Classification of Targets Using Statistical Features from Range FFT of mmWave FMCW Radars

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Heat stress tolerance in peas (Pisum sativum L.): Current status and way forward

In the era of climate change, the overall productivity of pea (Pisum sativum L.) is being threatened by several abiotic stresses including heat stress (HS). HS causes severe yield losses by adversely affecting several traits in peas. A reduction in pod yield has been reported from 11.1% to 17.5% when mean daily temperature increase from 1.4 to 2.2°C. High-temperature stress (30.5-33°C) especially during reproductive phase is known to drastically reduce both seed yield and germination. HS during germination and early vegetative stage resulted in poor emergence and stunted plant growth along with detrimental effects on physiological functions of the pea plant. To combat HS and continue its life cycle, plants use various defense strategies including heat escape, avoidance or tolerance mechanisms. Ironically, the threshold temperatures for pea plant and its responses are inconsistent and not yet clearly identified. Trait discovery through traditional breeding such as semi leaflessness (afila), upright growing habit, lodging tolerance, lower canopy temperature and small seeded nature has highlighted their utility for greater adaptation under HS in pea. Screening of crop gene pool and landraces for HS tolerance in a targeted environment is a simple approach to identify HS tolerant genotypes. Thus, precise phenotyping using modern phenomics tools could lead to increased breeding efficiency. The NGS (next generation sequencing) data can be associated to find the candidate genes responsible for the HS tolerance in pea. In addition, genomic selection, genome wide association studies (GWAS) and marker assisted selection (MAS) can be used for the development of HS tolerant pea genotypes. Additionally, development of transgenics could be an alternative strategy for the development of HS tolerant pea genotypes. This review comprehensively covers the various aspects of HS tolerance mechanisms in the pea plant, screening protocols, omic advances, and future challenges for the development of HS tolerant genotypes

Inactivation of Individual SeqA Binding Sites of the <i>E</i>. <i>coli</i> Origin Reveals Robustness of Replication Initiation Synchrony

<div><p>The <i>Escherichia coli</i> origin of replication, <i>oriC</i>, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to <i>oriC</i> are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites. The altered SeqA binding, however, does not affect initiation synchrony. Synchrony is also unaffected when individual SeqA sites are mutated. An apparent exception was one mutant where the mutation also changed an overlapping DnaA site. In this mutant, the observed asynchrony could be from altered DnaA binding, as selectively mutating this SeqA site did not cause asynchrony. These results reveal robust initiation synchrony against alterations of individual SeqA binding sites. The redundancy apparently ensures SeqA function in controlling replication in <i>E</i>. <i>coli</i>.</p></div

Sequence of <i>oriC</i> showing its major protein binding sites.

<p>(A) Sequence of <i>oriC</i> that includes the minimal region (coordinates 1–246) required for origin function. The coordinate 1 correspondence to 3923744 of gb|U00096.3|. The region includes several GATC sites (shown in red) which are methylated by the Dam methylase enzyme. There are three 13-mer repeats of AT rich sequences where the origin initially unwinds. The remainder of the origin has mainly 9-mer DnaA binding sites as well as sites for binding IHF and FIS proteins. DnaA sites have either high (R1, R2 and R4) or low (τ1, R5, τ2, I1, I2, C3, C2, I3 and C1) affinity for DnaA. The numbers #1–9 mark the GATC sites studied here. A TaqI site (TCGA) overlapping each of the nine GATC sites was created by converting their two upstream bases to TC (<u>NNGA</u>TC to <u>TCGA</u>TC). (B) A linear map of <i>oriC</i> features described above. The map also shows location of sites for restriction enzymes MboII and HphI that naturally occurs in <i>oriC</i>.</p

Cell cycle parameters of <i>oriC</i>-<i>zeo</i> mutants with mutated GATC^a

<p>Cell cycle parameters of <i>oriC</i>-<i>zeo</i> mutants with mutated GATC<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#t002fn001" target="_blank">^a</a></p

Cell cycle parameters of <i>oriC</i>-<i>FRT</i> mutants with TaqI sites^a.

<p>Cell cycle parameters of <i>oriC</i>-<i>FRT</i> mutants with TaqI sites<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#t001fn001" target="_blank">^a</a>.</p

Quantification of hemimethylated DNA level at different GATC sites of <i>oriC</i> in <i>seqA</i>⁺ and Δ<i>seqA</i> strains.

<p>(A) The top line shows a schematic map of <i>oriC</i> as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g001" target="_blank">Fig 1B</a>, which also includes the location of an <i>FRT</i> site that was present in all the strains used in this figure. The autorad is a representative Southern blot of genomic DNA from <i>seqA</i>+ and Δ<i>seqA</i> strains after digestion with TaqI. The blots were probed with a 220 bp PCR amplified fragment (uisng primers jj40+jj41) homologuous to the left flank of <i>oriC</i> (blue line). M represents <i>oriC</i> specific markers (1100bp, 460 bp, 330 bp and 240 bp). The arrows indicate the bands in which the test GATC sites were either fully methylated, thus resistant to TaqI digestion (upper bands), or HM and sensitive to TaqI digestion 50% of the time (lower bands), as explained in Figure A(A) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.s001" target="_blank">S1 File</a>. The intensity of the lower bands was multiplied by a factor of two to calculate the relative level of HM DNA (lower panel). [Note that the separation of the two bands is gradually narrowing because of shifting position of TaqI site within <i>oriC</i> with respect to the two TaqI sites that flank <i>oriC</i> (see also Figure A(B) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.s001" target="_blank">S1 File</a>). The upper fragment is generated when TaqI digests the two <i>oriC</i> flanking sites but not the TaqI site within <i>oriC</i>.] The black and gray bars represent mean HM DNA levels in <i>seqA</i>⁺ and Δ<i>seqA</i> strains, respectively, determined from three cultures innoculated with independent colonies (biological replicates). The error bar represents one standard deviation of the mean. Asterisks indicate pairwise comparisons that were statistically distinguishable (<i>P</i> < 0.05, students <i>t-</i>test). (B) Same as (A) except that the <i>oriC</i> probe was stripped off the blot, which was then reprobed with a PCR product (using primers jj193+jj194) from a region in <i>lacZ</i> (blue bar) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.ref028" target="_blank">28</a>]. The error bars represent variability in repeated measurements of the same blot (technical replicates).</p

Effect of GATC mutations in <i>oriC</i> on initiation synchrony and the level of HM DNA at the MboII site of <i>oriC</i>.

<p>(A) The top line shows a schematic map of <i>oriC</i> as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g001" target="_blank">Fig 1B</a> except for an additional GATC site (#3*) included in this study. In these experiments <i>oriC</i> was marked with a <i>zeo</i> drug^R cassette. GATC sites at positions #1–9 and #3* were individually mutated to GTTC and the mutant cells were analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g003" target="_blank">Fig 3A</a> before and after replication run-out. Cells were also studied after combining some of the mutations (#5–6 etc.). (B) HM DNA levels as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g002" target="_blank">Fig 2A</a> except that the levels were measured at the MboII site for all. The black bar represents the HM DNA level in the WT strain (<i>zeo</i> marked but without any GATC mutation within <i>oriC</i>) and the grey bars the same strain with individual or multiple GATC mutations. The error bars were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g002" target="_blank">Fig 2B</a>. Mutants whose HM DNA levels were significantly different from the WT (<i>p-</i>value <0.05) are marked by asterisks. The dashed line provides a visual aid for comparing HM DNA levels in different mutants relative to the WT.</p

Effect of DnaA titration on cell size, initiation synchrony and HM DNA level at <i>oriC</i>.

<p>(A) Cell size and chromosome content of <i>FRT</i> marked <i>oriC</i> cells without (WT) or with mutations creating TaqI sites #1–9. The cells had an R1 or R1-<i>datA</i> plasmid that was used as a vector control or for titrating DanA-ATP, respectively. Cells were grown at 37°C in supplemented 1X M63 medium up to an OD₆₀₀ ~ 0.15, and then processed either before or after replication run-out for flow cytometry to measure cell size by light scattering or chromosome content by fluorescence emission, respectively. The multimodal distribution of fluorescence profiles represent cells with different integral number of chromosomes as stated in the abscissa. (B) Southern blots of genomic DNA from cultures in (A) before replication run-out. Black and gray bars represent HM DNA levels in <i>seqA</i>⁺ strain in the presence of R1 and R1-<i>datA</i> plasmid, respectively. The details are otherwise same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166722#pone.0166722.g002" target="_blank">Fig 2A</a>. Note that band intensities were not quantified for the WT as no cut band was seen or expected because WT <i>oriC</i> does not have a TaqI site in the probed region. The difference in HM DNA levels in the presence of R1 and R1-<i>datA</i> plasmids was considered statistically significant in the case of mutants #6 and #7 only (<i>P</i> < 0.05).</p