Thrips (Thysanoptera: Thripidae) is a major insect pest for alfalfa which can result in decreased plant nutrients, low yields, and even plant death. To identify the differentially expressed genes and metabolites in response to thrips in alfalfa, a combination of metabolomics and transcriptomics was employed using alfalfa (Caoyuan No. 2) with and without thrips infestation. The results showed that the flavonoid biosynthesis and isoflavonoid biosynthesis pathways were the most significantly enriched pathways in response to thrips infection, as shown by the combined transcriptome and metabolome analysis. The transcriptome results showed that SA and JA signal transduction and PAPM-triggered immunity and the MAPK signaling pathway–plant pathways played a crucial role in thrips-induced plant resistance in alfalfa. In addition, we found that thrips infestation could also induce numerous changes in plant primary metabolism, such as carbohydrate and amino acid metabolism as compared to the control. Overall, our results described here should improve fundamental knowledge of molecular responses to herbivore-inducible plant defenses and contribute to the design of strategies against thrips in alfalfa.

Plants have evolved a series of inducible or constitutive defense mechanisms in response to herbivore attack. Constitutive plant defenses are morphological and biochemical traits of the plants themselves, regardless of the presence of herbivores. We bred an alfalfa variety (Caoyuan No.4) with high thrips resistance, but the mechanisms underlying Caoyuan No.4 resistance to thrips are not well understood. To explore the constitutive defense of Caoyuan No.4, the morphological and biochemical traits associated with constitutive defense to thrips in alfalfa were analyzed using a thrips-susceptible alfalfa accession (Caoyuan No.2) as a control. The results showed that Caoyuan No.4 had thicker palisade tissue and parenchyma tissue, wider collenchyma, phloem, cambium and lignin layer, and smaller epidermal cells and stomatal aperture compared to Caoyuan No.2. Moreover, Caoyuan No.4 showed more non-glandular trichomes in both leaves and stems, but less glandular trichomes and more wax in stems. In addition, the results of the widely targeted metabolomics analysis showed that metabolites related to flavonoid, isoflavonoid, flavone and flavonol biosynthesis, as well as cysteine and methionine metabolism, differed between CaoyuanNo.2 and Caoyuan No.4. These findings shed new light on the constitutive insect defense of plants associated with physical or biochemical traits and may provide convenient markers for breeding thrips-resistant alfalfa cultivars.

Stems are more important to forage quality than leaves in alfalfa. To understand lignin formation at different stages in alfalfa, lignin distribution, anatomical characteristics and transcriptome profile were employed using two alfalfa cultivars. The results showed that the in vitro true digestibility (IVTD) of stems in WL168 was significantly higher than that of Zhungeer, along with the significantly lower neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin contents. In addition, Zhungeer exhibited increased staining of the xylem areas in the stems of different developmental stages compared to WL168. Interestingly, the stems of WL168 appeared intracellular space from the stage 3, while Zhungeer did not. The comparative transcriptome analysis showed that a total of 1993 genes were differentially expressed in the stem between the cultivars, with a higher number of expressed genes in the stage 4. Of the differentially expressed genes, starch and sucrose metabolism as well as phenylpropanoid biosynthesis pathways were the most significantly enriched pathways. Furthermore, expression of genes involved in lignin biosynthesis such as PAL, 4CL, HCT, CAD, COMT and POD coincides with the anatomic characteristics and lignin accumulation. These results may help elucidate the regulatory mechanisms of lignin biosynthesis and improve forage quality in alfalfa.