Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks

Plants experiencing salt‐induced stress often reduce cytokinin levels during the early phases of stress‐response. Interestingly, we found that the cytokinin content in the apple rootstock “robusta” was maintained at a high level under salt stress. Through screening genes involved in cytokinin biosynthesis and catabolism, we found that the high expression levels of IPT5b in robusta roots were involved in maintaining the high cytokinin content. We identified a 42 bp deletion in the promoter region of IPT5b, which elevated IPT5b expression levels, and this deletion was linked to salt tolerance in robusta×M.9 segregating population. The 42 bp deletion resulted in the deletion of a Proline Response Element (ProRE), and our results suggest that ProRE negatively regulates IPT5b expression in response to proline. Under salt stress, the robusta cultivar maintains high cytokinin levels as IPT5b expression cannot be inhibited by proline due to the deletion of ProRE, leading to improve salt tolerance.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Overexpression of MsDREB6.2 results in cytokinin‐deficient developmental phenotypes and enhances drought tolerance in transgenic apple plants

Dehydration‐responsive element binding factors (DREBs) play important roles in plant growth, development, and stress signaling pathways in model plants. However, little is known about the function of DREBs in apple (Malus × domestica), a widely cultivated crop that is frequently threatened by drought. We isolated a DREB gene from Malus sieversii (Ledeb.) Roem., MsDREB6.2, and investigated its functions using overexpression analysis and c himeric re pressor gene‐s ilencing t echnology (CRES‐T). We identified possible target genes of the protein encoded by MsDREB6.2 using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). Overexpression of MsDREB6.2 increased the expression of a key cytokinin (CK) catabolism gene, MdCKX4a, which led to a significant reduction in endogenous CK levels, and caused a decrease in shoot:root ratio in transgenic apple plants. Overexpression of MsDREB6.2 resulted in a decrease in stomatal aperture and density and an increase in root hydraulic conductance (L₀), and thereby enhanced drought tolerance in transgenic plants. Furthermore, manipulating the level of MsDREB6.2 expression altered the expression of two aquaporin (AQP) genes. The effect of the two AQP genes on L₀ was further characterized using the AQP inhibitor HgCl₂. Based on these observations, we conclude that MsDREB6.2 enhances drought tolerance and that its function may be due, at least in part, to its influence on stomatal opening, root growth, and AQP expression. These results may have applications in apple rootstock breeding programs aimed at developing drought‐resistant apple varieties.     

Effects of varied water regimes on root length,dry matter partitioning and endogenous plant growth regulators in sunflower (Helianthus annuus L.)

Abstract

A field experiment was conducted to quantify the effect of varied water regimes on root length, partitioning of dry matter and plant growth regulators by using sunflower genotypes differing in maturity and drought tolerance. Significant depressing effect of drought stress was evident on traits (i.e., reproductive dry matter, leaf area index and cytokinin concentrations in leaves). However, root/shoot, reproductive/vegetative ratios and Abscisic acid (ABA) concentration were found to increase under drought stress. Drought stress also changed the dry matter accumulation pattern of genotypes. In most cases it reduced the days to reach the maximum peak showing early senescence.

ABA was identified as a multi-functional plant growth regulator under drought stress, causing early senescence of plants and translocation of assimilates to the roots and reproductive part while root growth under drought stress was explained by the indole-acetic acid (IAA) concentrations. Maintaining higher cytokinin contents were involved in accumulation of higher reproductive dry matter under drought stress. Although ABA and IAA were both involved in the development of defense responses during the adaptation and survival to drought stress but higher productivity under drought stress was only realized through maintaining higher cytokinin contents.     

A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana

A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.     

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.     

Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.     

A viral RNA silencing suppressor interferes with abscisic acid‐mediated signalling and induces drought tolerance in Arabidopsis thaliana

Cucumber mosaic virus (CMV) encodes the 2b protein, which plays a role in local and systemic virus movement, symptom induction and suppression of RNA silencing. It also disrupts signalling regulated by salicylic acid and jasmonic acid. CMV induced an increase in tolerance to drought in Arabidopsis thaliana. This was caused by the 2b protein, as transgenic plants expressing this viral factor showed increased drought tolerance, but plants infected with CMVΔ2b, a viral mutant lacking the 2b gene, did not. The silencing effector ARGONAUTE1 (AGO1) controls a microRNA‐mediated drought tolerance mechanism and, in this study, we noted that plants (dcl2/3/4 triple mutants) lacking functional short‐interfering RNA‐mediated silencing were also drought tolerant. However, drought tolerance engendered by CMV may be independent of the silencing suppressor activity of the 2b protein. Although CMV infection did not alter the accumulation of the drought response hormone abscisic acid (ABA), 2b‐transgenic and ago1‐mutant seeds were hypersensitive to ABA‐mediated inhibition of germination. However, the induction of ABA‐regulated genes in 2b‐transgenic and CMV‐infected plants was inhibited more strongly than in ago1‐mutant plants. The virus engenders drought tolerance by altering the characteristics of the roots and not of the aerial tissues as, compared with the leaves of silencing mutants, leaves excised from CMV‐infected or 2b‐transgenic plants showed greater stomatal permeability and lost water more rapidly. This further indicates that CMV‐induced drought tolerance is not mediated via a change in the silencing‐regulated drought response mechanism. Under natural conditions, virus‐induced drought tolerance may serve viruses by aiding susceptible hosts to survive periods of environmental stress.     

An apple sucrose transporter MdSUT2.2 is a phosphorylation target for protein kinase MdCIPK22 in response to drought

Sugars increase with drought stress in plants and accumulate in the vacuole. However, the exact molecular mechanism underlying this process is not clear yet. In this study, protein interaction and phosphorylation experiments were conducted for sucrose transporter and CIPK kinase in apple. The specific phosphorylation site of sucrose transporter was identified with mass spectrometry. Transgenic analyses were performed to characterize their biological function. It was found that overexpression of sucrose transporter gene MdSUT2.2 in apple plants promoted sugar accumulation and drought tolerance. MdSUT2.2 protein was phosphorylated at Ser³⁸¹ site in response to drought. A DUALmembrane system using MdSUT2.2 as bait through an apple cDNA library got a protein kinase MdCIPK22. Bimolecular fluorescence complementary (BiFC), pull‐down and co‐immunoprecipitation (Co‐IP) assays further demonstrated that MdCIPK22 interacted with MdSUT2.2. A series of transgenic analysis showed that MdCIPK22 was required for the drought‐induced phosphylation at Ser³⁸¹ site of MdSUT2.2 protein, and that it enhanced the stability and transport activity of MdSUT2.2 protein. Finally, it was found that MdCIPK22 overexpression promoted sugar accumulation and improved drought tolerance in an MdSUT2.2‐dependent manner in transgenic apple plants. MdCIPK22‐MdSUT2.2 regulatory module shed light on the molecular mechanism by which plant accumulates sugars and enhances tolerance in response to drought stress.     

The ubiquitin-binding protein MdRAD23D1 mediates drought response by regulating degradation of the proline-rich protein MdPRP6 in apple (Malus domestica)

RAD23 (RADIATION SENSITIVE23) proteins are a group of UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins that shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Drought stress is a major environmental constraint that limits plant growth and production, but whether RAD23 proteins are involved in this process is unclear. Here, we demonstrated that a shuttle protein, MdRAD23D1, mediated drought response in apple plants (Malus domestica). MdRAD23D1 levels increased under drought stress, and its suppression resulted in decreased stress tolerance in apple plants. Through in vitro and in vivo assays, we demonstrated that MdRAD23D1 interacted with a proline-rich protein MdPRP6, resulting in the degradation of MdPRP6 by the 26S proteasome. And MdRAD23D1 accelerated the degradation of MdPRP6 under drought stress. Suppression of MdPRP6 resulted in enhanced drought tolerance in apple plants, mainly because the free proline accumulation is changed. And the free proline is also involved in MdRAD23D1-mediated drought response. Taken together, these findings demonstrated that MdRAD23D1 and MdPRP6 oppositely regulated drought response. MdRAD23D1 levels increased under drought, accelerating the degradation of MdPRP6. MdPRP6 negatively regulated drought response, probably by regulating proline accumulation. Thus, “MdRAD23D1-MdPRP6” conferred drought stress tolerance in apple plants.