Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also ...been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coil-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1 -dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular "stand-by mode" by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1 -dependent leaf growth inhibition.
Summary
Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory ...networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells.
Here, direct incubation of leaf explants from the non‐medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High‐throughput LC‐MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells.
A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA‐treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin‐dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis‐(+)‐12‐oxophytodienoic acid, in synergy, could act as anticancer compounds.
Our work suggests a universal role for MeJA‐treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti‐cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments
Viruses of the genus
(family
) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous ...badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera
,
and
has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (
spp.). A diverse range of
bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.
Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated ...badnavirus sequences in some yam genomes. To date, only two badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4-7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed badnavirus infections occur relatively often in West African yam germplasm.
Badnaviruses (family
, genus
) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the ...detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (
spp.) plants are hosts to a diverse range of badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex 'fingerprints' composed of multiple sequences of
bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.
To date, several viruses of different genera have been reported to infect yam (
spp.). The full diversity of viruses infecting yam, however, remains to be explored. High-throughput sequencing (HTS) ...methods are increasingly being used in the discovery of new plant viral genomes. In this study, we employed HTS on yam to determine whether any undiscovered viruses were present that would restrict the international distribution of yam germplasm. We discovered a new virus sequence present in 31 yam samples tested and have tentatively named this virus "yam virus Y" (YVY). Twenty-three of the samples in which YVY was detected showed mosaic and chlorotic leaf symptoms, but
was also detected in these samples. Complete genome sequences of two YVY viral isolates were assembled and found to contain five open reading frames (ORFs). ORF1 encodes a large replication-associated protein, ORF2, ORF3 and ORF4 constitute the putative triple gene block proteins, and ORF5 encodes a putative coat protein. Considering the species demarcation criteria of the family
, YVY should be considered as a novel virus species in the family
. Further work is needed to understand the association of this new virus with any symptoms and yield loss and its implication on virus-free seed yam production.
•Sequence diversity show lack of geographical association between isolates from Ghana and Nigeria.•YMMV isolates from Ghana and Nigeria fall within four of the 11 monophyletic groups.•Need for ...stringent control of germplasm exchange as infection is mainly by infected tubers.
This study analyzed the genetic diversity of 18 Yam mild mosaic virus (YMMV, genus Potyvirus) isolates collected from field surveys in Ghana (N = 8) and Nigeria (N = 10) in 2012−13. The full coat protein (CP) encoding region of the virus genome was sequenced and used for comparison and phylogenetic analysis of the YMMV isolates available in the NCBI nucleotide database. The mean nucleotide (nt) diversity was 13.4% among the 18 isolates (17 from D. alata and one from D. rotundata), 11.4% within the isolates of Ghana and 7.4% within the isolates of Nigeria. The phylogenetic clustering of the 18 YMMV isolates did not show correlation with the country of origin, and they aligned with the reference sequences of four of the 11 YMMV monophyletic groups representing the cosmopolitan group and the African group of YMMV isolates. High sequence homology of 99% between the YMMV sequence from Nigeria (CP12-DaN6-1) and a previously reported sequence from Togo (GenBank Accession Number AF548514) suggests a prevalence of seed-borne virus spread within the region. Understanding YMMV sequence diversity in West Africa aid in the improvement of diagnostic assays necessary for virus indexing and seed certification.
Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value ...chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain
cell suspension cultures is devised and presented in detail.
cell cultures were directly transformed with
for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence
peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.
Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key ...factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA).
The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation.
The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.
Since the first discovery of badnaviruses (family
, genus
) in yam (
spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase ...(RT)-ribonuclease H (RNaseH) sequences have been characterised ( Kenyon
, 2008 ; Bousalem
, 2009 ), but only a few complete
bacilliform virus (DBV) genome sequences have been reported ( Phillips
, 1999 ; Seal and Muller, 2007; Bömer
, 2016 and 2017; Sukal
, 2017 ; Umber
, 2017 ). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses ( Bömer
, 2016 ). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome ( Turaki
, 2017 ). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes.
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