Correction to "Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics".

[This corrects the article DOI: 10.1021/acssuschemeng.2c01160.].

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor.

BACKGROUND: The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. RESULTS: We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. CONCLUSIONS: Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls.

A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants.

The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.

Identification and characterization of an epi-allele of FIE1 reveals a regulatory linkage between two epigenetic marks in rice.

DNA methylation and histone H3 Lys 9 dimethylation (H3K9me2) are important epigenetic repression marks for silencing transposons in heterochromatin and for regulating gene expression. However, the mechanistic relationship to other repressive marks, such as histone H3 Lys 27 trimethylation (H3K27me3) is unclear. FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1) encodes an Esc-like core component of the Polycomb repressive complex 2, which is involved in H3K27me3-mediated gene repression. Here, we identify a gain-of-function epi-allele (Epi-df) of rice (Oryza sativa) FIE1; this allele causes a dwarf stature and various floral defects that are inherited in a dominant fashion. We found that Epi-df has no changes in nucleotide sequence but is hypomethylated in the 5' region of FIE1 and has reduced H3K9me2 and increased H3K4me3. In Epi-df, FIE1 was ectopically expressed and its imprinting was disrupted. FIE1 interacted with rice Enhancer of Zeste homologs, consistent with its role in H3K27me3 repression. Ectopic expression of FIE1 in Epi-df resulted in alteration of H3K27me3 levels in hundreds of genes. In summary, this work identifies an epi-allele involved in H3K27me3-mediated gene repression that itself is highly regulated by DNA methylation and histone H3K9me2, thereby shedding light on the link between DNA methylation and histone methylation, the two important epigenetic marks regulating rice development.

Brassinosteroids regulate grain filling in rice.

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO(2) assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.

Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors.

Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture.

Expression of human nuclear receptors in plants for the discovery of plant-derived ligands.

Plants have the potential to produce a wide array of secondary metabolites that have utility as drugs to treat human diseases. To tap this potential, functional human nuclear receptors have been expressed in plants to create in planta screening assays as a tool to discover natural product ligands. Assays have been designed and validated using 3 nuclear receptors: the estrogen receptor (ER), the androgen receptor (AR), and the heterodimeric retinoid X receptor-alpha plus thyroid hormone receptor-beta (RXRA/THRB). Nuclear receptor-reporter constructs have been expressed in plants to detect the presence of natural ligands that are produced de novo in several plant species during different stages of development, in various tissues, and in response to different stress elicitors. Screening experiments with ER, AR, and RXRA/THRB have been conducted, leading to the identification of plant sources of natural product ligands of human nuclear receptors. This in planta screen has led to the identification of previously unreported ER ligands, providing evidence of the complementary value of this approach to current in vitro high-throughput screening assays.

Association of interleukin-10 A-592C and T-819C polymorphisms with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is the most common form of diabetes. The association of low interleukin (IL)-10 production capacity with the metabolic syndrome and T2DM was recently established. Approximately 75% of the variation in IL-10 secretion capacity in humans derives from genetic factors that contribute to disease susceptibility. Based on the facts that IL-10 secretion ability is tightly controlled at the transcription level and the low production capacity of IL-10 is associated with T2DM, it seemed tempting to investigate if polymorphisms in the IL-10 gene promoter contribute to T2DM. IL-10 promoter polymorphisms at positions -592 and -819 among 370 consecutive patients with T2DM seen at the Department of Internal Medicine, Chung Shan Medical University Hospital, were examined using polymerase chain reaction restriction fragment length polymorphism. Though no significant association was detected between either the A-592C (p=0.088) or T-819C (p=0.160) polymorphism and T2DM, significantly more T2DM subjects carried -592*C (34.28%, p=0.027) and -819*C (32.57%, p<0.001) alleles, which were associated with high levels of IL-10 production. Nevertheless, no association was observed between these two polymorphisms and biochemical markers for T2DM. Our study suggests that IL-10 genetic polymorphisms may play a specific role(s) in determining diabetic susceptibility, but do not seem to be important in the clinical manifestations of diabetes.