Yingping Cao

Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin

*White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation was evaluated. *White lupin plants were treated with the NO donor sodium nitroprusside (SNP) and scavenger or inhibitor of NO synthase under conditions of P deficiency (0 muM) or P sufficiency (50 muM). *Phosphorus deficiency enhanced NO production in primary and lateral root tips, with a greater increase in cluster roots than in noncluster roots. NO concentrations decreased with cluster root development from the pre-emergent stage, through the juvenile stage, to the mature stage. The P deficiency-induced increase in NO production was inhibited by antagonists of NO synthase and xanthine oxidoreductase, suggesting the involvement of these enzymes in NO production. SNP markedly increased the number of cluster roots. Citrate exudation from different root segments in P-deficient roots was positively correlated with endogenous root NO concentrations. *These findings demonstrate differential patterns of NO production in white lupin, depending on root zone, developmental stage and P nutritional status. NO appears to play a regulatory role in the formation of cluster roots and citrate exudation in white lupin under conditions of P deficiency.

Comprehensive analysis of CCCH zinc finger family in poplar (Populus trichocarpa)

BackgroundCCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date.ResultsIn the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X11-C-X6-C-X3-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs (81 of 90, 90 %) are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue-specific expression and responses to drought stress treatment of 12 selected Populus CCCH genes.ConclusionsThis study provides the first systematic analysis of the Populus CCCH proteins. Comprehensive genomic analyses suggested that segmental duplications contribute significantly to the expansion of Populus CCCH gene family. Transcriptome profiling provides first insights into the functional divergences among members of Populus CCCH gene family. Particularly, some CCCH genes may be involved in wood development while others in drought tolerance regulation. Our results presented here may provide a starting point for the functional dissection of this family of potential RNA-binding proteins.

Increased lipid productivity and TAG content in Nannochloropsis by heavy-ion irradiation mutagenesis

One mutant (HP-1) with higher growth rate was obtained from Nannochloropsis oceanica IMET1 by heavy-ion irradiation mutagenesis. Compared to the wild type, the biomass accumulation and maximum growth rate of HP-1 were individually increased by 19% and 6%, and its lipid productivity was increased by 28% from 211 to 271 mg L(-1) d(-1). Subsequently analysis indicated photosynthetic efficiency of HP-1 was higher than that of wild type during cultivation. Further, lipid composition analysis indicated TAG content of HP-1 was 14% higher, while polar lipid content was 15% lower than that of wild type. Moreover, fatty acid profiles analysis revealed no significant variation was found between the two strains. The mutant is discussed in terms of its comparative advantage over the wild type with respect to its potential utilization for biodiesel production. Owing to its higher lipid productivity and TAG content, HP-1 could be considered as a valuable candidate for microalgal biodiesel production.

MlWRKY12, a novel Miscanthus transcription factor, participates in pith secondary cell wall formation and promotes flowering

WRKY proteins play crucial roles in various plant processes. An AtWRKY12 homologous gene, named MlWRKY12, was isolated from Miscanthus lutarioriparius. The MlWRKY12 gene encodes a WRKY transcription factor belonging to the group IIc subfamily. MlWRKY12 is a nuclear protein. Gene expression pattern analysis revealed a relatively high MlWRKY12 expression level in rhizomes, stems and leaf sheaths. In situ hybridization analysis further demonstrated that MlWRKY12 was expressed in vascular bundle sheath, sclerenchyma and parenchyma tissues. The heterologous expression of MlWRKY12 in an atwrky12 background mutant successfully rescued the phenotype of pith cell walls caused by the defect of AtWRKY12. Most strikingly, the transgenic Arabidopsis plants overexpressing MlWRKY12 exhibited early flowering. The transcript abundance of flowering related genes was measured by quantitative RT-PCR analysis, suggesting that overexpression of MlWRKY12 in Arabidopsis had a significant impact on the expression level of CONSTANS (CO). Moreover, the expression levels of FLOWERING LOCUS T (FT), LFY (LEAFY), APETALA1 (AP1), CAULIFLOWER (CAL) and FRUITFULL (FUL) were upregulated in transgenic plants. These results demonstrated the conserved function of MlWRKY12 existing in secondary cell wall formation of monocotyledonous species and implied a possible impact of MlWRKY12 on flowering control.

Switchgrass SBP-box transcription factors PvSPL1 and 2 function redundantly to initiate side tillers and affect biomass yield of energy crop

BackgroundSwitchgrass (Panicum virgatum L.) is a dedicated lignocellulosic feedstock for bioenergy production. The SQUAMOSA PROMOTER-BINDING PROTEIN (SBP-box)-LIKE transcription factors (SPLs) change plant architecture and vegetative-to-reproductive phase transition significantly, and as such, they are promising candidates for genetic improvement of switchgrass biomass yield. However, the genome-wide identification and functional characterization of SPL genes have yet to be investigated in herbaceous energy crops.ResultsWe identified 35 full-length SPL genes in the switchgrass genome. The phylogenetic relationship and expression pattern of PvSPLs provided baseline information for their function characterization. Based on the global overview of PvSPLs, we explored the biological function of miR156-targeted PvSPL1 and PvSPL2, which are closely related members of SPL family in switchgrass. Our results showed that PvSPL1 and PvSPL2 acted redundantly to modulate side tiller initiation, whereas they did not affect phase transition and internode initiation. Consistently, overexpression of the miR156-resistant rPvSPL2 in the miR156-overexpressing transgenic plants greatly reduced tiller initiation, but did not rescue the delayed flowering and increased internode numbers. Furthermore, suppression of PvSPL2 activity in switchgrass increased biomass yield and reduced lignin accumulation, which thereby elevated the total amount of solubilized sugars.ConclusionsOur results indicate that different miR156-targeted PvSPL subfamily genes function predominantly in certain biological processes in switchgrass. We suggest that PvSPL2 and its paralogs can be utilized as the valuable targets in molecular breeding of energy crops for developing novel germplasms with high biofuel production.

Arabidopsis C3H14 and C3H15 have overlapping roles in the regulation of secondary wall thickening and anther development

Plant tandem CCCH zinc finger (TZF) proteins play diverse roles in developmental and adaptive processes. Arabidopsis C3H14 has been shown to act as a potential regulator of secondary wall biosynthesis. However, there is lack of direct evidence to support its functions in Arabidopsis. It is demonstrated here that C3H14 and its homologue C3H15 redundantly regulate secondary wall formation and that they additionally function in anther development. Plants with double, but not single, T-DNA mutants for C3H14 or C3H15 have few pollen grains and thinner stem secondary walls than the wild type. Plants homozygous for c3h14 and heterozygous for c3h15 [c3h14 c3h15(±)] have slightly thinner secondary walls than plants heterozygous for c3h14 and homozygous for c3h15 [c3h14(±) c3h15], and c3h14(±) c3h15 have lower fertility. Overexpression of C3H14 or C3H15 led to increased secondary wall thickness in stems and the ectopic deposition of secondary walls in various tissues, but did not affect anther morphology. Transcript profiles from the C3H14/15 overexpression and c3h14 c3h15 plants revealed marked changes in the expression of many genes associated with cell wall metabolism and pollen formation. Subcellular localization and biochemical analyses suggest that C3H14/15 might function at both the transcriptional and post-transcriptional levels.

Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar

Wood biomass is mainly made of secondary cell walls, whose formation is controlled by a multilevel network. The tandem CCCH zinc finger (TZF) proteins involved in plant secondary wall formation are poorly understood. Two TZF genes, PdC3H17 and PdC3H18, were isolated from Populus deltoides and functionally characterized in Escherichia coli, tobacco, Arabidopsis and poplar. PdC3H17 and PdC3H18 are predominantly expressed in cells of developing wood, and the proteins they encode are targeted to cytoplasmic foci. Transcriptional activation assays showed that PdMYB2/3/20/21 individually activated the PdC3H17 and PdC3H18 promoters, but PdMYB3/21 were most significant. Electrophoretic mobility shift assays revealed that PdMYB3/21 bound directly to the PdC3H17/18 promoters. Overexpression of PdC3H17/18 in poplar increased secondary xylem width and secondary wall thickening in stems, whereas dominant repressors of them had the opposite effects on these traits. Similar alteration in secondary wall thickening was observed in their transgenic Arabidopsis plants. qRT-PCR results showed that PdC3H17/18 regulated the expression of cellulose, xylan and lignin biosynthetic genes, and several wood-associated MYB genes. These results demonstrate that PdC3H17 and PdC3H18 are the targets of PdMYB3 and PdMYB21 and are an additional two components in the regulatory network of secondary xylem formation in poplar.

Cell wall polysaccharide distribution in Miscanthus lutarioriparius stem using immuno-detection

Key messageCell wall polysaccharides’ occurrences in two internodes of different development stages inM. lutarioripariusstem were analyzed and three major differences between them were identified by cell wall polysaccharide probes.AbstractDeposition and modification of cell wall polysaccharides during stem development affect biomass yield of the Miscanthus energy crop. The distribution patterns of cell wall polysaccharides in the 2nd and the 11th internodes of M. lutarioriparius stem were studied using in situ immunofluorescence assay. Crystalline cellulose and xylan were present in most of the stem tissues except phloem, where xyloglucan was the major composition of hemicellulose. The distribution of pectin polysaccharides varied in stem tissues, particularly in vascular bundle elements. Xylogalacturonan, feruloylated-1,4-β-d-galactan and (1,3)(1,4)-β-glucans, however, were insufficient for antibodies binding in both internodes. Furthermore, the distribution of cell wall polysaccharides was differentiated in the two internodes of M. lutarioriparius. The significant differences in the pattern of occurrence of long 1,5-α-l-arabinan chain, homogalacturonan and fucosylated xyloglucans epitope were detected between the two internodes. In addition, the relationships between probable functions of polysaccharides and their distribution patterns in M. lutarioriparius stem cell wall were discussed, which would be helpful to understand the growth characteristics of Miscanthus and identify potential targets for either modification or degradation.

Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3

Root hairs are tip-growing long tubular outgrowths of specialized epidermal cells, and are important for nutrient and water uptake and interaction with the soil microflora. Here we characterized two poplar cellulose synthase-like D (CSLD) genes, PdCSLD5 and PdCSLD6, the most probable orthologs to the Arabidopsis AtCSLD3/KOJAK gene. Both PdCSLD5 and PdCSLD6 are strongly expressed in roots, including in the root hairs. Subcellular localization experiments showed that these two proteins are located not only in the polarized plasma membrane of root hair tips, but also in Golgi apparatus of the root hair and non-hair-forming cells. Overexpression of these two poplar genes in the atcsld3 mutant was able to rescue most of the defects caused by disruption of AtCSLD3, including root hair morphological changes, altered cell wall monosaccharide composition, increased non-crystalline β-1,4-glucan and decreased crystalline cellulose contents. Taken together, our results provide evidence indicating that PdCSLD5 and PdCSLD6 are functionally conserved with AtCSLD3 and support a role for PdCSLD5 and PdCSL6 specifically in crystalline cellulose production in poplar root hair tips. The results presented here also suggest that at least part of the mechanism of root hair formation is conserved between herbaceous and woody plants.

Structural analysis of galactoarabinan from duckweed.

A highly branched galactoarabinan named DAG1 (Mw∼4.0×10(4) Da) was purified from Lemna aequinoctialis 6000 via 70% (v/v) ethanol extraction, followed by size-exclusion chromatography on Bio-Gel P2 and Superdex 75. Methylation analysis showed that DAG1 consisted of t-Araf, (1→5)-Araf, (1→2,5)-Araf, (1→3)-Galp, and (1→3,6)-Galp in a relative proportion of approximately 6:4:3:3:3, suggesting an arabinogalactan/galactoarabinan polysacchairde. With the aid of arabinan degrading enzymes, the structure of DAG1 repeating unit was further characterized by ELISA with specific monoclonal antibodies and Yariv reagent assay. Analyses indicated that the proposed repeating unit of DAG1 had a backbone composed of seven α-(1→5)-L-arabinofuranose residues where branching occurred at O-2 with either terminal arabinoses or arabinogalactan side chain. The arabinogalactan side chain was composed of six β-(1→3)-D-galactopyranose residues, half of which were ramified at O-6 with terminal arabinoses and the last galactose was terminated with arabinose.