A High-Throughput Screening System for Populus Wood-Associated Transcription Factors and Its Application to Lignin Regulation

Wood formation of trees is a complex and costly developmental process, whose regulatory network is involved in the protein-protein and protein-DNA interactions. To detect such interactions in wood development, we developed a high-throughput screening system with 517 Gal4-AD-wood-associated transcription factors (TFs) library from Populus alba × P. glandulosa cv “84K.” This system can be used for screening the upstream regulators and interacting proteins of targets by mating-based yeast-one hybrid (Y1H) and yeast-two-hybrid (Y2H) method, respectively. Multiple regulatory modules of lignin biosynthesis were identified based on this Populus system. Five TFs interacted with the 500-bp promoter fragment of PHENYLALANINE AMMONIA-LYASE 2 (PAL2), the first rate-limiting enzyme gene in the lignin biosynthesis pathway, and 10 TFs interacted with PaMYB4/LTF1, a key regulator of lignin biosynthesis. Some of these interactions were further validated by EMSA and BiFC assays. The TF-PaPAL2 promoter interaction and TF-PaMYB4 interaction revealed a complex mechanism governing the regulation of lignin synthesis in wood cells. Our high-throughput Y1H/Y2H screening system may be an efficient tool for studying regulatory network of wood formation in tree species.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant–pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Transcriptomic analysis of melon/squash graft junction reveals molecular mechanisms potentially underlying the graft union development

Oriental melon (Cucumis melo var. makuwa Makino) has become a widely planted horticultural crop in China especially in recent years and has been subjected to the grafting technique for the improvement of cultivation and stress resistance. Although grafting has a long history in horticulture, there is little known about the molecular mechanisms of the graft healing process in oriental melon. This study aims to reveal the molecular changes involved in the graft healing process. In the present work, anatomical observations indicated that the 2, 6, and 9 DAG were three critical stages for the graft healing and therefore, were selected for the subsequent high-throughput RNA-seq analysis. A total of 1,950 and 1,313 DEGs were identified by comparing IL vs. CA and CA vs. VB libraries, respectively. More DEGs in the melon scion exhibited abundant transcriptional changes compared to the squash rootstock, providing increased metabolic activity and thus more material basis for the graft healing formation in the scion. Several DEGs were enriched in the plant hormone signal transduction pathway, phenylpropanoid biosynthesis, and carbon metabolism. In addition, the results showed that concentrations of IAA, GA3, and ZR were induced in the graft junctions. In conclusion, our study determined that genes involved in the hormone-signaling pathway and lignin biosynthesis played the essential roles during graft healing. These findings expand our current understandings of the molecular basis of the graft junction formation and facilitate the improvement and success of melon grafting in future production.

The SlHB8 Acts as a Negative Regulator in Stem Development and Lignin Biosynthesis

The stem is an important organ in supporting plant body, transporting nutrients and communicating signals for plant growing. However, studies on the regulation of stem development in tomato are rather limited. In our study, we demonstrated that SlHB8 negatively regulated tomato stem development. SlHB8 belongs to homeo domain-leucine zipper Class III gene family transcription factors and expressed in all the organs examined including root, stem, leaves, flower, and fruit. Among these tissues, SlHB8 showed stable high expression level during tomato stem development. Overexpression of SlHB8 gene decreased stem diameter with inhibited xylem width and xylem cell layers, while loss of function of SlHB8gene increased the stem diameter and xylem width. The contents of lignin were decreased both in leaves and stems of SlHB8 overexpression plants. RNA-seq analysis on the stems of wild type and SlHB8 transgenic plants showed that the 116 DEGs (differential expressed genes) with reversible expression profiles in SlHB8-ox and SlHB8-cr plants were significantly enriched in the phenylpropanoid biosynthesis pathway and plant-pathogen pathway which were related to lignin biosynthesis and disease resistance. Meanwhile, the key genes involved in the lignin biosynthesis pathway such as SlCCR (cinnamoyl-CoA reductase), SlCYP73A14/C4H (cinnamate 4-hydroxylase), SlC3H (coumarate 3-hydroxylase) and SlCAD (cinnamoyl alcohol dehydrogenase) were down-regulated in both stem and leaves of SlHB8 overexpression plants, indicating a negative regulatory role of SlHB8 in the lignin biosynthesis and stem development.

Whole-genome sequencing of Acer catalpifolium reveals evolutionary history of endangered species

Acer catalpifolium is an endangered species restricted to remote localities of West China. Understanding the genomic content and evolution of A. catalpifolium is essential to conservation efforts of this rare and ecologically valuable plant. Here, we report a high-quality genome of A. catalpifolium consisting of ∼654 Mbps and ∼35,132 protein-coding genes. We detected 969 positively-selected genes in two Acer genomes compared with four other eudicots, 65 of which were transcription factors. We hypothesize that these positively-selected mutations in transcription factors might affect their function and thus contribute to A. catalpifolium’s decline-type population. We also identified 179 significantly expanded gene families compared to 12 other eudicots, some of which are involved in stress responses, such as the FRS-FRF family. We inferred that A. catalpifolium has experienced gene family expansions to cope with environmental stress in its evolutionary history. Finally, 109 candidate genes encoding key enzymes in the lignin biosynthesis pathway were identified in A. catalpifolium; of particular note were the large range and high copy number of cinnamyl alcohol dehydrogenase genes. The chromosome-level genome of A. catalpifolium presented here may serve as a fundamental genomic resource for better understanding endangered Acer species, informing future conservation efforts.

Network Analysis of Different Exogenous Hormones on the Regulation of Deep Sowing Tolerance in Maize Seedlings

The planting method of deep sowing can make the seeds make full use of water in deep soil, which is considered to be an effective way to respond to drought stress. However, deep sowing will affect the growth and development of maize (Zea mays L.) at seedling stage. To better understand the response of maize to deep sowing stress and the mechanism of exogenous hormones [Gibberellin (GA3), Brassinolide (BR), Strigolactone (SL)] alleviates the damaging effects of deep-sowing stress, the physiological and transcriptome expression profiles of seedlings of deep sowing sensitive inbred line Zi330 and the deep-tolerant inbred line Qi319 were compared under deep sowing stress and the conditions of exogenous hormones alleviates stress. The results showed that mesocotyl elongated significantly after both deep sowing stress and application of exogenous hormones, and its elongation was mainly through elongation and expansion of cell volume. Hormone assays revealed no significant changes in zeatin (ZT) content of the mesocotyl after deep sowing and exogenous hormone application. The endogenous GA3 and auxin (IAA) contents in the mesocotyl of the two inbred lines increased significantly after the addition of exogenous GA3, BR, and SL under deep sowing stress compared to deep sowing stress, while BR and SL decreased significantly. Transcriptome analysis showed that the deep seeding stress was alleviated by GA3, BR, and SLs, the differentially expressed genes (DEGs) mainly included cellulose synthase, expansin and glucanase, oxidase, lignin biosynthesis genes and so on. We also found that protein phosphatase 2C and GA receptor GID1 enhanced the ability of resist deep seeding stress in maize by participating in the abscisic acid (ABA) and the GA signaling pathway, respectively. In addition, we identified two gene modules that were significantly related to mesocotyl elongation, and identified some hub genes that were significantly related to mesocotyl elongation by WGCNA analysis. These genes were mainly involved in transcription regulation, hydrolase activity, protein binding and plasma membrane. Our results from this study may provide theoretical basis for determining the maize deep seeding tolerance and the mechanism by which exogenous hormones regulates deep seeding tolerance.

Homo- and Hetero-Dimers of CAD Enzymes Regulate Lignification and Abiotic Stress Response in Moso Bamboo

Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.

PmMYB4, a Transcriptional Activator from Pinus massoniana, Regulates Secondary Cell Wall Formation and Lignin Biosynthesis

Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine (Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4, which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-lignified stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes (PmPAL and PmCCoAOMT) involved in SCW biosynthesis. In addition, PmMYB4-OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees.

Transcriptomic Characterization of the Effects of Selenium on Maize Seedling Growth

Selenium (Se) is a trace mineral element in soils that can be beneficial to plants in small amounts. Although maize is among the most economically important crops, there are few reports on the effects of Se on maize seedling growth at the molecular level. In this study, the growth of maize seedlings treated with different concentrations of Na2SeO3 was investigated, and the physiological characteristics were measured. Compared with the control, a low Se concentration promoted seedling growth, whereas a high Se concentration inhibited it. To illustrate the transcriptional effects of Se on maize seedling growth, samples from control plants and those treated with low or high concentrations of Se were subjected to RNA sequencing. The differentially expressed gene (DEG) analysis revealed that there were 239 upregulated and 106 downregulated genes in the low Se treatment groups, while there were 845 upregulated and 1,686 downregulated DEGs in the high Se treatment groups. Both the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses showed a low concentration of the Se-stimulated expression of “DNA replication” and “glutathione (GSH) metabolism”-related genes. A high concentration of Se repressed the expression of auxin signal transduction and lignin biosynthesis-related genes. The real-time quantitative reverse transcription PCR (qRT-PCR) results showed that in the low Se treatment, “auxin signal transduction,” “DNA replication,” and lignin biosynthesis-related genes were upregulated 1.4- to 57.68-fold compared to the control, while, in the high Se concentration treatment, auxin signal transduction and lignin biosynthesis-related genes were downregulated 1.6- to 16.23-fold compared to the control. Based on these transcriptional differences and qRT-PCR validation, it was found that a low dosage of Se may promote maize seedling growth but becomes inhibitory to growth at higher concentrations. This study lays a foundation for the mechanisms underlying the effects of Se on maize seedling growth.