Conditional up-regulation of cytokinin status increases growth and survival of sugarcane in water-limited conditions

Abstract

Water deficit (water stress) is a major cause of yield loss across all crops. It is the single largest crop productivity constraint in sugarcane in most of sugarcane producing countries and is attracting considerable research interest. Several biological factors have been implicated to water stress responses in plants. Hormones, particularly abscisic acid (ABA), ethylene, cytokinins (CK) and gibberellins are emerging as key regulators of water stress responses. Here, I explored the effect of artificial elevation of CK levels on the response of young sugarcane plants to water stress. Focus of the research was on the onset of senescence and change in photosynthesis that typify water stress responses of plants. Understanding the mechanistic basis of responses of sugarcane to water stress may allow developing strategies for crop improvement and breeding. I studied the effects of modifying cytokinin (CK) levels in water-stressed sugarcane (Saccharum officinarum L.) via two strategies; exogenous supply of synthetic cytokinin N6-benzyladenine (BA) and conditional or constitutional up-regulation of CK biosynthesis in 113 independent transgenic sugarcane lines that were generated as part of this thesis research. Young plants were exposed to water stress in the glasshouse by maintaining soil moisture at 50% field capacity via daily irrigation. Exogenous application of CK occurred via foliar spray or a root drench, while conditional up-regulation of endogenous CK levels under water stress conditions was achieved by expressing the gene encoding CK biosynthesis regulatory enzyme, 2-isopentenyltransferase (IPT), via senescence-associated (SAG12) or abscisic acid-responsive (RAB17) promoters. Maize Ubi promoter was used for constitutive expression of IPT transgene. A popular Australian sugarcane variety Q208A was used for this study. All promotors elevated endogenous CK levels under water stress conditions in the majority of the transgenic lines created. Responses to water stress was studied by quantifying biomass, root/shoot allocation, leaf area, photosynthesis parameters, hormone profiles and gene expression to discern the effects of impaired water relations on carbon fixation and the basis of CKinduced growth improvement. Increased CK levels, via exogenous supply of BA or stress-induced expression of IPT, strongly improved sugarcane growth and survival under water stress. On average, plants with elevated CK-levels retained 30-40% more chlorophyll and 40-50% more green leaf area than wild-type plants at the end of 50 days of growth under water deficit conditions.Compared with control plants, CK-supplied and IPT-transgenic plants maintained higher photosynthetic rates and stomatal conductance, and achieved greater biomass under water stress.External supply of CK significantly increased growth under water stressed and non-stressed ii conditions in wild type plants compared to those that did not receive CK. Transgenic line RAB25showed the greatest improvement in biomass production. Some transgenic lines, including RAB2...

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“…Cytokinin was first discovered in the early 1940s when coconut milk was added to aid cell division in tobacco plants [74]. All CKs are adenine derivatives and mostly occur as either free compounds, glucosides, or ribosides in the plant root system, particularly the root apex [44,75]. According to [44], CK biosynthesis occurs through biochemical modification of dimethylallyl diphosphate, which is initiated through the transference of the isopentenyl moiety from dimethylallyl diphosphate to the N6 position of adenosine triphosphate, catalyzed by isopentenyl transferases.…”

“…All CKs are adenine derivatives and mostly occur as either free compounds, glucosides, or ribosides in the plant root system, particularly the root apex [44,75]. According to [44], CK biosynthesis occurs through biochemical modification of dimethylallyl diphosphate, which is initiated through the transference of the isopentenyl moiety from dimethylallyl diphosphate to the N6 position of adenosine triphosphate, catalyzed by isopentenyl transferases. These form the isopentenyl transferases and the isoprene side chain, which is subsequently trans-hydroxylated by cytochrome P450 (CYP450) to yield zeatin ribosides [44].…”

“…According to [44], CK biosynthesis occurs through biochemical modification of dimethylallyl diphosphate, which is initiated through the transference of the isopentenyl moiety from dimethylallyl diphosphate to the N6 position of adenosine triphosphate, catalyzed by isopentenyl transferases. These form the isopentenyl transferases and the isoprene side chain, which is subsequently trans-hydroxylated by cytochrome P450 (CYP450) to yield zeatin ribosides [44]. The metabolic storage and transport of CK is not yet fully understood, however, it is hypothesized that transport takes place via the vascular tissue (particularly the xylem), from the roots to the shoots of the plant [76].…”

“…The most common molecular mechanisms of GA signaling in plants is through the GA receptor, Gibberellin Insensitive Dwarf 1 (GID1) [44,82]. Upon binding, the receptors undergo conformation changes that favor the binding of DELLA proteins, a group of nuclear transcriptional regulators that repress GA signaling and plant growth [44,82].…”

“…The most common molecular mechanisms of GA signaling in plants is through the GA receptor, Gibberellin Insensitive Dwarf 1 (GID1) [44,82]. Upon binding, the receptors undergo conformation changes that favor the binding of DELLA proteins, a group of nuclear transcriptional regulators that repress GA signaling and plant growth [44,82]. Homeostasis and regulation of the GA biosynthetic pathway depends on the developmental and environmental signals, specifically the genes GA 20 -oxidases, GA 3oxidase, and GA 2 -oxidases [44].…”