Systemic acquired resistance (SAR) in plants is a defense response that provides resistance against a wide range of pathogens at the whole-plant level following primary infection. Although the molecular mechanisms of SAR have been extensively studied in recent years, the role of phosphorylation that occurs in systemic leaves of SAR-induced plants is poorly understood. We used a data-independent acquisition (DIA) phosphoproteomics platform based on high-resolution mass spectrometry in an Arabidopsis thaliana model to identify phosphoproteins related to SAR establishment. A total of 8011 phosphorylation sites from 3234 proteins were identified in systemic leaves of Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) and mock locally inoculated plants. A total of 859 significantly changed phosphoproteins from 1119 significantly changed phosphopeptides were detected in systemic leaves of Psm ES4326 locally inoculated plants, including numerous transcription factors and kinases. A variety of defense response-related proteins were found to be differentially phosphorylated in systemic leaves of Psm ES4326 locally inoculated leaves, suggesting that these proteins may be functionally involved in SAR through phosphorylation or dephosphorylation. Significantly changed phosphoproteins were enriched mainly in categories related to response to abscisic acid, regulation of stomatal movement, plant–pathogen interaction, MAPK signaling pathway, purine metabolism, photosynthesis-antenna proteins, and flavonoid biosynthesis. A total of 28 proteins were regulated at both protein and phosphorylation levels during SAR. RT-qPCR analysis revealed that changes in phosphorylation levels of proteins during SAR did not result from changes in transcript abundance. This study provides comprehensive details of key phosphoproteins associated with SAR, which will facilitate further research on the molecular mechanisms of SAR.

Background The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species. Results SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers. Conclusion All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

APETALA3 (AP3) and PISTILLATA (PI) are B‐class MADS‐box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3‐6 and pi‐1 homozygous mutant and in wild‐type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C‐terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.