GhRabA4c coordinates cell elongation via regulating actin filament-dependent vesicle transport.

Plant cell expands via a tip growth or diffuse growth mode. In plants, RabA is the largest group of Rab GTPases that regulate vesicle trafficking. The functions of RabA protein in modulating polarized expansion in tip growth cells have been demonstrated. However, whether and how RabA protein functions in diffuse growth plant cells have never been explored. Here, we addressed this question by examining the role of GhRabA4c in cotton fibers. GhRabA4c was preferentially expressed in elongating fibers with its protein localized to endoplasmic reticulum and Golgi apparatus. Over- and down-expression of GhRabA4c in cotton lead to longer and shorter fibers, respectively. GhRabA4c interacted with GhACT4 to promote the assembly of actin filament to facilitate vesicle transport for cell wall synthesis. Consistently, GhRabA4c-overexpressed fibers exhibited increased content of wall components and the transcript levels of the genes responsible for the synthesis of cell wall materials. We further identified two MYB proteins that directly regulate the transcription of GhRabA4c Collectively, our data showed that GhRabA4c promotes diffused cell expansion by supporting vesicle trafficking and cell wall synthesis.

Morphological, physiological and molecular assessment of cotton for drought tolerance under field conditions.

Climate change could be an existential threat to many crops. Drought and heat stress are becoming harder for cultivated crops. Cotton in Pakistan is grown under natural high temperature and low moisture, could be used as a source of heat and drought tolerance. Therefore, the study was conducted to morphological, physiological and molecular characterization of cotton genotypes under field conditions. A total of 25 cotton genotypes were selected from the gene pool of Pakistan based on tolerance to heat and drought stress. In field trail, the stress related traits like boll retention percentage, plant height, number of nodes and inter-nodal distance were recorded. In physiological assessment, traits such as photosynthesis rate, stomatal conductance, transpiration rate, leaf temperature, relative water content and excised leaf water loss were observed. At molecular level, a set of 19 important transcription factors, controlling drought/heat stress tolerance (HSPCB, GHSP26, HSFA2, HSP101, HSP3, DREB1A, DREB2A, TPS, GhNAC2, GbMYB5, GhWRKY41, GhMKK3, GhMPK17, GhMKK1, GhMPK2, APX1, HSC70, ANNAT8, and GhPP2A1) were analyzed from all genotypes. Data analyses depicted that boll retention percentage, photosynthesis, stomatal conductance, relative water content under the stress conditions were associated with the presence of important drought & heat TF/genes which depicts high genetic potential of Pakistani cotton varieties against abiotic stress. The variety MNH-886 appeared in medium plant height, high boll retention percentage, high relative water content, photosynthesis rate, stomatal conductance, transpiration rate and with maximum number transcription factors under study. The variety may be used as source material for heat and drought tolerant cotton breeding. The results of this study may be useful for the cotton breeders to develop genotype adoptable to environmental stresses under climate change scenario.

The Transcriptional Landscape and Hub Genes Associated with Physiological Responses to Drought Stress in Pinus tabuliformis.

Drought stress has an extensive impact on regulating various physiological, metabolic, and molecular responses. In the present study, the Pinus tabuliformis transcriptome was studied to evaluate the drought-responsive genes using RNA- Sequencing approache. The results depicted that photosynthetic rate and H2O conductance started to decline under drought but recovered 24 h after re-watering; however, the intercellular CO2 concentration (Ci) increased with the onset of drought. We identified 84 drought-responsive transcription factors, 62 protein kinases, 17 transcriptional regulators, and 10 network hub genes. Additionally, we observed the expression patterns of several important gene families, including 2192 genes positively expressed in all 48 samples, and 40 genes were commonly co-expressed in all drought and recovery stages compared with the control samples. The drought-responsive transcriptome was conserved mainly between P. tabuliformis and A. thaliana, as 70% (6163) genes had a homologous in arabidopsis, out of which 52% homologous (3178 genes corresponding to 2086 genes in Arabidopsis) were also drought response genes in arabidopsis. The collaborative network exhibited 10 core hub genes integrating with ABA-dependent and independent pathways closely conserved with the ABA signaling pathway in the transcription factors module. PtNCED3 from the ABA family genes had shown significantly different expression patterns under control, mild, prolonged drought, and recovery stages. We found the expression pattern was considerably increased with the prolonged drought condition. PtNCED3 highly expressed in all drought-tested samples; more interestingly, expression pattern was higher under mild and prolonged drought. PtNCED3 is reported as one of the important regulating enzymes in ABA synthesis. The continuous accumulation of ABA in leaves increased resistance against drought was due to accumulation of PtNCED3 under drought stress in the pine needles.

Genome-Wide Association Analysis Reveals Loci and Candidate Genes Involved in Fiber Quality Traits Under Multiple Field Environments in Cotton (Gossypium hirsutum).

Fiber length, fiber strength, and fiber micronaire are the main fiber quality parameters in cotton. Thus, mining the elite and stable loci/alleles related to fiber quality traits and elucidating the relationship between the two may accelerate genetic improvement of fiber quality in cotton. Here, genome-wide association analysis (GWAS) was performed for fiber quality parameters based on phenotypic data, and 56,010 high-quality single nucleotide polymorphisms (SNPs) using 242 upland cotton accessions under 12 field environments were obtained. Phenotypic analysis exhibited that fiber length (FL) had a positive correlation with fiber strength (FS) and had a negative correlation with fiber micronaire (Mic). Genetic analysis also indicated that FL, FS, and Mic had high heritability of more than 80%. A total of 67 stable quantitative trait loci (QTLs) were identified through GWAS analysis, including 31 for FL, 21 for FS, and 22 for Mic. Of them, three pairs homologous QTLs were detected between A and D subgenomes, and seven co-located QTLs with two fiber quality parameters were found. Compared with the reported QTLs, 34 co-located with previous studies, and 33 were newly revealed. Integrated with transcriptome analysis, we selected 256, 244, and 149 candidate genes for FL, FS, and Mic, respectively. Gene Ontology (GO) analysis showed that most of the genes located in QTLs interval of the three fiber quality traits were involved in sugar biosynthesis, sugar metabolism, microtubule, and cytoskeleton organization, which played crucial roles in fiber development. Through correlation analysis between haplotypes and phenotypes, three genes (GH_A05G1494, GH_D11G3097, and GH_A05G1082) predominately expressed in fiber development stages were indicated to be potentially responsible for FL, FS, and Mic, respectively. The GH_A05G1494 encoded a protein containing SGS-domain, which is related to tubulin-binding and ubiquitin-protein ligase binding. The GH_D11G3097 encoded 20S proteasome beta subunit G1, and was involved in the ubiquitin-dependent protein catabolic process. The GH_A05G1082 encoded RAN binding protein 1 with a molecular function of GTPase activator activity. These results provide new insights and candidate loci/genes for the improvement of fiber quality in cotton.

Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants.

Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.

Impact of heat stress responsive factors on growth and physiology of cotton (Gossypium hirsutum L.).

Pakistan ranked highest with reference to average temperatures in cotton growing areas of the world. The heat waves are becoming more intense and unpredictable due to climate change. Identification of heat tolerant genotypes requires comprehensive screening using molecular, physiological and morphological analysis. Heat shock proteins play an important role in tolerance against heat stress. In the current study, eight heat stress responsive factors, proteins and genes (HSFA2, GHSP26, GHPP2A, HSP101, HSC70-1, HSP3, APX1 and ANNAT8) were evaluated morphologically and physiologically for their role in heat stress tolerance. For this purpose, cotton crop was grown at two temperature conditions i.e. normal weather and heat stress at 45 °C. For molecular analysis, genotypes were screened for the presence or absence of heat shock protein genes. Physiological analysis of genotypes was conducted to assess net photosynthesis, stomatal conductance, transpiration rate, leaf-air temperature and cell membrane stability under control as well as high temperature. The traits photosynthesis, cell membrane stability, leaf-air temperature and number of heat stress responsive factors in each genotypes showed a strong correlation with boll retention percentage under heat stress. The genotypes with maximum heat shock protein genes such as Cyto-177, MNH-886, VH-305 and Cyto-515 showed increased photosynthesis, stomatal conductance, negative leaf-air temperature and high boll retention percentage under heat stress condition. These varieties may be used as heat tolerant breeding material.

Quantitative trait loci (QTL) mapping for physiological and biochemical attributes in a Pasban90/Frontana recombinant inbred lines (RILs) population of wheat (Triticum aestivum) under salt stress condition.

Salt stress causes nutritional imbalance and ion toxicity which affects wheat growth and production. A population of recombinant inbred lines (RILs) were developed by crossing Pasban90 (salt tolerant) and Frontana (salt suceptible) for identification of quantitative trait loci (QTLs) for physiological traits including relative water content, membrane stability index, water potential, osmotic potential, total chlorophyll content, chlorophyll a, chlorophyll b and biochemical traits including proline contents, superoxide dismutase, sodium content, potassium content, chloride content and sodium/potassium ratio by tagging 202 polymorphic simple sequence repeats (SSR) markers. Linkage map of RILs comprised of 21 linkage group covering A, B and D genome for tagging and maped a total of 60 QTLs with major and minor effect. B genome contributed to the highest number of QTLs under salt stress condition. Xgwm70 and Xbarc361 mapped on chromosome 6B was linked with Total chlorophyll, water potential and sodium content. The increasing allele for all these QTLs were advanced from parent Pasban90. Current study showed that Genome B and D had more potentially active genes conferring plant tolerance against salinity stress which may be exploited for marker assisted selection to breed salinity tolerant high yielding wheat varieties.

Manure storage operations mitigate nutrient losses and their products can sustain soil fertility and enhance wheat productivity.

Livestock manure is a valuable source of nutrients for plants. However, poor handling practices during storage resulted in nutrient losses from the manure and decrement in its nitrogen (N) fertilizer value. We explored the influence of divergent storage methods on manure chemical composition, carbon (C) and N losses to the environment as well as fertilizer value of storage products after their application to the wheat. Fresh buffalo manure (FM) was subjected to different storage operations for a period of ∼6 months, (i) fermentation by covering with a plastic sheet (CM) (ii) placed under the roof (RM) (iii) heap was unturned (SM) to remain stacked at an open space and (iv) manure heap turned monthly (TM) to make compost. During storage, 8, 24, 45 and 46% of the initial Ntotal was lost from CM, RM, SM, and TM, respectively. The respective C losses from these treatments were 16, 34, 47 and 44% of the initial C content. After stored manures application to the wheat crop, mineral N in the soil remained 27% higher in CM (14.1 vs. 11.1 kg ha-1) and 3% (10.8 vs. 11.1 kg ha-1) lower in SM compared to FM treatment. In contrast, microbial biomass C and N was 35 (509 vs.782 mg C kg-1 soil) and 25% (278 vs.370 mg N kg-1 soil) lower in CM than FM treatment, respectively indicating lower N immobilization of CM in the soil. These findings could result in the highest grain yield (5166 kg ha-1) and N uptake (117 kg ha-1) in CM and the lowest in SM treatments (3105 and 61 kg ha-1, respectively). Similarly, wheat crop recovered 44, 15 and 13% N from CM, TM and SM, respectively. Hence, management operations play a critical role in conserving N during storage phase and after stored manure application to the field. Among the studied operations, storing animal manure under an impermeable plastic sheet is a much better and cheaper option for decreasing N losses during storage and improving wheat yield when incorporated into the soil. Therefore, by adopting this manure storage technique, farmers can improve the agro-environmental value of animal manure in Pakistan.