Dynamic formation and transcriptional regulation mediated by phytohormones during chalkiness formation in rice.

BACKGROUND: Rice (Oryza sativa L.) Chalkiness, the opaque part in the kernel endosperm formed by loosely piled starch and protein bodies. Chalkiness is a complex quantitative trait regulated by multiple genes and various environmental factors. Phytohormones play important roles in the regulation of chalkiness formation but the underlying molecular mechanism is still unclear at present. RESULTS: In this research, Xiangzaoxian24 (X24, pure line of indica rice with high-chalkiness) and its origin parents Xiangzaoxian11 (X11, female parent, pure line of indica rice with high-chalkiness) and Xiangzaoxian7 (X7, male parent, pure line of indica rice with low-chalkiness) were used as materials. The phenotype, physiological and biochemical traits combined with transcriptome analysis were conducted to illustrate the dynamic process and transcriptional regulation of rice chalkiness formation. Impressively, phytohormonal contents and multiple phytohormonal signals were significantly different in chalky caryopsis, suggesting the involvement of phytohormones, particularly ABA and auxin, in the regulation of rice chalkiness formation, through the interaction of multiple transcription factors and their downstream regulators. CONCLUSION: These results indicated that chalkiness formation is a dynamic process associated with multiple genes, forming a complex regulatory network in which phytohormones play important roles. These results provided informative clues for illustrating the regulatory mechanisms of chalkiness formation in rice.

Auxin Extraction and Purification Based on Recombinant Aux/IAA Proteins.

BACKGROUND: Indole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones. RESULTS: Since the AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) proteins show higher specific binding capability with auxin, recombinant IAA1, IAA7 and IAA28 proteins were used as sorbents to develop an IAA extraction and purification method. A GST tag was used to solidify the recombinant protein in a column. Aux/IAA proteins solidified in a column have successfully trapped trace IAA in aqueous solutions. The IAA7 protein showed higher IAA binding capability than the other proteins tested. In addition, expression of the IAA7 protein in Drosophila Schneider 2 (S2) cells produced better levels of binding than IAA7 expressed in E. coli. CONCLUSION: This work validated the potential of Aux/IAA proteins to extract and purify IAA from crude plant extracts once we refined the techniques for these processes.

Genome-wide identification and characterization of auxin response factor (ARF) family genes related to flower and fruit development in papaya (Carica papaya L.).

BACKGROUND: Auxin and auxin signaling are involved in a series of developmental processes in plants. Auxin Response Factors (ARFs) is reported to modulate the expression of target genes by binding to auxin response elements (AuxREs) and influence the transcriptional activation of down-stream target genes. However, how ARF genes function in flower development and fruit ripening of papaya (Carica papaya L.) is largely unknown. In this study, a comprehensive characterization and expression profiling analysis of 11 C. papaya ARF (CpARF) genes was performed using the newly updated papaya reference genome data. RESULTS: We analyzed CpARF expression patterns at different developmental stages. CpARF1, CpARF2, CpARF4, CpARF5, and CpARF10 showed the highest expression at the initial stage of flower development, but decreased during the following developmental stages. CpARF6 expression increased during the developmental process and reached its peak level at the final stage of flower development. The expression of CpARF1 increased significantly during the fruit ripening stages. Many AuxREs were included in the promoters of two ethylene signaling genes (CpETR1 and CpETR2) and three ethylene-synthesis-related genes (CpACS1, CpACS2, and CpACO1), suggesting that CpARFs might be involved in fruit ripening via the regulation of ethylene signaling. CONCLUSIONS: Our study provided comprehensive information on ARF family in papaya, including gene structures, chromosome locations, phylogenetic relationships, and expression patterns. The involvement of CpARF gene expression changes in flower and fruit development allowed us to understand the role of ARF-mediated auxin signaling in the maturation of reproductive organs in papaya.

Erratum to: Model of Cation Transportation Mediated by High-Affinity Potassium Transporters (HKTs) in Higher Plants.

[This corrects the article DOI: 10.1186/s12575-014-0013-3.].

Model of Cation Transportation Mediated by High-Affinity Potassium Transporters (HKTs) in Higher Plants.

Trk/Ktr/HKT transporters probably were evolved from simple K(+) channels KcsA. HKT transporters, which mediate Na(+)-uniport or Na(+)/K(+)-symport, maintain K(+)/Na(+) homeostasis and increase salinity tolerance, can be classified into three subfamilies in higher plants. In this review, we systematically analyzed the characteristics of amino acids sequences and physiological functions of HKT transporters in higher plant. Furthermore, we depicted the hypothetical models of cations selection and transportation mediated by HKT transporters according to the highly conserved structure for the goal of better understanding the cations transportation processes.

Transcriptome Analysis Reveals Novel Insights into the Hyperaccumulator Phytolacca acinosa Roxb. Responses to Cadmium Stress.

Cadmium (Cd) is a highly toxic heavy metal that causes serious damage to plant and human health. Phytolacca acinosa Roxb. has a large amount of aboveground biomass and a rapid growth rate, and it has been identified as a novel type of Cd hyperaccumulator that can be harnessed for phytoremediation. However, the molecular mechanisms underlying the response of P. acinosa to Cd2+ stress remain largely unclear. In this study, the phenotype, biochemical, and physiological traits of P. acinosa seeds and seedlings were analyzed under different concentrations of Cd2+ treatments. The results showed higher Cd2+ tolerance of P. acinosa compared to common plants. Meanwhile, the Cd2+ content in shoots reached 449 mg/kg under 10 mg/L Cd2+ treatment, which was obviously higher than the threshold for Cd hyperaccumulators. To investigate the molecular mechanism underlying the adaptability of P. acinosa to Cd stress, RNA-Seq was used to examine transcriptional responses of P. acinosa to Cd stress. Transcriptome analysis found that 61 genes encoding TFs, 48 cell wall-related genes, 35 secondary metabolism-related genes, 133 membrane proteins and ion transporters, and 96 defense system-related genes were differentially expressed under Cd2+ stress, indicating that a series of genes were involved in Cd2+ stress, forming a complex signaling regulatory mechanism. These results provide new scientific evidence for elucidating the regulatory mechanisms of P. acinosa response to Cd2+ stress and new clues for the molecular breeding of heavy metal phytoremediation.

Simultaneous splicing of multiple DNA fragments in one PCR reaction.

BACKGROUND: Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. RESULTS: We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5-10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification. CONCLUSIONS: Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match.

Multiple High-Affinity K+ Transporters and ABC Transporters Involved in K+ Uptake/Transport in the Potassium-Hyperaccumulator Plant Phytolacca acinosa Roxb.

Potassium is an important essential element for plant growth and development. Long-term potassium deprivation can lead to a severe deficiency phenotype in plants. Interestingly, Phytolacca acinosa is a plant with an unusually high potassium content and can grow well and complete its lifecycle even in severely potassium deficient soil. In this study, we found that its stems and leaves were the main tissues for high potassium accumulation, and P. acinosa showed a strong ability of K+ absorption in roots and a large capability of potassium accumulation in shoots. Analysis of plant growth and physiological characteristics indicated that P. acinosa had an adaptability in a wide range of external potassium levels. To reveal the mechanism of K+ uptake and transport in the potassium-hyperaccumulator plant P. acinosa, K+ uptake-/transport-related genes were screened by transcriptome sequencing, and their expression profiles were compared between K+ starved plants and normal cultured plants. Eighteen members of HAK/KT/KUPs, ten members of AKTs, and one member of HKT were identified in P. acinosa. Among them, six HAKs, and two AKTs and PaHKT1 showed significantly different expression. These transporters might be coordinatively involved in K+ uptake/transport in P. acinosa and lead to high potassium accumulation in plant tissues. In addition, significantly changed expression of some ABC transporters indicated that ABC transporters might be important for K+ uptake and transport in P. acinosa under low K+ concentrations.

The Copy Number Variation of OsMTD1 Regulates Rice Plant Architecture.

Copy number variation (CNV) may have phenotypic effects by altering the expression level of the gene(s) or regulatory element(s) contained. It is believed that CNVs play pivotal roles in controlling plant architecture and other traits in plant. However, the effects of CNV contributing to special traits remain largely unknown. Here we report a CNV involved in rice architecture by modulating tiller number and leaf angle. In the genome of Oryza sativa ssp. japonica cv. Nipponbare, we found a locus Loc_Os08g34249 is derived from a 13,002-bp tandem duplication in the nearby region of OsMTD1, a gene regulating tillering in rice. Further survey of 230 rice cultivars showed that the duplication occurred in only 13 japonica rice cultivars. Phenotypic investigation indicated that this CNV region may contribute to tiller number. Moreover, we revealed that OsMTD1 not only influences rice tiller number and leaf angle, but also represses pri-miR156f transcription in the CNV region. Intriguingly, this CNV performs function through both the dosage and position effects on OsMTD1 and pri-miR156f. Thus, our work identified a CNV and revealed a molecular regulatory basis for its effects on plant architecture, implying this CNV may possess importance and application potential in molecular breeding in rice.

Chlorophyll deficiency in the maize elongated mesocotyl2 mutant is caused by a defective heme oxygenase and delaying grana stacking.

BACKGROUND: Etiolated seedlings initiate grana stacking and chlorophyll biosynthesis in parallel with the first exposure to light, during which phytochromes play an important role. Functional phytochromes are biosynthesized separately for two components. One phytochrome is biosynthesized for apoprotein and the other is biosynthesized for the chromophore that includes heme oxygenase (HO). METHODOLOGY/PRINCIPAL FINDING: We isolated a ho1 homolog by map-based cloning of a maize elongated mesocotyl2 (elm2) mutant. cDNA sequencing of the ho1 homolog in elm2 revealed a 31 bp deletion. De-etiolation responses to red and far-red light were disrupted in elm2 seedlings, with a pronounced elongation of the mesocotyl. The endogenous HO activity in the elm2 mutant decreased remarkably. Transgenic complementation further confirmed the dysfunction in the maize ho1 gene. Moreover, non-appressed thylakoids were specifically stacked at the seedling stage in the elm2 mutant. CONCLUSION: The 31 bp deletion in the ho1 gene resulted in a decrease in endogenous HO activity and disrupted the de-etiolation responses to red and far-red light. The specific stacking of non-appressed thylakoids suggested that the chlorophyll biosynthesis regulated by HO1 is achieved by coordinating the heme level with the regulation of grana stacking.

[High performance liquid chromatographic determination of internal hormones in inter-subspecific hybrid rice].

A simple, rapid and perfect extraction and determination method for the internal hormones (gibberellin acid (GA3), indole-3-acetic acid (IAA), zeatin (Z), abscisic acid (ABA)) in inter-subspecific hybrid rice with reversed-phase high performance liquid chromatography has been developed. The chromatographic conditions were as follows: a Waters C18 column (4.6 mm i.d. x 250 mm, 5 microns), an SPD-6AV detector set at 254 nm and the mobile phase was a mixture of methanol, water and acetic acid (45:54.2:0.8, volume ratio). The lower detection limits were 0.5 mg/L for GA3, 0.1 mg/L for IAA, 0.3 mg/L for Z and 0.03 mg/L for ABA.