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Red clover (Trifolium pratense) is a perennial legume with high feeding and medicinal value attributed to its abundant isoflavone content. Previous studies reported that R2R3-MYB transcription factors are involved in the biosynthesis of isoflavones; however, their specific role in red clover remains poorly understood. Through comprehensive genome-wide and transcriptome analyses, a total of 138 TpR2R3-MYB genes were identified and classified into 30 distinct subgroups within a phylogenetic tree. Importantly, six of these subgroups showed associations with isoflavone biosynthesis in red clover. The majority of segmental duplication events (Ka/Ks < 1) indicated that the TpR2R3-MYB gene underwent strong purifying selection during evolution. The qRT-PCR analysis demonstrated high expression levels of TpMYB79 and TpMYB53 in Minshan red clover at full flowering stage, consistent with the trend for isoflavone content determination, suggesting that TpMYB79 and TpMYB53 might be important regulators of isoflavone biosynthesis in red clover. Additionally, we observed nucleus and vacuole membrane localization of TpMYB53 and TpMYB79, with TpMYB53 primarily exerting transcriptional activation through its C-terminal activation motifs while TpMYB79 exhibited no transcriptional activity. These findings provided a foundation for the study of the biological function of R2R3-MYB transcription factors in red clover., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Revealing the genetic basis for stress-resistant traits in extremophile plants will yield important information for crop improvement. Zygophyllum xanthoxylum, an extant species of the ancient Mediterranean, is a succulent xerophyte that can maintain a favorable water status under desert habitats; however, the genetic basis of this adaptive trait is poorly understood. Furthermore, the phylogenetic position of Zygophyllales, to which Z. xanthoxylum belongs, remains controversial. In this study, we sequenced and assembled the chromosome-level genome of Z. xanthoxylum. Phylogenetic analysis showed that Zygophyllales and Myrtales form a separated taxon as a sister to the clade comprising fabids and malvids, clarifying the phylogenetic position of Zygophyllales at whole-genome scale. Analysis of genomic and transcriptomic data revealed multiple critical mechanisms underlying the efficient osmotic adjustment using Na+ and K+ as "cheap" osmolytes that Z. xanthoxylum has evolved through the expansion and synchronized expression of genes encoding key transporters/channels and their regulators involved in Na+/K+ uptake, transport, and compartmentation. It is worth noting that ZxCNGC1;1 (cyclic nucleotide-gated channels) and ZxCNGC1;2 constituted a previously undiscovered energy-saving pathway for Na+ uptake. Meanwhile, the core genes involved in biosynthesis of cuticular wax also featured an expansion and upregulated expression, contributing to the water retention capacity of Z. xanthoxylum under desert environments. Overall, these findings boost the understanding of evolutionary relationships of eudicots, illustrate the unique water retention mechanism in the succulent xerophyte that is distinct from glycophyte, and thus provide valuable genetic resources for the improvement of stress tolerance in crops and insights into the remediation of sodic lands., Competing Interests: Conflict of interest statement. The authors declare no potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genetic counseling students with minoritized identities have reported experiencing microaggressions throughout graduate training, including from fieldwork supervisors. However, the impacts of these fieldwork experiences have not been thoroughly investigated. As supervision is known to be integral to genetic counseling students' skill development and success, the purpose of this qualitative study was to explore the impact of microaggressions on student training, with a specific focus on the supervisory working alliance. To achieve this goal, we conducted 11 interviews with recent genetic counseling graduates (2019-2021) who reported experiencing at least one microaggression from a fieldwork supervisor during graduate school training. Purposive sampling was used to prioritize interviewees who identified as underrepresented in the field due to race, ethnicity, gender identity, sexual orientation, and/or disability status. All interviewees were initially recruited as part of a larger mixed-methods study investigating the frequency and types of microaggressions genetic counseling students experience from fieldwork supervisors. Interview questions explored the time period before a microaggression event, during the event, and after. Qualitative thematic analysis resulted in four themes, three of which are presented in this paper: (1) Impact of microaggressions, (2) Barriers to reporting microaggressions, and (3) Experience reporting microaggressions. Microaggressions from supervisors were shown to impair the psychological well-being of participants and hinder learning opportunities. These experiences led participants to question their choice of profession and avoid time in clinic, ultimately constraining the development of strong supervisory working alliances. Some participants did not report microaggressions due to fear of negative repercussions, and those who did described defensive responses which harmed students' relationships with program leadership. This study reveals opportunities for supervisors to improve student training conditions by centering students' feelings and experiences, increasing open and honest communication, and extending psychosocial tools to supervision. Additionally, graduate programs are encouraged to establish structured reporting protocols for students and evaluate current shortcomings in equity and inclusion initiatives., (© 2024 The Authors. Journal of Genetic Counseling published by Wiley Periodicals LLC on behalf of National Society of Genetic Counselors.)

While research has shown that genetic counseling students with minoritized racial or ethnic identities face microaggressions throughout graduate training, quantitative data regarding the frequency of these experiences have not been reported. The purpose of this mixed-methods study was to investigate the frequency and types of microaggressions experienced by graduates of accredited genetic counseling programs in the United States during fieldwork rotations. A quantitative survey was administered to assess how frequently 14 different types of microaggressions occurred in interactions with supervisors. Survey responses were analyzed using situation-based coding (the number of different types of microaggressions experienced) and frequency-based coding (the sum of participants' weighted Likert answers). Select survey respondents with minoritized identities were interviewed to better contextualize and categorize microaggression experiences. Analysis of 87 survey responses revealed that participants with minoritized racial and ethnic identities experience significantly more types of microaggressions (t(61) = 2.77; p = 0.007) at a significantly higher frequency (t(55) = 2.67; p = 0.010) than their white counterparts. Participants who identified as part of the disability community were also found to experience significantly more types of microaggressions (t(10) = 3.25; p = 0.009) at a significantly higher frequency (t(9) = 2.32; p = 0.045) than those who did not. Qualitative analysis of 11 interviews revealed that microaggressions from supervisors included offensive and inappropriate comments, unequal treatment, cultural intolerance, and disparaging feedback. Overall, our data present evidence that students with minoritized racial and ethnic identities and students with disabilities are subjected to a variety of inequitable, exclusionary, and harmful interactions. As a result, we recommend that all supervisors receive training about recognizing and preventing microaggressions to ensure that students are provided with an equitable and inclusive training experience, regardless of identity., (© 2024 The Authors. Journal of Genetic Counseling published by Wiley Periodicals LLC on behalf of National Society of Genetic Counselors.)

Key Message: The content of flavonoids could increase in A. canescens under saline conditions. Overexpression of AcCHI in transgenic A. thaliana promotes flavonoid biosynthesis, thereby functioning in the tolerance of transgenic plants to salt and osmotic stress by maintaining ROS homeostasis. Atriplex canescens is a halophytic forage shrub with excellent adaptation to saline environment. Our previous study showed that a large number of genes related to the biosynthesis of flavonoids in A. canescens were significantly up-regulated by NaCl treatments. However, it remains unclear whether flavonoids are involved in A. canescens response to salinity. In this study, we found that the accumulation of flavonoids significantly increased in either the leaves or roots of A. canescens seedling under 100 and 300 mM NaCl treatments. Correspondingly, AcCHS, AcCHI and AcF3H, which encode three key enzymes (chalcone synthases (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), respectively) of flavonoids biosynthesis, were significantly induced in the roots or leaves of A. canescens by 100 or 300 mM NaCl. Then, we generated the transgenic Arabidopsis thaliana overexpressing AcCHI and found that transgenic plants accumulated more flavonoids through enhancing the pathway of flavonoids biosynthesis. Furthermore, overexpression of AcCHI conferred salt and osmotic stress tolerance in transgenic A. thaliana. Contrasted with wild-type A. thaliana, transgenic lines grew better with greater biomass, less H 2 O 2 content as well as lower relative plasma permeability in either salt or osmotic stress conditions. In conclusion, our results indicate that flavonoids play an important role in A. canescens response to salt stress through reactive oxygen species (ROS) scavenging and the key enzyme gene AcCHI in flavonoids biosynthesis pathway of A. canescens has the potential to improve the stress tolerance of forages and crops., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Aims: Desert plants possess excellent water-conservation capacities to survive in extreme environments. Cuticular wax plays a pivotal role in reducing water loss through plant aerial surfaces. However, the role of cuticular wax in water retention by desert plants is poorly understood., Methods: We investigated leaf epidermal morphology and wax composition of five desert shrubs from north-west China and characterized the wax morphology and composition for the typical xerophyte Zygophyllum xanthoxylum under salt, drought and heat treatments. Moreover, we examined leaf water loss and chlorophyll leaching of Z. xanthoxylum and analysed their relationships with wax composition under the above treatments., Key Results: The leaf epidermis of Z. xanthoxylum was densely covered by cuticular wax, whereas the other four desert shrubs had trichomes or cuticular folds in addition to cuticular wax. The total amount of cuticular wax on leaves of Z. xanthoxylum and Ammopiptanthus mongolicus was significantly higher than that of the other three shrubs. Strikingly, C31 alkane, the most abundant component, composed >71 % of total alkanes in Z. xanthoxylum, which was higher than for the other four shrubs studied here. Salt, drought and heat treatments resulted in significant increases in the amount of cuticular wax. Of these treatments, the combined drought plus 45 °C treatment led to the largest increase (107 %) in the total amount of cuticular wax, attributable primarily to an increase of 122 % in C31 alkane. Moreover, the proportion of C31 alkane within total alkanes remained >75 % in all the above treatments. Notably, the water loss and chlorophyll leaching were reduced, which was negatively correlated with C31 alkane content., Conclusion: Zygophyllum xanthoxylum could serve as a model desert plant for study of the function of cuticular wax in water retention because of its relatively uncomplicated leaf surface and because it accumulates C31 alkane massively to reduce cuticular permeability and resist abiotic stressors., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Heat stress has adverse effects on the growth and reproduction of plants. Zygophyllum xanthoxylum, a typical xerophyte, is a dominant species in the desert where summer temperatures are around 40 °C. However, the mechanism underlying the thermotolerance of Z. xanthoxylum remained unclear., Results: Here, we characterized the acclimation of Z. xanthoxylum to heat using a combination of physiological measurements and transcriptional profiles under treatments at 40 °C and 45 °C, respectively. Strikingly, moderate high temperature (40 °C) led to an increase in photosynthetic capacity and superior plant performance, whereas severe high temperature (45 °C) was accompanied by reduced photosynthetic capacity and inhibited growth. Transcriptome profiling indicated that the differentially expressed genes (DEGs) were related to transcription factor activity, protein folding and photosynthesis under heat conditions. Furthermore, numerous genes encoding heat transcription shock factors (HSFs) and heat shock proteins (HSPs) were significantly up-regulated under heat treatments, which were correlated with thermotolerance of Z. xanthoxylum. Interestingly, the up-regulation of PSI and PSII genes and the down-regulation of chlorophyll catabolism genes likely contribute to improving plant performance of Z. xanthoxylum under moderate high temperature., Conclusions: We identified key genes associated with of thermotolerance and growth in Z. xanthoxylum, which provide significant insights into the regulatory mechanisms of thermotolerance and growth regulation in Z. xanthoxylum under high temperature conditions., (© 2023. The Author(s).)

Atriplex canescens is a representative halophyte with excellent tolerance to salt. Previous studies have revealed certain physiological mechanisms and detected functional genes associated with salt tolerance. However, knowledge on the ROS scavenging system and regulatory mechanisms in this species when adapting to salinity is limited. Therefore, this study further analyzed the transcriptional changes in genes related to the ROS scavenging system and important regulatory mechanisms in A. canescens under saline conditions using our previous RNA sequencing data. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation revealed that the differentially expressed genes (DEGs) were highly enriched in signal transduction- and reactive oxygen species-related biological processes, including "response to oxidative stress", "oxidoreductase activity", "protein kinase activity", "transcription factor activity", and "plant hormone signal transduction". Further analyses suggested that the transcription abundance of many genes involved in SOD, the AsA-GSH cycle, the GPX pathway, PrxR/Trx, and the flavonoid biosynthesis pathway were obviously enhanced. These pathways are favorable for scavenging excessive ROS induced by salt and maintaining the integrity of the cell membrane. Meanwhile, many vital transcription factor genes (WRKY, MYB, ZF, HSF, DREB, and NAC) exhibited increased transcripts, which is conducive to dealing with saline conditions by regulating downstream salt-responsive genes. Furthermore, a larger number of genes encoding protein kinases (RLK, CDPK, MAPK, and CTR1) were significantly induced by saline conditions, which is beneficial to the reception/transduction of salt-related signals. This study describes the abundant genetic resources for enhancing the salt tolerance in salt-sensitive plants, especially in forages and crops.

Competing Interests: The authors report no conflicts of interest.

Atriplex canescens is a C 4 shrub with excellent adaptation to saline and arid environments. Our previous study showed that the secretion of excessive Na + into leaf salt bladders is a primary strategy in salt tolerance of A. canescens and external 100 mM NaCl can substantially stimulate its growth. To investigate whether NaCl could facilitate Atriplex canescens response to drought stress, five-week-old seedlings were subjected to drought stress (30% of field water capacity) in the presence or absence of additional 100 mM NaCl. The results showed that, under drought stress, the addition of NaCl could substantially improve the growth of A. canescens by increasing leaf relative water content, enhancing photosynthetic activity and inducing a significant declined leaf osmotic potential (Ψs). The addition of NaCl significantly increased Na + concentration in leaf salt bladders and the Na + contribution to leaf Ψs, while had no adverse effects on K + accumulation in leaf laminae. Therefore, the large accumulation of Na + in salt bladders for enhancing osmotic adjustment (OA) ability is a vital strategy in A. canescens responding to drought stress. In addition, the concentration of free proline, bataine and soluble sugars exhibited a significant increase in the presence of NaCl under drought stress, and the betaine contribution to leaf Ψs was significantly increased by additional NaCl compared with that under drought treatment alone, suggesting that compatible solutes are also involved in OA in addition to functioning as protectants to alleviate water deficit injury., Competing Interests: Declaration of competing interest The authors declare that there are not any potential conflicts of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Through self-reflection, self-education, and with a learning mindset each of us has embarked on a personal path to understand the impact of racism in our personal and professional lives. This personal work is ongoing, though it was through our individual paths that led us to engage in dialogue on race and racism at the 38th National Society of Genetic Counselors Annual Conference. We initially did not know each other; however, we were drawn by a mutual desire to further the conversation and sought connection with each other after the Conversations Around Diversity Platform Presentations. Through sustained, open dialogue we created a brave space for sharing our emotional and intellectual responses to the conference. Through this dialogue and through written reflections, we recognized an emboldened urgency to author a joint reflection on our shared responsibility as genetic counseling training program leaders to use our privilege in service to our students and future students. We have the evidence that we are not a diverse profession. We have more evidence now than we did before that our profession performs poorly with regards to inclusivity. Our inability to acknowledge, address, and discuss racism and other forms of oppression is damaging to each of us individually and as a group of professionals. We owe it to ourselves, our students, our patients, and colleagues to name our learned biases and behaviors, own them and interrupt them., (© 2020 National Society of Genetic Counselors.)

Background: Atriplex canescens is a typical C 4 secretohalophyte with salt bladders on the leaves. Accumulating excessive Na + in tissues and salt bladders, maintaining intracellular K + homeostasis and increasing leaf organic solutes are crucial for A. canescens survival in harsh saline environments, and enhanced photosynthetic activity and water balance promote its adaptation to salt. However, the molecular basis for these physiological mechanisms is poorly understood. Four-week-old A. canescens seedlings were treated with 100 mM NaCl for 6 and 24 h, and differentially expressed genes in leaves and roots were identified, respectively, with Illumina sequencing., Results: In A. canescens treated with 100 mM NaCl, the transcripts of genes encoding transporters/channels for important nutrient elements, which affect growth under salinity, significantly increased, and genes involved in exclusion, uptake and vacuolar compartmentalization of Na + in leaves might play vital roles in Na + accumulation in salt bladders. Moreover, NaCl treatment upregulated the transcripts of key genes related to leaf organic osmolytes synthesis, which are conducive to osmotic adjustment. Correspondingly, aquaporin-encoding genes in leaves showed increased transcripts under NaCl treatment, which might facilitate water balance maintenance of A. canescens seedlings in a low water potential condition. Additionally, the transcripts of many genes involved in photosynthetic electron transport and the C 4 pathway was rapidly induced, while other genes related to chlorophyll biosynthesis, electron transport and C 3 carbon fixation were later upregulated by 100 mM NaCl., Conclusions: We identified many important candidate genes involved in the primary physiological mechanisms of A. canescens salt tolerance. This study provides excellent gene resources for genetic improvement of salt tolerance of important crops and forages.

HKT1 and SOS1 are two key Na⁺ transporters that modulate salt tolerance in plants. Although much is known about the respective functions of HKT1 and SOS1 under salt conditions, few studies have examined the effects of HKT1 and SOS1 mutations on the expression of other important Na⁺ and K⁺ transporter genes. This study investigated the physiological parameters and expression profiles of AtHKT1;1 , AtSOS1 , AtHAK5 , AtAKT1 , AtSKOR , AtNHX1 , and AtAVP1 in wild-type (WT) and athkt1;1 and atsos1 mutants of Arabidopsis thaliana under 25 mM NaCl. We found that AtSOS1 mutation induced a significant decrease in transcripts of AtHKT1;1 (by 56⁻62% at 6⁻24 h), AtSKOR (by 36⁻78% at 6⁻24 h), and AtAKT1 (by 31⁻53% at 6⁻24 h) in the roots compared with WT. This led to an increase in Na⁺ accumulation in the roots, a decrease in K⁺ uptake and transportation, and finally resulted in suppression of plant growth. AtHKT1;1 loss induced a 39⁻76% (6⁻24 h) decrease and a 27⁻32% (6⁻24 h) increase in transcripts of AtSKOR and AtHAK5 , respectively, in the roots compared with WT. At the same time, 25 mM NaCl decreased the net selective transport capacity for K⁺ over Na⁺ by 92% in the athkt1;1 roots compared with the WT roots. Consequently, Na⁺ was loaded into the xylem and delivered to the shoots, whereas K⁺ transport was restricted. The results indicate that AtHKT1;1 and AtSOS1 not only mediate Na⁺ transport but also control ion uptake and the spatial distribution of Na⁺ and K⁺ by cooperatively regulating the expression levels of relevant Na⁺ and K⁺ transporter genes, ultimately regulating plant growth under salt stress.

Puccinellia tenuiflora is a typical salt-excluding halophytic grass with excellent salt tolerance. Plasma membrane Na + /H + transporter SOS1, HKT-type protein and tonoplast Na + /H + antiporter NHX1 are key Na + transporters involved in plant salt tolerance. Based on our previous research, we had proposed a function model for these transporters in Na + homeostasis according to the expression of PtSOS1 and Na + , K + levels in P. tenuiflora responding to salt stress. Here, we analyzed the expression patterns of PtSOS1 , PtHKT1;5 , and PtNHX1 in P. tenuiflora under 25 and 150 mM NaCl to further validate this model by combining previous physiological characteristics. Results showed that the expressions of PtSOS1 and PtHKT1;5 in roots were significantly induced and peaked at 6 h under both 25 and 150 mM NaCl. Compared to the control, the expression of PtSOS1 significantly increased by 5.8-folds, while that of PtHKT1;5 increased only by 1.2-folds in roots under 25 mM NaCl; on the contrary, the expression of PtSOS1 increased by 1.4-folds, whereas that of PtHKT1;5 increased by 2.2-folds in roots under 150 mM NaCl. In addition, PtNHX1 was induced instantaneously under 25 mM NaCl, while its expression was much higher and more persistent in shoots under 150 mM NaCl. These results provide stronger evidences for the previous hypothesis and extend the model which highlights that SOS1, HKT1;5, and NHX1 synergistically regulate Na + homeostasis by controlling Na + transport systems at the whole-plant level under both lower and higher salt conditions. Under mild salinity, PtNHX1 in shoots compartmentalized Na + into vacuole slowly, and vacuole potential capacity for sequestering Na + would enhance Na + loading into the xylem of roots by PtSOS1 through feedback regulation; and consequently, Na + could be transported from roots to shoots by transpiration stream for osmotic adjustment. While under severe salinity, Na + was rapidly sequestrated into vacuoles of mesophyll cells by PtNHX1 and the vacuole capacity became saturated for sequestering more Na + , which in turn regulated long-distance Na + transport from roots to shoots. As a result, the expression of PtHKT1;5 was strongly induced so that the excessive Na + was unloaded from xylem into xylem parenchyma cells by PtHKT1;5.

Salinization, desertification, and soil nutrient deprivation are threatening the production of alfalfa ( Medicago sativa L.) in northern China. We have previously generated T 0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop this excellent breeding material into the new forage cultivar, stress tolerance, productivity, and forage quality of T 1 transgenic alfalfa (GM) were assessed in this study. The GM inherited the traits of salt and drought tolerance from T 0 generation. Most importantly, co-overexpression of ZxNHX and ZxVP1-1 enhanced the tolerance to Pi deficiency in GM, which was associated with more Pi accumulation in plants. Meanwhile, T 1 transgenic alfalfa developed a larger root system with increased root size, root dry weight and root/shoot ratio, which may be one important reason for the improvement of phosphorus nutrition and high biomass accumulation in GM under various conditions. GM also accumulated more crude protein, crude fiber, crude fat, and crude ash than wild-type (WT) plants, especially under stress conditions and in the field. More interestingly, the crude fat contents sharply dropped in WT (by 66-74%), whereas showed no change or decreased less in GM, when subjected to salinity, drought or low-Pi. Our results indicate that T 1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 shows stronger stress tolerance, higher productivity and better forage quality. This study provides a solid foundation for creating the alfalfa cultivars with high yield, good quality and wide adaptability on saline, dry, and nutrient-deprived marginal lands of northern China.

Atriplex canescens (fourwing saltbush) is a C4 perennial fodder shrub with excellent resistance to salinity. However, the mechanisms underlying the salt tolerance in A. canescens are poorly understood. In this study, 5-weeks-old A. canescens seedlings were treated with various concentrations of external NaCl (0-400 mM). The results showed that the growth of A. canescens seedlings was significantly stimulated by moderate salinity (100 mM NaCl) and unaffected by high salinity (200 or 400 mM NaCl). Furthermore, A. canescens seedlings showed higher photosynthetic capacity under NaCl treatments (except for 100 mM NaCl treatment) with significant increases in net photosynthetic rate and water use efficiency. Under saline conditions, the A. canescens seedlings accumulated more Na(+) in either plant tissues or salt bladders, and also retained relatively constant K(+) in leaf tissues and bladders by enhancing the selective transport capacity for K(+) over Na(+) (ST value) from stem to leaf and from leaf to bladder. External NaCl treatments on A. canescens seedlings had no adverse impact on leaf relative water content, and this resulted from lower leaf osmotic potential under the salinity conditions. The contribution of Na(+) to the leaf osmotic potential (Ψs) was sharply enhanced from 2% in control plants to 49% in plants subjected to 400 mM NaCl. However, the contribution of K(+) to Ψs showed a significant decrease from 34% (control) to 9% under 400 mM NaCl. Interestingly, concentrations of betaine and free proline showed significant increase in the leaves of A. canescens seedlings, these compatible solutes presented up to 12% of contribution to Ψs under high salinity. These findings suggest that, under saline environments, A. canescens is able to enhance photosynthetic capacity, increase Na(+) accumulation in tissues and salt bladders, maintain relative K(+) homeostasis in leaves, and use inorganic ions and compatible solutes for osmotic adjustment which may contribute to the improvement of water status in plant.

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens-mediated transformation. Compared with wild-type (WT) plants, transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water-deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water-use efficiency than WT plants. Our results indicate that co-expression of tonoplast NHX and H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants.

Lotus corniculatus L. is an important legume for forage, but is sensitive to salinity and drought. To develop salt- and drought-resistant L. corniculatus, ZxNHX and ZxVP1-1 genes encoding tonoplast Na+/H+ antiporter and H+-pyrophosphatase (H+-PPase) from a succulent xerophyte Zygophyllum xanthoxylum L., which is well adapted to arid environments through accumulating Na+ in its leaves, were transferred into this forage. We obtained the transgenic lines co-expressing ZxNHX and ZxVP1-1 genes (VX) as well as expressing ZxVP1-1 gene alone (VP). Compared with wild-type, both VX and VP transgenic lines grew better at 200mM NaCl, and also exhibited higher tolerance and faster recovery from water-deficit stress: these performances were associated with more Na+, K+ and Ca2+ accumulation in their leaves and roots, which caused lower leaf solute potential and thus retained more water. Moreover, the transgenic lines maintained lower relative membrane permeability and higher net photosynthesis rate under salt or water-deficit stress. These results indicate that expression of tonoplast Na+/H+ antiporter and H+-PPase genes from xerophyte enhanced salt and drought tolerance of L. corniculatus. Furthermore, compared with VP, VX showed higher shoot biomass, more cations accumulation, higher water retention, lesser cell membrane damage and higher photosynthesis capacity under salt or water-deficit condition, suggesting that co-expression of ZxVP1-1 and ZxNHX confers even greater performance to transgenic L. corniculatus than expression of the single ZxVP1-1.

The plasma membrane Na+/H+ antiporter (SOS1) was shown to be a Na+ efflux protein and also involved in K+ uptake and transport. PtSOS1 was characterised from Puccinellia tenuiflora (Griseb.) Scribn. et Merr., a monocotyledonous halophyte that has a high selectivity for K+ over Na+ by roots under salt stress. To assess the contribution of PtSOS1 to the selectivity for K+ over Na+, the expression levels of PtSOS1 and Na+, K+ accumulations in P. tenuiflora exposed to different concentrations of NaCl, KCl or NaCl plus KCl were analysed. Results showed that the expression levels of PtSOS1 in roots increased significantly with the increase of external NaCl (25-150mM), accompanied by an increase of selective transport (ST) capacity for K+ over Na+ by roots. Transcription levels of PtSOS1 in roots and ST values increased under 0.1-1mM KCl, then declined sharply under 5-10mM KCl. Under 150mM NaCl, PtSOS1 expression levels in roots and ST values at 0.1mM KCl was significantly lower than that at 5mM KCl with the prolonging of treatment time. A significant positive correlation was found between root PtSOS1 expression levels and ST values under various concentrations of NaCl, KCl or 150mM NaCl plus 0.1 or 5mM KCl treatments. Therefore, it is proposed that PtSOS1 is the major component of selective transport capacity for K+ over Na+ and hence, salt tolerance of P. tenuiflora. Finally, we hypothesise a function model of SOS1 in regulating K+ and Na+ transport system in the membrane of xylem parenchyma cells by sustaining the membrane integrity; it also appears that this model could reasonably explain the phenomenon of Na+ retrieval from the xylem when plants are exposed to severe salt stress.

Zygophyllum xanthoxylum, a C(3) woody species, is a succulent xerophyte that is well adapted to arid environments. Our previous investigations showed that Na(+) has a positive effect on the growth of Z. xanthoxylum under drought conditions, which was closely related to high Na(+) accumulation in leaves. To reveal the physiological mechanisms underlying how Na(+) accumulation improves the drought resistance of Z. xanthoxylum, 3-week-old seedlings were treated with a series of additional external NaCl concentrations (5-150 mM) in sand culture experiments. Seedlings were also subjected to water deficit (30% of field water capacity) in the presence or absence of additional NaCl (50 mM) in pot experiments. The results indicated that 50 mM NaCl could mitigate deleterious impacts of water deficit on the growth of Z. xanthoxylum, by improving the relative water content, inducing a significant drop in leaf water potential and, concomitantly, increasing leaf turgor pressure and chlorophyll concentrations resulting in an enhancement of overall plant photosynthetic activity (i.e., photosynthetic rate and water use efficiency). Furthermore, NaCl (50 mM) could alleviate the inhibitory effect of water deficit on the activity of photosystem II in Z. xanthoxylum. The contribution of Na(+) to the total osmotic potential varied from 8% in the control to 13% in plants subjected to water deficit and, surprisingly, to 28% in plants grown in the presence of 50 mM NaCl under water deficit; however, the contribution of K(+) significantly decreased from 13 to 8%. These findings suggest that, under arid environments, Z. xanthoxylum is able to accumulate a high concentration of Na(+) in its leaves and use it directly for osmotic adjustment, which was coupled with an improvement in leaf hydration and photosynthetic activity.

Sodium (Na(+)) has been found to play important roles in the adaptation of xerophytic species to drought conditions. The tonoplast Na(+)/H(+) antiporter (NHX) proved to be involved in the compartmentalization of Na(+) into vacuoles from the cytosol. In this study, a gene (ZxNHX) encoding tonoplast Na(+)/H(+) antiporter was isolated and characterized in Zygophyllum xanthoxylum, a succulent xerophyte growing in desert areas of northwest China. The results revealed that ZxNHX consisted of 532 amino acid residues with a conserved binding domain ((78)LFFIYLLPPI(87)) for amiloride and shared high similarity (73-81%) with the identified tonoplast Na(+)/H(+) antiporters in other plant species. Semi-quantitative RT-PCR analysis showed that the mRNA level of ZxNHX was significantly higher in the leaf than in stem or root. The transcript abundance of ZxNHX in Z. xanthoxylum subjected to salt (5-150 mM NaCl) or drought (50-15% of field water capacity (FWC)) was 1.4-8.4 times or 2.3-4.4 times that of plants grown in the absence of NaCl or 70% of FWC, respectively. Leaf Na(+) concentration in plants exposed to salt or drought was 1.7-5.2 times or 1.5-2.2 times that of corresponding control plants, respectively. It is clear that there is a positive correlation between up-regulation of ZxNHX and accumulation of Na(+) in Z. xanthoxylum exposed to salt or drought. Furthermore, Z. xanthoxylum accumulated larger amounts of Na(+) than K(+) in the leaf under drought conditions, even in low salt soil. In summary, our results suggest that ZxNHX encodes a tonoplast Na(+)/H(+) antiporter and plays important roles in Na(+) accumulation and homeostasis of Z. xanthoxylum under salt and drought conditions., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

To investigate the effects of amiloride, which is an inhibitor of Na(+)/H(+) antiporter (NHX), on Na(+) accumulation and transport at the whole plant level, in this study, 3-week-old Zygophyllum xanthoxylum plants were exposed to 25, 50 and 100 mM NaCl with or without 0.5 mM amiloride. After 72 h of treatment, dry weight, tissue water content, Na(+) and K(+) concentrations, and transcript levels of ZxNHX were determined. The results indicated that, under salt conditions, leaf Na(+) concentration, total Na(+) quantity and root net Na(+) uptake rate in plants treated with amiloride are significantly lower than those in control plants. Amiloride remarkably increased Na(+) proportion in stem and decreased Na(+) proportion in leaf of Z. xanthoxylum. Furthermore, our results showed that the transcript levels of ZxNHX are down-regulated by amiloride. It is clear that the inhibition of vacuolar Na(+)/H(+) antiporter by amiloride could disrupt Na(+) accumulation of leaf, and reduce Na(+) uptake by root and Na(+) transport from stem to leaf, thus resulting in the growth inhibition of Z. xanthoxylum exposed to salt.

Salinity is an increasing problem for agricultural production worldwide. The result of low-affinity Na(+) uptake is toxic to the cytoplasm of most crop plants. Nevertheless, the pathways for this low-affinity Na(+) uptake are still uncertain. In this work we used ²²Na(+) isotope tracing technology to investigate factors associated with determination of root ²²Na(+) influx in the salt accumulation halophyte Suaeda maritima. We found that a 2 min of exposure to the ²²Na(+) labeled uptake solution was optimal for determining ²²Na(+) influx into excised roots of S. maritima and that 7 min of blotting is suitable in ²²Na(+) influx experiments. ²²Na(+) influx did not increase linearly with the increasing external Na(+) concentration, in the range tested, of 2 to 300 mM NaCl. But root ²²Na(+) influx and root Na(+) concentration were well correlated. ²²Na(+) influx into excised roots of S. maritima was not, however, well correlated with the plant size. All the above results indicated further that this ²²Na(+) isotope influx procedure is a good method for quantify Na(+) uptake rate by the roots of the salt accumulation halophyte.