Transcription factors act as master switches of transcriptional reprogramming, inducing diverse protective mechanisms in response to abiotic stresses.We identified a large number of TFs responsive to dehydration, and 287 TFs were responsive to rehydration from among the DTGs . Among these TFs, the bHLH, MYB, and WRKY families were the largest groups during both dehydration and rehydration . Members of these three TF families are implicated in stress responses in model plants. In Arabidopsis, bHLH-type genes AtMYC2 and AtAIB are involved in ABA signaling, and overexpression of AtMYC2 or AtAIB enhances the drought tolerance of transgenic plants.Multiple MYB genes have also been implicated in stress responses. For example, AtMYB2 is involved in the ABA-dependent drought tolerance pathway,and AtMYB108 is associated with both biotic and abiotic stress responses.WRKY TFs are well known for their involvement in the regulation of plant development and in response to abiotic stresses.Functional analyses of rice OsWRKY11 and soybean GmWRKY54 demonstrated that WRKYs are involved in drought signaling pathways.Our results revealed that 9 SBP and 5 TCP type TFs also showed a response to dehydration in M. flabellifolia . Further functional characterization of these TFs may shed light on their roles in desiccation tolerance. PKs are essential signaling regulators in the acquisition of desiccation tolerance. Among the DTGs,vertical grow system we identified strikingly large numbers that respond to dehydration and rehydration .
PKs may play a key regulatory role in drought stress adaptation in M. flabellifolia, as corroborated by comparison of our data with Arabidopsis and chrysanthemum drought stress expression profile data.In Arabidopsis, only 121 PKs were found to be responsive to osmotic stress.Xu et al. identified only 229 differentially expressed PKs during drought stress in chrysanthemum, a species with a large and complex polyploid genome and high heterozygosity.Protein family distribution analyses showed that three receptorlike kinase type families – leucine-rich repeat kinases, domain of unknown function DUF, and receptor like cytoplasmic kinases – were the largest groups of DTGs in both the dehydration and rehydration treatments . RLKs constitute the largest PK family in plants and have been implicated in the regulation of meristem proliferation, organ specification, reproduction, and hormone signal transduction.Drought transcript profiling in Arabidopsis has revealed that the transcript abundance of many RLK genes peaks 1 h after the start of drought treatment, indicating that RLKs may also be part of a rapid drought response.Several functional studies have also confirmed roles of RLKs in drought tolerance.In Arabidopsis, an LRR kinase, receptor protein kinase 1 , is induced by ABA. Repression of RPK1 decreases sensitivity to ABA, suggesting that RPK1 is involved in ABA perception.A DUF type kinase, CRK, and a receptor-like cytoplasmic type kinase, ARCK1, form a complex that negatively controls ABA and osmotic stress signal transduction.
In addition,DTGs encoding mitogen-activated protein kinases were identified during dehydration stress in M. flabellifolia . MAPK cascades function in transducing environmental and developmental cues to intracellular responses.In Arabidopsis, several MAPKs are involved in abiotic stress signaling pathways including MEKK1,MPK1,MPK3,MPK4,MPK6,MAP9,and MAP12.The transcript abundance of homologs of all these MAPKs, apart from MPK6 and MAP12, was altered in response to dehydration in M. flabellifolia . To identify early responsive regulatory genes, the expression pattern of differentially transcribed TFs and PKs was analyzed by hierarchical clustering . The transcript abundance of 53 TFs and 91 PKs peaked early in dehydration , indicating that broad regulatory networks are quickly employed to coordinate global transcriptional reprogramming during desiccation of M. flabellifolia. Among the up-regulated TFs during early dehydration, the strongest induction was observed for genes encoding putative DREB family members . The DREB TFs are well-known regulators of ABA-independent drought stress signaling pathways in other plant species.In addition, a total of nine genes encoding WRKY family members were included among the up-regulated TFs in early dehydration, making the WRKY family the biggest group of early dehydration up-regulated TFs . Furthermore, among the up-regulated PKs, the LRK10 L kinase family is one of the largest groups in during early dehydration , suggesting that LRK10 L kinases may be important upstream regulators of rapid responses to water deficit. For example, the transcript abundance of a DTG encoding an LRK10 L kinase increased nearly 120-fold in early dehydration .
LRK10 L genes have previously been reported to be associated with resistance to pathogen infection.Functional characterization of these LRK10 L kinases will be an important step toward elucidating their roles in desiccation tolerance.Various genetically encodable reporters have been developed to monitor gene expression, protein subcellular localization, protein stability, hormonal signaling, and impacts of environmental signals. The green fluorescent protein and its derivatives such as RFP, mCherry, and YFP have many applications as reporters for gene expression or as fusion proteins. Although GFP is easy to use, it needs light sources to visualize the fluorescence signals. The β-glucuronidase reporter has been widely used in plants for monitoring gene expression patterns and as a reporter for hormonal signaling. For example, DR5-GUS transgenic lines are commonly used to monitor auxin distribution and auxin signaling. Luciferase is another broadly used reporter in both animals and plants. Both GUS and luciferase require the addition of expensive substrates X-Gluc and luciferin, respectively. Whereas the traditional reporters have been very useful, they have limitations. Fluorescent proteins are often monitored under a microscope, rendering it less useful in analyzing plants in natural growing fields or analyzing large samples such as a tree. GUS staining is invasive and often requires sacrifice of the plants. Luciferase can be used non-invasively, but it requires a special camera and spraying the expensive substrate. It is also not very practical to use them in fields. GUS and luciferase may not be optimal for sterile conditions such as tissue culture because addition of substrates increases the chance for contamination of microbes. Therefore, there is a need to develop new reporter systems that can be widely used to monitor cellular activities noninvasively, continuously, and cost effectively. For the past few years,strawberry pots gene editing has been widely used in basic research and crop improvement. A visible marker for transgenes will greatly accelerate the isolation of edited plants that no longer harbor the gene editing machinery. Plants produce many colorful compounds that potentially can serve as reporters. For example, anthocyanins display bright red-blue colors and anthocyanin-producing rice plants have been used to generate interesting patternsin rice field. However, synthesis of anthocyanins requires multiple enzymes and varies greatly among different plants. It is difficult to use anthocyanin biosynthesis pathways as a universal visible reporter. Betalains are a class of plant natural products derived from the aminoacid tyrosine. The bright red color seen in beets, dragon fruit, Swiss chard, and other plants is resulted from accumulation of betalains. Biosynthesis of betalains has been well studied and only needs three enzymatic reactions to convert tyrosine into betalain. Tyrosine is first hydroxylated on the benzene ring, resulting in L-3,4-dihydroxyphenylalanine . The reaction is catalyzed by the P450 oxygenase CYP76AD1 . L-DOPA can be further oxidized into cyclo-DOPA by CYP76AD1 . Alternatively, LDOPA is catalyzed by L-DOPA 4,5-dioxygenase into betalamic acid, which is subsequently condensed with cyclo-DOPA into betanidin. The condensation reaction does not require an enzyme . Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the colorful betalain . Betalain has a very bright red color, which potentially can serve as a reporter to track gene expression or to visualize transgenic events. Because every cell contains the amino-acid tyrosine, exogenous application of tyrosine to tissues may not be required. We hypothesized that betalain would be a more convenient reporter than the aforementioned reporters. It is visible to naked eyes without any needs for special equipment. It does not require processing samples and it allows continuously monitoring events throughout the life cycle of an organism. Moreover, it is applicable to large plants grown under normal field conditions. Herein, we synthesize an artificial open reading frame named RUBY that when expressed can produce all of the enzymes required for betalain biosynthesis. We show that RUBY is a very effective marker for noninvasively selecting transformation events in both rice and Arabidopsis. Moreover, we show that RUBY can be used to visualize gene expression without any chemical treatments or special equipment, providing useful tools for visualizing gene expression in large plants under natural field growth conditions.Heterologous expression of CYP76AD1, DODA in tobacco, and other plants demonstrated that the betalain biosynthetic pathway can be re-constituted in plant cells. In order to use betalain as a visual reporter, we need to effectively co-express the entire pathway using a single promoter. We organized CYP76AD1, DODA, and Glucosyltransferase into a single open reading frame . The stop codons of CYP76AD1 and DODA were removed. The three genes were linked by sequences that encode 2A peptides. Upon transcription, the single transcript, which includes the coding regions of the three enzymes, produced the three separate enzymes through either 2A-mediated self-cleavage or ribosomal “skipping”.
The 2A system enables the expression of multiple proteins under the control of a single promoter. We name the 2A-linked unit of CYP76AD1, DODA, and Glucosyltransferase RUBY . RUBY can be expressed when a promoter is placed in front of it. The expression pattern and level of a particular gene may be inferred from the red color of betalain if the gene’s promoter is used to drive RUBY expression.We first placed RUBY under the control of Cauliflower Mosaic Virus 35S promoter, which is a widely used constitutively strong promoter. To test whether RUBY can produce functional enzymes for betalain synthesis, we infiltrated tobacco leaves with Agrobacteria that contain RUBY-expressing plasmid.Transient expression of RUBY led to the production of betalain in tobacco leaves, suggesting that the synthetic open reading frame RUBY can produce the functional enzymes for the synthesis of betalain. Moreover, we observed that betalain was not transported from the spots of Agrobacterium-infiltration spots to other leaves of the plant .We transformed the 35S:RUBY construct into Arabidopsis using Agrobacterium-mediated floral dipping. Two days after floral dipping, we noticed that the transformed plants displayed patches of red color , indicating that the RUBY cassette was functionally expressed and that RUBY may be used to monitor transient Arabidopsis transformation. Once the seeds from the Agrobacterium-dipped plants were harvested, transgenic seeds could be easily differentiated from non-transgenic seeds . The transformed seeds had a dark red color , demonstrating that RUBY can be used as a visual selection marker for transgenic events in Arabidopsis. We previously used mCherry as a very effective marker to select transgenic events , which requires a dissecting microscope with fluoresence capability. RUBY is a better option because it does not require special equipment. The 35S:RUBY plants produced sufficient amount of betalain to become visually evident . Consistent with previous reports that CaMV 35S promoter is constitutively active, we observed red color in all tissues throughout the plant life cycle . We also expressed RUBY reporter under the control of the Maize UBIQUITIN promoter, which has been widely used to over express genes in monocots. Similar to 35S:RUBY plants, UBQ:RUBY plants were also visibly red in leaves, stem, and flowers . These results clearly demonstrated that RUBY could be expressed in Arabidopsis and that our RUBY reporter was able to functionally re-constitute the betalain biosynthetic pathway. We expressed RUBY using the seed specific At2S3 promoter, which we previously used to drive mCherry expression in Arabidopsis to facilitate the selection of transgenes. As shown in Fig. 2c, the transgenic plants were indistinguishable from wild type plants. When we checked the seeds in a silique from an At2S3:RUBY T1 plant, RUBY-expressing seeds displayed strong red color, whereas the non-transgenic seeds were green . RUBY can be conveniently used to select single T-DNA insertion events by analyzing the ratio of red seeds to green seeds, which should be ~3:1 for single insertions. The At2S3:RUBY results demonstrate that RUBY could be an effective marker for Arabidopsis transformation. Furthermore, betalain was not widely transported from the sites of synthesis to other tissues as we did not see any red color in leaves . We also expressed RUBY under the control of the Arabidopsis YUC4 promoter . YUC4, which encodes a key enzyme in auxin biosynthesis, was shown to express in small regions of embryos, leaves, and flowers. GUS signals were observed in leaf tips and apical region of a gynoecium in YUC4 promoter:GUS transgenic plants. We observed similar patterns of betalain production in YUC4:RUBY lines .Unlike Arabidopsis, rice and many other plants are transformed through tissue culture and the formation of calli, which are often mosaic.