Functional Analysis Of CDNAs Associated With Flesh Lignification In Loquat Fruit And Small RNAs Associated With Auxin Response Factors In Tomato

Part 1: The ripening fruit of two loquat (Eriobotrya japonica LindI. cv.Luoyangqing & Baisha) cultivars with different levels of lignin accumulation providean intriguing example of lignification in flesh tissue. Increase in firmness as a resultof lignification in ripening red-fleshed Luoyangqing (LYQ) fruit was confirmed,whereas white-fleshed Baisha (BS) fruit softened without lignification. Six cDNAsassociated with the lignification pathway i.e. EjPAL1 (phenylalanine ammonia lyase,PAL, EC 4.3.1.5), EjPAL2, Ej4CL (4-coumarate: coenzyme A ligase, 4CL, EC6.2.1.12), EjCAD1 (cinnamyl alcohol dehydrogenase, CAD, EC 1.1.1.195), EjCAD2and EjPOD (peroxidase, POD), were cloned from flesh tissue of LYQ fruit.Expression profiles of the six corresponding genes differed greatly in different tissues,and during fruit development and ripening in both LYQ and BS cultivars. Associatedactivities of PAL, 4CL, CAD, and POD enzymes were also measured. CAD and PODenzyme activities and the expression level of EjCAD1 and EjPOD genes were mostclosely associated temporally with lignification of loquat flesh tissue. Levels ofEjCAD1 transcripts were particularly aligned with changes in lignification duringripening as modified either by ethylene treatment or low temperature conditioning.The two PAL genes showed different expression patterns during fruit development,with EjPAL1 strongly expressed in mature fruit and EjPAL2 only expressed in earlystages of development. In addition, EjCAD1 expression was stimulated by lowtemperature and may contribute to chilling injury in the fruit. Our integrated data onlignin, monolignol precursors, and associated enzymes and genes, provide aconsistent model of fruit lignification.Part 2: Two endogenous trans-acting small regulatory RNAs, microRNAs(miRNA, miR160) and trans-acting interfering short RNA (tasiRNA, TAS3) intomato (Solanum lycopersicum Mill. cv. MicroTom) were identified in this paper. Intomato, miR160 was highly very conserved whereas TAS3 showed high diversity intheir nucleotide sequences. Transcript level of miR160 were found higher in root,flower and the early development stage ovary while those of TAS3 were mostlyexpressed in flower, especially in the sepal and ovary but were trace in immature fruitand stamen. In silico analysis showed, miR160 targeted three auxin response factor (ARF) genes, ARF10, ARF16, and ARF17 in both Arabidopsis and tomato, and TAS3targeted three other ARF genes, ARF2, ARF3, and ARF4. Cleavage of ARF10, ARF16,and ARF17 by miR160 and cleavage of ARF4 by TAS3 were confirmed by 5'-RACEin tomato. Tomato transformed with sense strand of miR160 precusor resulted in thedown-regulation of ARF10 and induced fused sepal and fruit with aborted seeds,while over-accumulation of TAS3 precursor resulted in down-regulation of ARF4 anddark green immature fruit. Such data suggested an important role that both miR160and TAS3 may play roles in the early stage of tomato fruit development by regulatingtheir target ARF genes and also others.