The roles of cotton (Gossypium hirsutum) aquaporins in cell expansion

Zhu, Youmin

Title: The roles of cotton (Gossypium hirsutum) aquaporins in cell expansion
Creator: Zhu, Youmin
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2016
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: Aquaporins (AQPs), with a major function as water channels, may play important roles in cell expansion. However, the roles of GhAQPs (Gh, Gossypium hirsutum) in cell expansion have not been clearly clarified. To address the roles of GhAQPs in plant cell expansion, this study aimed at identifying candidate GhAQPs involved in cotton fibre expansion by exploring their expression patterns and sub–cellular localization, examining their physiological roles in vivo, and exploring the cellular and molecular basis of the observed GhAQPs–mediated phenotypes. EST contigs were analysed by searching GhAQPs with high EST numbers in the cotton EST database (www.cottondb.com) to generate a preliminary selection of eight candidate GhAQPs possibly involved in cotton fibre elongation. These were four GhPIPs (GhPIP1;2, GhPIP1;3, GhPIP2;3, GhPIP2;4) and four GhTIPs (GhTIP1;1, GhTIP1;2, GhTIP1;4, GhTIP2;1). Semi–quantitative transcript analyses reviewed that GhAQPs were highly expressed during fibre expansion (5–15 days after anthesis, DAA) and relatively lower during the post expansion phase (~20 DAA onwards), which was generally consistent with temporal changes in rates of fibre elongation. These GhAQPs were also highly expressed in other expanding organs and more lowly expressed in expanded organs. GhPIP2;3 and GhTIP1;2 exhibited the highest transcript levels of all candidates, suggesting that GhPIP2;3 and GhTIP1;2 played a major role in cell expansion of cotton fibre and other organs, and thus were targeted as fibre expansion representatives for more detailed study. GhAQPs fused with green fluorescent protein (GFP) constructs were transformed into WT Arabidopsis by floral dipping to determine the intracellular localization of GhAQPs. In addition, a parallel experiment was designed in which GhAQPs fused with red fluorescent protein (RFP) constructs were transformed into WT Arabidopsis and Arabidopsis transformed by a tonoplast marker ShMTP (magnesium transporter proteins)–GFP. For GhPIP2;3 and GhTIP1;2, a putative plasma membrane or tonoplast intracellular localization was discovered, respectively. There is a strong link between AQP localization and their functions. The cell membrane localization of GhPIP2;3 and GhTIP1;2 implied that these water channels would increase the permeability of the cell membranes, which reinforces their role in cell expansion. To examine the physiological role of identified GhAQPs in vivo, a complementation and an overexpression experiments were performed by transforming GhPIP2;3/GhTIP1;2 into Arabidopsis (a time–saving model to study GhAQPs’ function) T–DNA insertion lines atpip2;3–1/attip1;3 and WT respectively. The mutants atpip2;3–1 and attip1;3 exhibit reduced root length compared to WT when grown in the standard ½ MS medium or a medium containing 100 mM NaCl and reduced pollen tube length when grown in the standard medium or a medium without nitrogen (NO₃–) respectively. In this study, overexpression of GhPIP2;3 in pip2;3–1 and overexpression of GhTIP1;2 in tip1;3 respectively complemented the mutants’ short root and pollen tube lengths respectively in the standard medium or in other media (osmotic/salt stress and without nitrogen), which demonstrated that GhPIP2;3 and GhTIP1;2 really functioned as a PIP and a TIP respectively in planta (translated into functional proteins after transformation). Overexpression of GhPIP2;3 and GhTIP1;2 in WT Arabidopsis exhibited phenotypes of increased root/root cortex cell and pollen tube lengths respectively in ½ MS medium alone as well as modified ½ MS media causing osmotic/salt stress and nitrogen deficiency, suggesting that GhPIP2;3 and GhTIP1;2 played important roles in cell expansion. During cell expansion, GhAQPs were found to confer salt tolerance by increasing water transport and diluting salt ions. It was also observed that the Arabidopsis root length didn’t show significant differences between their growth under NaCl and KCl stresses except the mutant showed shorter root length in 100 mM NaCl medium compared with in 100 mM KCl medium, suggesting Cl– might be the major ion performing toxicity and leading to the root reduction while Na+ toxicity also played some roles in root reduction. Three lines overexpressing GhPIP2;3 in WT showed significantly less reduced root length compared with WT in ½ MS + 100 mM NaCl medium compared with in ½ MS + 200 mM sorbitol medium, suggesting overexpression of GhPIP2;3 increased salt tolerance. In exploring the molecular basis of the observed increased root and pollen tube elongation in the lines overexpressing GhPIP2;3 and GhTIP1;2 respectively, a new phenomenon was found that overexpression of GhPIP2;3 and GhTIP1;2 could increase the expression of some sugar transporters (ERD6 for GhPIP2;3, ERDL6 and STP11 for GhTIP1;2) and K⁺ transporters (KC1 and SKOR for GhPIP2;3) (except that SWEET17 was decreased in overexpression of GhPIP2;3 lines, as decreasing the expression of SWEET17 might help to maintain cytoplasmic sugar homeostasis or maintain the solutes concentration in the vacuole during cell expansion). The roles of GhPIP2;3 and GhTIP1;2 in cell expansion might be due to their water transport activity combined with their impact on elevating sugar and K⁺ transporter expression that lowered cell water potentials with the overall effect of increasing turgor pressure to drive cell expansion. Another new discovery was that root hair length of the lines overexpressing GhPIP2;3 in WT were significantly increased compared with WT grown on MS medium containing 100 mM NaCl (½ MS + 100 mM NaCl). Meanwhile, three lines overexpressing GhPIP2;3 in pip2;3–1 background complemented the short root hair phenotype in the mutant in the ½ MS medium, and three lines overexpressing GhPIP2;3 in WT increased root hair length compared with WT in the ½ MS medium. At a molecular level overexpression of GhPIP2;3 was found to increase expression of root hair elongation related genes including AUX1 (auxin transport), ETR1 (ethylene receptor), Myosin XIK (Myosin XI) and EPC1 (Glycosyltransferase), and some hair density related genes such as IAA17 (Repressor of auxin–responsive transcription), PRP3 (Proline–rich cell wall protein), RHD6 (Protein ROOT HAIR DEFECTIVE6) and RHL1 (Topoisomerase subunits). These findings suggested that overexpression of GhPIP2;3 might increase root hair elongation and hair density by inducing expression of these root hair elongation related genes (in addition to increasing cell turgor for hair elongation by enhancing water transport) and root hair density related genes. The molecular basis for the role of GhPIP2;3 on salt tolerance was also explored. Among the salt tolerance genes chosen from NCBI and published papers, three salt tolerance genes, Rd29A (Responsive to desiccation 29A), SAT32 (Protein salt tolerance 32) and SOS1 (Sodium/hydrogen exchanger 7) were found to be higher expressed in the lines overexpressing GhPIP2;3 than in WT. It was proposed that overexpression of GhPIP2;3 increased salt tolerance probably by a combination of increased water transport, increased root hair length and density, overlaid by enhancing expression of salt tolerance genes. Furthermore, one interesting discovery was that overexpression of GhPIP2;3 increased stomatal density, which might increase CO₂ assimilation, then contributed to transpiration and photosynthesis.
Subject: cotton; aquaporin; cell expansion
Identifier: http://hdl.handle.net/1959.13/1322560
Identifier: uon:24603
Language: eng
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