Arabidopsis with determinate growth pattern did not show a similar increase in shoot growth when transformed with the same construct. To conclude, expression of an Arabidopsis CKX6 ortholog of chickpea in the root endodermal cells of chickpea produced a robust increase in root network and improved tolerance to long‐term drought without any apparent negative effect on the shoot. Lower cytokinin and ABA levels in the roots of the transgenic lines might have helped the loading of nutrients in the vascular system. After entry into the root, water and nutrients move radially to enter the central vascular system after crossing a restrictive endodermal cell layer. While the decrease in the stomatal conductance, transpiration rate and CO2 assimilation rate in the control plants was 90%, 87% and 83%, respectively, the transgenic lines showed a decrease of 46%–73% for stomatal conductance, 43%–73% for transpiration rate and 18%–51% in CO2 assimilation rate (Figure 7e–g). CKX activity was assessed in soil‐grown 30 dpg plants, and an increase of 2.1–3.7‐fold over the untransformed plant was observed only in the root, while CKX activity in the shoot tissue (not shown) was not altered (Figure 3f). Bombshell. Although ABA level increased in both wild‐type and transgenic chickpea lines under drought, ABA level in the transgenic lines was 18%–50% less than that of the wild type. Figure S1 Phylogenetic analysis of CKX proteins in Arabidopsis, Medicago and chickpea. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. RWC of third and fourth leaves from the top of the plants of same age was measured under control and 40 day‐drought conditions. The dry mature plants (155 days old) were uprooted carefully and manually. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 20% off our IPA mixed case. Functional analysis of CKX6 gene in legume crops has not been reported so far. Figure S7 Nodule phenotype and total seed protein estimation in transgenic plants. Over two decades, cytokinin oxidase/dehydrogenase (CKX) has been used extensively as a tool for studying the role of cytokinin in plant development in Arabidopsis. LP, MC and PM are supported by SRF & JRF fellowship from Council of Scientific and Industrial Research (CSIR, Govt. Higher leaf RWC together with lower ABA level might have contributed to higher carbon assimilation under long‐term drought conditions. HK, SKG and VD raised transgenic plants and assisted manuscript preparation. In contrast, we have observed a limited but significant increase in shoot biomass and seed yield in transgenic chickpea lines. For subcellular localization of CaCKX6, M13_F and M13_R were used to amplify PCR product from pENTR clone of CaCKX6 and cloned to pEG101 Gateway binary vector through LR clonase reaction. Its finish is edgy & uncompromising and leaves for an expressive, almost spicy palate. Additionally, legumes develop a symbiotic relationship with rhizobia and form root nodules allowing them to acquire fixed nitrogen. A decrease in the lateral root number might be one of the reasons for decreased root biomass (Figure 7a–d). Cultivated chickpea genotypes show an indeterminate growth habit. The other two CKX6‐like proteins of these two legume species (XP_00451481.1 of chickpea and XP_003599606.1 of Medicago) showed the presence of extra twenty amino acids at their N‐terminus. Nobody will agree with this, but I much prefer double to be for penalties after a 1NT opening, not that I would double in this situation, although it could be the winning move. Learn the good & bad for 250,000+ products. I see the crap people overcall 1NT with. Stomatal conductance, CO2 assimilation rate and transpiration rate of the third leaf from the top of all the lines showed a decrease in drought; however, decrease in these parameters was significantly less in the transgenic lines. Portable photosynthesis system LI‐6400 XT (LI‐COR Biosciences, Inc.) was used for measurement of photosynthesis‐related parameters. Shoot biomass decreased significantly as expected. Acetylene reduction assay (ARA) was performed as described previously (Mandal and Sinharoy, 2018; Oke and Long, 1999). As the relative moisture content was higher deep in the soil, the acquisition of more resources by a deeper root system might be one of the reasons for improved tolerance. Research Progress on the Roles of Cytokinin in Plant Response to Stress. Draught Lines is a publication of Origlio Beverage. The plates were kept at 22 °C temperature in dark. Chickpea transgenic plants followed the similar phenotypes of transgenic Arabidopsis and showed up to 1.8‐fold increase in root length and lateral root numbers in 10 dpg stage when grown in soilrite pots in controlled growth chamber (Figure 3a–c). Seven CKX isoforms of Arabidopsis showed differential tissue‐specific expression, subcellular protein localization and substrate specificity (Werner et al., 2003). The relative decrease in RWC in the wild‐type plants was more than those of the transgenic lines (Figure S8b). The mechanism of initiation and elongation of LR is well studied in Arabidopsis. The metal concentrations were measured using inductively coupled plasma emission spectrometry (ICP‐MS; PerkinElmer Élan 9000‐USA). Daily Goals. Three biological replicates for each genotype were analysed. Similar search resulted in the identification of nine CKX‐encoding genes in the annotated Medicago truncatula genome assembly (Young et al., 2011). Table S1 List of primers used in this study. The ability of the root system to adjust in response to various abiotic stresses is an important aspect of the plant’s performance (Smith and De Smet, 2012). The chickpea WRKY protein that showed the highest sequence homology with AtWRKY6 has been annotated as CaWRKY31 (XP_004507670.1). This raised a parallel argument emphasizing the role of environment on sink activity as the limiting factor for tissue growth in which demand of assimilate usage, in addition to the assimilation, is ultimately responsible for net primary production at favourable growth conditions where the plant is not resource‐starved (Körner, 2015; Marcelis, 1996; Sonnewald and Fernie, 2018). of India). Expression of AtCKX1 and AtCKX3 under predominantly root‐expressing promoters of AtWRKY6 or AtPYK10 caused increased root growth without compromising shoot growth (Werner et al., 2010). Appendix S3 Histochemical GUS staining and protein localization, RNA isolation, RT‐qPCR, CKX activity assay and Phytohormone assay. For instance, chickpea cultivars with higher root length density are more drought‐tolerant and high yielding under terminal drought (Kashiwagi et al., 2006; Singh et al., 2016). However, information about CKX gene of an edible legume crops and their application to improve agronomic traits is lacking. If you do not receive an email within 10 minutes, your email address may not be registered, Perfect for any occation. All authors have read and approved the final manuscript. The net assimilation rate, internal CO2 concentration, stomatal conductance and transpiration rate were all recorded from four biological replicates and three technical replicates. After 48 h, these explants were washed with autoclaved MilliQ water containing cefotaxime 250 mg/L to remove the excess bacterial cells. Our American IPA is fully loaded with a juggernaut of hops that will send earthquakes crashing through your taste buds. The root system is highly plastic in its development and adaptation to variable environmental conditions such as uneven water and nutrient distribution. Higher shoot biomass without changes in plant height was observed due to an increase in the number of nodes and secondary branches in the soil‐grown matured plants (Figure 4e). To explore subcellular localization of CaCKX6 protein, the CaCKX6‐YFP construct was agroinfiltrated in Nicotiana benthamiana leaves along with endoplasmic reticulum (ER) marker (CD3‐959 mCherry) and a plasma membrane (PM) marker (CD3‐1007 mCherry) (Nelson et al., 2007). Spatio‐temporal regulation of cytokinin is very important during nodule development. A significant increase (1.7–2.2‐fold) in the expression of CaCKX6 was observed by RT‐qPCR in the root, while expression of the transgene in the shoot was not detected (Figure 3d–e). 100 %18 gCarbs. CaCKX6 cDNA was amplified from total mRNA by RT‐PCR using primers (CaCKX6_F and CaCKX6_R) and cloned after CaWRKY31 promoter between Sma1 and Sac1 sites in pBI 101.2 binary vector. The transformed soil‐grown 6 dpg old chickpea plants showed expression of the reporter protein activity only in the root (both primary and the lateral root). This observation raised a long‐debated issue of carbon partitioning between vegetative and reproductive growth, and between shoot and root. Starting with all of the familiar Loose Cannon aromas and flavors, aging this beer in Jameson casks added healthy notes of vanilla, oak, and warming whiskey that transformed Loose Cannon into a completely new experience. Roots were carefully lifted from the soil pots and cautiously washed to remove bound soil particles. Integration of single copy transgene in the genomic DNA was verified by Southern blot using a 196‐base probe designed with the sequence from the junction between CaWRKY31 5’UAS and CaCKX6 CDS (Figure S5, Appendix S2). We expressed chickpea cytokinin oxidase 6 (CaCKX6) under the control of a chickpea root‐specific promoter of CaWRKY31 in Arabidopsis thaliana and chickpea having determinate and indeterminate growth patterns, respectively, to study the effect of cytokinin depletion on root growth and drought tolerance. II. To investigate in planta expression pattern of CaWRKY31 promoter in chickpea, an efficient chickpea transformation protocol was established by modifying published methods (Bhatnagar‐Mathur et al., 2009; Chakraborti et al., 2006; Jayanand et al., 2003; Sarmah et al., 2004; Sharma et al., 2006). Personalized health review for User added: Loose cannon IPA: 220 calories, nutrition grade (N/A), problematic ingredients, and more. Grafting of AtCKX1‐overexpressing tobacco shoot on wild‐type root and vice‐versa did not alter the phenotypes of roots and shoots derived from the other plants indicating that the effect of cytokinin degradation is limited within the CKX1‐expressing tissue only (Werner et al., 2010). Seeds were sown in PVC pipes (50 cm height/ 20 cm diameter) containing soil and sand mixture (50:50/v:v) having a relative soil moisture content of 14%–17% throughout the pipe in growth chamber maintained with parameters mentioned above. The authors are grateful to the DBT‐eLibrary Consortium (DeLCON) for providing access to e‐Resources. Root‐to‐shoot biomass ratio was increased by up to 1.7‐fold in transgenic chickpea lines (Figure 4f). Transgenic lines produced an equivalent amount of ethylene suggesting root‐specific cytokinin degradation in the transgenic chickpea lines did not affect N2‐fixation in chickpea (Figure 5c). Enhancing the root network by local manipulation of cytokinin level might be an effective approach along with conventional breeding to alleviate yield loss in chickpea and other indeterminate legume crops.

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