The overlooked functions of trichomes: Water absorption and metal detoxication.

Trichomes are epidermal outgrowths on plant shoots. Their roles in protecting plants against herbivores and in the biosynthesis of specialized metabolites have long been recognized. Recently, studies are increasingly showing that trichomes also play important roles in water absorption and metal detoxication, with these roles having important implications for ecology, the environment, and agriculture. However, these two functions of trichomes have been largely overlooked and much remains unknown. In this review, we show that the trichomes of 37 plant species belonging to 14 plant families are involved in water absorption, while the trichomes of 33 species from 13 families are capable of sequestering metals within their trichomes. The ability of trichomes to absorb water results from their decreased hydrophobicity compared to the remainder of the leaf surface as well as the presence of special structures for collecting and absorbing water. In contrast, the metal detoxication function of trichomes results not only from the good connection of their basal cells to the underlying vascular tissues, but also from the presence of metal-chelating ligands and transporters within the trichomes themselves. Knowledge gaps and critical future research questions regarding these two trichome functions are highlighted. This review improves our understanding on trichomes.

The Stem Cell Niche in Leaf Axils Is Established by Auxin and Cytokinin in Arabidopsis.

Plants differ from most animals in their ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems harboring new stem cell niches. In seed plants, this is achieved by axillary meristems, which are established in the axil of each leaf base and develop into lateral branches. Here, we describe the initial processes of Arabidopsis thaliana axillary meristem initiation. Using reporter gene expression analysis, we find that axillary meristems initiate from leaf axil cells with low auxin through stereotypical stages. Consistent with this, ectopic overproduction of auxin in the leaf axil efficiently inhibits axillary meristem initiation. Furthermore, our results demonstrate that auxin efflux is required for the leaf axil auxin minimum and axillary meristem initiation. After lowering of auxin levels, a subsequent cytokinin signaling pulse is observed prior to axillary meristem initiation. Genetic analysis suggests that cytokinin perception and signaling are both required for axillary meristem initiation. Finally, we show that cytokinin overproduction in the leaf axil partially rescue axillary meristem initiation-deficient mutants. These results define a mechanistic framework for understanding axillary meristem initiation.

Auxin depletion from leaf primordia contributes to organ patterning.

Stem cells are responsible for organogenesis, but it is largely unknown whether and how information from stem cells acts to direct organ patterning after organ primordia are formed. It has long been proposed that the stem cells at the plant shoot apex produce a signal, which promotes leaf adaxial-abaxial (dorsoventral) patterning. Here we show the existence of a transient low auxin zone in the adaxial domain of early leaf primordia. We also demonstrate that this adaxial low auxin domain contributes to leaf adaxial-abaxial patterning. The auxin signal is mediated by the auxin-responsive transcription factor MONOPTEROS (MP), whose constitutive activation in the adaxial domain promotes abaxial cell fate. Furthermore, we show that auxin flow from emerging leaf primordia to the shoot apical meristem establishes the low auxin zone, and that this auxin flow contributes to leaf polarity. Our results provide an explanation for the hypothetical meristem-derived leaf polarity signal. Opposite to the original proposal, instead of a signal derived from the meristem, we show that a signaling molecule is departing from the primordium to the meristem to promote robustness in leaf patterning.

An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation.

Gene regulatory networks (GRNs) control development via cell type-specific gene expression and interactions between transcription factors (TFs) and regulatory promoter regions. Plant organ boundaries separate lateral organs from the apical meristem and harbor axillary meristems (AMs). AMs, as stem cell niches, make the shoot a ramifying system. Although AMs have important functions in plant development, our knowledge of organ boundary and AM formation remains rudimentary. Here, we generated a cellular-resolution genomewide gene expression map for low-abundance Arabidopsis thaliana organ boundary cells and constructed a genomewide protein-DNA interaction map focusing on genes affecting boundary and AM formation. The resulting GRN uncovers transcriptional signatures, predicts cellular functions, and identifies promoter hub regions that are bound by many TFs. Importantly, further experimental studies determined the regulatory effects of many TFs on their targets, identifying regulators and regulatory relationships in AM initiation. This systems biology approach thus enhances our understanding of a key developmental process.

HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

Transcriptome analysis of Taraxacum kok-saghyz reveals the role of exogenous methyl jasmonate in regulating rubber biosynthesis and drought tolerance.

Taraxacum kok-saghyz has been identified as one of the most promising alternative rubber crops, with laticifer cells that produce high-quality rubber. To uncover the underlying molecular mechanisms regulating natural rubber biosynthesis under MeJA induction, a reference transcriptome was constructed from nine samples of T. kok-saghyz. MeJA treatment was applied for 0 h (control), 6 h, and 24 h. A total of 7452 differentially expressed genes (DEGs) were identified in response to MeJA stress, relative to the control. Functional enrichment showed that these DEGs were primarily related to hormone signaling, defensive responses, and secondary metabolism. Combined analysis of the DEGs induced by MeJA and high-expression genes in laticifer cells further identified seven DEGs related to natural rubber biosynthesis that were upregulated in latex tissue, suggesting that these candidate genes could prove valuable in studying the mechanism of MeJA-mediated natural rubber biosynthesis. In addition, 415 MeJA-responsive DEGs were from several transcription factor families associated with drought resistance. This study helps to elucidate the mechanism of natural rubber biosynthesis in T. kok-saghyz in response to MeJA stress and identifies key candidate MeJA-induced DEGs in laticifer tissue, as well as a candidate drought-response target gene, whose knowledge will promote the breeding of T. kok-saghyz in the aspect of rubber yields and quality, and drought tolerance.

Genome-wide analysis of the SWEET genes in Taraxacum kok-saghyz Rodin: An insight into two latex-abundant isoforms.

Taraxacum kok-saghyz Rodin (Tk) is a promising alternative rubber-producing grass. However, low biomass and rubber-producing capability limit its commercial application. As a carbon source transporter in plants, sugar will eventually be exported transporters (SWEETs) have been reported to play pivotal roles in diverse physiological events in the context of carbon assimilate transport and utilization. Theoretically, SWEETs would participate in Tk growth, development and response to environmental cues with relation to the accumulation of rubber and biomass, both of which rely on the input of carbon assimilates. Here, we identified 22 TkSWEETs through homology searching of the Tk genomes and bioinformatics analyses. RNA-seq and qRT-PCR analysis revealed these TkSWEETs to have overlapping yet distinct tissue expression patterns. Two TkSWEET isofroms, TkSWEET1 and TkSWEET12 expressed substantially in the latex, the cytoplasm of rubber-producing laticifers as well as the rubber source. As revealed by the transient expression analysis using Tk mesophyll protoplasts, both TkSWEET1 and TkSWEET12 were located in the plasma membrane. Heterologous expressions of the two TkSWEETs in a yeast mutant revealed that only TkSWEET1 exhibited apparent sugar transport activities, with a preference for monosaccharides. Interestingly, TkSWEET12, the latex-predominant TkSWEET isoform, seemed to have evolved from a tandem duplication event that results in a cluster of six TkSWEET genes with the TkSWEET12 therein, suggesting its specialized roles in the laticifers.

Chromosome-level genomes of three key Allium crops and their trait evolution.

Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.

Trehalose 6-Phosphate/SnRK1 Signaling Participates in Harvesting-Stimulated Rubber Production in the Hevea Tree.

Trehalose 6-phosphate (T6P), the intermediate of trehalose biosynthesis and a signaling molecule, affects crop yield via targeting sucrose allocation and utilization. As there have been no reports of T6P signaling affecting secondary metabolism in a crop plant, the rubber tree Hevea brasiliensis serves as an ideal model in this regard. Sucrose metabolism critically influences the productivity of natural rubber, a secondary metabolite of industrial importance. Here, we report on the characterization of the T6P synthase (TPS) gene family and the T6P/SNF1-related protein kinase1 (T6P/SnRK1) signaling components in Hevea laticifers under tapping (rubber harvesting), an agronomic manipulation that itself stimulates rubber production. A total of fourteen TPS genes were identified, among which a class II TPS gene, HbTPS5, seemed to have evolved with a function specialized in laticifers. T6P and trehalose increased when the trees were tapped, this being consistent with the observed enhanced activities of TPS and T6P phosphatase (TPP) and expression of an active TPS-encoding gene, HbTPS1. On the other hand, SnRK1 activities decreased, suggesting the inhibition of elevated T6P on SnRK1. Expression profiles of the SnRK1 marker genes coincided with elevated T6P and depressed SnRK1. Interestingly, HbTPS5 expression decreased significantly with the onset of tapping, suggesting a regulatory function in the T6P pathway associated with latex production in laticifers. In brief, transcriptional, enzymatic, and metabolic evidence supports the participation of T6P/SnRK1 signaling in rubber formation, thus providing a possible avenue to increasing the yield of a valuable secondary metabolite by targeting T6P in specific cells.

Transcript and Protein Profiling Provides Insights Into the Molecular Mechanisms of Harvesting-Induced Latex Production in Rubber Tree.

Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping. Conspicuous stimulation on latex production is observed for the first few tappings conducted on virgin (untapped before) or resting (tapped before but no tapping for a period) rubber trees. To understand the underlying mechanisms, an integrative analysis of the latex transcriptome and proteome was conducted on virgin or resting Hevea trees for the first five tappings. A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified by silver-staining cDNA-AFLP, with 217 exhibiting patterns of upregulated, 180 downregulated and 108 irregularly-regulated. Meanwhile, 117 two dimensional gel electrophoresis DE-protein spots were isolated and subjected to mass spectrometry analysis, with 89 and 57 being successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively. About 72.5% DE-TDFs and 76.1% DE-proteins were functionally annotated and categorized. Noteworthily, most of the DE-TDFs implicated in sugar transport and metabolism as well as rubber biosynthesis were upregulated by the tapping treatment. The importance of sugar metabolism in harvesting-induced latex production was reinforced by the identification of abundant relevant DE-protein spots. About 83.8% of the randomly selected DE-TDFs were validated for expression patterns by semi-quantitative RT-PCR, and an 89.7% consistency for the 29 latex regeneration-related DE-TDFs examined by quantitative RT-PCR analysis. In brief, our results reveal extensive physiological and molecular changes in Hevea laticifers incurred by the tapping treatment, and the vast number of DE genes and proteins identified here contribute to unraveling the gene regulatory network of tapping-stimulated latex production.

Ethylene response factors regulate expression of HbSUT3, the sucrose influx carrier in laticifers of Hevea brasiliensis.

Natural rubber is an important industrial raw material and is commercially produced by rubber trees (Hevea brasiliensis). The sucrose transporter HbSUT3 plays an essential role in rubber production. Its expression in latex (cytoplasm of rubber-producing laticifers) is induced by bark treatment with Ethrel, an ethylene releaser, and the inducing effect correlates well with Ethrel-stimulated rubber yield increase. However, the mechanisms of ethylene induction on HbSUT3 expression are not known. Here, five Ethylene Response Factor (ERF) genes were identified from the cDNA library of Hevea latex by yeast one-hybrid screening with the promoter of HbSUT3 gene as bait. As revealed in a tobacco (Nicotiana tabacum) protoplast transient expression system, these HbERFs were mainly localized in the nucleus and four of them exhibited apparent transactivation activity. Of the five HbERF genes, HbERF-IXc4 was the most frequently screened in yeast one-hybrid, accounting for 65% of the ERF clones obtained. Moreover, among the five HbERFs, HbERF-IXc4 showed the strongest transactivation capacity when expressed in tobacco protoplast, the highest transcript abundance in latex and a close expressional correlation with its target gene, HbSUT3, in response to the Ethrel treatment. Taken together, our results indicate that ERFs, especially HbERF-IXc4, are critically involved in the activation of HbSUT3 expression in latex after Ethrel treatment on Hevea bark, and thus the stimulated latex yield.

Characterization of Sugar Contents and Sucrose Metabolizing Enzymes in Developing Leaves of Hevea brasiliensis.

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants.

BIL1-mediated MP phosphorylation integrates PXY and cytokinin signalling in secondary growth.

Vascular cambium proliferation in plants is crucial for the generation of vascular tissues and for mechanical strength. Phytohormones and mobile peptides are key regulators of vascular cambial activity during secondary growth; however, the signalling cross-talk underlying their coordinated action is largely unknown. Here, we reveal that BIN2-LIKE 1 (BIL1), a glycogen synthase kinase 3, integrates the PHLOEM INTERCALATED WITH XYLEM/tracheary element differentiation inhibitory factor (TDIF) RECEPTOR (PXY/TDR) module into MONOPTEROS/AUXIN RESPONSE FACTOR 5 (MP/ARF5) transcription factor action during secondary growth. BIL1-mediated phosphorylation of MP/ARF5 enhances its negative effect on vascular cambial activity, which upregulates the negative regulators of cytokinin signalling ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7) and ARR15. PXY/TDR inhibits BIL1 activity, which attenuates the effect of MP/ARF5 on ARR7 and ARR15 expression, thus increasing vascular cambial activity. Together, these results suggest that BIL1 is a key mediator that links peptide signalling with auxin-cytokinin signalling for the maintenance of cambial activity.

Spatial specificity of auxin responses coordinates wood formation.

Spatial organization of signalling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signalling but its exact role and spatial organization is obscure. Here we show that, while auxin signalling levels increase in differentiating cambium descendants, a moderate level of signalling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by directly attenuating the activity of the stem cell-promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signalling on distinct cambium features by specific signalling components and allow the conceptual integration of plant stem cell systems with distinct anatomies.

Characterization of the rubber tree metallothionein family reveals a role in mitigating the effects of reactive oxygen species associated with physiological stress.

Metallothioneins (MTs) as reactive oxygen species (ROS) scavengers play important roles in stress response and heavy metal homeostasis. In Hevea brasiliensis (the para rubber tree that is the source of commercial natural rubber) and in other trees, the functions of MTs are not well understood. Latex exudes when the rubber tree is tapped. The flow of latex and its regeneration can be enhanced by tapping, wounding and ethylene treatment, all of which produce ROS as a by-product. Here, we show the presence of four MT genes in H. brasiliensis, comprising three Type 2 (HbMT2, -2a and -2b) and one Type 3 (HbMT3L) isoforms, representing one of the smallest MT gene families among angiosperms. The four HbMTs exhibited distinct tissue expression patterns: HbMT2 and HbMT3L mainly in leaves, HbMT2a specifically in flowers and HbMT2b in diverse tissues. The expression of HbMT2b, an isoform present in latex, decreased significantly in the latex following the stress-inducing treatments of tapping, wounding and ethephon (an ethylene generator). The expressions of the leaf-abundant isoforms, HbMT2 and -3L were up-regulated following pathogenic fungus infection and high-temperature stress, but down-regulated by low-temperature stress. These reactions were consistent with multiple defense- and hormone-responsive cis-acting elements in the HbMT promoters. Nine transcription factors were shown to implicate in the high-temperature responsiveness of HbMT2 and -3L in leaves. Overexpression of HbMT2 in Escherichia coli enhanced the bacterium's tolerance to heavy metals and ROS, consistent with its predicted role as an ROS scavenger. Taken together, our results, along with other relevant studies, suggest an important role of HbMTs in latex regeneration as well as species adaptation via the regulation of ROS homeostasis.

A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree).

Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (-70 degrees C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.

Cell polarity in plants: the Yin and Yang of cellular functions.

Spatial organization is fundamental for the performance of living organisms and is reflected in a distinct distribution of structures and molecules down to the subcellular level. In particular, eukaryotic cells harbor a vast range of possibilities for distributing organelles, the cytoskeleton or the extracellular matrix in an active and highly regulated manner. An asymmetric or polar distribution is rather the rule than the exception and often reflects a particular position or orientation of a cell within a multicellular body. Here, we highlight recent insights into the regulation of cell polarity in plants and reveal the interactive nature of underlying molecular processes.

The SWEET gene family in Hevea brasiliensis - its evolution and expression compared with four other plant species.

SWEET proteins play an indispensable role as a sugar efflux transporter in plant development and stress responses. The SWEET genes have previously been characterized in several plants. Here, we present a comprehensive analysis of this gene family in the rubber tree, Hevea brasiliensis. There are 36 members of the SWEET gene family in this species, making it one of the largest families in plant genomes sequenced so far. Structure and phylogeny analyses of these genes in Hevea and in other species demonstrated broad evolutionary conservation. RNA-seq analyses revealed that SWEET2, 16, and 17 might represent the main evolutionary direction of SWEET genes in plants. Our results in Hevea suggested the involvement of HbSWEET1a, 2e, 2f, and 3b in phloem loading, HbSWEET10a and 16b in laticifer sugar transport, and HbSWEET9a in nectary-specific sugar transport. Parallel studies of RNA-seq analyses extended to three other plant species (Manihot esculenta, Populus trichocarpa, and Arabidopsis thaliana) produced findings which implicated MeSWEET10a, 3a, and 15b in M. esculenta storage root development, and the involvement of PtSWEET16b and PtSWEET16d in P. trichocarpa xylem development. RT-qPCR results further revealed that HbSWEET10a, 16b, and 1a play important roles in phloem sugar transport. The results from this study provide a foundation not only for further investigation into the functionality of the SWEET gene family in Hevea, especially in its sugar transport for latex production, but also for related studies of this gene family in the plant kingdom.

Mechanical regulation of organ asymmetry in leaves.

How appendages, such as plant leaves or animal limbs, develop asymmetric shapes remains a fundamental question in biology. Although ongoing research has revealed the genetic regulation of organ pattern formation, how gene activity ultimately directs organ shape remains unclear. Here, we show that leaf dorsoventral (adaxial-abaxial) polarity signals lead to mechanical heterogeneity of the cell wall, related to the methyl-esterification of cell-wall pectins in tomato and Arabidopsis. Numerical simulations predicate that mechanical heterogeneity is sufficient to produce the asymmetry seen in planar leaves. Experimental tests that alter pectin methyl-esterification, and therefore cell wall mechanical properties, support this model and lead to polar changes in gene expression, suggesting the existence of a feedback mechanism for mechanical signals in morphogenesis. Thus, mechanical heterogeneity within tissue may underlie organ shape asymmetry.