Biology and function of the OsALMT1 gene in rice (Oryza sativa L.)
thesisposted on 2023-05-27, 10:20 authored by Liu, J
Members of the aluminium (Al)-activated malate transporter (ALMT) gene family encode transmembrane proteins which function as anion channels and perform multiple functions involving anion transport. The TaALMT1 gene from wheat was the first member identified in plants because it controlled the major mechanism of aluminium (Al) resistance in that species. TaALMT1 mediates the Al-activated efflux of malate from roots which chelates the toxic Al3‚ÄövÖ‚à´ and prevents it from inhibiting root growth. Other ALMTs characterised to date show a range of functions related to Al resistance, guard cell function, mineral nutrition, grain development and germination, vacuolar malate balance and fruit acidity. The rice (Oryza sativa L.) genome contains nine members of the ALMT family but no functional information is available for any of these genes. This study characterised OsALMT1 and provides the first detailed analysis of an ALMT gene in rice. OsALMT1 expression in roots and leaves was examined by qRT-PCR under various treatments and results showed it was responsive to salt and osmotic stress, dehydration, low Cl-, various light treatments, as well as several hormones (abscisic acid, indol-3 acetic acid, and salicylic acid). The cDNA of OsALMT1 was predicted to encode a protein with a typical ALMT structure of six trans-membrane regions and a long hydrophilic C-terminus end. The cDNA was fused with the green fluorescence protein (GFP) and transient expression in leek (Allium ampeloprasum) tissues and tobacco (Nicotiana benthamiana) leaves demonstrated that OsALMT1 localised to the plasma membrane. Tissue specific expression of OsALMT1 was examined by using 2496 bp of sequence upstream of the coding region to drive GFP expression in transgenic rice plants. OsALMT1 was widely expressed in roots and shoots. Expression was prominent in vascular tissues (stele of roots and vascular bundles of leaves), in the root apices, in emerging lateral roots and root hairs, the collar of the leaf, regions of the rachillas, the nucellar projection of developing grain and embryo and in various flower parts. Rice cultivar Nipponbare‚ÄövÑvp was transformed with the OsALMT1 gene using the Agrobacterium method and several independent T2 or T3 rice homozygous lines showing approximately 40-fold higher OsALMT1 expression were. Transgenic lines with reduced OsALMT1 expression were also generated with RNAi and two independent homozygous lines with expression levels 10 to 20% of wild type plants were used to investigate gene function. Transgenic rice plants over-expressing OsALMT1 tended to have smaller roots and shoots than null lines and develop brown spots on the leaves which were consistent with manganese toxicity. Those transgenic plants also showed a constitutive release of malate and fumarate from their roots and had higher concentrations of malate in the xylem sap. The malate efflux was increased by high light intensity and inhibited by niflumic acid, salicylic acid and high concentrations of GABA. The transgenic plants showed greater tolerance to Al toxicity than null lines and sometimes accumulated higher concentrations of boron, manganese and other elements in the leaves and grain. In longer-term growth studies transgenic lines with increased OsALMT1 expression tended to be more sensitive to osmotic stress, low light and manganese toxicity and they tended to be more resistant to low Cl- treatments and to B toxicity. Transgenic lines with reduced OsALMT1 expression tended to be more sensitive to low Cl- conditions and to low light. In conclusion, OsALMT1 is a plasma membrane-localised ion channel that is widely expressed throughout rice plants. It is permeable to the organic anions malate and fumarate and it is predicted to be permeable to borate anions as well. OsALMT1 expression in roots and shoots is responsive to several abiotic stresses and hormone treatments which indicates a central role in plant metabolism. We conclude that OsALMT1 is primarily a malate transporter that performs multiple functions including osmotic adjustment and the maintenance of electroneutrality.
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