Phenylalanine ammonia-lyase (PAL, E.C.4.3.1.5) catalyzes the benzene propane metabolism and is the most extensively studied enzyme of the phenylpropanoid pathway. However, the role of PAL genes in Astragalus membranaceus, a non-model plant showing high capability toward abiotic stress, is less studied. Here, we cloned AmPAL and found that it encodes a protein that resides in the cytoplasmic membrane. The mRNA of AmPAL was strongly induced by NaCl or NaHCO₃ treatment, especially in the root. Overexpressing AmPAL in Nicotiana tabacum resulted in higher PAL enzyme activities, lower levels of malondialdehyde (MDA), and better root elongation in the seedlings under stress treatment compared to the control plants. The protective role of AmPAL under saline-alkali stress was also observed in 30-day soil-grown plants, which showed higher levels of superoxide dismutase (SOD), proline, and chlorophyll compared to wild-type N. Tabacum. Collectively, we provide evidence that AmPAL is responsive to multiple abiotic stresses and that manipulating the expression of AmPAL can be used to increase the tolerance to adverse environmental factors in plants.

Drought and high salinity greatly affect plant growth and development. WRKY transcription factors play a key role in plant tolerance to abiotic stress, but the functions of WRKYs in the ornamental monocotyledon Iris laevigata remain largely unexplored. In this study, we cloned IlWRKY70 and found that it is a Group III WRKY localized in the nucleus. The expression of IlWRKY70 was induced by NaCl and PEG-6000, which reached peaks (4.38 and 5.65 times) after 3 h and 1 h, respectively. The exogenous overexpression of IlWRKY70 in N. tabacum significantly improved the resistance under NaCl and drought treatments, as evidenced by higher germination rates, longer root lengths, and increased fresh weights compared to those of control plants. In addition, transgenic seedlings showed significantly reduced wilting, higher photosynthetic performance, higher Fv/Fm and chlorophyll content, and lower stomatal conductance. Moreover, transgenic lines showed higher antioxidant enzymatic activities, lower reactive oxygen species (ROS), and lower malondialdehyde contents. Accordingly, we also found higher expressions of antioxidant defense genes, including SOD, CAT, and POD, in transgenic lines compared to controls under salt and drought stresses. Thus, IlWRKY70 enhances the abilities of salt and drought tolerances in plants, at least partially, via ROS regulation and can be used for breeding I. laevigata possessing enhanced salt and drought resistances.

Iris laevigata is ideal for gardening and landscaping in northeast China because of its beautiful flowers and strong cold resistance. However, the short length of flowering time (2 days for individual flowers) greatly limits its applications. Molecular breeding and engineering hold high potential for producing I. laevigata of desirable flowering properties. A prerequisite is to identify and characterize key flowering control genes, the identity of which remains largely unknown in I. laevigata due to the lack of genome information. To fill this knowledge gap, we used sequencing data of the I. laevigata transcriptome to identify MADS-box gene-encoding transcription factors that have been shown to play key roles in developmental processes, including flowering. Our data revealed 41 putative MADS-box genes, which consisted of 8 type I (5 Mα and 3 Mβ, respectively) and 33 type II members (2 MIKC* and 31 MIKC^C, respectively). We then selected IlSEP3 and IlSVP for functional studies and found that both are localized to the nucleus and that they interact physically in vitro. Ectopic expression of IlSEP3 in Arabidopsis resulted in early flowering (32 days) compared to that of control plants (36 days), which could be mediated by modulating the expression of FT, SOC1, AP1, SVP, SPL3, VRN1, and GA20OX. By contrast, plants overexpressing IlSVP were phenotypically similar to that of wild type. Our functional validation of IlSEP3 was consistent with the notion that SEP3 promotes flowering in multiple plant species and indicated that IlSEP3 regulates flowering in I. laevigata. Taken together, this work provided a systematic identification of MADS-box genes in I. laevigata and demonstrated that the flowering time of I. laevigata can be genetically controlled by altering the expression of key MADS-box genes.

Pigments in cyclamen (Cyclamen purpurascens) endows flowers with great ornamental and medicinal values. However, little is known about the biosynthetic pathways of pigments, especially anthocyanins, in cyclamen flowers. Herein, anthocyanins profiling and RNA-Seq were used to decipher the molecular events using cyclamen genotypes of red (HXK) or white (BXK) flowers. We found that red cyclamen petals are rich in cyanidin-3-O-rutinoside, cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-rutinoside, quercetin-3-O-glucoside, and ruti. In addition, our transcriptomics data revealed 3589 up-regulated genes and 2788 down-regulated genes comparing the BXK to HXK. Our rich dataset also identified eight putative key genes for anthocyanin synthesis, including four chalcone synthase (CHS, g13809_i0, g12097_i0, g18851_i0, g36714_i0), one chalcone isomerase (CHI, g26337_i0), two flavonoid 3-hydroxylase (F3′H, g14710_i0 and g15005_i0), and one anthocyanidin synthase (ANS, g18981_i0). Importantly, we found a 2.5 order of magnitude higher expression of anthocyanin 3-O-glucosyltransferase (g8206_i0), which encodes a key gene in glycosylation of anthocyanins, in HXK compared to BXK. Taken together, our multiomics approach demonstrated massive changes in gene regulatory networks and anthocyanin metabolism in controlling cyclamen flower color.

Iris laevigata Fisch. is a perennial aquatic plant with strong cold resistance and showy flowers. However, the flowering period of a single flower is only two days, greatly limiting its potential use in landscaping and the cutting-flower industry. In addition, I. laevigata is often challenged with various abiotic stresses including high salinity and drought in its native habitat. Thus, breeding novel cultivars with prolonged flowering time and higher resistance to abiotic stress is of high importance. In this study, we first performed genome-wide identification of WRKYs, key transcription factors in modulating flowering time and abiotic stress responses. We overexpressed IlWRKY22 in Arabidopsis thaliana and found the flowering time was delayed in the transgenic plants. Molecular characterization further revealed that IlWRKY22 promotes the expression of CO and GA20OX genes in the photoperiod and gibberellin pathways, respectively, and inhibits the expression of SPL3 in the aging pathway. We also created transgenic Nicotiana tabacum overexpressing IlWRKY22, which showed significantly improved resistance to both salt and drought compared to control plants. Specifically, the photosynthetic rate, maximum potential quantum efficiency of Photosystem II (Fv/Fm), and chlorophyll content were higher in the transgenic plants, which was accompanied with higher antioxidant enzyme activity and lower levels of reactive oxygen species. Thus, our study revealed a unique dual function of IlWRKY22, an excellent candidate gene for breeding novel I. laevigata cultivars of desirable traits.