An alfalfa cDNA library induced by salt stress was constructed by suppression subtraction hybridization (SSH) technology. Total RNA from 10-day-old seedlings was used as a "driver," and total RNA from seedlings induced by salt was used as a "tester". One hundred and nineteen clones identified as positive clones by reverse Northern dot-blotting resulted in 82 uni-ESTs comprised of 16 contigs and 66 singletons. Blast analysis of deduced protein sequences revealed that 51 ESTs had identity similar to proteins with known function, while 24 could not be annotated at all. Most of the annotated sequences were homologous to genes involved in abiotic or biotic stress in plants. Among these proteins, beta-amylase, fructose-1,6-bisphosphate, aldolase, and sucrose synthase are related to osmolyte synthesis; a CCCH-type zinc finger protein, DNA binding protein, His-Asp phosphotransfer protein, and the RelA/SpoT protein partake in transcription regulation and signal transduction; and ribulose-l,5-bisphosphate carboxylase/oxygenase, chlorophyll a/b binding proteins, and an early light-inducible proteins are related to photosynthesis. In addition, several ESTs, similar to genes from other plant species, closely involved in salt stress were isolated from alfalfa, such as an aquaporin protein, a late embryogenesis-abundant protein, and glutathione peroxidase.

Homogentisate solanesyltransferase (HST) plays an important role in plastoquinone (PQ) biosynthesis and acts as the electron acceptor in the carotenoids and abscisic acid (ABA) biosynthesis pathways. We isolated and identified a T-DNA insertion mutant of the HST gene that displayed the albino and dwarf phenotypes. PCR analyses and functional complementation also confirmed that the mutant phenotypes were caused by disruption of the HST gene. The mutants also had some developmental defects, including trichome development and stomata closure defects. Chloroplast development was also arrested and chlorophyll (Chl) was almost absent. Developmental defects in the chloroplasts were consistent with the SDS-PAGE result and the RNAi transgenic phenotype. Exogenous gibberellin (GA) could partially rescue the dwarf phenotype and the root development defects and exogenous ABA could rescue the stomata closure defects. Further analysis showed that ABA and GA levels were both very low in the pds2-1 mutants, which suggested that biosynthesis inhibition by GAs and ABA contributed to the pds2-1 mutants' phenotypes. An early flowering phenotype was found in pds2-1 mutants, which showed that disruption of the HST gene promoted flowering by partially regulating plant hormones. RNA-sequencing showed that disruption of the HST gene resulted in expression changes to many of the genes involved in flowering time regulation and in the biosynthesis of PQ, Chl, GAs, ABA and carotenoids. These results suggest that HST is essential for chloroplast development, hormone biosynthesis, pigment accumulation and plant development.

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A suppression subtraction hybridization (SSH) cDNA library had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA (Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared by blast revealed high homology with zinc finger protein of other plants. Sequence comparison showed that there were five conserved typical zinc finger motifs, and one sugar transfer protein signature. The calculated molecular weight of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13. The MsZFN localized in nucleus. Under normal growth conditions, differential expression of MsZFN exhibited that the expression was the highest in leaf and the lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and reached its maximum at 30 min.