Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana.

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H2O2, as indicated by fluorescence probes, BES-So-AM and CM-H2DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H2O2 scavenger) confirmed that both the superoxide and H2O2 were requisites for stomatal closure. Co-localization of the superoxide and H2O2 in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H2O2 generation in mitochondria. In contrast, MD elevated superoxide/H2O2 levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.

Melatonin-mediated regulation of autophagy is independent of ABA under drought stress in sensitive variety of Gossypium hirsutum L.

Autophagy is a highly conserved process that plays a crucial role in adaptation of plants to stress conditions. Melatonin and abscisic acid (ABA) share an antagonistic relationship; however, both are reported to elevate autophagy individually. Here, we report that melatonin alleviates drought stress effects like wilting and stunted growth in 18-day-old plants of drought-sensitive variety of cotton (Gossypium hirsutum L.) and improves the plant growth, chlorophyll content, photosynthetic efficiency, and sugar metabolism and transport. Melatonin priming increased the endogenous melatonin content (5.02-times) but decreased the ABA (2.63-times) by reducing NCED3 expression as compared to unprimed plants under drought. Also, elevated expression of ATG8c and ATG8f correlated with higher lipidated-ATG8 levels and modulation of RAPTOR1 suggesting a higher occurrence of autophagy and regulation of plant growth in primed stressed plants. Additionally, decreased TPS63 and increased TPP22 expression could have lowered the accumulation of trehalose-6-P (T6P) in primed stressed plants thus contributing to autophagy progression. Priming also enhanced the expression of MAPK6 and RAF18, and increased the transcript/protein levels of SnRK2.6 and KIN10, which is pointing towards melatonin's beneficial effect on autophagy under drought. Despite higher ABA content, elevated TPS63 and downregulated TPP22 could have hindered autophagy induction in unprimed stressed plants. Although fluridone treatment reduced the ABA content, the expression of SnRK2.6 and KIN10 remained unaltered in fluridone-treated and untreated primed plants indicating the ABA-independent expression. These results suggest that the melatonin-mediated activation of MAPK contributes to the ABA-independent activation of SnRK2, consequently, SnRK1 and autophagy under drought.

Complete genome sequencing and construction of full-length infectious cDNA clone of papaya ringspot virus-HYD isolate and its efficient in planta expression.

Papaya ringspot virus (PRSV) is a devastating Potyvirus that causes papaya ringspot disease in Carica papaya plantations globally. In this study, the complete genome sequence of a PRSV isolate from Shankarpalli, Telangana, India, was reported and designated as PRSV-HYD (KP743981.1). The genome is a single-stranded positive-sense RNA comprising 10,341 nucleotides. Phylogenetic analysis revealed that PRSV-HYD is closely related to PRSV Pune (Aundh) isolate with 92 and 95% nucleotide and amino acid sequence identity, respectively. To develop infectious cDNA (icDNA), the complete nucleotide sequence of PRSV-HYD was cloned between the right and left borders in the binary vector pCB301 using BglII and XmaI restriction sites. Cauliflower mosaic virus (CaMV) double promoter (35S) was fused at the 5'-end and Avocado sunblotch viroid (ASBVd) ribozyme (RZ) sequence was fused to the 3' end to generate an authentic 3' viral end in the transcribed mRNAs. The icDNA generated was mobilized into the Agrobacterium tumefaciens EHA 105, and the agrobacterial cultures were infiltrated into the natural host C. papaya and a non-host Nicotiana benthamiana plants; both did not show any symptoms. In RT-PCR analysis of RNAs isolated from N. benthamiana, we could detect viral genes as early as 3 days and continued up to 28 days post infiltration. Alternatively, virion particles were purified from agroinfiltrated N. benthamiana plants and introduced into C. papaya by mechanical inoculation as well as by pinprick method. In both cases, we could see visible systemic symptoms similar to that of wild type by 40 days. Additionally, we studied the expression patterns of the genes related to plant defense, transcription factors (TFs), and developmental aspects from both C. papaya and N. benthamiana.

Melatonin induces drought tolerance by modulating lipoxygenase expression, redox homeostasis and photosynthetic efficiency in Arachis hypogaea L.

Melatonin (N-acetyl-5-hydroxy tryptamine), a multipotent biomolecule is well known for its ability to confer tolerance to several abiotic and biotic stresses. The regulation of melatonin-mediated drought tolerance in drought-distinguished varieties can be different due to discriminating redox levels. The present study was focused on assessing the effects of melatonin priming against polyethylene glycol (PEG)-induced stress with respect to the antioxidant system, photosynthetic parameters, lipoxygenase expression, JA and ABA levels in drought-sensitive (Kadiri-7) and drought-tolerant (Kadiri-9) varieties. Exogenous melatonin alleviated the drought stress effects in sensitive variety (Kadiri-7) by increasing the endogenous melatonin content with an improved antioxidant system and photosynthetic attributes. The primed stressed plants of the sensitive variety exhibited reduced expression and activity of the chlorophyll degrading enzymes (Chl-deg PRX, pheophytinase and chlorophyllase) with a concomitant increase in chlorophyll content in comparison to unprimed controls. Interestingly, melatonin priming stimulated higher expression and activity of lipoxygenase (LOX) as well as enhanced the expression of genes involved in the synthesis of jasmonic acid (JA) including its content in drought stressed plants of the sensitive variety. The expression of NCED3 (involved in ABA-biosynthesis) was upregulated while CYP707A2 (ABA-degradation) was downregulated which corresponded with higher ABA levels. Contrastingly, priming caused a decrease in endogenous melatonin content under drought stress in tolerant variety (Kadiri-9) which might be due to feedback inhibition of its synthesis to maintain intracellular redox balance and regulate better plant metabolism. Furthermore, the higher endogenous melatonin content along with improved antioxidant system, photosynthetic efficiency and LOX expression associated with the increased levels of JA and ABA in unprimed stressed plants of the tolerant variety (Kadiri-9) is pointing towards the effectiveness of melatonin in mediating drought stress tolerance. Overall, exogenous melatonin alleviated the adverse effects of drought stress in sensitive variety while having no add-on effect on drought stress responses of tolerant variety which is inherently equipped to withstand the given duration of drought stress treatment.

Melatonin Mediated Differential Regulation of Drought Tolerance in Sensitive and Tolerant Varieties of Upland Cotton (Gossypium hirsutum L.).

Melatonin (N-acetyl-5-methoxytryptamine), a biomolecule with multifunctional phyto-protectant activities, enhances the tolerance to broad-spectrum biotic and abiotic stresses in plants. However, little information is available on the effect of melatonin on different morpho-physiological, biochemical, and molecular parameters during drought stress incidence in varieties contrastingly differing in their tolerance levels. The present study is aimed at investigating the drought stress responses of drought-sensitive (var. L-799) and drought-tolerant (var. Suraj) varieties after exogenous melatonin priming and gaining mechanistic insights into drought tolerance in upland cotton (Gossypium hirsutum). Melatonin-priming enhanced the tolerance of L-799 to drought stress by modulating the antioxidant system, with increased photosynthetic activity, water-use efficiency, and nitrogen metabolism. Higher endogenous melatonin content and upregulated expression of candidate stress-responsive genes in primed L-799 suggested their involvement in drought tolerance. The higher expression of autophagosome marker [lipidated (ATG8-PE)] in melatonin-primed drought-stressed plants of L-799 also indicated the role of autophagy in alleviating drought stress. Interestingly, melatonin-priming did not show pronounced differences in the different parameters studied during the presence or absence of drought stress in Suraj. In conclusion, this study showed that melatonin plays an important role in mitigating drought stress effects by modulating several physiological, biochemical, and molecular processes, with the key regulatory factor being the plant tolerance level that serves as the switch that turns the priming effects on/off.