Neighbor Detection Induces Organ-Specific Transcriptomes, Revealing Patterns Underlying Hypocotyl-Specific Growth.

In response to neighbor proximity, plants increase the growth of specific organs (e.g., hypocotyls) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing these bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we separately analyzed gene expression patterns in the major light-sensing organ (cotyledons) and in rapidly elongating hypocotyls of Arabidopsis thaliana PIFs initiate transcriptional reprogramming in both organs within 15 min, comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. We show that cotyledon-derived auxin is both necessary and sufficient to initiate hypocotyl growth, but we also provide evidence for the functional importance of the local PIF-induced response. With time, the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between different developmentally regulated growth processes and responses to the environment in different organs.

YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance.

Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

An expression analysis of the ascorbate biosynthesis enzyme VTC2.

The vtc2 mutant of Arabidopsis thaliana is vitamin C deficient and is defective in the ascorbate biosynthesis enzyme GDP-L-Galactose phosphorylase/L -Galactose guanylyltransferase. The connection between the VTC2 gene and this enzyme has only recently been established, and little is known about the molecular characteristics of the VTC2 gene. In this paper, the expression of the VTC2 gene was characterized on both the RNA and the protein level. The VTC2 gene was expressed in all developmental stages, and the mRNA was expressed at a higher level in green tissues than in the root. VTC2 mRNA expression was induced strongly by exposing dark-grown seedlings to light, to levels higher than found in light-grown seedlings. A VTC2:GUS fusion protein was detected only in green tissues. Unexpectedly, a VTC2:YFP fusion protein was found not only in the cytosol, but also in the nucleus, which suggests that GDP-L-Galactose phosphorylase/L: -Galactose guanylyltransferase might be a dual-function protein, which has both enzymatic and regulatory function.

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants.

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.

Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts.

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress.

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.

Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate-deficient mutant of Arabidopsis.

The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.

PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts.

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.

PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition.

Feedback de-excitation (qE) regulates light harvesting in plants to prevent inhibition of photosynthesis when light absorption exceeds photosynthetic capacity. Although the mechanism of qE is not completely understood, it is known to require a low thylakoid lumen pH, de-epoxidized xanthophylls, and the photosystem II protein PsbS. During a short-term 4-h exposure to excess light, three PsbS- and qE-deficient Arabidopsis thaliana mutants that differed in xanthophyll composition were more photoinhibited than the wild type. The extent of photoinhibition was the same in all of the mutants, suggesting that qE capacity rather than xanthophyll composition is critical for photoprotection in short-term high light, in contrast to longer-term high light conditions (days) when additional antioxidant roles of specific xanthophylls are evident. Plants with a 2-fold increase in qE capacity were generated by overexpression of PsbS, demonstrating that the level of PsbS limits the qE capacity in wild-type Arabidopsis. These results are consistent with the idea that variations in PsbS expression are responsible for species-specific and environmentally induced differences in qE capacity observed in nature. Furthermore, plants with higher qE capacity were more resistant to photoinhibition than the wild type. Increased qE was associated with decreased photosystem II excitation pressure and changes in the fractional areas of chlorophyll a fluorescence lifetime distributions, but not the lifetime centers, suggesting that qE protects from photoinhibition by preventing overreduction of photosystem II electron acceptors. Engineering of qE capacity by PsbS overexpression could potentially yield crop plants that are more resistant to environmental stress.

Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo.

As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from violaxanthin. The enzyme responsible for the conversion of violaxanthin to zeaxanthin is violaxanthin de-epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2. During exposure to high light (1,500 micromol photons m(-2) s(-1)), vtc2-2 plants initially grown in low light (150 micromol photons m(-2) s(-1)) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of violaxanthin to zeaxanthin induced by high light was slower in vtc2-2, and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit violaxanthin de-epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.