Effects of N application methods on cotton yield and fertilizer N recovery efficiency in salinity fields with drip irrigation under mulch film using 15N tracing technique.

Introduction: Drip irrigation under mulch film promotes a non-uniform salinity distribution in salt fields. The effect of different N application methods on the growth and yield of cotton under drip irrigation under mulch film conditions in eastern coastal saline-alkaline soils in China remain remained unclear. Methods: A randomized complete block design was used in the experiment. Three N application methods were assigned: N applied under mulch film (low-salinity area; UM), N applied between mulch films (high-salinity area; BM), and half N applied under mulch film and half between mulch films (HUHB). Results: Plant height, photosynthesis, Chl content, boll load, biomass, boll weight and boll density under UM were all significantly higher than those under the other two treatments. The N absorption of UM was higher than in the other two treatments, which might be attributed to the expression of GHNRT1.5 and GHNRT2.1. The net NO3- influx in the roots in UM increased significantly compared with that in BM. The yield and FNRE of UM were 3.9% and 9.1%, respectively, and were 26.52% and 90.36% higher than under HUHB and BM treatments. Discussion: UM not only improved cotton yield but also alleviated the pollution of N residue on drip irrigation under mulch film conditions in salt areas.

C-terminally encoded peptides act as signals to increase cotton root nitrate uptake under nonuniform salinity.

Soil salinity is often heterogeneous in saline fields. Nonuniform root salinity increases nitrate uptake into cotton (Gossypium hirsutum) root portions exposed to low salinity, which may be regulated by root portions exposed to high salinity through a systemic long-distance signaling mechanism. However, the signals transmitted between shoots and roots and their precise molecular mechanisms for regulating nitrate uptake remain unknown. Here, we showed that nonuniform root salinity treatment using split-root systems increases the expression of C-TERMINALLY ENCODED PEPTIDE (GhCEP) genes in high-saline-treated root portions. GhCEP peptides originating in high-saline-treated root portions act as ascending long-distance mobile signals transported to the shoots to promote the expression of CEP DOWNSTREAM (GhCEPD) genes by inducing the expression of CEP receptor (GhCEPR) genes. The shoot-derived GhCEPD polypeptides act as descending mobile signals transported to the roots through the phloem, increasing the expression of nitrate transport genes NITRATE TRANSPORTER 1.1 (GhNRT1.1), GhNRT2.1, and GhNRT1.5 in nonsaline-treated root portions, thereby increasing nitrate uptake in the nonsaline-treated root portions. This study indicates that GhCEP and GhCEPD signals are transported between roots and shoots to increase nitrate uptake in cotton, and the transport from the nonsaline root side is in response to nonuniform root salinity distribution.

Light induced shoot-sourced transcription factor HY5 regulates the nitrate uptake of cotton by shoot-to-root signal transport.

Elongated hypocotyls 5 (HY5) is a transcription factor that can be induced by illumination and promotes nitrate uptake in Arabidopsis. However, whether GhHY5 regulates nitrate uptake in cotton is unknown. In this study, the cotton seedlings growing in light and dark conditions were treated with 15N-labeled nutrient solution to study whether the GhHY5 regulates nitrate uptake in cotton. The results showed that the 15N content and GhNRT1.1 expression in the light condition were higher than that in the dark condition, indicating that light induced the expression of GhNRT1.1 and subsequently promoted N uptake. Additionally, the expression of GhHY5 in the leaf and root of cotton was induced by light and the expression pattern of GhHY5 in the root was similar to that of GhNRT1.1. Furthermore, when the GhHY5 expression in the root was reduced, the 15N content and GhNRT1.1 expression were both decreased, indicating that the GhNRT1.1 expression was regulated by GhHY5. The root expression of GhHY5 was decreased in the grafted seedlings which the GhHY5 in the shoot was silenced by VIGS or the seedlings which the hypocotyl was girdled, but the expression of GhHY5 on one side root of the grafted cotton seedling was not changed if the GhHY5 was silenced on the other side root. Thus, we proposed that the light induced shoot-derived GhHY5 gene or GhHY5 protein may be transported from the xylem to the root, regulating the expression of GhHY5 and GhNRT1.1, and thus regulating N uptake at the root of cotton.

Seeding depth and seeding rate regulate apical hook formation by inducing GhHLS1 expression via ethylene during cotton emergence.

Apical hook formation is essential for the emergence and stand establishment of cotton plants. Searching for agronomic measures to regulate apical hook formation and clarifying its mechanism are important for full stand establishment in cotton. In this study, cotton seeds were sown at varying seeding rates or depths in sand to determine if and how apical hook formation was regulated by seeding rates or depths. The results showed that deep seeding or low seeding rates increased mechanical pressure and then increased ethylene content by increasing GhACO1 and GhACS2 expression to improve apical hook formation. Silencing of the GhACO1 and GhACS2 genes or exogenous application of 1-methylcyclopropene (1-MCP) decreased the ethylene content and inhibited apical hook formation in the cotton seedlings. Deep seeding, a low seeding rate, or 1-amino cyclopropane-1-carboxylic acid (ACC) treatment increased the expression of GhHLS1 and GhPIF3 genes, but their expression was decreased in theVIGS-ACO1 and VIGS-ACS2 seedlings. Silencing of the GhHLS1 and GhPIF3 genes inhibited apical hook formation, although the expression of GhACO1 and GhACS2 was unchanged. GhPIF3 may act upstream of GhHLS1, as the expression of GhPIF3 in the VIGS-HLS1 seedlings was unchanged, while the expression of GhHLS1 in the VIGS-PIF3 seedlings decreased. These results suggested that raised mechanical pressure could increase ethylene content by inducing GhACO1 and GhACS2 gene expression, which promoted apical hook formation by increasing the expression of GhHLS1. Therefore, adjusting the mechanical pressure through changing the seeding depth or seeding rate is an important means to regulate apical hook formation and emergence.

Leaf-Derived Jasmonate Mediates Water Uptake from Hydrated Cotton Roots under Partial Root-Zone Irrigation.

Partial root-zone irrigation (PRI), a water-saving technique, improves water uptake in hydrated roots by inducing specific responses that are thought to be regulated by signals originating from leaves; however, this signaling is poorly understood. Using a split-root system and polyethylene glycol 6000 to simulate PRI in cotton (Gossypium hirsutum), we showed that increased root hydraulic conductance (L) and water uptake in the hydrated roots may be due to the elevated expression of cotton plasma membrane intrinsic protein (PIP) genes. Jasmonate (jasmonic acid [JA] and jasmonic acid-isoleucine conjugate [JA-Ile]) content and the expression of three JA biosynthesis genes increased in the leaves of the PRI plants compared with those of the polyethylene glycol-free control. JA/JA-Ile content also increased in the hydrated roots, although the expression of the three JA genes was unaltered, compared with the control. The JA/JA-Ile contents in leaves increased after the foliar application of exogenous JA and was followed by an increase in both JA/JA-Ile content and L in the hydrated roots, whereas the silencing of the three JA genes had the opposite effect in the leaves. Ring-barking the hydrated hypocotyls increased the JA/JA-Ile content in the leaves but decreased the JA/JA-Ile content and L in the hydrated roots. These results suggested that the increased JA/JA-Ile in the hydrated roots was mostly transported from the leaves through the phloem, thus increasing L by increasing the expression of GhPIP in the hydrated roots under PRI. We believe that leaf-derived JA/JA-Ile, as a long-distance signal, positively mediates water uptake from the hydrated roots of cotton under PRI.

Global gene expression in cotton (Gossypium hirsutum L.) leaves to waterlogging stress.

Cotton is sensitive to waterlogging stress, which usually results in stunted growth and yield loss. To date, the molecular mechanisms underlying the responses to waterlogging in cotton remain elusive. Cotton was grown in a rain-shelter and subjected to 0 (control)-, 10-, 15- and 20-d waterlogging at flowering stage. The fourth-leaves on the main-stem from the top were sampled and immediately frozen in liquid nitrogen for physiological measurement. Global gene transcription in the leaves of 15-d waterlogged plants was analyzed by RNA-Seq. Seven hundred and ninety four genes were up-regulated and 1018 genes were down-regulated in waterlogged cotton leaves compared with non-waterlogged control. The differentially expressed genes were mainly related to photosynthesis, nitrogen metabolism, starch and sucrose metabolism, glycolysis and plant hormone signal transduction. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that most genes related to flavonoid biosynthesis, oxidative phosphorylation, amino acid metabolism and biosynthesis as well as circadian rhythm pathways were differently expressed. Waterlogging increased the expression of anaerobic fermentation related genes, such as alcohol dehydrogenase (ADH), but decreased the leaf chlorophyll concentration and photosynthesis by down-regulating the expression of photosynthesis related genes. Many genes related to plant hormones and transcription factors were differently expressed under waterlogging stress. Most of the ethylene related genes and ethylene-responsive factor-type transcription factors were up-regulated under water-logging stress, suggesting that ethylene may play key roles in the survival of cotton under waterlogging stress.

Non-uniform salinity in the root zone alleviates salt damage by increasing sodium, water and nutrient transport genes expression in cotton.

Non-uniform salinity alleviates salt damage through sets of physiological adjustments in Na+ transport in leaf and water and nutrient uptake in the non-saline root side. However, little is known of how non-uniform salinity induces these adjustments. In this study, RNA sequencing (RNA-Seq) analysis shown that the expression of sodium transport and photosynthesis related genes in the non-uniform treatment were higher than that in the uniform treatment, which may be the reason for the increased photosynthetic (Pn) rate and decreased Na+ content in leaves of the non-uniform salinity treatment. Most of the water and nutrient transport related genes were up-regulated in the non-saline root side but down-regulated in roots of the high-saline side, which might be the key reason for the increased water and nutrient uptake in the non-saline root side. Furthermore, the expression pattern of most differentially expressed transcription factor and hormone related genes in the non-saline root side was similar to that in the high-saline side. The alleviated salt damage by non-uniform salinity was probably attributed to the increased expression of salt tolerance related genes in the leaf and that of water and nutrient uptake genes in the non-saline root side.

Establishment of New Split-root System by Grafting.

A new split-root system was used to simulate non-uniform salt, drought or nutrient deficiency stress in the root zone, in which the root system was divided into two or more equal portions. Here, we established a split-root system by grafting of cotton seedlings. In contrast to the conventional split-root, the main roots of the new system remained intact, which provided a better system for studying cotton response to unequal treatment in the root zone. The new system was suitable for plant growth in nutrient solution and the two root systems can fully be immerged in the nutrient solution.

H2O2 and ABA signaling are responsible for the increased Na+ efflux and water uptake in Gossypium hirsutum L. roots in the non-saline side under non-uniform root zone salinity.

Non-uniform root salinity increases the Na(+)efflux, water use, and growth of the root in non-saline side, which may be regulated by some form of signaling induced by the high-salinity side. However, the signaling and its specific function have remained unknown. Using a split-root system to simulate a non-uniform root zone salinity in Gossypium hirsutum L., we showed that the up-regulated expression of sodium efflux-related genes (SOS1, SOS2, PMA1, and PMA2) and water uptake-related genes (PIP1 and PIP2) was possibly involved in the elevated Na(+) efflux and water use in the the roots in the non-saline side. The increased level of indole acetic acid (IAA) in the non-saline side was the likely cause of the increased root growth. Also, the abscisic acid (ABA) and H2O2 contents in roots in the non-saline side increased, possibly due to the increased expression of their key biosynthesis genes, NCED and RBOHC, and the decreased expression of ABA catabolic CYP707A genes. Exogenous ABA added to the non-saline side induced H2O2 generation by up-regulating the RBOHC gene, but this was decreased by exogenous fluridone. Exogenous H2O2 added to the non-saline side reduced the ABA content by down-regulating NCED genes, which can be induced by diphenylene iodonium (DPI) treatment in the non-saline side, suggesting a feedback mechanism between ABA and H2O2.Both exogenous ABA and H2O2 enhanced the expression of SOS1, PIP1;7 ,PIP2;2, and PIP2;10 genes, but these were down-regulated by fluridone and DPI, suggesting that H2O2 and ABA are important signals for increasing root Na(+) efflux and water uptake in the roots in the non-saline side.

Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development.

Many secondary metabolites have insecticidal efficacy against pests and may be affected by abiotic stress. However, little is known of how plants may respond to such stress as pertains the growth and development of pests. The objective of this study was to determine if and how salt stress on cotton plants affects the population dynamics of aphids. The NaCl treatment (50 mM, 100 mM, 150 mM and 200 mM) increased contents of gossypol in cotton by 26.8-51.4%, flavonoids by 22.5-37.6% and tannic by 15.1-24.3% at 7-28 d after salt stress. Compared with non-stressed plants, the population of aphids on 150 and 200 mM NaCl stressed plants was reduced by 46.4 and 65.4% at 7d and by 97.3 and 100% at 14 days after infestation. Reductions in aphid population were possibly attributed to the elevated secondary metabolism under salt stress. A total of 796 clones for aphids transcriptome, 412 clones in the positive- library (TEST) and 384 clones in the reverse-library (Ck), were obtained from subtracted cDNA libraries and sequenced. Gene ontology (GO) functional classification and KEGG pathway analysis showed more genes related to fatty acid and lipid biosynthesis, and fewer genes related to carbohydrate metabolism, amino acid metabolism, energy metabolism and cell motility pathways in TEST than in Ck library, which might be the reason of aphids population reduction. A comparative analysis with qRT-PCR indicated high expression of transcripts CYP6A14, CYP6A13, CYP303A1, NADH dehydrogenase and fatty acid synthase in the TEST group. However, CYP307A1 and two ecdysone-induced protein genes were down regulated. The results indicate that genes of aphids related to growth and development can express at a higher level in reaction to the enhanced secondary metabolism in cotton under salinity stress. The expression of CYP307A1 was positively correlated with the population dynamics of aphids since it was involved in ecdysone synthesis.

Gene expression profiles deciphering leaf senescence variation between early- and late-senescence cotton lines.

Leaf senescence varies greatly among genotypes of cotton (Gossypium hirsutium L), possibly due to the different expression of senescence-related genes. To determine genes involved in leaf senescence, we performed genome-wide transcriptional profiling of the main-stem leaves of an early- (K1) and a late-senescence (K2) cotton line at 110 day after planting (DAP) using the Solexa technology. The profiling analysis indicated that 1132 genes were up-regulated and 455 genes down-regulated in K1 compared with K2 at 110 DAP. The Solexa data were highly consistent with, and thus were validated by those from real-time quantitative PCR (RT-PCR). Most of the genes related to photosynthesis, anabolism of carbohydrates and other biomolecules were down-regulated, but those for catabolism of proteins, nucleic acids, lipids and nutrient recycling were mostly up-regulated in K1 compared with K2. Fifty-one differently expressed hormone-related genes were identified, of which 5 ethylene, 3 brassinosteroid (BR), 5 JA, 18 auxin, 8 GA and 1 ABA related genes were up-regulated in K1 compared with K2, indicating that these hormone-related genes might play crucial roles in early senescence of K1 leaves. Many differently expressed transcription factor (TF) genes were identified and 11 NAC and 8 WRKY TF genes were up-regulated in K1 compared with K2, suggesting that TF genes, especially NAC and WRKY genes were involved in early senescence of K1 leaves. Genotypic variation in leaf senescence was attributed to differently expressed genes, particularly hormone-related and TF genes.

Effects of non-uniform root zone salinity on water use, Na+ recirculation, and Na+ and H+ flux in cotton.

A new split-root system was established through grafting to study cotton response to non-uniform salinity. Each root half was treated with either uniform (100/100 mM) or non-uniform NaCl concentrations (0/200 and 50/150 mM). In contrast to uniform control, non-uniform salinity treatment improved plant growth and water use, with more water absorbed from the non- and low salinity side. Non-uniform treatments decreased Na(+) concentrations in leaves. The [Na(+)] in the '0' side roots of the 0/200 treatment was significantly higher than that in either side of the 0/0 control, but greatly decreased when the '0' side phloem was girdled, suggesting that the increased [Na(+)] in the '0' side roots was possibly due to transportation of foliar Na(+) to roots through phloem. Plants under non-uniform salinity extruded more Na(+) from the root than those under uniform salinity. Root Na(+) efflux in the low salinity side was greatly enhanced by the higher salinity side. NaCl-induced Na(+) efflux and H(+) influx were inhibited by amiloride and sodium orthovanadate, suggesting that root Na(+) extrusion was probably due to active Na(+)/H(+) antiport across the plasma membrane. Improved plant growth under non-uniform salinity was thus attributed to increased water use, reduced leaf Na(+) concentration, transport of excessive foliar Na(+) to the low salinity side, and enhanced Na(+) efflux from the low salinity root.

Cloning and functional characterization of a cation-chloride cotransporter gene OsCCC1.

Potassium (K+) and chloride (Cl-) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K+ and Cl-, it remains unclear if they actively use K+-coupled Cl- cotransporters (KCC), as used in animals, to transport K+ and Cl-. We have cloned an Oryza sativa cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K--Cl- cotransporters than to other cation-Cl- cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na+, K+, and Cl- contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K+ and Cl- than the latter but that they did not differ in terms of Na+ contents, suggesting OsCCC1 may be involved in K+ and Cl- transport. Results from different tests indicated that the OsCCC1 plays a significant role in K+ and Cl- homeostasis and rice plant development.