Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H2O2, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date.

/pdf/lignins-natural-polymers-from-oxidative-coupling-of-4-oxcvb84q0y.pdf

Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Senescence is a complex, highly regulated, developmental phase in the life of a leaf that results in the co-ordinated degradation of macromolecules and the subsequent mobilization of components to other parts of the plant. The application of molecular biology techniques to the study of leaf senescence has, in the last few years, enabled the isolation and characterization of a large range of cDNA clones representing genes that show increased expression in senescing leaves. The analysis of these genes and identification of the function of the encoded proteins will allow a picture of the complex processes that take place during senescence to be assembled. To date, genes encoding degradative enzymes such as proteases and nucleases, enzymes involved in lipid and carbohydrate metabolism and enzymes involved in nitrogen mobilization have all been identified as senescenceenhanced genes. A variety of other genes of no obvious senescence-related function have also been identified; their role in senescence may be less predictable and, possibly, more interesting. The combined action of several internal and external signals may be involved in the induction of senescence. Analysis of the regulatory mechanisms controlling the expression of senescence-induced genes will allow the signalling pathways that are involved in the regulation of senescence to be elucidated. Experiments with transgenic plants and mutants are already shedding light on the role played by cytokinins and ethylene in regulating senescence in leaves.

The molecular biology of leaf senescence

Plant peroxidases (class III peroxidases) are present in all land plants. They are members of a large multigenic family. Probably due to this high number of isoforms, and to a very heterogeneous regulation of their expression, plant peroxidases are involved in a broad range of physiological processes all along the plant life cycle. Due to two possible catalytic cycles, peroxidative and hydroxylic, peroxidases can generate reactive oxygen species (ROS) (•OH, HOO•), polymerise cell wall compounds, and regulate H2O2 levels. By modulating their activity and expression following internal and external stimuli, peroxidases are prevalent at every stage of plant growth, including the demands that the plant meets in stressful conditions. These multifunctional enzymes can build a rigid wall or produce ROS to make it more flexible; they can prevent biological and chemical attacks by raising physical barriers or by counterattacking with a large production of ROS; they can be involved in a more peaceful symbiosis. They are finally present from the first hours of a plant’s life until its last moments. Although some functions look paradoxical, the whole process is probably regulated by a fine-tuning that has yet to be elucidated. This review will discuss the factors that can influence this delicate balance.

/pdf/peroxidases-have-more-functions-than-a-swiss-army-knife-49s5dfpkwk.pdf

Peroxidases have more functions than a Swiss army knife

The plant cuticle is an extracellular hydrophobic layer that covers the aerial epidermis of all land plants, providing protection against desiccation and external environmental stresses. The past decade has seen considerable progress in assembling models for the biosynthesis of its two major components, the polymer cutin and cuticular waxes. Most recently, two breakthroughs in the long-sought molecular bases of alkane formation and polyester synthesis have allowed construction of nearly complete biosynthetic pathways for both waxes and cutin. Concurrently, a complex regulatory network controlling the synthesis of the cuticle is emerging. It has also become clear that the physiological role of the cuticle extends well beyond its primary function as a transpiration barrier, playing important roles in processes ranging from development to interaction with microbes. Here, we review recent progress in the biochemistry and molecular biology of cuticle synthesis and function and highlight some of the major questions that will drive future research in this field.

/pdf/the-formation-and-function-of-plant-cuticles-1f70fxx5eo.pdf

The Formation and Function of Plant Cuticles

3Core Research for Evolutional Science and Technology (CREST), Chiyoda-ku, Tokyo, 101-0062 Japan ; Class III plant peroxidase (POX), a plant-specific oxidoreductase, is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms. POXs exist as isoenzymes in individual plant species, and each isoenzyme has variable amino acid sequences and shows diverse expression profiles, suggesting their involvement in various physiological processes. Indeed, studies have provided evidence that POXs participate in lignification, suberization, auxin catabolism, wound healing and defense against pathogen infection. Little, however, is known about the signal transduction for inducing expression of the pox genes. Recent studies have provided information on the regulatory mechanisms of wound- and pathogen-induced expression of some pox genes. These studies suggest that pox genes are induced via different signal transduction pathways from those of other known defense-related genes.

/pdf/a-large-family-of-class-iii-plant-peroxidases-1plxqcbkk0.pdf

A large family of class III plant peroxidases.

The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.

/pdf/a-tomato-peroxidase-involved-in-the-synthesis-of-lignin-and-3493eqronk.pdf

A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin

A cDNA clone encoding a daylily (Hemerocallis spp.) thiolprotease (SEN11), whose expression is strongly up-regulated in flower tepal senescence, has been isolated. The amino acid sequence, deduced from the nucleotide sequence, showed highest similarity to plant thiolproteases of Vigna mungo, Phaseolus vulgaris and Hemerocallis (SEN102), and contains a putative ER retention signal that has been described in Vigna mungo. SEN102 and SEN11 transcripts were not detectable in flower buds at the opening stage, but two peaks of transcripts were seen after 9 h and 19 h, in both petals and sepals, when wilting symptoms were apparent. The pattern of protease activity migrating on a 26.3 kDa protein was similar to the SEN102 and SEN11 transcript profiles. These two genes were also expressed in stamens and leaves, but their transcripts were undetectable in carpels and rhizomes. The expression of SEN102 was lower in the senescent leaf than in the green leaf. The pattern of expression of these genes suggests their involvement in the protein hydrolysis occurring in tepals at the late senescence stage, whereas in leaves they could be involved in the constitutive protein turnover machinery. Exogenous gibberellic acid application to cut flowers increased transcripts of both genes.

/pdf/analysis-of-the-expression-of-two-thiolprotease-genes-from-1l5pk109cr.pdf

Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence

The flowers of daylily (Hemerocallis x hybrida cv. Cradle Song) open at midnight, start to senesce 12 h later, and are completely senescent by the following midnight. Differential screening of a cDNA library constructed from tepals of flowers showing incipient senescence revealed 25 clones that were strongly up-regulated in senescent tepals. Re-screening and interactive Southern analysis of these clones revealed 3 families of up-regulated clones. Transcripts of one clone, SEN10, were not detectable at midnight, but increased dramatically as senescence proceeded. The derived amino acid sequence of the full-length cDNA (SEN102) has strong homology with cysteine proteases that have been reported from other plant tissues. The sequence contains a secretory signal peptide and a probable prosequence upstream of the mature protein. Amino acids critical to the active site and structure of cysteine proteases are conserved, and the C-terminus of the polypeptide has a unique putative endoplasmic reticulum retention signal -RDEL.

http://ucce.ucdavis.edu/files/datastore/234-936.pdf

Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers.

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato (Lycopersicon esculentum) leaves for 6 h to 12 g l -1 NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

GDSL and SGNH hydrolases are lipases involved in a wide range of functions, behaving in many cases as bifunctional enzymes. In this work, the isolation and characterization of AgaSGNH, a cDNA encoding a member of the SGNH-hydrolase superfamily from young leaf epidermis of the monocot Agave americana L., is reported. The protein possesses a typical signal peptide at its N-terminus that allows its secretion to the epidermis cell wall, as verified by immunolocalization experiments. In addition, the AgaSGNH sequence contains a His-Leu-Gly-Ala-Glu (HLGAE) motif which is similar to that observed in other plant acyltransferases. Expression levels by northern blot and in situ localization of the corresponding mRNA, as well as the immunolocalization of the protein in Agave young leaves indicate that the protein is specifically present in the epidermal cells. The detailed study performed in different parts of the Agave leaf confirms two aspects: first, the expression of AgaSGNH is limited to the epidermis, and second, the maximum mRNA levels are found in the epidermis of the youngest zones of the leaf which are especially active in cutin biosynthesis. These levels dramatically decrease in the oldest zone of the leaf, where the presence of AgaSGNH mRNA is undetectable, and the biosynthesis of different cuticle components is severely reduced. These data could be compatible with the hypothesis that AgaSGNH could carry out both the hydrolysis and the transfer, from an activated acyl-CoA to a crescent cutin in Agave americana leaves and, therefore, be involved in the still unknown mechanism of plant cutin biosynthesis.

Consuelo Guerrero

Papers

A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin

Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence

Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers.

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Isolation, characterization, and localization of AgaSGNH cDNA: a new SGNH-motif plant hydrolase specific to Agave americana L. leaf epidermis