Molecular mechanism of mulberry response to drought stress revealed by complementary transcriptomic and iTRAQ analyses.

BACKGROUND: The use of mulberry leaves has long been limited to raising silkworms, but with the continuous improvement of mulberry (Morus alba) resource development and utilization, various mulberry leaf extension products have emerged. However, the fresh leaves of mulberry trees have a specific window of time for picking and are susceptible to adverse factors, such as drought stress. Therefore, exploring the molecular mechanism by which mulberry trees resist drought stress and clarifying the regulatory network of the mulberry drought response is the focus of the current work. RESULTS: In this study, natural and drought-treated mulberry grafted seedlings were used for transcriptomic and proteomic analyses (CK vs. DS9), aiming to clarify the molecular mechanism of the mulberry drought stress response. Through transcriptome and proteome sequencing, we identified 9889 DEGs and 1893 DEPs enriched in stress-responsive GO functional categories, such as signal transducer activity, antioxidant activity, and transcription regulator activity. KEGG enrichment analysis showed that a large number of codifferentially expressed genes were enriched in flavonoid biosynthesis pathways, hormone signalling pathways, lignin metabolism and other pathways. Through subsequent cooperation analysis, we identified 818 codifferentially expressed genes in the CK vs. DS9 comparison group, including peroxidase (POD), superoxide dismutase (SOD), aldehyde dehydrogenase (ALDHs), glutathione s-transferase (GST) and other genes closely related to the stress response. In addition, we determined that the mulberry gene MaWRKYIII8 (XP_010104968.1) underwent drought- and abscisic acid (ABA)-induced expression, indicating that it may play an important role in the mulberry response to drought stress. CONCLUSIONS: Our research shows that mulberry can activate proline and ABA biosynthesis pathways and produce a large amount of proline and ABA, which improves the drought resistance of mulberry. MaWRKYIII8 was up-regulated and induced by drought and exogenous ABA, indicating that MaWRKYIII8 may be involved in the mulberry response to drought stress. These studies will help us to analyse the molecular mechanism underlying mulberry drought tolerance and provide important gene information and a theoretical basis for improving mulberry drought tolerance through molecular breeding in the future.

Natural population re-sequencing detects the genetic basis of local adaptation to low temperature in a woody plant.

KEY MESSAGE: Total of 14 SNPs associated with overwintering-related traits and 75 selective regions were detected. Important candidate genes were identified and a possible network of cold-stress responses in woody plants was proposed. Local adaptation to low temperature is essential for woody plants to against changeable climate and safely survive the winter. To uncover the specific molecular mechanism of low temperature adaptation in woody plants, we sequenced 134 core individuals selected from 494 paper mulberry (Broussonetia papyrifera), which naturally distributed in different climate zones and latitudes. The population structure analysis, PCA analysis and neighbor-joining tree analysis indicated that the individuals were classified into three clusters, which showed forceful geographic distribution patterns because of the adaptation to local climate. Using two overwintering phenotypic data collected at high latitudes of 40°N and one bioclimatic variable, genome-phenotype and genome-environment associations, and genome-wide scans were performed. We detected 75 selective regions which possibly undergone temperature selection and identified 14 trait-associated SNPs that corresponded to 16 candidate genes (including LRR-RLK, PP2A, BCS1, etc.). Meanwhile, low temperature adaptation was also supported by other three trait-associated SNPs which exhibiting significant differences in overwintering traits between alleles within three geographic groups. To sum up, a possible network of cold signal perception and responses in woody plants were proposed, including important genes that have been confirmed in previous studies while others could be key potential candidates of woody plants. Overall, our results highlighted the specific and complex molecular mechanism of low temperature adaptation and overwintering of woody plants.

A salicylic acid inducible mulberry WRKY transcription factor, MiWRKY53 is involved in plant defence response.

KEY MESSAGE: MiWRKY53 is expressed in response to various stresses and hormones. Although it is localized in the nucleus, it shows no transcriptional activation. Role of SA-mediated plant defence response is demonstrated. WRKY transcription factors are one the largest gene families in plants involved in almost every process in plants including development, physiological processes, and stress response. Salicylic acid (SA) is key regulator of biotic stress against various pathogens in plants acting via its multiple mechanisms to induce defence response. Herein, we have identified and functionally validated WRKY53 from mulberry (Morus indica var. K2). MiWRKY53 expressed differentially in response to different stress and hormonal treatments. MiWRKY53 belongs to group III of WKRY gene family, localized in nucleus, and lacks transcriptional activation activity in yeast. Hormone responsive behaviour of MiWRKY53 Arabidopsis overexpression (OE) transgenics preferentially was noted in root growth assay in response to Salicylic acid (SA). Arabidopsis overexpression plants also displayed alteration in leaf phenotype having wider leaves than the wild-type plants. PR-1 transcripts were higher in MiWRKY53 Arabidopsis OE plants and they displayed resistance towards biotrophic pathogen Pseudomonas syringae PstDC3000. MiWRKY53 Mulberry OE transgenics also depicted SA-responsive behaviour. Several hormones and stress-related cis-acting elements were also identified in the 1.2-Kb upstream regulatory region (URR) of MiWRKY53. Functional characterization of full-length promoter region revealed that it is induced by SA and further analysis of deletion constructs helped in the identification of minimal promoter responsible for its inducibility by SA. Altogether, the findings from this study point towards the SA preferential behaviour of MiWRKY53 and its function as regulator of plant defence response through SA-mediated mechanisms.

Accumulation and translocation of food chain in soil-mulberry (Morus alba L.)-silkworm (Bombyx mori) under single and combined stress of lead and cadmium.

In recent years, heavy metal pollution has caused immeasurable harm to the environment. As an emerging technology, phytoremediation technology has gained a place in the treatment of heavy metal pollution with its unique advantages. This study analyzes the toxic effects of mulberry (Morus alba) seeds, seedling growth and silkworm under heavy metal stress of lead (Pb) and cadmium (Cd), and explore the accumulation and migration of Pb and Cd in the soil-mulberry tree-silkworm system. The main results were as follows: (1) Seed germination and potted seedling experiments were conducted under heavy metal Pb and Cd stresses, and it was found that Pb and Cd had inhibitory effects on mulberry seed germination, growth and photosynthesis of mulberry seedlings, and as the concentration of heavy metals increased, the stronger the inhibitory effect. Moreover, Pb and Cd have a synergistic effect under compound stress. (2) The accumulation and transfer rules of Pb and Cd ions in mulberry were different. The content of Pb in mulberry was root > leaf > stem and the content of Cd was root > stem > leaf. The combined stress promoted the transfer of Pb and Cd from the underground part to the aerial portion of mulberry. (3) The silkworm feeds on mulberry leaves contaminated with heavy metals in this experiment and found that: with the increase of silkworm feeding, the heavy metal content in the silkworm body increased significantly, but the content remained in the silkworm body was less, most of it was excreted with silkworm excrement. Combined stress has no significant effect on the detoxification mechanism of silkworm. It is indispensable to think of the synergistic effect of heavy metals on plants germination when seeds are used for phytoremediation.

In vivo photoprotection mechanisms observed from leaf spectral absorbance changes showing VIS-NIR slow-induced conformational pigment bed changes.

Regulated heat dissipation under excessive light comprises a complexity of mechanisms, whereby the supramolecular light-harvesting pigment-protein complex (LHC) shifts state from light harvesting towards heat dissipation, quenching the excess of photo-induced excitation energy in a non-photochemical way. Based on whole-leaf spectroscopy measuring upward and downward spectral radiance fluxes, we studied spectrally contiguous (hyperspectral) transient time series of absorbance A(λ,t) and passively induced chlorophyll fluorescence F(λ,t) dynamics of intact leaves in the visible and near-infrared wavelengths (VIS-NIR, 400-800 nm) after sudden strong natural-like illumination exposure. Besides light avoidance mechanism, we observed on absorbance signatures, calculated from simultaneous reflectance R(λ,t) and transmittance T(λ,t) measurements as A(λ,t) = 1 - R(λ,t) - T(λ,t), major dynamic events with specific onsets and kinetical behaviour. A consistent well-known fast carotenoid absorbance feature (500-570 nm) appears within the first seconds to minutes, seen from both the reflected (backscattered) and transmitted (forward scattered) radiance differences. Simultaneous fast Chl features are observed, either as an increased or decreased scattering behaviour during quick light adjustment consistent with re-organizations of the membrane. The carotenoid absorbance feature shows up simultaneously with a major F decrease and corresponds to the xanthophyll conversion, as quick response to the proton gradient build-up. After xanthophyll conversion (t = 3 min), a kinetically slower but major and smooth absorbance increase was occasionally observed from the transmitted radiance measurements as wide peaks in the green (~ 550 nm) and the near-infrared (~ 750 nm) wavelengths, involving no further F quenching. Surprisingly, in relation to the response to high light, this broad and consistent VIS-NIR feature indicates a slowly induced absorbance increase with a sigmoid kinetical behaviour. In analogy to sub-leaf-level observations, we suggest that this mechanism can be explained by a structure-induced low-energy-shifted energy redistribution involving both Car and Chl. These findings might pave the way towards a further non-invasive spectral investigation of antenna conformations and their relations with energy quenching at the intact leaf level, which is, in combination with F measurements, of a high importance for assessing plant photosynthesis in vivo and in addition from remote observations.

Photochemistry and proteomics of mulberry (Morus alba L.) seedlings under NaCl and NaHCO3 stress.

In order to explore the response and adaptation mechanisms of photosynthesis of the leaves of mulberry (Morus alba L.) seedlings to saline-alkali stress. Photosynthetic activity, and the response of related proteomics of M. alba seedling leaves under NaCl and NaHCO3 stress were studied by using chlorophyll fluorescence and gas exchange technique combined with TMT proteomics. The results showed that NaCl stress had no significant effect on photosystem II (PSII) activity in M. alba seedling leaves. In addition, the expressions of proteins of the PSII oxygen-evolving complex (OEE3-1 and PPD4) and the LHCII antenna (CP24 10A, CP26, and CP29) were increased, and the photosystem I (PSI) activity in the leaves of M. alba seedlings was increased, as well as expressions of proteins, such as PsaF, PsaG, PsaH, PsaL, PsaN, and Ycf4. Under NaHCO3 stress, the activity of PSII and PSI and the expression of their protein complexes and the electron transfer-related proteins significantly decreased. NaCl stress had little effect on RuBP regeneration during dark reaction in the leaves and the expressions of glucose synthesis related proteins and net photosynthetic rate (Pn) did not decrease significantly. The leaves could adapt to NaCl stress by reducing stomatal conductance (Gs) and increasing water use efficiency (WUE). Under NaHCO3 stress, the expression of dark reaction-related proteins was mostly down-regulated, while Gs was reduced, which indicated that non-stomatal factors can be responsible for inhibition of carbon assimilation.

Physiological and Proteomic Responses of Mulberry Trees (Morus alba. L.) to Combined Salt and Drought Stress.

Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.

Physiological and Transcriptomic Changes during the Early Phases of Adventitious Root Formation in Mulberry Stem Hardwood Cuttings.

The initiation and induction of root primordia are of great importance for adventitious root (AR) formation in cutting propagation of horticultural and forestry crops. However, the underlying mechanisms orchestrating these early phases of AR formation remain largely unexplored. Here, we investigated the physiological and transcriptomic changes during the early AR phases in mulberry stem hardwood cuttings. The results showed that the concentrations of soluble proteins increased, whereas concentrations of soluble sugars and starch were decreased. Indole-3-acetic acid (IAA) and zeatin had a rapid transit peak at 6 h after planting (hAP) and declined thereafter. The activities of peroxidase and catalase persistently increased and indole-3-acetic acid oxidase was maintained at a higher stable level from 0 hAP, while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels. The comparative transcriptome identified 4276 common genes that were differentially regulated at -6, 0 and 54 hAP. They were separated into five clusters with distinct biological functions such as defense response and photosynthesis. Considerable common genes were assigned to pathways of sugar metabolism, mitogen-activated protein kinase, and circadian rhythm. The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses, hormone signaling, energy metabolism, starch metabolism, and circadian rhythm. These findings demonstrate the positive effect of auxin on AR induction, and uncovered the crucial roles of stress responses, hormone signaling and circadian rhythm in coordinating the physiological changes during the early phases of AR formation in mulberry stem hardwood cuttings.

Structures of Bordered Pits Potentially Contributing to Isolation of a Refilled Vessel from Negative Xylem Pressure in Stems of Morus australis Poir.: Testing of the Pit Membrane Osmosis and Pit Valve Hypotheses.

Two hypotheses have been proposed to explain the mechanism preventing the refilling vessel water from being drained to the neighboring functional vessels under negative pressure. The pit membrane osmosis hypothesis proposes that the xylem parenchyma cells release polysaccharides that are impermeable to the intervessel pit membranes into the refilling vessel; this osmotically counteracts the negative pressure, thereby allowing the vessel to refill. The pit valve hypothesis proposes that gas trapped within intervessel bordered pits isolates the refilling vessel water from the surrounding functional vessels. Here, using the single-vessel method, we assessed these hypotheses in shoots of mulberry (Morus australis Poir.). First, we confirmed the occurrence of xylem refilling under negative pressure in the potted mulberry saplings. To examine the pit membrane osmosis hypothesis, we estimated the semi-permeability of pit membranes for molecules of various sizes and found that the pit membranes were not semi-permeable to polyethylene glycol of molecular mass <20,000. For the pit valve hypothesis, we formed pit valves in the intervessel pits in the short stem segments and measured the maximum liquid pressure up to which gases in bordered pits were retained. The threshold pressure ranged from 0.025 to 0.10 MPa. These values matched the theoretical value calculated from the geometry of the pit chamber (0.0692-0.101 MPa). Our results suggest that gas in the pits is retained by surface tension, even under substantial positive pressure to resolve gases in the refilling vessel, whereas the molecule size required for the pit membrane osmosis mechanism in mulberry would be unrealistically large.

Life span and structure of ephemeral root modules of different functional groups from a desert system.

The terminal branch orders of plant root systems have been proposed as short-lived 'ephemeral' modules specialized for resource absorption. The occurrence of ephemeral root modules has so far only been reported for a temperate tree species and it is unclear if the concept also applies to other woody (shrub, tree) and herb species. Fine roots of 12 perennial dicotyledonous herb, shrub and tree species were monitored for two growing seasons using a branch-order classification, sequential sampling and rhizotrons in the Taklamakan desert. Two root modules existed in all three plant functional groups. Among the first five branch orders, the first two (perennial herbs, shrubs) or three (trees) root orders were ephemeral and had a primary anatomical structure, high nitrogen (N) concentrations, high respiration rates and very short life spans of 1-4 months, whereas the last two branch orders in all functional groups were perennial, with thicker diameters, no or collapsed cortex, distinct secondary growth, low N concentrations, low respiration rates, but much longer life spans. Ephemeral, short-lived root modules and long-lived, persistent root modules seem to be a general feature across many plant functional groups and could represent a basic root system design.

Phenotypic and Transcriptomic Analyses of Autotetraploid and Diploid Mulberry (Morus alba L.).

Autopolyploid plants and their organs are often larger than their diploid counterparts, which makes them attractive to plant breeders. Mulberry (Morus alba L.) is an important commercial woody plant in many tropical and subtropical areas. In this study, we obtained a series of autotetraploid mulberry plants resulting from a colchicine treatment. To evaluate the effects of genome duplications in mulberry, we compared the phenotypes and transcriptomes of autotetraploid and diploid mulberry trees. In the autotetraploids, the height, breast-height diameter, leaf size, and fruit size were larger than those of diploids. Transcriptome data revealed that of 21,229 expressed genes only 609 (2.87%) were differentially expressed between diploids and autotetraploids. Among them, 30 genes were associated with the biosynthesis and signal transduction of plant hormones, including cytokinin, gibberellins, ethylene, and auxin. In addition, 41 differentially expressed genes were involved in photosynthesis. These results enhance our understanding of the variations that occur in mulberry autotetraploids and will benefit future breeding work.

Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress.

The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress.

Beyond seasonal climate: statistical estimation of phenological responses to weather.

Phenological events, such as the timing of flowering or insect emergence, are influenced by a complex combination of climatic and non-climatic factors. Although temperature is generally considered most important, other weather events such as frosts and precipitation events can also influence many species' phenology. Non-climatic variables such as photoperiod and site-specific habitat characteristics can also have important effects on phenology. Forecasting phenological shifts due to climate change requires understanding and quantifying how these multiple factors combine to affect phenology. However, current approaches to analyzing phenological data have a limited ability for quantifying multiple drivers simultaneously. Here, we use a novel statistical approach to estimate the combined effects of multiple variables, including local weather events, on the phenology of several taxa (a tree, an insect, and a fungus). We found that thermal forcing had a significant positive effect on each species, frost events delayed the phenology of the tree and butterfly, and precipitation had a positive effect on fungal fruiting. Using data from sites across latitudinal gradients, we found that these effects are remarkably consistent across sites once latitude and other site effects are accounted for. This consistency suggests an underlying biological response to these variables that is not commonly estimated using data from field observations. This approach's flexibility will be useful for forecasting ongoing phenological responses to changes in climate variability in addition to seasonal trends.

Transcriptomic and metabolomic analyses reveal the importance of ethylene networks in mulberry fruit ripening.

Mulberry (Morus alba L.) is a climacteric and highly perishable fruit. Ethylene has been considered to be an important trigger of fruit ripening process. However, the role of ethylene in the mulberry fruit ripening process remains unclear. In this study, we performed a comprehensive analysis of metabolomic and transcriptomic data of mulberry fruit and the physiological changes accompanying the fruit ripening process. Our study revealed that changes in the accumulation of specific metabolites at different stages of fruit development and ripening were closely correlated to transcriptional changes as well as underlying physiological changes and the development of taste biomolecules. The ripening of mulberry fruits was highly associated with the production of endogenous ethylene, and further application of exogenous ethylene assisted the ripening process. Transcriptomic analysis revealed that differential expression of diverse ripening-related genes was involved in sugar metabolism, anthocyanin biosynthesis, and cell wall modification pathways. Network analysis of transcriptomics and metabolomics data revealed that many transcription factors and ripening-related genes were involved, among which ethylene-responsive transcription factor 3 (MaERF3) plays a crucial role in the ripening process. The role of MaERF3 in ripening was experimentally proven in a transient overexpression assay in apples. Our study indicates that ethylene plays a vital role in modulating mulberry fruit ripening. The results provide a basis for guiding the genetic manipulation of mulberry fruits towards sustainable agricultural practices and improve post-harvest management, potentially enhancing the quality and shelf life of mulberry fruits for sustainable agriculture and forestry.

Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees.

The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.

The impact of boron nutrient supply in mulberry (Morus alba) response to metabolomics, enzyme activities, and physiological parameters.

Boron (B) is essential for normal and healthy plant growth. Therefore, Boron stress is a common abiotic stress that limits plant growth and productivity. However, how mulberry copes with boron stress remains unclear. In this study, seedlings of the Morus alba cultivar, Yu-711, were treated with five different concentrations of boric acid (H3BO3), including deficient (0 and 0.02 mM), sufficient (0.1 mM) and toxic (0.5 and 1 mM) levels. Physiological parameters, enzymatic activities and non-targeted liquid chromatography-mass spectrometry (LC-MS) technique were employed to evaluate the effects of boron stress on the net photosynthetic rate (Pn), chlorophyll content, stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci) and metabolome signatures. Physiological analysis revealed that Boron deficiency and toxicity induced a decline in Pn, Ci, Gs, Tr, and chlorophyll content. Also, enzymatic activities, including catalase (CAT) and superoxide dismutase (SOD), decreased, while POD activity increased in response to Boron stress. Osmotic substances such as soluble sugars, soluble proteins, and proline (PRO) presented elevated levels under all Boron concentrations. Metabolome analysis indicated that differential metabolites, including amino acids, secondary metabolites, carbohydrates, and lipids, played a key role in Yu-711's response to Boron stress. These metabolites were mainly involved in amino acid metabolism, biosynthesis of other secondary metabolites, lipid metabolism, metabolism of cofactors and vitamins, and metabolism of other amino acids pathways. Our findings reveal the various metabolites pathways in mulberry response to boron nutrient supply and may serve as fundamental knowledge in breeding resistance mulberry plants, so that it can cope with climate changes.

Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress.

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.

Response of the Morus bombycis growing season to temperature and its latitudinal pattern in Japan.

Changes in leaf phenology lengthen the growing season length (GSL, the days between leaf budburst and leaf fall) under the global warming. GSL and the leaf phenology response to climate change is one of the most important predictors of climate change effect on plants. Empirical evidence of climatic effects on GSL remains scarce, especially at a regional scale and the latitudinal pattern. This study analyzed the datasets of leaf budburst and fall phenology in Morus bombycis (Urticales), which were observed by the agency of the Japan Meteorological Agency (JMA) from 1953 to 2005 over a wide range of latitudes in Japan (31 to 44° N). In the present study, single regression slopes of leaf phenological timing and air temperature across Japan were calculated and their spatial patterns using general linear models were tested. The results showed that the GSL extension was caused mainly by a delay in leaf fall phenology. Relationships between latitude and leaf phenological and GSL responses against air temperature were significantly negative. The response of leaf phenology and GSL to air temperature at lower latitudes was larger than that at higher latitudes. The findings indicate that GSL extension should be considered with regards to latitude and climate change.

Direct evidence of drought stress memory in mulberry from a physiological perspective: Antioxidative, osmotic and phytohormonal regulations.

Drought stress commonly happens more than once during the life cycle of perennial trees. Stress memory endows better capacity to cope with repeated stresses for plants, while the underlying mechanisms are not fully elucidated. In this study, 2-month-old saplings of two mulberry cultivars (Husang32 and 7307 of Morus multicaulis) with or without an early soil water deficit were subjected to subsequent drought for 9 days. The shoot height growth, biomass production, stable carbon isotope discrimination, phytohormones, reactive oxygen species (ROS), osmotic substances and antioxidant enzymes were analyzed after the first and the second drought, respectively. Drought priming saplings sustained comparable or slightly higher biomass accumulation under the second drought than those non-priming. They also exhibited decreased levels of soluble sugars, free proline and soluble proteins, lower accumulation of malonaldehyde (MDA) and superoxide anion (O2•-), reduced activities of superoxide dismutase (SOD) and peroxidase (POD) compared to non-priming plants. Moreover, cultivar Husang32 exhibited elevated abscisic acid (ABA) and jasmonic acid (JA) where 7307 displayed opposite changes. PCA suggests that MDA, H2O2, free proline, SOD and POD in roots, and ROS, soluble sugars and glutamate reductase in leaves are dominant factors influenced by stress memory. ABA and JA in leaves also play important roles in exerting drought imprints. Collectively, stress memory can confer mulberry resistance to recurrent drought via combined regulations of antioxidative protection, osmotic adjustment and phytohormonal responses.

Diurnal Response of Photosystem I to Fluctuating Light Is Affected by Stomatal Conductance.

Upon a sudden transition from low to high light, electrons transported from photosystem II (PSII) to PSI should be rapidly consumed by downstream sinks to avoid the over-reduction of PSI. However, the over-reduction of PSI under fluctuating light might be accelerated if primary metabolism is restricted by low stomatal conductance. To test this hypothesis, we measured the effect of diurnal changes in stomatal conductance on photosynthetic regulation under fluctuating light in tomato (Solanum lycopersicum) and common mulberry (Morus alba). Under conditions of high stomatal conductance, we observed PSI over-reduction within the first 10 s after transition from low to high light. Lower stomatal conductance limited the activity of the Calvin-Benson-Bassham cycle and aggravated PSI over-reduction within 10 s after the light transition. We also observed PSI over-reduction after transition from low to high light for 30 s at the low stomatal conductance typical of the late afternoon, indicating that low stomatal conductance extends the period of PSI over-reduction under fluctuating light. Therefore, diurnal changes in stomatal conductance significantly affect the PSI redox state under fluctuating light. Moreover, our analysis revealed an unexpected inhibition of cyclic electron flow by the severe over-reduction of PSI seen at low stomatal conductance. In conclusion, stomatal conductance can have a large effect on thylakoid reactions under fluctuating light.