutant plants displays no alterations, and their lignin content and composition remain unchanged. On the other hand, the ccoaomt1 comt mutant displays phenotypic and lignin alterations similar to those already described for the comt mutant. Although stems from the three mutants display a similar increase of hemicelluloses, the effect on cell wall degradability varies, the cell walls of ccoaomt1 being the most degradable. This suggests that the positive effect of lignin reduction on cell wall degradability of comt and ccoaomt1 comt mutants is counteracted by changes occurring in lignin composition, such as the decreased S/G ratio. In addition, the role of the flavonolignin unit derived from tricin in cell wall degradability is also discussed.

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize.

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes.

Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.

Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves.

AtMYB7, a new player in the regulation of UV-sunscreens in Arabidopsis thaliana.

The phenylpropanoid metabolic pathway provides a wide variety of essential compounds for plants. Together with sinapate esters, in Brassicaceae species, flavonoids play an important role in protecting plants against UV irradiation. In this work we have characterized Arabidopsis thaliana AtMYB7, the closest homolog of AtMYB4 and AtMYB32, described as repressors of different branches of phenylpropanoid metabolism. The characterization of atmyb7 plants revealed an induction of several genes involved in flavonol biosynthesis and an increased amount of these compounds. In addition, AtMYB7 gene expression is repressed by AtMYB4. As a consequence, the atmyb4 mutant plants present a reduction of flavonol contents, indicating once more that AtMYB7 represses flavonol biosynthesis. Our results also show that AtMYB7 gene expression is induced by salt stress. Induction assays indicated that AtMYB7 represses several genes of the flavonoid pathway, DFR and UGT being early targets of this transcription factor. The results obtained indicate that AtMYB7 is a repressor of flavonol biosynthesis and also led us to propose AtMYB4 and AtMYB7 as part of the regulatory mechanism controlling the balance of the main A. thaliana UV-sunscreens.

Grass phenylpropanoids: regulate before using!

The phenylpropanoid pathway is responsible for the synthesis of lignin as well as a large number of compounds of fundamental importance for the biology of plants. Over the years, important knowledge has accumulated on how dicotyledoneous plants control various branches of phenylpropanoid accumulation, but comparable information on the grasses is lagging significantly behind. In addition to playing fundamental roles in biotic and abiotic interactions, phenylpropanoids in the grasses play a very important function in the reinforcement of cell wall components. Understanding how phenylpropanoid metabolism is controlled in the grasses has been complicated by recent genome duplications, the difficulties in making transgenic plants and the absence of mutants in many genes. Recent studies in a particular subgroup of R2R3-MYB transcription factors suggest that they might play a central role in regulating a small set of phenylpropanoid genes, opening the door for the identification of other related regulators, and perhaps also finding out which combinations of biosynthesis genes function in particular cell types for the formation of specific compounds. This information will be essential for the rational metabolic engineering of this pathway, either to increase biomass or decrease phenolic accumulation for better accessibility of polysaccharides for forage quality and biofuel production.

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Deepening into the proteome of maize cells habituated to the cellulose biosynthesis inhibitor dichlobenil.

Cellulose biosynthesis inhibitors, such as dichlobenil (DCB), have become a valuable tool for the analysis of structural and compositional plasticity of plant cell walls. By stepwise increasing the concentration of DCB in the culture medium, we obtained maize cells able to cope with DCB through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of feruloylated arabinoxylans. Recently we demonstrated that the expression of several Cellulose Synthase and phenylpropanoid-related genes is altered in DCB-habituated cells. In addition, by using a proteomic approach we identified several proteins induced or repressed in DCB-habituated cells. After a more in-depth analysis, some new proteins induced (two inhibitors TAXI-IV, an α-1,4-glucan-protein synthase, and a pectinesterase inhibitor) or repressed (a chaperonin 60, a fructokinase-1 and a spermidine synthase 1) were identified, and their possible role in the context of DCB-habituation is discussed.

ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux.

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.

Unraveling the biochemical and molecular networks involved in maize cell habituation to the cellulose biosynthesis inhibitor dichlobenil.

The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil (DCB) were investigated. DCB affects the synthesis of cellulose both in active and stationary growth phases and alters the expression of several CesA genes. Of these, ZmCesA5 and ZmCesA7 seem to play a major role in habituating cells to growth in the presence of DCB. As a consequence of the reduction in cellulose, the expression of several genes involved in the synthesis of hydroxycinnamates is increased, resulting in cell walls with higher levels of ferulic and p-coumaric acids. A proteomic analysis revealed that habituation to DCB is linked to modifications in several metabolic pathways. Finally, habituated cells present a reduction in glutathione S-transferase detoxifying activity and antioxidant activities. Plant cell adaptation to the disturbance of such a crucial process as cellulose biosynthesis requires changes in several metabolic networks, in order to modify cell wall architecture and metabolism, and survive in the presence of the inhibitor. Some of these modifications are described in this paper.

Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling.

Intracellular redox status is a critical parameter determining plant development in response to biotic and abiotic stress. Thioredoxin (TRX) and glutathione are key regulators of redox homeostasis, and the TRX and glutathione pathways are essential for postembryonic meristematic activities. Here, we show by associating TRX reductases (ntra ntrb) and glutathione biosynthesis (cad2) mutations that these two thiol reduction pathways interfere with developmental processes through modulation of auxin signaling. The triple ntra ntrb cad2 mutant develops normally at the rosette stage, undergoes the floral transition, but produces almost naked stems, reminiscent of the phenotype of several mutants affected in auxin transport or biosynthesis. In addition, the ntra ntrb cad2 mutant shows a loss of apical dominance, vasculature defects, and reduced secondary root production, several phenotypes tightly regulated by auxin. We further show that auxin transport capacities and auxin levels are perturbed in the mutant, suggesting that the NTR-glutathione pathways alter both auxin transport and metabolism. Analysis of ntr and glutathione biosynthesis mutants suggests that glutathione homeostasis plays a major role in auxin transport as both NTR and glutathione pathways are involved in auxin homeostasis.

The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana.

The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway.

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana.

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.

Down-regulation of the maize and Arabidopsis thaliana caffeic acid O-methyl-transferase genes by two new maize R2R3-MYB transcription factors.

The maize (Zea mays L.) caffeic acid O-methyl-transferase (COMT) is a key enzyme in the biosynthesis of lignin. In this work we have characterized the involvement of COMT in the lignification process through the study of the molecular mechanisms involved in its regulation. The examination of the maize COMT gene promoter revealed a putative ACIII box, typically recognized by R2R3-MYB transcription factors. We used the sequence of known R2R3-MYB factors to isolate five maize R2R3-MYB factors (ZmMYB2, ZmMYB8, ZmMYB31, ZmMYB39, and ZmMYB42) and study their possible roles as regulators of the maize COMT gene. The factors ZmMYB8, ZmMY31, and ZmMYB42 belong to the subgroup 4 of the R2R3-MYB family along with other factors associated with lignin biosynthesis repression. In addition, the induction pattern of ZmMYB31 and ZmMYB42 gene expression on wounding is that expected for repressors of the maize COMT gene. Arabidopsis thaliana plants over-expressing ZmMYB31 and ZmMYB42 down-regulate both the A. thaliana and the maize COMT genes. Furthermore, the over-expression of ZmMYB31 and ZmMYB42 also affect the expression of other genes of the lignin pathway and produces a decrease in lignin content of the transgenic plants.

AtPng1p. The first plant transglutaminase.

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.